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-treate