Agrobacterium

Proc Natl Acad Sci U S A, 1995 Jan 3, 92(1), 230 - 4
Intracellular Agrobacterium can transfer DNA to the cell nucleus of the host plant; Escudero J et al.; Agrobacterium tumefaciens is a Gram-negative, soil-borne bacterium responsible for the crown gall disease of plants . The galls result from genetic transformation of plant cells by the bacteria . Genes located on the transferred DNA (T-DNA), which is part of the large tumor-inducing (Ti) plasmid of Agrobacterium, are integrated into host plant chromosomes and expressed . This transfer requires virulence (vir) genes that map outside the T-DNA on the Ti plasmid and that encode a series of elaborate functions that appear similar to those of interbacterial plasmid transfer . It remains a major challenge to understand how T-DNA moves from Agrobacterium into the plant cell nucleus, in view of the complexity of obstacles presented by the eukaryotic host cell . Specific anchoring of bacteria to the outer surface of the plant cell seems to be an important prelude to the mobilization of the T-DNA/protein complex from the bacterial cell to the plant cell . However, the precise mode of infection is not clear, although a requirement of wounded cells has been documented . By using a microinjection approach, we show here that the process of T-DNA transfer from the bacteria to the eukaryotic nucleus can occur entirely inside the plant cell . Such transfer is absolutely dependent on induction of vir genes and a functional virB operon . Thus, A . tumefaciens can function as an intracellular infectious agent in plants.

Proc Natl Acad Sci U S A, 1994 Aug 16, 91(17), 8000 - 4
Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus; Tinland B et al.; Transferred DNA (T-DNA) is transferred as a single-stranded derivative from Agrobacterium to the plant cell nucleus . This conclusion is drawn from experiments exploiting the different properties of single- and double-stranded DNA to perform extrachromosomal homologous recombination in plant cells . After transfer from Agrobacterium to plant cells, T-DNA molecules recombined much more efficiently if the homologous sequences were of opposite polarity than if they were of the same polarity . This observation reflects the properties of single-stranded DNA; single-stranded DNA molecules of opposite polarity can anneal directly, whereas single-stranded DNA molecules of the same polarity first have to become double stranded to anneal . Judging from the relative amounts of single- to double-stranded T-DNA derivatives undergoing recombination, we infer that the T-DNA derivatives enter the plant nucleus in their single-stranded form.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1488 - 92
T-DNA transfer to maize cells: histochemical investigation of beta-glucuronidase activity in maize tissues; Shen WH et al.; Agrobacterium tumefaciens is routinely used to engineer desirable genes into dicotyledonous plants . However, the economically important graminaceous plant maize is refractory to tumor induction by inoculation with virulent strains of A . tumefaciens . Currently, the only clearcut evidence for transferred DNA (T-DNA) transport from Agrobacterium to maize comes from agroinfection . To study T-DNA transfer from Agrobacterium to maize cells in a virus-free system, we used here the beta-glucuronidase (GUS; EC 3.2.1.31) gene as a marker . GUS expression was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium carrying the GUS gene . Agrobacterium virulence mutants, incapable of transferring T-DNA to dicot tissue, were shown to be deficient in eliciting GUS expression in maize . Hence, expression of the T-DNA-located GUS gene in maize cells is strictly dependent on Agrobacterium-mediated DNA transfer . Histochemical staining of maize shoots revealed GUS expression located mainly in the leaves and the coleoptile.

Proc Natl Acad Sci U S A, 1991 Dec 1, 88(23), 10426 - 30
virF, the host-range-determining virulence gene of Agrobacterium tumefaciens, affects T-DNA transfer to Zea mays; Jarchow E et al.; The monocotyledonous plant Zea mays does not develop tumors after inoculation with Agrobacterium tumefaciens and is thus defined as nonhost . Agroinfection, Agrobacterium-mediated delivery of maize streak virus, demonstrates that transferred DNA (T-DNA) transfer to the plant does occur . Nopaline-type Agrobacterium strains such as C58 are efficient in the transfer process whereas the octopine-type strain A6 is unable to transfer T-DNA to maize . This phenotypic difference maps to the tumor-inducing (Ti) plasmid but not to the T-DNA . Steps preceding T-DNA transfer, such as attachment and induction of the virulence genes, were shown to take place in the octopine strain . The nopaline-plasmid-specific locus tzs and the octopine-plasmid-specific locus pinF (virH) are not involved in the strain specificity . However, mutations in the virF locus rendered the octopine strain agroinfectious on maize, whereas such virF-defective octopine strains, when complemented by virF on a plasmid, completely lost their agroinfectivity . We propose that VirF, known to increase the host range of the bacteria in other systems, acts as an inhibitor of T-DNA transfer to maize.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9350 - 4
Activity of the Agrobacterium T-DNA transfer machinery is affected by virB gene products; Ward JE Jr et al.; The oriT (origin of transfer) sequence and mob (mobilization) genes of plasmid RSF1010 can functionally replace transfer DNA (T-DNA) borders to generate an RSF1010 intermediate transferable to plants through activities of the tumor-inducing (Ti)-plasmid virulence (vir) genes of Agrobacterium tumefaciens . Because the Ti plasmid virB gene products are hypothesized to form a membrane-localized T-DNA transport apparatus, we investigated whether specific virB genes were needed for RSF1010 transfer . Here we report that transformation of Nicotiana tabacum leaf explants by an RSF1010-derivative plasmid (pJW323) requires the essential virulence genes virB9, virB10, and virB11, consistent with the hypothesis that both the T-DNA and RSF1010 transfer intermediates utilize the same transport machinery . Further, while pJW323 is transferred into plant cells by Agrobacterium strains harboring both pJW323 and pTiA6, the initiation of crown gall tumors (i.e., T-DNA transfer) is greatly suppressed . Coordinate overexpression of the virB9, virB10, and virB11 gene products relieves pJW323-mediated oncogenic suppression and restores tumorigenicity, but does not increase the transfer frequency of pJW323 into plant cells . We propose that the virB9, virB10, and virB11 gene products function coordinately and stoichiometrically to enhance DNA transfer in a fashion specific for the T-DNA intermediate.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6181 - 5
Maize oleosin is correctly targeted to seed oil bodies in Brassica napus transformed with the maize oleosin gene; Lee WS et al.; Oleosins are small hydrophobic abundant proteins localized in the oil bodies of plant seeds . An oleosin gene from the monocotyledonous maize (Zea mays L.) was transferred into the dicotyledonous Brassica napus L . using Agrobacterium-mediated transformation . The maize oleosin gene was placed under the control of either its own promoter/terminator or the promoter/terminator of a Brassica seed storage protein (napin) gene . Southern blot analyses of individual transformed plants suggested that the oleosin gene from either construct was incorporated into the Brassica chromosomes without appreciable structural alterations . The amount of construct incorporated was from 1 to >10 copies per haploid genome, depending on the individual transformant . Maize oleosin mRNA and protein were detected only in the transformants containing the napin gene promoter/terminator constructs; these transformants were studied further . Northern blot analyses of RNA isolated from different tissues and seeds of different developmental stages indicated that the maize oleosin mRNA was present only in the maturing seed . Approximately 1% of the total protein in mature seed was represented by maize oleosin . Subcellular fractionation of the mature seed revealed that 90% or more of the maize oleosin, as well as the Brassica oleosin, was localized in the oil bodies . The results show that a monocotyledonous oleosin possesses sufficient targeting information for its proper intracellular transport in a dicotyledon and also suggest that the napin gene promoter/terminator of Brassica, or equivalent seed storage protein regulatory elements of other plant species, may be used to express genes for the genetic engineering of seed oils.

Proc Natl Acad Sci U S A, 1990 Sep, 87(17), 6684 - 8
Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides; Shimoda N et al.; Most virulence (vir) genes of Agrobacterium tumefaciens that are required for the formation of crown gall tumors are expressed in response to such plant signal molecules as acetosyringone and lignin precursors . The phenolic signals are transduced through a receptor VirA protein in the inner membrane of the bacterial cell . The expression of these genes triggers the transfer of a specific DNA segment, called transferred DNA (T-DNA), from the Ti plasmid to plant cells, and its integration into their nuclear DNA . We show here that a group of aldoses (L-arabinose, D-xylose, D-lyxose, D-glucose, D-mannose, D-idose, D-galactose, and D-talose) can markedly enhance acetosyringone-dependent expression of vir genes when the concentration of acetosyringone is limited (10 microM) but does not enhance the expression of noninducible genes . Likewise, a 2-deoxy-D-glucose, a nonmetabolized sugar, is also effective . When a deletion was introduced into the virA gene in the region encoding the periplasmic portion of the VirA protein, enhancement by glucose disappeared, but vir expression was induced by acetosyringone in this mutant . These results suggest that these sugars directly enhance a signaling process initiated by phenolic inducers that results in an increase in expression of the vir genes.

Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4368 - 72
A nontransformable Triticum monococcum monocotyledonous culture produces the potent Agrobacterium vir-inducing compound ethyl ferulate; Messens E et al.; Exudates of dicotyledonous plants contain specific phenolic signal molecules, such as acetosyringone, which serve as potent inducers for the expression of the virulence (vir) regulon of the phytopathogen Agrobacterium tumefaciens . This induction activates the Agrobacterium T-DNA transfer process to initiate the genetic transformation of target plant cells . Wounded and metabolically active plant cells are particularly susceptible to Agrobacterium infection, and these cells specifically produce vir-inducing molecules . Most monocotyledonous, as opposed to dicotyledonous, species are resistant to Agrobacterium transformation . One hypothesis for this resistance is that nonsusceptible monocotyledonous cells fail to produce vir signal molecules and, thus, are not recognized by Agrobacterium as transformation targets . Here we demonstrate that monocotyledonous cells make such molecules, and, furthermore, we purify the inducer produced by a Triticum monococcum suspension culture that is resistant to Agrobacterium infection . This molecule is shown to correspond to ethyl ferulate {C12H14O4; 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid ethyl ester}, to be more active for vir induction at low concentrations than acetosyringone, and to be produced in quantities giving significant levels of induction . Thus, at least for the wheat cell line used in this study, monocotyledonous resistance to Agrobacterium transformation must result from a block to a step of the T-DNA transfer process subsequent to vir induction.

Proc Natl Acad Sci U S A, 1990 May, 87(10), 3879 - 83
Corn metabolites affect growth and virulence of Agrobacterium tumefaciens; Sahi SV et al.; Homogenates of corn seedlings inhibit both growth of Agrobacterium tumefaciens and induction of its Ti plasmid virulence (vir) genes by acetosyringone (AS) . The heat-labile inhibitor has been identified as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), present in 2-week-old seedlings (B73) at a concentration of 1.5 mM or greater . A concentration of 0.3 mM DIMBOA is sufficient to block growth of A . tumefaciens completely for 220 hr . DIMBOA at 0.1 mM concentration completely inhibited vir gene induction by 100 microM AS and reduced growth rate by 50% . Thus, DIMBOA can be expected to have a significant effect on attempts to transform corn by using A . tumefaciens as a vector.

J Exp Bot, 2001 Nov, 52(364), 2089 - 95
An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens; Le VQ et al.; An efficient and reproducible procedure for the transformation of white spruce (Picea glauca {Moench} Voss) embryogenic tissues was developed using A . tumefaciens-mediated gene transfer . Rapidly dividing white spruce embryogenic tissues were co-cultivated with disarmed A . tumefaciens strains containing additional copies of the virulence regions from plasmid PToK47 . The plasmid pBi121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the beta-glucuronidase (uidA) reporter gene, was used as binary vector . The highest frequency of transformation (15 transformed tissues g(-1) FW of treated embryogenic tissue) was obtained with 5-d-old tissues grown in liquid medium and co-cultivated with Agrobacterium for 2 d in the same medium but containing 50 microM acetosyringone . Recovery of kanamycin-resistant tissues was improved when tissues were first grown for 10 d on a timentin-containing medium (400 mg l(-1)), to prevent bacterial overgrowth, before application of the selection pressure . After 6 weeks on kanamycin-selection medium, resistant tissues were obtained and showed stable uidA expression . The presence of the transgenes was demonstrated by PCR analysis and their integration into the genome was confirmed by Southern hybridization . Transgenic plants were regenerated from transformed tissues within 4 months after co-culture.

Transgenic Res, 2001 Aug, 10(4), 363 - 71
Transgenic radish (Raphanus sativus L . longipinnatus Bailey) by floral-dip method--plant development and surfactant are important in optimizing transformation efficiency; Curtis IS et al.; Transgenic radish (Raphanus sativus L . longipinnatus Bailey) plants were produced from the progeny of plants which were dipped into a suspension of Agrobacterium carrying both the beta-glucuronidase (gusA) gene and a gene for resistance to the herbicide Basta (bar) between T-DNA border sequences . The importance of development of the floral-dipped plant and presence of surfactant in the inoculation medium were evaluated in terms of transgenic plant production . Plants dipped at the primary bolt stage of growth, into a suspension of Agrobacterium containing 0.05% (v/v) Silwet L-77 resulted in optimum transformation efficiency, with 1.4% from 1110 seeds . The presence of Pluronic F-68 or Tween 20 in the inoculation medium was beneficial towards transgenic plant output compared to treatments without surfactant . Putative transformed T1 plants were efficiently selected by spraying with 0.03% (v/v) Basta and all herbicide-resistant plants tested positive for GUS activity when analysed both histochemically and fluorometrically . Southern analysis revealed that both the gusA and bar genes integrated into the genome of transformed plants and segregated as dominant Mendelian traits . These results demonstrate that radish can be genetically modified for the improvement of this important vegetable crop.

J Bacteriol, 2001 Nov, 183(21), 6234 - 43
Identification of the partitioning site within the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1; Bartosik D et al.; The replicator region of composite plasmid pTAV1 of Paracoccus versutus (included in mini-replicon pTAV320) belongs to the family of repABC replicons commonly found in plasmids harbored by Agrobacterium and Rhizobium spp . The repABC replicons encode three genes clustered in an operon, which are involved in partitioning (repA and repB) and replication (repC) . In order to localize the partitioning site of pTAV320, the two identified incompatibility determinants of this mini-replicon (inc1, located in the intergenic sequence between repB and repC; and inc2, situated downstream of the repC gene) were PCR amplified and used together with purified RepB fusion protein (homologous to the type B partitioning proteins binding to the partitioning sites) in an electrophoretic mobility shift assay . The protein bound only inc2, forming two complexes in a protein concentration-dependent manner . The inc2 region contains two long (14-bp) repeated sequences (R1 and R2) . Disruption of these sequences completely eliminates RepB binding ability . R1 and R2 have sequence similarities with analogous repeats of another repABC replicon of plasmid pPAN1 of Paracoccus pantotrophus DSM 82.5 and with centromeric sequences of the Bacillus subtilis chromosome . Excess RepB protein resulted in destabilization of the inc2-containing plasmid in Escherichia coli . On the other hand, the inc2 region could stabilize another unstable replicon in P . versutus when RepA and RepB were delivered in trans, proving that this region has centromere-like activity . Thus, it was demonstrated that repA, repB, and inc2 constitute a functional system for active partitioning of pTAV320.

Tsitol Genet, 2001 Jan-Feb, 35(1), 22 - 7
{Changes of gene expression in Solanum tuberosum L . as a result of transgenes}; Totskii VN et al.; Potato plants of various cultivars, transformed using Agrobacterium tumefaciens with pGV941 plasmid, differed from control plants in glyphosate herbicide tolerance, tryptophane content, intensity of callusogenesis, microtuber formation in vitro and multimolecular forms of peroxidase (EC 1.11.1.7) and superoxide dismutase (EC 1.15.1.11) . The results demonstrate the influence of alien DNA on structural gene expression in transgenic plants.

Yi Chuan Xue Bao, 2001, 28(9), 877 - 83
{Genetic analysis of T1 progeny of transgenic tobacco with metallothionein gene and metallothionein domain mutant alpha alpha gene}; Zhang XY et al.; The chimeric gene containing a cloned mouse metallothionein processed gene or a cloned mouse metallothionein domain mutant alpha alpha gene was respectively introduced into tobacco (Nicotiana tobacum L . cv . NC89) on a disarmed Ti-plasmid of Agrobacterium tumefaciens . T1 seeds from self-fertilized transgenic tobacco were germinated on media containing cadmium or herbicide PPT . The PPT tolerance trait followed Mendelian inheritance and co-segregated with heavy metal tolerance . Meanwhile Southern blot and Western blot verified the existence of the MT gene and alpha alpha mutant gene in the T1 generation plants which keep tolerance to heavy metal . All the results demonstrated the stable integration and inheritance of exotic genes . In addition, assay of the root length and fresh weight of T1 seedlings indicate that transgenic tobacco plants with alpha alpha mutant gene still have a little higher tolerance than that with matural MT gene.

Mol Microbiol, 2001 Sep, 41(6), 1283 - 93
The carboxy-terminus of VirE2 from Agrobacterium tumefaciens is required for its transport to host cells by the virB-encoded type IV transport system; Simone M et al.; Agrobacterium tumefaciens transfers DNA from the resident 'tumour-inducing' (Ti) plasmid into plant cells, where it can be stably integrated into the plant genome, ultimately resulting in crown gall tumour formation . The mobilized DNA molecule is a single-stranded intermediate with VirD2 covalently bound to its 5' end . Successful transport of the transferred DNA (T-DNA) and integration of the DNA into the genome requires that additional proteins be transported to the plant as well, including the single-stranded (ss)DNA-binding protein, VirE2 . The transport of these two different substrates occurs as a result of the activities of a type IV secretion system encoded by the virB operon . Although the substrates have been identified, the mechanism of their transport remains unknown . In the experiments described here, a region in one of these substrates, VirE2, necessary for transport is identified . The addition of a C-terminal FLAG epitope tag to VirE2, or the deletion of its C-terminal 18 amino acids, renders it non-functional in A . tumefaciens . However, transgenic plants expressing either of these virE2 genes respond to virE2 mutants of A . tumefaciens by forming wild-type tumours . These results indicate that this region of VirE2 is necessary for the protein to be transported into the plant cells, but is not necessary for its function within the plant . Additionally, these studies demonstrate that mutant forms of VirE2 lacking this region do not disrupt the activities of the VirB transporter and support the hypothesis that VirE2 and the VirD2 T-strand are transported independently, even when they co-exist in the same cell.

Phytochemistry, 2001 Oct, 58(4), 595 - 8
Flavonoids from Glycyrrhiza pallidiflora hairy root cultures; Li W et al.; Glycyrrhiza pallidiflora hairy roots were induced from axenic young plants by direct infection with Agrobacterium rhizogenes . The chemical constituents were then investigated after mass culture . The isoflavone, licoagroisoflavone and the coumestan, licoagroside C, were isolated along with seven known flavonoids . Their structures were determined on the basis of spectroscopic evidence.

Plant Mol Biol, 2001 Aug, 46(6), 695 - 703
A library of Arabidopsis 35S-cDNA lines for identifying novel mutants; LeClere S et al.; We have developed a system to over-express or co-suppress random cDNAs in Arabidopsis thaliana upon Agrobacterium tumefaciens-mediated transformation . We constructed a binary vector containing a novel Arabidopsis cDNA library driven by the cauliflower mosaic virus (CaMV) 35S promoter . The vector, 35SpBARN, offers in terra selection with glufosinate ammonium (BASTA) and the ability to identify the cDNA insert using PCR with flanking primers . We introduced this overexpression library into Arabidopsis and selected over 30,000 transformants . A random sample of 50 T1 plants was analyzed to determine the quality of the cDNA library in planta . About 90% of T1 plants in the collection have inserts, the average insert size is ca . 1.1 kb, and ca . 43% of these inserts appear to encode full-length proteins . T1 plants were screened for visible abnormalities, and one mutant, V5, was chosen for further study . This mutant displays a pale green phenotype, and its transgene contains a partial petH cDNA encoding chloroplast ferredoxin-NADP+ reductase (EC 1.18.1.2) . This construct co-suppresses the endogenous petH transcript . We recapitulated the mutant phenotype by expressing either the full-length or truncated petH cDNA from the CaMV 35S promoter in wild-type Arabidopsis . Our results indicate that co-suppressing endogenous genes can cause dominant phenotypes as expected . As we have also used the 35SpBARN vector to successfully over-express other transcripts in planta, we predict that this system will be generally useful for identifying genes that yield phenotypes upon over-expression as well.

DNA Res, 2001 Aug 31, 8(4), 141 - 52
Genome analysis of Agrobacterium tumefaciens: construction of physical maps for linear and circular chromosomal DNAs, determination of copy number ratio and mapping of chromosomal virulence genes; Suzuki K et al.; The phytopathogenic bacterium Agrobacterium tumefaciens is unique in that it possesses both linear and circular DNA chromosomes in addition to a plant-tumor-inducing (Ti) plasmid . We analyzed the two chromosomal DNA molecules in strain MAFF301001, whose Ti plasmid has already been sequenced completely . Physical maps of the chromosomal DNAs were constructed by Southern hybridization experiments using Pme I and Swa I fragments and short fragments bridging the Swa I fragments with special care to avoid any missing fragment . Hybridization with 16S rDNA probe showed one rDNA locus on the linear chromosome and two loci on the circular chromosome . For this bacterium to be pathogenic, not only Ti plasmid but also chromosomal genes are required . The chromosomal virulence (chv) genes (chvA, chvB, chvD, chvE, chvG, chvH, and chvI) and the chromosomal genes affecting the virulence {acvB, pgm(exoC), glgP, miaA, and ros} were successfully mapped onto 5 different regions in the chromosomal physical maps . These chv genes and the chromosomal genes affecting the virulence other than pgm and glgP were found on the circular chromosome, whereas the pgm and glgP genes were located on the linear chromosome . In contrast to the large terminal inverted repeats of Streptomyces linear chromosomal DNA, no hybridization signal was detected between left and right terminal fragments of the linear A . tumefaciens chromosome . Quantitative analysis of DNA fragments indicated that the copy numbers of the two chromosomal DNAs and the Ti plasmid are identical.

C R Acad Sci III, 2001 Oct, 324(10), 915 - 22
{Current concepts on the pathogenicity of phytopathogenic bacteria}; Boucher C et al.; What are the molecular determinants that make a bacterium a plant pathogen? In the last 10-20 years, important progress has been made in answering this question . In the early 20th century soon after the discovery of infectious diseases, the first studies of pathogenicity were undertaken . These early studies relied mostly on biochemistry and led to the discovery of several major pathogenicity determinants, such as toxins and hydrolytic enzymes which govern the production of major disease symptoms . From these pioneering studies, a simplistic view of pathogenicity arose . It was thought that only a few functions were sufficient to transform a bacterium into a pathogen . This view rapidly changed when modern techniques of molecular genetics were applied to analyse pathogenicity . Modern analyses of pathogenicity determinants took advantage of the relatively simple organization of the haploid genome of pathogenic bacteria . By creating non-pathogenic mutants, a large number of genes governing bacterium-host interactions were identified . These genes are required either for host colonization or for the production of symptoms . Even though the role of motility and chemotaxis in these processes is still unclear, it is clear that a strong attachment of Agrobacterium to plant cells is a prerequisite for efficient plant transformation and disease . Other important pathogenicity factors identified with a molecular genetic approach include hydrolytic enzymes such as pectinases and cellulases which not only provide nutrients to the bacteria but also facilitate pathogen invasion into host tissues . The precise role of exopolysaccharide in pathogenicity is still under discussion, however it is has been established that it is crucial for the induction of wilt symptoms caused by Ralstonia solanacearum . Trafficking of effector proteins from the invading bacterium into the host cell emerged recently as a new central concept . In plant pathogenic bacteria, protein translocation takes place through the so-called 'type II secretion machinery' encoded by hrp genes in the bacterium . These genes are present in representatives of all the major groups of Gram negative plant pathogenic bacteria except Agrobacterium . Most of these genes have counterparts in pathogens of mammals (including those of human) and they also play a central role in pathogenicity . Additionally, recent evidence suggests that a 'type IV secretion machinery' injects bacterial proteins into host cells . This machinery, originally found to be involved in the transfer of t-DNA from Agrobacterium into plant cells, was recently shown to translocate pathogenicity proteins in pathogens of mammals such as Helicobacter pylori and Brucella . Discovery of the trafficking of proteins from the pathogen into host cells revolutionized our conception of pathogenicity . First, it rather unexpectedly established the conservation of basic pathogenicity strategies in plant and animal pathogens . Second, this discovery changes our ideas about the overall strategy (or mechanism) of pathogenicity, although we still think the end result is exploitation of host cell nutritive components . Rather than killing the host cell from outside, we envision a more subtle approach in which pathogens inject effector proteins into the host cell to effect a change in host cell biology advantageous to the pathogen . Identification of the effector proteins, of their function and of the corresponding molecular targets in the host is a new challenge which will contribute to the conception of new strategies to control diseases.

C R Acad Sci III, 2001 Oct, 324(10), 905 - 14
{Discovery of phytopathogenic bacteria 100 years ago: transatlantic controversies and polemics}; Paulin JP et al.; The demonstration of a bacterial cause of some plant diseases has been claimed few years after it was commonly recognized that bacteria were able to cause diseases of human and animal . Nevertheless, some sharp controversies took place, between German and American specialists (1897-1901), before the existence of bacterial diseases of plants was accepted by all phytopathologists . Nowadays, about 350 bacteria are described, which infect plants: they are pathovars, or subspecies, belonging to 21 genera . Bacterial diseases of plants can be classified into three major categories according to the type of symptoms shown by the infected plant: necrosis and wilt, soft-rot, tumour . The interaction between bacteria and plant cells is usually established from the apoplast, although some bacteria are xylem or phloem limited . This interaction involves an original protein secretion system (which is also described in bacteria pathogenic for animals), hydrolytic enzymes (pectinases, cellulases), toxins and/or phytohormones . Bacteria of one group (Agrobacterium) modify the plant metabolism after gene transfer from a plasmid . On the economic and social point of view, these diseases may be limiting factors of some key-productions (rice, cassava) . In addition, they play a role in reducing the quality of agricultural products (reduced growth, spots on leaves and fruits) . Control of bacterial diseases is limited . It relies usually on a combination of prophylaxy, chemical applications, and use of resistant genotypes.

J Bacteriol, 2001 Oct, 183(20), 5813 - 25
Role of Agrobacterium VirB11 ATPase in T-pilus assembly and substrate selection; Sagulenko E et al.; The VirB11 ATPase is a subunit of the Agrobacterium tumefaciens transfer DNA (T-DNA) transfer system, a type IV secretion pathway required for delivery of T-DNA and effector proteins to plant cells during infection . In this study, we examined the effects of virB11 mutations on VirB protein accumulation, T-pilus production, and substrate translocation . Strains synthesizing VirB11 derivatives with mutations in the nucleoside triphosphate binding site (Walker A motif) accumulated wild-type levels of VirB proteins but failed to produce the T-pilus or export substrates at detectable levels, establishing the importance of nucleoside triphosphate binding or hydrolysis for T-pilus biogenesis . Similar findings were obtained for VirB4, a second ATPase of this transfer system . Analyses of strains expressing virB11 dominant alleles in general showed that T-pilus production is correlated with substrate translocation . Notably, strains expressing dominant alleles previously designated class II (dominant and nonfunctional) neither transferred T-DNA nor elaborated detectable levels of the T-pilus . By contrast, strains expressing most dominant alleles designated class III (dominant and functional) efficiently translocated T-DNA and synthesized abundant levels of T pilus . We did, however, identify four types of virB11 mutations or strain genotypes that selectively disrupted substrate translocation or T-pilus production: (i) virB11/virB11* merodiploid strains expressing all class II and III dominant alleles were strongly suppressed for T-DNA translocation but efficiently mobilized an IncQ plasmid to agrobacterial recipients and also elaborated abundant levels of T pilus; (ii) strains synthesizing two class III mutant proteins, VirB11, V258G and VirB11.I265T, efficiently transferred both DNA substrates but produced low and undetectable levels of T pilus, respectively; (iii) a strain synthesizing the class II mutant protein VirB11.I103T/M301L efficiently exported VirE2 but produced undetectable levels of T pilus; (iv) strains synthesizing three VirB11 derivatives with a four-residue (HMVD) insertion (L75.i4, C168.i4, and L302.i4) neither transferred T-DNA nor produced detectable levels of T pilus but efficiently transferred VirE2 to plants and the IncQ plasmid to agrobacterial recipient cells . Together, our findings support a model in which the VirB11 ATPase contributes at two levels to type IV secretion, T-pilus morphogenesis, and substrate selection . Furthermore, the contributions of VirB11 to machine assembly and substrate transfer can be uncoupled by mutagenesis.

Biomol Eng, 2001 Oct 15, 18(3), 87 - 94
Production of secretory IgA antibodies in plants; Larrick JW et al.; Functional antibodies produced in tobacco plants were first reported over a decade ago (1989) . The basic protocol used to generate these 'plantibodies' involved the independent cloning of H and L chain antibody genes in Agrobacterium tumefaciens vectors, the transformation of plant tissue in vitro with the recombinant bacterium, the reconstitution of whole plants expressing individual chains, and their sexual cross . In a 'Mendelian' fashion, a fully assembled and functional antibody was recovered from plant tissue in some double-transgenic plants . In mammalian cells, the antibody H and L chains are produced as precursor proteins that are translocated into the endoplasmic reticulum (ER), under the guidance of signal sequences . Within the ER, the signal peptides are proteolytically cleaved, and several stress proteins act as chaperonins to bind the unassembled antibody chains, and direct subsequent folding and tetramer formation . A similar process occurs in plant cells, and expression can be directed via signal sequences (even of foreign origin) into the aqueous environment of the apoplasm, or to be accumulated in other specific plant tissues, including tubers, fruit, or seed . Plants can facilely assemble secretory IgA, which is comprised of four chains, H and L chains, J chain and secretory component . Plant 'bioreactors' are expected to yield over 10 kg of therapeutic antibody/acre in tobacco, maize, soybean, and alfalfa {(Ann . NY Acad . Sci.)721(1994)235; (Biotechnol . Bioeng.)20(1999)135} . Compared with conventional steel tank bioreactors using mammalian cells, or microorganisms, the costs of GMP plantibodies are expected to perhaps one tenth . The differences in glycosylation patterns of plant and mammalian cell produced antibodies apparently have no effect on antigen-binding or specificity, but there is some concern about potential immunogenicity in humans . N-linked glycans of plants differ from human by having fucose-linked alpha 1,3 and the sugar xylose . No adverse effects or human anti-mouse antibodies (HAMA) have been observed in >40 patients receiving topical oral application of a plant produced secretory IgA specific to Streptococcus mutans, for the control of caries {(Nat . Med.)4(1998)601} . The progressive improvement of expression vectors for plantibodies, and purification strategies, as well as the increase in transformable crop species, is expected to lead to almost limitless availability of inexpensive (even edible forms of) recombinant immunoglobulins free of human pathogens for human and animal therapy, and for novel industrial applications (e.g . catalytic antibodies).

J Biotechnol, 2001 Oct 4, 91(2-3), 223 - 36
Characterisation of Phaseolus symbionts isolated from Mediterranean soils and analysis of genetic factors related to pH tolerance; Priefer UB et al.; The ultimate objective of PhIMED, in which two European (Germany, Italy) and two Mediterranean (Morocco, Egypt) countries collaborate, is to improve the cultivation of French bean (Phaseolus vulgaris) under arid and semi-arid conditions by analysing and enhancing stress tolerance of the nitrogen fixing rhizobial microsymbionts . Rhizobial strains nodulating P . vulgaris (RP strains) isolated from areas in Morocco frequently subjected to drought were analysed for their salt and pH tolerance and their phylogenetic relationship . Strain RP163, exhibiting high nodulation efficiency and a broad pH tolerance was mutagenised by Tn5 and mutants unable to grow on extreme pH media were isolated . Some of the mutants affected in low pH tolerance were found to be mutated in genes related to cobalmin biosynthesis and in succinate dehydrogenase (sdhA) . In a parallel approach, promoters and genes inducible under extreme pH values were identified in Rhizobium leguminosarum bv . viciae VF39, among them gabT, which encodes the GABA transaminase and which is induced under acidic conditions . The same gene is present and similarly regulated in RP163 . The actSR gene region was cloned from VF39, sequenced and mutants generated in this region were found to be impaired in growth at low pH, but also under neutral conditions . The Agrobacterium rhizogenes 'promintron' promoter, reported to be activated in stationary phase, was found to be also strongly induced under acidic conditions in rhizobia and it is currently being characterised to construct a system allowing the expression of stress tolerance genes in bacteroids and free-living bacteria.

J Biotechnol, 2001 Oct 4, 91(2-3), 155 - 68
Genetic diversity of rhizobia isolated from Astragalus adsurgens growing in different geographical regions of China; Gao J et al.; The genetic diversity among 95 isolates from Astragalus adsurgens was investigated using molecular biological methods . All of the isolates and 24 reference strains could be differentiated by AFLP, REP-, ERIC- and BOX-PCR fingerprinting analysis . By cluster analysis of the data, 31 AFLP and 38 Rep-PCR genomic groups were delineated, indicating considerable genetic diversity among the isolates . Fifty-four representative strains were further analyzed by RFLP of PCR-amplified 16S and 23S rDNA, revealing 26 rDNA genotypes among the isolates . The phylogenetic relationship of the isolates was determined by partial sequencing of 16S rRNA genes of 16 strains . The results suggest that the A . adsurgens rhizobia belong to the genera Agrobacterium, Mesorhizobium, Rhizobium and Sinorhizobium.

J Gen Virol, 2001 Oct, 82(Pt 10), 2549 - 58
Cloning and sequence analysis of an infectious clone of Citrus yellow mosaic virus that can infect sweet orange via Agrobacterium-mediated inoculation; Huang Q et al.; Citrus yellow mosaic virus (CYMV), a member of the family Caulimoviridae, genus Badnavirus, causes citrus mosaic disease, a disease that occurs commonly in India . The CYMV genome has been cloned and its complete nucleotide sequence determined . Its DNA genome is 7559 bp in length and contains six putative open reading frames (ORFs), all on the plus-strand of the genome and each capable of encoding proteins with a molecular mass of greater than 10 kDa . ORF 3, the largest ORF, encodes a putative polyprotein for functions involved in virus movement, assembly and replication . The other ORFs encode proteins whose exact functions are not completely understood . The genome also contains a plant tRNA(met)-binding site, which may serve as a primer for minus-strand DNA synthesis, in its intergenic region . Phylogenetic analysis of the badnaviruses revealed that CYMV is most closely related to Cacao swollen shoot virus . It was demonstrated that a construct containing 1.4 copies of the cloned CYMV genome could infect sweet orange via Agrobacterium-mediated inoculation.

J Interferon Cytokine Res, 2001 Aug, 21(8), 595 - 602
Expression of two subtypes of human IFN-alpha in transgenic potato plants; Ohya K et al.; Plant expression systems have advantages over other in vitro expression systems in terms of low production costs and low risk of contamination by animal viruses or bacterial endotoxins . In this study, cDNA encoding two subtypes of human interferon-alpha2b and 8 (HuIFN-alpha2b and HuIFN-alpha8) were introduced into potato plants (Solanum tuberosum) using Agrobacterium-mediated transformation . Transcription and translation of the inserted HuIFN-alpha cDNA were confirmed by Northern blot analysis and ELISA, respectively . Bioactivity of the products was assayed by inhibition of vesicular stomatitis virus (VSV) replication on a human amniotic cell line . However, because of the presence of substances in potato tissue extracts that were toxic to animal cells, successful demonstration of IFN bioactivity in the transformants was achieved only after removal of such substances by dialysis . The maximum level of IFN activity in plant extracts was 560 IU/g of tissue . These results indicated that the HuIFN-alpha gene introduced into the potato plant was correctly translated and transcribed in plant cells . This report for the first time shows that biologically active animal cytokines with potential pharmaceutical applications could be expressed in transgenic potato plants.

Genetika, 2001 Jul, 37(7), 888 - 92
{Cloning and identification of an Agrobacterium radiobacter 5D-1 chromosome fragment involved in control of nitrogen metabolism, biosynthesis of indolylacetic acid and replication of ColE1 plasmids}; Kameneva SV et al.; Pleiotropic chromosomal mutations were earlier identified in saprophytic associative bacterium Agrobacterium radiobacter 5D-1 . The mutations changed nitrogen metabolism, disturbed synthesis of indolylacetic acid (IAA), and conferred the ability to sustain replication of ColE1 plasmid derivatives, which are not normally maintained in bacteria other than Escherichia . The mutations were designated Nr (Nitrogen metabolism) and assigned to a single cluster on an A . radiobacter genetic map . A 420-bp fragment AGH23.1.1 was cloned from an agrobacterial genomic library . Introduced in the Nr mutants as a part of a pUC18-based recombinant plasmid, the AGH23.1.1 fragment complemented the Nr mutations with respect to nitrogen metabolism and IAA biosynthesis, but transformants still sustained replication of ColE1 plasmids . Transformation with the linear AGH23.1.1 fragment was due to substitution of a mutant allele of the nr gene with its wild-type counterpart as a result of recombination and completely restored the wild type in the Nr mutants, including the inability to maintain ColE1 plasmids . The AGH23.1.1 fragment and its flanking regions were sequenced . The established sequence was shown to contain two open reading frames (ORFs) coding for proteins with unknown functions . Thus, the cloned fragment contained a gene(s) that controls nitrogen metabolism and IAA synthesis and prevent replication of ColE1 plasmids in A . radiobacter cells . Possible variants of the genetic control of these processes are considered.

Arch Virol, 2001 Jul, 146(7), 1337 - 53
Transgenic resistance in potato plants expressing potato leaf roll virus (PLRV) replicase gene sequences is RNA-mediated and suggests the involvement of post-transcriptional gene silencing; Vazquez Rovere C et al.; Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant . Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants . However, details of the protecting mechanism were not reported, so far . The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively . To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv . Kennebec) by Agrobacterium tumefaciens-mediated transformation . Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated . Field trial infection confirmed that resistant transgenic events were obtained . Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.

Mol Microbiol, 2001 Sep, 41(5), 1173 - 85
Co-evolution of the agrocinopine opines and the agrocinopine-mediated control of TraR, the quorum-sensing activator of the Ti plasmid conjugation system; Oger P et al.; Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is controlled by a hierarchical system in which opines, substrates produced by crown gall tumours, induce a quorum-sensing system . The cascade results from the control of expression of traR, the quorum-sensing activator, by a regulator responsive to the opine . In the two cases studied to date, the gene arrangements responsible for the cascade differ remarkably, suggesting that considerable diversity exists among the many Ti-like plasmids in the agrobacteria . In this study, we demonstrated that the novel Ti plasmid pTiChry5 is induced to transfer at high frequency by extracts from tumours initiated by strain Chry5 . The purified inducer had the chemical and biological properties of agrocinopines C and D, a set of sugar phosphodiester opines known to induce transfer of another Ti plasmid, pTiBo542 . The T-region of pTiChry5 contained a gene whose product, called Acs(Chry5), is virtually identical to the agrocinopine C+D synthase from the T-region of pTiBo542 . The two genes are less closely related to acs of pTiC58, which is responsible for the production of agrocinopines A+B, a similar but not identical set of phosphodiester opines by tumours induced by strain C58 . Agrocinopines A+B induce transfer of pTiC58 but did not induce transfer of pTi(Chry5) . A single copy of traR was identified at the 11 o'clock region of pTi(Chry5), where it is part of a two-gene operon called arc(Chry5) . Although altered by deletions, arc(Chry5) is related to the five-gene arc operon that controls the expression of traR on pTiC58 . Expression of traR(Chry5) was induced by agrocinopines C+D and the opines isolated from Chry5 tumours but not by agrocinopines A+B . A mutation in traR(Chry5) abolished transfer, and transfer was restored by complementation in trans . We conclude that the agrocinopine opines and the corresponding opine-meditated conjugal regulatory regions of pTiChry5 and pTiC58 share a common origin, but that the opine signals for the two Ti plasmids have evolved divergently through changes in the opine synthase enzymes . The alterations in the opines, in turn, necessitated a co-evolutionary change in the opine recognition systems responsible for controlling expression of the traR genes on these two types of Ti plasmids.

Infect Immun, 2001 Oct, 69(10), 6495 - 502
Intracellular induction of the Bartonella henselae virB operon by human endothelial cells; Schmiederer M et al.; One of the more recently identified bacterial exportation systems is the type IV secretion mechanism, which is characterized by a multiprotein complex that spans the inner and outer bacterial membranes and contains a pilin component . The most thoroughly studied type IV secretion system is encoded by the virB operon of Agrobacterium tumefaciens . In Bartonella henselae, 8 of the 10 virB operon genes share extensive homology and arrangement with the virB operon of A . tumefaciens . Sequencing of the region upstream of the B . henselae virB2 gene revealed a region with sequence homology to the vir box of A . tumefaciens . This possible promoter region was cloned upstream of the green fluorescent protein reporter gene in the promoterless vector pANT3 and used to transform B . henselae . Minimal reporter gene expression was seen in the transformed bacteria cultivated in the absence of host cells, but expression was strongly induced in intracellular bacteria cultivated with human microvascular endothelial cells . Deletion of an 87-bp fragment, which contained the putative vir box from the 5' end of the promoter region, diminished intracellular induction of the reporter gene . Host cell induction of the 17-kDa antigen gene, which replaces virB5 in B . henselae, was also demonstrated at the protein level using specific antiserum . Thus, expression of the virB genes of B . henselae is induced in bacteria, which have invaded host cells, through a mechanism that may be similar to the environment-sensing mechanism found in the virB operon of A . tumefaciens.

Genome, 2001 Aug, 44(4), 549 - 58
Molecular variation in plant cell populations evolving in vitro in different physiological contexts; Bogani P et al.; Previous work has shown the fixation of context-specific random amplified polymorphic DNA (RAPD) patterns in tomato cell cultures grown for 2 years in different hormonal contexts . In this work, RAPD sequences were characterised and RAPD-derived molecular markers used for a further study of variation between and within auto- and auxo-trophic tomato cultures grown in different hormonal equilibria . Results were then compared with those obtained using microsatellite markers located in noncoding regions of differentiation- and hormone-related genes and with those obtained with the external transcribed spacer (ETS) from tomato rDNA . Hybridisation of RAPDs on a tomato genomic DNA bank, or on total DNA after enzymatic digestion, suggested that the markers were repetitive in nature . Sequence analysis . however, showed that the homology between different fragments was due mainly to the presence of homo-AT nucleotide stretches . Moreover, a series of computational methods, such as an information-theory algorithm coupled with AG estimates, suggested that the RAPD fragments isolated in our experiments are noncoding . The amplification of SSR-containing RAPD-derived markers, and of other SSRs located in noncoding regions of tomato functional genes, consistently showed polymorphism between auxo- and auto-trophic somaclones (the latter being either habituated or transgenic for Agrobacterium tumefaciens oncogenes) but not within these same clones . Differences were also found between auxotrophic clones and the differentiated tissue . These findings were confirmed by restriction fragment length polymorphism (RFLP) analysis with the REII repetitive element of the ETS from tomato rDNA, which was isolated during this study . The results obtained suggest a possible role for physiological context in the selection of RAPD patterns during the evolution of tomato cells with different endogenous hormonal equilibria . The results are discussed in terms of a possible role for variation in noncoding regions of hormone-related genes in the adaptation to different physiological contexts.

J Gravit Physiol, 1997 Oct, 4(3), 5 - 14
Agravitropic behaviour of roots of rapeseed (Brassica napus L.) transformed by Agrobacterium rhizogenes; Odegaard E et al.; Transgenic hairy roots of Brassica napus (cv . Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots . The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes . The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia . Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected . The transformed nature of the roots was confirmed by Southern blot analyses . The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings . While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground . The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy . At the ultrastructural level no major differences could be detected between the roots studied . There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type . Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.

Planta, 1996 Sep, 200(1), 119 - 24
The response to auxin of rapeseed (Brassica napus L.) roots displaying reduced gravitropism due to transformation by Agrobacterium rhizogenes; Legue V et al.; It has recently been documented that, compared to untransformed controls, the roots of oilseed rape (Brassica napus L . CV CrGC5) seedlings transformed by Agrobacterium rhizogenes A4 show a reduced gravitropic reaction (Legue et al . 1994, Physiol Plant 91: 559-566) . After stimulation at 90 degrees C or 135 degrees, the transformed root tips curve . but never reach a vertical orientation . In the present study, we investigated the causes of reduced gravitropic bending observed in stimulated transformed root tips . First, we localized the gravitropic curvature in normal and in transformed roots after 1.5 h of stimulation . The cells involved in root curvature (target cells) corresponded at the cellular level to the apical part of the zone of increasing cell length . In transformed roots grown in the vertical position, these cells showed a reduction in cell length compared to controls . Because auxin is considered to be the gravitropic mediator, the response of normal and transformed roots to exogenous auxin was studied . Indole-3-acetic acid (IAA) was applied along the first 3 mm using resin beads loaded with the hormone . In comparison to normal roots, transformed roots showed reduced bending toward the bead at all points of bead application . Moreover, the cells which responded to IAA corresponded to the target cells involved in the gravitropic reaction . The level of endogenous IAA was lower in transformed roots . Thus, it was concluded that the modified behavior of transformed roots during gravitropic stimulation could be due to differences either in IAA levels or in reactivity of the target cells to the message from the cap.

Transgenic Res, 1996 Sep, 5(5), 325 - 35
Transgenic white clover . Studies with the auxin-responsive promoter, GH3, in root gravitropism and lateral root development; Larkin PJ et al.; We report improved method for white clover (Trifolium repens) transformation using Agrobacterium tumefaciens . High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed . Transgenic plants were recovered routinely from over 50% of treated cotyledons . The bar gene and phosphinothricin selection was shown to be a more effective selection system than nptII (kanamycin selection) or aadA (spectinomycin selection) . White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (beta-glucuronidase) to study the involvement of auxin in root development . Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied . The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered . Expression of the GH3:GUS fusion was not enhanced by other phytohormones . A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues . In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated . For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated . Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development . Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature . These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.

Microgravity Sci Technol, 1995 Feb, 7(4), 336 - 8
Enhancing effects of simulated microgravity on Agrobacterium-infected frequency of tobacco callus; Dong LY et al.; Under the condition of rotation-induced gravity compensation, the time course of interaction between Agrobacteriun tumefaciens and tobacco callus was investigated by means of a scanning electronic microscope . Resulted from repeated experiments, it was found that callus induced from tobacco leaves under simulated microgravity was easier to be infected by A . tumefaciens than controls . Analyses with a scanning electronic microscope indicated that A . tumefaciens were instantly detected on the surface of cell in the first 5 min, that is, A . tumefaciens are liable to interrecognize with callus cell upon contact with each other . With the proceeding of co-culture, the infection efficiency of A . tumefaciens was correspondingly increased . When the time reached 6 h, the fiber was formed between A . tumefaciens and callus cell . In our experiment, the erecting rotating state was taken as the control to exclude the interference of rotating . In this case, A . tumefaciens did not adsorb on calli until 3 h of co-culture, and fiber was only observed as late as 16 h . Statistic data showed that A . tumefaciens-infected frequency of the callus under the action of microgravity was elevated to 176% over that of control.

Proc Natl Acad Sci U S A, 2001 Sep 11, 98(19), 10954 - 9 Epub 2001 Sep 04.
Plant gene expression response to Agrobacterium tumefaciens; Ditt RF et al.; To elucidate the nature of plant response to infection and transformation by Agrobacterium tumefaciens, we compared the cDNA-amplified fragment length polymorphism (AFLP) pattern of Agrobacterium- and mock-inoculated Ageratum conyzoides plant cell cultures . From 16,000 cDNA fragments analyzed, 251 (1.6%) were differentially regulated (0.5% down-regulated) 48 h after cocultivation with Agrobacterium . From 75 strongly regulated fragments, 56 were already regulated 24 h after cocultivation . Sequence similarities were obtained for 20 of these fragments, and reverse transcription-PCR analysis was carried out with seven to confirm their cDNA-AFLP differential pattern . Their sequence similarities suggest a role for these genes in signal perception, transduction, and plant defense . Reverse transcription-PCR analysis indicated that four genes involved in defense response are regulated in a similar manner by nonpathogenic bacteria, whereas one gene putatively involved in signal transduction appeared to respond more strongly to Agrobacterium . A nodulin-like gene was regulated only by Agrobacterium . These results demonstrate a rapid plant cell response to Agrobacterium infection, which overlaps a general response to bacteria but also has Agrobacterium-specific features.

Mol Gen Mikrobiol Virusol, 2001, (3), 13 - 5
{Formation of TRA-dependent surface structures in Agrobacterium tumefaciens and their absence in R1 (deltatraR) mutant}; Tugarova AV et al.; Agrobacteria have Ti plasmid DNA delivering systems for the transfer to recipient cells by the conjugation mechanism . This transfer is absolutely dependent on induction tra genes . It is not clear which tra-dependent surface (extracellular) proteins (structures) are involved in the transport mechanism and whether these proteins also play a role in the contact formation . SDS-PAGE electrophoresis of proteins released from the cell showed disappearance of 63 and 67 kD proteins in R1(delta traR) strain, which were found in the growth medium and triton extract from the outer membrane of Ti plasmid-harboring A . tumefaciens R10 strains . The traR defective mutant did not express these proteins and had a higher hemagglutination and flocculation capacity than the wild strain . On the other hand, the wild strain showed D-galactose and N-acetyl-galactosamine specific hemagglutination which was not shown by traR mutant . Motility and chemotactic behavior of traR mutant in semisolid medium were defective . As a rule, one (or rarely two) thread-like connections in vir(-) and tra(+) conditions were observed on the agrobacterial cell surface . SDS pretreatment of agrobacterial cells had a significant effect on the expression of tra-dependent surface structures.

Tsitologiia, 2001, 43(6), 537 - 43
{Modern aspects of studying phytohormones . Cytokinins}; Ivanova AB et al.; The review presents current data on mechanisms of cytokinin action in plants . By analogy with the first part (Ivanova et al., 1999), in which general principles of phytohormone action and cardinal trends of phytohormone investigations were examined, here the relevant information on mechanisms of action of auxins and gibberellins has been given, and taking cytokines as example an attempt has been done to summarize the literature data on the number of questions offered for analysing hormones of high animals (Gudwin, Merser, 1986) . The review demonstrates that mechanisms of cytokine action at the cellular level are not known in many cases . One of the most significant factors in the action of phytohormones of this class on plants is their concentration, determined by their synthesis, transportation and further chemical conversions . This paper points to a poor knowledge of the relative role of these processes in regulation of cytokinin contents and their distribution among plant organs . Two possible ways of studying cytokinin action at the present day stage of investigations have been designated: 1) revealing the cytokinin expressed genes and establishing mechanisms of their action; 2) estimation of endogenous cytokinin alteration and the influence of this alteration on definite processes in the cell with the help of ipt-gene from t-DNA of Agrobacterium tumefaciens.

Plant J, 2001 Aug, 27(4), 367 - 71
Biolistic transformation of Arabidopsis root hairs: a novel technique to facilitate map-based cloning; Kemp A et al.; The final stage of map-based gene isolation is complementation of the mutant phenotype with wild-type DNA to determine the exact location of the gene of interest . This usually involves Agrobacterium tumefaciens-mediated transformation, which is reliable and produces stable transformants . However, the process of Agrobacterium transformation may take up to three months to complete . If the mutant phenotype can be seen in a single cell, and the wild-type copy of the gene can act cell autonomously, then complementation of the whole plant is not strictly necessary . We have developed a technique for the biolistic transformation of Arabidopsis thaliana root hairs, and used this to test large insert clones for complementation of two recessive mutant phenotypes, a procedure that takes less than a day . Our results show that biolistic transformation can be used with transient assays to conduct rapid tests for complementation by large insert clones.

Appl Environ Microbiol, 2001 Sep, 67(9), 4305 - 15
A second quorum-sensing system regulates cell surface properties but not phenazine antibiotic production in Pseudomonas aureofaciens; Zhang Z et al.; The root-associated biological control bacterium Pseudomonas aureofaciens 30-84 produces a range of exoproducts, including protease and phenazines . Phenazine antibiotic biosynthesis by phzXYFABCD is regulated in part by the PhzR-PhzI quorum-sensing system . Mutants defective in phzR or phzI produce very low levels of phenazines but wild-type levels of exoprotease . In the present study, a second genomic region of strain 30-84 was identified that, when present in trans, increased beta-galactosidase activity in a genomic phzB::lacZ reporter and partially restored phenazine production to a phzR mutant . Sequence analysis identified two adjacent genes, csaR and csaI, that encode members of the LuxR-LuxI family of regulatory proteins . No putative promoter region is present upstream of the csaI start codon and no lux box-like element was found in either the csaR promoter or the 30-bp intergenic region between csaR and csaI . Both the PhzR-PhzI and CsaR-CsaI systems are regulated by the GacS-GacA two-component regulatory system . In contrast to the multicopy effects of csaR and csaI in trans, a genomic csaR mutant (30-84R2) and a csaI mutant (30-84I2) did not exhibit altered phenazine production in vitro or in situ, indicating that the CsaR-CsaI system is not involved in phenazine regulation in strain 30-84 . Both mutants also produced wild-type levels of protease . However, disruption of both csaI and phzI or both csaR and phzR eliminated both phenazine and protease production completely . Thus, the two quorum-sensing systems do not interact for phenazine regulation but do interact for protease regulation . Additionally, the CsaI N-acylhomoserine lactone (AHL) signal was not recognized by the phenazine AHL reporter 30-84I/Z but was recognized by the AHL reporters Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136(pCF240) . Inactivation of csaR resulted in a smooth mucoid colony phenotype and formation of cell aggregates in broth, suggesting that CsaR is involved in regulating biosynthesis of cell surface components . Strain 30-84I/I2 exhibited mucoid colony and clumping phenotypes similar to those of 30-84R2 . Both phenotypes were reversed by complementation with csaR-csaI or by the addition of the CsaI AHL signal . Both quorum-sensing systems play a role in colonization by strain 30-84 . Whereas loss of PhzR resulted in a 6.6-fold decrease in colonization by strain 30-84 on wheat roots in natural soil, a phzR csaR double mutant resulted in a 47-fold decrease . These data suggest that gene(s) regulated by the CsaR-CsaI system also plays a role in the rhizosphere competence of P . aureofaciens 30-84.

Curr Genet, 2001 Jul, 39(5-6), 388 - 93
Efficient Agrobacterium tumefaciens-mediated gene disruption in the phytopathogen Mycosphaerella graminicola; Zwiers LH et al.; Agrobacterium tumefaciens-mediated transformation has been successfully applied to the wheat pathogen Mycosphaerella graminicola . Both protoplasts and intact cells have been transformed to hygromycin B resistance . Furthermore, A . tumefaciens-mediated transformation using homologous DNA originating from the M . graminicola ABC transporter gene MgAtr2 resulted in the efficient generation of disruption mutants . In 44% of the transformants, disruption of MgAtr2 was achieved and transformants resulted from the integration of a single copy of the transforming DNA . These results indicate that A . tumefaciens-mediated transformation is a useful tool to generate targeted gene disruption in the phytopathogen M . graminicola, where gene targeting by conventional methods is hardly possible.

Phytochemistry, 2001 Sep, 58(1), 137 - 42
Structure and characterization of the crown gall opines heliopine, vitopine and ridéopine; Chilton WS et al.; The crown gall opines heliopine from tumors induced by octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N2-(1'R-carboxyethyl)-L-glutamine . Tumors produced by strains S4 and T2 do not contain octopine or lysopine, but they do contain heliopine and the new opine rideopine identified as N-(4'-aminobutyl)-D-glutamic acid . Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic rideopine as well as on rideopine lactam as sole carbon source . While octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks . Heliopine catabolism by octopine strains is not induced by octopine.

Planta, 2001 May, 213(1), 29 - 36
In situ localization of endogenous cytokinins during shooty tumor development on Eucalyptus globulus Labill; Azmi A et al.; Our previous results demonstrated that endogenous cytokinins are involved in the shooty potential of tumors initiated on Eucalyptus globulus plantlets inoculated with Agrobacterium tumefaciens strain 82.139 {A . Azmi et al . (1997a) Plant Sci 127: 81-90} . In order to investigate whether or not these hormones are distributed homogeneously in the tumors prior to the onset of bud regeneration, decapitated hypocotyls were inoculated with the strain C58pMP90/T139 GUS-INT harboring the wild transferred DNA (T-DNA) of strain 82.139 tagged with the beta-glucuronidase (gus)-reporter gene . In situ immunolocalization of zeatin, dihydrozeatin and isopentenyladenine was performed in the developing tumors and combined with the histo-enzymological beta-glucuronidase assay . It was found that the expression of the T-DNA was restricted to only some small areas located deeply in the tumors . These sites were also provided with a high cytokinin signal while the untransformed parts of the tumors displayed a weaker signal, except in the early differentiating tracheary elements . The regenerated buds were untransformed and originated from superficial parts of the tumors provided with a moderate signal for cytokinins . The method of colocalization of both cytokinins and gus expression developed here might be helpful for further studies concerning the role of these hormones in controlling gene expression at cell and tissue levels.

Planta, 2001 May, 213(1), 11 - 9
Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L; Langhans M et al.; Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L . and, for comparison, in stems of Cucurbita pepo L . and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue . The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies . Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM . Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types . Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms . The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs . The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

Nucleic Acids Res, 2001 Sep 1, 29(17), 3685 - 93
Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells; Ferrando A et al.; Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling . The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs . However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo . Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells . This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.

Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 288 - 92
{Construction of transgenic rice populations by inserting the maize transponson Ac/Ds and genetic analysis for several mutants}; Zhu ZG et al.; An efficient and rapid gene transformation system of rice mediated by Agrobacterium tumefaciens was used . Calli induced from immature and mature embryos of Zhonghua No . 11, a japonic rice variety, were cultured with the A . tumefaciens strain EHA105 harboring the superbinary plasmid pDsBar1300 or pUBITs separately, and more than 400 independent transgenic lines inserted Ds element or Ac fragment were obtained . Some visible mutants in T0 or T1 generation were found, consisting of disease resistance, albino, dwarf, male sterile, chlorosis, early heading, late heading, stripe, etc . From the phenotype analysis, a few mutants such as dwarf and male sterile seemed to be linked to the Basta resistance and the transposon.

Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 273 - 7
{Studies on transgenic tobacco plants expressing two kinds of insect resistant genes}; Zhao CY et al.; A synthetic Bt cry1Ac gene fussed with a secretary signal coding sequences at 5' end and a modified gna gene were used to construct a plant expression vector pBSGS1M+ and this vector was transferred into tobacco (Nicotiana tabacum L.) by Agrobacterium-mediated transformation method . Results of PCR, Southern blot and Slot blot analysis indicated that both the chimeric Bt cry1Ac and gna genes were integrated into the genomes of transformed plants . Western blot analysis indicated that at least the cry1Ac protein was produced in transgenic plants . Upon insect bioassay using cotton bollworm (Heliothis armigera Hubner), the mortality of insect larvae on 60% regenerated plants reached 100% in 5 days post infestation and the growth of the survived larvae was seriously inhibited; The results from insect bioassay with peach aphid (Myzus persicae) showed that the transgenic plants were aphid-resistant, evidenced by a 50%-60% reduction in aphid population density, even over 80% for some individual transgenic plants . These results reflect that the modification of the two insect resistant genes and construction of the expression vector are correct and could be valuable for later application in crop breeding for insect resistance.

Sheng Wu Gong Cheng Xue Bao, 2001 May, 17(3), 264 - 8
{The primary role of central region of HC-pro of potato Y potyvirus in synergism of plant viruses}; Lu RF et al.; Five deleted mutants of HC-Pro gene of Chinese isolate of potato Y potyvirus (PVY-C) were obtained by PCR mutation, and their plant expression vectors were constructed . They were transformed into tobacco K326 (Nicotina tabacum cv . K326) mediated by Agrobacterium . PCR and Southern blot analysis revealed that PVY-C HC-Pro gene and its deleted mutants were integrated into tobacco genome, and Western blot analysis showed that they were all expressed in transgenic tobacco plants . Furthermore, infection test demonstrated that the central region of PVY-C HC-Pro can mediate synergism of PVY-C/cucumber mosaic cucumovirus (CMV) and PVY-C/potato X potexvirus (PVX), identifying that it is functional domain in synergism.

J Bacteriol, 2001 Sep, 183(18), 5343 - 51
The Brucella suis homologue of the Agrobacterium tumefaciens chromosomal virulence operon chvE is essential for sugar utilization but not for survival in macrophages; Alvarez-Martinez MT et al.; Brucella strains possess an operon encoding type IV secretion machinery very similar to that coded by the Agrobacterium tumefaciens virB operon . Here we describe cloning of the Brucella suis homologue of the chvE-gguA-gguB operon of A . tumefaciens and characterize the sugar binding protein ChvE (78% identity), which in A . tumefaciens is involved in virulence gene expression . B . suis chvE is upstream of the putative sugar transporter-encoding genes gguA and gguB, also present in A . tumefaciens, but not adjacent to that of a LysR-type transcription regulator . Although results of Southern hybridization experiments suggested that the gene is present in all Brucella strains, the ChvE protein was detected only in B . suis and Brucella canis with A . tumefaciens ChvE-specific antisera, suggesting that chvE genes are differently expressed in different Brucella species . Analysis of cell growth of B . suis and of its chvE or gguA mutants in different media revealed that ChvE exhibited a sugar specificity similar to that of its A . tumefaciens homologue and that both ChvE and GguA were necessary for utilization of these sugars . Murine or human macrophage infections with B . suis chvE and gguA mutants resulted in multiplication similar to that of the wild-type strain, suggesting that virB expression was unaffected . These data indicate that the ChvE and GguA homologous proteins of B . suis are essential for the utilization of certain sugars but are not necessary for survival and replication inside macrophages.

J Bacteriol, 2001 Sep, 183(18), 5302 - 10
DnaK chaperone-mediated control of activity of a sigma(32) homolog (RpoH) plays a major role in the heat shock response of Agrobacterium tumefaciens; Nakahigashi K et al.; RpoH (Escherichia coli sigma(32) and its homologs) is the central regulator of the heat shock response in gram-negative proteobacteria . Here we studied salient regulatory features of RpoH in Agrobacterium tumefaciens by examining its synthesis, stability, and activity while increasing the temperature from 25 to 37 degrees C . Heat induction of RpoH synthesis occurred at the level of transcription from an RpoH-dependent promoter, coordinately with that of DnaK, and followed by an increase in the RpoH level . Essentially normal induction of heat shock proteins was observed even with a strain that was unable to increase the RpoH level upon heat shock . Moreover, heat-induced accumulation of dnaK mRNA occurred without protein synthesis, showing that preexisting RpoH was sufficient for induction of the heat shock response . These results suggested that controlling the activity, rather than the amount, of RpoH plays a major role in regulation of the heat shock response . In addition, increasing or decreasing the DnaK-DnaJ chaperones specifically reduced or enhanced the RpoH activity, respectively . On the other hand, the RpoH protein was normally stable and remained stable during the induction phase but was destabilized transiently during the adaptation phase . We propose that the DnaK-mediated control of RpoH activity plays a primary role in the induction of heat shock response in A . tumefaciens, in contrast to what has been found in E . coli.

Biochemistry, 2001 Aug 28, 40(34), 10169 - 78
Identification of functionally important amino-terminal arginines of Agrobacterium tumefaciens ADP-glucose pyrophosphorylase by alanine scanning mutagenesis; Gomez-Casati DF et al.; Treatment of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase with the arginyl reagent phenylglyoxal resulted in complete desensitization to fructose 6-phosphate (F6P) activation, and partial desensitization to pyruvate activation . The enzyme was protected from desensitization by ATP, F6P, pyruvate, and phosphate . Alignment studies revealed that this enzyme contains arginine residues in the amino-terminal region that are relatively conserved in similarly regulated ADP-glucose pyrophosphorylases . To functionally evaluate the role(s) of these arginines, alanine scanning mutagenesis was performed to generate the following enzymes: R5A, R11A, R22A, R25A, R32A, R33A, R45A, and R60A . All of the enzymes, except R60A, were successfully expressed and purified to near homogeneity . Both the R5A and R11A enzymes displayed desensitization to pyruvate, partial activation by F6P, and increased sensitivity to phosphate inhibition . Both the R22A and R25A enzymes exhibited reduced V(max) values in the absence of activators, lower apparent affinities for ATP and F6P, and reduced sensitivities to phosphate . The presence of F6P restored R22A enzyme activity, while the R25A enzyme exhibited only approximately 1.5% of the wild-type activity . The R32A enzyme displayed an approximately 11.5-fold reduced affinity for F6P while exhibiting behavior identical to that of the wild type with respect to pyruvate activation . Both the R33A and R45A enzymes demonstrated a higher activity than the wild-type enzyme in the absence of activators, no response to F6P, partial activation by pyruvate, and desensitization to phosphate inhibition . These altered enzymes were also insensitive to phenylglyoxal . The data demonstrate unique functional roles for these arginines and the presence of separate subsites for the activators.

Infect Immun, 2001 Sep, 69(9), 5786 - 93
Towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a Mannheimia haemolytica A1 leukotoxin 50 fusion protein; Lee RW et al.; Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly on Mannheimia haemolytica A1 leukotoxin (Lkt) . In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated . Derivatives of the M . haemolytica Lkt in which the hydrophobic transmembrane domains were removed were made . Lkt66 retained its immunogenicity and was capable of eliciting an antibody response in rabbits that recognized and neutralized authentic Lkt . Genes encoding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation . Constructs were screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco . The fusion construct pBlkt50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for transcription, was selected and introduced into white clover by Agrobacterium tumefaciens-mediated transformation . Transgenic lines of white clover were recovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis . Lkt50-GFP was found to be stable in clover tissue after drying of the plant material at room temperature for 4 days . An extract containing Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt . This is the first demonstration of the expression of an M . haemolytica antigen in plants and paves the way for the development of transgenic plants expressing M . haemolytica antigens as an edible vaccine against bovine pneumonic pasteurellosis.

J Bacteriol, 2001 Sep, 183(17), 5058 - 66
Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases; van Hylckama Vlieg JE et al.; Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides . Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity . The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity . The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively . A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs) . Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases . The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases . A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding . The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide . The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.

Plant J, 2001 Jul, 27(2), 171 - 8
Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system; Corneille S et al.; Incorporation of a selectable marker gene during transformation is essential to obtain transformed plastids . However, once transformation is accomplished, having the marker gene becomes undesirable . Here we report on adapting the P1 bacteriophage CRE-lox site-specific recombination system for the elimination of marker genes from the plastid genome . The system was tested by the elimination of a negative selectable marker, codA, which is flanked by two directly oriented lox sites (>codA>) . Highly efficient elimination of >codA> was triggered by introduction of a nuclear-encoded plastid-targeted CRE by Agrobacterium transformation or via pollen . Excision of >codA> in tissue culture cells was frequently accompanied by a large deletion of a plastid genome segment which includes the tRNA-ValUAC gene . However, the large deletions were absent when cre was introduced by pollination . Thus pollination is our preferred protocol for the introduction of cre . Removal of the >codA> coding region occurred at a dramatic speed, in striking contrast to the slow and gradual build-up of transgenic copies during plastid transformation . The nuclear cre gene could subsequently be removed by segregation in the seed progeny . The modified CRE-lox system described here will be a highly efficient tool to obtain marker-free transplastomic plants.

Mol Microbiol, 2001 Jul, 41(2), 379 - 91
Natural transformation competence in Helicobacter pylori is mediated by the basic components of a type IV secretion system; Hofreuter D et al.; Helicobacter pylori (Hp), a Gram-negative bacterial pathogen and aetiologic agent of gastroduodenal disease in humans, is naturally competent for genetic transformation . Natural competence in bacteria is usually correlated with the presence of type IV pili or type IV pilin-like proteins, which are absent in Hp . Instead, we recently identified the comB operon in Hp, carrying four genes tentatively designated as orf2, comB1, comB2 and comB3 . We show here that all ComB proteins and the 37-amino-acid Orf2 peptide display significant primary sequence and structural homology/identity to the basic components of a type IV secretion apparatus . ComB1, ComB2 and ComB3, now renamed ComB8, ComB9 and ComB10, correspond to the Agrobacterium tumefaciens VirB8, VirB9 and VirB10 proteins respectively . The peptide Orf2 carries a lipoprotein motif and a second cysteine residue homologous to VirB7, and was thus designated ComB7 . The putative ATPase ComB4, encoded by the open reading frame hp0017 of strain 26695, corresponds to virB4 of the A . tumefaciens type IV secretion system . A Hp comB4 transposon insertion mutant was totally defective in natural transformation . By complementation of a Hp DeltacomB deletion mutant, we demonstrate that each of the proteins from ComB8 to ComB10 is absolutely essential for the development of natural transformation competence . The putative lipoprotein ComB7 is not essential, but apparently stabilizes the apparatus and modulates the transformation efficiency . Thus, pathogenic type I Hp strains contain two functional independent type IV transport systems, one for protein translocation encoded by the cag pathogenicity island and one for uptake of DNA by natural transformation . The latter system indicates a possible novel mechanism for natural DNA transformation in bacteria.

Appl Environ Microbiol, 2001 Aug, 67(8), 3655 - 64
Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR; Tan Z et al.; In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice . Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth . In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment . 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii . Rhizobium sp . (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter) . Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides) . The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B . elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp . (Chamaecytisus) strain BTA-1 . It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance . Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays . Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation . Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.

Can J Microbiol, 2001 Jun, 47(6), 495 - 502
Analysis of the genetic region encoding a novel rhizobiocin from Rhizobium leguminosarum bv . viciae strain 306; Venter AP et al.; Cross-testing of a number of strains of Rhizobium leguminosarum for bacteriocin production revealed that strain 306 produced at least two distinct bacteriocins . Further analysis involving plasmid transfer to Agrobacterium and other hosts demonstrated that there were bacteriocin determinants on plasmids pRle306b and pRle306c, as well as a third bacteriocin . The bacteriocin encoded by pRle306b was indistinguishable from the bacteriocin encoded by strain 248, whereas the bacteriocin encoded by plasmid pRle306c had a distinctive spectrum of activity against susceptible strains, as well as different physical properties from other bacteriocins that we have studied in our lab . Two mutants altered in production of the pRle306c bacteriocin were generated by transposon Tn5 mutagenesis, and the DNA flanking the transposon inserts in these mutants was cloned and characterized . DNA sequence analysis suggested that the pRle306c bacteriocin was a large protein belonging to the RTX family, and that a type I secretion system involving an ABC type transporter was required for export of the bacteriocin . A mutant unable to produce this bacteriocin was unaltered in its competitive properties, both in broth and in nodulation assays, suggesting that the bacteriocin may not play a major role in determining the ecological success of this strain.

Biotechniques, 2001 Jul, 31(1), 132 - 4, 136-40
Quantitative real-time PCR assay for determining transgene copy number in transformed plants; Ingham DJ et al.; The development of transgenic events can be limited by many factors . These include expression levels, insert stability and inheritance, and the identification of simple insertion events . All of the factors can be related to the copy number of the transgene . Traditionally, copy number has been determined by laborious blotting techniques . We have developed an alternative approach that utilizes the fluorogenic 5' nuclease (TaqMan) assay to quantitatively determine transgene copy level in plants . Using this assay, hundreds of samples can be analyzed per day in contrast to the low throughput encountered with traditional methods . To develop the TaqMan copy number assay, we chose to utilize our highly efficient Agrobacterium-mediated transformation system of maize . This transformation procedure generates predominantly low copy number insertion events, which simplified assay development . We have also successful applied this assay to other crops and transformation systems.

Mol Cells, 2001 Jun 30, 11(3), 326 - 33
Production of transgenic male sterile tobacco plants with the cDNA encoding a ribosome inactivating protein in Dianthus sinensis L; Cho HJ et al.; The ribosome inactivating protein (RIP) gene from D . sinensis was used as a cytotoxin gene to induce male sterility in tobacco plants . The TA29 promoter, obtained by PCR amplification from tobacco, was fused to the RIP cDNA, and the chimaeric molecule was then introduced into tobacco plants by Agrobacterium-mediated transformation . Out of twenty-one independent transformants, twenty transgenic tobacco plants exhibited male sterility . Southern blot analysis revealed that four of the transgenic plants contained a single copy of the RIP gene, while the rest of the transgenic tobacco plants had two to four copies of the gene . The transgenic male sterile plants set seeds normally when pollinated with pollens from untransformed control plants, indicating that the RIP gene does not affect the pistil development . Furthermore, the seed yield of the transgenic plant was similar to that of the untransformed, self-pollinated control plant . A light microscopic observation of anther cross sections clearly showed that the tapetal tissue of the anther was selectively and completely destroyed causing male sterility . This study suggests that the RIP gene can be used as a cytotoxin gene for induction of male sterility in the plant.

Plant Physiol, 2001 Jul, 126(3), 930 - 8
Silencing on the spot . Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system; Johansen LK et al.; The Agrobacterium-mediated transient expression assay in intact tissues has emerged as a rapid and useful method to analyze genes and gene products in plants . In many cases, high levels of active protein can be produced without the need to produce transgenic plants . In this study, a series of tools were developed to enable strong or weak induction of RNA silencing and to suppress RNA silencing in the absence of stable transgenes . Transient delivery of a gene directing production of a double-stranded green fluorescent protein (GFP) transcript rapidly induced RNA silencing of a codelivered GFP reporter gene, effectively preventing accumulation of GFP protein and mRNA . RNA silencing triggered by the strong dsGFP inducer was partially inhibited by the tobacco etch virus silencing suppressor, P1/HC-Pro . In the absence of the strong double-stranded GFP inducer, the functional GFP gene served as a weak RNA silencing inducer in the transient assay, severely limiting accumulation of the GFP mRNA over time . The weak silencing induced by the GFP gene was suppressed by P1/HC-Pro . These results indicate RNA silencing can be triggered by a variety of inducers and analyzed entirely using transient gene delivery systems . They also indicate that RNA silencing may be a significant limitation to expression of genes in the Agrobacterium-mediated transient assay but that this limitation can be overcome by using RNA silencing suppressors.

Physiol Plant, 2001 Jun, 112(2), 223 - 232
Overexpression of a heterologous sam gene encoding S-adenosylmethionine synthetase in flax (Linum usitatissimum) cells: Consequences on methylation of lignin precursors and pectins; Lamblin F et al.; The Arabidopsis thaliana sam1 gene encoding S-adenosylmethionine synthetase (EC 2.5.1.6) was transferred to flax (Linum usitatissimum) cells via Agrobacterium tumefaciens . This enzyme catalyses the conversion of methionine to S-adenosylmethionine (SAM), the major methyl group donor in living cells . The aim of this work was to study the consequences of an increased SAM-synthetase (SAM-S) activity in transgenic cell lines on both the production of mono- and dimethoxylated lignin monomers and the degree of methylesterification of pectins . Hypocotyls were cocultivated with Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the pO35SSAM binary vector carrying the sam1 gene under the control of the 35S promoter and the nptII gene for selection of putative transformed cells . Most of the transgenic cell lines exhibited a significant (up to 3.2-fold) increase in SAM-S activity compared to the controls . The results showed that for the cell lines analysed this transformation had no effect on caffeic acid O-methyltransferase (COMT, EC 2.1.1.68) in vitro activity, degree of methoxylation of lignin precursors or lignin deposition, pectin methyltransferase (PMT, EC 2.1.1) in vitro activity, but led to an increase of pectin methylesterification in friable and fast-growing transgenic cell lines.

Mol Microbiol, 2001 Jul, 41(1), 199 - 205
Cloning of the genes for a 4-sulphocatechol-oxidizing protocatechuate 3,4-dioxygenase from Hydrogenophaga intermedia S1 and identification of the amino acid residues responsible for the ability to convert 4-sulphocatechol; Contzen M et al.; The genes for a protocatechuate 3,4-dioxygenase (P34O-II) with the ability to oxidize 4-sulphocatechol were cloned from the 4-aminobenzenesulphonate(sulphanilate)-degrading bacterium Hydrogenophaga intermedia strain S1 (DSMZ 5680) . Sequence comparisons of the deduced amino acid sequences of both subunits of the P34O-II from H . intermedia S1 (PcaH-II and PcaG-II) with those of another P34O-II, previously obtained from Agrobacterium radiobacter S2, and the corresponding sequences from the protocatechuate 3,4-dioxygenases from other bacterial genera demonstrated that seven amino acid residues, which were conserved in all previously known P34Os (P34O-Is), were different in both P34O-IIs . According to previously published structural data for the P34O of Pseudomonas putida only two of these amino acid residues were located near the catalytical centre . The respective amino acid residues were mutated in the P34O-I from A . radiobacter S2 by site-specific mutagenesis, and it was found that a single amino acid exchange enabled the protocatechuate converting P34O also to oxidize 4-sulphocatechol.

Cell Res, 2001 Jun, 11(2), 149 - 55
Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer; Huang JQ et al.; Immature embryos of rice varieties "Xiushuill" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene) . The resistant calli were transferred onto the differentiation medium and plants were regenerated . The transformation frequency reached 56% approximately 72% measured as numbers of Geneticin (G418)-resistant calli produced and 36% approximately 60% measured as numbers of transgenic plants regenerated, respectively . PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome . Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38% approximately 61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16% approximately 75% . The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

Cell Res, 2001 Jun, 11(2), 142 - 8
HAL1 mediate salt adaptation in Arabidopsis thaliana; Yang SX et al.; The yeast HAL1 gene was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation with vacuum infiltration under the control of CaMV 35S promoter . Thirty-three individual kanamycin resistant plants were obtained from 75,000 seeds . Southern blotting analysis indicated that HAL1 gene had been integrated into all of the transgenic plants' genomes . The copy number of HAL1 gene in transgenic plants was mostly 1 to 3 by Southern analysis . Phenotypes of transgenic plants have no differences with wild type plants . Several samples of transformants were self-pollinated, and progenies from transformed and non-transformed plants (controls) were evaluated for salt tolerance and gene expression . Measurement of concentrations of intracellular K+ and Na+ showed that transgenic lines were able to retain less Na+ than that of the control under salt stress . Results from different tests indicated the expression of HAL1 gene promotes a higher level of salt tolerance in vivo in the transgenic Arabidopsis plants.

Fitoterapia, 2000 Sep, 71(5), 547 - 52
Evaluation of antitumor activity of some medicinal plants of Bangladesh by potato disk bioassay; Haque N et al.; The antitumor activity of the ethanolic extracts of 12 medicinal plants of Bangladesh, including the vincristine-vinblastine producing Catharanthus roseus was studied using the potato disk bioassay technique . Among these, 10 plant extracts at 25.0-microgram/disc exhibited significant inhibition of crown gall tumors caused by Agrobacterium tumefaciens.

Plant Sci, 2001 Jul, 161(2), 239 - 247
Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L . Wilczek) - a recalcitrant grain legume; Jaiwal PK et al.; Agrobacterium-mediated transformation of Vigna radiata L . Wilczek has been achieved . Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B(5) basal medium supplemented with 5x10(-6) M BAP, 2.5x10(-6) M each of 2,4-D and NAA and 50 mg l(-1) kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing beta-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes . Transformed calli were found resistant to kanamycin up to 1000 mg(.)l(-1) . Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively . Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis . Transgenic calli could not regenerate shoots on B(5) or B(5) containing different cytokinins or auxins alone or in combination . However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B(5) medium containing 6-benzylaminopurine (5x10(-7) M) and 75 mg l(-1) kanamycin . The putative transformed shoots were rooted on B(5)+indole-3-butyric acid (5x10(-6) M) within 10-14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction . The stamens, pollen grains and T(0) seeds showed GUS activity . Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T(0) plants and their seeds.

Plant Sci, 2001 Mar, 160(4), 713 - 721
Isolation of a flax pectin methylesterase promoter and its expression in transgenic tobacco; Roger D et al.; Pectin methylesterases (PME) catalyze the de-esterification of methoxylated pectins in plant cell walls . We have isolated a 1.9 kb regulatory region upstream from the Lupme3 coding sequence of Linum usitatissimum L . (flax) using a 'Polymerase Chain Reaction (PCR) walking' strategy . Two 5' truncated deletion fragments (1.5 and 0.44 kb) of this potential promoter sequence were inserted upstream of the gus reporter gene in order to study their expression in transgenic plants . These constructs were transferred into Nicotiana tabacum, a heterologous system using Agrobacterium tumefaciens . Expression of the reporter gene was analyzed in regenerated transgenic plants and calli to study the promoter activities of these sequences . This expression was observed in calli with both constructs . In contrast, expression in organs was only detected in tobacco plants transformed with the largest (1.5 kb) construct . This long fragment triggered expression in roots and immature or vitrified leaves . Expression in both organs was localized in the vasculature, but also detected in the root meristem . These results are the first evidence, to our knowledge, of the spatial and temporal regulation of a specific pme promoter of flax . Localization of Lupme3 promoter activity in vascular tissues of immature organs provides an insight into the role of this PME isoform in cell elongation and differentiation.

Plant Sci, 2001 Mar, 160(4), 691 - 698
Effects of ipt gene expression on the physiological and chemical characteristics of Artemisia annua L; Sa G et al.; An isopentenyl transferase gene (ipt) from T-DNA was transferred into Artemisia annua L . via Agrobacterium tumefaciens . The ipt gene was placed in a binary vector under the control of the CaMV 35S promoter . Leaf explants were infected with A . tumefaciens LBA4404 containing pBIipt to induce the buds . Nineteen shoot lines were selected, which were resistant to kanamycin . Polymerase chain reactions and Southern blotting confirmed that at least five shoot lines contained the foreign gene . The results of RT-PCR and Northern blotting analyses suggested that the foreign ipt gene of the transgenic shoot was expressed . Cytokinins, chlorophyll and artemisinin contents were found increased at different degree . Content of cytokinins (iPA and iP) was elevated 2- to 3-fold, chlorophyll increased 20-60% and artemisinin increased 30-70% compared with the control plants, respectively . A direct correlation was found between the contents of cytokinins, chlorophyll and artemisinin . This may be the first report on the relationship between endogenous cytokinin content and the production of secondary metabolites in plants.

Tree Physiol, 2001 Jul, 21(10), 665 - 72
Inducible expression of the heterologous PAL2 promoter from bean in white pine (Pinus strobus) transgenic cells; Levee V et al.; To elucidate heterologous promoter function in gymnosperms, we introduced the bean phenylalanine ammonia-lyase-beta-glucuronidase (PAL2-GUS) gene fusion into white pine (Pinus strobus L.) . Over 15 lines were produced and integration of Agrobacterium T-DNA was confirmed by Southern analysis . Induction of the reporter gene was detected in all of the lines tested following UV illumination . In contrast, a weak but constant induction was seen in only a few lines following treatment with salicylic acid (SA) or jasmonic acid (JA) . However, pretreatment of suspension cultures with SA or JA enhanced the induction of PAL2-GUS expression by UV irradiation . This specific enhancement or potentiation was reduced by 50% by treating the cells with indomethacin, an inhibitor of phospholipase activity, suggesting that the observed potentiation of UV induction involves the octadecanoid pathway . The UV induction was completely abolished by treating the cells with okadaic acid, an inhibitor of phosphatase activity . Thus, the induction of the heterologous PAL2 promoter from bean is consistent with the induction of phenylalanine ammonia-lyase (PAL) in angiosperms . Furthermore, our findings suggest that conifers, although phylogenetically distant to angiosperms, share some conserved promoter elements and some signal transduction mechanisms for UV-light perception.

Prikl Biokhim Mikrobiol, 2001 May-Jun, 37(3), 369 - 73
{Use of highly dispersed materials for culturing and isolation of granular Agrobacterium radiobacter preparations }; Kurdish IK et al.; The effects of synthetic and natural high-dispersion materials on the growth of Agrobacterium radiobacter were studied . Natural minerals montmorillonite and palygorskite (10 g/l nutrient medium) were more potent than high-dispersion silica and its modified forms in stimulating growth of Agrobacterium radiobacter . The interaction of Agrobacterium radiobacter with clay minerals increased the survival rate of bacteria at supraoptimal temperatures . We elaborated new granular bacterial preparation, which enhanced the productivity of cucumbers by 12-15%.

Prikl Biokhim Mikrobiol, 2001 May-Jun, 37(3), 349 - 54
{Isolation of genetically modified potato plant containing the gene of defensive peptide from Amaranthus}; Liapkova NS et al.; The plants of potato (Solanum tuberosum L., var . Desire) have been transformed with a pH22Kneo vector carrying the gene ac2, encoding the fungicidal peptide (defensin) from the seed of amaranth (Amaranthus caudatus L.) . The transformation involved co-cultivation of potato stem explants (excised from aseptically grown plants) and Agrobacterium tumefaciens on solid MS medium . Factors affecting in vitro regeneration of the explants and the transformation efficiency were optimized . Regenerated potato plants harboring the amaranth defensin gene were selected by two traits, growth and ability to form roots on kanamycin-supplemented MS medium . The transgenic state was confirmed PCR analysis of ac2 in tissues of the kanamycin-resistant plants . The transgenic organisms thus obtained differed from the original ambiol-treated plants in growth patterns and proton translocation across the plasma membrane of the tuber cells.

Plant Mol Biol, 2001 May, 46(2), 215 - 27
Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis; Greco R et al.; A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation . The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci . Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification . New transpositions were generated at a frequency of 15-50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny . The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function . Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes . A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random . Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice.

Transgenic Res, 2001 Jun, 10(3), 237 - 45
Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders; Zheng SJ et al.; Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes . However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern . Furthermore, genomic DNA blot hybridization is a time-consuming method . Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization . Using AL-PCR we proved that T-DNA was integrated into the A . cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA 105 (pCAMBIA 1301) . The AL-PCR patterns obtained were specific and reproducible for a given transgenic line . The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present . Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants . After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail . For example it was shown that upon T-DNA integration a 66 bp genomic sequence was deleted, and no filler DNA was inserted . Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration.

Genes Genet Syst, 2001 Apr, 76(2), 131 - 9
The transposition pattern of the Ac element in tobacco cultured cells; Semiarti E et al.; We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells . We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI . Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element . The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated . These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters . Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated . The genomic DNAs of these sublines were isolated and digested with MluI . Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis . Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome . These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome . In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.

Genes Genet Syst, 2001 Apr, 76(2), 121 - 30
Sequence characterization of the vir region of a nopaline type Ti plasmid, pTi-SAKURA; Hattori Y et al.; We isolated a crown gall tumor-inducing nopaline type Ti plasmid from Agrobacterium tumefaciens on a Sakura Japanese cherry tree, and designated it as pTi-SAKURA . By primer walking sequencing with long PCR and a newly developed PCR subcloning technique for long insert DNA, we completed DNA sequencing of the most important functional unit, the virulence (vir) region of pTi-SAKURA, which is indispensable for T-DNA transfer into the plant's chromosomes . By homology searches with the vir genes of other bacterial plasmids, we identified 11 open reading frames (orfs) and 31 genes and 11 vir box, which are 6 bp regulatory sequences . In total, 26 vir genes, including the putative virF and virK and the main vir region, were present as the vir gene cluster . The presence of vir box, GC content, codon usage and expression analysis in these genes led us to propose a new vir region.

Hereditas, 2000, 133(3), 229 - 33
Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene; Yu TT et al.; Creeping bentgrass (Agrostis palustris Huds.) is a cool season grass widely used on putting greens in golf courses . Transformation of creeping bentgrass has been conducted using microprojectile bombardment and protoplast electroporation . The objective of our study is to develop an alternative and more efficient approach in transforming the grass using Agrobacterium (strain EHA 101) . This technique was effective in transforming 40-day old calli derived from mature seeds cultured on MS medium supplemented with 2,4-D, kinetin, and sucrose . Dozens of transgenic plants have been produced from two independent transformed calli . Presence of functional green fluorescence protein (GFP) was detected in leaves, stems, and roots of transgenic seedlings . Four putative transgenic plants and two control plants were randomly chosen and analyzed by Southern blot analysis . Bands corresponding to the GFP gene were clearly shown in transgenic plants . These results indicated that Agrobacterium transformation can successfully be applied to creeping bentgrass.

J Exp Bot, 2001 May, 52(358), 1135 - 42
Factors influencing Agrobacterium-mediated transient expression of uidA in wheat inflorescence tissue; Amoah BK et al.; A critical step in the development of Agrobacterium tumifaciens-mediated transformation is the establishment of optimal conditions for T-DNA delivery into tissue from which whole plants can be regenerated . The efficient transformation of inflorescence tissue from 'Baldus', a commercial wheat variety, using the Agrobacterium strain AGLI harbouring the binary vector pAL156 is reported here . The effects of various factors on delivery and the transient expression of the uidA gene were studied including the duration of preculture, vacuum infiltration, the effect of sonication treatments, and Agrobacterium cell density . Optimal T-DNA delivery (as measured by uidA activity) was obtained from inflorescence tissues precultured for 21 d and sonicated . Increasing Agrobacterium cell density, the duration of inoculation/co-cultivation, and vacuum pressure, up to a threshold, increased uidA expression . The investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of immature wheat inflorescence tissue.

EMBO J, 2001 Jul 2, 20(13), 3596 - 607
VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity; Tzfira T et al.; T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium . This event is thought to be mediated by two bacterial proteins, VirD2 and VirE2, which are associated with the transported T-DNA molecule . While VirD2 is imported into the nuclei of plant, animal and yeast cells, nuclear uptake of VirE2 occurs most efficiently in plant cells . To understand better the mechanism of VirE2 action, a cellular interactor of VirE2 was identified and its encoding gene cloned from Arabidopsis . The identified plant protein, designated VIP1, specifically bound VirE2 and allowed its nuclear import in non-plant systems . In plants, VIP1 was required for VirE2 nuclear import and Agrobacterium tumorigenicity, participating in early stages of T-DNA expression.

Org Lett, 2001 Jan 11, 3(1), 41 - 3
Highly enantioselective and regioselective biocatalytic azidolysis of aromatic epoxides; Spelberg JH et al.; {figure: see text} The halohydrin dehalogenase from Agrobacterium radiobacter AD1 catalyzed the highly enantioselective and beta-regioselective azidolysis of (substituted) styrene oxides . By means of kinetic resolutions the remaining epoxide and the formed azido alcohol could be obtained in very high ee . In a large scale conversion, the decrease in yield and selectivity due to the uncatalyzed chemical side reaction could be overcome by slow addition of azide.

Microbiology, 2001 Jul, 147(Pt 7), 1815 - 24
Genetic and biochemical characterization of an enantioselective amidase from Agrobacterium tumefaciens strain d3; Trott S et al.; An enantioselective amidase was purified to homogeneity from Agrobacterium tumefaciens d3 . The enzyme has a molecular mass of about 490000 Da and is composed of identical subunits with a molecular mass of about 63000 Da . The purified enzyme converted racemic 2-phenylpropionamide to the corresponding S-acid with an enantiomeric excess (ee) value >95% at almost 50% conversion of the racemic amide . The purified enzyme was digested with trypsin and the amino acid sequences of the N terminus and different tryptic peptides determined . These amino acid sequences were used to clone the encoding gene . Finally, a 9330 bp DNA fragment was sequenced and the amidase gene identified . The deduced amino acid sequence showed homology to other enantioselective amidases from different bacterial genera . No indications of a structural coupling of the amidase gene with the genes for a nitrile hydratase could be found on the cloned DNA fragment . The amidase gene was encoded by an approximately 500 kb circular plasmid in A . tumefaciens d3 . The amidase was heterologously expressed in Escherichia coli and, as well as 2-phenylpropionamide, was shown to hydrolyse alpha-chloro- and alpha-methoxyphenylacetamide and 2-methyl-3-phenylpropionamide highly enantioselectively . Some amino acids within a highly conserved region common amongst all known enantioselective amidases ('amidase signature') were changed by site-specific mutagenesis and significant changes in the relative activities with different amides observed.

Mol Phylogenet Evol, 2001 Jul, 20(1), 100 - 10
The horizontal transfer of Agrobacterium rhizogenes genes and the evolution of the genus Nicotiana; Intrieri MC et al.; With the aim of understanding better the distribution and evolution of Agrobacterium rhizogenes genes transferred in the genus Nicotiana, 42 species were screened for presence of rolB, rolC, ORF13, and ORF14 . The transferred sequences were then compared within the genus and with current bacterial sequences . The results obtained showed the presence of at least one bacterial gene in 15 species belonging to different subgenera . Sequence analyses supported the hypothesis of coevolution of bacterial and plant sequences, thus suggesting a possible role for the transferred genes in the early events of Nicotiana species differentiation . The high level of conservation of Agrobacterium sequences and the dependence of their expression from the plant physiological context along with previous data suggesting their involvement in the determination of the plant hormonal balance were all consistent with this hypothesis . The results are finally discussed also as to their relevance for the hypothesis of mono and multi ancient infection by Agrobacterium .

Z Naturforsch {C}, 2001 May-Jun, 56(5-6), 375 - 81
Transformation of Catalpa ovata by Agrobacterium rhizogenes and phenylethanoid glycosides production in transformed root cultures; Wysokinska H et al.; Transformed root cultures of Catalpa ovata were established following shoots infection with four agropine strains of Agrobacterium rhizogenes . Frequency of root formation was dependent on the bacterial strain and the presence of acetosyringone in the incubation medium . It is the first report concerning the possibility of transforming Catalpa ovata by A . rhizogenes . Both transformed and untransformed root cultures of C . ovata were studied for their growth and phenylethanoid glycoside production . As with the roots of intact plants, cis- and trans-verbascoside as well as martynoside were produced in transformed and untransformed root cultures of C . ovata . In hairy roots, total (cis + trans) verbascoside production could be stimulated up to three-fold of that of roots of 6-month-old plants grown in a greenhouse, by using an appropriate root line cultured in liquid 1/2 B5 Gamborg medium containing indole-3-butyric acid (0.1 mg/l) in the dark but not light conditions . Transformed and untransformed root cultures of C . ovata were also found to have 10 times higher martynoside production than roots of intact plants.

Mikrobiol Z, 2000 Jul-Aug, 62(4), 29 - 37
{Ability of representatives of Pantoea agglomerans, as well as Bacillus subtilis and some Pseudomonas species to suppress the development of phytopathgenic bacteria and micromycetes in regulating plant growth}; Romanenko VM et al.; The ability of representatives of Pantoea agglomerans (Erwinia herbicola (Lohnis) Dye {21}), Bacillus subtilis and some species of Pseudomonas genus to inhibit the growth of phytopathogenic bacteria and micromycetes and to regulate the growth of plants has been comparatively studied . The ability to inhibit the growth of mycellium of phytopathogenic Fusarium avenaceum, F . gibbosum, F . oxysporum was found out in all of 13 investigated strains of P . agglomerans, while the growth of F . culmorum is inhibited by 2 strains and Bipolaris sorokiniana is inhibited by 7 strains . The strains of P . agglomerans and Bacillus subtilis inhibit the growth of mycellium of these mycromycetes to the greater extent than the representatives of Pseudomonas genus . The mycellium growth of B . sorokiniana is better inhibited by B . subtilis and representatives of Pseudomonas genus . Besides the antifungal action 8 strains of P . agglomerans manifested the antagonistic activity in respect to phytopathogenic Agrobacterium tumefaciens and representatives of genera Clavibacter, Erwinia, Pseudomonas, Xanthomonas and also in respect to the microflora which is present in the cabbage and wheat seeds . The strains have been revealed which, parallel with high antagonistic activity in respect to phytopathogenic micromycetes and bacteria, stimulate the seed germination and increase the weight of the cabbage and wheat sprouts.

J Bacteriol, 2001 Jul, 183(14), 4296 - 304
Structural and functional characterization of IS679 and IS66-family elements; Han CG et al.; A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171 . IS679 has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called tnpA, tnpB, and tnpC) . A plasmid carrying a composite transposon (Tn679) with the kanamycin resistance gene flanked by an intact IS679 sequence and an IS679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS679 . A transposition assay done with a mating system showed that Tn679 could transpose at a high frequency to the F plasmid derivative used as the target . On transposition, Tn679 duplicated an 8-bp sequence at the target site . Tn679 derivatives with a deletion in each ORF of IS679 did not transpose, finding indicative that all three IS679 ORFs are essential for transposition . The tnpA and tnpC products appear to have the amino acid sequence motif characteristic of most transposases . A homology search of the databases found that a total of 25 elements homologous to IS679 are present in Agrobacterium, Escherichia, Rhizobium, Pseudomonas, and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria . We found that these elements belong to the IS66 family, as do other elements, including nine not previously reported . Almost all of the elements have IRs similar to those in IS679 and, like IS679, most appear to have duplicated an 8-bp sequence at the target site on transposition . These elements have three ORFs corresponding to those in IS679, but many have a mutation(s) in an ORF(s) . In almost all of the elements, tnpB is located in the -1 frame relative to tnpA, such that the initiation codon of tnpB overlaps the TGA termination codon of tnpA . In contrast, tnpC, separated from tnpB by a space of ca . 20 bp, is located in any one of three frames relative to tnpB . No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.

Virology, 2001 Jun 20, 285(1), 71 - 81
Long-distance movement and replication maintenance functions correlate with silencing suppression activity of potyviral HC-Pro; Kasschau KD et al.; The tobacco etch potyviral protein, HC-Pro, is a multifunctional proteinase required for long-distance movement in plants and maintenance of genome replication at the single-cell level . It also functions in a counterdefensive capacity as a suppressor of posttranscriptional gene silencing (PTGS) . To determine whether the requirements for HC-Pro during long distance movement and replication maintenance are due to the silencing suppressor function of the protein, a series of HC-Pro alanine scanning and other site-directed mutants were analyzed . Using a transient silencing suppression assay in Agrobacterium-injected leaf tissue, several suppression-defective mutants were identified . Each of six HC-Pro mutations, which were shown previously to confer long-distance movement and replication maintenance defects, conferred PTGS suppression defects . Interestingly, the genes encoding these defective HC-Pro derivatives were themselves susceptible targets of PTGS, resulting in low levels of mRNA and protein accumulation . Mutations that inactivated the proteinase domain active site had no effect on PTGS suppression function . The results are consistent with the hypothesis that the role of HC-Pro in long-distance movement and genome replication depends on PTGS suppression function and that this function is independent of HC-Pro proteolytic activity .

J Exp Bot, 2001 Apr, 52(357), 845 - 50
A simple protocol for transient gene expression in ripe fleshy fruit mediated by Agrobacterium; Spolaore S et al.; Fleshy fruits represent a very important economic resource and, therefore, they are an ideal target for biotechnological ameliorations . However, because of their physiological and anatomical characteristics, ripe fleshy fruits represent an extremely difficult material for transient gene expression assays aimed at the study of gene promoters in a short time . To this purpose, a fast and efficient Agrobacterium-mediated transient gene expression system was developed for ripe fleshy fruits . A beta-glucuronidase reporter gene interrupted by an intron was used in order to prevent the possible expression of GUS activity by the Agrobacterium cells . The contemporary use of another reporter gene was used to check the transformation efficiency . This method is based on the injection of an Agrobacterium suspension into the fruits, and allows both qualitative and quantitative assays in a wide range of fruits to be carried out.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1023 - 6
Agrobacterium larrymoorei sp . nov., a pathogen isolated from aerial tumours of Ficus benjamina; Bouzar H et al.; Tumorigenic Agrobacterium strains isolated from tumours growing on pruned branches of Ficus benjamina have previously been shown to have unique opine metabolism and sufficient 16S rRNA sequence differences to suggest that they belong to a new species . DNA-DNA hybridization results confirmed that these strains represent a new species and Agrobacterium larrymoorei sp . nov . (type strain ATCC 51759T = CFBP 5473T = NCPPB 4096T) is proposed as the name for the species.

Gene, 2001 May 30, 270(1-2), 245 - 52
Characterization of a putative RND-type efflux system in Agrobacterium tumefaciens; Peng WT et al.; Sequencing of a 7277 bp fragment adjacent to the chvH locus of Agrobacterium tumefaciens revealed four open reading frames (ORFs), designated ameR, ameA, ameB and ameC, respectively . These ORFs exhibit amino acid similarities to components of Resistance-Nodulation-Cell Division (RND) type efflux systems . AmeA and AmeB show high homology to membrane fusion proteins (MFP) and RND-type transporters, whereas AmeC shows similarity to NodT and other members of outer membrane factor families . Mutations of the ameA and ameB genes did not affect the susceptibility profile of the wild-type strain to several detergents and antibiotics . In contrast, mutations of the ameC gene dramatically affected the susceptibility of the strain to these same inhibitory compounds . RT-PCR analysis demonstrated that the ameABC genes form an operon . In addition, ameC gene has its own promoter gene located in the intergenic region between ameB and ameC . Mapping upstream of ameA is ameR, which encodes a protein that shows similarity especially at its N-terminal end to the TetR family of bacterial transcriptional regulators . AmeR negatively regulates expression of the ameABC operon . A mutation in ameR increased the resistance of the cells to several antimicrobial agents . This regulatory locus appears to be in the same operon as a gene located upstream which codes for a product that has high similarity to numerous 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) synthetases . The possible role of the putative efflux pump coded by the ame genes is discussed.

Plasmid, 2001 May, 45(3), 222 - 6
Characterization and sequence analysis of the replicator region of the novel plasmid pALC1 from Paracoccus alcaliphilus; Bartosik D et al.; The replicator region of a low-copy-number plasmid, pALC1, of Paracoccus alcaliphilus JCM 7364 was cloned in a form of the minireplicon pALC100 (3.6 kb) . The host range of the minireplicon embraces several species of genus Paracoccus, as well as Agrobacterium tumefaciens, Rhizobium leguminosarum, and Rhodobacter sphaeroides (all belonging to alpha-Proteobacteria), but not Escherichia coli . The complete nucleotide sequence of the replicator region (2276 bp) revealed the presence of one complete open reading frame coding for the 28.4-kDa protein (RepA) with similarity to replication proteins of plasmid pSW500 of Erwinia stewartii and pVS1 of Pseudomonas fluorescens . The iteron-like region was identified upstream of the repA gene and consisted of two clusters of repeated sequences (17 bp long) separated by a putative DnaA box . Analysis of the predicted amino acid sequence of two adjacent incomplete ORFs suggests the localization of repA between genes involved in conjugation (traG) and partitioning (parA) within the pALC1 genome.

Enzyme Microb Technol, 2001 Jun 7, 28(9-10), 806 - 814
Effect of cell membrane of Agrobacterium radiobacter on enhancing D-amino acids production from racemic hydantoins; Lee C et al.; An interesting phenomenon was observed that the existence of the intact cell membrane can enhance the D-amino acids production from D,L-5-substituted hydantoins by reacting with the whole cells of Agrobacterium radiobacter . Two intracellular enzymes were involved in the reaction process . The first enzyme D-hydantoinase converted hydantoins to carbamoyl derivatives which were further converted to D-amino acids by D-amidohydrolase . The amount of D-amino acids produced from hydantoins by the intact cells were 1.8-2.4 fold higher than the toluene treated cells . In addition, by using the intact cells the amount of D-amino acids produced from hydantoins was about 10 fold higher than that produced directly from carbamoyl derivatives . The relatively lower permeability of cell membrane to the reaction intermediate carbamoyl derivatives was confirmed by a simple mathematical model to be the main factor for the better performance of the intact cells for D-amino acid production . Besides, the low intracellular enzymes activities also contributed to the effect of intact cell membrane on enhancing the D-amino acid production.

Indian J Exp Biol, 2000 Nov, 38(11), 1147 - 51
Colonization of arbuscular-mycorrhizal fungi on Ri T-DNA transformed roots in synthetic medium; Abdul-Khaliq et al.; Hairy root culture of tomato (Lycopersicon esculantum L.) was induced with three strains of Agrobacterium rhizogenes namely A4, ATCC 15834 and LBA 9402 . The best response in terms of growth of hairy root was observed with A . rhizogenes strain A4 and LBA 9402 followed by ATCC 15834 . Hairy roots were maintained on Murashige and Skoog (MS) medium but it could also grow on minimal (M) medium . Spores of Gigaspora margarita were isolated by wet sieving and decanting method and further recovered by sucrose density gradient method . A new method for surface sterilization of spores has been described which is simpler than the methods described earlier . Surface sterilized spores of G . margarita were used for inoculation of transformed roots grown on M medium as it was found more favourable for germination and growth of spores . During co-cultivation, mycorrhizal spore germination and its penetration into root cortex were observed . Inter and intracellular mycelial spread and formation of arbuscules were also observed in the cortical region of transformed roots of this plant.

Indian J Exp Biol, 2000 Nov, 38(11), 1086 - 91
Polyamines and plant alkaloids; Ghosh B; Naturally occurring alkaloids are nitrogenous compounds that constitute the pharmacogenically active basic principles of flowering plants . Alkaloids are classified into several biogenically related groups . Tobacco alkaloids are metabolised from polyamines and diamines putrescine and cadaverine . N-methyl transferase is the first enzyme in alkaloid biosynthetic pathway which drives the flow of nitrogen away from polyamine biosynthesis to alkaloid biosynthesis . Arginine decarboxylase has been suggested to be primarily responsible for providing putrescine for nicotine synthesis . Tryptophan is the precursor of indole alkaloids . However, the biosynthetic pathway of tropane and isoquinoline alkaloids are not clear . Genes for several key biosynthetic enzymes like arginine decarboxylase, ornithine decarboxylase, putrescine N-methyl transferase and spermidine synthase, hyoscyamine 6 beta hydroxylase,tryptophan decarboxylase etc have been cloned from different plant species . These genes are regulated by plant hormones, light, different kinds of stress and elicitors like jasmonates and their strong expression is primarily in the cultured roots . In view of this, the axenic hairy root cultures induced by Agrobacterium rhizogenes have been utilised to synthesise secondary metabolites . The current development in the knowledge of alkaloid biosynthesis, particularly molecular analysis, has been discussed in this review that may help to open up new avenues of investigation for the researchers.

Mol Biotechnol, 2001 Feb, 17(2), 109 - 17
Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.); Zhang CL et al.; Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic plants . The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants . It was envisaged that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate "escapes" and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance genes in field-released crops . The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for enhanced fluorescence under blue light . This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet via Agrobacterium-mediated transformation . Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several days after cocultivation . Stably transformed calli, which showed green fluorescence at a range of densities, were obtained at frequencies of 3-11% after transferring the inoculated cultures to selection media . Cocultivated shoot explants or embryogenic calli were regularly monitored under the microscope with blue light when they were transferred to media without selective agents . Green fluorescent shoots were obtained at frequencies of 2-5% . It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has potential for the development of selectable marker-free transgenic sugar beet.

J Bacteriol, 2001 Jul, 183(13), 4079 - 89
Efficient vir gene induction in Agrobacterium tumefaciens requires virA, virG, and vir box from the same Ti plasmid; Krishnamohan A et al.; The vir genes of octopine, nopaline, and L,L-succinamopine Ti plasmids exhibit structural and functional similarities . However, we observed differences in the interactions between octopine and nopaline vir components . The induction of an octopine virE(A6)::lacZ fusion (pSM358cd) was 2.3-fold higher in an octopine strain (A348) than in a nopaline strain (C58) . Supplementation of the octopine virG(A6) in a nopaline strain with pSM358 did not completely restore virE(A6) induction . However, addition of the octopine virA(A6) to the above strain increased virE(A6) induction to a level almost comparable to that in octopine strains . In a reciprocal analysis, the induction of a nopaline virE(C58)::cat fusion (pUCD1553) was two- to threefold higher in nopaline (C58 and T37) strains than in octopine (A348 and Ach5) and L,L-succinamopine (A281) strains . Supplementation of nopaline virA(C58) and virG(C58) in an octopine strain (A348) harboring pUCD1553 increased induction levels of virE(C58)::cat fusion to a level comparable to that in a nopaline strain (C58) . Our results suggest that octopine and L,L-succinamopine VirG proteins induce the octopine virE(A6) more efficiently than they do the nopaline virE(C58) . Conversely, the nopaline VirG protein induces the nopaline virE(C58) more efficiently than it does the octopine virE(A6) . The ability of Bo542 virG to bring about supervirulence in tobacco is observed for an octopine vir helper (LBA4404) but not for a nopaline vir helper (PMP90) . Our analyses reveal that quantitative differences exist in the interactions between VirG and vir boxes of different Ti plasmids . Efficient vir gene induction in octopine and nopaline strains requires virA, virG, and vir boxes from the respective Ti plasmids.

J Bacteriol, 2001 Jul, 183(13), 3919 - 30
The Agrobacterium tumefaciens rnd homolog is required for TraR-mediated quorum-dependent activation of Ti plasmid tra gene expression; Luo ZQ et al.; Conjugal transfer of Agrobacterium tumefaciens Ti plasmids is regulated by quorum sensing via TraR and its cognate autoinducer, N-(3-oxo-octanoyl)-L-homoserine lactone . We isolated four Tn5-induced mutants of A . tumefaciens C58 deficient in TraR-mediated activation of tra genes on pTiC58DeltaaccR . These mutations also affected the growth of the bacterium but had no detectable influence on the expression of two tester gene systems that are not regulated by quorum sensing . In all four mutants Tn5 was inserted in a chromosomal open reading frame (ORF) coding for a product showing high similarity to RNase D, coded for by rnd of Escherichia coli, an RNase known to be involved in tRNA processing . The wild-type allele of the rnd homolog cloned from C58 restored the two phenotypes to each mutant . Several ORFs, including a homolog of cya2, surround A . tumefaciens rnd, but none of these genes exerted a detectable effect on the expression of the tra reporter . In the mutant, traR was expressed from the Ti plasmid at a level about twofold lower than that in NT1 . The expression of tra, but not the growth rate, was partially restored by increasing the copy number of traR or by disrupting traM, a Ti plasmid gene coding for an antiactivator specific for TraR . The mutation in rnd also slightly reduced expression of two tested vir genes but had no detectable effect on tumor induction by this mutant . Our data suggest that the defect in tra gene induction in the mutants results from lowered levels of TraR . In turn, production of sufficient amounts of TraR apparently is sensitive to a cellular function requiring RNase D.

J Bacteriol, 2001 Jul, 183(13), 3855 - 65
Activities of virE1 and the VirE1 secretion chaperone in export of the multifunctional VirE2 effector via an Agrobacterium type IV secretion pathway; Zhao Z et al.; Agrobacterium tumefaciens uses a type IV secretion system to deliver oncogenic nucleoprotein particles and effector proteins, such as the multifunctional VirE2 protein, to plant cells . In this study, we examined the function of virE1 and its product, the VirE1 secretion chaperone, in mediating VirE2 export . A nonpolar virE1 null mutant accumulated low levels of VirE2, and trans expression of virE1 in this mutant only partially restored VirE2 abundance . Deletion of virE1 did not affect transcription but decreased translation of virE2, as shown by analysis of lacZ transcriptional and translational fusions . VirE2 was stable for a prolonged period, more than 6 h, when it was expressed in cis with virE1, and it exhibited half-lives of about 2 h when it was expressed in trans with virE1 and less than 10 min when it was expressed in the absence of virE1, as shown by pulse-chase experiments . VirE1 stabilized VirE2 via an interaction with a domain near the N terminus of VirE2, as shown by analyses of VirE2 truncation and insertion mutants synthesized in A . tumefaciens . VirE1 self-association was demonstrated by using bacteriophage lambda cI repressor fusion and pull-down assays, and evidence of VirE1 homomultimerization in vivo was obtained by native polyacrylamide gel electrophoresis and gel filtration chromatography . A putative VirE1-VirE2 complex with a molecular mass of about 70 to 80 kDa was detected by gel filtration chromatography of extracts from wild-type cells, whereas higher-order VirE2 complexes or aggregates were detected in extracts from a virE1 mutant . Taken together, our findings show that virE1 contributes in several ways to VirE2 export:(i) virE1 regulates efficient virE2 translation in the context of expression from the native P(virE) promoter; (ii) the VirE1 secretion chaperone stabilizes VirE2, most probably via an interaction with an N-terminal domain; and (iii) VirE1 forms a VirE1-VirE2 complex with a predicted 2:1 stoichiometry that inhibits assembly of higher-order VirE2 complexes or aggregates.

Phytochemistry, 2001 Jun, 57(3), 365 - 71
Ginsenoside production in different phenotypes of Panax ginseng transformed roots; Mallol A et al.; Transformed roots were obtained after the inoculation of sterile root discs of Panax ginseng C.A . Meyer with Agrobacterium rhizogenes A4 . The established hairy root lines displayed three morphological phenotypes when cultured on hormone-free liquid Schenk and Hildebrandt medium . Most of the cultures showed the characteristic traits of hairy roots (HR-M), while others were either callus-like (C-M) or thin (T-M) without branching . The growth rate of the transformed root lines was always higher than that of untransformed roots, showing that the genetic changes caused by the A . rhizogenes transformation conditioned a higher biomass formation . When considering the different transformed root phenotypes, we can observe that the highest ginsenoside production was achieved by HR-M root lines, closely followed by C-M ones, whereas the lowest yield was reached by T-M root phenotype . The study of the integration of the TL-DNA and TR-DNA fragments of the pRiA4 in the root genome showed that the aux1 gene was always detected in HR-M and C-M root phenotypes which presented the highest biomass and ginsenoside productions . This fact suggests a significant role of aux genes in the morphology of Panax ginseng transformed roots . The ginsenoside pattern of transformed roots varied according to their morphology, although the ginsenoside contents of the Rg group was always higher than that of the Rb group . From our results, we can infer the potential of some root phenotypes of Panax ginseng hairy root cultures for an improved ginsenoside production.

Bioorg Med Chem Lett, 2001 May 21, 11(10), 1339 - 42
Powerful probes for glycosidases: novel, fluorescently tagged glycosidase inhibitors; Hermetter A et al.; Amino-1,2,5-trideoxy-2,5-imino-D-mannitol was fluorescently tagged by reaction with dansyl chloride at N-1 or by attachment of a dansyl amide bearing spacer to this centre . Compounds obtained are highly potent inhibitors of beta-glucosidase exhibiting Ki values in the single figure nanomolar range . The 1-N-dansyl substituted inhibitor was successfully exploited for binding studies with beta-glucosidase from Agrobacterium sp . employing fluorescence spectrometric methods.

Mol Plant Microbe Interact, 2001 Jun, 14(6), 793 - 803
Intracellular accumulation of mannopine, an opine produced by crown gall tumors, transiently inhibits growth of Agrobacterium tumefaciens; Kim KS et al.; pYDH208, a cosmid clone from the octopine-mannityl opine-type tumor-inducing (Ti) plasmid pTi15955 confers utilization of mannopine (MOP) and agropine (AGR) on Agrobacterium tumefaciens strain NT1 . NT1 harboring pYDH208 with an insertion mutation in mocC, which codes for MOP oxidoreductase, not only fails to utilize MOP as a sole carbon source, but also was inhibited in its growth by MOP and AGR . In contrast, the growth of mutants with insertions in other tested moc genes was not inhibited by either opine . Growth of strains NT1 or UIA5, a derivative of C58 that lacks pAtC58, was not inhibited by MOP, but growth of NT1 or UIA5 harboring pRE10, which codes for the MOP transport system, was inhibited by the opine . When a clone expressing mocC was introduced, the growth of strain NT1(pRE10) was not inhibited by MOP, although UIA5(pRE10) was still weakly inhibited . In strain NT1(pRE10, mocC), santhopine (SOP), produced by the oxidation of MOP by MocC, was further degraded by functions encoded by pAtC58 . These results suggest that MOP and, to a lesser extent, SOP are inhibitory when accumulated intracellularly . The growth of NT1(pRE10), as measured by turbidity and viable cell counts, ceased upon the addition of MOP but restarted in a few hours . Regrowth was partly the result of the outgrowth of spontaneous MOP-resistant mutants and partly the adaptation of cells to MOP in the medium . Chrysopine, isochrysopine, and analogs of MOP in which the glutamine residue is substituted with other amino acids were barely taken up by NT1(pRE10) and were not inhibitory to growth of the strain . Sugar analogs of MOP were inhibitory, and those containing sugars in the D form were more inhibitory than those containing sugars in the L form . MOP analogs containing hexose sugars were more inhibitory than those containing sugars with three, four, or five carbon atoms . Mutants of NT1(pRE10) that are resistant to MOP arose in the zone of growth inhibition . Genetic and physiological analyses indicate that the mutations are located on pRE10 and abolish uptake of the opine.

Mol Plant Microbe Interact, 2001 Jun, 14(6), 695 - 700
Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations; Boisson-Dernier A et al.; Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization . An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues . For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M . truncatula . This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest . M . truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti . Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines . M . truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells . Our results suggest that M . truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.

Mikrobiologiia, 2001 Mar-Apr, 70(2), 275 - 82
{Study of extracellular structures of Agrobacterium involved in bacterial and plant interactions}; Chumakov MI et al.; Agrobacterial cells produced straight microfibrils not only when in contact with wheat seedling roots, but also when in contact with each other . After 2 h of incubation, agrobacterial cells were found to form aggregates, in which the cells were in contact either directly or through thick straight microfibrils (bridges) of an unknown composition . The majority of the microfibrils were susceptible to attack by cellulase, although some of them showed resistance to this enzyme . Like the wild-type flagellated agrobacteria, their bald mutants produced long straight microfibrils . The cells surface structures of agrobacteria were examined by labeling them immunocytochemically with colloidal gold conjugated antibodies against O-specific lipopolysaccharides, Vir proteins, and cellulase . Agrobacterial cells treated with acetosyringone and brought into contact were found to contain subpolar and polar cell surface structures . Antibodies against the VirB2 protein were able to interact with a tuft of thin microfibrils located on one pole of the agrobacterial cell, whose vir genes were induced by acetosyringone, but were unable to interact with the surface structures of the agrobacterial cells aggregated in liquid medium in the absence of wheat seedlings.

Mikrobiologiia, 2001 Mar-Apr, 70(2), 263 - 9
{Processes of plant colonization by Methylobacterium strains and some bacterial properties }; Romanovskaia VA et al.; The pink-pigmented facultative methylotrophic bacteria (PPFMB) of the genus Methylobacterium are indespensible inhabitants of the plant phyllosphere . Using maize Zea mays as a model, the ways of plant colonization by PPFMB and some properties of the latter that might be beneficial to plants were studied . A marked strain, Methylobacterium mesophilicum APR-8 (pULB113), was generated to facilitate the detection of the methylotrophic bacteria inoculated into the soil or applied to the maize leaves . Colonization of maize leaves by M . mesophilicum APR-8 (pULB113) occurred only after the bacteria were applied onto the leaf surface . In this case, the number of PPFMB cells on inoculated leaves increased with plant growth . During seed germination, no colonization of maize leaves with M . mesophilicum cells occurred immediately from the soil inoculated with the marked strain . Thus, under natural conditions, colonization of plant leaves with PPFMB seems to occur via soil particle transfer to the leaves by air . PPFMB monocultures were not antagonistic to phytopathogenic bacteria . However, mixed cultures of epiphytic bacteria containing Methylobacterium mesophilicum or M . extorquens did exhibit an antagonistic effect against the phytopathogenic bacteria studied (Xanthomonas camprestris, Pseudomonas syringae, Erwinia carotovora, Clavibacter michiganense, and Agrobacterium tumifaciens) . Neither epiphytic and soil strains of Methylobacterium extorquens, M . organophillum, M . mesophilicum, and M . fujisawaense catalyzed ice nucleation . Hence, they cause no frost injury to plants . Thus, the results indicate that the strains of the genus Methylobacterium can protect plants against adverse environmental factors.

Curr Microbiol, 2001 Jun, 42(6), 398 - 402
Toxicity and mutagenesis of chrysotile asbestos to Agrobacterium radiobacter; Yoshida N et al.; The mutation of Agrobacterium radiobacter cells exposed to chrysotile asbestos was examined by the random amplified polymorphic DNA (RAPD) method . Approximately 1.4 kbp of DNA in A . radiobacter, which was not amplified strongly in the cells that were not exposed to asbestos, was amplified in the cells that were exposed to asbestos . Mutation in genomic DNA of A . radiobacter was found to be induced by asbestos . Specific DNA that was amplified by asbestos present in PCR products and that which exists latently in genomic DNA were cloned, and these sequences were then determined and compared . It was shown that one of the mutations by the asbestos in the A . radiobacter occurred only in the primer annealed region and was a point mutation or deletion.

Appl Environ Microbiol, 2001 Jun, 67(6), 2617 - 21
Natural transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in soil; Demaneche S et al.; Little information is available concerning the occurrence of natural transformation of bacteria in soil, the frequency of such events, and the actual role of this process on bacterial evolution . This is because few bacteria are known to possess the genes required to develop competence and because the tested bacteria are unable to reach this physiological state in situ . In this study we found that two soil bacteria, Agrobacterium tumefaciens and Pseudomonas fluorescens, can undergo transformation in soil microcosms without any specific physical or chemical treatment . Moreover, P . fluorescens produced transformants in both sterile and nonsterile soil microcosms but failed to do so in the various in vitro conditions we tested . A . tumefaciens could be transformed in vitro and in sterile soil samples . These results indicate that the number of transformable bacteria could be higher than previously thought and that these bacteria could find the conditions necessary for uptake of extracellular DNA in soil.

Mol Biol Evol, 2001 Jun, 18(6), 907 - 16
Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer; Suominen L et al.; Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants . In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed . Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process . Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots . This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R . galegae . Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R . galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon . Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R . galegae, S . meliloti, and R . leguminosarum biovars viciae and trifolii . This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R . galegae close to agrobacteria and separate from most other rhizobia . The topology of the nodA tree was similar to that of the corresponding host plant tree . Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.

J Bacteriol, 2001 Jun, 183(12), 3704 - 11
Reconstitution of acetosyringone-mediated Agrobacterium tumefaciens virulence gene expression in the heterologous host Escherichia coli; Lohrke SM et al.; The ability to utilize Escherichia coli as a heterologous system in which to study the regulation of Agrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes . We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E . coli containing a constitutively active virG gene {virG(Con)} . Here we show that an RpoA hybrid containing the N-terminal 247 residues from E . coli and the C-terminal 89 residues from A . tumefaciens was able to significantly express virBp::lacZ in E . coli in a VirG(Con)-dependent manner . Utilization of lac promoter-driven virA and virG in combination with the A . tumefaciens rpoA construct resulted in significant inducer-mediated expression of the virBp::lacZ fusion, and the level of virBp::lacZ expression was positively correlated to the copy number of the rpoA construct . This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A . tumefaciens . Furthermore, the effect of sugars on vir gene expression was observed only in the presence of the chvE gene, suggesting that the glucose-binding protein of E . coli, a homologue of ChvE, does not interact with the VirA molecule . We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A . tumefaciens strain A348 from which the virA clone was derived . These data support the notion that VirA directly senses the phenolic inducer . However, the overall level of expression of the vir genes in E . coli is less than what is observed in A . tumefaciens, suggesting that additional gene(s) from A . tumefaciens may be required for the full expression of virulence genes in E . coli.

J Bacteriol, 2001 Jun, 183(12), 3642 - 51
VirB7 lipoprotein is exocellular and associates with the Agrobacterium tumefaciens T pilus; Sagulenko V et al.; Agrobacterium tumefaciens transfers oncogenic T-DNA and effector proteins to plant cells via a type IV secretion pathway . This transfer system, assembled from the products of the virB operon, is thought to consist of a transenvelope mating channel and the T pilus . When screened for the presence of VirB and VirE proteins, material sheared from the cell surface of octopine strain A348 was seen to possess detectable levels of VirB2 pilin, VirB5, and the VirB7 outer membrane lipoprotein . Material sheared from the cell surface of most virB gene deletion mutants also possessed VirB7, but not VirB2 or VirB5 . During purification of the T pilus from wild-type cells, VirB2, VirB5, and VirB7 cofractionated through successive steps of gel filtration chromatography and sucrose density gradient centrifugation . A complex containing VirB2 and VirB7 was precipitated from a gel filtration fraction enriched for T pilus with both anti-VirB2 and anti-VirB7 antiserum . Both the exocellular and cellular forms of VirB7 migrated as disulfide-cross-linked dimers and monomers when samples were electrophoresed under nonreducing conditions . A mutant synthesizing VirB7 with a Ser substitution of the lipid-modified Cys15 residue failed to elaborate the T pilus, whereas a mutant synthesizing VirB7 with a Ser substitution for the disulfide-reactive Cys24 residue produced very low levels of T pilus . Together, these findings establish that the VirB7 lipoprotein localizes exocellularly, it associates with the T pilus, and both VirB7 lipid modification and disulfide cross-linking are important for T-pilus assembly . T-pilus-associated VirB2 migrated in nonreducing gels as a monomer and a disulfide-cross-linked homodimer, whereas cellular VirB2 migrated as a monomer . A strain synthesizing a VirB2 mutant with a Ser substitution for the reactive Cys64 residue elaborated T pilus but exhibited an attenuated virulence phenotype . Dithiothreitol-treated T pilus composed of native VirB2 pilin and untreated T pilus composed of the VirB2C64S mutant pilin distributed in sucrose gradients more predominantly in regions of lower sucrose density than untreated, native T pili . These findings indicate that intermolecular cross-linking of pilin monomers is not required for T-pilus production, but cross-linking does contribute to T-pilus stabilization.

J Bacteriol, 2001 Jun, 183(12), 3636 - 41
Functional analysis of the Agrobacterium tumefaciens T-DNA transport pore protein VirB8; Kumar RB et al.; The VirB8 protein of Agrobacterium tumefaciens is essential for DNA transfer to plants . VirB8, a 237-residue polypeptide, is an integral membrane protein with a short N-terminal cytoplasmic domain . It interacts with two transport pore proteins, VirB9 and VirB10, in addition to itself . To study the role of these interactions in DNA transfer and to identify essential amino acids of VirB8, we introduced random mutations in virB8 by the mutagenic PCR method . The putative mutants were tested for VirB8 function by the ability to complement a virB8 deletion mutant in tumor formation assays . After multiple rounds of screening 13 mutants that failed to complement the virB8 deletion mutation were identified . Analysis of the mutant strains by DNA sequence analysis, Western blot assays, and reconstruction of new point mutations led to the identification of five amino acid residues that are essential for VirB8 function . The substitution of glycine-78 to serine, serine-87 to leucine, alanine-100 to valine, arginine-107 to proline or alanine, and threonine-192 to methionine led to the loss of VirB8 activity . When introduced into the wild-type strain, virB8(S87L) partially suppressed the tumor forming ability of the wild-type protein . Analysis of protein-protein interaction by the yeast two-hybrid assay indicated that VirB8(R107P) is defective in interactions with both VirB9 and VirB10 . A second mutant VirB8(S87L) is defective in interaction with VirB9.

Mol Genet Genomics, 2001 Mar, 265(1), 32 - 42
Tnt1 transposition events are induced by in vitro transformation of Arabidopsis thaliana, and transposed copies integrate into genes; Courtial B et al.; Tissue culture has been shown to induce the transposition of plant transposable elements; their insertion at novel sites results in somaclonal variation . Introduction of the tobacco retrotransposon Tnt1 into Arabidopsis thaliana by co-cultivation of root explants with Agrobacterium tumefaciens induces its transposition at a high frequency, but no transposed copies are found in plants transformed by the in planta procedure . Transposition occurs in the transformed root cells or in the calli derived from them, allowing the regeneration of transformed plants with up to 26 transposed copies of Tnt1 . Analysis of Tnt1 integration sites in Arabidopsis shows that the Tnt1 endonuclease does not show any cleavage-site specificity at the sequence level . The insertion sites are unlinked and distributed on all five Arabidopsis chromosomes . The fact that the majority of the integration sites are located in coding regions, and none in repeated sequences, demonstrates the potential of Tnt1 as a tool for gene tagging.

Proc Natl Sci Counc Repub China B, 2001 Apr, 25(2), 119 - 27
Generation of PCR-based DNA fragments for specific detection of Streptomyces saraceticus N45; Kong LR et al.; Streptomyces saraceticus strain N45, a saprophytic Gram-positive bacteria, has been shown to harbor high chitinase activity . Due to its potential use in biological control, the cloning of chitinase genes and the development of methods to quickly and precisely detect its presence have become necessary . In this study, PCR-based random amplified polymorphic DNA (RAPD) and PCR strategies were used to amplify random DNA fragments from the genome of S . saraceticus N45 . Three amplified DNA fragments, 417, 523 and 655 bp in length, were further isolated, subcloned and sequenced . Nest primers were designed from terminal ends of these three fragments and used for further PCR reactions . A single specific band was produced from the genomic DNA of S . saraceticus N45 for each nest primer pair . These three single bands were S . saraceticus N45 specific and were not amplified from other species of Streptomyces or bacteria, such as Ralstonia solanacearum, Agrobacterium tumefaciens, E . coli, Bacillus subtilis and Xanthomonas campestris pv . campestris . Through detection of the coexistence of these three fragments in PCR reaction using DNA or bacterial cells directly, the presence of S . saraceticus N45 can be confirmed . Further Southern analysis indicated that these three DNA fragments were specifically present in the S . saraceticus N45 genome in a single copy manner, and therefore, that they can potentially be used as markers for identification of S . saraceticus N45.

J Gen Virol, 2001 Jun, 82(Pt 6), 1517 - 27
Mutational analysis of the proteinase function of Potato leafroll virus; Sadowy E et al.; cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2 . Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells . Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513-515 of the replicase to VHD abolished PLRV replication . Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein . Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other . Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.

Membr Cell Biol, 2000, 14(3), 309 - 31
Transfer of genetic information from agrobacteria to bacterial and plant cells: membrane and supramembrane structures involved in transfer; Chumakov MI; The review deals with the supramembrane and membrane structures involved in the initial contact (attachment) of an agrobacterial cell with a bacterial or plant cell during the transfer of the agrobacterial genetic information . The relationships between the donor cell attachment to the recipient cell surface and the infection and conjugation processes are discussed . Experimental data on the recently found agrobacterial pili and surface protein rhicadhesin, which are involved in the conjugative transfer of the plasmid between agrobacteria, are considered . The role of adhesive and conjugative pili of E . coli in the initial and tight contacts is analyzed in the context of the recently proved similarity between the mechanisms of agrobacterial transformation in plants and conjugative transfer in bacteria . Possible involvement of the pilus in the conjugative transfer of agrobacterial DNA across the membranes of donor and recipient (bacterial and plant) cells is discussed.

Chem Biol, 2001 May, 8(5), 437 - 43
Directed evolution of new glycosynthases from Agrobacterium beta-glucosidase: a general screen to detect enzymes for oligosaccharide synthesis; Mayer C et al.; BACKGROUND: Oligosaccharide synthesis is becoming increasingly important to industry as diverse therapeutic roles for these molecules are discovered . The chemical synthesis of oligosaccharides on an industrial scale is often prohibitively complex and costly . An alternative, that of enzymatic synthesis, is limited by the difficulty of obtaining an appropriate enzyme . A general screen for enzymes that catalyze the synthesis of the glycosidic bond would enable the identification and engineering of new or improved enzymes . RESULTS: Glycosynthases are nucleophile mutants of retaining glycosidases that efficiently catalyze the synthesis of the glycosidic linkage by condensing an activated glycosyl fluoride donor with a suitable acceptor sugar . A novel agar plate-based coupled-enzyme screen was developed (using a two-plasmid system) and used to select an improved glycosynthase from a library of mutants . CONCLUSIONS: Plate-based coupled-enzyme screens of this type are extremely valuable for identification of functional synthetic enzymes and can be applied to the evolution of a range of glycosyl transferases.

Plant Mol Biol, 2001 Mar, 45(4), 377 - 85
Transient GFP expression in Nicotiana plumbaginifolia suspension cells: the role of gene silencing, cell death and T-DNA loss; Weld R et al.; The transient nature of T-DNA expression was studied with a gfp reporter gene transferred to Nicotiana plumbaginifolia suspension cells from Agrobacterium tumefaciens . Individual GFP-expressing protoplasts were isolated after 4 days' co-cultivation . The protoplasts were cultured without selection and 4 weeks later the surviving proto-calluses were again screened for GFP expression . Of the proto-calluses initially expressing GFP, 50% had lost detectable GFP activity during the first 4 weeks of culture . Multiple T-DNA copies of the gfp gene were detected in 10 of 17 proto-calluses lacking visible GFP activity . The remaining 7 cell lines contained no gfp sequences . Our results confirm that transiently expressed T-DNAs can be lost during growth of somatic cells and demonstrate that transiently expressing cells frequently integrate multiple T-DNAs that become silenced . In cells competent for DNA uptake, cell death and gene silencing were more important barriers to the recovery of stably expressing transformants than lack of T-DNA integration.

Plant Physiol, 2001 May, 126(1), 278 - 88
Temperature-sensitive alleles of RSW2 link the KORRIGAN endo-1,4-beta-glucanase to cellulose synthesis and cytokinesis in Arabidopsis; Lane DR et al.; An 8.5-kb cosmid containing the KORRIGAN gene complements the cellulose-deficient rsw2-1 mutant of Arabidopsis . Three temperature-sensitive alleles of rsw2 show single amino acid mutations in the putative endo-1,4-beta-glucanase encoded by KOR . The F1 from crosses between kor-1 and rsw2 alleles shows a weak, temperature-sensitive root phenotype . The shoots of rsw2-1 seedlings produce less cellulose and accumulate a short chain, readily extractable glucan resembling that reported for rsw1 (which is defective in a putative glycosyltransferase required for cellulose synthesis) . The double mutant (rsw2-1 rsw1) shows further reductions in cellulose production relative to both single mutants, constitutively slow root growth, and enhanced temperature-sensitive responses that are typically more severe than in either single mutant . Abnormal cytokinesis and severely reduced birefringent retardation in elongating root cell walls of rsw2 link the enzyme to cellulose production for primary cell walls and probably cell plates . The Rsw2(-) phenotype generally resembles the Kor(-) and cellulose-deficient Rsw1(-) phenotypes, but anther dehiscence is impaired in Rsw2-1(-) . The findings link a second putative enzyme activity to cellulose synthesis in primary cell walls of Arabidopsis and further increases the parallels to cellulose synthesis in Agrobacterium tumefaciens where the celA and celC genes are required and encode a putative glycosyltransferase and an endo-1,4-beta-glucanase related to RSW1 and KOR, respectively.

Planta, 2001 Apr, 212(5-6), 864 - 71
In vivo evidence that Ids3 from Hordeum vulgare encodes a dioxygenase that converts 2'-deoxymugineic acid to mugineic acid in transgenic rice; Kobayashi T et al.; We proposed that an Fe-deficiency-induced gene, Ids3 (Iron deficiency specific clone no . 3), from barley (Hordeum vulgare L.) roots encodes a dioxygenase that catalyzes the hydroxylation step from 2'-deoxymugineic acid (DMA) to mugineic acid (MA) . To prove this hypothesis, we introduced the Ids3 gene into rice (Oryza sativa L.), which lacks Ids3 homologues and secretes DMA, but not MA . Transgenic rice plants, carrying either Ids3 cDNA or a barley genomic DNA fragment (20 kb) containing Ids3, were obtained using Agrobacterium-mediated transformation . Ids3 cDNA under the control of the cauliflower mosaic virus 35S promoter was constitutively expressed in both the roots and the leaves of the transgenic rice, regardless of Fe nutrition status . In contrast, in the roots of transformants carrying a barley genomic fragment, transcripts of Ids3 were markedly increased in response to Fe deficiency . Slight expression of Ids3 was also observed in the leaves of the Fe-deficient plants . Western blot analysis confirmed the induction of Ids3 in response to Fe deficiency in the roots of the transformants carrying a genomic fragment . These expression patterns indicate that the 5'-flanking region of Ids3 works as a strong Fe-deficiency-inducible promoter in rice, as well as in barley . Both kinds of transgenic rice secreted MA in addition to DMA under Fe-deficient conditions, but wild-type rice secreted only DMA . This is in vivo evidence that IDS3 is the "MA synthase" that converts DMA to MA.

Planta Med, 2001 Apr, 67(3), 249 - 53
Decreased scopolamine yield in field-grown Duboisia plants regenerated from hairy roots; Roig Celma C et al.; Hairy root cultures were obtained from hybrid clones of Duboisia myoporoides x D . leichhardtii following transformation by Agrobacterium rhizogenes strain A4 . Shoots spontaneously regenerating from the hairy root cultures were rooted and transferred to soil . The plants displayed typical morphological alterations known as hairy root syndrome to varying degrees . PCR analysis confirmed that all transformed plants contained the rolA, rolB and rolC genes, irrespective of the degree of morphological alterations . A field test of the transformed regenerated plants revealed that those plants displaying the strongest hairy root syndrome symptoms had the highest content of the tropane alkaloid scopolamine . However, the overall scopolamine and hyoscyamine yield of all transformed plants was clearly reduced compared to untransformed control plants . These results demonstrate that the A . rhizogenes-transformed plants tested in this study do not provide a viable alternative to agricultural farming of hybrid clones of D . myoporoides x D . leichhardtii obtained by conventional breeding.

J Bacteriol, 2001 Jun, 183(11), 3310 - 7
ChvD, a chromosomally encoded ATP-binding cassette transporter-homologous protein involved in regulation of virulence gene expression in Agrobacterium tumefaciens; Liu Z et al.; A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the Agrobacterium tumefaciens VirB8 protein was carried out . This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon screen as a potential regulator of virG expression, chvD . In this report, the cloning of the entire chvD gene is described and the gene is sequenced and characterized . Insertion of a promoterless lacZ gene into the chvD locus greatly attenuated virulence and vir gene expression . Compared to that of the wild-type strain, growth of the chvD mutant was reduced in rich, but not minimal, medium . Expression of chvD, as monitored by expression of beta-galactosidase activity from the chvD-lacZ fusion, occurred in both rich and minimal media as well as under conditions that induce virulence gene expression . The ChvD protein is highly homologous to a family of ATP-binding cassette transporters involved in antibiotic export from bacteria and has two complete Walker box motifs . Molecular genetic analysis demonstrated that disruption of either Walker A box, singly, does not inactivate this protein's effect on virulence but that mutations in both Walker A boxes renders it incapable of complementing a chvD mutant strain . Constitutive expression of virG in the chvD mutant strain restored virulence, supporting the hypothesis that ChvD controls virulence through effects on virG expression.

Plant Cell, 2001 May, 13(5), 1035 - 46
Overexpression of the tobacco Tsi1 gene encoding an EREBP/AP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco; Park JM et al.; Using mRNA differential display analysis, we isolated a salt-induced transcript that showed a significant sequence homology with an EREBP/AP2 DNA binding motif from oilseed rape plants . With this cDNA fragment as a probe, cDNA clone Tsi1 (for Tobacco stress-induced gene1) was isolated from a tobacco cDNA library . RNA gel blot analysis indicated that transcripts homologous with Tsi1 were induced not only in NaCl-treated leaves but also in leaves treated with ethephon or salicylic acid . Transient expression analysis using a Tsi1::smGFP fusion gene in BY-2 cells indicated that the Tsi1 protein was targeted to the nucleus . Fusion protein of Tsi1 with GAL4 DNA binding domain strongly activated transcription in yeast, and the transactivating activity was localized to the 13 C-terminal amino acids of Tsi1 . Electrophoretic mobility shift assays revealed that Tsi1 could bind specifically to the GCC and the DRE/CRT sequences, although the binding activity to the former was stronger than that to the latter . Furthermore, Agrobacterium-mediated transient expression and transgenic plants expressing Tsi1 demonstrated that overexpression of the Tsi1 gene induced expression of several pathogenesis-related genes under normal conditions, resulting in improved tolerance to salt and pathogens . These results suggest that Tsi1 might be involved as a positive trans-acting factor in two separate signal transduction pathways under abiotic and biotic stress.

Arch Pharm Res, 2001 Apr, 24(2), 109 - 13
Anticoagulant activity of sulfoalkyl derivatives of curdlan; Lee KB et al.; Curdlan is a natural beta-1,3-glucan produced by Agrobacterium biovar 1 . In this study, the anticoagulant activity of sulfoalkyl derivatives of curdlan was investigated by carrying out activated partial thromboplastin time (APTT) assay and compared with that of o-sulfonated curdlan . Approximately 100-fold higher concentration of o-sulfonated curdlan than heparin was required to obtain the same level of the clotting time . Anticoagulant activity of curdlan derivatives was dependent on the degree of sulfation in prolonging the clotting time . However, the chain length of the substituent did not play a role in prolonging the clotting time . The curdlan derivatives enhanced thrombin inhibition by mediating through antithrombin III . The inhibition of thrombin by o-sulfonated curdlan was found to be approximately 10-fold weaker than that by heparin.

Mol Plant Microbe Interact, 2001 May, 14(5), 629 - 38
Genetic mapping and functional analysis of the tomato Bs4 locus governing recognition of the Xanthomonas campestris pv . vesicatoria AvrBs4 protein; Ballvora A et al.; Xanthomonas campestris pv . vesicatoria is the causal agent of bacterial spot disease on pepper (Capsicum spp.) and tomato (Lycopersicon spp.) . Analysis of 17 different Lycopersicon accessions with avrBs4-expressing X . campestris pv . vesicatoria strains identified 15 resistant and two susceptible tomato genotypes . Genetic analysis revealed that AvrBs4 recognition in tomato is governed by a single locus, designated Bs4 (bacterial spot resistance locus no . 4) . Amplified fragment length polymorphism and bulked DNA templates from resistant and susceptible plants were used to define a 2.6-cM interval containing the Bs4 locus . A standard tomato mapping population was employed to localize Bs4-linked markers on the short arm of chromosome 5 . Investigation of X . campestris pv . vesicatoria hrp mutant strains revealed that AvrBs4 secretion and avirulence activity are hrp dependent . Agrobacterium-based delivery of the avrBs4 gene into tomato triggered a plant response that phenotypically resembled the hypersensitive response induced by avrBs4-expressing X . campestris pv . vesicatoria strains, suggesting symplastic perception of the avirulence protein . Mutations in the avrBs4 C-terminal nuclear localization signals (NLSs) showed that NLSs are dispensable for Bs4-mediated recognition . Our data suggest that tomato Bs4 and pepper Bs3 employ different recognition modes for detection of the highly homologous X . campestris pv . vesicatoria avirulence proteins AvrBs4 and AvrBs3.

Biosci Biotechnol Biochem, 2001 Mar, 65(3), 658 - 61
Purification and characterization of mannose isomerase from Agrobacterium radiobacter M-1; Hirose J et al.; A mannose isomerase from Agrobacterium radiobacter M-1 (formerly Pseudomonas sp . MI) was purified to electrophoretic homogeneity and characterized . A cell-free extract was separated by ammonium sulfate fractionation, Butyl-Toyopearl 650M, DEAE-Sepharose and hydroxylapatite column chromatography . Its molecular mass was estimated to be 44 kDa by SDS-PAGE and 90 kDa by gel filtration, in which the enzyme is most likely a dimer composed of two identical subunits . The purified enzyme had an optimum pH at 8.0, an optimum temperature at 60 degrees C, a pI of 5.2 and a Km of 20 mM, and specifically converted D-mannose and D-lyxose to ketose . The N-terminal amino acid sequence was identified.

Yi Chuan Xue Bao, 2001, 28(4), 352 - 8
{Improvement of transformation frequency of rice mediated by Agrobacterium}; Yi ZL et al.; The factors influencing the frequency of rice transformation mediated by Agrobacterium have been investigated by using 16 commercially important indica and japonica rice cultivars or lines . The main results were as following: For most rice CC medium was the best for both callus initiation and subculture . With supplement of 2.5-5 mg/L ABA the quality of calli can be improved . The concentration of selective agent for Indica rice callus was lower than that for japonica rice callus . Agrobacterium tumefaciens strain EHA105 was more efficient than LBA4404 and AGL1 for rice transformation . The inhibitive effect of cefotaxime to Agrobacterium was better than that of carbenicillin . The partial desiccation treatment after co-cultivation was beneficial to inhibit the growth of Agrobacterium and increase transformation efficiency . A stable and efficient Agrobacterium-mediated transformation system has been established in ten different rice cultivars and fertile transgenic plants have been obtained.

Yi Chuan Xue Bao, 2001, 28(4), 345 - 51
{Amplification and analysis of T-DNA flanking sequences in transgenic rice}; Fang J et al.; Rice transformation mediated by Agrobacterium tumefaciens has technically matured to some extent, but the mechanics of T-DNA integration in transgenic rice remains largely unknown . Using thermal asymmetric interlaced PCR (TAIL-PCR), we analyzed the flanking sequences of T-DNAs in transgenic rice plants, in which the resistance gene for rice bacterial blight disease, Xa21, had been integrated stably . Sequence analysis of 24 fragments amplified by TAIL-PCR showed that of them 14 were rice genomic DNA, 9 contained vector backbone sequences, and one was a fragment of the exogenous gene Xa21 . The characteristics of the 14 rice genomic DNA sequences at T-DNA integrated sites are significantly different from those of the reported rice genomic sequences into which exogenous genes were integrated through direct DNA transformation methods . T-DNA border sequences integrated in rice genome have similar features as those integrated in dicotyledonous genomes . In the backbone containing flanking sequences (37.5%, 9/24) vector backbones appeared in different types.

Nat Biotechnol, 2001 May, 19(5), 466 - 9
Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes; Takahashi M et al.; One of the widest ranging abiotic stresses in world agriculture arises from low iron (Fe) availability due to high soil pH, with 30% of arable land too alkaline for optimal crop production . Rice is especially susceptible to low iron supply, whereas other graminaceous crops such as barley are not . A barley genomic DNA fragment containing two naat genes, which encode crucial enzymes involved in the biosynthesis of phytosiderophores, was introduced into rice using Agrobacterium-mediated transformation and pBIGRZ1 . Phytosiderophores are natural iron chelators that graminaceous plants secrete from their roots to solubilize iron in the soil . The two transgenes were expressed in response to low iron nutritional status in both the shoots and roots of rice transformants . Transgenic rice expressing the two genes showed a higher nicotianamine aminotransferase activity and secreted larger amounts of phytosiderophores than nontransformants under iron-deficient conditions . Consequently, the transgenic rice showed an enhanced tolerance to low iron availability and had 4.1 times greater grain yields than that of the nontransformant rice in an alkaline soil.

Bioorg Med Chem Lett, 2001 Apr 23, 11(8), 1063 - 4
Novel, lipophilic derivatives of 2,5-dideoxy-2,5-imino-D-mannitol (DMDP) are powerful beta-glucosidase inhibitors; Wrodnigg TM et al.; Novel derivatives of the D-glucosidase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol bearing lipophilic aliphatic or aromatic amides attached to C-1 have been found to inhibit beta-glucosidase from Agrobacterium sp . in the nanomolar range . One of them, a coumarin derivative, ranks amongst the most active compounds in the class of reversible glycosidase inhibitors of the iminoalditol type.

J Bacteriol, 2001 May, 183(10), 3065 - 75
The CcrM DNA methyltransferase of Agrobacterium tumefaciens is essential, and its activity is cell cycle regulated; Kahng LS et al.; DNA methylation is now recognized as a regulator of multiple bacterial cellular processes . CcrM is a DNA adenine methyltransferase found in the alpha subdivision of the proteobacteria . Like the Dam enzyme, which is found primarily in Escherichia coli and other gamma proteobacteria, it does not appear to be part of a DNA restriction-modification system . The CcrM homolog of Agrobacterium tumefaciens was found to be essential for viability . Overexpression of CcrM is associated with significant abnormalities of cell morphology and DNA ploidy . Mapping of the transcriptional start site revealed a conserved binding motif for the global response regulator CtrA at the -35 position; this motif was footprinted by purified Caulobacter crescentus CtrA protein in its phosphorylated state . We have succeeded in isolating synchronized populations of Agrobacterium cells and analyzing their progression through the cell cycle . We demonstrate that DNA replication and cell division can be followed in an orderly manner and that flagellin expression is cyclic, consistent with our observation that motility varies during the cell cycle . Using these synchronized populations, we show that CcrM methylation of the chromosome is restricted to the late S phase of the cell cycle . Thus, within the alpha subdivision, there is a conserved cell cycle dependence and regulatory mechanism controlling ccrM expression.

J Nat Prod, 2001 Apr, 64(4), 531 - 2
A new metabolite from the marine bacterium Vibrio angustum S14; de Nys R et al.; The new metabolite {1-(2'-methylpropoxy)-2-hydroxy-2-methylpropoxy}butane was isolated from the cell-free culture supernatant of the marine bacterium Vibrio angustum S14 as part of studies investigating the role of chemical signals in prokaryote--prokaryote and prokaryote--eukaryote interactions . The structure was elucidated by interpretation of its high-field NMR and mass spectrometric data . {1-(2'-Methylpropoxy)-2-hydroxy-2-methylpropoxy}butane induced the acylated homoserine lactone (AHL) reporter system in Agrobacterium tumefaciens and bioluminescence in Vibrio harveyi.

Curr Genet, 2001 Feb, 39(1), 35 - 9
Transformation of the cultivated mushroom Agaricus bisporus (Lange) using T-DNA from Agrobacterium tumefaciens; Mikosch TS et al.; Agrobacterium tumefaciens is known to transfer parts of its tumor-inducing plasmid, the T-DNA, to plants, yeasts and filamentous fungi . We have used this system to transform germinating basidiospores and vegetative mycelium of a commercial strain of the cultivated basidiomycete Agaricus bisporus . Analysis of transformants shows that the T-DNA integrates at random sites into the host genome and that the selection marker is stable during mitosis and meiosis . The Agrobacterium system allows the transformation of both homokaryons and heterokaryons of A . bisporus . Also, both karyotypes of an heterokaryon can be transformed simultaneously . Furthermore, this is the first report on the transformation of vegetative mycelium of a commercial strain of A . bisporus.

Biotechnol Prog, 2001 Mar-Apr, 17(2), 247 - 51
Stable genetic transformation of Eschscholzia californica expressing synthetic green fluorescent proteins; Lee J et al.; An efficient protocol is described for the stable genetic transformation of Eschscholzia californica (California poppy) using Agrobacterium tumefaciens as a vector . We have employed the disarmed A . tumefaciens LBA4404 encoding a synthetic green fluorescent protein reporter gene that is further controlled by an enhanced cauliflower mosaic virus 35S promoter . Stably transformed E . californica cells appear 3 weeks after initial cocultivation of A . tumefaciens with poppy leaves, stems, or roots . Transformed poppy calli were visualized by exposure to long-wave UV or blue light and analyzed in detail by fluorescent microscopy and laser-scanning microscopy . Moreover, green fluorescent calli have been maintained through continual subculture and grow well either on Gamborg's B5 agarose or liquid medium.

FEMS Microbiol Ecol, 2001 May, 35(3), 277 - 285
Stress-induced proteins of Agrobacterium tumefaciens; Rosen R et al.; The pattern of proteins produced by bacteria represents the physiological state of the organism as well as the environmental conditions encountered . Environmental stress induces the expression of several regulons encoding stress proteins . Extensive information about the proteins which constitute these regulons (or stimulons) and their control is available for very few bacteria, such as the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli (gamma-proteobacteria) and is minimal for all other bacteria . Agrobacterium tumefaciens is a Gram-negative plant pathogen of the alpha-proteobacteria, which constitutes the main tool for plant recombinant genetics . Our previous studies on the control of chaperone-coding operons indicated that A . tumefaciens has unique features and combines regulatory elements from both B . subtilis and E . coli . Therefore, we examined the patterns of proteins induced in A . tumefaciens by environmental changes using two-dimensional gel electrophoresis and dual-channel image analysis . Shifts to high temperature, oxidative and mild acid stresses stimulated the expression of 97 proteins . The results indicate that most of these stress-induced proteins (80/97) were specific to one stress stimulon . Only 10 proteins appear to belong to a general stress regulon.

Virus Res, 2001 May, 75(1), 29 - 34
Restoration of secondary hairpin II is associated with restoration of infectivity of a non-viable recombinant viroid; Candresse T et al.; Mutagenesis and/or construction of recombinants by exchange of genomic regions between parental molecules constitute powerful tools for the study of viroids . However, a large proportion of such modifications results in molecules, which have lost their infectivity . Such is the case for a recombinant viroid named CECS, obtained by replacing the right half of a citrus exocortis viroid (CEVd) by the same region from chrysanthemum stunt viroid (CSVd) . In an effort to recover viable infectious progeny from this recombinant, tomato plants were inoculated with an Agrobacterium strain carrying a dimer of the CECS viroid in positive orientation under the control of the CaMV 35S promoter . About 20% of the plants treated in this way were found to be infected with a replicating viroid, which was further propagated . Sequence analysis of six cloned full-length cDNAs derived from progeny molecules revealed the presence of mutations as compared with the parental CECS sequence . However, only two types of mutations were consistently recovered in all progeny molecules, the addition of a G in a string of four at positions 70-73, a mutation frequently observed in CEVd isolates and mutations leading to the restoration of the correct base pairing in secondary hairpin II . These results show that agro-infection is a suitable technique for the recovery of viable molecules from non-infectious viroid mutants and confirm that the ability to form secondary hairpin II is a prerequisite for viroid infectivity.

Mol Plant Microbe Interact, 2001 Apr, 14(4), 577 - 9
Novel constructions to enable the integration of genes into the Agrobacterium tumefaciens C58 chromosome; Lee LY et al.; We constructed several versatile sets of vectors that can be used to introduce any gene into the pgl/picA locus of the Agrobacterium tumefaciens C58 chromosome without affecting T-DNA transfer . One set contains a fragment containing the lacIq and lacZ genes and a multiple cloning site from pBluescriptII SK(+) inserted into a PstI site between the pgl and picA genes on an incPalpha plasmid . The resulting plasmid contains eight unique restriction endonuclease sites and the ability to use blue-white screening for the presence of an insert . A second plasmid also contains a beta-lactamase gene within this locus and provides a convenient ampicillin-carbenicillin resistance marker for the selection of genes integrated into the chromosome following double homologous recombination (homogenotization) . A third plasmid contains, in addition to the lacZ, lacIq, and beta-lactamase genes within the pgl/picA locus, a sacRB gene cassette within the vector to counterselect against the presence of the vector within A . tumefaciens . To test this system, we introduced a wild-type virD2 gene into the A . tumefaciens chromosome at the pgl/picA locus . When a Ti plasmid harboring a deletion of virD2 was in this strain, the integrated virD2 gene complemented the virD2 deletion and the resulting transformation phenotype was identical to that resulting from A . tumefaciens strains harboring a wild-type virD2 gene located on a replicating plasmid.

Mol Microbiol, 2001 Apr, 40(2), 414 - 21
TrlR, a defective TraR-like protein of Agrobacterium tumefaciens, blocks TraR function in vitro by forming inactive TrlR:TraR dimers; Chai Y et al.; Octopine-type Ti plasmids of Agrobacterium tumefaciens require the quorum-sensing proteins TraR and TraI and the diffusible pheromone 3-oxooctanoyl homoserine lactone (AAI) to regulate genes required for conjugal transfer . TraR activity is inhibited by a protein called TrlR, which closely resembles amino acids 1-181 of TraR but is truncated as a result of a shift in the reading frame at codon 182 . This frameshift does not affect synthesis of the amino-terminal domain, which is thought to bind autoinducer and mediate protein dimerization, but abolishes translation of the carboxyl-terminal, DNA-binding domain . In this study, we show that TrlR, like TraR, requires AAI for solubility when overexpressed in Escherichia coli . TrlR bound one molecule of AAI per protein monomer, supporting the prediction that the amino-terminal domain of TraR contains the AAI binding site . Purified TrlR blocked TraR for both specific DNA binding and transcription of a tra promoter, supporting previous studies performed with whole cells . When TrlR and a TraR fusion protein were co-expressed in E . coli, these proteins readily formed heterodimeric complexes that were inactive in DNA-binding activity . These data support the hypotheses that (i) the amino-terminal half of TraR binds AAI and mediates protein dimerization; (ii) both DNA-binding domains in a TraR dimer are required for stable DNA binding; and (iii) TrlR blocks TraR by direct protein-protein interactions.

Mol Microbiol, 2001 Apr, 40(2), 294 - 305
Type IV secretion: intercellular transfer of macromolecules by systems ancestrally related to conjugation machines; Christie PJ; Bacterial conjugation systems are highly promiscuous macromolecular transfer systems that impact human health significantly . In clinical settings, conjugation is exceptionally problematic, leading to the rapid dissemination of antibiotic resistance genes and other virulence traits among bacterial populations . Recent work has shown that several pathogens of plants and mammals - Agrobacterium tumefaciens, Bordetella pertussis, Helicobacter pylori and Legionella pneumophila - have evolved secretion pathways ancestrally related to conjugation systems for the purpose of delivering effector molecules to eukaryotic target cells . Each of these systems exports distinct DNA or protein substrates to effect a myriad of changes in host cell physiology during infection . Collectively, secretion pathways ancestrally related to bacterial conjugation systems are now referred to as the type IV secretion family . The list of putative type IV family members is increasing rapidly, suggesting that macromolecular transfer by these systems is a widespread phenomenon in nature.

Transgenic Res, 2001 Apr, 10(2), 143 - 55
Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation; McCormac AC et al.; The co-transformation of a single plant genome with two independent T-DNA regions provides opportunities for genetic separation in subsequent generations . In an effective strategy, co-delivery events must form a high proportion of the total transformed population . In this study, using the model plant species tobacco (Nicotiana tabacum), it was shown that the frequency of co-transformation within a given To population could be as high as 100% and this was found to be dependent, at least in part, on designing the plasmid vectors so that the kbp size of the first selected T-DNA region was >2-fold that of the designated T-DNA region for co-transfer . Overall, 40-50% of To lines demonstrated the capacity for segregational separation of co-transformed T-DNA regions . Hence, the estimate of the required number of total transformants for such an independent strategy may seem to be as little as 2-fold that for a conventional, single T-DNA strategy, but we strongly temper such estimates with indications that high co-transformation frequencies may be associated with a higher incidence of linkage . In this co-transformation study we used a single (Agrobacterium) strain system in which a single binary plasmid contained either two or three T-DNA regions, each with a selectable marker . This arrangement could reveal that 'read-through' events within the Agrobacterium cells, resulting in the co-transfer of adjacent T-DNA regions as a single linked unit, accounted for up to 20% of co-transformed plant lines . Such read-through co-delivery appeared to be more frequent from the 'supervirulent' EHA101 A . tumefaciens strain, compared to the 'ordinary' LBA4404 strain . By using the binary plasmid with three selectable T-DNA regions, we have been able to consider the frequency of co-integration of a third independent T-DNA within a T0 subpopulation of co-transformants . This was found to be higher than expected . These observations were applied to the co-transfer of (unwanted) plasmid backbone sequences and showed that screening against such sequences may add a significant factor in achieving the desired, final genotype.

Transgenic Res, 2001 Apr, 10(2), 121 - 32
Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato; Frary A et al.; The efficiency of the binary bacterial artificial chromosome (BIBAC) vector for Agrobacterium-mediated stable transfer of high molecular weight DNA into plants was tested in tomato . Several variables affecting transformation efficiency were examined including insert size, Agrobacterium genetic background, and the presence of additional copies of the virG, virE1 and virE2 genes . It was found that a helper plasmid containing extra copies of virG was an absolute requirement for obtaining tomato transformants with the BIBAC . MOG101 with the virG helper plasmid was found to be the most efficient strain for transfer of high molecular weight DNA (150 kb) . Selected high molecular weight DNA transformants were advanced several generations (up to the R4) to assess T-DNA stability . This analysis showed that the T-DNA was stably maintained and inherited through several meioses regardless of whether it was in the hemizygous or homozygous state . Expression of a selectable marker gene within the T-DNA was also examined through several generations and no gene silencing was observed . Thus, the BIBAC is a useful system for transfer of large DNA fragments into the plant genome.

Transgenic Res, 2001 Apr, 10(2), 105 - 12
Genetic transformation of major wine grape cultivars of Vitis vinifera L; Iocco P et al.; We have developed an Agrobacterium-mediated transformation system for a number of important grapevine cultivars used in wine production . Transgenic plants were obtained for the seven cultivars: Cabernet Sauvignon, Shiraz, Chardonnay, Riesling, Sauvignon Blanc, Chenin Blanc and Muscat Gordo Blanco . Embryogenic callus was initiated from anther filaments and genotypic differences were observed for initiation and subsequent proliferation with Chardonnay responding most favourably to culture conditions . The transformation system allowed the recovery of germinating transgenic embryos 10-12 weeks after Agrobacterium inoculation and plants within 18 weeks . Examination of the expression patterns of the green fluorescent protein gene under the control of the CAMV35S promoter in leaf tissue of transgenic plants showed that for up to 35% of plants the pattern was not uniform . The successful transformation of a genetically diverse group of wine grape cultivars indicates that the transformation system may have general application to an even wider range of Vitis vinifera cultivars.

Tree Physiol, 2000 Jul, 20(13), 901 - 7
Regeneration of phenotypically normal English elm (Ulmus procera) plantlets following transformation with an Agrobacterium tumefaciens binary vector; Gartland JS et al.; A transformation system was developed for English elm (Ulmus procera Salisbury) using Agrobacterium tumefaciens C58 pMP90 p35SGUS/INTRON, allowing for the transfer of foreign genes and regeneration of phenotypically normal elm plantlets . The PCR analysis indicated that both nptII and uidA genes were stably inserted in the plant genome . beta-Glucuronidase histochemical and fluorimetric assays revealed expression of the uidA gene in the shoots, leaves, stems and roots of regenerated transgenic plants . The DNA-DNA hybridizations confirmed the presence of the uidA gene in regenerant plants . Factors influencing successful transformation and regeneration of elms included: identifying gene-transfer-proficient Agrobacterium strains for use with elms; developing an infection protocol allowing T-DNA transfer while retaining the ability to remove inciting bacteria; and identifying selection conditions to eliminate non-transformed material and choice of regeneration medium to allow shoot production . The potential utility of an effective elm transformation and regeneration system in the control of Dutch elm disease is discussed.

Biosci Biotechnol Biochem, 2001 Feb, 65(2), 383 - 8
A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential; Murata M et al.; Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples . Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO . Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene . Four KM-resistant callus lines were obtained from 356 leaf explants . Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM . One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus . The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus.

Plant Sci, 2001 Apr, 160(5), 889 - 898
Transformation of peanut (Arachis hypogaea L.) with tobacco chitinase gene: variable response of transformants to leaf spot disease; Rohini VK et al.; Fertile transgenic plants of peanut (Arachis hypogaea L.) cv . TMV-2 expressing tobacco chitinase and neomycin phosphotransferase (npt II) genes were generated using an Agrobacterium tumefaciens (LBA4404/pBI121-pBTex)-mediated transformation system . A tissue culture-independent method wherein embryo in the mature seed is inoculated and reared into single plant transformant was used for transformation . Southern blot analysis of genomic DNA isolated from T(0) transformants and progeny plants (T(1)) demonstrated that the transgenes are stably integrated in the genome of transgenic peanut plants and inherited by the offspring . The expression of the heterologous chitinase gene driven by CaMV 35S promoter led to a high level of activity in some of the transgenic plants . Small-scale field tests indicated increased ability of these plants to resist the fungal pathogen Cercospora arachidicola (the causal organism of the leaf spot or Tikka disease of peanut), which is an important peanut pathogen . These results suggest that a heterologous chitinase gene was functional in peanut and expressed in healthy plants . The study also shows that peanut plants containing transgenically increased activity of chitinase were resistant to attack by the fungal pathogen C . arachidicola to different degrees . The strategy employed may be useful for the control of other fungal diseases of the crop.

Plant Sci, 2001 Apr, 160(5), 869 - 875
Arginine decarboxylase transgene expression and analysis of environmental stress tolerance in transgenic rice; Roy M et al.; Arginine decarboxylase (ADC) cDNA from oat (Avena sativa L.) was introduced into rice (Oryza sativa L.) genome by an Agrobacterium-mediated transformation method . Expression of the ADC transgene under the control of an ABA-inducible promoter led to stress-induced upregulation of ADC activity and polyamine accumulation in transgenic rice plants . Second-generation (Rl) transgenic rice plants showed an increase in biomass under salinity-stress conditions, as compared to the non-transformed control plants.

Plant Sci, 2001 Apr, 160(5), 837 - 845
Transformation of Japanese persimmon (Diospyros kaki Thunb.) with apple cDNA encoding NADP-dependent sorbitol-6-phosphate dehydrogenase; Gao M et al.; Japanese persimmon (Diospyros kaki Thunb . cv Jiro) was transformed with apple (Malus x domestica Borkh.) cDNA encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) by an Agrobacterium-mediated leaf-disc transformation system . Integration and expression of the transgene were confirmed by genomic DNA blot and immunoblot analyses . Sorbitol accumulation in five of six transgenic plants obtained was confirmed by GC-MS . The amount of sorbitol in the leaves of transgenic plants varied from 14.5 to 61.5 micromol g(-1) fr wt(-1) . Sorbitol was not found in leaves of non-transformed 'Jiro' or the line PS7 that produced S6PDH protein with no S6PDH activity . Eventually, two transformed lines producing high (PS1) and medium (PS6) amounts of sorbitol, one control transformed line (PS7), and non-transformed 'Jiro' were selected and evaluated for salt-stress tolerance . Under NaCl stress, the activity of photosystem II in leaves was determined in terms of the ratio of the variable (Fv) to the maximum (Fm) fluorescence of chlorophyll . The rate of decline in Fv/Fm under NaCl stress was lower in PS1 than the other three lines, suggesting that PS1 is more tolerant to NaCl stress than the other three lines . The factors that caused enhanced salt stress tolerance in PS1 are discussed in relation to sorbitol biosynthesis and its growth.

Planta, 2001 Feb, 212(3), 404 - 15
Proteomic analysis reveals a novel set of cell wall proteins in a transformed tobacco cell culture that synthesises secondary walls as determined by biochemical and morphological parameters; Blee KA et al.; A cell suspension culture of a tobacco (Nicotiana tabacum L . cv . Petit Havana) cell line derived from a cultivar transformed with the Tcyt gene from Agrobacterium, which leads to high endogenous levels of cytokinin, has been established . This cell line shows increased cell aggregation, elongated cells and a 5-fold increase in wall thickness . If allowed to carry on growing it can form a single mass without shedding cells into the medium . When analysed at an earlier growth stage, these cultures were found to produce improved levels of vascular nodule formation than in other systems that employ exogenous cytokinin . This differentiation was optimised with respect to sucrose and auxin signals in order to induce maximum production of cells with thickened walls and a morphology characteristic of fibre cells and tracheids, in addition to cells that remain meristematic . In order to establish the validity of this system for studying secondary wall formation, the walls and associated biosynthetic changes were analysed in these cells by chemical analysis of the walls, changes in activities of enzymes of xylan and monolignol synthesis, and expression of mRNAs coding for enzymes of lignin biosynthesis . The wall composition of the transformed cells was compared with that determined for primary walls from a typical untransformed tobacco cell line . Recovery of wall material was 50% greater in the transformed culture . In this material a major difference was found in the pectin fraction where there was a distinct difference in size distribution together with a lower level of methylation for the transformed line, which may be related to increased adhesiveness . There were increased amounts of xylan, although the ratio of xyloglucan to xylan content was not substantially different due to the mixture of cell types . There was also an increase in cellulose and phenolic components . Increased activity of enzymes involved in the synthesis of xylan as a marker for the secondary wall occurred around the time of tracheid differentiation and coincided with a broad peak of cinnamyl alcohol dehydrogenase activity . The expression of mRNAs coding for enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, catechol O-methyl transferase was relatively constitutive in the cultures while transcripts of ferulate 5-hydroxylase, cinnamoyl CoA-reductase, cinnamyl alcohol dehydrogenase and lignin peroxidase were induced . The walls of the transformed cells also showed considerable differences in the subset of extractable proteins from that found in primary walls of tobacco when these were subjected to proteomic analysis . Many of these proteins appear to be novel and not present in primary walls . However an Mr-32,000 chitinase, an Mr-34,000 peroxidase, an Mr-65,000 polyphenoloxidase/laccase and possibly an Mr-68,000 xylanase could be identified as well as structural proteins.

Biochimie, 2001 Feb, 83(2), 177 - 86
The form of chromosomal DNA molecules in bacterial cells; Bendich AJ; The circular concept of the bacterial chromosome was based initially on experiments involving conjugation mapping and autoradiographic imaging of DNA . This view was then supported by DNA fragment mapping, genome sequencing, and the analysis of linear DNA produced by a single cleavage of chromosomal DNA . A circular chromosome is also indicated by the existence of a mechanism for segregating dimeric chromosomes produced by recombination and the replication of DNA on both sides of the replication terminus . The evidence for circularity is reviewed here and found to be compatible with either a circular or a linear chromosomal DNA molecule . Moving pictures of ethidium-stained DNA revealed most chromosomal DNA as a rosette form with loops emanating from a dense node or as a network of strands lacking a node . This description applies to Escherichia coli, Agrobacterium tumefaciens, Pyrococcus endeavorii, Vibrio cholerae, and both the linear-mapping chromosome of Streptomyces lividans and its circular-mapping derivative . Networks without nodes were found for two linear-mapping Borrelia species . For the E . coli chromosome, open-form circles of various sizes were found only at extremely low frequency . The node of the rosette was reduced in size or eliminated in recA mutants, as well as by treatment with either ribonuclease, topoisomerase IV, 1 M NaCl, or lysozyme . A model is presented for the bacterial chromosome in which the DNA is compacted by many points of strand association (including recombination junctions, tangles and knots) created during the repair of DNA damage that occurs many times in each chromosome replication cycle.

Mol Plant Microbe Interact, 2001 Mar, 14(3), 422 - 5
A bacterial artificial chromosome library of Lotus japonicus constructed in an Agrobacterium tumefaciens-transformable vector; Men AE et al.; We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage . We used vector PCLD04541, which allows direct plant transformation by BACs . The average insert size is 94 kb . Clones were stable in Escherichia coli and Agrobacterium tumefaciens.

Mol Plant Microbe Interact, 2001 Mar, 14(3), 405 - 11
Biological activity of the rolB-like 5' end of the A4-orf8 gene from the Agrobacterium rhizogenes TL-DNA; Otten L et al.; The iaaM gene from different plant-associated bacteria encodes a tryptophan monooxygenase (IaaM) that catalyzes the synthesis of indole-3-acetamide (IAM), a precursor of indole-3-acetic acid (IAA) . Unlike the IaaM proteins from other bacteria, Agrobacterium spp . T-DNA-encoded IaaM proteins carry a 200 amino acid N-terminal extension with low homology to various members of the RolB protein family . This family is composed of 18 highly divergent T-DNA-encoded proteins, the basic functions of which are still largely undetermined . Deletion of the 5' rolB-like extension of the iaaM gene from Agrobacterium tumefaciens strain Ach5 did not lead to a reduction in IAM synthesis in plants . When expressed in tobacco, the rolB-like fragment did not affect growth or morphology . An iaaM homolog (A4-orf8) from the TL-DNA of Agrobacterium rhizogenes strain A4 also was investigated . Neither the full-size A4-orf8 gene nor the 5'-truncated form induced detectable IAM synthesis . Plants expressing the rolB-like part of the A4-orf8 gene, however, were dwarfed and mottled to various extents and synthesized abnormally high amounts of glucose, fructose, sucrose, and starch.

J Mol Biol, 2001 Mar 30, 307(3), 771 - 84
The complete nucleotide sequence of a plant root-inducing (Ri) plasmid indicates its chimeric structure and evolutionary relationship between tumor-inducing (Ti) and symbiotic (Sym) plasmids in Rhizobiaceae; Moriguchi K et al.; The Ri (root-inducing) plasmid in Agrobacterium rhizogenes and Ti (tumor-inducing) plasmid in Agrobacterium tumefaciens have provided the fundamental basis for the construction of plant vectors and transgenic plants . Recently, the determination of the first complete nucleotide sequence of the Ti plasmid (pTi-SAKURA) has been successful . To understand the general structure of these oncogenic T-DNA transfer plasmids, the whole nucleotide sequence of a mikimopine-type Ri plasmid, pRi1724, was analyzed . The plasmid is 217,594 bp in size, and has 173 open reading frames (ORFs) in total, which are asymmetrically distributed . Except for 27 ORFs, which are unknown, 173 ORFs were classified into 12 groups as follows: three for DNA replication, nine for plasmid modification, 22 for conjugation, 26 for virulence, 11 for T-DNA gene, 19 for mikimopine/mikimopine-lactam transport, ten for an unknown opine metabolism, seven for transcriptional regulator, five for sugar transport, five for glycerol metabolism, four for chemoreceptor and 32 for others . The elucidated chimeric structure of pRi1724 interestingly indicates that the evolution of Rhizobiaceae plasmids seems to have kept interactions among the plasmids; especially, the genes and elements for a conjugal transfer of pRi1724 had clearly closer kinship to those of a Sym (symbiotic) plasmid, pNGR234a in Rhizobium sp . than those of Ti plasmids . By using sequencing and Northern analysis, we examined the metabolic pathway and gene expression of mikimopine, which is probably an Ri-specific opine .

Indian J Exp Biol, 2000 May, 38(5), 493 - 8
Agrobacterium mediated transformation of Vigna sesquipedalis Koern (asparagus bean); Ignacimuthu S; Agrobacterium mediated transformation of Vigna sesquipedalis was achieved using cotyledonary node explants prepared from 5 days old seedlings germinated on B5 basal medium, and transformed using Agrobacterium tumefaciens strain EHA101, carrying the phosphinothricin-N-acetyltransferase gene and neomycin-3-phosphotransferase-II gene as selectable markers and GUS gene as a screenable marker . Gene transfer was achieved by inoculation of cotyledonary node explants with a bacterial suspension and a further cocultivation with Agrobacterium suspension for 3 days on B5 basal medium . Only 10% of the explants were transformed with EHA101 and exhibited transient expression of GUS genes, while 2% of shoots exhibited stable integration of genes and developed into plants . Transgenic character of tissues was confirmed by GUS assay and Southern analysis . Histological analysis of GUS gene expression directly after cocultivation revealed a high competence of subepidermal cell layers of cotyledonary node and associated cotyledons for transformation with Agrobacterium.

Genome, 2001 Feb, 44(1), 104 - 10
An efficient method for the physical mapping of transgenes in barley using in situ hybridization; Salvo-Garrido H et al.; The genetic transformation of crops by particle bombardment and Agrobacterium tumefaciens systems have the potential to complement conventional plant breeding programmes . However, before deployment, transgenic plants need to be characterized in detail, and physical mapping is an integral part of this process . Therefore, it is important to have a highly efficient method for transgene detection by fluorescence in situ hybridization (FISH) . This study describes a new approach, which provides efficient control of probe length and labelling, both of which play an important role in in situ hybridization of transgenes . The approach is based on reducing the size of the plasmid prior to labelling by nick translation, rather than using the whole or linearized plasmid, or varying the amounts of DNaseI in the nick translation mixture . This provided much more efficient labelling of the probe, which yielded optimal hybridization . minimal fluorescent background, and accurate physical location of the transgene.

Proc Natl Acad Sci U S A, 2001 Mar 27, 98(7), 4136 - 41 Epub 2001 Mar 20.
Complete genome sequence of Caulobacter crescentus; Nierman WC et al.; The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes . This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events . With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach . Two-component signal transduction proteins are known to play a significant role in cell cycle progression . Genome analysis revealed that the C . crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far . Another regulatory mechanism involved in cell cycle progression is DNA methylation . The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions . The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat . Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations . C . crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

Vaccine, 2001 Mar 21, 19(17-19), 2749 - 55
Oral immunisation of naive and primed animals with transgenic potato tubers expressing LT-B; Lauterslager TG et al.; The efficacy of edible vaccines produced in potato tubers was examined in mice . Transgenic plants were developed by Agrobacterium tumefaciens-mediated transformation . The antigen selected was the non-toxic B subunit of the Escherichia coli enterotoxin (recLT-B) . A synthetic gene coding for recLT-B was made and optimised for expression in potato tubers and accumulation in the endoplasmic reticulum . Introduction of this gene under control of the tuber-specific patatin promoter in potato plants resulted in the production of functional, i.e . Gm1-binding, recLT-B pentamers in tubers . Selected tubers containing about 13 microg of recLT-B per gram fresh weight were used for immunisation . Subcutaneous immunisation with an extract of recLT-B tubers yielded high antibody titres in serum that were similar to those obtained with bacterial recLT-B . The efficacy of oral administration of recLT-B tubers was determined by measuring mucosal and systemic immune responses in naive and primed mice . Animals were primed by subcutaneous injection of an extract of recLT-B tuber plus adjuvant . Naive and primed mice were fed 5 g of tubers ( approximately 65 microg of recLT-B) or were intubated intragastrically with 0.4 ml of tuber extract ( approximately 2 microg of recLT-B) . In naive mice, feeding recLT-B tubers or intubation of tuber extract did not induce detectable anti-LT antibody titres . In primed animals, however, oral immunisation resulted in significant anti-LT IgA antibody responses in serum and faeces . Intragastric intubation of tuber extract revealed higher responses than feeding of tubers.These results indicate clearly that functional recLT-B can be produced in potato tubers, that this recombinant protein is immunogenic and that oral administration thereof elicits both systemic and local IgA responses in parentally primed, but not naive, animals.

Vaccine, 2001 Mar 21, 19(17-19), 2735 - 41
The green revolution: plants as heterologous expression vectors; Koprowski H et al.; Agrobacterium tumefaciens mediated gene transfer into the plant genome laid the groundwork for new procedures aimed at crop improvement, including resistance to pathogens, increased product yield, modified oil content, and resistance to environmental stress conditions . New developments in molecular plant virology have led to the generation of plant-based systems for transient expression of foreign sequences using plant virus vectors . In the last decade both transgenic plants and plant virus vectors have been used increasingly to produce a wide range of biomedical reagents, including vaccine antigens, in a safe and economically feasible manner . These new plant-based technologies have enormous potential for a variety of applications, including the oral delivery of vaccine antigens.

Biotechnol Bioeng, 2001 Apr 5, 73(1), 44 - 54
Effect of mass transfer limitations on the enzymatic kinetic resolution of epoxides in a two-liquid-phase system; Baldascini H et al.; Optically active epoxides can be obtained by kinetic resolution of racemic mixtures using enantioselective epoxide hydrolases . To increase the productivity of the conversion of sparingly aqueous soluble epoxides, we investigated the use of a two-phase aqueous/organic system . A kinetic model which takes into account interphase mass transfer, enzymatic reaction, and enzyme inactivation was developed to describe epoxide conversion in the system by the epoxide hydrolase from Agrobacterium radiobacter . A Lewis cell was used to determine model parameters and results from resolutions carried out in the Lewis cell were compared to model predictions to validate the model . It was found that n-octane is a biocompatible immiscible solvent suitable for use as the organic phase . Good agreement between the model predictions and experimental data was found when the enzyme inactivation rate was fitted . Simulations showed that mass transfer limitations have to be avoided in order to maximize the yield of enantiomerically pure epoxide . Resolution of a 39 g/L solution of racemic styrene oxide in octane was successfully carried out in an emulsion batch reactor to obtain (S)-styrene oxide in high enantiomeric excess (>95% e.e.) with a yield of 30% .

Curr Biol, 2001 Feb 20, 11(4), 258 - 62
Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein; Schrammeijer B et al.; The infection of plants by Agrobacterium tumefaciens leads to the formation of crown gall tumors due to the transfer of a nucleoprotein complex into plant cells that is mediated by the virulence (vir) region-encoded transport system (reviewed in {1-5}) . In addition, A . tumefaciens secretes the Vir proteins, VirE2 and VirF, directly into plant cells via the same VirB/VirD4 transport system {6}, and both assist there in the transformation of normal cells into tumor cells . The function of the 22 kDa VirF protein is not clear . Deletion of the virF gene in A . tumefaciens leads to diminished virulence {7, 8} and can be complemented by the expression of the virF gene in the host plant . This finding indicates that VirF functions within the plant cell {8} . Here, we report that the VirF protein is the first prokaryotic protein with an F box by which it can interact with plant homologs of the yeast Skp1 protein . The presence of the F box turned out to be essential for the biological function of VirF . F box proteins and Skp1p are both subunits of a class of E3 ubiquitin ligases referred to as SCF complexes . Thus, VirF may be involved in the targeted proteolysis of specific host proteins in early stages of the transformation process.

J Microbiol Methods, 2001 Apr, 44(3), 239 - 51
Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics; Ravn L et al.; In the process of evaluating the role of acylated homoserine lactones (AHLs) in food-spoiling Gram-negative bacteria, we have combined a range of bacterial AHL monitor systems to determine the AHL-profile and the kinetics of AHL-production . AHL production from 148 strains of Enterobacteriaceae isolated from foods was tested using Escherichia coli pSB403 (LuxR), Agrobacterium tumefaciens A136 (TraR) and both induction and inhibition of Chromobacterium violaceum CV026 (CviR) . All strains except one was found to produce AHL(s) . In no case could a single monitor system identify more than 64% of the Enterobacteriaceae as AHL-producers, showing that the simultaneous use of monitor strains is required in the process of screening bacterial populations for AHL-production . AHLs from 20 selected strains were profiled by thin layer chromatography . Most strains produced more than one AHL with 3-N-oxo-hexanoyl homoserine lactone being the most prominent . It was found that the simultaneous use of monitor strains in the top-layer was necessary for the detection of (presumably) all the AHLs . An agar well-diffusion assay based on A . tumefaciens pDZLR4 was used for quantifying AHLs from bacterial supernatants and enabled an assessment of the kinetics of AHL-production of 3 strains (Serratia proteamaculans strain B5a, Erwinia carotovora ATCC 39048 and V . fischeri strain MJ-1) . As expected, the production of AHL (OHHL) and luminescence in Vibrio fischeri strain MJ-1 increased faster than growth indicating up-regulation of the AHL regulated phenotype and auto-induction of AHL production . In contrast, production kinetics of AHL (OHHL) in the two Enterobacteriaceae indicated lack of auto-induction.

J Mol Biol, 2001 Feb 16, 306(2), 251 - 61
Crystal structure and site-directed mutagenesis studies of N-carbamoyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter reveals a homotetramer and insight into a catalytic cleft; Wang WC et al.; The N-carbamoyl-D-amino-acid amidohydrolase (D-NCAase) is used on an industrial scale for the production of D-amino acids . The crystal structure of D-NCAase was solved by multiple isomorphous replacement with anomalous scattering using xenon and gold derivatives, and refined to 1.95 A resolution, to an R-factor of 18.6 % . The crystal structure shows a four-layer alpha/beta fold with two six-stranded beta sheets packed on either side by two alpha helices . One exterior layer faces the solvent, whereas the other one is buried and involved in the tight intersubunit contacts . A long C-terminal fragment extends from a monomer to a site near a dyad axis, and associates with another monomer to form a small and hydrophobic cavity, where a xenon atom can bind . Site-directed mutagenesis of His129, His144 and His215 revealed strict geometric requirements of these conserved residues to maintain a stable conformation of a putative catalytic cleft . A region located within this cleft involving Cys172, Glu47, and Lys127 is proposed for D-NCAase catalysis and is similar to the Cys-Asp-Lys site of N-carbamoylsarcosine amidohydrolase . The homologous active-site framework of these enzymes with distinct structures suggests convergent evolution of a common catalytic mechanism.

Mol Gen Mikrobiol Virusol, 2001, (1), 13 - 29
{Transfer of T-DNA from agrobacteria into plant cells through cell walls and membranes}; Chumakov MI; Discusses probable routes of agrobacterial penetration through the plant integumental tissues, cell wall, and plant cell plasmodesma . Analyzes the contribution of extracellular structures of agrobacteria in penetration through barriers of a plant cell, primary contact (adhesion), and during DNA transfer from bacterial (E . coli, A . tumefaciens) to recipient (bacterial or plant) cells . Discusses the relationship between donor cell adhesion to recipient cell surface and the infectious and conjugation processes . Considers the probable role of piles in conjugative transfer of agrobacterial DNA through membranes of donor and recipient (bacterial and plant) cells . Analyzes the contribution of the plant cell cytoskeleton to T-DNA transfer . Suggests a model of transport of T-DNA-VirD2 complex and VirE2 proteins through independent channels consisting of vir-coded proteins.

Indian J Exp Biol, 2000 Jan, 38(1), 6 - 17
Transgenic strategies for genetic improvement of Basmati rice; Jain RK et al.; Transgenic approach offers an attractive alternative to conventional techniques for the genetic improvement of Basmati rice because they enable the introduction of one or more genes into a leading cultivar without affecting its genetic background . During the last ten years, a rapid progress has been made towards the development of transformation methods in rice . Several transformation methods including Agrobacterium, biolistic, and DNA uptake by protoplasts, have been employed to produce transgenic rice . An array of useful genes is now available and many of these have already been transferred in rice to improve the resistance against biotic and abiotic stresses . In Basmati rice, a beginning has already been made regarding the development of tissue culture protocols, transformation methods and production of useful transgenic plants . The application and future prospects of transformation technology to engineer the resistance against insect pests (stem borer, leaf folder, brown plant hopper, gall midge), fungal diseases (blast, bakanae/foot, rot), bacterial diseases (bacterial leaf blight, sheath blight), abiotic stresses (salinity and drought) and improved nutritional quality (accumulation of provitamin A and essential amino acids in endosperm) in Basmati rice, have been addressed.

Appl Environ Microbiol, 2001 Mar, 67(3), 1070 - 5
The effect of the Agrobacterium tumefaciens attR mutation on attachment and root colonization differs between legumes and other dicots; Matthysse AG et al.; Infections of wound sites on dicot plants by Agrobacterium tumefaciens result in the formation of crown gall tumors . An early step in tumor formation is bacterial attachment to the plant cells . AttR mutants failed to attach to wound sites of both legumes and nonlegumes and were avirulent on both groups of plants . AttR mutants also failed to attach to the root epidermis and root hairs of nonlegumes and had a markedly reduced ability to colonize the roots of these plants . However, AttR mutants were able to attach to the root epidermis and root hairs of alfalfa, garden bean, and pea . The mutant showed little reduction in its ability to colonize these roots . Thus, A . tumefaciens appears to possess two systems for binding to plant cells . One system is AttR dependent and is required for virulence on all of the plants tested and for colonization of the roots of all of the plants tested except legumes . Attachment to root hairs through this system can be blocked by the acetylated capsular polysaccharide . The second system is AttR independent, is not inhibited by the acetylated capsular polysaccharide, and allows the bacteria to bind to the roots of legumes.

Curr Opin Plant Biol, 2001 Apr, 4(2), 111 - 7
Arabidopsis gene knockout: phenotypes wanted; Bouche N et al.; Gene knockout is considered to be a major component of the functional genomics toolbox, and is aimed at revealing the function of genes discovered through large-scale sequencing programs . In the past few years, several Arabidopsis populations mutagenized with insertion elements, such as the T-DNA of Agrobacterium or transposons, have been produced . These large populations are routinely screened for insertions into specific genes, allowing mass-isolation of knockout lines . Although many Arabidopsis knockouts have already been obtained, few of them have been reported to present informative phenotypes that provide a direct clue to gene function . Although functional redundancy explains the lack of phenotypical alterations in some cases, it also appears that many mutations are conditional and/or do not alter plant morphology even in the presence of severe physiological defects . Consequently, gene knockout per se is not sufficient to assess gene function and must be integrated into a more global approach for determining biological functions.

Plant Cell, 2001 Feb, 13(2), 369 - 83
Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins; Ziemienowicz A et al.; To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro . Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA . For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2 . A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei . Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function . RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells . We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore . Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.

Gene, 2001 Jan 24, 263(1-2), 49 - 58
Mikimopine synthase (mis) gene on pRi1724; Suzuki K et al.; By determination of the nucleotide sequence adjacent to the right border of T-DNA of the mikimopine-type Ri plasmid (pRi1724) in Agrobacterium rhizogenes, a new open reading frame (ORF) encoding 318 amino acids was found . A transcript of 1.35 kb derived from this ORF was observed in hairy roots of Ajuga reptans by northern blotting analysis . Including its own promoter and terminator, this ORF was isolated from the pRi1724 T-DNA and introduced into tobacco plants by the Agrobacterium-binary vector system . Since mikimopine, an opine and a stereoisomer of cucumopine, was accumulated in all organs of the transgenic tobacco plants, the new ORF was deduced to be the mikimopine synthase gene . For comparison, the nucleotide sequence of cucumopine synthase encoded on pRi2659 was also determined . No homology was found between mikimopine synthase and cucumopine synthase at the nucleotide, but partial homology was found at the amino acid level . Mikimopine synthase and cucumopine synthase produced by a protein expression system using E . coli catalyzed the synthesis of mikimopine and cucumopine from L-histidine and alpha-ketoglutaric acid, requiring NADH as a cofactor . These synthesized opines were identified by paper electrophoresis, TLC and HPLC analyses . The synthesized mikimopine or cucumopine could be degraded by A . rhizogenes strains harboring Ri plasmids encoding the respective catabolic enzyme.

Planta, 2001 Jan, 212(2), 215 - 21
The production of an inducible antisense alternative oxidase (Aox1a) plant; Potter FJ et al.; Plant mitochondria contain an alternative oxidase (AOX) acting as a terminal electron acceptor of the alternative pathway in the electron transport chain . Here we describe the production of inducible antisense Aox1a plants of Arabidopsis thaliana (L.) Heynh . and the procedures used to determine the resulting alternative pathway activity . The Arabidopsis Aox1a cDNA sequence was cloned behind a copper-inducible promoter system in the antisense orientation . Arabidopsis thaliana (Columbia) plants were transformed by in-planta vacuum infiltration with Agrobacterium containing the antisense construct . Whole-leaf ethanol production was used as a measure to investigate alternative pathway activity in the presence of antimycin A . After 24 h, leaves from the copper-induced, antisense line F1.1 produced up to 8.8 times more ethanol (via aerobic fermentation) than the non-induced and wild-type leaves, indicating effective cytochrome pathway inhibition by antimycin A and a decreased alternative pathway activity in induced F1.1 leaves . Transgene expression studies also revealed no expression in non-induced leaves and up until 24 h post-induction . Copper-induced transgenic leaves were less susceptible to alternative pathway inhibition than non-induced transgenic leaves, as seen via tissue-slice respiratory studies, and mitochondrial respiration, using F1.1 cell cultures, also supported this . These results demonstrate the successful production of a transgenic line of Arabidopsis in which the alternative pathway activity can be genetically manipulated with an inducible antisense system.

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 89 - 103
A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al . 1998 as new combinations: Rhizobium radiobacter, R . rhizogenes, R . rubi, R . undicola and R . vitis; Young JM et al.; Rhizobium, Agrobacterium and Allorhizobium are genera within the bacterial family Rhizobiaceae, together with Sinorhizobium . The species of Agrobacterium, Agrobacterium tumefaciens (syn . Agrobacterium radiobacter), Agrobacterium rhizogenes, Agrobacterium rubi and Agrobacterium vitis, together with Allorhizobium undicola, form a monophyletic group with all Rhizobium species, based on comparative 16S rDNA analyses . Agrobacterium is an artificial genus comprising plant-pathogenic species . The monophyletic nature of Agrobacterium, Allorhizobium and Rhizobium and their common phenotypic generic circumscription support their amalgamation into a single genus, Rhizobium . Agrobacterium tumefaciens was conserved as the type species of Agrobacterium, but the epithet radiobacter would take precedence as Rhizobium radiobacter in the revised genus . The proposed new combinations are Rhizobium radiobacter, Rhizobium rhizogenes, Rhizobium rubi, Rhizobium undicola and Rhizobium vitis.

Virus Genes, 2001 Jan, 22(1), 73 - 83
Expression of immunogenic Puumala virus nucleocapsid protein in transgenic tobacco and potato plants; Kehm R et al.; Transgenic plants, expressing recombinant proteins, are suitable alternatives for the production of relevant immunogens . In the present study, the expression of Puumala virus nucleocapsid protein in tobacco and potato plants (Nicotiana tabacum and Solanum tuberosum) and its immunogenicity was investigated . After infection of leaf discs of SR1 tobacco and tuber discs of potato cv . "Desiree" with the Agrobacterium strain LBA4404 (pAL4404, pBinAR-PUU-S) containing the 1302 bp cDNA sequence of S-RNA segment of a Puumala virus, transgenic tobacco and potato plants expressed the Puumala virus nucleocapsid protein under control of the cauliflower 35S promoter . The recombinant proteins were found to be identical to the authentic Puumala virus nucleocapsid protein as analyzed by immunoblotting . Expression of the nucleocapsid protein was investigated over four plant generations (P to F4) and found to be stable (1 ng/3 microg dried leaf tissue) . Transgenic tobacco plants were smaller compared to controls . The transformed potato plants were morphologically similar to control plants and produced tubers as the control potatoes . The S-antigen was expressed at a level of 1 ng protein/5 microg and 1 ng protein/4 microg dried leaf and root tissues, respectively, and remained stable in the first generation of vegetatively propagated potato plants . The immunogenicity of the Puumala virus nucleocapsid protein expressed in Nicotiana tabacum and Solanum tuberosum was investigated in New Zealand white rabbits . They were immunized with leaf extracts from transgenic tobacco and potato plants, and the serum recognized Puumala virus nucleocapsid protein . Transgenic plants expressing hantaviral proteins can thus be used for the development of cost-effective diagnostic systems and for alternative vaccination strategies.

Yi Chuan Xue Bao, 2000, 27(11), 992 - 8
{Production of herbicide-resistant rice with transforming heterogene}; Wu AZ et al.; Using pAHC20 (containing Bar gene), pWRG1515 (containing GUS gene and hygromycin phosphotransferase gene), and pCAMBIA3300 RG with Bar gene and snowdrop lectin (GNA) gene as donor DNA, the micro-adventitious shoots and the calli induced from mature embryos of Oryza sativa 87203, Eyi105, Shangnong aromatic glutinous rice as recipients were transformed with particle bombardment and Agrobacterium tumefaciens strain LBA4404 containing pAL4404, respectively . After chosen with phosphinothricin and antibiotic, GUS detection and PCR analysis, The results showed that the foreign genes had been transformed microprojectile-mediated to Oryza sativa Eyi105, the regeneration plants were obtained, and, 5 transgenic calli of Oryza sativa Eyi105 were obtained with Agrobacterium-mediated transformation.

Yi Chuan Xue Bao, 2000, 27(11), 982 - 91
{Establishment of a method for GUS gene transferring into wheat (Triticum astivum L.) embryos by low energy ion beam implantation}; Wu LF et al.; Physical parameters influencing transformation of wheat mediated by low energy ion beam, including type of ion, parameters of ion energy, dose and dose rate, were studied . Ar+ was regarded as suitable ions implanted in transformation . 20-25 keV of energy, 4.68 x 10(16) ions/cm2 of dose, 2.6 x 10(15) ions/cm2 of dose rate were chosen as appropriate implantation parameters . The suitable culture conditions for induction and growth of callus and the optimal selection scheme were established, After implantation and selection, resistant calli and hygromycin-resistant plantlets were obtained from three varieties . Molecular analysis data proved that GUS gene had integrated into the wheat genome . The mature embryo transformation efficiency of wherat variety Yangmai 5, Yangmai 158, Wanmai 32 reached 9.5%, 10.8%, 11.2% measured in produced hygromycin-resistant callus and 1.4%, 3.4%, 1.7% measured in regenerated plants, respectively, This experiment provides a basis for further investigation of wheat transformation system . Low energy ion beam mediated transformation can be extended to other plant recalcitrant to Agrobacterium tumefaciens as soon as methodological parameters are optimized.

Yi Chuan Xue Bao, 2000, 27(11), 953 - 8
{The research on the expression of rabbit defensin (NP-1) gene in transgenic tomato}; Zhang XH et al.; Rabbit defensin NP-1 is one kind of alpha-defensins . It is composed of 33 amino acids . It was firstly extracted from polymorphonnuclear neutrophile of rabbits . It displayed resistance to bacteria, fungi and virus, especially high resistance to bacteria . In our experiments NP-1 gene was constructed into a plant expression vector . Eight transgenic plants containing the rabbit defensin gene (NP-1) were obtained through agrobacterium-mediated transformation . The transgenic plants were analysized by PCR, Southern hybridization, Northern dot blot hybridization and in vitro microbicidical activity against E . coli and Fusarium oxysporum . The results showed that NP-1 gene was transformed into the tomato, and the transgene displayed physiological-level expression . The transgenic tomato also showed resistance to pathogen Fusarium oxysporum in vivo . Our experiments paved a way for pathogen resistance breeding of tomato.

Transgenic Res, 2000 Dec, 9(6), 477 - 86
Active expression of the ubiA gene from E . coli in tobacco: influence of plant ER-specific signal peptides on the expression of a membrane-bound prenyltransferase in plant cells; Boehm R et al.; The ubiA gene from E . coli codes for 4-hydroxybenzoate: polyprenyldiphosphate 3-polyprenyltransferase, an integral membrane protein involved in ubiquinone biosynthesis . This prokaryotic membrane protein was stably expressed in tobacco using Agrobacterium tumefaciens-mediated transformation . Transgenic lines containing a direct fusion of the ubiA structural gene to a 35S-derived promoter gave very low enzyme activity levels (average 0.16 pkat/mg) . Inclusion of an N-terminal ER-specific signal peptide from a lectin gene from Phaseolus vulgaris resulted in an average activity of 1.08 pkat/mg in the transgenic tobacco lines . The additional inclusion of a C-terminal HDEL tetrapeptide, responsible for the retention of proteins in the endoplasmic reticulum of eukaryotic cells, increased the activity to 18.6 pkat/mg . When the promotor of this construct was changed from the 35S derivative to the recently described very strong plant promoter (ocs)3mas, the activity increased further to 128.6 pkat/mg . The most active tobacco line showed activities of the introduced enzyme which exceeded those of wild-type E . coli (the source of ubiA) by a factor of 1100 . These results demonstrate the efficacy of plant ER-specific signal peptides for the active expression of a prokaryotic membrane protein in plants.

Transgenic Res, 2000 Dec, 9(6), 471 - 6
Floral spray transformation can efficiently generate Arabidopsis transgenic plants; Chung MH et al.; In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis . The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis . The transgene construct was developed in binary vectors and mobilized into Agrobacterium . When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip . More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation . The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods . The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.

Transgenic Res, 2000 Dec, 9(6), 405 - 15
Efficient production of transgenic cassava using negative and positive selection; Zhang P et al.; In order to improve the efficiency of cassava (Manihot esculenta Crantz) transformation, two different selection systems were assessed, a positive one based on the use of mannose as the selective agent, and a negative one based on hygromycin resistance encoded by an intron-containing hph gene . Transgenic plants selected on mannose or hygromycin were regenerated for the first time from embryogenic suspensions cocultivated with Agrobacterium . After the initial selection using mannose and hygromycin, 82.6% and 100% of the respective developing embryogenic callus lines were transgenic . A system allowing plant regeneration from only transgenic lines was designed by combining chemical selection with histochemical GUS assays . In total, 12 morphologically normal transgenic plant lines were produced, five using mannose and seven using hygromycin . The stable integration of the transgenes into the nuclear genome was verified using PCR and Southern analysis . RT-PCR and northern analyses confirmed the transgene expression in the regenerated plants . A rooting test on mannose containing medium was developed as an alternative to GUS assays in order to eliminate escapes from the positive selection system . Our results show that transgenic cassava plants can be obtained by using either antibiotic resistance genes that are not expressed in the micro-organisms or an antibiotic-free positive selection system.

Farmaco, 2000 Jun-Jul, 55(6-7), 492 - 4
Isolation and bioactivities of epidioxysterol from the tunicate Cynthia savignyi; Abourriche A et al.; From a hexane extract of the tunicate Cynthia savignyi, collected in Morocco, epidioxysterol or 5,8-alpha-epidioxy-5alpha-cholest-6-en-3beta-ol has been isolated . This is the first example of epidioxysterol found in the tunicate C . savignyi . The structure of epidioxysterol has been characterised by NMR data (1H, 13C and 2D) . Epidioxysterol possesses antifungal activity against three tomato pathogenic fungi: Botrytis cinerea, Fusarium oxysporum and Verticillium albo atrum and antibacterial activity against Agrobacterium tumefaciens, Escherichia coli, Staphylococcusfaecalis, Staphylococcus aureus and Pseudomonas aeruginosa and cytotoxicity against Artemia salina larvae.

Plant Mol Biol, 2000 Dec, 44(6), 789 - 98
Agrobacterium-mediated sorghum transformation; Zhao ZY et al.; Agrobacterium tumefaciens was used to genetically transform sorghum . Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants . The Agrobacterium strain used was LBA4404 carrying a 'Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells . A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments . A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines . Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos . Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome . Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening . This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants . The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.

Plant Mol Biol, 2000 Nov, 44(4), 543 - 57
Binding of cell type-specific nuclear proteins to the 5'-flanking region of maize C4 phosphoenolpyruvate carboxylase gene confers its differential transcription in mesophyll cells; Taniguchi M et al.; C4-type phosphenolpyruvate carboxylase (C4PEPC) acts as a primary carbon assimilatory enzyme in the C4 photosynthetic pathway . The maize C4PEPC gene (C4Ppc1) is specifically expressed in mesophyll cells (MC) of light-grown leaves, but the molecular mechanism responsible for its cell type-specific expression has not been characterized . In this study, we introduced a chimeric maize C4Ppc1 5'-flanking region/beta-glucuronidase (GUS) gene into maize plants by Agrobacterium-mediated transformation . Activity assay and histochemical staining showed that GUS is almost exclusively localized in leaf MC of transgenic maize plants . This observation suggests that the introduced 5' region of maize C4Ppc1 contains the necessary cis element(s) for its specific expression in MC . Next, we investigated whether the 5' region of the maize gene interacts with nuclear proteins in a cell type-specific manner . By gel shift assays with nuclear extracts prepared from MC or bundle sheath cells (BSC), cell type-specific DNA-protein interactions were detected: nuclear factors PEP(Ib) and PEP(Ic) are specific to MC whereas PEP(Ia) and PEP(IIa) are specific to BSC . Light alters the binding activity of these factors . These interactions were not detected in the assay with nuclear extract prepared from root, or competed out by oligonucleotides corresponding to the binding sites for the maize nuclear protein, PEP-I, which is known to bind specifically to the promoter region of C4Ppc1 . The results suggest that novel cell type-specific positive and negative nuclear factors bind to the maize C4Ppc1 5'-flanking region and regulate its differential transcription in MC in a light-dependent manner.

Mol Plant Microbe Interact, 2001 Jan, 14(1), 98 - 103
Construction of a derivative of Agrobacterium tumefaciens C58 that does not mutate to tetracycline resistance; Luo ZQ et al.; Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker . Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain . By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance . The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

Genetika, 2000 Dec, 36(12), 1725 - 8
{Effect of azacytidine on expression of traits concomitant with tumor formation in the radish (Raphanus sativus) in vitro}; Matveeva TV et al.; The effect of azacytidine, a demethylating agent, on the expression of traits concomitant with tumor formation was studied in inbred radish (Rhaphanus sativus) lines carrying genomic sequences homologous to the tmr/tml genes of Agrobacteriium tumefaciens . AzaC was found to have no effect on the traits studied, which provided evidence that the capacity for tumor formation in radish lines does not depend on the level of methylation of these sequences.

Wei Sheng Wu Xue Bao, 1997 Oct, 37(5), 335 - 43
{A study on taxonomy of Rhizobia isolated from Astragalus sp}; Wang S et al.; Thirty-six strains isolated from root nodule of Astragalus spp., in comparison with 31 reference strains of Rhizobium, Bradyrhizobium and Sinorhizobium species, and some other strains isolated from legumes in Xinjiang and Hainian Province, were classified by performing numberical taxonomy, DNA-DNA hybrization and partial 16S rRNA gene sequencing . Results of herichical analysis showed most of trains isolated from Astragalus spp . fell into subgroups 8 and 9, Also the DNA homolgy among strains of subgroups 8 and 9 are more than 70%, with the exception of strains CA8593 and SX044, and the DNA homology between strain CA8561, JL84 and type strains of all described rhizobial species are less than 56% . These results indicated that these two subgroups 8 and 9 were unique DNA homologous groups, distinguishing from all described rhizobial species . Sequencing of partial 16S rRNA gene showed that cetrostrain CA8561 of subgroup 8 is phylogenetically far from all know species of Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium and Agrobacterium, and it is a unique geneline . The cetrostrain JL84 of subgroup 9 has a unique position in the phylogenetic branch consisted of species of Rhizobium and Agrobacterium.

Genome Biol . 2000;1(4):REVIEWS3001 . Epub 2000 Oct 13.
Higher plant cellulose synthases; Richmond T; SUMMARY: Cellulose, an aggregate of unbranched polymers of beta-1,4-linked glucose residues, is the major component of wood and thus paper, and is synthesized by plants, most algae, some bacteria and fungi, and even some animals . The genes that synthesize cellulose in higher plants differ greatly from the well-characterized genes found in Acetobacter and Agrobacterium sp . More correctly designated as 'cellulose synthase catalytic subunits', plant cellulose synthase (CesA) proteins are integral membrane proteins, approximately 1,000 amino acids in length . The sequences for more than 20 full-length CesA genes are available, and they show high similarity to one another across the entire length of the encoded protein, except for two small regions of variability . There are a number of highly conserved residues, including several motifs shown to be necessary for processive glycosyltransferase activity . No crystal structure is known for cellulose synthase proteins, and the exact enzymatic mechanism is unknown . There are a number of mutations in cellulose synthase genes in the model organism Arabidopsis thaliana . Some of these mutants show altered morphology due to the lack of a properly developed primary or secondary cell wall . Others show resistance to well-characterized cellulose biosynthesis inhibitors.

Proc Natl Acad Sci U S A, 2001 Feb 13, 98(4), 1871 - 6 Epub 2001 Jan 30.
Genetic transformation of HeLa cells by Agrobacterium; Kunik T et al.; Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species . In nature, however, Agrobacterium also may encounter organisms belonging to other kingdoms such as insects and animals that feed on the infected plants . Can Agrobacterium, then, also infect animal cells? Here, we report that Agrobacterium attaches to and genetically transforms several types of human cells . In stably transformed HeLa cells, the integration event occurred at the right border of the tumor-inducing plasmid's transferred-DNA (T-DNA), suggesting bona fide T-DNA transfer and lending support to the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells . Collectively, our results suggest that Agrobacterium can transport its T-DNA to human cells and integrate it into their genome.

Biochem Soc Trans, 2000 Dec, 28(6), 969 - 72
Transgenic oil palm: production and projection; Parveez GK et al.; Oil palm is an important economic crop for Malaysia . Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids and novel products to ensure the sustainability of the palm oil industry . Establishment of a reliable transformation and regeneration system is essential for genetic engineering . Biolistic was initially chosen as the method for oil palm transformation as it has been the most successful method for monocotyledons to date . Optimization of physical and biological parameters, including testing of promoters and selective agents, was carried out as a prerequisite for stable transformation . This has resulted in the successful transfer of reporter genes into oil palm and the regeneration of transgenic oil palm, thus making it possible to improve the oil palm through genetic engineering . Besides application of the Biolistics method, studies on transformation mediated by Agrobacterium and utilization of the green fluorescent protein gene as a selectable marker gene have been initiated . Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement . Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics . The efforts in oil palm genetic engineering are thus not targeted as commodity palm oil . Due to the long life cycle of the palm and the time taken to regenerate plants in tissue culture, it is envisaged that commercial planting of transgenic palms will not occur any earlier than the year 2020.

Biochem Soc Trans, 2000 Dec, 28(6), 938 - 40
High-oleic acid Australian Brassica napus and B . juncea varieties produced by co-suppression of endogenous Delta12-desaturases; Stoutjesdijk PA et al.; Genetic engineering methods have been used successfully to modify the fatty acid profile of elite Australian germplasm of Brassica napus and B . juncea . Co-suppression plasmids carrying oleate desaturase genes from each species have been constructed and transferred into Australian elite breeding lines of B . napus and B . juncea using Agrobacterium tumifaciens plant-transformation techniques . Modifications to existing Brassica transformation protocols and the use of an intron-interrupted hygromycin-resistance gene as the selectable marker have resulted in improved transformation efficiencies . Silencing of the endogenous oleate desaturase genes has resulted in substantial increases in oleic acid levels, up to 89% in B . napus and 73% in B . juncea.

Biochem Soc Trans, 2000 Dec, 28(6), 790 - 1
Polyprenols in hairy roots of Coluria geoides; Skorupinska-Tudek K et al.; Long-chain polyisoprenoid alcohols built from several up to more than 100 isoprenoid units are common constituents of all living organisms . They were found mostly in plants, bacteria, yeasts and mammalian cells . In vitro hairy root culture of Coluria geoides was obtained from plants transformed with Agrobacterium rhizogenes . Growth was optimal at 0.75% (w/v) glucose and at 22 degrees C . Dry samples of roots were extracted and lipid content was analysed by HPLC . According to our estimation, polyprenols are accumulated in roots of C . geoides cultivated in vitro as a mixture of several prenologues with the dominating prenol composed of 16 isoprenoid units . The content of polyprenols in tissue was approx . 300 microg/g of dry weight.

Plant J, 2001 Jan, 25(2), 159 - 67
Nicotianamine synthase gene expression differs in barley and rice under Fe-deficient conditions; Higuchi K et al.; Nicotianamine (NA) is an intermediate in the biosynthetic pathway of the mugineic acid family phytosiderophores (MAs), which are crucial components of the iron acquisition apparatus of graminaceous plants . In non-graminaceous plants, NA is thought to be an essential chelator for metal cation homeostasis . Thus NA plays a key role in Fe metabolism and homeostasis in all higher plants . Nicotianamine synthase (NAS, EC 2.5.1.43) catalyzes the trimerization of S-adenosylmethionine to form one molecule of NA . Barley, a plant that is resistant to Fe deficiency, secretes large amounts of MAs, whereas rice, a plant that is susceptible to Fe deficiency, secretes only small amounts . In this study we isolated a genomic fragment containing HvNAS1 from barley and three rice cDNA clones, osnas1, osnas2 and osnas3, from Fe-deficient rice roots . We also isolated a genomic fragment containing both OsNAS1 and OsNAS2 . In contrast to barley, in which Fe deficiency induces the expression of NAS genes only in roots, Fe deficiency in rice induced NAS gene expression in both roots and chlorotic leaves . The amounts of endogenous NA in both the roots and leaves were higher than in barley . We introduced barley genomic DNA fragments containing HvNAS1 with either 9 or 2 kb of the 5'-flanking region into rice, using Agrobacterium-mediated transformation . Fe deficiency induced HvNAS1 expression in both roots and leaves of the transgenic rice, as occurs with rice NAS genes . Barley and rice NAS genes are compared in a discussion of alteration of the NAS genes during adaptation to Fe deficiency.

Lett Appl Microbiol, 2001 Jan, 32(1), 57 - 61
Detection of Vibrionaceae in mussels and in their seawater growing area; Croci L et al.; AIMS: The seasonal trend and frequency of detection of Vibrionaceae in seawater samples and in molluscs collected in the Adriatic Sea was measured . METHODS AND RESULTS: Over a 2-year period, 726 bacterial strains were isolated, of which 46.9% belonged to the Vibrio genus, 29.8% to the Aeromonas genus and the remaining 23.3% was made up of the Pseudomonas, Flavobacterium, Pasteurella, Agrobacterium and Ochrobacterium genera . Many of the isolated strains were shown to produce toxins . CONCLUSION: The Vibrio genus, which was isolated more often than the other genera, was particularly prevalent in summer (54.4% of the total number of bacteria isolated during this season), while it was scarce in the winter months.

Plant Sci, 2001 Feb 5, 160(3), 433 - 439
Transformation of the apple rootstock M.9/29 with the rolB gene and its influence on rooting and growth; Zhu L et al.; To improve the rooting ability, the dwarfing apple rootstock M.9/29 was transformed with the rolB gene by Agrobacterium-mediated gene transfer . The use of sorbitol in the induction medium resulted in a successful transformation, while the use of sucrose failed to give any transformants . Totally 14 putative clones, named ARB1-14, were obtained from ten different leaves . Polymerase chain reaction (PCR) and Southern analyses confirmed that all the clones contained the nptII and rolB genes, while only four of them contained the intact gus gene . The in vitro rooting test showed that all the tested clones rooted to 83-100% on the hormone free rooting medium, while only 1% for the control plants . The root number of the transgenic clones ranged from 3.5 to 9, while the control plants produced only one root . Growth analysis showed that the clone ARB9 and ARB10 had a significant reduced node number and stem length compared with the control plants . However, the relative growth rate (RGR) of the tested clones was similar to that of the control plants, indicating that RGR is not directly related to dwarfism of a plant . The clone ARB10 also showed a significant reduced internode length compared with the control plants . The root length and root morphology did not differ between the transgenic clones and the untransformed control plants.

J Biotechnol, 2001 Jan 23, 85(1), 35 - 40
The effect of nitrate and ammonium concentrations on growth and alkaloid accumulation of Atropa belladonna hairy roots; Bensaddek L et al.; The growth, the alkaloid production, as well as the scopolamine/hyoscyamine ratio of two clones of belladonna hairy roots were studied . The effects of nitrate and ammonium concentrations on these cultures were investigated . A rise in ammonium concentration caused the decline of the hairy roots, while nitrate had a marked effect on the alkaloid content . The alkaloid production obtained with 15.8 mM of NO3- and 20.5 mM of NH4+ was 1.2-1.4 times higher than that obtained when the roots were grown in the standard Murashige and Skoog medium (MS medium, 39.5 mM of NO3- and 20.5 mM of NH4+) . The nitrate and ammonium concentrations in the culture medium also had a strong influence on the scopolamine/hyoscyamine ratio . When nitrate or ammonium concentrations were raised, that ratio also was increased 2-3-fold . The hairy root clones originating from transformations with two distinct strains of Agrobacterium had similar responses.

Plant Sci, 2001 Jan 5, 160(2), 341 - 353
Transgenic crop plants expressing synthetic cry9Aa gene are protected against insect damage; Kuvshinov V V et al.; A synthetic gene sequence of cry9Aa was made to achieve high expression levels in a plant cell . Tobacco, potato, cauliflower and turnip rape plants were transformed with this synthetic gene driven by the double 35S promoter using Agrobacterium tumefaciens LBA4404 . The presence and expression of the synthetic cry9Aa gene was evaluated in Southern, Northern and Western analysis and with insect bioassays . The expression of the gene in tobacco plants reached a level of 5 pg of mRNA per 1 microg of total RNA and 0.3% of soluble protein or 1.4 microg of Cry9Aa protein per 1 g of leaf material . The expression level in the other species was three to ten times lower . Tobacco plants were also transformed with a truncated native cry9Aa gene construct and with a translational fusion construct of the truncated native cry9Aa and the uidA (GUS) gene sequence . The constructs were transformed in tobacco plants under the control of the same promoter as the synthetic cry9Aa . The expression level of the native cry9Aa gene constructs ranged from 0.03 to 1 pg of cry9Aa mRNA per 1 microg of total RNA . The protein was undetectable in Western analysis . In comparison to the native constructs the expression level of the synthetic cry9Aa gene was five to ten times higher at the mRNA level and at least 50 times higher at the translational level . Bioassays against Plutella xylostella performed with transgenic cauliflower showed high insecticidal activity of the plants expressing the synthetic cry9Aa gene.

Plant Sci, 2001 Jan 5, 160(2), 259 - 264
Establishment of hairy root cultures of Ammi majus; Krolicka A et al.; Axenically grown Ammi majus plantlets were inoculated with seven different Agrobacterium rhizogenes strains . Hairy root lines were established only after inoculation with the two agropine strains: A4 and LBA9402 . The growth rate of hairy root cultures was about thirty times faster than that of callus and cell suspension cultures . Polymerase chain reaction with primers for the genes rolB and rolC confirmed the integration of the T-DNA fragment of Ri plasmid of A . rhizogenes to the genome of hairy roots obtained after transformation by both Agrobacterium strains . The furanocoumarins (psoralen, xanthotoxine, bergapten and imperatorin) usually found in seeds of A . majus were not detected in callus, cell suspension and hairy root cultures using Gas chromatography-mass spectrometry (GC-MS) . However, umbelliferone, a precursor of furanocoumarins, was detected in callus, cell suspension and hairy root cultures . The umbelliferone content in extracts of hairy root cultures, obtained after transformation by A4, was similar to that determined in A . majus seeds (19 microg/g DW) and higher than those obtained for cell suspension and callus cultures (2 and 9 microg/g DW, respectively).

Protein Expr Purif, 2001 Feb, 21(1), 170 - 5
Two-step purification of d(-)-specific carbamoylase from Agrobacterium tumefaciens AM 10; Sareen D et al.; A simple, economical and rapid affinity chromatography procedure with red dye as a ligand has been described for the two-step purification of a relatively thermostable d(-)-carbamoylase from the cell-free extract of Agrobacterium tumefaciens AM 10 . The enzyme was purified 232-fold to homogeneity with a recovery of 30% in the presence of 2 mM dithiothreitol . The specific activity of the enzyme was 7.88 U/mg protein . The enzyme is a dimer with a native molecular mass of 67 kDa and a subunit relative molecular mass of 38 kDa . The isoelectric point of the enzyme was found to be 5.83 . The K(m) values for N-carbamoyl-dl-methionine and N-carbamoyl-d-phenylglycine were 3.84 and 5.0 mM, respectively .

Infect Immun, 2001 Feb, 69(2), 865 - 8
Gene discovery through genomic sequencing of Brucella abortus; Sanchez DO et al.; Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human . We generated DNA random sequences from the genome of B . abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen . The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10(-5)) to sequences deposited in the GenBank databases . Among them, 925 represent putative novel genes for the Brucella genus . Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function . Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function . A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship . Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation . We have also identified several B . abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli . Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery.

Appl Environ Microbiol, 2001 Feb, 67(2), 654 - 64
Iron-binding compounds from Agrobacterium spp.: biological control strain Agrobacterium rhizogenes K84 produces a hydroxamate siderophore; Penyalver R et al.; Iron-binding compounds were produced in various amounts in response to iron starvation by a collection of Agrobacterium strains belonging to the species A . tumefaciens, A . rhizogenes, and A . vitis . The crown gall biocontrol agent A . rhizogenes strain K84 produced a hydroxamate iron chelator in large amounts . Production of this compound, and also of a previously described antibiotic-like substance called ALS84, occurred only in cultures of strain K84 grown in iron-deficient medium . Similarly, sensitivity to ALS84 was expressed only when susceptible cells were tested in low-iron media . Five independent Tn5-induced mutants of strain K84 affected in the production of the hydroxamate iron chelator showed a similar reduction in the production of ALS84 . One of these mutants, M8-10, was completely deficient in the production of both agents and grew poorly compared to the wild type under iron-limiting conditions . Thus, the hydroxamate compound has siderophore activity . A 9.1-kb fragment of chromosomal DNA containing the Tn5 insertion from this mutant was cloned and marker exchanged into wild-type strain K84 . The homogenote lost the ability to produce the hydroxamate siderophore and also ALS84 . A cosmid clone was isolated from a genomic library of strain K84 that restored to strain M8-10 the ability to produce of the siderophore and ALS84, as well as growth in iron-deficient medium . This cosmid clone contained the region in which Tn5 was located in the mutant . Sequence analysis showed that the Tn5 insert in this mutant was located in an open reading frame coding for a protein that has similarity to those of the gramicidin S synthetase repeat superfamily . Some such proteins are required for synthesis of hydroxamate siderophores by other bacteria . Southern analysis revealed that the biosynthetic gene from strain K84 is present only in isolates of A . rhizogenes that produce hydroxamate-type compounds under low-iron conditions . Based on physiological and genetic analyses showing a correlation between production of a hydroxamate siderophore and ALS84 by strain K84, we conclude that the two activities share a biosynthetic route and may be the same compound.

Plant Cell, 2000 Dec, 12(12), 2383 - 2394
Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis; Borevitz JO et al.; Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures . Unfortunately, these complex natural products usually occur at very low abundance and with restricted tissue distribution, thereby hindering their evaluation . Here, we report a novel approach for enhancing the accumulation of natural products based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mosaic virus 35S enhancer sequences at its right border . Among approximately 5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development . This upregulation of pigmentation reflected a dominant mutation that resulted in massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocyanins that were responsible for the purple color . These phenotypes, caused by insertion of the viral enhancer sequences adjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic controls regulating the accumulation of specific natural products during plant development . Our findings suggest a functional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation of massively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes.

Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 485 - 90 Epub 2001 Jan 09.
An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells; Dumas F et al.; Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer . T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2 . The VirE2 protein is highly induced on contact of A . tumefaciens with a plant host and has been reported to act in late steps of transfer . One of its previously demonstrated functions is binding to the single-stranded (ss) T-DNA and protecting it from degradation . Recent experiments suggest other functions of the protein . A combination of planar lipid bilayer experiments, vesicle swelling assays, and DNA transport experiments demonstrated that VirE2 can insert itself into artificial membranes and form channels . These channels are voltage gated, anion selective, and single-stranded DNA-specific and can facilitate the efficient transport of single-stranded DNA through membranes . These experiments demonstrate a VirE2 function as a transmembrane DNA transporter, which could have applications in gene delivery systems.

Yi Chuan Xue Bao, 2000, 27(4), 331 - 7
{Regeneration of HNP transgenic alfalfa plants by Agrobacterium mediated gene transfer}; Lu DY et al.; Transgenic plants regenerated from cotyledons of M . sativa L . infected using Agrobacterium tumefaciens A281 with plasmid pBF649 containing a gene encoding protein of high sulfur-amino acid content (HNP) were obtained successfully . The plants grew and fertiled well in field . Cotyledon explants were better recipient for transformation of M . sativa L . Environment of suitable temperature (15 degrees C) and high humidity on high viability of the plants transplanted into soil were essential conditions.

J Exp Bot, 2000 Dec, 51(353), 1961 - 8
Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones; Chateau S et al.; Many plant species and/or genotypes are highly recalcitrant to Agrobacterium-mediated genetic transformation, and yet little is known about this phenomenon . Using several Arabidopsis: genotypes/ecotypes, the results of this study indicated that phytohormone pretreatment could overcome this recalcitrance by increasing the transformation rate in the known recalcitrant genotypes . Transient expression of a T-DNA encoded ss-glucuronidase (GUS) gene and stable kanamycin resistance were obtained for the ten ARABIDOPSIS: genotypes tested as well as for the mutant uvh1 (up to 69% of petioles with blue spots and up to 42% resistant calli) . Cultivation of Arabidopsis: tissues on phytohormones for 2-8 d before co-cultivation with Agrobacterium tumefaciens significantly increased transient GUS gene expression by 2-11-fold and stable T-DNA integration with petiole explants . Different Arabidopsis ecotypes revealed differences in their susceptibility to Agrobacterium-mediated transformation and in their type of reaction to pre-cultivation (three types of reactions were defined by gathering ecotypes into three groups) . The Arabidopsis uvh1 mutant described as defective in a DNA repair system showed slightly lower competence to transformation than did its progenitor Colombia . This reduced transformation competence, however, could be overcome by 4-d pre-culture with phytohormones . The importance of pre-cultivation with phytohormones for genetic transformation is discussed.

J Exp Bot, 2000 Dec, 51(353), 1951 - 60
Vascularization is a general requirement for growth of plant and animal tumours; Ullrich CI et al.; Solid-tumour growth in animals as in humans depends on angiogenesis . Tumours that fail to induce the formation of new blood vessels do not enlarge beyond a few millimetres in diameter . Plant tumours induced by Agrobacterium tumefaciens can reach diameters of more than 100 mm, thus raising the question of how they are sufficiently supplied with nutrients and water . Until recently, these rapidly growing tumours were considered unorganized or partly organized masses . However, in analogy to animal and human tumours, growth of leaf and stem tumours depends on neovascularization . Plant tumour cells induce the formation of a sophisticated vascular network consisting of water-conducting vessels and assimilate-transporting sieve elements . Similar to animal and human tumours that overexpress angiogenic growth factors, plant tumours overexpress the T-DNA-encoded vascularization-promoting growth factors auxin and cytokinin upon AGROBACTERIUM: infection . High auxin levels induce ethylene emission from the tumours, which has a strong impact on tumour and host stem, as well as on root structure and function . Ethylene apparently stimulates abscisic acid synthesis in the leaves above the tumour, which reduces transpiration and thus protects the host plant from rapid wilting . Hence, for the elucidation of phytohormone-dependent vascular development in plants, such tumours are regarded as an excellent model system . The comparison of analogous requirement of neovascularization for tumour growth in plants, as in animals and humans, is discussed in terms of interdisciplinary strategies of possible prevention and therapy.

Virus Res, 2000 Nov, 71(1-2), 63 - 9
The use of transgenic fruit trees as a resistance strategy for virus epidemics: the plum pox (sharka) model; Ravelonandro M et al.; Sharka or plum pox, caused by Plum pox virus (PPV: genus Potyvirus; Family Potyviridae), is the most serious disease of Prunus . Most cultivated Prunus species are highly susceptible and conventional breeding has not produced highly resistant and commercially acceptable varieties . Success in developing virus-resistant herbaceous crops through genetic engineering led us to investigate this approach for resistance to PPV . Our programme aims to develop a biotechnological approach to PPV control that is effective and shown to be environmentally safe . The programme began with the cloning of the PPV coat protein (CP) gene and the development of a transformation system for plum (Prunus domestica) . The CP construct was first tested in Nicotiana benthamiana in which it proved effective in producing transgenic plants with varying levels of CP expression . Some of these plants, particularly low PPV CP expressers, were resistant to PPV, or recovered from initial infection . Based on these results plum was transformed using the Agrobacterium tumefaciens system and both low and high PPV CP-expressing transgenic plum lines were obtained . These were inoculated with PPV by bud grafts in the greenhouse . Line C-5 proved to be highly resistant . It contained multiple copies of the insert, produced low levels of PPV CP mRNA, no detectable CP and the insert appeared to be methylated . These characteristics all suggest that the resistance of the C-5 clone is based on post-transcriptional gene silencing (PTGS) . Field tests of C-5 and other transgenic lines in Poland, Romania and Spain have demonstrated that such trees when inoculated by bud-grafts allow a low level of PPV multiplication, from which they rapidly recover . C-5 plants exposed to natural infection for 3 years did not become infected, whereas control trees were infected in the first year . Hybrid plums having the C-5 PPV CP insert inherited from C-5 are virus-resistant, demonstrating the usefulness of C-5 as a parent in developing new PPV-resistant plum varieties . Research is in progress on the biorisks of PPV CP transgenic plants . Gene constructs that either produce no CP or CP that cannot be transmitted by aphids have been developed, tested in N . benthamiana and transferred to plum . Studies have begun on the potential for synergistic interactions between the PPV CP gene and the other common viruses of Prunus spp . In the future we will be participating in investigating the toxicity or/and the allergenicity of transgenic fruit products and, more importantly, transgenic lines will be developed that express transgenes only in vegetative parts of the plant and not in the fruit.

Appl Environ Microbiol, 2001 Jan, 67(1), 65 - 74
Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2; Mougel C et al.; Ecology and biodiversity studies of Agrobacterium spp . require tools such as selective media and DNA probes . Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria . The known biodiversity within the genus was taken into account when the selectivity of K(2)TeO(3) was analyzed and its potential for isolating Agrobacterium spp . directly from soil was evaluated . A K(2)TeO(3) concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria . Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies . The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K(2)TeO(3)-amended medium . The bona fide agrobacterium colonies growing on media amended with K(2)TeO(3) were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes . Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2 . Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies . Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes . All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria . Direct colony counting of agrobacterial populations could be done . We found 10(3) to 10(4) agrobacteria . g of dry soil(-1) in a silt loam bulk soil cultivated with maize . All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.

Transgenic Res, 2000, 9(4-5), 245 - 260; discussion 243
A transgenic perspective on plant functional genomics; Pereira A; Transgenic crops are very much in the news due to the increasing public debate on their acceptance . In the scientific community though, transgenic plants are proving to be powerful tools to study various aspects of plant sciences . The emerging scientific revolution sparked by genomics based technologies is producing enormous amounts of DNA sequence information that, together with plant transformation methodology, is opening up new experimental opportunities for functional genomics analysis . An overview is provided here on the use of transgenic technology for the functional analysis of plant genes in model plants and a link made to their utilization in transgenic crops . In transgenic plants, insertional mutagenesis using heterologous maize transposons or Agrobacterium mediated T-DNA insertions, have been valuable tools for the identification and isolation of genes that display a mutant phenotype . To discover functions of genes that do not display phenotypes when mutated, insertion sequences have been engineered to monitor or change the expression pattern of adjacent genes . These gene detector insertions can detect adjacent promoters, enhancers or gene exons and precisely reflect the expression pattern of the tagged gene . Activation tag insertions can mis-express the adjacent gene and confer dominant phenotypes that help bridge the phenotype gap . Employment of various forms of gene silencing technology broadens the scope of recovering knockout phenotypes for genes with redundant function . All these transgenic strategies describing gene-phenotype relationships can be addressed by high throughput reverse genetics methods that will help provide functions to the genes discovered by genome sequencing . The gene functions discovered by insertional mutagenesis and silencing strategies along with expression pattern analysis will provide an integrated functional genomics perspective and offer unique applications in transgenic crops.

J Gen Virol, 2001 Jan, 82(Pt 1), 53 - 8
Complete nucleotide sequence and host range of South African cassava mosaic virus: further evidence for recombination amongst begomoviruses; Berrie LC et al.; Complete nucleotide sequences of the DNA-A (2800 nt) and DNA-B (2760 nt) components of a novel cassava-infecting begomovirus, South African cassava mosaic virus (SACMV), were determined and compared with various New World and Old World begomoviruses . SACMV is most closely related to East African cassava mosaic virus (EACMV) in both its DNA-A (85% with EACMV-MH and -MK) and -B (90% with EACMV-UG2-Mld and EACMV-UG3-Svr) components; however, percentage sequence similarities of less than 90% in the DNA-A component allowed SACMV to be considered a distinct virus . One significant recombination event spanning the entire AC4 open reading frame was identified; however, there was no evidence of recombination in the DNA-B component . Infectivity of the cloned SACMV genome was demonstrated by successful agroinoculation of cassava and three other plant species (Phaseolus vulgaris, Malva parviflora and Nicotiana benthamiana) . This is the first description of successful infection of cassava with a geminivirus using Agrobacterium tumefaciens.

Curr Opin Microbiol, 2000 Dec, 3(6), 643 - 8
The role of the T-pilus in horizontal gene transfer and tumorigenesis; Kado CI; The T-pilus is a flexuous filamentous appendage that is essential for Agrobacterium tumefaciens virulence . T-pilus subunits are derived from a VirB2-processing reaction that generates cyclized polypeptide subunits . The T-pilus filament has a diameter of 10 nm and contains a lumen approximately 2 nm in diameter . Biogenesis of the T-pilus requires all 11 VirB proteins, but not the VirD4 protein, which is used in conjugal plasmid transfer . VirB4 and VirB11 are two ATPases that may form homohexameric rings within the transport apparatus, which is composed of VirB6-10 proteins.

Enzyme Microb Technol, 2001 Jan 2, 28(1), 100 - 105
The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza; Chen H et al.; Hairy root cultures of Salvia miltiorrhiza transformed with Agrobacterium rhizogenes ATCC 15834 produced a tiny amount of tanshinones and a constituent level of phenolic acids under normal growth conditions . Upon elicitation with yeast elicitor, the production of both phenolic acids and tanshinones was enhanced . For example, the contents of two phenolic acids, rosmarinic acid and lithospermic acid B were elevated from 1.24% and 2.59% to 2.89% and 2.98% of dry wt, respectively while the intracellular content of cryptotanshinone increased from 0.001% to as much as 0.096% of dry wt . Yeast elicitor also improved the growth of hairy roots (from 3.9 g/l to 7.3 g/l on a dry wt basis) . Liquid chromatography-mass spectrometry (LC-MS) was developed for simultaneous detection and identification of phenolic acids and tanshinones in the extracts of S . miltiorrhiza . Rosmarinic acid, lithospermic acid B, cryptotanshinone, tanshinone I, tanshinone IIA and tanshinone IIB were identified by comparison with standards available . Dihydrotanshinone I and methylenetanshiquinone were tentatively identified by the molecular weights and the elution comparable with the literature . An unknown compound with a molecular weight of 280 was found in yeast-elicitor treated hairy root cultures, which was one of the major tanshinones induced.

J Bacteriol, 2001 Jan, 183(1), 36 - 45
The chvH locus of Agrobacterium encodes a homologue of an elongation factor involved in protein synthesis; Peng WT et al.; The virulence of Agrobacterium tumefaciens depends on both chromosome- and Ti plasmid-encoded gene products . In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation . Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in Escherichia coli . The analysis of vir-lacZ and vir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain . The E . coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH and efp are functionally homologous . As expected, ChvH exerts its activity at the posttranscriptional level . Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A . tumefaciens . We constructed a chvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment . On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14778 - 83
An alternative cytokinin biosynthesis pathway; Astot C et al.; Studies of de novo cytokinin biosynthesis in isopentenyltransferase (ipt)-transformed Arabidopsis thaliana, involving in vivo deuterium labeling and mass spectrometry, showed that the biosynthetic rate of zeatinriboside-5'-monophosphate was around 66-fold higher than that of isopentenyladenosine-5'-monophosphate (iPMP), the proposed primary product of the Agrobacterium ipt . Double tracer analysis, using {(2)H(6)} isopentenyladenosine and deuterium oxide, provided evidence for an alternative, iPMP-independent, biosynthetic pathway for zeatin-type cytokinins, present in both ipt-expressing and wild-type Arabidopsis thaliana . Reduction of the biosynthetic flux in the alternative pathway by use of mevastatin, an inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase, indicated a terpenoid origin for the side-chain precursor of the iPMP independent pathway.

Ophthalmologe, 2000 Oct, 97(10), 703 - 7
{Sterilization of phacoemulsification and vitrectomy instruments.Contamination and evaluation}; Mino de Kaspar H et al.; BACKGROUND: Contamination of automated surgical equipment is widely disregarded as a potential source of perioperative infection . We investigated the possibility of contamination of the aspiration fluid by the vacuum control manifold (VCM) . The normal, unsterile internal VCM was compared with a modified external VCM that was regularly disinfected . MATERIALS AND METHODS: We investigated 37 aspiration fluid specimens from routine cataract and vitrectomy operations performed with automated evacuation systems . There were 25 specimens from three automated evacuation systems equipped with an internal VCM (experimental groups) and 12 specimens from one system equipped with a modified external VCM (control group) . No hygiene procedures were used with the hidden internal VCM, but the modified external VCM was regularly rinsed and filled with 70% isopropanol overnight . Specimens were collected under sterile conditions, centrifuged, cultured for bacterial growth on blood agar and MacConkey agar for 24-48 h at 37 degrees C, and analyzed microbiologically . RESULTS: Aspiration fluids of irrigation/aspiration systems used for intraocular surgery were found to be severely contaminated with bacteria originating from the VCM . In all aspiration fluid specimens from internal VCM systems, 2(+)-4+ bacterial growth was found . Stenotrophomonas maltophilia (17), Comamonas acidovorans (8), and Agrobacterium radiobacter (13) were found most frequently . All specimens from the modified external VCM system remained sterile . There was a significant difference with regard to the frequency of contamination of the aspiration fluid between experimental and control groups (P = 0.0001, chi 2) . CONCLUSIONS: We found that the aspiration fluid of common phaco- and vitrectomy systems was strongly contaminated by bacteria originating from the internal VCM . The technical modification of an external VCM allows easy disinfection and prevents contamination of the aspiration fluid.

Plant Cell Physiol, 2000 Sep, 41(9), 1030 - 7
Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil; Koyama H et al.; The gene for mitochondrial citrate synthase (CS) was isolated from Daucus carota (DcCS) and introduced into Arabidopsis thaliana (strain WS) using Agrobacterium tumefaciens-mediated transformation . Characteristics of citrate excretion were compared between T3 transgenic plants, which were derived from the initial transgenic plants by self-fertilization and homozygous for DcCS, and the control plants that had no DcCS . The highest CS activity 0.78 micromol protein min(-1) exhibited by the transgenic plants was about threefold greater than that found in the control plants (0.23-0.28 micromol protein min(-1)) . Western analysis of the transgenic plants showed two CS signals corresponding to signals obtained from both D . carota and A . thaliana . Thus, it appears that the CS polypeptides by ectopic expression of DcCS were processed into the mature form and localized in the mitochondria of A . thaliana . The signal corresponding to the mature form of DcCS were greater in the transgenic plants having higher levels of CS activity . When the transgenic plants were grown in Al-phosphate media, a correlation between the levels of CS activity and the amounts of citrate excreted into the medium . The highest value (5.1 nmol per plant) was about 2.5-fold greater than that from control plants (1.9 nmol per plant) . Both growth and P accumulation were greater in transgenic plants with high CS activity than that in control plants when they were grown on an acid soil where the availability of phosphate was low due to the formation of Al-phosphate . It appears that the overexpression of CS in A . thaliana improves the growth in phosphorous limited soil as a result of enhanced citrate excretion from the roots.

Mol Biotechnol, 2000 Sep, 16(1), 53 - 65
Plant transformation technology . Developments and applications; Newell CA; Plant transformation has its roots in the research on Agrobacterium that was being undertaken in the early 1980s . The last two decades have seen significant developments in plant transformation technology, such that a large number of transgenic crop plants have now been released for commercial production . Advances in the technology have been due to development of a range of Agrobacterium-mediated and direct DNA delivery techniques, along with appropriate tissue culture techniques for regenerating whole plants from plant cells or tissues in a large number of species . In addition, parallel developments in molecular biology have greatly extended the range of investigations to which plant transformation technology can be applied . Research in plant transformation is concentrating now not so much on the introduction of DNA into plant cells, but rather more on the problems associated with stable integration and reliable expression of the DNA once it has been integrated.

Curr Infect Dis Rep, 2000 Feb, 2(1), 73 - 77
Novel Approaches to Oral Vaccines: Delivery of Antigens by Edible Plants; Yu J et al.; Advances in genetic engineering in the past decade have accelerated the expression, in plants, of foreign proteins with industrial and pharmaceutical value . Antigens from infectious bacterial or viral diseases have been introduced into plants through plant virus-mediated infection or Agrobacterium tumefaciens-mediated stable transformation methods . Oral immunization with transgenic plant tissues that contain vaccine antigen proteins stimulates both systemic and mucosal immune responses in animals . Plant-based vaccines can provide significant levels of protection against challenge by viral or bacterial pathogens.

Membr Cell Biol, 2000, 14(2), 199 - 203
Formation of extracellular structures containing VirB2 proteins in mating cultures of agrobacteria; Kurbanova IV et al.; Using transmission electron immunomicroscopy, VirB2 protein has been revealed at the surface of acetosyringon-treated A . tumefaciens cells . VirB2 was seen within long flexible and short structures localized at the opposite poles of the cells . These structures were not observed in cells not treated with acetosyringon and in agrobacterial cells treated with this reagent but carrying no Ti-plasmid . Labeled complexes {antibodies to virB2 protein + (A protein + colloidal gold)} bound to pili at a certain periodicity.

J Agric Food Chem, 2000 Nov, 48(11), 5243 - 8
Transgenic apple (Malus x domestica) shoot showing low browning potential; Murata M et al.; Transgenic apple shoots were prepared from leaf disks by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistance gene and antisense polyphenol oxidase (PPO) DNA . Four transgenic apple lines that grew on the medium containing 50 microgram/mL KM were obtained . They contained the KM resistance gene and grew stably on the medium for >3 years . Two transgenic shoot lines containing antisense PPO DNA in which PPO activity was repressed showed a lower browning potential than a control shoot.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13324 - 9
Molecular signals required for type III secretion and translocation of the Xanthomonas campestris AvrBs2 protein to pepper plants; Mudgett MB et al.; Strains of Xanthomonas campestris pv . vesicatoria (Xcv) carrying avrBs2 are specifically recognized by Bs2 pepper plants, resulting in localized cell death and plant resistance . Agrobacterium-mediated transient expression of the Xcv avrBs2 gene in plant cells results in Bs2-dependent cell death, indicating that the AvrBs2 protein alone is sufficient for the activation of disease resistance-mediated cell death in planta . We now provide evidence that AvrBs2 is secreted from Xcv and that secretion is type III (hrp) dependent . N- and C-terminal deletion analysis of AvrBs2 has identified the effector domain of AvrBs2 recognized by Bs2 pepper plants . By using a truncated Pseudomonas syringae AvrRpt2 effector reporter devoid of type III signal sequences, we have localized the minimal region of AvrBs2 required for type III secretion in Xcv . Furthermore, we have identified the region of AvrBs2 required for both type III secretion and translocation to host plants . The mapping of AvrBs2 sequences sufficient for type III delivery also revealed the presence of a potential mRNA secretion signal.

Proc Natl Acad Sci U S A, 2000 Nov 21, 97(24), 13401 - 6
Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway; Llave C et al.; Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread . The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes . The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems . HC-Pro inactivated PTGS in plants containing a preexisting silenced beta-glucuronidase (GUS) transgene . PTGS in this system was associated with both small RNA molecules (21-26 nt) corresponding to the 3' proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3' end of the GUS transgene . Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation . These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

Plant Sci, 2000 Nov 6, 159(2), 289 - 299
Temporal and spatial activity of a promoter from a pea enzyme inhibitor gene and its exploitation for seed quality improvement; Welham T et al.; The promoter from one of the two seed-expressed genes encoding trypsin/chymotrypsin inhibitors (TI) has been isolated and characterised in transgenic pea lines, following its re-introduction by Agrobacterium-mediated transformation, as a TI promoter-beta-glucuronidase (GUS) gene fusion . The promoter from this gene (TI1) directed expression of GUS enzyme at late stages of embryogenesis, comparable to those determined for activity of the homologous native TI genes . GUS expression was detected in roots of plants subjected to drought stress conditions, indicating that the TI1 gene, normally seed-specific in its expression, can be induced under these conditions . A second gene construct utilised the TI1 gene promoter to direct expression of an antisense TI gene . Seed TI activities in some lines transformed with this construct were reduced significantly . A limitation of the pea transformation methodology for antisense manipulations, in particular, is the observed frequency of non-transmission of transgenes from primary transformants (up to 80%).

Plant Sci, 2000 Nov 6, 159(2), 273 - 280
Transformation of Antirrhinum majus L . by a rol-type multi-auto-transformation (MAT) vector system; Minlong C et al.; A total of 11 independent beta-glucuronidase (GUS) positive hairy roots were induced following co-cultivation of leaf explants of Antirrhinum majus L . with Agrobacterium tumefaciens strain GV2260 containing rol-type multi-auto-transformation (MAT) vector pNPI702 . A total of 326 adventitious shoots were regenerated from the hairy root lines on 1/2 MS medium without plant growth regulators at 25 degrees C under a 16 h/day photoperiod condition 4 months after infection of the A . tumefaciens GV2260 . The absence of the rol genes in five plants was verified by polymerase chain reaction (PCR) and Southern blot analysis . Acclimatized transformants exhibited normal phenotypes in height and in the morphology of leaves and flowers . Furthermore, the GUS gene was strongly expressed in the leaves, inflorescence of the transformed plant, and the progeny . This result demonstrates that the rol-type MAT vector can be used to study gene functions controlling the morphogenesis of Antirrhinum majus plants.

Plant Sci, 2000 Nov 6, 159(2), 183 - 189
The roles of Rirol and Ngrol genes in hairy root induction in Nicotiana debneyi; Aoki S et al.; The function of Rirol genes in TL-DNA of the Ri plasmid of Agrobacterium rhizogenes has been previously studied in Nicotiana tabacum and Daucus carota, but it was reported that these plants have a TL-DNA-similar sequence in their genome . We investigated the function of Rirol genes in N . debneyi by infection with A . tumefaciens harboring these genes, because the genome of N . debneyi does not contain a TL-DNA-similar sequence . The single gene RirolB induced adventitious roots in N . debneyi . Introduction of a DNA fragment that contained RirolB, RirolC, RiORF13 and RiORF14 resulted in more intense and earlier root formation than that of RirolB . Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) in the genome of Nicotiana glauca that are similar in sequence to Rirol genes were also examined . In contrast with Rirol genes, Ngrol genes did not induce adventitious roots on leaf segments of N . debneyi . Further infection analysis revealed that one of the reasons for this diversity of their functions might be the difference in the rolB region between the sequence of bacteria and plants . The difference in function between the genes of plants and bacteria is analyzed and the molecular evolution of Ngrol genes is discussed.

Plant J, 2000 Nov, 24(3), 413 - 9
Metabolic engineering of beta-carotene and lycopene content in tomato fruit; Rosati C et al.; Ripe tomato fruits accumulate large amounts of the red linear carotene, lycopene (a dietary antioxidant) and small amounts of its orange cyclisation product, beta-carotene (pro-vitamin A) . Lycopene is transformed into beta-carotene by the action of lycopene beta-cyclase (beta-Lcy) . We introduced, via Agrobacterium-mediated transformation, DNA constructs aimed at up-regulating (OE construct) or down-regulating (AS construct) the expression of the beta-Lcy gene in a fruit-specific fashion . Three transformants containing the OE construct show a significant increase in fruit beta-carotene content . The fruits from these plants display different colour phenotypes, from orange to orange-red, depending on the lycopene/beta-carotene ratio . Fruits from AS transformants show up to 50% inhibition of beta-Lcy expression, accompanied by a slight increase in lycopene content . Leaf carotenoid composition is unaltered in all transformants . In most transformants, an increase in total carotenoid content is observed with respect to the parental line . This increase occurs in the absence of major variations in the expression of endogenous carotenoid genes.

Plant J, 2000 Oct, 24(2), 275 - 83
A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant pathogens; Takken FL et al.; We have devised a novel, high-throughput functional cloning method to isolate cDNAs from plant pathogens of which the products elicit a hypersensitive response (HR) in plants . Copy DNA, made from RNA isolated from the tomato pathogen Cladosporium fulvum grown under nutrient-limiting conditions in vitro, was cloned into a binary, potato virus X (PVX)-based expression vector and transformed to Agrobacterium tumefaciens . 9600 colonies were individually toothpick-inoculated onto leaflets of tomato plants resistant to C . fulvum . Four cDNAs were identified whose expression induced formation of a necrotic lesion around the inoculation site . One of these clones, specifically inducing HR on tomato plants carrying the Cf-4 resistance gene, encodes race-specific elicitor AVR4 . The other three cDNAs, inducing a non-genotype-specific HR, encode a protein highly homologous to bZIP, basic transcription factors . To determine whether this approach has general applicability, part of the library was also inoculated onto Nicotiana tabacum var . Samsun NN, which is not a host for C . fulvum . Four independent HR-inducing cDNAs were identified which all encode ECP2, an extracellular protein of C . fulvum known to induce necrosis in certain Nicotiana species . These observations confirm that this functional screening method is a versatile strategy to identify cDNAs of pathogens that encode (race-specific) elicitors and other HR-inducing proteins.

Science, 2000 Nov 3, 290(5493), 979 - 82
VirB/D4-dependent protein translocation from Agrobacterium into plant cells; Vergunst AC et al.; The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease . In addition, several Virulence proteins must somehow be transported to fulfill a function in planta . Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells . Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA.

Chemosphere, 2000 Dec, 41(12), 1873 - 9
Biodegradation of 2,4,6-trichlorophenol in the presence of primary substrate by immobilized pure culture bacteria; Wang CC et al.; In this study, pure strains that are capable of utilizing 2,4,6-trichlorophenol have been isolated from the mixed culture grown on substrates containing chlorophenolic compounds . Studies have been carried out on the capability of these isolated pure strains in suspended and immobilized forms to decompose 2,4,6-trichlorophenol . Additionally, the influence of primary substrates (e.g., phenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol) on the decomposition of 2,4,6-trichlorophenol by the isolated pure strains grown in immobilized form is also investigated . The results are: Through bacterial isolation and identification, three pure strains have been obtained: Pseudomonas spp . strain 01, Pseudomonas spp . strain 02 and Agrobacterium spp . Whether in suspended or immobilized forms, all strains have poor removal efficiencies of 2,4,6-trichlorophenol . However, addition of 200 mg/l phenol will enable the immobilized Pseudomonas spp . strain 01, and Pseudomonas spp . strain 02 to achieve 65% and 48% removal of 2,4,6-trichlorophenol, respectively . Addition of phenol will assist the immobilized Pseudomonas spp . strain 02 in achieving removal of 2,4,6-trichlorophenol but the removal efficiency is not good if the phenol concentration is too low . The optimum phenol concentration should be between 200 and 400 mg/l.

Mol Plant Microbe Interact, 2000 Nov, 13(11), 1275 - 9
Agroinfiltration of Cauliflower mosaic virus gene VI elicits hypersensitive response in Nicotiana species; Palanichelvam K et al.; Cauliflower mosaic virus strain W260 induces hypersensitive response (HR) in Nicotiana edwardsonii and systemic cell death in N . clevelandii . In contrast, the D4 strain of Cauliflower mosaic virus evades the host defenses in Nicotiana species; it induces chlorotic primary lesions and a systemic mosaic in both hosts . Previous studies with chimeric viruses had indicated that gene VI of W260 was responsible for elicitation of HR or cell death . To prove conclusively that W260 gene VI is responsible, we inserted gene VI of W260 and D4 into the Agrobacterium tumefaciens binary vector pKYLX7 . Agroinfiltration of these constructs into the leaves of N . edwardsonii and N . clevelandii revealed that gene VI of W260 elicited HR in N . edwardsonii 4 to 5 days after infiltration and cell death in N . clevelandii approximately 9 to 12 days after infiltration . In contrast, gene VI of D4 did not elicit HR or cell death in either Nicotiana species . A frameshift mutation introduced into gene VI of W260 abolished its ability to elicit HR or cell death in both Nicotiana species, demonstrating that the elicitor is the gene VI protein.

Biotechniques, 2000 Oct, 29(4), 832 - 6, 838-43
Transgenic plants and biosafety: science, misconceptions and public perceptions; Stewart CN Jr et al.; One usually thinks of plant biology as a non-controversial topic, but the concerns raised over the biosafety of genetically modified (GM) plants have reached disproportionate levels relative to the actual risks . While the technology of changing the genome of plants has been gradually refined and increasingly implemented, the commercialization of GM crops has exploded . Today's commercialized transgenic plants have been produced using Agrobacterium tumefaciens-mediated transformation or gene gun-mediated transformation . Recently, incremental improvements of biotechnologies, such as the use of green fluorescent protein (GFP) as a selectable marker, have been developed . Non-transformation genetic modification technologies such as chimeraplasty will be increasingly used to more precisely modify germplasm . In spite of the increasing knowledge about genetic modification of plants, concerns over ecological and food biosafety have escalated beyond scientific rationality . While several risks associated with GM crops and foods have been identified, the popular press, spurred by colorful protest groups, has left the general public with a sense of imminent danger . Reviewed here are the risks that are currently under research . Ecological biosafety research has identified potential risks associated with certain crop/transgene combinations, such as intra- and interspecific transgene flow, persistence and the consequences of transgenes in unintended hosts . Resistance management strategies for insect resistance transgenes and non-target effects of these genes have also been studied . Food biosafety research has focused on transgenic product toxicity and allergenicity . However, an estimated 3.5 x 10(12) transgenic plants have been grown in the U.S . in the past 12 years, with over two trillion being grown in 1999 and 2000 alone . These large numbers and the absence of any negative reports of compromised biosafety indicate that genetic modification by biotechnology poses no immediate or significant risks and that resulting food products from GM crops are as safe as foods from conventional varieties . We are increasingly convinced that scientists have a duty to conduct objective research and to effectively communicate the results--especially those pertaining to the relative risks and potential benefits--to scientists first and then to the public . All stakeholders in the technology need more effective dialogues to better understand risks and benefits of adopting or not adopting agricultural biotechnologies.

Appl Environ Microbiol, 2000 Nov, 66(11), 5099 - 103
Genetic characterization of soybean rhizobia in Paraguay; Chen LS et al.; The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s . Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow . Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time . Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Parana and Itapua, was undertaken . A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains . Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities . However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia . Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q . However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants.

Plant Mol Biol, 2000 Jul, 43(4), 495 - 502
The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation; van der Fits L et al.; This paper describes a so-called ternary transformation system for plant cells . We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species . For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies . Analysis of stably transformed C . roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome . Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A . tumefaciens strains in combination with standard binary vectors.

J Biotechnol, 2000 Oct 13, 83(3), 199 - 210
Root hairiness: effect on fluid flow and oxygen transfer in hairy root cultures; Shiao TL et al.; The effect of root hairiness on fluid flow and oxygen transfer in hairy root cultures was investigated using wild-type, transgenic and root-hair mutants of Arabidopsis thaliana . The root hair morphologies of the A . thaliana lines were hairless, short hairs, moderately hairy (wild-type) and excessively hairy, and these morphologies were maintained after transformation of seedlings with Agrobacterium rhizogenes . Filtration experiments were used to determine the permeability of packed beds of roots; permeability declined significantly with increasing root hairiness as well as with increasing biomass density . Hairy roots of wild-type A . thaliana grew fastest with a doubling time of 6.9 days, but the hairless roots exhibited the highest specific oxygen uptake rate . In experiments using a gradientless packed bed reactor with medium recirculation, the liquid velocity required to eliminate external mass transfer boundary layer effects increased with increasing root hairiness, reflecting the greater tendency towards liquid stagnation near the surface of roots covered with hairs . External critical oxygen tensions also increased with increasing root hairiness, ranging from 50% air saturation for hairless roots to ca . 150% air saturation for roots with excessive root hairs . These results are consistent with root hairs providing a significant additional resistance to oxygen transfer to the roots, indicating that very hairy roots are more likely than hairless roots to become oxygen-limited in culture . This investigation demonstrates that root hairiness is an important biological parameter affecting the performance of root cultures and suggests that control over root hair formation, either by use of genetically modified plant lines or manipulation of culture conditions, is desirable in large-scale hairy root systems.

Chem Biol, 2000 Aug, 7(8), 611 - 21
At the maize/Agrobacterium interface: natural factors limiting host transformation; Zhang J et al.; BACKGROUND: Agrobacterium tumefaciens has been successfully harnessed as the only natural vector for the incorporation of foreign genes into higher plants, but its use in the grain crops is often limited . Low transformation efficiency has been partly attributed to a failure in the initial events in the transformation process, specifically in the capacity of the VirA/VirG two-component system to induce expression of the virulence genes . RESULTS: Here we show that the root exudate of Zea mays seedlings specifically inhibits virulence gene expression, determine that 2-hydroxy-4,7-dimethoxybenzoxazin-3-one (MDIBOA), which constitutes > 98% of the organic exudate of the roots of these seedlings, is the most potent and specific inhibitor of signal perception in A . tumefaciens-mediated gene transfer yet discovered, and develop a model that is able to predict the MDIBOA concentration at any distance from the root surface . Finally, variants of A . tumefaciens resistant to MDIBOA-mediated inhibition of vir gene expression have been selected and partially characterized . CONCLUSIONS: These results suggest a strategy in which a plant may resist pathogen invasion by specifically blocking virulence gene activation and yet ensure that the 'resistance factor' does not accumulate to levels sufficient to impose toxicity and selection pressure on the pathogen . The data further establish that naturally occurring inhibitors directed against signal perception by the VirA/VirG two-component regulatory system can play an important role in host defense . Finally, selected variants resistant to specific MDIBOA inhibition may now be used to extend the transformation efficiency of maize and possibly other cereals.

Mol Plant Microbe Interact, 2000 Oct, 13(10), 1081 - 91
A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid; Palanichelvam K et al.; Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy) . Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58 . Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5 . These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines . The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment . The two T-regions are separated by approximately 15 kb of plasmid DNA . Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL . Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency . All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines . Moreover, A . thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency . These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants . A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events . This plasmid, called pKPSF2, lacks both of the known T-regions and their borders . pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

Zhongguo Zhong Yao Za Zhi, 1997 May, 22(5), 274 - 5, 318
{The plant regeneration of Salvia miltiorrhiza Bge . transformed by Agrobacterium}; Zhang Y et al.; The hairy roots and crown galls of Salvia miltiorrhiza were obtained by infecting plant with A . rhizogenes (strain 15834, LBA 9402) and A . tumefaciens (strain C58) . The transformed plants were regenerated light and transplanted form cultural medium into soil successfully . The plants transformed by A . rhizogenes have characteristics of short stems and develop hairy roots, those and trans formed by A . tumefaciens grow vigorously featuring, longer stems and well developed roots . Both biomass production and tanshenone content are higher than the original plant.

Plant Cell Physiol, 2000 Aug, 41(8), 911 - 9
Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon; Boehm R et al.; The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb . et Zucc . (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures . We have now manipulated the biosynthetic pathway leading to shikonin in L . erythrorhizon by introduction of the bacterial gene ubiA . This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis . Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis . The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER) . Constructs with different constitutive promoters were introduced into L . erythrorhizon using Agrobacterium rhizogenes-mediated transformation . In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg(-1) . Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50 . However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation . This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.

Chin J Biotechnol, 1999, 15(4), 203 - 10
Construction of plant vectors with high level expression of B.t . toxin gene and studies on their expression behavior in transgenic tobaccos; Chunlin S et al.; Breeding pest-resistant plants using plant genetic engineering technique is an effective strategy in integrated pest management (IPM) . Increasing the expression level of foreign insecticidal protein by using a strong promoter is a useful method . In this work, a plant expression vector, pBinMoBc, was constructed . It contained the CryIA(c) gene under the control of a chimeric OM promoter and the omega factor . As a control, another vector, pBinoBc, was also constructed in this study . pBinoBc carrying CryIA(c) gene was under the control of a CaMV 35S promoter . The vectors were transferred into tobacco plants via Agrobacterium-mediated transformation . ELISA assay showed that the average expression level of the CryIA(c) gene in pBinMoBc transgenic tobacco plants is 2.44-times that in pBinoBc transgenic tobacco plants . The expression of B.t . toxin in individual plant can be up to 0.255% of total soluble proteins . Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal effects than pBinoBc transgenic tobacco plants . The above results showed that the chimeric OM promoter is a stronger promoter than the CaMV 35S promoter that was widely used in plant genetic engineering . This is very useful in pest-resistant plant genetic engineering.

Transgenic Res, 2000 Jun, 9(3), 195 - 203
Ribozyme-mediated resistance to rice dwarf virus and the transgene silencing in the progeny of transgenic rice plants; Han S et al.; A hammerhead ribozyme (Rz) with long hybridizing arms targeting the mRNA of rice dwarf virus (RDV) segment 5 and a mutated nonfunctional ribozyme (mRz) were constructed . As predicted, Rz transcribed in vitro cleaved the target mRNA of RDV segment 5 into two fragments of 138 and 238 nucleotides in length . The Rz and mRz genes were each placed under the control of the CaMV 35S promoter and used to transform Japonica rice variety 'Tongling No . 1' via Agrobacterium tumefaciens . A total of 32 independent lines containing Rz or mRz was obtained as demonstrated by Southern blot analysis . Challenge inoculation with RDV viruliferous leafhoppers (Nephotettix cincticeps) showed that T1 plants containing the Rz transgene displayed high resistance or delayed and attenuated viral symptoms . In contrast, transgenic lines expressing mRz showed severe symptoms similar to the control plants transformed with the vector alone . These results suggest that Rz confers RDV resistance in transgenic rice . Genomic DNA PCR analysis confirmed that all of the examined T6 progeny plants contained the Rz transgene . However, accumulation of the Rz transcripts was detectable by RT-PCR only in the plants that were resistant to RDV . This suggested that loss of RDV resistance in progeny plants containing the Rz transgene may result from silencing of the Rz transgene.

Appl Microbiol Biotechnol, 2000 Sep, 54(3), 348 - 53
Overproduction of D-hydantoinase and carbamoylase in a soluble form in Escherichia coli; Chao YP et al.; The production of D-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli . We initiated a systematic study aimed at overproducting these two proteins in a soluble form . As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES . In contrast, the production of a high level of D-hydantoinase in an active form can be achieved at low temperature (25 degrees C) or by the coproduction of DnaJ/DnaK . Overall, with such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity . In addition, by fusion with thioredoxin, D-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK . These results indicate the success of our approaches to overproducing D-hydantoinase and carbamoylase in a soluble form in E . coli.

J Bacteriol, 2000 Nov, 182(21), 6123 - 9
Characterization of the genes for two protocatechuate 3, 4-dioxygenases from the 4-sulfocatechol-degrading bacterium Agrobacterium radiobacter strain S2; Contzen M et al.; The genes for two different protocatechuate 3,4-dioxygenases (P34Os) were cloned from the 4-sulfocatechol-degrading bacterium Agrobacterium radiobacter strain S2 (DSMZ 5681) . The pcaH1G1 genes encoded a P34O (P34O-I) which oxidized protocatechuate but not 4-sulfocatechol . These genes were part of a protocatechuate-degradative operon which strongly resembled the isofunctional operon from the protocatechuate-degrading strain Agrobacterium tumefaciens A348 described previously by D . Parke (FEMS Microbiol . Lett . 146:3-12, 1997) . The second P34O (P34O-II), encoded by the pcaH2G2 genes, was functionally expressed and shown to convert protocatechuate and 4-sulfocatechol . A comparison of the deduced amino acid sequences of PcaH-I and PcaH-II, and of PcaG-I and PcaG-II, with each other and with the corresponding sequences from the P34Os, from other bacterial genera suggested that the genes for the P34O-II were obtained by strain S2 by lateral gene transfer . The genes encoding the P34O-II were found in a putative operon together with two genes which, according to sequence alignments, encoded transport proteins . Further downstream from this putative operon, two open reading frames which code for a putative regulator protein of the IclR family and a putative 3-carboxymuconate cycloisomerase were identified.

Annu Rev Microbiol, 2000, 54, 187 - 219
Nucleic acid transport in plant-microbe interactions: the molecules that walk through the walls; Tzfira T et al.; Many microbes "genetically invade" plants by introducing DNA or RNA molecules into the host cells . For example, plant viruses transport their genomes between host cells, whereas Agrobacterium spp . transfer T-DNA to the cell nucleus and integrate it into the plant DNA . During these events, the transported nucleic acids must negotiate several barriers, such as plant cell walls, plasma membranes, and nuclear envelopes . This review describes the microbial and host proteins that participate in cell-to-cell transport and nuclear import of nucleic acids during infection by plant viruses and Agrobacterium spp . Possible molecular mechanisms by which these transport processes occur are discussed.

Appl Environ Microbiol, 2000 Oct, 66(10), 4510 - 3
A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus; Chen X et al.; We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus . Salient features of this procedure include cocultivation of Agrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter . This method offers new prospects for the genetic manipulation of this commercially important mushroom species.

Appl Environ Microbiol, 2000 Oct, 66(10), 4247 - 52
Substrate specificity of atrazine chlorohydrolase and atrazine-catabolizing bacteria; Seffernick JL et al.; Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp . strain ADP . Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear . Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs . Purified AtzA from Pseudomonas sp . strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine . AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups . Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA . AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group . However, when both amino groups were unalkylated, no reaction occurred . Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp . strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D . All showed identical substrate specificity to purified AtzA from Pseudomonas sp . strain ADP . Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp . strain ADP . This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.

Plant Sci, 2000 Oct 16, 159(1), 7 - 19
An efficient method for the production of transgenic plants of peanut (Arachis hypogaea L.) through Agrobacterium tumefaciens-mediated genetic transformation; Sharma KK et al.; Cotyledon explants from mature peanut seeds (Arachis hypogaea L.) were optimized to obtain adventitious shoot buds with high frequencies (>90%) . Efficient transformation of these cotyledons by using Agrobacterium tumefaciens strain C58 carrying neomycin phosphotransferase II (nptII) and ss-glucuronidase (GUS; uidA), or coat protein gene of the Indian peanut clump virus (IPCVcp) and nptII on binary vectors (pBI121; pROKII:IPCVcp) led to the production of a large percentage (55%) of transgenic plants . Transformed individuals were obtained through selection on medium containing 125 mg l(-1) kanamycin . A large number of independently transformed plants (over 75) were successfully transplanted to the glasshouse . Integration of the transgenes and stable genetic transformants in the progeny were assessed by PCR amplification of 700-bp fragment of nptII and 585-bp of IPCVcp genes, and Southern blot hybridizations in the T1 generation of transgenic plants . Analysis of 35 transgenic plants of T1 generation from the progeny of a single transformation event suggested the segregation of a single copy insert in a 3:1 Mendelian ratio . On an average, 120-150 days were required between the initiation of explant transformation and transfer of rooted plants to the greenhouse . The cotyledon regeneration system proved to be an excellent vehicle for the production of a large number of independently transformed peanut plants . Shoot formation was rapid and prolific, and a large proportion of these shoots developed into fertile plants . The method reported here provides new opportunities for the crop improvement of peanut via genetic transformation.

J Nat Prod, 2000 Sep, 63(9), 1249 - 52
Effects of the rol C gene on hairy root: induction development and tropane alkaloid production by Atropa belladonna; Bonhomme V et al.; Two series of Atropa belladonna hairy root lines were obtained, the first one transformed via Agrobacterium tumefaciens harboring rol C and npt II genes, and the other transformed with rol ABC and npt II genes . Thirteen hairy root lines were obtained and selected on hormone-free medium . The transformation was confirmed by PCR analysis, and these root lines were first examinated for their growth rate . Then hyoscyamine and scopolamine production was measured after 3 and 4 weeks of culture to evaluate the possible role of rol C gene in tropane alkaloid formation . The rol C gene alone played a significant role in the hairy root growth rate (17-fold increase) . However this effect was much lower than that induced by the rol ABC genes together (75-fold increase) . In contrast, the rol C gene alone was as efficient as the rol ABC genes together (mean value of total alkaloids: 0.36% dry weight, i.e., 12-fold times more than in untransformed roots) to stimulate the biosynthesis of tropane alkaloids in A . belladonna hairy root cultures.

Int J Biol Macromol, 2000 Aug 28, 27(5), 319 - 26
Structure of succinoglycan from an infectious strain of Agrobacterium radiobacter; Evans LR et al.; The exopolysaccharide produced by a cystic fibrosis clinical isolate of Agrobacterium radiobacter was shown by monosaccharide and methylation analyses, degradation with succinoglycanase and NMR analysis to be a succinoglycan with the structure shown below . (S)-pyruvic acid is found stoichiometrically as 4,6-O-ketal substituent of terminal glucose . Succinic acid is present in 40% of the repeating units and it is attached to O-6 of the 3-linked glucose next to the pyruvate carrying sugar . Some evidence is found that a small amount of succinic acid (ca . 6% of the total) is linked to O-6 of another undetermined glucose . {structure: see text}

Plant Sci, 2000 Sep 8, 158(1-2), 1 - 18
Rice transformation for crop improvement and functional genomics; Tyagi AK et al.; Although several japonica and some indica varieties of rice have already been transformed, there is significant scope for improvement in the technology for transformation of economically important indica varieties . Successful transformation of rice employing Agrobacterium and recent advances in direct gene transfer by biolistics, evidenced by transfer of multiple genes, have removed some of the serious impediments in the area of gene engineering . The transfer of genes for nutritionally important biosynthetic pathway has provided many opportunities for performing metabolic engineering . Other useful genes for resistance against pests, diseases and abiotic stresses have also been transferred to rice . But the limited knowledge about important target genes requires rapid progress in the field of functional genomics . Transgenic rice system can be applied to isolate new genes, promoters, and enhancers and their functions could be unravelled . The combination of novel regulatory systems for targeted expression and useful new genes should pave the way for improvement of rice and other cereals.

Genetika, 2000 Jul, 36(7), 932 - 41
{Production of transgenic rape plants (Brassica napus L.) using Agrobacterium tumefaciens}; Radchuk VV et al.; The procedure for genetic transformation of two spring and one winter rapeseed cultivars was developed . No-paline strains of Agrobacterium tumefaciens GV3101 and EHA105 were shown to be preferable for gene transfer, as compared to the octopine strain GV2260 . With two types of plant explants, the segments of hypocotyls and cotyledons, transformation was successful; however, its efficiency was somewhat higher with the fragments of hypocotyls . Analysis of regenerated plants by PCR and Southern blotting confirmed the presence of the nptII and nisA genes in transformants . RNA analysis by Northern blotting showed expression of the nisA gene in transformed shoots . The transgenes were inherited in T2 as Mendelian traits . The effect of biotic and abiotic factors on the efficiency of genetic transformation in rapeseed is discussed.

Infect Immun, 2000 Oct, 68(10), 5716 - 23
Identification and characterization of the Brucella abortus phosphoglucomutase gene: role of lipopolysaccharide in virulence and intracellular multiplication; Ugalde JE et al.; Smooth lipopolysaccharide (LPS) of Brucella abortus has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear . While the protective properties of LPS against complement are well accepted, there is still some controversy about the capacity of rough mutants to replicate intracellularly . The B . abortus phosphoglucomutase gene (pgm) was cloned, sequenced, and disrupted . The gene has a high index of identity to Agrobacterium tumefaciens pgm but is not part of the glycogen operon . A B . abortus null mutant lacks LPS O antigen but has an LPS core with an electrophoretic profile undistinguishable from that of the wild-type core, suggesting that glucose, galactose, or a derivative of these sugars may be part of the linkage between the core and the O antigen . This mutant is unable to survive in mice but replicates in HeLa cells, indicating that the complete LPS is not essential either for invasion or for intracellular multiplication . This behavior suggests that the LPS may play a role in extracellular survival in the animal, probably protecting the cell against complement-mediated lysis, but is not involved in intracellular survival.

Pharmazie, 2000 Aug, 55(8), 561 - 7
Medicinal plant biotechnology: the Apiaceae family as the example of rapid development; Ekiert H; This article reviews the results of research representative of different trends in biotechnological studies of the Apiaceae species . These species are a well-known source of many important herbal products . A number of investigations focus on the biosynthesis of secondary metabolites in plant in vitro cultures . There are also numerous reports concerning the methods of plant micropropagation, especially using somatic embryogenesis . The papers dealing with the usage of the enzymatic potential of plant cells cultured in vitro, in biotransformation processes are less copious . Several studies are related to genetic engineering, exploring mainly the possibility of plant transformation with Agrobacterium rhizogenes to obtain hairy root cultures . The present review demonstrates the significance of biotechnological methods in the studies of medicinal plants taking species of the Apiaceae family as an example.

J Biotechnol, 2000 Aug 25, 81(2-3), 151 - 8
Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium rhizogenes 15834 and Agrobacterium tumefaciens containing rol A, B, C genes only; Bonhomme V et al.; Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rol ABC and npt II genes . Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation . A great diversity has been observed in the growth rate of these 13 root lines . The root biomass increased up to 75 times . The total alkaloid contents were similar in the root lines obtained by infection with A . rhizogenes 15834 and A . tumefaciens rol ABC . The last ones accumulated between 4 (1.1 mg g(-1) DW) and 27 (8 mg g(-1) DW) times more alkaloids than the intact roots (0.3 mg g(-1) DW) . This work has shown that the rol ABC genes were sufficient to increase tropane alkaloid production in A . belladonna hairy root cultures.

J Bacteriol, 2000 Oct, 182(19), 5486 - 94
oriT-directed cloning of defined large regions from bacterial genomes: identification of the Sinorhizobium meliloti pExo megaplasmid replicator region; Chain PS et al.; We have developed a procedure to directly clone large fragments from the genome of the soil bacterium Sinorhizobium meliloti . Specific regions to be cloned are first flanked by parallel copies of an origin of transfer (oriT) together with a plasmid replication origin capable of replicating large clones in Escherichia coli but not in the target organism . Supplying transfer genes in trans specifically transfers the oriT-flanked region, and in this process, site-specific recombination at the oriT sites results in a plasmid carrying the flanked region of interest that can replicate in E . coli from the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector) . We have used this procedure with the oriT of the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the S . meliloti pExo megaplasmid . Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium Agrobacterium tumefaciens . The nucleotide sequence of this fragment revealed a replicator region including homologs of the repA, repB, and repC genes from other Rhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.

Clin Experiment Ophthalmol, 2000 Jun, 28(3), 201 - 4
Invasive strains of Pseudomonas aeruginosa are able to cause epithelial cell cytotoxicity that is dependent on bacterial cell density; Zhu H et al.; The purpose of this study was to investigate the hypothesis that quorum-sensing systems are involved in the ability of invasive Pseudomonas aeruginosa strains to cause corneal epithelial cell death . Two invasive strains, 6294 and PAOI, were co-cultured with human corneal epithelial cells at different bacterial concentrations (10(5), 10(7) and 10(9) CFU/mL) . Cytotoxicity was measured using a cytotoxicity assay kit . The levels of autoinducer in the supernatant were examined using a reporter strain of Agrobacterium tumefaciens (A136) . Protease production was also monitored . Cytotoxicity of both strains was dependent on bacterial density; a moderate to high concentration of bacterial cells (10(7) and 10(9) CFU/mL) caused 70% to 94% loss of cell variability . Cytotoxicity was significantly correlated with enhanced autoinducer and protease production (r>0.95, P<0.05).These results indicate that the invasive strains regulate the production of virulence factors and, in turn, induce chronic dose-related cytotoxicity.

Yi Chuan Xue Bao, 2000, 27(5), 428 - 33
{Cloning of E . coli mtl-D gene and its expression in transgenic Balizhuangyang (Populus)}; Liu FH et al.; E . coli 1-phosphate mannitol dehydrogenase gene (mtl-D) was cloned using PCR method . Sequence analysis showed that the gene was the same as the published one except that codon CAT at position 416 was replaced by AAA and resulted in a Lys residue instead of His . This gene (mtl-D) was inserted in a binary vector and transformed into populas via agrobacteria . Several transgenic plants grow very well in 0.6% NaCl while controls can not survive even in 0.4% NaCl . PCR analysis and Northern blotting indicated that foreign gene was integrated into the genome of transgenic plant and transcribed successfully.

Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 173 - 8
{Molecular identification of intergeneric somatic hybrid plants between alfalfa and sainfoin}; Xu ZQ et al.; Somatic hybrid plants between alfalfa and sainfoin were regenerated by protoplast fusion and culture . DNA samples of the hybrid plants, hydroxyproline-resistant sainfoin plants, alfalfa cell line transformed with Agrobacterium tumefaciens 702 were isolated with a new and simple method . The hybridity was identified by random amplified polymorphic DNAs and Southern hybridization . Significant differences can be seen in the sequences amplified, which are specific for each parent/primer combination under the amplification conditions used . In 20 random oligonucleotide primers used, six could amplified more DNA fragments and had better polymorphisms . The results suggested that besides containing nuclear substances of two parents, the hybrid genome was inclined to eliminate sainfoin chromosome with DNA reconstruction . However, the somatic genome also could produce the sainfoin-specified DNA fragments which further confirmed by Southern hybridization . The hybrids were asymmetric and had certain regeneration ability just because the intervention of sainfoin DNA.

Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 142 - 6
{Studies on transgenic oilseed rape (Brassica napus) plants transformed with beta-1,3-glucanase and chitinase genes and its resistance to Sclerotinia sclerotiorium}; Lan HY et al.; By Agrobacterium-mediated method, the cotyledonary petiole of good quality rape variety H165 was transformed with plant expression vector pBLGC which constitutively express beta-1,3-glucanase and chitinase genes . We obtained some Kanamycin(Kan)-resistant regenerational green shoots, with these shoots, PCR identification was conducted . Results showed that 30% green shoots which grew in medium of Kan 15 mg/L and 53% green shoots in Kan 25 mg/L had positive reaction . We also made dot blot analysis with those green shoots, some of them gave positive signal, indicating that the foreign genes had integrated into rape genome . Fungal challenge of these transgenic plants showed that some plants were much more resistant to Sclerotinia sclerotiorium than non-transgenic control plants.

Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 137 - 41
{Introduction of wide spectrum rice bacterial blight resistance gene Xa21 into two-line genic male sterile rice variety Pei'ai 64S}; Zhao B et al.; Agrobacterium-mediated transformation of two-line genic male sterile Indica rice variety Pei'ai 64S was conducted using a cloned gene, Xa21, as the foreign gene and mature embryo calli as the recipients . A total of 46 transgenic plants had been obtained . The PCR analysis and Southern blotting showed the integration of Xa21 gene into the genome the transgenic plants . Results of inoculation with philippine race 6 of Xanthomonas oryzae pv . oryzae indicated that most of transgenic plants obtained high resistance to rice bacterial blight disease (Xoo) . Analyses of T1 plants of the tested transgenic lines showed that integrated Xa21 gene could be steadily inherited and segregated in a 3:1 ratio.

Sheng Wu Gong Cheng Xue Bao, 2000 Mar, 16(2), 129 - 33
{Insect-resistant transgenic poplar expressing AaIT gene}; Wu NF et al.; Insect-specific scorpion neurotoxin AaIT gene inserted into a binary vector was transferred into a hybrid poplar clone N-106(P . deltoides x P . simonii) growing in the Southern of China . We obtained sixty-two regenerated plants by Agrobacterium tumefaciens transferring system . PCR and PCR-Southern analysis showed that AaIT gene was incorporated into the genome of some recovered poplar plants . One of the transformed plants named A5 was significantly resistant to feeding by first instar larvae of Lymantria dispar, compared with the untransformed control plant . It caused a decrease in leaf consumption by larvae, a lower larval weight gain and a higher larval motality rate of Lymantria dispar . ELISA analysis proved that AaIT gene was expressed in this transfomed poplar plant.

Mol Plant Microbe Interact, 2000 Sep, 13(9), 1019 - 21
Rhizobium etli CE3 carries vir gene homologs on a self-transmissible plasmid; Bittinger MA et al.; RosR is a transcriptional regulator important for determining cell-surface characteristics and nodulation competitiveness in Rhizobium etli CE3 . We identified a 15-kb region that contains genes with similarity to members of the virB, virC, virG, and virE operons of Agrobacterium tumefaciens and demonstrated that RosR directly regulates one operon in this region . These genes were located on plasmid pa of R . etli CE3, which is self-transmissible between R . etli and A . tumefaciens.

Mol Plant Microbe Interact, 2000 Sep, 13(9), 911 - 21
Resistance of tomato and pepper to T3 strains of Xanthomonas campestris pv . vesicatoria is specified by a plant-inducible avirulence gene; Astua-Monge G et al.; Tomato race 3 (T3) of Xanthomonas campestris pv . vesicatoria (Xcv) elicits a hypersensitive response (HR) in leaves of Lycopersicon esculentum near-isogenic line (NIL) 216 and pepper genotypes . One cosmid clone (35 kb) selected from a genomic library of a T3 strain induced an HR in all resistant plants . A 1.5-kb active subclone containing the putative avirulence (avr) gene, designated avrXv3, was sequenced . The avrXv3 gene encodes a 654-bp open reading frame (ORF) with no homology to any known gene . Expression studies with a fusion of this gene and uidA indicated that avrXv3 is plant inducible and controlled by the hypersensitivity and pathogenicity (hrp) regulatory system . Mutational analysis and transcription activation assays revealed that AvrXv3 has transcription activation activity in yeast, and that the putative domain responsible for that activity is located at the C terminus of the AvrXv3 protein . Agrobacterium tumefaciens-mediated transient expression confirmed the direct role of AvrXv3 in eliciting the HR in tomato NIL 216 and supported the hypothesis that Avr proteins must be present inside the plant host cell to trigger the HR.

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