Mol Gen Mikrobiol Virusol, 2000, (3), 26 - 31 {Formation of extracellular structures in conjugated cultures of agrobacteria}; Kurbanova IV et al.; Electron microscopy of noncentrifugated agrobacterial cells on a nitrocellulose membrane labeled with colloid gold-conjugated antibodies to VirB1 showed that the labeled complex bound to acetosyringone (AS)-induced cells but failed to form red-colored stains during incubation with Ti aplasmid cells . Supramembrane structures of AS-treated A . tumefaciens cells were for the first time visualized by transmission electron microscopy . Colloid gold labeling of VirB2-specific antibodies showed that VirB2 proteins produce long thin pilus structures emerging at the poles of AS-induced agrobacterial cells but never on the surface untreated with AS and Ti-plasmid-free agrobacterial cells . As a rule, one (or rarely two) thread-like connections and bridges were observed between the cells at the primary contact stage . The bridges were not destroyed by SDS, did not react with VirB2-specific antibodies, and remained visible at 30 degrees C . Visible close contacts between mating bacteria did not cease after SDS treatment . SDS pretreatment of donor cells or a mating cell suspension significantly modified the efficiency of pTd33 plasmid transfer from donor to recipient agrobacterial cells . In the presence of AS the optimal temperature for transfer was 25 degrees C . The frequency of plasmid pTd33 transfer from A . tumefaciens via vir-dependent pathway decreased 2-4-fold due to increase of temperature from 19.25 to 31 degrees C. Plant J, 2000 Sep, 23(5), 687 - 95 Development of an efficient maintenance and screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector; Liu YG et al.; Three large-insert genomic DNA libraries of common wheat, Triticum aestivum cv . Chinese Spring, were constructed in a newly developed transformation-competent artificial chromosome (TAC) vector, pYLTAC17, which accepts and maintains large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens . The vector contains the cis sequence required for Agrobacterium-mediated gene transfer into grasses . The average insert sizes of the three genomic libraries were approximately 46, 65 and 120 kbp, covering three haploid genome equivalents . Genomic libraries were stored as frozen cultures in a 96-well format, each well containing approximately 300-600 colonies (12 plates for small library, four for medium-size library and four for large library) . In each of the libraries, approximately 80% of the colonies harbored genomic DNA inserts of >50 kbp . TAC clones containing gene(s) of interest were identified by the pooled PCR technique . Once the target TAC clones were isolated, they could be immediately transferred into grass genomes with the Agrobacterium system . Five clones containing the thionin type I genes (single copy per genome), corresponding to each of the three genomes (A, B and D), were successfully selected by the pooled PCR method, in addition to an STS marker (aWG464; single copy per genome) and CAB (a multigene family) . TAC libraries constructed as described here can be used to isolate genomic clones containing target genes, and to carry out genome walking for positional cloning. Chromosoma, 2000 Jul, 109(4), 287 - 97 Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event; Vergunst AC et al.; Cre recombinase was used to mediate recombination between a chromosomally introduced loxP sequence in Arabidopsis thaliana (35S-lox-cre) and transferred DNA (T-DNA) originating from Agrobacterium tumefaciens (plox-npt), carrying a single loxP sequence . Constructs were designed for specific Cre-mediated recombination between the two lox sites, resulting in restoration of neomycin phosphotransferase (nptII) expression at the target locus . Kanamycin resistant (Km(r)) recombinants were obtained with an efficiency of about 1% compared with random integration . Molecular analyses confirmed that these were indeed due to recombination between the lox sites of the target and introduced T-DNA . However, polymerase chain reaction analysis revealed that these reflected site-specific integration events only in a minority (4%) . The other events were classified as translocations/inversions (71%) or deletions (25%), and were probably caused by site-specific recombination between a randomly integrated T-DNA and the original target locus . We studied some of these events in detail, including a Cre-mediated balanced translocation event, which was characterized by a combination of molecular, genetic and cytogenetic experiments (fluorescence in situ hybridization to spread pollen mother cells at meiotic prophase I) . Our data clearly demonstrate that Agrobacterium-mediated transfer of a targeting T-DNA with a single lox site allows the isolation of multiple chromosomal rearrangements, including translocation and deletion events . Given that the complete sequence of the Arabidopsis genome will have been determined shortly this method has significant potential for applications in functional genomics. Plant Cell Physiol, 2000 Jul, 41(7), 831 - 9 Jasmonate induction of putrescine N-methyltransferase genes in the root of Nicotiana sylvestris; Shoji T et al.; Nicotine alkaloids are synthesized in the root of Nicotiana species, and their synthesis increases after insect attack, wounding and jasmonate treatment of the leaf . Putrescine N-methyltransferase (PMT) catalyzes the first committed step in nicotine biosynthesis . The expression patterns of the three Nicotiana sylvestris PMT genes (NsPMT1, NsPMT2, and NsPMT3) are reported in this study . Transcripts of the NsPMT genes were detected only in the root, and were up-regulated by methyl jasmonate treatment . When the 5'-flanking regions of NsPMT1, NsPMT2, and NsPMT3 were fused independently to beta-glucuronidase reporter gene and introduced into N . sylvestris by Agrobacterium-mediated transformation, all introduced transgenes were expressed in the cortex, endodermis, and xylem in the root, as well as upregulated by methyl jasmonate treatment . These qualitatively similar patterns of expression for the NsPMT genes are achieved with only 0.25 kb of their conserved 5'-flanking regions, which contained no known jasmonate-responsive elements. Res Microbiol, 2000 Jul-Aug, 151(6), 487 - 91 Genetic competence in Helicobacter pylori: mechanisms and biological implications; Hofreuter D et al.; Helicobacter pylori is naturally competent for genetic transformation . The comB locus, consisting of the open reading frames orf2, comB1, comB2, and comB3, is involved in natural transformation competence . Homologies of the ComB proteins with components of the type IV secretion apparatus (VirB9 and VirB10) from the Ti plasmid of Agrobacterium tumefaciens, as well as proteins involved in conjugation of plasmids RP1 and RP4, suggest a similar organization of DNA import (transformation) in H . pylori with well-known DNA export systems. Mol Gen Genet, 2000 Jul, 263(6), 939 - 47 Genetic analysis of the signal-sensing region of the histidine protein kinase VirA of Agrobacterium tumefaciens; Toyoda-Yamamoto A et al.; The membrane-bound sensor protein kinase VirA of Agrobacterium tumefaciens detects plant phenolic substances, which induce expression of vir genes that are essential for the formation of the crown gall tumor . VirA also responds to specific monosaccharides, which enhance vir expression . These sugars are sensed by the periplasmic domain of VirA that includes the region homologous to the chemoreceptor Trg, and the phenolics are thought to be detected by a part of the cytoplasmic linker domain, while the second transmembrane domain (TM2) is reported to be nonessential . To define regions of VirA that are essential for signal sensing, we introduced base-substitution and deletion mutations into coding regions that are conserved among the respective domains of VirA proteins from various Agrobacterium strains, and examined the effects of these mutations on vir induction and tumorigenicity . The results show that the Trg-homologous region in the periplasmic domain is not essential for the enhancement of vir gene expression by sugars . Most mutations in the TM2 domain also failed to influence enhancement by sugars and reduced the level of vir induction, but a mutation in the TM2 region adjacent to the cytoplasmic linker abolished induction of the vir genes . In the linker domain, sites essential for vir induction by phenolics were scattered over the entire region . We propose that a topological feature formed by the linker domain and at least part of the TM2 may be crucial for activation of a membrane-anchored VirA protein . Complementation analysis with two different VirA mutants suggested that intermolecular phosphorylation between VirA molecules occurs in vivo, and that two intact periplasmic regions in a VirA dimer are required for the enhancement of vir induction by sugars. Transgenic Res, 2000 Apr, 9(2), 103 - 13 Transgenic Trifolium repens with foliage accumulating the high sulphur protein, sunflower seed albumin; Christiansen P et al.; With the aim of increasing the rumen-protected level of the sulphur amino acids cysteine and methionine in Trifolium repens, we introduced the coding sequence of the sunflower seed albumin (SSA) into T . repens by Agrobacterium tumefaciens-mediated transformation . The SSA gene was modified such that the protein would be localised to the endoplasmic reticulum (ER) . Four different T-DNA constructions all containing the SSA gene driven by either the promoter of a gene encoding the small subunit of ribulose bisphosphate carboxylase (Rubisco) from Arabidopsis thaliana (Assu), the promoter of the gene encoding the small subunit of Rubisco of Medicago sativa (Lssu), or the Cauliflower Mosaic Virus 35S promoter (CaMV35S), were transferred to T . repens cv . Haifa . Transgenic T0-plants and inter-transgenic hybrids were analysed for the level of SSA accumulation in the leaves by western blotting . The highest observed level of SSA accumulation was 0.1% of total extractable leaf protein . We observed that the promoter had a substantive effect on the level of SSA accumulation with Assu > CaMV35S > Lssu . Results from the inter-transgenic hybrids showed that the capacity to synthesise SSA was inherited . However the level of SSA accumulation in the leaves generally appears not to be additive with extra transgenic loci . During this work, we attempted to improve the efficiency of A . tumefaciens-mediated transformation of T . repens using the SAAT-method (Sonication Assisted Agrobacterium-mediated Transformation) on cotyledons of T . repens . T-DNA transfer was in general not enhanced by sonication compared to traditional A . tumefaciens-mediated transformation . Furthermore, Southern blot analyses of plants regenerated from the same cotyledon after A . tumefaciens treatment and under selection, indicated that multiple shoots were usually derived from the same transformation event . We concluded from these results that only one plant from each A . tumefaciens-treated cotyledon should be taken to avoid transgenic clones. Microbiol Res, 2000 Jul, 155(2), 113 - 21 Effect of associative bacteria on element composition of barley seedlings grown in solution culture at toxic cadmium concentrations; Belimov AA et al.; The response of barley seedlings to inoculation with associative rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10 and Flavobacterium sp . L30 was studied in hydroponic and quartz sand cultures in the presence of 50 microM CdCl2 . Cadmium caused severe inhibition in the growth and uptake of nutrient elements by the plants . Inoculation with the bacteria slightly stimulated root length and biomass of hydroponically grown Cd-treated seedlings . The bacteria increased the content of nutrients such as P, Mg, Ca, Fe, Mn and Na in roots and or shoots of the plants grown in the absence of Cd . Positive changes in the element composition caused by the bacteria were less pronounced in Cd-treated plants, whereas the total amount of nutrients taken by the inoculated plants was generally increased significantly . The content of Cd in the inoculated plants was unchanged, except increased in roots upon addition of A . lipoferum 137 . Inoculation did not affect the activity of peroxidase, alpha-mannosidase, phosphodiesterae, alpha-galactosidase, and concentration of sulfhydryl compounds used as biochemical markers of stress in plant roots . The results showed that associative bacteria were capable of decreasing partially the toxicity of Cd for the barley plants through the improvement in uptake of nutrient elements. Plant Cell, 2000 Aug, 12(8), 1319 - 29 Evidence for a role of the N terminus and leucine-rich repeat region of the Mi gene product in regulation of localized cell death; Hwang CF et al.; The tomato Mi gene confers resistance against root-knot nematodes and potato aphids . Chimeric constructs of the functional gene, Mi-1 . 2, with a homolog, Mi-1.1, were produced, and their phenotypes were examined in Agrobacterium rhizogenes-transformed roots . Exchange of the leucine-rich repeat (LRR) region of Mi-1.1 into Mi-1.2 resulted in the loss of ability to confer nematode resistance, as did substitution of a 6-amino acid sequence from the Mi-1.1 LRR into Mi-1.2 . Introduction of the Mi-1.2 LRR-encoding region into Mi-1.1 resulted in a lethal phenotype, as did substitution of the fragment encoding the N-terminal 161 amino acids of Mi-1.1 into Mi-1.2 . Transient expression of the latter two chimeric constructs in Nicotiana benthamiana leaves produced localized cell death . The cell death caused by the N-terminal exchange was suppressed by coinfiltration with a construct expressing the N-terminal 161 amino acids of Mi-1.2 . The phenotypes of these and other constructs indicate that the LRR region of Mi-1.2 has a role in signaling localized cell death and that the N-terminal 161 amino acids have a role in regulating this death. J Exp Bot, 2000 Jun, 51(347), 1167 - 9 Sequence analysis of the vir-region from Agrobacterium tumefaciens octopine Ti plasmid pTi15955; Schrammeijer B et al.; The nucleotide sequence of 42 775 bp of the vir-region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955 is reported here . Although the nucleotide sequences of several parts of this region from this or closely related plasmids have been published previously, the present work establishes for the first time the complete arrangement of all the essential virulence genes and their intergenic regions of an octopine Ti plasmid . The disruption of some of the intergenic areas by insertion (IS) elements is typical for the octopine Ti plasmids . Several new ORFs were identified, including ORFs immediately downstream of virD4 and virE2, which probably represent new genes involved in virulence. J Exp Bot, 2000 Jun, 51(347), 1005 - 16 Agrobacterium rhizogenes-mediated transformation of opium poppy, Papaver somniferum l., and California poppy, Eschscholzia californica cham., root cultures; Park SU et al.; An efficient protocol for the establishment of transgenic opium poppy (Papaver somniferum L.) and California poppy (Eschscholzia californica Cham.) root cultures using A . grobacterium rhizogenes is reported . Five strains of A . rhizogenes were tested for their ability to produce hairy roots on wounded opium poppy seedlings and California poppy embryogenic calli . Three of the strains induced hairy root formation on both species, whereas two others either caused the growth of tumorigenic calli or produced no response . To characterize the putative transgenic roots further, explant tissues were co-cultivated with the most effective A: rhizogenes strain (R1000) carrying the pBI121 binary vector . Except for the co-cultivation medium, all formulations included 50 mg l(-1) paromomycin to select for transformants and 200 mg l(-1) timentin to eliminate the Agrobacterium . Four weeks after infection, paromomycin-resistant roots appeared on 92-98% of explants maintained on hormone-free medium . Isolated hairy roots were propagated in liquid medium containing 1.0 mg l(-1) indole-3-acetic acid to promote rapid growth . Detection of the neomycin phosphotransferase gene, high levels of beta-glucuronidase (GUS) transcripts and enzyme activity, and GUS histochemical localization confirmed the integrative transformation of root cultures . Transgenic roots grew faster than wild-type roots, and California poppy roots grew more rapidly than those of opium poppy . With the exception of a less compact arrangement of epidermal cells and more root hairs, transformed roots of both species displayed anatomical features and benzylisoquinoline alkaloid profiles that were virtually identical to those of wild-type roots . Transgenic root cultures of opium poppy and California poppy are a simple, reliable and well-defined model system to investigate the molecular and metabolic regulation of benzylisoquinoline alkaloid biosynthesis, and to evaluate the genetic engineering potential of these important medicinal plants. J Food Prot, 2000 Aug, 63(8), 1123 - 32 Classification of a bacterial isolate, from pozol, exhibiting antimicrobial activity against several gram-positive and gram-negative bacteria, yeasts, and molds; Ray P et al.; A bacterial isolate, designated CS93, capable of producing a broad-spectrum antimicrobial compound(s) effective against gram-positive and gram-negative bacteria, yeasts, and molds was isolated from pozol, a fermented maize product . This strain was phenotypically similar to another pozol isolate that was previously designated as Agrobacterium azotophilium by other investigators . By using biochemical, phenotypic, and 16S rRNA sequence analysis, both pozol isolates were identified as members of the genus Bacillus, possibly a variant of Bacillus subtilis . While the antimicrobial compound(s) was initially produced only on a solid medium, parameters were identified for production in broth . The compound(s) was heat stable (121 degrees C for 15 min), exhibited activity over a wide pH range (pH 3 to pH 11), and was inactivated by pronase E . The antimicrobial compound(s) was bactericidal and bacteriolytic against Escherichia coli V517, bacteriostatic against Micrococcus luteus, and fungistatic against Saccharomyces cerevisiae . The inhibitory compound(s) could possibly serve as a food biopreservative. Plant Cell Physiol, 2000 Jun, 41(6), 811 - 6 A rapid and efficient system of Agrobacterium infection-mediated transient gene expression in rice Oc cells and its application for analysis of the expression and antisense suppression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene; Yang H et al.; A rapid and efficient system for Agrobacterium infection-mediated transient gene expression in rice has been developed . Using this system, transient expression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene was analyzed . The results suggest that the Agrobacterium infection-mediated transient gene expression system is as efficient in rice Oc cells as in tobacco BY-2 cells and might be useful for rapid analysis not only of foreign gene expression, but also of antisense gene suppression. Plant Cell Physiol, 2000 Jun, 41(6), 743 - 9 Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen; Lee HJ et al.; Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides . However, Bacillus subtilis Protox is known to be resistant to the herbicides . In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B . subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation . The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots . Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies . The expression levels of B . subtilis Protox mRNA appeared to correlate with the copy number . Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines . The transgenic plants expressing the B . subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation . The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm . In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed. Plant Cell Physiol, 2000 Jun, 41(6), 733 - 42 Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants; Nakagawa Y et al.; Two kinds of T-DNA constructs, I-RS/dAc-I-RS and Hm(R)Ds, carrying a non-autonomous transposable element of Ac of maize were introduced into rice plants by Agrobacterium-mediated gene transfer . Six transgenic rice plants identified as containing a single copy of the element were crossed with two transgenic rice plants carrying a gene for Ac transposase under the control of the cauliflower mosaic virus 35S promoter . In F2 progenies, excision of the element was detected by PCR analysis and re-integration of the element was investigated by Southern blot analysis . The frequency of the excision of the element was found to vary from 0 to 70% depending on the crossing combination . The frequency of the number of individual transposition events out of the total number of F2 plants with germinal excision was 44% in one crossing combination and 38% in the other . In the most efficient case, 10 plants with independent transposition were obtained out of the 49 F2 plants tested . Linkage analysis of the empty donor site and the transposed Ds-insertion site in F3 plants demonstrated that one of five Ds-insertion sites was not linked to the empty donor site . The transgenic rice obtained in this study can be used for functional genomics of rice. J Bacteriol, 2000 Sep, 182(17), 4849 - 55 A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication; Sieira R et al.; As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems . The B . abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs) . Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found . Gene reporter studies demonstrated that the B . abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth . A B . abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication . Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus . Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B . abortus virulence . It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B . abortus to an endoplasmic reticulum-related replication compartment. Phytochemistry, 2000 Jun, 54(5), 517 - 23 Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography; Mateus L et al.; Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A . tumefaciens strain C58C1pRTGus104 . The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis . Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry . Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages . Significant differences appeared between the alkaloid contents of the different clones . In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively . The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine . The aerial parts of H . muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids. Kokuritsu Iyakuhin Shokuhin Eisei Kenkyusho Hokoku, 1999, (117), 148 - 54 High production of ginsenosides by transformed root cultures of Panax ginseng: effect of basal medium and Agrobacterium rhizogenes strains; Shu W et al.; Successful transformation of Panax ginseng was achieved when petiole segments were infected with Agrobacterium rhizogenes ATCC 15834 and MAFF 03-01724 . Transformed roots were obtained after galls developed at infected sites . The root morphology, growth and ginsenoside productivity of roots transformed with different bacterial strains differed, and the roots from A . rhizogenes ATCC 15834 grew better and produced much more ginsenosides . Using the ATCC transformed root clone, various liquid culture media were tested to determine the optimum culture medium for ginsenoside production . The root growth was optimum in phytohormone-free Gamborg B5 liquid medium, however highest content of ginsenosides (a total of five ginsenosides 1.88% dry weight) was obtained when the roots were cultured in half-macro-salt strength Gamborg B5 liquid medium . Growth of the roots over a period of 8 weeks showed that their fresh and dry weight continued to increase . The ginsenoside Rb1 content was optimum after 5 weeks of culture . Ginsenoside Rc content began to decrease slightly after the third week of culture . Ginsenosides Rd and Rg1 contents fluctuated, while ginsenoside Re content continued to rise throughout the 8 weeks of culture . Ginsenoside production, however, did not peak within the 8 weeks of culture. Mol Cell Biol, 2000 Sep, 20(17), 6317 - 22 Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro; Ziemienowicz A et al.; Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species . In the plant cell, the transferred DNA (T-DNA) is integrated into the genome . An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process . The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro . We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system . The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts . This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase . We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction . Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target . T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP. J Biotechnol, 2000 Jul 28, 81(1), 45 - 53 Increased ability of transgenic plants expressing the bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb, and Zn; Grichko VP et al.; Transgenic tomato plants Lycopersicon esculentum (Solanaceae) cv . Heinz 902 expressing the bacterial gene 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, under the transcriptional control of either two tandem 35S cauliflower mosaic virus promoters (constitutive expression), the rolD promoter from Agrobacterium rhizogenes (root specific expression) or the pathogenesis related PRB-1b promoter from tobacco, were compared to non-transgenic tomato plants in their ability to grow in the presence of Cd, Co, Cu, Mg, Ni, Pb, or Zn and to accumulate these metals . Parameters that were examined include metal concentration and ACC deaminase activity in both plant shoots and roots; root and shoot development; and leaf chlorophyll content . In general, transgenic tomato plants expressing ACC deaminase, especially those controlled by the PRB-1b promoter, acquired a greater amount of metal within the plant tissues, and were less subject to the deleterious effects of the metals on plant growth than were non-transgenic plants. Plant Sci, 2000 Jul 28, 156(2), 235 - 244 Analysis of transgenic grapevine (Vitis rupestris) and Nicotiana benthamiana plants expressing an Arabis mosaic virus coat protein gene; Spielmann A et al.; A disarmed LBA4404 strain of Agrobacterium tumefaciens harboring a binary vector which contained chimeric genes encoding the neomycin phosphotransferase (npt II) and the coat protein (CP) of Arabis mosaic nepovirus (ArMV) was used in co-cultivation experiments with leaf discs of Nicotiana benthamiana and somatic embryos of the grapevine rootstock cultivar Vitis rupestris . Transgenic N . benthamiana expressing the ArMV CP gene were regenerated and six independent lines were characterized . Enzyme-linked immunosorbent assay (ELISA) performed on leaf tissue demonstrated the accumulation of the ArMV CP in five of the six lines analyzed . Immunosorbent electron microscopy (ISEM) studies revealed the presence of virion-like isometric particles (VLPs) reacting to a rabbit antiserum specific to ArMV virions . ArMV-CP expressing transgenic N . benthamiana lines showed protection against ArMV expressed as a delay in infection and a reduction of the percentage of infected plants . Four independent transgenic lines of V . rupestris transformed with the ArMV CP gene were regenerated and characterized . In contrast to N . benthamiana, transgenic V . rupestris did not accumulate the ArMV CP at levels detectable by ELISA and no VLPs could be observed by ISEM . Northern blot analysis showed that the ArMV CP mRNA was expressed at lower level in V . rupestris compared with N . benthamiana . The reason for this difference in transgene expression and/or mRNA stability between grapevine and N . benthamiana is unclear, but the genetic state of the transgene(s) (homozygous in N . benthamiana versus hemizygous in V . rupestris) may have an effect on gene expression. Mol Biotechnol, 1999 Dec 1, 13(2), 165 - 70 T-DNA transfer to maize plants; Shen WH et al.; Agrobacterium-mediated transformation is the method of choice to engineer desirable genes into plants . Here we describe a protocol for demonstrating T-DNA transfer from Agrobacterium into the economically important graminaceous plant maize . Expression of the T-DNA-located GUS gene was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium. Mol Biotechnol, 1999 Nov, 13(1), 67 - 72 Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment; Maenpaa P et al.; Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues . For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported . In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique. Biotechnol Prog, 2000 Jul-Aug, 16(4), 564 - 70 Production of D-amino acid using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and N-carbamoylase; Park JH et al.; We developed a fully enzymatic process employing D-hydantoinase and N-carbamoylase for the production of D-amino acid from 5'-monosubstituted hydantoin . For the comparison of the reaction systems using two sequential enzymes, D-hydantoinase of Bacillus stearothermophilus SD1 and N-carbamoyl-D-amino acid amidohydrolase (N-carbamoylase) of Agrobacterium tumefaciens NRRL B11291 were separately expressed in each host cell and coexpressed in the same host cell . A high level and constitutive expression of both enzymes in Escherichia coli in a soluble form was achieved using a promoter derived from B . stearothermophilus SD1 . The expression levels of both enzymes ranged from 17% to 23% of the total soluble protein, depending on the expression system . In the case of employing separately expressed enzymes, the product yield of D-hydroxyphenylglycine from D,L-p-hydroxyphenylhydantoin and productivity were 71% and 2.57 mM/g-cell/h in 15 h, respectively . The accumulation of N-carbamoyl-D-hydroxyphenylglycine was significant over the reaction time . On the other hand, use of coexpressed enzymes resulted in 98% product yield of D-hydroxyphenylglycine in 15 h, minimizing the level of intermediates in the reaction mixture . The productivity of coexpressed whole cell reaction was estimated to be 6.47 mM/g-cell/h in 15 h . The coexpressed system was tested for an elevated concentration of D,L-p-hydroxyphenylhydantoin, and efficient production can be achieved. Can J Microbiol, 2000 Jul, 46(7), 600 - 6 Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms; Oehrle NW et al.; Internally seedborne microorganisms are those surviving common surface sterilization procedures . Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum . Bacterial isolates from surface-sterilized soybean seed, cv . Williams 82 and cv . Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis . Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G . The effects of this antibiotic on seedling development and on B . japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here . Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development . No differences in nodulation kinetics, nitrogen fixation onset or rates were observed . However, the number of B . japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment . Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B . japonicum to the soybean root, without affecting plant development. Syst Appl Microbiol, 2000 Jun, 23(2), 238 - 44 Phylogenetic analysis of 23S rRNA gene sequences of . Agrobacterium, Rhizobium and Sinorhizobium strains; Pulawska J et al.; The phylogenetic relationship among twelve Agrobacterium, four Rhizobium, and two Sinorhizobium strains originating from various host plants and geographical regions was studied by analysis of the 23S rDNA sequences . The study included Agrobacterium strains belonging to biovars 1, 2 (with tumor- or hairy-root inducing and non-pathogenic strains), A . vitis, A . rubi; representative species of the Rhizobium genus: R . galegae, R . leguminosarum and R . tropici and Sinorhizobium meliloti strains . The phylogenetic analysis showed that within Agrobacterium, the biovar designation was reflected in the 23S rDNA similarity and that strains of Agrobacterium and Rhizobium are closely related to each other . The results suggest that the taxonomic definition of Agrobacterium and Rhizobium should be considered for revision and that the Agrobacterium-biovar identity is probably a reliable taxonomic trait. Plant J, 2000 Jul, 23(2), 279 - 84 Transgenic tobacco plants co-expressing Agrobacterium iaa and ipt genes have wild-type hormone levels but display both auxin- and cytokinin-overproducing phenotypes; Eklof S et al.; Transgenic tobacco lines simultaneously expressing the Agrobacterium iaaM, iaaH and ipt genes, obtained by crossing lines expressing ipt with lines expressing iaaM and iaaH, were used to study in planta interactions between auxin and cytokinins . All phenotypic traits of the respective parental lines characteristic of cytokinin and auxin overproduction were present in the cross . Indole-3-acetic acid (IAA) and combined zeatin riboside (ZR) and zeatin riboside-5'-monophosphate (ZRMP) contents were analysed by mass spectrometry in young, developing leaves from the cross, the parental lines and the wild type . Unexpectedly, hormone levels in the cross were very similar to wild-type levels . Thus IAA levels in the cross were much lower throughout vegetative development than in the parental IAA overproducing line, although expression of the bacterial IAA biosynthesis genes was not reduced . The results suggest that effects on apical dominance, adventitious root formation, leaf morphology and other traits commonly +/- associated with IAA and cytokinin overproduction, and observed in the iaa E ipt cross, cannot be explained solely by analysis of auxin and cytokinin contents in individual organs . As traits associated with both hormones are expressed in close spatial and temporal proximity, it is likely that cellular resolution of hormone contents is essential to explain physiological responses to auxins and cytokinins. Genetics, 2000 Aug, 155(4), 1875 - 87 The maternal chromosome set is the target of the T-DNA in the in planta transformation of Arabidopsis thaliana; Bechtold N et al.; In planta transformation methods are now commonly used to transform Arabidopsis thaliana by Agrobacterium tumefaciens . The origin of transformants obtained by these methods has been studied by inoculating different floral stages and examining gametophytic expression of an introduced beta-glucuronidase marker gene encoding GUS . We observed that transformation can still occur after treating flowers where embryo sacs have reached the stage of the third division . No GUS expression was observed in embryo sacs or pollen of plants infiltrated with an Agrobacterium strain bearing a GUS gene under the control of a gametophyte-specific promoter . To identify the genetic target we used an insertion mutant in which a gene essential for male gametophytic development has been disrupted by a T-DNA bearing a Basta resistance gene (B(R)) . In this mutant the B(R) marker is transferred to the progeny only by the female gametes . This mutant was retransformed with a hygromycin resistance marker and doubly resistant plants were selected . The study of 193 progeny of these transformants revealed 25 plants in which the two resistance markers were linked in coupling and only one plant where they were linked in repulsion . These results point to the chromosome set of the female gametophyte as the main target for the T-DNA. Appl Environ Microbiol, 2000 Aug, 66(8), 3499 - 505 Expression of a functional antizearalenone single-chain Fv antibody in transgenic Arabidopsis plants; Yuan Q et al.; The efficacy of cloning a recombinant mycotoxin antibody in plants was tested using Arabidopsis as a model . An antizearalenone single-chain Fv (scFv) DNA fragment was first cloned in the newly constructed phage display vector (pEY.5) and then recloned in the plant transformation vector pKYLX71::35S(2) . After transformation, constructs of antizearalenone scFv were introduced into immature Arabidopsis seeds via Agrobacterium tumefaciens mediation by vacuum infiltration . Only plants transformed with the construct containing a PR-1b signal peptide sequence produced transgenic offspring . The antizearalenone scFv "plantibody" from these transgenic plants bound zearalenone with a high affinity (50% inhibitory concentration, 11.2 ng/ml) that was comparable to that of bacterially produced scFv antibody and the parent monoclonal antibody (MAb) . By electron microscopic immunogold labeling, the presence of antizearalenone scFv was detected mainly in the cytoplasm and only occasionally outside the cell . Like bacterially produced scFv antibody, antizearalenone scFv plantibody exhibited greater sensitivity to methanol destabilization than did the parent MAb . The sensitivity of antizearalenone scFv plantibody to acidic disassociation was similar to the sensitivities of bacterially produced scFv antibody and MAb . Expression of specific plantibodies in crops might be useful for neutralizing mycotoxins in animal feeds and for reducing mycotoxin-associated plant diseases. Bone Marrow Transplant, 2000 Jul, 26(1), 101 - 4 Agrobacterium yellow group: bacteremia and possible septic arthritis following peripheral blood stem cell transplantation; Chalandon Y et al.; A 47-year-old male patient developed sepsis and monoarticular arthritis following autologous stem cell transplantation for recurrent Hodgkin's disease . Blood cultures were positive for Agrobacterium yellow group . The knee pain and swelling responded promptly to the institution of empirical broad-spectrum antibiotics . Recurrent bacteremia developed necessitating Hickman line removal for eventual resolution of the infection . Transplant physicians should be aware of this unusual pathogen and the potential for both persistent line-related sepsis and possible septic arthritis. Biosci Biotechnol Biochem, 2000 Jun, 64(6), 1255 - 62 Purification and characterization of a novel lactonohydrolase from Agrobacterium tumefaciens; Kataoka M et al.; A novel lactonohydrolase, catalyzing the stereospecific hydrolysis of L-pantoyl lactone to L-pantoic acid, was purified 2,400-fold to apparent homogeneity with a 1.96% overall recovery from Agrobacterium tumefaciens AKU 316 through a purification procedure including ammonium sulfate fractionation, and column chromatographies on DEAE-Sephacel, phenyl-Sepharose CL-4B, Sephacryl S-200, Mono-Q and alkyl-Superose . The relative molecular mass of the native enzyme estimated on high-pressure gel permeation chromatography was 62,000 Da, and the subunit molecular mass was estimated to 26,500 Da on SDS-polyacrylamide gel electrophoresis . The enzyme hydrolyzes several aromatic lactones, such as 3,4-dihydrocoumarin and homogentisic acid lactone, other than L-pantoyl lactone . The Km and Vmax for L-pantoyl lactone were 3.59 mM and 13.7micromol/min/mg, respectively . The enzymatic activity was inhibited by several chelating reagents, Fe2+, Sn2+, Pb2+, and Fe3+. Genetika, 2000 Jun, 36(6), 792 - 8 {Effect of a selective agent and a plant intron on the effectiveness of transformation and expression of heterologous genes in the pear (Pyrus communis L.)}; Lebedev VG et al.; The effect of selective agents on the efficiency of Agrobacterium-mediated transformation of pear was shown . The transformation frequency of the pear stock PS no . 217 by a binary vector carrying the nptII gene conferring kanamycin resistance was 0.4 or 3.1%, while the hpt gene for hygromycin resistance used as a selective marker increased transformation frequency to 6.2 (11.5%) . In addition, upon selection on hygromycin B, the proportion of pseudotransgenic regenerants considerably decreased . In four transformation experiments, twenty independent clones were recovered, and their transgenic nature was confirmed by PCR, histochemical, and fluorometric analyses of GUS activity . The presence of introns in the coding region of a heterologous gene was shown to influence the efficiency and stability of transgene expression in plant tissues . Fluorometric determination of GUS activity conducted for a period of two years demonstrated a threefold increase in transgene expression in the case that an intron-containing construct was used for transformation . The expression level was rather stable across several years . The transformation procedure developed may be used for successful expression of heterologous genes controlling agronomic characters in pear plants. Trends Microbiol, 2000 Aug, 8(8), 361 - 9 The T-pilus of Agrobacterium tumefaciens; Lai EM et al.; T-pilus biogenesis uses a conserved transmembrane nucleoprotein- and protein-transport apparatus for the transport of cyclic T-pilin subunits to the Agrobacterium cell surface . T-pilin subunits are processed from full-length VirB2 pro-pilin into a cyclized peptide, a rapid reaction that is Agrobacterium specific and can occur in the absence of Ti-plasmid genes. Trends Microbiol, 2000 Aug, 8(8), 354 - 60 Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells; Christie PJ et al.; Several bacterial pathogens utilize conjugation machines to export effector molecules during infection . Such systems are members of the type IV or 'adapted conjugation' secretion family . The prototypical type IV system is the Agrobacterium tumefaciens T-DNA transfer machine, which delivers oncogenic nucleoprotein particles to plant cells . Other pathogens, including Bordetella pertussis, Legionella pneumophila, Brucellaspp . and Helicobacter pylori, use type IV machines to export effector proteins to the extracellular milieu or the mammalian cell cytosol. J Bacteriol, 2000 Aug, 182(16), 4505 - 11 VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens; Hapfelmeier S et al.; VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants . Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors . Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively . Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels . Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly . In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished . This effect could not be restored by expression of VirB6 in trans . Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain . VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required. Planta Med, 2000 Jun, 66(5), 452 - 7 Production of amarogentin in root cultures of Swertia chirata; Keil M et al.; Conventional and Agrobacterium rhizogenes-transformed root cultures were studied with respect to growth and amarogentin content following cultivation in various growth media . The fastest growth rate was observed using Nitsch medium . The best amarogentin content was obtained after cultivation in root culture (RC) medium for which the slowest growth rate was noticed . Addition of sucrose at 6% and 9% (w/v), respectively, also resulted in better growth rates and increased total but unaltered relative amarogentin content compared to 3% (w/v) sucrose . No change in amarogentin content was observed upon addition of elicitors, putative precursors of amarogentin biosynthesis, and plant growth hormones with the exception of salicylic acid and chitosan: at 100 mM salicylic acid a reduction and at 25 mg/L chitosan an increase of amarogentin were observed at significant levels . The cultivation of S . chirata roots in a 2-L stirred-tank bioreactor was successful only with a stainless-steel mesh fitted inside the culture vessel for immobilization of the roots . A 15-fold enhancement of amarogentin content in the medium was achieved by a root permeabilisation treatment using Tween 20 at 1.3% (v/v) final concentration in the bioreactor. Planta Med, 2000 Jun, 66(5), 448 - 51 The regulation of solasodine production by Agrobacterium rhizogenes-transformed roots of Solanum aviculare; Argolo AC et al.; Transgenic roots of Solanum spp . containing extra copies of an hmgr gene derived from Artemisia annua have been obtained via transformation with Agrobacterium rhizogenes . Hairy root clones of Solanum aviculare which were transgenic for hmgr typically grew faster than those which did not contain extra copies of this gene and also accumulated up to 4.2 times more solasodine when grown under dark, but not light, conditions . The implications of these findings with respect to the regulation of solasodine production in Solanum spp . are considered. DNA Res, 2000 Jun 30, 7(3), 157 - 63 Analysis of unique variable region of a plant root inducing plasmid, pRi1724, by the construction of its physical map and library; Moriguchi K et al.; Ri plasmids in Agrobacterium rhizogenes specifically induce the hairy root syndrome on various dicotyledonous plants . Its T-DNA transfer system as well as those of Ti plasmids have successfully provided the fundamental technique to introduce exogenous genes into plants . To study the Ri genome structure, we constructed a complete BamHI physical map and a lambda library of pRi1724 of A . rhizogenes strain 1724 . By using these, we carried out the complete sequence of the 74-kb region between the right border of T-DNA and tra operon, which is the highly variable region (VAR) among Ri and Ti plasmids . As a result, we found three kinds of putative ABC-type transport operons, histidine utilization operon, glycerol utilization operon and two chemoreceptor genes . In addition, a virulence-related gene, tzs was located independently of the vir region. Structure Fold Des, 2000 Jul 15, 8(7), 729 - 37 Crystal structure of N-carbamyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases; Nakai T et al.; BACKGROUND: N-carbamyl-D-amino acid amidohydrolase (DCase) catalyzes the hydrolysis of N-carbamyl-D-amino acids to the corresponding D-amino acids, which are useful intermediates in the preparation of beta-lactam antibiotics . To understand the catalytic mechanism of N-carbamyl-D-amino acid hydrolysis, the substrate specificity and thermostability of the enzyme, we have determined the structure of DCase from Agrobacterium sp . strain KNK712 . RESULTS: The crystal structure of DCase has been determined to 1.7 A resolution . The enzyme forms a homotetramer and each monomer consists of a variant of the alpha + beta fold . The topology of the enzyme comprises a sandwich of parallel beta sheets surrounded by two layers of alpha helices, this topology has not been observed in other amidohydrolases such as the N-terminal nucleophile (Ntn) hydrolases . CONCLUSIONS: The catalytic center could be identified and consists of Glu46, Lys126 and Cys171 . Cys171 was found to be the catalytic nucleophile, and its nucleophilic character appeared to be increased through general-base activation by Glu46 . DCase shows only weak sequence similarity with a family of amidohydrolases, including beta-alanine synthase, aliphatic amidases and nitrilases, but might share highly conserved residues in a novel framework, which could provide a possible explanation for the catalytic mechanism for this family of enzymes. Mol Cells, 2000 Jun 30, 10(3), 263 - 8 Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae; Yeo ET et al.; In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1 . Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation . The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation . The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development . However, the plants recovered from these negative growth effects when grown in a soil mixture . The TPS1 transgenic potato plants showed significantly increased drought resistance . These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance. J Bacteriol, 2000 Aug, 182(15), 4137 - 45 Self-assembly of the Agrobacterium tumefaciens VirB11 traffic ATPase; Rashkova S et al.; The Agrobacterium tumefaciens VirB11 ATPase is a component of a type IV transporter dedicated to T-DNA delivery to plant cells . In this study, we tested a prediction from genetic findings that VirB11 self-associates in vivo . A chimeric protein composed of VirB11 fused to the DNA binding domain of lambda cI repressor protein formed dimers, as shown by immunity of Escherichia coli to lambda superinfection . An allele encoding VirB11 fused at its C terminus to the green fluorescent protein (GFP) exerted strong negative dominance when synthesized in wild-type A . tumefaciens cells . Dominance was suppressed by overproduction of native VirB11, suggestive of titrating or competitive interactions between VirB11 and VirB11::GFP . In support of the titration model, a complex of native VirB11 and VirB11::GFP was recovered by precipitation with anti-GFP antibodies from detergent-solubilized A . tumefaciens cell extracts . VirB11 was shown by cI repressor fusion and immunoprecipitation assays to interact with VirB11 derivatives encoded by (i) 11 dominant negative alleles, (ii) recessive alleles bearing codon substitutions or deletions in the Walker A nucleotide binding motif, and (iii) alleles corresponding to the 5' and 3' halves of virB11 . Further immunoprecipitation studies showed a hybrid protein composed of the N-terminal half of VirB11 fused to GFP interacted with mutant proteins exerting dominant effects and with a recessive Walker A deletion mutant (Delta GKT174-176) . By contrast, a hybrid protein composed of the C-terminal half fused to GFP interacted with mutants exerting dominant effects but not the Walker A mutant protein . Together, these studies establish that VirB11 assembles as homomultimers in vivo via domains residing in each half of the protein . Furthermore, ATP binding appears to be critical for C-terminal interactions required for assembly of productive homomultimers. Parasitol Res, 2000 Jun, 86(6), 444 - 52 Molecular genetic characterization and subcellular localization of Theileria annulata mitochondrial heat-shock protein 70; Schnittger L et al.; A Theileria annulata mitochondrial heat-shock protein of the 70-kDa family (Tamthsp70) was isolated by screening of the cDNA library of a T . annulata-infected bovine lymphoblastoid cell line with an antibody raised against T . annulata schizonts . The Tamthsp70 coding sequence was found to be most closely related to a previously reported mitochondrial hsp70 gene of Eimeria tenella exhibiting a similarity of 67% with mitochondrial hsp70 genes of eukaryotic plants (Pisum sativum, Phaseolus vulgaris) and with dnaK proteins of prokaryotes (Rhizobium meliloti, Agrobacterium tumefaciens) . The Tamthsp70 mRNA is expressed within the sporozoite, schizont, and merozoite stages of the parasite, which suggests that it is constitutively transcribed throughout the life cycle . The gene encodes a polypeptide of 681 amino acids and exhibits a mitochondrial targeting sequence and several sequence motifs common to mitochondrial hsp70 and prokaryotic dnaK proteins . The protein level of the Tamthsp70 protein after heat shock decreased slightly during the exposure of infected cells to a temperature of 42 degrees C in comparison with cells cultured at 37 degrees C . By immunofluorescence the protein was located in the area in which the schizonts reside within infected cells . Immunoelectron microscopy showed that the hsp70 protein was predominantly localized in the mitochondria of the parasites . However, it was also found in small amounts in the cytoplasm of the parasite and host cell . This indicates (1) that Tamthsp70 is very probably translated in the parasite cytoplasm and then transported across the mitochondrial membrane into the mitochondrial matrix and (2) that it is transported across the parasite membrane into the host-cell cytoplasm. Eur J Drug Metab Pharmacokinet, 1999 Oct-Dec, 24(4), 303 - 8 A study of the production of essential oils in chamomile hairy root cultures; Maday E et al.; The active substances in chamomile (Matricaria recutita L.) belong to chemically different structural types . The largest group of medically important compounds forming the essential oils are primarily chamazulene, (-)-alpha-bisabolol, bisabololoxides, bisabolonoxide A, trans-beta-farnesene, alpha-farnesene, spathulenol and the cis/trans-en-in-dicycloethers . Flavonoids, coumarins, mucilages, mono- and oligosaccharides also have pharmacological effects . We studied the production of essential oils in genetically transformed cultures . Sterile juvenile chamomile plants were infected with A4-Y strains of Agrobacterium rhizogenes . They are known plant pathogens and are capable of inducing so-called hairy roots . The transfer DNA segment of the Ri-virulence plasmid of A . rhizogenes becomes integrated in the genome of the plant cells . The isolated hairy roots grow rapidly on hormone-free media . In order to obtain bacteria-free media, we cultured the transformed roots on Murashige-Skoog (MS) medium supplemented with carbenicillin (800 mg/l) . To study the production of essential oils, the clones were propagated on liquid and solid MS and Gamborg (B5) media, respectively . According to gas chromatography, the composition of the essential oil of hairy root cultures on different media was found to be similar, but differing in proportion . The main component of the essential oil which was identified by gas chromatography and mass spectrometry was trans-beta-farnesene, as in the intact roots. Plant Mol Biol, 2000 Apr, 42(6), 883 - 97 Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.); Takakura Y et al.; A flower-predominant cDNA for a gene, termed OsChia 1;175, was isolated from a cDNA library of rice pistils . Northern blot and RT-PCR analyses revealed that the OsChia 1;175 gene is highly expressed in floral organs (pistils, stamens and lodicules at the heading stage) but not or at an extremely low level in vegetative organs . OsChia 1;175 encodes a protein that consists of 340 amino acid residues, and the putative mature protein shows 52% to 63% amino acid identity to class I chitinases of rice or other plants . The phylogenetic tree shows that the OsChia 1;175 protein is a new type of plant class I chitinase in rice . The expression of OsChia 1;175 in vegetative organs is not induced by several chemicals, UV, and wounding . The soluble putative mature OsChia 1;175 protein expressed in Escherichia coli exhibited chitinase activity in the assay with colloidal chitin as a substrate . Genomic Southern analysis revealed that the OsChia 1;175 gene was organized as a low-copy gene family . The rice genomic library was screened and a genome clone corresponding to OsChia 1;175 was isolated . The transcription start sites of the OsChia 1;175 gene were mapped by primer extension analysis . The 1.2 kb putative promoter region of the OsChia 1;175 gene was fused to the GUS (beta-glucuronidase) gene, and this chimeric gene was introduced to rice by Agrobacterium-mediated transformation . The flower-predominant gene expression was identified also in the transgenic rice plants . The high promoter activity was detected in the stigmas, styles, stamens and lodicules in transgenic plants . The possible functions of OsChia 1;175 are discussed. Plant Mol Biol, 2000 Apr, 42(6), 819 - 32 pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation; Hellens RP et al.; Binary Ti vectors are the plasmid vectors of choice in Agrobacterium-mediated plant transformation protocols . The pGreen series of binary Ti vectors are configured for ease-of-use and to meet the demands of a wide range of transformation procedures for many plant species . This plasmid system allows any arrangement of selectable marker and reporter gene at the right and left T-DNA borders without compromising the choice of restriction sites for cloning, since the pGreen cloning sites are based on the well-known pBluescript general vector plasmids . Its size and copy number in Escherichia coli offers increased efficiencies in routine in vitro recombination procedures . pGreen can replicate in Agrobacterium only if another plasmid, pSoup, is co-resident in the same strain . pSoup provides replication functions in trans for pGreen . The removal of RepA and Mob functions has enabled the size of pGreen to be kept to a minimum . Versions of pGreen have been used to transform several plant species with the same efficiencies as other binary Ti vectors . Information on the pGreen plasmid system is supplemented by an Internet site through which comprehensive information, protocols, order forms and lists of different pGreen marker gene permutations can be found. Plant Physiol, 2000 Jul, 123(3), 1069 - 76 Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene; Cho HJ et al.; A cDNA clone that encodes a feedback-insensitive anthranilate synthase (AS), ASA2, isolated from a 5-methyl-tryptophan (Trp) (5MT)-resistant tobacco cell line under the control of the constitutive cauliflower mosaic virus 35S promoter, was introduced into the forage legume Astragalus sinicus by Agrobacterium rhizogenes with kanamycin selection . The 35S-ASA2 gene was expressed constitutively as demonstrated by northern-blot hybridization analyses and the presence of feedback-insensitive AS . Hairy root lines transformed with 35S-ASA2 grew in concentrations of up to 100 microM 5MT, whereas the controls were completely inhibited by 15 microM 5MT . Expression of the feedback-insensitive ASA2 resulted in a 1.3- to 5.5-fold increase in free Trp . Kinetic studies of the AS activity demonstrate the Trp feedback alterations and indicate that the ASA2 alpha-subunit can interact with the native A . sinicus beta-subunit to form an active enzyme . The ASA2 transcript and high free Trp were also detected in the leaves, stems, and roots of plants regenerated from the transformed hairy roots . Thus, we show for the first time that ASA2 can be used to transform plants of a different species to increase the levels of the essential amino acid Trp and impart 5MT resistance. Plant Physiol, 2000 Jul, 123(3), 895 - 904 Female reproductive tissues are the primary target of Agrobacterium-mediated transformation by the Arabidopsis floral-dip method; Desfeux C et al.; The floral-dip method for Agrobacterium-mediated transformation of Arabidopsis allows efficient plant transformation without need for tissue culture . To facilitate use with other plant species, we investigated the mechanisms that underlie this method . In manual outcrossing experiments, application of Agrobacterium tumefaciens to pollen donor plants did not produce any transformed progeny, whereas application of Agrobacterium to pollen recipient plants yielded transformants at a rate of 0.48% . Agrobacterium strains with T-DNA carrying gusA (encoding beta-glucuronidase {GUS}) under the control of 35S, LAT52, or ACT11 promoters revealed delivery of GUS activity to developing ovules, whereas no GUS staining of pollen or pollen tubes was observed . Transformants derived from the same seed pod contained independent T-DNA integration events . In Arabidopsis flowers, the gynoecium develops as an open, vase-like structure that fuses to form closed locules roughly 3 d prior to anthesis . In correlation with this fact, we found that the timing of Agrobacterium infection was critical . Transformants were obtained and GUS staining of ovules and embryo sacs was observed only if the Agrobacterium were applied 5 d or more prior to anthesis . A 6-fold higher rate of transformation was obtained with a CRABS-CLAW mutant that maintains an open gynoecium . Our results suggest that ovules are the site of productive transformation in the floral-dip method, and further suggest that Agrobacterium must be delivered to the interior of the developing gynoecium prior to locule closure if efficient transformation is to be achieved. Yi Chuan Xue Bao, 2000, 27(2), 158 - 64 {Cloning and expression of trehalose synthase genes in Escherchia coli}; Dai XY et al.; Escherichia coli trehalose synthase otsBA genes have been cloned by using transposon Mu in vivo . The cloning frequency was 1.45 x 10(-3)/Kanr transductant . Genetic complements, endonuclase digestion and partial nucleotide sequencing analysis of these clones indicated that otsBA gene was on plasmid Mud5005 . Subcloning 2.87 kb DNA fragment onto expression plasmids with higher or lower copy number and transforming into E . coli recipient strain FF4050, which have otsBA genes deleted respectively, the transformed strains obtained ability of growth on medium containing 0.5 mol/L NaCl and increased trehalose synthesis under high osmotic pressure . Production and accumulation of trehalose may play an important role in crop breeding . These studies will lay an important foundation for constructing an expression vector with the otsBA genes and transformation to tobacco mediated by Agrobacterium, and obtaining ability for drought resistance and high osmotic pressure tolerance. Plant J, 2000 Jun, 22(6), 553 - 60 Intron-tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells; Ferrando A et al.; Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo . Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells . We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium . Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection . This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells. Plant J, 2000 Jun, 22(6), 543 - 51 In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves; Yang Y et al.; A convenient, Agrobacterium-mediated transient expression assay has been evaluated for rapid analysis of plant promoters and transcription factors in vivo . By simple infiltration of Agrobacterium cells carrying appropriate plasmid constructs into tobacco leaves in planta, reproducible expression assays could be conducted in as little as 2-3 days without using expensive equipment (e.g . biolistic gun or electroporation apparatus) or complicated procedures (e.g . preparation of protoplasts) . Biotic and abiotic treatments could be applied to the intact plant to examine their influence on promoter activity and gene expression . Using this method, we have tested the stress-responsive as-1 and heat shock elements, yeast GAL4 transactivation system, two promoters of pathogenesis-related (PR) genes as well as a heat shock promoter . Through deletion analyses of tobacco PR1a promoter and a novel myb1 promoter, we have also successfully identified the cis-regulatory regions in these promoters that are responsive to salicylic acid treatment or tobacco mosaic virus infection . Together, our results demonstrate that Agrobacterium-mediated transient expression is a simple and efficient method for in vivo assays of plant promoters and transcription factors. Plant J, 2000 Jun, 22(6), 531 - 41 Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium; Trieu AT et al.; Two rapid and simple in planta transformation methods have been developed for the model legume Medicago truncatula . The first approach is based on a method developed for transformation of Arabidopsis thaliana and involves infiltration of flowering plants with a suspension of Agrobacterium . The second method involves infiltration of young seedlings with Agrobacterium . In both cases a proportion of the progeny of the infiltrated plants is transformed . The transformation frequency ranges from 4.7 to 76% for the flower infiltration method, and from 2.9 to 27.6% for the seedling infiltration method . Both procedures resulted in a mixture of independent transformants and sibling transformants . The transformants were genetically stable, and analysis of the T2 generation indicates that the transgenes are inherited in a Mendelian fashion . These transformation systems will increase the utility of M . truncatula as a model system and enable large-scale insertional mutagenesis . T-DNA tagging and the many adaptations of this approach provide a wide range of opportunities for the analysis of the unique aspects of legumes. Br Med Bull, 2000, 56(1), 62 - 73 Genetically modified crops: methodology, benefits, regulation and public concerns; Halford NG et al.; The genetic modification of crop plants from the methodology involved in their production through to the current debate on their use in agriculture are reviewed . Techniques for plant transformation by Agrobacterium tumefaciens and particle bombardment, and for the selection of transgenic plants using marker genes are described . The benefits of currently available genetically modified (GM) crops in reducing waste and agrochemical use in agriculture, and the potential of the technology for further crop improvement in the future are discussed . The legal requirements for containment of novel GM crops and the roles of relevant regulatory bodies in ensuring that GM crops and food are safe are summarized . Some of the major concerns of the general public regarding GM crops and food: segregation of GM and non-GM crops and cross-pollination between GM crops and wild species, the use of antibiotic resistance marker genes, the prevention of new allergens being introduced in to the food chain and the relative safety of GM and non-GM foods are considered . Finally, the current debate on the use of GM crops in agriculture and the need for the government, scientists and industry to persevere with the technology in the face of widespread hostility is studied. Microbiology, 2000 Jul, 146 ( Pt 7), 1735 - 42 Osmotic regulation of cyclic 1,2-beta-glucan synthesis; de Iannino NI et al.; In contrast to what happens in AGROBACTERIUM: tumefaciens and RHIZOBIUM: meliloti, synthesis of periplasmic cyclic 1,2-beta-glucan in BRUCELLA: spp . was not inhibited when bacteria were grown in media of high osmolarity . Studies performed with crude membrane preparations showed that cyclic 1,2-beta-glucan synthetase of BRUCELLA: spp . was not inhibited by 0.5 M KCl or potassium glutamate; concentrations that completely inhibit the osmosensitive enzymes of A . tumefaciens A348 or R . meliloti 102F34, respectively encoded by the chvB or ndvB genes . The BRUCELLA: abortus cyclic 1, 2-beta-glucan synthetase gene (cgs) was introduced into A . tumefaciens A1011 chvB and R . meliloti GRT21s ndvB mutants . Synthesis of cyclic 1,2-beta-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (0.25 M NaCl or 0.5 M mannitol) . On the other hand, when the A . tumefaciens cyclic 1, 2-beta-glucan synthetase gene was introduced into the R . meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-beta-glucan synthesis when grown in high-osmolarity media . However, the same gene introduced into a B . abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity . In vitro studies with crude membranes isolated from recombinant strains revealed that BRUCELLA: cyclic 1, 2-beta-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in AGROBACTERIUM: or RHIZOBIUM: backgrounds . It was concluded that the lack of effect of high osmolarity on 1,2-beta-glucan synthesis in BRUCELLA: is due to two convergent mechanisms: a) the presence of a cyclic 1, 2-beta-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1, 2-beta-glucan synthetase. Appl Environ Microbiol, 2000 Jul, 66(7), 2882 - 7 Isolation and characterization of 2,3-dichloro-1-propanol-degrading rhizobia; Effendi AJ et al.; 2,3-Dichloro-1-propanol is more chemically stable than its isomer, 1, 3-dichloro-2-propanol, and is therefore more difficult to degrade . The isolation of bacteria capable of complete mineralization of 2, 3-dichloro-1-propanol was successful only from enrichments at high pH . The bacteria thus isolated were found to be members of the alpha division of the Proteobacteria in the Rhizobium subdivision, most likely Agrobacterium sp . They could utilize both dihaloalcohol substrates and 2-chloropropionic acid . The growth of these strains in the presence of 2,3-dichloro-1-propanol was strongly affected by the pH and buffer strength of the medium . Under certain conditions, a ladder of four active dehalogenase bands could be visualized from this strain in activity gels . The enzyme involved in the complete mineralization of 2,3-dichloro-1-propanol was shown to have a native molecular weight of 114,000 and consisted of four subunits of similar molecular weights. Yi Chuan Xue Bao, 1999, 26(6), 711 - 4 {Introduction of rabbit defensin NP-1 gene into poplar (P . tomentosa) by Agrobacterium-mediated transformation}; Zhao SM et al.; Rabbit defensin NP-1 possesses a broad resistant spectrum to pathogens . In this work, we have transferred the NP-1 gene into poplar plants by Agrobacterium-mediated transformation . PCR amplification and Southern analysis showed that rabbit defensin NP-1 gene was integrated into the poplar genome . The transformation efficiency is about 15.6% . Antimicrobial activity test showed that the extract of transgenic plants inhibited the growth of the tested microbes. DNA Cell Biol, 2000 Jun, 19(6), 377 - 82 The gene encoding the 17-kDa antigen of Bartonella henselae is located within a cluster of genes homologous to the virB virulence operon; Padmalayam I et al.; A Bartonella henselae genomic A library was screened with antiserum generated in mice against live B . henselae . One of the immunoreactive clones expressed a 17-kDa antigen that was characterized previously as an immunodominant protein of B . henselae . Sequence analysis of the recombinant clone, pBHIM-2, revealed that the open reading frame (ORF) encoding the 17-kDa antigen was situated between homologs of virB4 and virB6, two genes that belong to the virB operon . The virB operon has been associated with the transfer of oncogenic T-DNA in Agrobacterium tumefaciens and with secretion of the pertussis toxin in Bordetella pertussis . Downstream of the virB6 gene within pBHIM-2 was a partial open reading frame that was homologous to the virB8 gene . Rescreening of the library by plaque hybridization using probes specific to the 5' and 3' ends of the pBHIM-2 insert resulted in the isolation of recombinant clones containing additional virB genes . Assembly of the sequences obtained from the recombinant clones revealed that eight of the open reading frames encode homologs of the VirB proteins . The homology and colinearity with the virB genes suggest that the gene encoding the 17-kDa antigen is expressed within the virB locus of B . henselae. Mol Plant Microbe Interact, 2000 Jul, 13(7), 787 - 90 The ORF8 gene product of Agrobacterium rhizogenes TL-DNA has tryptophan 2-monooxygenase activity; Lemcke K et al.; The open reading frame 8 (ORF8) is located on the TL-DNA of the phytopathogenic soil bacterium Agrobacterium rhizogenes strain A4 . The predicted ORF8 protein has a particular structure and is possibly a natural fusion protein . The N-terminal domain shows homology to the A . rhizogenes rolB protein and may modulate the auxin responsiveness of host cells . The C terminus has up to 38% homology to tryptophan 2-monooxygenases (t2m) . We show that ORF8 overexpressing plants contain a fivefold higher concentration of indole-3-acetamide (IAM) than untransformed plants . Protein extracts from seedlings and Escherichia coli overexpressing ORF8 show significantly higher turnover rates of tryptophan to IAM than negative controls . We conclude that the ORF8 gene product has tryptophan 2-monooxygenase activity. Mol Plant Microbe Interact, 2000 Jul, 13(7), 715 - 23 Nodule-expressed Cyp15a cysteine protease genes map to syntenic genome regions in Pisum and Medicago spp; Vincent JL et al.; PsCyp15a is a gene that encodes a vacuolar cysteine protease expressed in wilt-induced shoots of Pisum sativum (pea) and in root nodules . To further the understanding of nodular PsCyp15a expression, a region 5' to the coding sequence of the gene was cloned . Varying lengths of 5' untranslated sequence were fused with the uidA coding region and introduced from Agrobacterium rhizogenes into "hairy roots" of Vicia hirsuta . In this transgenic root nodulation assay, a promoter sequence of 900 bp was sufficient to give an expression pattern indistinguishable from that obtained in pea nodules by in situ hybridization . An orthologue of PsCyp15a was cloned from nodule mRNA of Medicago sativa and a corresponding gene identified in M . truncatula was also shown to express strongly in nodules . With molecular mapping techniques, it was demonstrated that these genes map to a syntenic genome location in pea and Medicago spp., but the map positions of the Cyp15a genes cannot be correlated with existing nodulation mutants. Plasmid, 2000 Jul, 44(1), 1 - 11 pT3.2I, the smallest plasmid of Thiobacillus T3.2; Aparicio T et al.; The whole nucleotide sequence of pT3.2I, the smallest plasmid of the acidophilic bacterium Thiobacillus T3.2, has been determined . pT3.2I is 15,390 bp long with a 53.7% GC content . Different regions can be defined in it: one 2569-bp putative insertion sequence similar to other insertion sequences of some Agrobacterium Ti plasmids; and a longer sequence, which occurs in two almost identical copies, differing only in a 1-bp deletion (6406 and 6405 bp) . Several open reading frames and some smaller sequences were found in this duplicated region: ORFA and ORFG, encoding a putative polyol dehydrogenase and a putative RepA replication protein, respectively, an 83-bp sequence which could code for an antisense RNA, and a 36-bp region highly homologous to ori sequences of ColE2- and ColE3-related plasmids . Another putative gene, ORFH, is only present in the longer copy of this region (it is deleted in the short copy) and might encode a 90-amino-acid polypeptide which could act as a second replication protein, RepB . Based on sequence comparisons, pT3 . 2I can be related to plasmids in the pColE2-CA42 incB incompatibility group . Planta, 2000 May, 210(6), 1018 - 22 Expansion of transgenic tobacco protoplasts expressing pumpkin ascorbate oxidase is more rapid than that of wild-type protoplasts; Kato N et al.; When pumpkin (Cucurbita spp., cv . Ebisu Nankin) ascorbate oxidase cDNA was introduced into cultured cells of tobacco BY-2 (Nicotiana tabacum L . cv . Bright Yellow No . 2) by Agrobacterium-mediated transformation, the transgenic cells expressed and secreted the recombinant pumpkin ascorbate oxidase into the culture medium . These transgenic cells showed no morphological difference from wild-type cells . However, in the presence of applied hormones protoplasts prepared from the transgenic cells elongated more rapidly than those of wild-type cells . We propose that ascorbate oxidase may play a key role in the regulation of cell expansion perhaps by controlling transport processes through the plasma membrane, but not by affecting the cell wall. Biotechniques . 2000 Jun;28(6):1128 1130, 1132, 1134 passim. Determination of transgene repeat formation and promoter methylation in transgenic plants; Kumar S et al.; The integration of transgenes into a plant host genome following Agrobacterium tumefaciens-mediated or direct transformation may occur as a single copy or in the form of tandem repeats . The latter has been associated with promoter methylation and silencing of transgenes . Thus, the early screening of such transgenic plants is desirable for ruling out future repeat-dependent transgene instability . We developed a simple PCR-based method in which primer pairs were specifically designed so that amplifications could only be obtained if the transgene was present in the form of multiple inserts in a transgenic line . The method was established using 35S-rolC transgenic aspen lines showing morphologically visible transgenic silencing . Later, it was possible to screen independent transgenic lines showing no visible marker gene expression . Furthermore, a method was developed in which positive PCR amplification was indicative of promoter methylation . The results were consistent and reproducible across different independent transgenic lines . The methods were quick, reliable, consistent and reproducible, and can be useful for routine screening of transgene silencing in lines derived from many different systems. Plant Physiol, 2000 Jun, 123(2), 531 - 42 Cambial-region-specific expression of the Agrobacterium iaa genes in transgenic aspen visualized by a linked uidA reporter gene; Tuominen H et al.; The level of indole-3-acetic acid (IAA) was locally modified in cambial tissues of transgenic aspen (Populus tremula L . x Populus tremuloides Michx.) . We also demonstrate the use of a linked reporter gene to visualize the expression of the iaa genes . The rate-limiting bacterial IAA-biosynthetic gene iaaM and the reporter gene for beta-glucuronidase (GUS), uidA, were each fused to the cambial-region-specific Agrobacterium rhizogenes rolC promoter and linked on the same T-DNA . In situ hybridization of the iaaM gene confirmed that histochemical analysis of GUS activity could be used to predict iaaM gene expression . Moreover, quantitative fluorometric analysis of GUS activity allowed estimation of the level of de novo production of IAA in transgenic lines carrying a single-copy insert of the iaaM, uidA T-DNA . Microscale analysis of the IAA concentration across the cambial region tissues showed an increase in IAA concentration of about 35% to 40% in the two transgenic lines, but no changes in the radial distribution pattern of IAA compared with wild-type plants . This increase did not result in any changes in the developmental pattern of cambial derivatives or the cambial growth rate, which emphasizes the importance of the radial distribution pattern of IAA in controlling the development of secondary xylem, and suggests that a moderate increase in IAA concentration does not necessarily stimulate growth. FEMS Microbiol Lett, 2000 Jun 15, 187(2), 175 - 8 A single amino acid substitution beyond the C2H2-zinc finger in Ros derepresses virulence and T-DNA genes in Agrobacterium tumefaciens; Archdeacon J et al.; Ros is a chromosomally-encoded repressor containing a novel C2H2 zinc finger in Agrobacterium tumefaciens . Ros regulates the expression of six virulence genes and an oncogene on the Ti plasmid . Constitutive expression of these genes occurs in the spontaneous mutant 4011R derived from the octopine strain Ach-5, resulting in T-DNA processing in the absence of induction, and in the biosynthesis of cytokinin . Interestingly, the mutation in 4011R is an Arg to Cys conversion at amino acid residue 125 near the C-terminus well outside the zinc finger of Ros . Yet, Ros bearing this mutation is unable to bind to the Ros-box and is unable to complement other ros mutants. Transgenic Res, 2000 Feb, 9(1), 71 - 8 Expression of full-length bioactive antimicrobial human lactoferrin in potato plants; Chong DK et al.; A cDNA fragment encoding human lactoferrin (hLF) linked to a plant microsomal retention signal peptide (SEK-DEL) was stably integrated into the Solanum tuberosum genome by Agrobacterium tumefaciens-mediated leaf disk transformation methods . The lactoferrin gene was expressed under control of both the auxin-inducible manopine synthase (mas) P2 promoter and the cauliflower mosaic virus (CaMV) 35S tandem promoter . The presence of the hLF cDNA in the genome of regenerated transformed potato plants was detected by polymerase chain reaction amplification methods . Full-length hLF protein was identified by immunoblot analysis in tuber tissue extracts from the transformed plants by immunoblot analysis . The hLF produced in transgenic plant tissues migrated during polyacrylamide gel electrophoresis as a single band with an approximate molecular mass equal to hLF . Auxin activation of the mas P2 promoter increased lactoferrin expression levels in transformed tuber and leaf tissues to approximately 0.1% of total soluble plant protein . Antimicrobial activity against four different human pathogenic bacterial strains was detected in extracts of lactoferrin-containing potato tuber tissues . This is the first report of synthesis of full length, biologically active hLF in edible plants. Transgenic Res, 2000 Feb, 9(1), 11 - 9 Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns; Fu X et al.; Whole plasmids are used in both Agrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s) . This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements . We, therefore, bombarded rice tissue with two constructs: a plasmid containing the bar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimal bar gene expression cassette (promoter, open reading frame and terminator) . We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed . Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements . Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements . We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs . We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linear hpt and gusA cassettes . We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers . Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors. Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7545 - 50 Transferred DNA (T-DNA)-associated proteins of Agrobacterium tumefaciens are exported independently of virB; Chen L et al.; The transfer of T-DNA from Agrobacterium to plant cells is mediated by a system which involves the virB operon of the Ti plasmid . We report that VirE2 and VirD2, two T-DNA-associated proteins, as well as VirF, a protein known to be secreted into plant cells, are present in the periplasm and supernatant fractions of growing cells of Agrobacterium as are VirJ and ChvE, two known periplasmic proteins . Two cytoplasmic proteins, Ros and chloramphenicol acetyl transferase, and a VirE2green fluorescent protein construct were not detected in the above fraction . Export of VirE2 into the culture supernatant did not require any Ti plasmid genes, except for VirE1, a specific chaperone for VirE2 . The levels of the VirE2 and VirD2 proteins in the supernatant increased significantly when cells were grown at 19 degrees C as compared with 28 degrees C . When Agrobacterium expressed the oncogenic suppressive activity protein (Osa), VirE2 and VirF proteins could not be detected in the supernatant or the periplasm and the level of VirD2 was greatly reduced . However, oncogenic suppressive activity protein did not block the accumulation of VirJ and ChvE in the periplasm . Our data suggest that VirD2, VirE2, and VirF are transported across the cytoplasmic membrane by a specific pathway, independent of virB . Thus, transfer of the T-complex of Agrobacterium may take place in two steps, the first mediated by an unidentified pathway and the second by the virB system. J Bacteriol, 2000 Jun, 182(12), 3437 - 45 The N- and C-terminal portions of the Agrobacterium VirB1 protein independently enhance tumorigenesis; Llosa M et al.; Genetic transformation of plants by Agrobacterium tumefaciens is mediated by a virulence (vir)-specific type IV secretion apparatus assembled from 11 VirB proteins and VirD4 . VirB1, targeted to the periplasm by an N-terminal signal peptide, is processed to yield VirB1*, comprising the C-terminal 73 amino acids . The N-terminal segment, which shares homology with chicken egg white lysozyme as well as lytic transglycosylases, may provide local lysis of the peptidoglycan cell wall to create channels for transporter assembly . Synthesis of VirB1* followed by its secretion to the exterior of the cell suggests that VirB1* may also have a role in virulence . In the present study, we provide evidence for the dual roles of VirB1 in tumorigenesis as well as the requirements for processing and secretion of VirB1* . Complementation of a virB1 deletion strain with constructs expressing either the N-terminal lysozyme-homologous region or VirB1* results in tumors intermediate in size between those induced by a wild-type strain and a virB1 deletion strain, suggesting that each domain has a unique role in tumorigenesis . The secretion of VirB1* translationally fused to the signal peptide indicates that processing and secretion are not coupled . When expressed independently of all other vir genes, VirB1 was processed and VirB1* was secreted . When restricted to the cytoplasm by deletion of the signal peptide, VirB1 was neither processed nor secreted and did not restore virulence to the virB1 deletion strain . Thus, factors that mediate processing of VirB1 and secretion of VirB1* are localized in the periplasm or outer membrane and are not subject to vir regulation. J Bacteriol, 2000 Jul, 182(13), 3705 - 16 Genetic and environmental factors affecting T-pilin export and T-pilus biogenesis in relation to flagellation of Agrobacterium tumefaciens; Lai EM et al.; The T pilus, primarily composed of cyclic T-pilin subunits, is essential for the transmission of the Ti-plasmid T-DNA from Agrobacterium tumefaciens to plant cells . Although the virB2 gene of the 11-gene virB operon was previously demonstrated to encode the full-length propilin, and other genes of this operon have been implicated as members of a conserved transmembrane transport apparatus, the role of each virB gene in T-pilin synthesis and transport and T-pilus biogenesis remained undefined . In the present study, each virB gene was examined and was found to be unessential for T-pilin biosynthesis, except virB2, but was determined to be essential for the export of the T-pilin subunits and for T-pilus formation . We also find that the genes of the virD operon are neither involved in T-pilin export nor T-pilus formation . Critical analysis of three different virD4 mutants also showed that they are not involved in T-pilus biogenesis irrespective of the A . tumefaciens strains used . With respect to the environmental effects on T-pilus biogenesis, we find that T pili are produced both on agar and in liquid culture and are produced at one end of the A . tumefaciens rod-shaped cell in a polar manner . We also report a novel phenomenon whereby flagellum production is shut down under conditions which turn on T-pilus formation . These conditions are the usual induction with acetosyringone at pH 5.5 of Ti-plasmid vir genes . A search of the vir genes involved in controlling this biphasic reaction in induced A . tumefaciens cells revealed that virA on the Ti plasmid is involved and that neither virB nor virD genes are needed for this reaction . The biphasic reaction therefore appears to be mediated through a two-component signal transducing system likely involving an unidentified vir gene in A . tumefaciens. Mol Microbiol, 2000 May, 36(3), 608 - 17 Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore; Kumar RB et al.; Agrobacterium tumefaciens transforms plants by transferring DNA to the plant cell nucleus . The VirB membrane proteins are postulated to form a pore for the transport of the DNA across the bacterial membranes . Immunofluorescence and immunoelectron microscopy were used to study the transport pore complex . Three likely components of the transport pore, VirB8, VirB9 and VirB10, localized primarily to the inner membrane, outer membrane and periplasm respectively . A significant amount of VirB10 was also found associated with the outer membrane . When expressed alone VirB9 and VirB10 were randomly distributed along the cell membrane . Subcellular location of both proteins changed dramatically in the presence of the other VirB proteins . Both proteins localized to fewer sites and most of the gold particles representing protein molecules were found in clusters suggesting that the two proteins are in a protein complex . VirB8, on the other hand, localized to clusters even in the absence of the other VirB proteins . To investigate the role of VirB8 in the formation of VirB9 and VirB10 protein complexes, we studied the effect of deletion of virB8 on the subcellular location of VirB9 and VirB10 . In a virB8 deletion mutant both proteins were distributed randomly on the cell membrane indicating that VirB8 is essential for complex assembly. J Infect Dis, 2000 Jun, 181(6), 2106 - 10 Epub 2000 Jun 05. Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens; Abuodeh RO et al.; Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis . A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A . tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia . This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia . Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy . A transformation frequency of 1 per 10(5) arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium: fungus ratio . This approach requires no special equipment that might complicate biocontainment . Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure . A . tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C . immitis and perhaps other fungi of medical importance. Appl Environ Microbiol, 2000 Jun, 66(6), 2365 - 71 Prey range characterization, ribotyping, and diversity of soil and rhizosphere Bdellovibrio spp . isolated on phytopathogenic bacteria; Jurkevitch E et al.; Thirty new Bdellovibrio strains were isolated from an agricultural soil and from the rhizosphere of plants grown in that soil . Using a combined molecular and culture-based approach, we found that the soil bdellovibrios included subpopulations of organisms that differed from rhizosphere bdellovibrios . Thirteen soil and seven common bean rhizosphere Bdellovibrio strains were isolated when Pseudomonas corrugata was used as prey; seven and two soil strains were isolated when Erwinia carotovora subsp . carotovora and Agrobacterium tumefaciens, respectively, were used as prey; and one tomato rhizosphere strain was isolated when A . tumefaciens was used as prey . In soil and in the rhizosphere, depending on the prey cells used, the concentrations of bdellovibrios were between 3 x 10(2) to 6 x 10(3) and 2.8 x 10(2) to 2.3 x 10(4) PFU g(-1) . A prey range analysis of five soil and rhizosphere Bdellovibrio isolates performed with 22 substrate species, most of which were plant-pathogenic and plant growth-enhancing bacteria, revealed unique utilization patterns and differences between closely related prey cells . An approximately 830-bp fragment of the 16S rRNA genes of all of the Bdellovibrio strains used was obtained by PCR amplification by using a Bdellovibrio-specific primer combination . Soil and common bean rhizosphere strains produced two and one restriction patterns for this PCR product, respectively . The 16S rRNA genes of three soil isolates and three root-associated isolates were sequenced . One soil isolate belonged to the Bdellovibrio stolpii-Bdellovibrio starrii clade, while all of the other isolates clustered with Bdellovibrio bacteriovorus and formed two distantly related, heterogeneous groups. Biosci Biotechnol Biochem, 2000 Apr, 64(4), 845 - 7 Development of a simple and efficient method for transformation of buckwheat plants (Fagopyrum esculentum) using Agrobacterium tumefaciens; Kojima M et al.; Apical meristems of seedlings of buckwheat (Fagopyrum esculentum var . Shinano No . 1) were pricked with a needle and inoculated with Agrobacterium tumefaciens (LBA4404, pBI121) . The inoculated seedlings were grown to maturation and allowed to pollinate randomly to set the seeds (T1 plants) . The transformation efficiency of the T1 plants was estimated by germination in the presence of geneticin (20 microg/ml) and by detection of beta-glucuronidase (GUS) gene with PCR, indicating that 36% and 70% of the T1 plants were transformed, respectively . Four plants taking on a mutated morphology were selected from T1 plants which were transformed with the method using A . tumefaciens harboring a modified pBI121 for plasmid rescue . Southern blot analysis of plasmids rescued from the 4 T1 plants demonstrated that each plasmid contained a different flanking DNA of the buckwheat genome, an evidence that T-DNA was integrated in different sites of the genomic DNA among the 4 T1 plants. Mol Plant Microbe Interact, 2000 Jun, 13(6), 658 - 65 Determination of the T-DNA transfer and the T-DNA integration frequencies upon cocultivation of Arabidopsis thaliana root explants; De Buck S et al.; Using the Cre/lox recombination system, we analyzed the extent to which T-DNA transfer to the plant cell and T-DNA integration into the plant genome determine the transformation and cotransformation frequencies of Arabidopsis root cells . Without selection for transformation competence, the stable transformation frequency of shoots obtained after cocultivation and regeneration on nonselective medium is below 0.5% . T-DNA transfer and expression occur in 5% of the shoots, indicating that the T-DNA integrates in less than 10% of the transiently expressing plant cells . A limited fraction of root cells, predominantly located at the wounded sites and in the pericycle, are competent for interaction with agrobacteria and the uptake of a T-DNA, as demonstrated by histochemical GUS staining . When selection for transformation competence is applied, the picture is completely different . Then, approximately 50% of the transformants show transient expression of a second, nonselected T-DNA and almost 50% of these cotransferred T-DNAs are integrated into the plant genome . Our results indicate that both T-DNA transfer and T-DNA integration limit the transformation and cotransformation frequencies and that plant cell competence for transformation is based on these two factors. Mol Plant Microbe Interact, 2000 Jun, 13(6), 649 - 57 Evidence for Agrobacterium-induced apoptosis in maize cells; Hansen G; Agrobacterium spp . can genetically transform most dicotyledonous plant cells whereas many monocot species are recalcitrant to Agrobacterium-mediated transformation . One major obstacle is that co-cultivation of Agrobacterium spp . with plant tissues often results in cell death . Report here is that, in maize tissues, this process resembles apoptosis, with characteristic DNA cleavage into oligonucleosomal fragments and morphological changes . Two anti-apoptotic genes from baculovirus, p35 and iap, had the ability to prevent the onset of apoptosis triggered by Agrobacterium spp . in maize tissues . p35 is reported to act as a direct inhibitor of a certain class of proteases (caspase) whereas i.a.p . may act upstream to prevent their activation . This evidence raises the possibility that caspase-like proteases may also be involved in the apoptotic pathway in plant cells. Biochemistry, 2000 May 9, 39(18), 5600 - 13 Tyrosine residues serve as proton donor in the catalytic mechanism of epoxide hydrolase from Agrobacterium radiobacter; Rink R et al.; Epoxide hydrolase from Agrobacterium radiobacter catalyzes the hydrolysis of epoxides to their diols via an alkyl-enzyme intermediate . The recently solved X-ray structure of the enzyme shows that two tyrosine residues (Tyr152 and Tyr215) are positioned close to the nucleophile Asp107 in such a way that they can serve as proton donor in the alkylation reaction step . The role of these tyrosines, which are conserved in other epoxide hydrolases, was studied by site-directed mutagenesis . Mutation of Tyr215 to Phe and Ala and mutation of Tyr152 to Phe resulted in mutant enzymes of which the k(cat) values were only 2-4-fold lower than for wild-type enzyme, whereas the K(m) values for the (R)-enantiomers of styrene oxide and p-nitrostyrene oxide were 3 orders of magnitude higher than the K(m) values of wild-type enzyme, showing that the alkylation half-reaction is severely affected by the mutations . Pre-steady-state analysis of the conversion of (R)-styrene oxide by the Y215F and Y215A mutants showed that the 1000-fold elevated K(m) values were mainly caused by a 15-40-fold increase in K(S) and a 20-fold reduction in the rate of alkylation . The rates of hydrolysis of the alkyl-enzyme intermediates were not significantly affected by the mutations . The double mutant Y152F+Y215F showed only a low residual activity for (R)-styrene oxide, with a k(cat)/K(m) value that was 6 orders of magnitude lower than with wild-type enzyme and 3 orders of magnitude lower than with the single tyrosine mutants . This indicates that the effects of the mutations were cumulative . The side chain of Gln134 is positioned in the active site of the X-ray structure of epoxide hydrolase . Mutation of Gln134 to Ala resulted in an active enzyme with slightly altered steady-state kinetic parameters compared to wild-type enzyme, indicating that Gln134 is not essential for catalysis and that the side chain of Gln134 mimics bound substrate . Based upon this observation, the inhibitory potential of various unsubstituted amides was tested, resulting in the identification of phenylacetamide as a competitive inhibitor with an inhibition constant of 30 microM. Arch Microbiol, 2000 Apr, 173(4), 311 - 5 Highly tumorigenic Agrobacterium tumefaciens strain from crown gall tumors of chrysanthemum; Ogawa Y et al.; A wild-type Agrobacterium tumefaciens strain CNI5 isolated from crown gall of chrysanthemum (Dendranthema grandiflora Tzvelev) was characterized . Strain CNI5 was classified into biovar 1, based on physiological and biochemical characteristics, and was resistant to ampicillin . Strain CNI5 induced tumors at a higher frequency and on a larger area of explants in most tested plant species, especially in chrysanthemum cultivars, than the octopine-type strain C58C1cmr (pTiB6S3) . Agropine and mannopine were detected in tumors induced by strain CNI5 and were specifically catabolized by this strain . Strain CNI5 harbored five plasmids including one plasmid that shared sequence similarity to TL-DNA of the octopine-type Ti plasmid and four cryptic plasmids. Plant Sci, 2000 Jun 29, 155(2), 179 - 185 Expression of a chimeric farnesyl diphosphate synthase gene in Artemisia annua L . transgenic plants via Agrobacterium tumefaciens-mediated transformation; Chen D et al.; An Agrobacterium tumefaciens-mediated transformation system was developed for Artemisia annua L . Using this system a cDNA encoding farnesyl diphosphate synthase (FDS placed under a CaMV 35S promoter) was transferred into A . annua via A . tumefaciens strain LB4404 . Leaf or leaf discs were used as explants to be infected with A . tumefaciens and an optimal concentration of 20 mg/l kanamycin was applied to select kanamycin resistant shoots . Forty-five lines of resistance kanamycin shoots transformed with FDS were established . Analysis of PCR showed that at least 20 shoots transformed with the FDS gene were PCR positive . Southern blot analysis suggested the foreign FDS gene had been integrated into the A . annua genome, and Northern blot analysis revealed that the foreign FDS gene expressed at the transcriptional level in five shoot lines (F-1, F-4, F-61, F-62 and F-73 shoot lines) . Analysis of artemisinin demonstrated that about 8 approximately 10 mg/g DW of artemisinin were then detected in transgenic plants regenerated from five shoot lines, this is about 2-3 times higher than that in the control. Mikrobiologiia, 2000 Jan-Feb, 69(1), 81 - 8 {Conjugative transfer of pTd33-plasmid between strains of Agrobacterium}; Chumakov MI et al.; Supramembrane structures that connect conjugating agrobacterial cells were visualized for the first time by transmission electron microscopy . The primary contact of cells during conjugation was shown to occur through the formation of long pili containing no VirB1 protein . Pretreatment of agrobacterial cells with acetosyringone resulted in a six- to tenfold increase in the transfer frequency of the plasmid pTd33 at 19-25 degrees C and had almost no effect at 30 degrees C . The transfer of the plasmid pTd33 from A . tumefaciens strain GV3101 to plasmid-free A . tumefaciens strain UBAPF-2 was 16 times decreased after the centrifugation of cells . The transfer efficiency of the plasmid pTd33 from A . tumefaciens strain LBA2525 (virB2::lacZ) to plasmid-free A . tumefaciens strain UBAPF-2 was one order of magnitude lower than the transfer from the wild-type A . tumefaciens strain GV3101 . Treatment of donor cells with 0.01% SDS before mating decreased the transfer efficiency by a factor of 26 . The role of pili in the establishment of contact between conjugating cells of agrobacteria is discussed. Plant Physiol, 2000 May, 123(1), 393 - 402 The yeast HAL1 gene improves salt tolerance of transgenic tomato; Gisbert C et al.; Overexpression of the HAL1 gene in yeast has a positive effect on salt tolerance by maintaining a high internal K(+) concentration and decreasing intracellular Na(+) during salt stress . In the present work, the yeast gene HAL1 was introduced into tomato (Lycopersicon esculentum Mill.) by Agrobacterium tumefaciens-mediated transformation . A sample of primary transformants was self-pollinated, and progeny from both transformed and non-transformed plants (controls) were evaluated for salt tolerance in vitro and in vivo . Results from different tests indicated a higher level of salt tolerance in the progeny of two different transgenic plants bearing four copies or one copy of the HAL1 gene . In addition, measurement of the intracellular K(+) to Na(+) ratios showed that transgenic lines were able to retain more K(+) than the control under salt stress . Although plants and yeast cannot be compared in an absolute sense, these results indicate that the mechanism controlling the positive effect of the HAL1 gene on salt tolerance may be similar in transgenic plants and yeast. Lett Appl Microbiol, 2000 May, 30(5), 406 - 10 Use of Agrobacterium expressing green fluorescent protein to evaluate colonization of sonication-assisted Agrobacterium-mediated transformation-treated soybean cotyledons; Finer KR et al.; Colonization and infection of soybean cotyledons by Agrobacterium tumefaciens and subsequent elimination of bacteria from cotyledons were monitored using bacteria expressing green fluorescent protein (GFP) . GFP provided a quick, non-destructive method to evaluate, in real time, Agrobacterium colonization of cotyledon surfaces as well as infection of internal cells . GFP was first detected 7 h following inoculation of the cotyledon . By 36 h, GFP expression was very intense, and was limited to the adaxial surface of the cotyledon . Expression of GFP also served as a useful indicator of successful elimination of the bacterium from plant tissue following antibiotic treatment. Appl Environ Microbiol, 2000 May, 66(5), 2133 - 8