Planta . 2005 Jan 15; {Epub ahead of print} Identification of pathogen-responsive regions in the promoter of a pepper lipid transfer protein gene (CALTPI) and the enhanced resistance of the CALTPI transgenic Arabidopsis against pathogen and environmental stresses; Jung HW et al.; The 5' flanking region of the CALTPI gene, which encodes a basic lipid transfer protein, was isolated and characterized from the genomic DNA of Capsicum annuum . Four different regions of the promoter sequence of the CALTPI gene were fused to the beta-glucuronidase (GUS) coding region . In an Agrobacterium-mediated transient expression assay, the transcriptional activations of the promoter deletions were examined in tobacco leaves after infection with Pseudomonas syringae pv . tabaci, and treatment with ethylene and salicylic acid . The -808 bp region of the CALTPI gene promoter sequence exhibited full promoter activity . The W-box and ERE-box elements, which are essential for induction by all signals, were localized in the region between -555 bp and -391 bp upstream of the translation initiation site . A CALTPI transgene was then introduced under the control of the 35S promoter into the Arabidopsis ecotype Col-0 . Transgenic Arabidopsis lines expressing the CALTPI gene developed rapidly compared to the wild-type plants, indicating that CALTPI may be involved in plant development . Overexpression of the CALTPI gene enhanced the resistance against infection by P . syringae pv . tomato and Botrytis cinerea . The transgenic plants expressing the CALTPI gene also showed high levels of tolerance to NaCl and drought stresses at various vegetative growth stages . No transcription of the PR-1, PR-2, PR-5, thionin, and RD29A genes was observed in untreated leaf tissues of the transgenic plants . The enhanced resistance to pathogen and environmental stresses in transgenic Arabidopsis correlated with the enhanced expression of the CALTPI gene. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 263 - 270 Phylogenetic analysis of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium on the basis of 16S rRNA gene and internally transcribed spacer region sequences; Kwon SW et al.; A total of 128 strains was isolated from more than 23 legume hosts in Korea . Phylogenetic relationships between these Korean isolates and reference strains of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were analysed using their 16S rRNA gene and internally transcribed spacer (ITS) region sequences . Among the Bradyrhizobium strains, dendrograms based on both the 16S rRNA gene and ITS region sequences produced two main groups . The ITS tree yielded at least two new clusters that were discernable from the seven previously delineated genospecies . Large discrepancies were revealed between phylogenetic dendrograms based on 16S rRNA gene and ITS region sequences for members of the genus Rhizobium, reflecting their taxonomic heterogeneity . The amalgamation of Rhizobium and former members of Agrobacterium was confirmed using the 16S rRNA tree . Phylogenetic analysis of ITS region sequences showed that the Rhizobium giardinii clade (group II) and the Rhizobium radiobacter/Rhizobium rubi clade (group III) could be tentatively recognized as groups that are separable from the core group (group I), which includes Rhizobium leguminosarum . Dendrograms based on the 16S rRNA gene and ITS region sequences of Mesorhizobium strains were highly conflicting due to the poor taxonomic resolution of the 16S rRNA gene sequences and the low confidence in the ITS dendrogram . Several Korean isolates within the genus Mesorhizobium are thought to represent novel taxa when considering their relatively low ITS region sequence similarities (<80 %) to the reference strains. Yi Chuan Xue Bao, 2004 Nov, 31(11), 1294 - 301 {Construction and expression of vector with aroA-in gene and its transformation in tobacco}; Zhao J et al.; Inteins are intervening protein sequences that undergo self-excision from precursor protein with concomitant joining of flanking sequences . Here, we demonstrated intein cis-splicing in Nicotiana tabacum nuclear genomes by using artificial cis Ssp DnaB and Rma DnaB intein . We want to test whether protein splicing can occur in higher eucaryotic cell,which would play an important role in transgene containment in transgenic plants . Glyphosate-resistant Salmonella typhimurium aroA gene was divided at position 235/236 aa within EPSPS by inverse PCR from pLEPSPS . Amplified gene products with artificial cis-Ssp DnaB/Rma DnaB intein and split-Ssp DnaB/Rma DnaB intein were inserted at position 235 of EPSPS respectively to construct plasmid pLEBC, pLERC, pLEBT and pLERT . Above four aroA-In gene fusions were ligated into pET-32 to obtain E . coli expression vectors termed pETLEBC, pETLEBT, pETLERC and pETLERT . E . coli DE3 cells containing individual recombinant plasmids described above were induced by IPTG to produce corresponding protein products . Detectable spliced EPSPS and unspliced precursor demonstrated that splicing occurred in bacteria . aroA-cis SSp DnaB and aroA-cis Rma DnaB were ligated into Agrobacterium tumefaciens binary vector pLYM . Then A . tumefaciens containing EPSPS-(cis) intein cassettes were used for leaf disk transformation in N . tabacum . Integration of aroA-In gene into plant genome was confirmed by genomic PCR analyses . To verify the expression of fusion genes at transcriptional level, RT-PCR analyses were performed and the expected products were identified . These results suggested that plant cells support expression of S . typhimurium aroA-In fusion gene in nulear genomes . Thus,we speculated the existence of protein-splicing activity in plant cells . This opens the possibility of applying intein trans-splicing technique to reduce/prevent gene transfer by way of pollen in transgenic plants. Vestn Ross Akad Med Nauk, 2004, (11), 50 - 5 {Designing (on the basis of transgenic tomato) of a candidate edible vaccine against hepatitis B and HIV}; Isolation and taxonomic affiliation of N-heterocyclic aromatic hydrocarbon-transforming bacteria; Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, Roskilde, Denmark, paw@milana.dkThe azaarenes (nitrogen-containing heterocyclic aromatic hydrocarbons) are products of incomplete combustion processes and thus are widely distributed with tar and oil products in the environment . Despite their adverse organoleptic, toxic, and carcinogenic characteristics, the biodegradability and fate of multi-ring azaarenes have received little attention . This work demonstrates the presence of genetically diverse azaarene-degrading bacteria in coal tar-contaminated soils . Thirty-eight bacterial strains able to transform the three-ring azaarenes, 5,6- and 7,8-benzoquinoline, phenanthridine, phenazine, or acridine, were isolated . Only seven of these strains grew in liquid medium on the specific azaarene compounds on which they were isolated using plates; and the rest transformed the azaarenes without growth . Taxonomic characterization by 16S ribosomal DNA sequencing revealed that our enrichment technique provided a diversity of 18 different azaarene-transforming bacterial species . Only a few strains were able to mineralize the homocyclic analogue, phenanthrene . Several of the isolates, e.g., Dyadobacter fermentans, Methylopila capsulata, and Agrobacterium tumefaciens, were related to genera relatively unknown with respect to the biodegradation of xenobiotic compounds . These strains can provide further information on the fate of azaarenes in the environment. Theor Appl Genet . 2005 Jan 14; {Epub ahead of print} Construction and characterization of a plant transformation-competent BIBAC library of the black Sigatoka-resistant banana Musa acuminata cv . Tuu Gia (AA); Ortiz-Vazquez E et al.; A plant transformation-competent binary bacterial artificial chromosome (BIBAC) library was constructed from Musa acuminata cv . Tuu Gia (AA), a black Sigatoka-resistant diploid banana . After digestion of high-molecular-weight banana DNA by HindIII, several methods of DNA size selection were tested, followed by ligation, using a vector/insert molar ratio of 4:1 . The library consists of 30,700 clones stored in 80 384-well microtiter plates . The mean insert size was estimated to be 100 kb, and the frequency of inserts with internal NotI sites was 61% . The majority of insert sizes fell into the range of 100+/-20 kb, making them suitable for Agrobacterium-mediated transformation . Only 1% and 0.9% of the clones contain chloroplast and mitochondrial DNA, respectively . This is the first BIBAC library for banana, estimated to represent five times its haploid genome (600 Mbp) . It was demonstrated by hybridization that the library contains typical members of resistance gene and defense gene families that can be used for transformation of disease susceptible banana cultivars for banana genetic improvement. Cell Mol Biol Lett, 2004, 9(4B), 903 - 17 Agrobacterium-mediated transformation of polyploid cereals . The efficiency of selection and transgene expression in wheat; Przetakiewicz A et al.; Three combinations of Agrobacterium tumefaciens strains and vectors were used in the transformation of selected Polish wheat cultivars . The combinations were: two hypervirulent strains, AGL1, containing the pDM805 binary plasmid, and EHA101, containing pGAH; and the common Agro strain LBA4404, harboring the super-binary pTOK233 vector . pDM805 contained bar under the control of Ubi1 promoter, pGAH had nptII under nos, and pTOK233 had hpt under 35S . Additionally, pDM805 and pTOK233 carried the gus reporter gene under the Act1 promoter or 35S promoter, respectively . The highest selection rate was 12.6% and was obtained with EHA101(pGAH) on a kanamycin-containing medium . Sixty-five of the plants grown on that medium were PCR positive . The second best combination was LBA4404(pTOK233) and kanamycin selection, which gave an average transformation rate of 2.3% . Phosphinothricin selection gave 1.0% transformation efficiency, while hygromycin, depending on the strain/vector used, gave from 0.2 to 0.4% . PCR tests in T(1)revealed that 67% of the lines showed a 3:1 segregation ratio, and 11% a 15:1 ratio, while in 22%, segregation was non-Mendelian . The high number of T(0)transgenic plants containing one copy of the transgene was confirmed via Southern blot analysis . Kanamycin resistance in the T(1)generation was very low; in some lines, all the progeny were kanamycin sensitive . GUS expression, only tested in young T(1)plants, was in agreement with Mendelian segregation in three out of the twelve tested . The factors influencing the efficiency of selection and transgene expression are discussed in this paper. Proc Natl Acad Sci U S A . 2005 Jan 11; {Epub ahead of print} Positive charge is an important feature of the C-terminal transport signal of the VirB/D4-translocated proteins of Agrobacterium; Vergunst AC et al.; Several human pathogens and the plant pathogen Agrobacterium tumefaciens use a type IV secretion system for translocation of effector proteins into host cells . How effector proteins are selected for transport is unknown, but a C-terminal transport signal is present in the proteins translocated by the A . tumefaciens VirB/D4 type IV secretion system . We characterized this signal in the virulence protein VirF by alanine scanning and further site-directed mutagenesis . The Cre recombinase was used as a reporter to measure the translocation efficiency of Cre-Vir fusions from A . tumefaciens to Arabidopsis . The data unambiguously showed that positive charge is an essential characteristic of the C-terminal transport signal . We increased the sensitivity of this translocation assay by modifying the Cre-induced readout in host cells from kanamycin resistance to GFP expression . This improvement allowed us to detect translocation of the VirD2 relaxase protein in the absence of transferred DNA, showing that attachment to the transferred DNA is not essential for transport by the VirB/D4 system . We also found another translocated effector protein, namely the VirD5 protein encoded by the tumor-inducing plasmid . According to secondary structure predictions, the C termini of all VirB/D4-translocated proteins identified so far are unstructured; however, they contain a characteristic hydropathic profile . Based on sequence alignments and mutational analysis of VirF, we conclude that the C-terminal transport signal for recruitment and translocation of effector proteins by the A . tumefaciens VirB/D4 system is hydrophilic and has a net positive charge with a consensus motif of R-X(7)-R-X-R-X-R-X-X(n)>. Planta Med, 2004 Dec, 70(12), 1174 - 1179 Nader BL, Taketa AT, Iturriaga G, Pereda-Miranda R, Villarreal ML. Transformed root cultures of Galphimia glauca (Malpighiaceae) were established by infecting cotyledons and hypocotyls with Agrobacterium rhizogenes ATCC 15 834 . Cotyledon-derived cell lines were grown in liquid B5 nutrient medium without phytohormones and have shown the typical hairy roots phenotype over two years of continuous subculturing . PCR analysis was used to confirm the integration of rol A and rol C genes into the plant genome . The transformed cultures synthesized three major norfriedelanes, the new glaucacetalins A - C ( 1 - 3), which were secreted into the nutrient medium . The structural elucidation of these in vitro produced metabolites was performed by the application of high resolution NMR techniques that proved them to be triterpenoids related to the known galphimines, the sedative principles of this plant species . These results suggest the possibility of further biotechnological exploration of sedative friedelane biosynthesis by in vitro plant organ cultures. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Dec, 30(6), 637 - 643 {Rapidly Obtaining the Markerless Transgenic Rice with Reduced Amylose Content by Co-transformation and Anther Culture.}; Shen GZ et al.; The plasmid p13W8 carrying antisense fragment of waxy gene and plasmid pCAMBIA1300 containing hpt gene were introduced into rice by Agrobacterium tumefaciens-mediated co-transformation, and 86 transgenic plants were obtained, 32 of them showed positive bands for antisense waxy gene by PCR analysis, the waxy-positive plant frequency is 37.2% . The segregation of antisense fragment of waxy gene and hpt gene was observed by PCR using hpt gene primers and waxy gene primers respectively in 29 T(1) population . One hundred and eighty-three plants containing only the antisense fragment of waxy gene were identified in 1 264 T(1) plants, the waxy-positive plant frequency is 14.4% . The amylose content of seeds derived from transgenic plants with only the antisense fragment of waxy gene were determined, varying degrees of reduction in amylose content were found in some plants . Four T(1) plants with reduced amylose content were selected through anther culture . Thirty-four anther culture plants seed normally, 23 of them were shown to contain only the antisense fragment of waxy gene by PCR analysis, and the amylose content was reduced to 5%-12% . It took only one and half years to obtain the stably inherited markerless transgenic rice with reduced amylose content by co-transformation and anther culture technique. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Dec, 30(6), 631 - 636 {Transgenic Soybean Obtained with Agrobacterium tumefaciens-mediated Transformation of Embryonic Tip of Soybean Mature Seeds.}; Liu HK et al.; Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains binary vector pCAMBIA2301, and cultured for 20 h . Our results showed that the T-DNA transfer efficiency reached up to 63.3% and the transformation efficiency reached up to 6.4%-12.1% . The effect of infection time on T-DNA delivery into soybean embryonic tips was determined . We also discuss the effects of days of co-cultivation to transient expression and the effects of different AS concentrations to transient expression of gus gene . These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation. Eukaryot Cell, 2005 Jan, 4(1), 111 - 20 Coccidioides posadasii Contains a Single 1,3-{beta}-Glucan Synthase Gene That Appears To Be Essential for Growth; Kellner EM et al.; 1,3-beta-Glucan synthase is responsible for the synthesis of beta-glucan, an essential cell wall structural component in most fungi . We sought to determine whether Coccidioides posadasii possesses genes homologous to known fungal FKS genes that encode the catalytic subunit of 1,3-beta-glucan synthase . A single gene, designated FKS1, was identified, and examination of its predicted protein product showed a high degree of conservation with Fks proteins from other filamentous fungi . FKS1 is expressed at similar levels in mycelia and early spherulating cultures, and expression decreases as the spherules mature . We used Agrobacterium-mediated transformation to create strains that harbor DeltaFKS1::hygB, a null allele of FKS1, and hypothesize that Fks1p function is essential, due to our inability to purify this allele away from a complementing wild-type FKS1 allele in a heterokaryotic strain . The heterokaryon appears normal with respect to growth rate and arthroconidium production; however, microscopic examination of strains with DeltaFKS1::hygB alleles revealed abnormal swelling of hyphal elements. Vnitr Lek, 1999 May, 45(5), 298 - 300 {Agrobacterium tumefaciens (radiobacter) as an infectious agent in an oncological patient}; Frankova H et al.; The authors submit the description of a 62-year-old patient with multiple myeloma where the causal agent of pyretic reactions was Agrobacterium tumefaciens (radiobacter) . It was a patient with an implanted venous port which was colonized by the above bacterium . This finding most probably has not been described so far in the Czech literature . In the English literature the authors found 36 cases . The authors draw attention to the possible higher incidence of future infections caused by organisms hitherto considered non-pathogenic for man, in particular in immunocompromised patients. Yi Chuan, 2004 Nov, 26(6), 991 - 6 {The Progress on T-DNA Integration Research.}; Yang JF et al.; The T-DNA integration is a critical step of the steady inheritance for foreign genes during the Agrobacterium tumefaciens-mediated transformation . There are many factors that influence the integration, including virulence proteins, host factors and so on.The article reviews the recent progress in these aspects, and also expatiates on the integration of T-DNA in host genome and distribution in the chromosomal level and the integration models. Yi Chuan, 2004 Nov, 26(6), 969 - 76 {Research progress on system of transgene in soybean.}; Wang P et al.; This review introduced the recent research progress on transgenic methods and system of receptor in soybean . The major obstacles of genetic transformation in soybean and possible approach for solving the problem were also discussed . Cotyledon node via Agrobacterium tumefaciens-mediated and immature cotyledon via particle bombardment were thought to be the efficient systems of genetic transformation . Three problems existed in genetic transformation of soybean . The first one was that the system of tissue culture needs to be further improved . The second was that efficiency of genetic transformation was still low and difficult to be repeated . The last one was that restricted genotypes of soybean have been transformed successfully as a receptor . The path of solving these problems need to set a new and high efficient system for tissue culture in soybean . Also the number of target gene to be transformed will be increased from single gene to several genes at same time. Yi Chuan, 2004 Nov, 26(6), 881 - 6 {Construction of expression vector with antisense rubisco activase gene and its genetic transformation in rice.}; Jin SH et al.; In this research, Rubisco activase gene (rca) was amplified using specific primers and inserted into pGEM T-easy vector, and then cut with EcoRI after confirming by sequencing . The fragment was subcloned into pBluescript KS+, digested with the enzyme BamHI and inserted into the binary expression vector pCAMBIA1301, and the resulting construction with antisense rca was named pCAMR02 . The pCAMR02 vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed to embryos of rice (Oryza . Sativa L.ssp.japonica) cultivar Zhonghua11 via Agrobacterium tumefaciens system . Plantlets were regenerated in vitro by resistance selection on medium containing various concentrations of hygromycin . Both GUS histochemical assays and PCR amplification demonstrated that antisense rca was integrated into T0 genomes and inherited to T1 . The measurement of phenotypes of transgenic rice plants with antisense rca showed that most of them could hardly survive at ambient CO2 concentration, even could not grow . The antisense plants that survived under natural condition were dwarf and grew slower than the wild-type controls, and their contents of RCA and Rubisco changed significantly . These plants generated in this experiment will be used to study the relationship between RCA and Rubisco and their regulation. Yi Chuan, 2004 Sep, 26(5), 695 - 700 {Studies of Somatic Embryogenesis and Genetic Transformation by Agrobacterium- mediated in Soybean.}; Wang P et al.; Somatic embryogenesis was induced and the regenerated plants were obtained by higher concentrations of auxins with immature cotyledon of 55 genotypes in soybean . Bivalent insect resistant genes were transformed into immature cotyledon of soybean which have high frequency of somatic embryogenesis via Agrobacterium-mediated . The results showed that 14 genotypes possessed high frequency of somatic embryogenesis (more than 40%) among soybean genotypes from Northeast area . 2147 immature cotyledons of 5 different soybean genotypes cultured in Northeast area were inoculated with LBA4404 (including pGBI121S4ABC plasmid) . 12 regenerated plants selected by Kanamicy gave positive PCR reaction. Yi Chuan, 2004 Jul, 26(4), 425 - 31 {Construction of the plant expression vector with hepatitis a capsid protein fusion gene and genetic transformation of citrus.sinensis osbeck.}; Hu R et al.; The use of edible plants for the production and delivery of vaccine proteins could provide an economical alternative to fermentation systems.The construction of the plant expression vector pBI121-A was reported, which contained a fusion gene encoding hepatitis A capsid proteins.The gene was located between the left and right Ti border sequences under the control of CaMV35S promoter.The vector was identified via PCR and restriction enzyme analysis and was introduced into Agrobacterium tumerifacience LBA4404.The transgenic Citrus plants were produced by Agrobacterium-mediated transformation of epicotyl segments.13 putatively transformed plants through the kanamycin selection were micrografted onto the seedlings.The presence and integration of the transgene had been verified by PCR analysis.The result showed that five transformants were integrated and the transformation efficiency was 4.1%. Yi Chuan, 2003 Sep, 25(5), 563 - 6 {Construction of a plant expression vector with tomato transcription factor gene pti5 and studies on transgenic tobaccos.}; Zhang YH et al.; The plasmid pBI121UCH1 carrying Pti5-VP16 gene under the control of the cauliflower mosaic virus 35s promoter was constructed.Leaf segments of tobacco SRI were infected by Agrobacterium tumefaciens EHA105 with plasmid pBI121UCH1,from which kanamycin resistant plants were obtained.PCR and Southern analysis proved that the Pti5-VP16 gene was integrated into the genomes of the tobacco plants.The disease resistance assay showed that the disease resistance was enhanced in the transgenic tobacco plants. Protein Pept Lett, 2005 Jan, 12(1), 69 - 73 Purification and properties of beta-alanine synthase from calf liver; Waldmann G et al.; beta-Alanine synthase (EC 3.5.1.6) catalyzes the conversion of N-carbamyl-beta-alanine to beta-alanine, ammonia and CO2 . The enzyme has been purified to apparent homogeneity from calf liver . The molecular size, pH optimum and substrate specificity have been determined . Sequence alignment of beta-alanine synthases with N-carbamyl-D-amino acid amidohydrolase from Agrobacter sp . revealed the conservation of a catalytically important triad Glu-Lys-Cys, most likely involved in the breakdown of N-carbamyl-beta-alanine. J Am Coll Nutr, 2004 Dec, 23(6), 763S - 7S Effect of magnesium on essential oil formation of genetically transformed and non-transformed chamomile cultures; Szoke E et al.; OBJECTIVE: The importance of chamomile (Chamomilla recutita) is widely known in classical and folk medicine, with the largest group of its effective substances forming the essential oil (chamazulene, alpha-bisabolol, trans-beta-farnesene, spathulenol, cis/trans-en-in-dicycloethers) . The increasing need for plant-derived high quality drugs cannot be provided by their collection in the wilderness . Method A: To preserve the genome of Szabadkigyo . wild type having high (-)-alpha-bisabolol content, we used biotechnological methods . RESULTS: The roots of organized culture contained beta-eudesmol, which we have identified in the intact roots . Our gas-chromatographic and mass-spectroscopic studies showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristics of the intact plant . We identified berkheyaradulene, geranyl-isovalerate and cedrol, as new components in these cultures . Magnesium (Mg) (370 and 740 mg/l MgSO(4)) has a positive effect on the growth of organized cultures and also on the quality and quantity of essential oil production . Method B: Another possible source of variants is available by the genetic transformation of organized cultures by infection with Agrobacterium rhisogenes . With this method, we cultivated chamomile infected by A4-Y clone and investigated the essential oil production by hairy root cultures cultivated on solid and liquid MS B-5 media . The main component of the essential oil of hairy root cultures was trans-beta-farnesene . Results: We identified alpha-selinene, as a new component in these hairy roots . We studied the growth rate of A4-Y clone on the cited media, containing MgSO(4) concentrations: 0; 185; 370 and 740 mg/l . The cultures grew most in medium containing 740 mg/l of MgSO(4) . Essential oil content was compared from hairy root cultures of different Mg containing media and measured by GC and GC-MS methods . Mg has a similar effect on hairy roots as on organized cultures. Shi Yan Sheng Wu Xue Bao, 2004 Oct, 37(5), 398 - 408 {Factors influencing agrobacterium-mediated transformation of maize elite inbred lines}; Huang XQ et al.; Using the maize elite inbred lines 9046, Qi319, 414, Mo17 as target genotypes, a highly efficient transformation system was developed based on the study of factors influencing the Agrobacterium-mediated maize transformation . The results showed that the immature embryos of 1.0-2.0 mm in length were optimal transformation explants . Inclusion of acetosyringone (200 micromol/L) and ascorbatic acid (50 mg/L) in both infection medium and co-cultivation medium led to a significantly increase in the transformation efficiency . However, high osmotic treatment on the explants before inoculation didn't improve transformation efficiency . Delaying selection was beneficial to the survival of resistant calli . Using the optimized transformation procedure, 42 PCR-positive transgenic plants were obtained from the 4 elite inbred lines and the frequency of PCR-positive plant ranged from 1.71%-4.09% . The integration of the transgenes into the maize nuclear genome was confirmed by PCR analysis using bar- and gus-specific primers and by Southern blot using gus- specific probe . Most of transgenic plants (71.4%) had one copy of T-DNA insert . The establishment of the transformation system in maize provides an efficient way for transferring useful foreign genes to maize plants. Shi Yan Sheng Wu Xue Bao, 2004 Oct, 37(5), 384 - 90 {Effect of sucrose concentration on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots}; Liang P et al.; Effects of sucrose concentrations on the growth and production of puerarin and isoflavones compounds in Pueraria phaseoloides hairy roots induced by Agrobacterium rhizogenes ATCC15834 were investigated . Changes of sucrose consumption in the medium during liquid culture were also determined . The results showed cultured for 16 days in MS medium with 5%, 4%, 3% and 2% sucrose, the proliferation times of dry weight of hairy roots were 11.7, 11.9, 10.1 and 5.9, respectively . 3% sucrose concentration in liquid medium was the best for accumulation of puerarin and isoflavones in the hairy roots . The highest content of puerarin, 5.147 mg/g DW, was obtained after 12 days of liquid culturing while the highest content of isoflavones, about 27.76 mg.g.DW, was gained after 16 days in culturing . Sucrose concentration decreased as hairy root growth proceeded . The growth rate and the content of soluble sugar in hairy roots of P . phaseoloides was directly proportional to the rate of sucrose utilization in the liquid medium during the whole culture . It was observed that the highest content of soluble sugar in hairy roots was at day 12 of liquid culture and sucrose in the liquid medium was used up at the end of 16 days of culture. Yi Chuan Xue Bao, 2004 Dec, 31(12), 1434 - 8 {Study on transformation of snowdrop lectin gene to chrysanthemum and aphid resistance of the transgenic plants}; Wang GL et al.; Agrobacterium-mediated transformation in chrysanthemum was studied to prevent the insect pest of aphid (Mizus persicae) . The gna gene was successfully transferred into chrysanthemum by leaf dish, and 93 transgenic clones were obtained . The highest transformation frequency 11.21% was achieved on the optimization facts, which were medium YEB with pH5.6, bacterial concentration OD600 = 0.4, precultivation for one day, cocultivation for four days, the cocultivation media supplemented with GA3 0.5 mg/L and leaf explants growed for 45 days . The results from PCR and FQ-PCR analysis confirmed that gna gene was integrated into the genome of chrysanthemum plants . The insect bioassay with aphid showed that the aphid resistance of different transgenic plants was difference, and the rate of aphid population inhibition of them were from 10% to 84% with an average rate of 39.4% . The leaf-extracts from different transgenic plants showed varying actinties in red-blood cell bioassay. Yi Chuan Xue Bao, 2004 Dec, 31(12), 1388 - 94 {Generation and identification of rice T-DNA insertional mutant lines}; Yan SY et al.; By using the matured embryos of Japonica rice variety Zhonghua No . 11 as explants, rice transformation was performed by Agrobacterium-mediated co-cultivation method, resulting in 1489 independent transgenic rice plants that carry a T-DNA insertion . Genomic DNA gel-blot and PCR analyses showed that 69.8% of the total lines contain the inserted T-DNA . The flanking sequence of T-DNA in transgenic rice plants was analyzed using Tail-PCR . In addition, we have evaluated 1066 T1 transgenic lines on heading days, plant height and panicles per hill, and found different types of mutants from a number of lines. Yi Chuan Xue Bao, 2004 Dec, 31(12), 1381 - 7 {Analysis of the transgenic rice plants derived from transformed anther calli}; Jiang S et al.; Rice calli derived from anther culture were used as recipient to transfer a rice blight resistance gene, Xa21, into a japonica rice variety, Taipei 309, via Agrobacterium-mediated transformation . Seven green transgenic plants, including one mixoploid, two haploid, and four diploid plants, were regenerated . PCR, Southern blot, FISH and blight resistance analysis all indicated that Xa21 gene has been integrated into the T0 plant genomes . T1 generations of the four diploid T0 plants were further investigated for resistance segregation . Chi2 test showed that two T1 populations segregated with a ratio of 3:1, indicating that a single copy of Xa21 gene was integrated into the genome, whereas the segregation ratios of the other two T1 populations were non-Mendelian . Therefore, the four diploid transgenic plants should be heterozygous diploids. J Agric Food Chem, 2005 Jan 12, 53(1), 57 - 61 Antibacterial Activity of Cuminum cyminum L . and Carum carvi L . Essential Oils; Iacobellis NS et al.; Essential oils extracted by hydrodistillation from fruits of Cuminum cyminum L . and Carum carvi L . were analyzed by gas chromatography (GC) and GC-mass spectrometry (MS) . The main components of C . cyminum oil were p-mentha-1,4-dien-7-al, cumin aldehyde, gamma-terpinene, and beta-pinene, while those of the C . carvi oil were carvone, limonene, germacrene D, and trans-dihydrocarvone . Antibacterial activity, determined with the agar diffusion method, was observed against Gram-positive and Gram-negative bacterial species in this study . The activity was particularly high against the genera Clavibacter, Curtobacterium, Rhodococcus, Erwinia, Xanthomonas, Ralstonia, and Agrobacterium, which are responsible for plant or cultivated mushroom diseases worldwide . In general, a lower activity was observed against bacteria belonging to the genus Pseudomonas . These results suggest the potential use of the above essential oils for the control of bacterial diseases. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Oct, 30(5), 541 - 5 {Prolonging the vase life of carnation "Mabel" through integrating repeated ACC oxidase genes into its genome.}; Yu YX et al.; Carnation (Dianthus caryophyllus L.) is one of the most important cut flowers . The cultivar "Mabel" of carnation was transformed with direct repeat gene of ACC oxidase, the key enzyme in ethylene synthesis, driven by the CaMV35S promoter mediated by Agrobacterium tumefacien . Hygromycin phosphotransferase (HPT) gene was used as selection marker . Leaf explants were pre-cultured on shoot-inducing medium for 2 d, then immersed in Agrobacterium suspension for 8-12 min . Co-cultivation was carried out on the medium (MS+BA 1.0 mg/L+NAA 0.3 mg/L +Acetosyringone 100 micromol/L, pH 5.8-6.0) for 3 d . After that transformants were obtained by transferring explants to selection medium supplemented with 5 mg/L hygromycin (Hyg) and 400 mg/L cefotaxime (Cef) . Southern blotting detection showed that a foreign gene was integrated into the carnation genome and 3 transgenic lines (T257, T299 and T273 line) obtained . Addition of acetosyringone and the time of co-culture were the main factors that influenced transformation frequency . After being transplanted to soil, transgenic plants were grew normally in greenhouse . Ethylene production of cut flower of transgenic T257 line was 95% lower than that of the control, and that of T299 line was reduced by 90% than that of the control, while that of transgenic T273 line has no of significantly different from control . Vase life of transgenic T257 line was 5 d longer than that of the control line at 25 degrees C. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Oct, 30(5), 517 - 22 {Overexpression of GST gene accelerates the growth of transgenic Arabidopsis under salt stress.}; Qi YC et al.; The Suaeda salsa glutathione s-transferase gene (GST) was inserted downstream of the 35S promoter in the plant expression vector pROK II and then was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens through floral dip method . Transformants were selected for their ability to grow on medium containing kanamycin . The fact that the GST gene had been transferred into the Arabidopsis thaliana genome was confirmed by the PCR-Southern blotting analysis . After cultivation, independent homozygous transgenic lines were obtained after selection of T(3) progenies on MS medium containing kanamycin . The expression of the gene transferred into the Arabidopsis thaliana was confirmed by Northern blotting . During salt stress, analysis of total glutathione (both oxidized and reduced type) and biomass of transgenic and wild Arabidopsis . The biomass of transgenic lines (GT) was slightly but significantly greater than that of wild type line (WT), and levels of oxidized glutathione (GSSG) were significantly higher in transgenic lines than in wild type . Therefore, overexpression of GST can increase Arabidopsis growth under salt stress, and this effect can be caused by oxidation of the reduced glutathione (GSH ). Plant Physiol, 2005 Jan, 137(1), 168 - 75 Epub 2004 Dec 23. Site preferences of insertional mutagenesis agents in Arabidopsis; Pan X et al.; We have performed a comparative analysis of the insertion sites of engineered Arabidopsis (Arabidopsis thaliana) insertional mutagenesis vectors that are based on the maize (Zea mays) transposable elements and Agrobacterium T-DNA . The transposon-based agents show marked preference for high GC content, whereas the T-DNA-based agents show preference for low GC content regions . The transposon-based agents show a bias toward insertions near the translation start codons of genes, while the T-DNAs show a predilection for the putative transcriptional regulatory regions of genes . The transposon-based agents also have higher insertion site densities in exons than do the T-DNA insertions . These observations show that the transposon-based and T-DNA-based mutagenesis techniques could complement one another well, and neither alone is sufficient to achieve the goal of saturation mutagenesis in Arabidopsis . These results also suggest that transposon-based mutagenesis techniques may prove the most effective for obtaining gene disruptions and for generating gene traps, while T-DNA-based agents may be more effective for activation tagging and enhancer trapping . From the patterns of insertion site distributions, we have identified a set of nucleotide sequence motifs that are overrepresented at the transposon insertion sites . These motifs may play a role in the transposon insertion site preferences . These results could help biologists to study the mechanisms of insertions of the insertional mutagenesis agents and to design better strategies for genome-wide insertional mutagenesis. EMBO J . 2004 Dec 23; {Epub ahead of print} The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation; Lacroix B et al.; To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell . Among these proteins, VirE3 is the only one whose biological function is completely unknown . Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway . In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import . The VirE2 nuclear import in turn is mediated by a plant protein, VIP1 . Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus . As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell. Izv Akad Nauk Ser Biol, 2004 Nov-Dec, (6), 698 - 707 {Colonization of wheat root hairs and roots by agrobacteria}; A surface loop of the potato leafroll virus coat protein is involved in virion assembly et al.; Department of Plant Pathology, 334 Plant Science, Cornell University, Ithaca, NY 14853, USATwo acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis . Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation . Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species . Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability . A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer . The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression . PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors . These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors. Mol Microbiol, 2005 Jan, 55(1), 115 - 24 The type IV secretion apparatus protein VirB6 of Agrobacterium tumefaciens localizes to a cell pole; Judd PK et al.; Summary Agrobacterium tumefaciens VirB proteins assemble a type IV secretion apparatus for the transfer of DNA and proteins to plant cells . To study the role of the VirB6 protein in the assembly and function of the type IV apparatus, we determined its subcellular location by immunofluorescence microscopy . In wild-type bacteria VirB6 localized to the cell poles but in the absence of the tumour-inducing plasmid it localized to random sites on the cell membranes . Five of the 11 VirB proteins, VirB7-VirB11, are required for the polar localization of VirB6 . We identified two regions of VirB6, a conserved tryptophan residue at position 197 and the extreme C-terminus, that are essential for its polar localization . Topology determination by PhoA fusion analysis placed both regions in the cell cytoplasm . Alteration of tryptophan 197 or the deletion of the extreme C-terminus led to the mislocalization of the mutant protein . The mutations abolished the DNA transfer function of the protein as well . The C-terminus of VirB6, in silico, can form an amphipathic helix that may encode a protein-protein interaction domain essential for targeting the protein to a cell pole . We previously reported that another DNA transfer protein, VirD4, localizes to a cell pole . To determine whether VirB6 and VirD4 localize to the same pole, we performed colocalization experiments . Both proteins localized to the same pole indicating that VirB6 and VirD4 are in close proximity and VirB6 is probably a component of the transport apparatus. Biotechnol Adv, 2005 Jan, 23(1), 3 - 39 Influence of rol genes in floriculture; Casanova E et al.; Traditionally, new traits have been introduced into ornamental plants through classical breeding . However, genetic engineering now enables specific alterations of single traits in already successful varieties . New or improved varieties of floricultural crops can be obtained by acting on floral traits, such as color, shape or fragrance, on vase life in cut-flower species, and on rooting potential or overall plant morphology . Overexpression of the rol genes of the Ri plasmid of Agrobacterium rhizogenes in plants alters several of the plant's developmental processes and affects their architecture . Both A . rhizogenes- and rol-transgenic plants display the "hairy-root phenotype", although specific differences are found between species and between transgenic lines . In general, these plants show a dwarfed phenotype, reduced apical dominance, smaller, wrinkled leaves, increased rooting, altered flowering and reduced fertility . Among the rol genes, termed rolA, B, C and D, rolC has been the most widely studied because its effects are the most advantageous in terms of improving ornamental and horticultural traits . In addition to the dwarfness and the increase in lateral shoots that lead to a bushy phenotype, rolC-plants display more, smaller flowers, and advanced flowering; surprisingly, these plants may have better rooting capacity and they show almost no undesirable traits . rolD, the least studied among the rol genes, offers promising applications due to its promotion of flowering . Although the biochemical functions of rol genes remain poorly understood, they are useful tools for improving ornamental flowers, as their expression in transgenic plants yields many beneficial traits. Plant J, 2005 Jan, 41(1), 162 - 74 High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions; Koroleva OA et al.; Summary We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells . The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization . This is an essential step towards full functional analysis . The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion . We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium . To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins . These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments . Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern . We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells . This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function. Planta . 2004 Dec 18; {Epub ahead of print} Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean; Piller KJ et al.; Enterotoxigenic Escherichia coli (ETEC) cause acute diarrhea in humans and farm animals, and can be fatal if the host is left untreated . As a potential alternative to traditional needle vaccination of cattle, we investigated the feasibility of expressing the major K99 fimbrial subunit, FanC, in soybean (Glycine max) for use as an edible subunit vaccine . As a first step in this developmental process, a synthetic version of fanC was optimized for expression in the cytosol and transferred to soybean via Agrobacterium-mediated transformation . Western analysis of T(0) events revealed the presence of a peptide with the expected mobility for FanC in transgenic protein extracts, and immunofluorescense confirmed localization to the cytosol . Two T(0) lines, which accumulated FanC to levels near 0.5% of total soluble protein, were chosen for further molecular characterization in the T(1) and T(2) generations . Mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing synthetic FanC developed significant antibody titers against bacterially derived FanC and produced antigen-specific CD4(+) T lymphocytes, demonstrating the ability of transgenic FanC to function as an immunogen . These experiments are the first to demonstrate the expression and immunogenicity of a model subunit antigen in the soybean system, and mark the first steps toward the development of a K99 edible vaccine to protect against ETEC. Planta, 2004 Oct, 219(6), 1042 - 9 Epub 2004 Jul 16. Efficient Agrobacterium tumefaciens-mediated transformation of soybeans using an embryonic tip regeneration system; Liu HK et al.; Here, we report the establishment of an efficient, in vitro, shoot organogenesis, regeneration system for soybeans {Glycine max (L.) Merr.} . Mature soybean seeds were soaked for 24 h, the embryonic tips were collected and cultured on MSB5 medium supplemented with 3.5 mg l(-1) N6-benzylaminopurine (BAP) for 24 h, and explants were transferred to MSB5 medium supplemented with 0.2 mg l(-1) BAP and 0.2 mg l(-1) indolebutyric acid . Use of embryonic tips yielded a higher regeneration frequency (87.7%) than regeneration systems using cotyledonary nodes (40.3%) and hypocotyl segments (56.4%) as starting materials . Regenerated embryonic tips were inoculated with Agrobacterium tumefaciens strain EHA105, which contains the binary vector pCAMBIA2301, and cultured for 20 h . Our results showed that the T-DNA transfer efficiency reached up to 78.2% and the transformation efficiency reached up to 15.8% . These data indicate that the embryonic tip regeneration system can be used for efficient, effective Agrobacterium-mediated transformation. Biotechnol Lett, 2004 Sep, 26(18), 1433 - 9 Expression of recombinant endostatin in Agrobacterium-inoculated leaf disks of Nicotiana tabacum var . Xanthi; Hong SH et al.; Recombinant endostatin was transiently expressed in Agrobacterium-inoculated leaf disks of Nicotiana tabacum var . Xanthi with a molecular size of 23 kDa . Expression of endostatin from a replicating vector based on tomato golden mosaic virus (TGMV) was 170% higher at the transcript level and double higher at the protein level than from a control vector of a non-replicating construct . Purified recombinant endostatin from tobacco leaf-disks has an anti-proliferative effect on bovine endothelial cells. Plant Mol Biol, 2004 Jul, 55(4), 531 - 9 Agrobacterium tumefaciens-mediated transformation of plants by the pTF-FC2 plasmid is efficient and strictly dependent on the MobA protein; Dube T et al.; In the transformation of plants by Agrobacterium tumefaciens the VirD2 protein has been shown to pilot T-DNA during its transfer to the plant cell nucleus . Other studies have shown that the MobA protein of plasmid RSF1010 is capable of mediating its transfer from Agrobacterium cells to plant cells by a similar process . We have demonstrated previously that plasmid pTF-FC2, which has some similarity to RSF1010, is also able to transfer DNA efficiently . In this study, we performed a mutational analysis of the roles played by A . tumefaciens VirD2 and pTF-FC2 MobA in DNA transfer-mediated by A . tumefaciens carrying pTF-FC2 . We show that MobA+/VirD2+ and MobA+/VirD2- strains were equally proficient in their ability to transfer a pTF-FC2-derived plasmid DNA to plants and to transform them . However, the MobA-/VirD2+ strain showed a DNA transfer efficiency of 0.03% compared with that of the other two strains . This sharply contrasts with our results that VirD2 can rather efficiently cleave the oriT sequence of pFT-FC2 in vitro . We therefore conclude that MobA plays a major VirD2-independent role in plant transformation by pTF-FC2. Plant Mol Biol, 2004 Jul, 55(4), 521 - 30 Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence; Chen JC et al.; Agrobacterium-mediated infection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petunia genes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes . Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues . Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers . The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers . Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers . Abundance of CHS transcripts in the white flowers was less than 4% of that in purple flowers on the same plant . Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers . Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct . We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1-aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5% and 20%, respectively) in infected flowers . Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues . These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes. Plant Mol Biol, 2004 Apr, 54(6), 931 - 41 Application of RNAi to confirm theobromine as the major intermediate for caffeine biosynthesis in coffee plants with potential for construction of decaffeinated varieties; Ogita S et al.; The caffeine biosynthetic pathway in coffee plants has been proposed to involve three distinct N -methyltransferases, xanthosine methyltransferase (XMT), 7- N -methylxanthine methyltransferase (MXMT; theobromine synthase), and 3,7-dimethylxanthine methyltransferase (DXMT; caffeine synthase) . We previously isolated all corresponding cDNAs designated as CaXMT1 , CaMXMT1 , CaMXMT2 and CaDXMT1 , respectively, and showed that caffeine was indeed synthesized in vitro by the combination of their gene products . In order to regulate caffeine biosynthesis in planta , we suppressed expression of CaMXMT1 by the double stranded RNA interference (RNAi) method . For this purpose, we first established a protocol for efficient somatic embryogenesis of Coffea arabica and C . canephora , and then Agrobacterium -mediated transformation techniques . The RNAi transgenic lines of embryogenic tissues derived from C . arabica and transgenic plantlets of C . canephora demonstrated a clear reduction in transcripts for CaMXMT1 in comparison with the control plants . Transcripts for CaXMT1 and CaDXMT1 were also reduced in the most cases . Both embryonic tissues and plantlets exhibited a concomitant reduction of theobromine and caffeine contents to a range between 30% and 50% of that of the control . These results suggest that the CaMXMT1 -RNAi sequence affected expression of not only CaMXMT1 itself, but also CaXMT1 and CaDXMT1 , and that, since the reduction in theobromine content was proportional to that for caffeine, it is involved in the major synthetic pathway in coffee plants . The results also indicate that the method can be practically applied to produce decaffeinated coffee plants. J Bacteriol, 2005 Jan, 187(1), 213 - 23 Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens; Wise AA et al.; The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants . VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG . Inducing signals include phenols, monosaccharides, and acidic pH . While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low . Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA . Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit . However, sugar enhancement of vir gene expression was vector dependent . virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA . Inhibition was conditional, depending on the induction medium and the virA allele tested . Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism. J Bacteriol, 2005 Jan, 187(1), 185 - 92 luxR homolog avhR in Agrobacterium vitis affects the development of a grape-specific necrosis and a tobacco hypersensitive response; Hao G et al.; The luxR homolog aviR in Agrobacterium vitis strain F2/5 was recently shown to be associated with induction of a hypersensitive response (HR) on tobacco and necrosis on grape plants, indicating that the responses are regulated by quorum sensing . We now report a second luxR homolog, avhR, whose disruption (mutant M1320) results in HR-negative and reduced grape necrosis phenotypes . The deduced AvhR protein has characteristic autoinducer binding and DNA binding domains and is unique among reported functional LuxR homologs in having substitutions at highly conserved Asp70, Trp57, and Trp85 residues, which are predicted to play important roles in autoinducer binding in TraR . M1320 was fully complemented with cloned avhR . The same array of N-acylhomoserine lactones (AHL) from F2/5, M1320, and complemented M1320 were observed; however, the signal strength from extracts of 6-day-old M1320 cultures was stronger than that of F2/5 . Cultures of F2/5 amended with AHL extracts from overnight and 6-day cultures of F2/5 and M1320 were not affected in ability to cause HR or necrosis . A region of about 14 kb flanking avhR was sequenced and compared with homologous regions of A . tumefaciens C58 and Sinorhizobium meliloti Rm1021 genomes . Gene order and homology are conserved between the species . A site-directed mutation in a putative gene that resides downstream of avhR and that has homology to genes belonging to the ATP-binding cassette transporter family did not affect HR or necrosis phenotypes . It was determined that avhR and aviR are expressed independently and that neither regulates the expression of a clpA homolog in F2/5. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Apr, 30(2), 173 - 8 {Glyphosate-resistant cotton (Gossypium hirsutum L.) Transformed with aroAM12 gene via Agrobacterium tumefaciens}; Xie LX et al.; A mutant, aroAM12, exhibiting resistance to glyphosate produced in a previous study using the staggered extension process with aroA genes from Salmonella typhimurium and Eschrichia coli . In this paper, we constructed a vector pGRA1300 carrying aroAM12 gene, comprising transit peptide of Arabidopsis EPSPS, under the control of the CaMV35S promoter and used as selectable marker for cotton plant (Gossypium hirsutum L.) transformation . Transgenic cottons with increased resistance to glyphosate were obtained by cotransformtion of hypocotyl segments with Agrobacterium tumefaciens and selected directly on medium containing glyphosate . Regeneration of glyphosate-resistant calli was carried out on a MS basic medium containing 2,4-D 0.1 mg/L, KT 0.1 mg/L, cefotaxime 500 mg/L and glyphosate 60 micromol/L . Globular embryos were induced and then developed by culturing on MSB (MS salts+B(5) vitamins) medium supplemented with asparagine 1 g/L and glutamine 2 g/L, but not containing hormone, for 40 d . The developed plantlets were then removed and cultured on an MS medium . After about 20 d, the deeply-rooted shoots were in soil . PCR analysis showed that the aroAM12 gene was present in all T(0) transgenic plants . The integration of the aroAM12 gene in the genomic DNA of cotton was further confirmed by Southern blot, which showed that the transgenic plants carried one or two copies of the aroAM12 genes . Western blot analysis showed that a 48-kD band of was detected in all T(0) transgenic plants . There was no apparent corelation between copy numbers and the expression level of the aroAM12 gene . Greenhouse screening for glyphosate resistance was performed to test 65 independent T(0) plants by spraying (three times) with an aqueous suspension at a dose corresponding to 9.317 kg/ha of Roundup (once every 5 d) . After 15 d, phenotype examination was carried out of the plants in comparison with untransformed control plants . Under these conditions, it was observed that the plants transformed with pGRA1300 showed high resistance to glyphosate whereas the control plants were all killed . The glyphosate resistance of T(1) generation was measured by spraying with Roundup, the numbers of glyphosate resistance and sensitive phenotypes showed Mendelian segregation ratio. Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Jun, 30(3), 351 - 8 {Promoter specificity of LE-ACS6 gene in LE-ACS6::GUS transgenic arabidopsis plants}; Fan R et al.; The LE-ACS6 gene encodes ACC synthase, the key enzyme of ethylene biosynthesis pathway . Accumulation of LE-ACS6 transcripts is concomitant with the system 1 ethylene production in the pre-climacteric tomato fruit, and both are down regulated by exogenous ethylene treatment . To elucidate the possible role of system 1 ethylene in plant development and investigate the promoter tissue specificity of LE-ACS6 gene, stable transformation of Arabidopsis with a LE-ACS6 promoter-GUS fusion construct by Agrobacterium method has been done . Histochemical localization of GUS activity and beta-glucuronidase enzyme assay in transgenic LE-ACS6::GUS plants showed strong expression of GUS in cotyledons and hypocotyls of 6 d seedlings, but no GUS activity was detected in roots . The GUS activity of 6 d seedling was increased significantly when treated with NAA 10(-4)mol/L . In 40 d rosette leaf LE-ACS6 promoter driven GUS gene was predominantly expressed in mature leaves . Lower level of GUS expression was detected in younger and older leaves . Wounding was found to increase GUS gene expression in transgenic leaves . Again, exogenous NAA was found to increase the GUS activity in wounded leaf tissue . Strong staining reaction was observed in the top part of rapidly growing stems . In different developmental stages of Arabidopsis seeds, "mature green" pods were strongly stained, but "ripening fruits" were not colored . These observations are concomitant with the system 1 ethylene production, suggesting a popular mechanism in regulating ethylene biosynthesis in different plants, at least in tomato and Arabidopsis. FEMS Microbiol Lett, 2004 Dec 15, 241(2), 207 - 14 Use of REMI and Agrobacterium-mediated transformation to identify pathogenicity mutants of the biocontrol fungus, Coniothyrium minitans; Rogers CW et al.; Restriction enzyme mediated integration (REMI) and Agrobacterium-mediated transformation (ATMT) were used to transform protoplasts or germinated conidia of the mycoparasite Coniothyrium minitans to hygromycin resistance . Using REMI, up to 32 transformants mug DNA(-1) were obtained, while 37.8 transformants 5x10(5) germlings(-1) were obtained using ATMT . Single-copy integrations occurred in 8% and 40% of REMI and ATMT transformants, respectively . A novel microtitre plate-based test was developed to expedite screening of 4000 REMI and ATMT C . minitans transformants . Nine pathogenicity mutants that displayed reduced or no pathogenicity on sclerotia of Sclerotinia sclerotiorum were identified. Fungal Genet Biol, 2005 Jan, 42(1), 9 - 19 Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori; Michielse CB et al.; In this study, the efficiency of gene replacement in Aspergillus awamori between Agrobacterium-mediated transformation and CaCl(2)/PEG-mediated transformation was compared . For the genes, pyrG and gfaA, it was found that the homologous recombination frequencies obtained by Agrobacterium-mediated transformation were 3- to 6-fold higher than the frequencies obtained with CaCl(2)/PEG protoplast transformation . For the pyrG gene, it was found that Agrobacterium-mediated transformation allowed an efficient homologous recombination with shorter DNA flanks than CaCl(2)/PEG protoplast transformation . Finally, the addition of the dominant amdS marker as a second selection marker to the gene replacement cassette led to a further 2-fold enrichment in transformants with gene replacement events, resulting in a gene replacement frequency of 55% . Based on the data it can be concluded that Agrobacterium-mediated transformation is an efficient tool for gene replacement and that the amdS gene can be successfully used as a second selection marker to select transformants with putative gene replacement. Transgenic Res, 2004 Oct, 13(5), 499 - 510 Expression of bioactive human interferon-gamma in transgenic rice cell suspension cultures; Chen TL et al.; We investigated the possibility of producing the therapeutic recombinant cytokine, Interferon-gamma (IFN-gamma), in transgenic rice cell (Oryza sativa, cultivar TNG67) suspension cultures . We tested expression of two vector constructs, each harboring an alphaAmy3 leader peptide and a C-terminus His 6 tag fused to a human IFN-gamma cDNA, one driven by a sucrose-starvation inducible promoter (rice alphaAmy3 promoter) and the other by a constitutive maize ubiquitin promoter, in rice cell suspensions, introduced via Agrobacterium tumefaciens . There was a significant difference in the amounts of recombinant IFN-gamma protein produced by the Ups and Amy cell lines, as cytosolic and secretory proteins respectively . Immunological analysis of IFN-gamma recombinant protein conferred a dose-dependent anti-dengue virus activity in human A549 cells, similar to the commercial product . We discuss the attractive attributes of using rice cell suspension system for the expression of therapeutic recombinant IFN-gamma. Transgenic Res, 2004 Oct, 13(5), 475 - 85 Expression of cold-tolerant pyruvate, orthophosphate dikinase cDNA, and heterotetramer formation in transgenic maize plants; Ohta S et al.; Maize is a typical C4 plant of the NADP-malic enzyme type, and its high productivity is supported by the C4 photosynthetic cycle, which concentrates atmospheric CO2 in the leaves . The plant exhibits superior photosynthetic ability under high light and high temperature, but under cold conditions the photosynthetic rate is significantly reduced . Pyruvate orthophosphate dikinase (PPDK), a key enzyme of the C4 pathway in maize, loses its activity below about 12 degrees C by dissociation of the tetramer and it is considered as one possible cause of the reduction in the photosynthetic rate of maize at low temperatures . To improve the cold stability of the enzyme, we introduced a cold-tolerant PPDK cDNA isolated from Flaveria brownii into maize by Agrobacterium-mediated transformation . We obtained higher levels of expression by using a double intron cassette and a chimeric cDNA made from F . bidentis and F . brownii with a maximum content of I mg/g fresh weight . In leaves of transgenic maize, PPDK molecules produced from the transgene were detected in cold-tolerant homotetramers or in heterotetramers of intermediate cold susceptibility formed with the internal PPDK . Simultaneous introduction of an antisense gene for maize PPDK generated plants in which the ratio of heterolologous and endogenous PPDK was greatly improved . Arrhenius plot analysis of the enzyme extracted from one such plant revealed that the break point was shifted about 3 degrees C lower than that of the wild type. Transgenic Res, 2004 Oct, 13(5), 451 - 61 Generation of marker-free transgenic maize by regular two-border Agrobacterium transformation vectors; Huang S et al.; By introducing additional T-DNA borders into a binary plasmid used in Agrobacterium-mediated plant transformation, previous studies have demonstrated that the marker gene and the gene of interest (GOI) can be carried by independent T-strands, which sometimes integrate in unlinked loci in the plant genome . This allows the recovery of marker-free transgenic plants through genetic segregation in the next generation . In this study, we have found that by repositioning the selectable marker gene in the backbone and leaving only the GOI in the T-DNA region, a regular two-border binary plasmid was able to generate marker-free transgenic maize plants more efficiently than a conventional single binary plasmid with multiple T-DNA borders . These results also provide evidence that both the right and left borders can initiate and terminate T-strands . Such non-canonical initiation and termination of T-strands may be the basis for the elevated frequencies of cotransformation and unlinked insertions. Plant J, 2004 Dec, 40(6), 870 - 81 Overexpression of tomato LeAGP-1 arabinogalactan-protein promotes lateral branching and hampers reproductive development; Sun W et al.; LeAGP-1 is a glycosylphosphatidylinositol (GPI)-anchored arabinogalactan-protein (AGP) in tomato (Lycopersicon esculentum) . Patterns of mRNA expression and protein localization for LeAGP-1 indicate that it likely functions in certain aspects of plant growth and development . To elucidate LeAGP-1 function(s), transgenic tomato plants expressing enhanced green fluorescent protein (GFP) fused to LeAGP-1 {GFP-LeAGP-1} or two LeAGP-1 variants, one lacking the C-terminal GPI-anchor domain {GFP-LeAGP-1DeltaC} and the other lacking the lysine-rich domain {GFP-LeAGP-1DeltaK}, under the control of the CaMV35S promoter were produced using Agrobacterium-mediated transformation . Transgenic T0 and T1 lines with high levels of both GFP-LeAGP-1 mRNA and protein: (i) were significantly shorter; (ii) were highly branched; (iii) produced more flower buds, but most of these flowers did not mature, resulting in less fruit production; and (iv) produced seeds that were significantly smaller than normal seeds . Overexpression of LeAGP-1DeltaK had a similar or even more pronounced effect on plant vegetative and reproductive growth, while the effect of LeAGP-1DeltaC overexpression on plant reproduction was minimal . These results indicate that the GPI anchor is critical for LeAGP-1 function . As the phenotype of GFP-LeAGP-1 overexpressing transgenic plants is similar to that of cytokinin-overproducing plants, mRNA expression patterns of LeAGP-1 under different hormone treatments were examined . Cytokinins upregulated LeAGP-1 mRNA expression, while auxins and ABA inhibited LeAGP-1 mRNA expression . Based on these results, GPI-anchored LeAGP-1 most likely functions in plant growth and development in concert with auxin/cytokinin signaling. Biotechnol Bioeng, 2005 Jan 20, 89(2), 188 - 94 Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor; Rodriguez M et al.; When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity . TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries . An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals . Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues . The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work . Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells . (c) 2004 Wiley Periodicals, Inc. J Exp Bot . 2004 Dec 6; {Epub ahead of print} A red beet (Beta vulgaris) UDP-glucosyltransferase gene induced by wounding, bacterial infiltration and oxidative stress; Sepulveda-Jimenez G et al.; Mechanical wounding, infiltration with P . syringae or A . tumefaciens, and exposure to an H2O2-generating system (Glc/Glc oxidase) induce betacyanin synthesis in red beet (Beta vulgaris) leaves . These conditions also induced the expression of BvGT, a gene encoding a glucosyltransferase (GT) from Beta vulgaris . BvGT has a high similarity to Dorotheanthus bellidiformis betanidin-5 GT involved in betacyanin synthesis . Furthermore, the transient expression of a BvGT antisense construct resulted in the reduction of BvGT transcript accumulation and betanin synthesis, suggesting a role for this gene product in betacyanin glucosylation . In addition, the NADPH oxidase inhibitor, diphenylene iodonium (DPI), inhibited the accumulation of the BvGT transcript in response to infiltration with Agrobacterium tumefaciens . Hence, this result suggests that ROS produced by a plasma membrane NADPH oxidase may act as a signal to induce BvGT expression, necessary for betanin synthesis after wounding and bacterial infiltration. Plant Cell Rep . 2004 Dec 3; {Epub ahead of print} Stable genetic transformation of castor (Ricinus communis L.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axes from mature seeds; Sujatha M et al.; A protocol for the transformation of castor embryo axes using the pCAMBIA vector 1304 in disarmed Agrobacterium tumefaciens strain EHA105 is presented . Co-cultivated explants were initially subjected to expansion and proliferation on MS medium with 0.5 mg l(-1) TDZ followed by three cycles of selection on medium with 0.5 mg l(-1) BA and increasing concentrations of hygromycin (20-40-60 mg l(-1)) . Selected shoot clusters were transferred to medium with 0.5 mg l(-1) BA for proliferation and 0.2 mg l(-1) BA for shoot elongation . Elongated shoots were rooted on half-strength MS medium with 2.0 mg l(-1) NAA . The presence and stable integration of the hpt gene was confirmed through PCR, RT-PCR, PCR-Southern blot, sequence analysis, Southern blot analysis and PCR analysis of progeny . Southern blot analysis of the primary transformants showed single copy integration and progeny analysis revealed monogenic inheritance of the introduced gene . This paper reports the first successful attempt at producing transgenic castor. Acta Microbiol Immunol Hung, 2004, 51(3), 321 - 32 Bacterial models for tumor development . Mini-review; Gyemant N et al.; The tumor-inducing effects of Agrobacterium, Bartonella and Helicobacter bacterial species are compared step by step . An analogy for the existence of these individual steps is considered in connection with the development of cancer . The transformations of eukaryotic cells occur in particular in the type IV secretion system, i.e . involving the simultaneous transmission of DNA and protein from bacterial cells to eukaryotic cells . Thus, transfected cells facilitate the indefinite growth of tissue cells and additionally produce growth factors, triggering further bacterial multiplication . The higher numbers of bacteria then produce more transfection and the cycle repeats as long as the host lives . The main limiting factor is the frequency of bacterial infection, while the secondary rate-limiting factors are the levels of transforming growth factors and factors triggering bacteria growth . CONCLUSIONS: Analogous processes are probably responsible for the tumor induction by the three different bacterial species; however, the critical points for eradication are different . The early eradication or limitation of B . henselae or H . pylori can prevent hemangiomas, stomach cancer and malignant cell proliferation . The crown gall formation by A . tumefaciens can only be avoided by prevention of the transforming activity of a single bacterial infection . Questions arise as to what is common in the three processes, and the nature of the rate-limiting step in the three different models . The frequency of transformation is the rate-limiting step, but the co-transmission of the DNA-protein complex is common in the three systems. Proc Natl Acad Sci U S A, 2004 Dec 7, 101(49), 17228 - 33 Epub 2004 Dec 7. Agrobacterium VirB10, an ATP energy sensor required for type IV secretion; Cascales E et al.; Bacteria use type IV secretion systems (T4SS) to translocate DNA and protein substrates to target cells of phylogenetically diverse taxa . Recently, by use of an assay termed transfer DNA immunoprecipitation (TrIP), we described the translocation route for a DNA substrate {T-DNA, portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells} of the Agrobacterium tumefaciens VirB/D4 T4SS in terms of a series of temporally and spatially ordered substrate contacts with subunits of the secretion channel . Here, we report that the bitopic inner membrane protein VirB10 undergoes a structural transition in response to ATP utilization by the VirD4 and VirB11 ATP-binding subunits, as monitored by protease susceptibility . VirB10 interacts with inner membrane VirD4 independently of cellular energetic status, whereas the energy-induced conformational change is required for VirB10 complex formation with an outer membrane-associated heterodimer of VirB7 lipoprotein and VirB9, as shown by coimmunoprecipitation . Under these conditions, the T-DNA substrate is delivered from the inner membrane channel components VirB6 and VirB8 to periplasmic and outer membrane-associated VirB2 pilin and VirB9 . We propose that VirD4 and VirB11 coordinate the ATP-dependent formation of a VirB10 "bridge" between inner and outer membrane subassemblies of the VirB/D4 T4SS, and that this morphogenetic event is required for T-DNA translocation across the A . tumefaciens cell envelope. Fitoterapia, 2004 Dec, 75(7-8), 737 - 9 Antibacterial activity of Cichorium intybus; Petrovic J et al.; Antibacterial activity of the water, ethanol and ethyl acetate extracts of Cichorium intybus was investigated . All the tested extracts showed antibacterial activity, the ethyl acetate extract being the most active . Water extract inhibits Agrobacterium radiobacter sp . tumefaciens, Erwinia carotovora, Pseudomonas fluorescens and P . aeruginosa. FEBS Lett, 2004 Nov 19, 577(3), 345 - 50 Transgenic plant-derived siRNAs can suppress propagation of influenza virus in mammalian cells; Zhou Y et al.; As an example of the cost-effective large-scale generation of small-interfering RNA (siRNAs), we have created transgenic tobacco plants that produce siRNAs targeted to the mRNA of the non-structural protein NS1 from the influenza A virus subtype H1N1 . We have investigated if these siRNAs, specifically targeted to the 5'-portion of the NS1 transcripts (5mNS1), would suppress viral propagation in mammalian cells . Agroinfiltration of transgenic tobacco with an Agrobacterium strain harboring a 5mNS1-expressing binary vector caused a reduction in 5mNS1 transcripts in the siRNA-accumulating transgenic plants . Further, H1N1 infection of siRNA-transfected mammalian cells resulted in significant suppression of viral replication . These results demonstrate that plant-derived siRNAs can inhibit viral propagation through RNA interference and could potentially be applied in control of viral-borne diseases. Protein Expr Purif, 2004 Dec, 38(2), 161 - 9 The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR; Boshoff A et al.; Molecular chaperones of the heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) play an important role in the folding and functioning of cellular protein machinery . The dnaK gene from the plant pathogen Agrobacterium tumefaciens RUOR was amplified using the polymerase chain reaction and the DnaK protein (Agt DnaK) was over-produced as a His-tagged protein in Escherichia coli . The Agt DnaK amino acid sequence was 96% identical to the A . tumefaciens C58 DnaK sequence and 65% identical to the E . coli DnaK sequence . Agt DnaK was shown to be able to functionally replace E . coli DnaK in vivo using complementation assays with an E . coli dnaK756 mutant strain and a dnaK52 deletion strain . Over-production and purification of Agt DnaK was successful, and allowed for further characterization of the protein . Kinetic analysis of the basal ATPase activity of purified Agt DnaK revealed a Vmax of 1.3 nmol phosphate released per minute per milligram DnaK, and a Km of 62 microM ATP . Thus, this is the first study to provide both in vivo and in vitro evidence that Agt DnaK has the properties of a molecular chaperone of the Hsp70 family. Mol Microbiol, 2004 Dec, 54(5), 1199 - 211 Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion; Atmakuri K et al.; Bacteria use type IV secretion systems (T4SS) to translocate DNA (T-DNA) and protein substrates across the cell envelope . By transfer DNA immunoprecipitation (TrIP), we recently showed that T-DNA translocates through the Agrobacterium tumefaciens VirB/D4 T4SS by forming close contacts sequentially with the VirD4 receptor, VirB11 ATPase, the inner membrane subunits VirB6 and VirB8 and, finally, VirB2 pilin and VirB9 . Here, by TrIP, we show that nucleoside triphosphate binding site (Walker A motif) mutations do not disrupt VirD4 substrate binding or transfer to VirB11, suggesting that these early reactions proceed independently of ATP binding or hydrolysis . In contrast, VirD4, VirB11 and VirB4 Walker A mutations each arrest substrate transfer to VirB6 and VirB8, suggesting that these subunits energize this transfer reaction by an ATP-dependent mechanism . By co-immunoprecipitation, we supply evidence for VirD4 interactions with VirB4 and VirB11 independently of other T4SS subunits or intact Walker A motifs, and with the bitopic inner membrane subunit VirB10 . We reconstituted substrate transfer from VirD4 to VirB11 and to VirB6 and VirB8 by co-synthesis of previously identified 'core' components of the VirB/D4 T4SS . Our findings define genetic requirements for DNA substrate binding and the early transfer reactions of a bacterial type IV translocation pathway. Biochim Biophys Acta, 2004 Nov 11, 1694(1-3), 219 - 34 Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems; Christie PJ; The translocation of DNA across biological membranes is an essential process for many living organisms . In bacteria, type IV secretion systems (T4SS) are used to deliver DNA as well as protein substrates from donor to target cells . The T4SS are structurally complex machines assembled from a dozen or more membrane proteins in response to environmental signals . In Gram-negative bacteria, the conjugation machines are composed of a cell envelope-spanning secretion channel and an extracellular pilus . These dynamic structures (i) direct formation of stable contacts-the mating junction-between donor and recipient cell membranes, (ii) transmit single-stranded DNA as a nucleoprotein particle, as well as protein substrates, across donor and recipient cell membranes, and (iii) mediate disassembly of the mating junction following substrate transfer . This review summarizes recent progress in our understanding of the mechanistic details of DNA trafficking with a focus on the paradigmatic Agrobacterium tumefaciens VirB/D4 T4SS and related conjugation systems. Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2003 - 12 Mesorhizobium septentrionale sp . nov . and Mesorhizobium temperatum sp . nov., isolated from Astragalus adsurgens growing in the northern regions of China; Gao JL et al.; Ninety-five rhizobial strains isolated from Astragalus adsurgens growing in the northern regions of China were classified into three main groups, candidate species I, II and III, based on a polyphasic approach . Comparative analysis of full-length 16S rRNA gene sequences of representative strains showed that candidate species I and II were Mesorhizobium, while candidate species III, which consisted of non-nodulating strains, was closely related to Agrobacterium tumefaciens . The phylogenetic relationships of the three candidate species and some related strains were also confirmed by the sequencing of glnA genes, which were used as an alternative chromosomal marker . The DNA-DNA relatedness was between 11.3 and 47.1 % among representative strains of candidate species I and II and the type strains of defined Mesorhizobium species . Candidate III had DNA relatedness of between 4.3 and 25.2 % with type strains of Agrobacterium tumefaciens and Agrobacterium rubi . Two novel species are proposed to accommodate candidate species I and II, Mesorhizobium septentrionale sp . nov . (type strain, SDW014(T)=CCBAU 11014(T)=HAMBI 2582(T)) and Mesorhizobium temperatum sp . nov . (type strain, SDW018(T)=CCBAU 11018(T)=HAMBI 2583(T)), respectively . At least two distinct nodA sequences were identified among the strains . The numerically dominant nodA sequence type was most similar to that from the Mesorhizobium tianshanense type strain and was identified in strains belonging to the two novel species as well as other, as yet, undefined genome types . Host range studies indicate that the different nodA sequences correlate with different host ranges . Further comparative studies with the defined Agrobacterium species are needed to clarify the taxonomic identity of candidate species III. Mol Biotechnol, 2004 Nov, 28(3), 175 - 83 Synthesis and assembly of anthrax lethal factor-cholera toxin B-subunit fusion protein in transgenic potato; Kim TG et al.; A DNA encoding the 27-kDa domain I of anthrax lethal factor protein (LF), was linked to the carboxyl terminus of the cholera toxin B-subunit (CTB-LF) . The CTB-LF fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated in vivo transformation methods and antibiotic-resistant plants were regenerated . The CTB-LF fusion gene was detected in transformed potato leaf genomic DNA by polymerase chain reaction (PCR)-mediated DNA amplification . Immunoblot analysis with anti-CTB and anti-LF primary antibodies verified the synthesis and assembly of biologically active CTB-LF fusion protein oligomers in transformed plant tuber tissues . Furthermore, the binding of CTB-LF fusion protein pentamers to intestinal epithelial cell membrane receptors measured by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) indicated that the CTB-LF fusion protein made up approx 0.002% of the total soluble tuber protein . Synthesis of CTB-LF monomers and their assembly into biologically active CTB-LF fusion protein pentamers in potato tuber tissues demonstrates the feasibility of using edible plants for production and delivery of adjuvanted LF protein for CTB-mediated immunostimulation of mucosal immune responses against anthrax toxin. Plant Cell Rep . 2004 Nov 5; {Epub ahead of print} Agrobacterium-mediated transformation of the wetland monocot Typha latifolia L . (Broadleaf cattail); Nandakumar R et al.; An Agrobacterium-mediated model transformation system was standardized for the wetland monocot Typha latifolia L . to achieve the long-term objective of introducing candidate genes for phytoremediation . Two binary plasmid vectors, pCAMBIA1301/EHA105 and pTOK233/LBA4404, both containing the gus (beta-glucuronidase) and hptII (hygromycin phosphotransferase II) genes, were used for transformation . Fifty-day-old 5 mg/l picloram-derived calli were cocultivated and selected on medium containing 20 mg/l or 40 mg/l hygromycin . Resistant calli were regenerated on medium supplemented with 5 mg/l 6-benzylaminopurine, with or without 20 mg/l or 40 mg/l hygromycin and with or without charcoal (10 g/l) . Transient GUS activity in explants ranged between 28% and 36% . Hygromycin-resistant calli, selected after 3 months, showed stable GUS expression . A total of 46 plants were regenerated and established in the greenhouse; 13 showed stable GUS expression . Cocultivation of dark culture-derived calli, directly selected on regeneration medium containing 20 mg/l hygromycin and rooted on medium with 20 mg/l hygromycin was the best protocol . The addition of charcoal did not have any effect on regeneration . PCR and Southern analyses of transgenic calli and transgenic plants confirmed the presence of the introduced genes . In conclusion, T . latifolia could be genetically transformed by Agrobacterium tumefaciens. Biotechnol Bioeng, 2004 Dec 20, 88(6), 722 - 9 Occurrence of circadian rhythms in hairy root cultures grown under controlled conditions; Lanoue A et al.; Hairy roots obtained by transformation via Agrobacterium rhizogenes provide an artificial plant material devoid of aerial parts with high growth on hormone-free media . Fundamental knowledge of hairy root physiology is essential to develop and control its culture . In contrast to shake-flask cultures, a bioreactor set-up combined with on-line data logging provides an efficient tool to study rapid physiological variations in hairy root cultures . Datura innoxia hairy roots were grown in a bioreactor equipped with on-line data analyses of pH, dissolved oxygen (pO2), conductivity, oxygen, and carbon dioxide . The experiments were done at a constant temperature and in the absence of light cues . The results obtained showed that the carbon dioxide evolution rate (CER) presented regular oscillations during the culture . Similar oscillations were also observed for the oxygen uptake rate (OUR) . These signals were treated mathematically to look for the existence of a rhythm . An autocorrelation function was used to detect any periodic components . The results demonstrate that hairy root respiration exhibited peaks of 1 day . These oscillations, having a period of about 24 h, were also observed in pH and conductivity signals, although not for the pO2 signal . The data acquired in the absence of hairy roots showed that the observed periodic behavior was not an artifact . No effect on rhythms was observed by the imposition of an external "day/night" cycle . The fact that oscillations persisted in the absence of external stimuli, with a free-running period of 24 h, suggests that a circadian rhythm exists in hairy roots of D . innoxia. Avian Dis, 2004 Sep, 48(3), 663 - 8 Immunization of chickens with VP2 protein of infectious bursal disease virus expressed in Arabidopsis thaliana; Wu H et al.; Transgenic plants represent a safe, effective, and inexpensive way to produce vaccines . The immunogenicity of VP2 protein of an infectious bursal disease (IBD) virus variant E isolate expressed in transgenic Arabidopsis thaliana was compared with a commercial vaccine in specific-pathogen-free broiler chickens . The VP2 coding sequence was isolated and integrated into A . thaliana genome by Agrobacterium tumefaciens-mediated transformation . Soluble VP2 expressed in transgenic plants was used to immunize chickens . Chickens receiving oral immunization with plant-derived VP2 at 1 and 3 wk of age had an antibody response using enzyme-linked immunosorbent assay and 80% protection against challenge infection at 4 wk . Chickens primed with a commercial vaccine at 1 wk followed by an oral booster with VP2 expressed in plants at 3 wk of age showed 90% protection . Chickens immunized with a commercial vaccine at 1 and 3 wk showed 78% protection . Results supported the efficacy of plant-produced VP2 as a vaccine against IBD. Microbiology, 2004 Nov, 150(Pt 11), 3857 - 66 Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58; Rondon MR et al.; Using the complete genome sequence from Agrobacterium tumefaciens C58, the authors identified a secondary metabolite gene cluster that encodes the biosynthesis of a metabolite with siderophore activity . Support for this conclusion came from genetic and regulatory analysis of the gene cluster, along with the purification of a metabolite from A . tumefaciens C58 with iron-chelating activity . Genetic analysis of mutant strains disrupted in this gene cluster showed that these strains grew more slowly than the wild-type strain in medium lacking iron . Additionally, the mutant strains failed to produce a chrome-azurol-S-reactive material in liquid or solid medium, and failed to produce the metabolite with iron-chelating characteristics that was identified in the wild-type strain . Addition of this purified metabolite to the growth medium of a mutant strain restored its ability to grow in iron-deficient medium . Furthermore, expression of this gene cluster was induced by growth under iron-limiting conditions, suggesting that expression of this gene cluster occurs when iron is scarce . These data are all consistent with the proposal that the proteins encoded by this gene cluster are involved in the production of a siderophore . Interestingly, these proteins show the highest level of amino acid similarity to proteins from a gene cluster found in the filamentous cyanobacterium Nostoc sp . PCC7120, rather than to known siderophore biosynthetic enzymes . Given these properties, it is proposed that the siderophore produced by A . tumefaciens C58 will have a unique chemical structure . Production of the siderophore was not required for virulence of A . tumefaciens when tested with a standard stem inoculation assay. Infect Control Hosp Epidemiol, 2004 Oct, 25(10), 885 - 7 Molecular typing of Agrobacterium species isolates from catheter-related bloodstream infections; Giammanco GM et al.; Agrobacterium isolates from intravenous catheters of three hospitalized patients were initially identified as A . tumefaciens, but inability to produce 3-ketolactose revealed that two of them were A . vitis . However, rDNA analysis correlated all of the isolates to A . tumefaciens . Pulsed-field gel electrophoresis analysis ascertained the nosocomial transmission of the infection. Planta . 2004 Oct 29; {Epub ahead of print} Expression of Escherichia coli branching enzyme in caryopses of transgenic rice results in amylopectin with an increased degree of branching; Kim WS et al.; Physiochemical properties of starch are dependent on several factors including the relative abundance of amylose and amylopectin, and the degree of branching of amylopectin . Utilizing Agrobacterium-mediated transformation, a construct containing the coding region of branching enzyme of Escherichia coli, under transcriptional control of the rice ( Oryza sativa L.) starch-branching enzyme promoter was introduced into rice cv . Nakdong . To enhance glgB expression, the first intron of rice starch-branching enzyme and the matrix attachment region (MAR) sequence from chicken lysozyme were included in the expression vector . Eleven independent transgenic rice plants were generated . Southern blot analysis indicated that the copy number of glgB integrated into transgenic rice varied from one to five . High-performance liquid chromatographic analysis of starch from transgenic lines revealed that amylopectin from transgenic lines exhibited greater branching than that of non-transgenic rice . The A/B1 ratio in amylopectin increased from 1.3 to 2.3 and the total branching ratio, A+B1/B-rest, increased from 6 to 12 in transgenic rice . The observed increase in the short-chain fractions with a degree of polymerization between 6 and 10 is expected to have a significant effect on retrogradation . Our study demonstrates that amylopectin branching can be altered in vivo, thus changing the physicochemical properties of starch. Chembiochem, 2004 Nov 5, 5(11), 1535 - 42 Integrating input from multiple signals: the VirA/VirG two-component system of Agrobacterium tumefaciens; Mukhopadhyay A et al.; Bacteria, fungi, and plants exploit histidine sensor kinase/response regulators to mobilize complex responses to inputs as diverse as environmental stimuli and hormonal regulation . More than 50 such two-component systems are found in many organisms, yet the mechanisms of signal perception, phosphotransfer regulation, and even the nature of the activating signals remain poorly defined . Here we resolve each phosphate transfer event in vivo for the Agrobacterium tumefaciens virulence two-component system VirA/VirG . The input signals for this system are known, and the complex autocatalytic regulation of the signaling components has been removed . Two separate and independent phosphotransfer events are resolved, an initial ATP-->sensorHis approximately PO(4)-->receiver approximately PO(4), that may be activated by xenognostic sugar/low pH, and a subsequent ATP-->His approximately PO(4)-->VirG approximately PO(4) that requires xenognostic phenol activation . The identification of these separate pathways places biochemical limits on the regulated steps in this two-component signal transduction module and further extends the model of how a single sensor is able to integrate multiple input stimuli. Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15742 - 7 Epub 2004 Oct 25. Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome; Lu R et al.; Viral infection in both plant and invertebrate hosts requires a virus-encoded function to block the RNA silencing antiviral defense . Here, we report the identification and characterization of three distinct suppressors of RNA silencing encoded by the approximately 20-kb plus-strand RNA genome of citrus tristeza virus (CTV) . When introduced by genetic crosses into plants carrying a silencing transgene, both p20 and p23, but not coat protein (CP), restored expression of the transgene . Although none of the CTV proteins prevented DNA methylation of the transgene, export of the silencing signal (capable of mediating intercellular silencing spread) was detected only from the F(1) plants expressing p23 and not from the CP- or p20-expressing F(1) plants, demonstrating suppression of intercellular silencing by CP and p20 but not by p23 . Thus, intracellular and intercellular silencing are each targeted by a CTV protein, whereas the third, p20, inhibits silencing at both levels . Notably, CP suppresses intercellular silencing without interfering with intracellular silencing . The novel property of CP suggests a mechanism distinct to p20 and all of the other viral suppressors known to interfere with intercellular silencing and that this class of viral suppressors may not be consistently identified by Agrobacterium coinfiltration because it also induces RNA silencing against the infiltrated suppressor transgene . Our analyses reveal a sophisticated viral counter-defense strategy that targets the silencing antiviral pathway at multiple steps and may be essential for protecting CTV with such a large RNA genome from antiviral silencing in the perennial tree host. Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15805 - 10 Epub 2004 Oct 25. Expression of antiapoptotic genes bcl-xL and ced-9 in tomato enhances tolerance to viral-induced necrosis and abiotic stress; Xu P et al.; D satellite RNA (satRNA) is a strain of cucumber mosaic virus (CMV) satRNA that induces an epidemic lethal disease in tomato . No natural resistance or tolerance has ever been found . Previously, we demonstrated the involvement of programmed cell death in disease development . Here, transgenic tomato plants expressing animal antiapoptotic genes bcl-xL and ced-9 were generated through agrobacterium-mediated transformation . High expression of bcl-xL or ced-9 affected plant growth and seed development . Inoculation of seedlings with CMV/D satRNA at T(1) and T(2) generations resulted in delayed cell-death symptoms or absence of symptoms . The degree of symptom suppression was correlated with increasing expression levels of the transgenes . Survival rates were compared among inoculated transgenic lines expressing bcl-xL, ced-9, and bcl-xL (G138A), a loss-of-function mutant of bcl-xL . More than 80% of the bcl-xL and ced-9 T(1) transgenic lines showed higher survival rates than the average for bcl-xL (G138A) transgenic lines . Total RNA extracted from surviving plants contained D satRNA, indicating systemic accumulation of D satRNA . Thus, expression of bcl-xL and ced-9 improved tolerance to, rather than resistance to, CMV/D satRNA infection . In addition, expression of bcl-xL and ced-9 specifically abrogated the formation of necrotic lesions, but not other symptoms, in tomato leaves during chilling at 4 degrees C . At 7 degrees C, temperature-induced leaf senescence was dramatically delayed in bcl-xL and ced-9 transgenic plants, and high levels of anthocyanins accumulated, possibly limiting oxidative stress . Hence, expression of these animal antiapoptotic genes improved plant survival under abiotic or biotic stress. Plant Cell Rep . 2004 Oct 21; {Epub ahead of print} Agrobacterium tumefaciens-mediated transformation of chickpea ( Cicer arietinum L.): gene integration, expression and inheritance; Polowick PL et al.; A reproducible method of Agrobacterium-mediated transformation was developed for Cicer arietinum (chickpea) . Initial explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed . The plasmid contained a bi-functional fusion gene conferring both beta-glucuronidase and neomycin phosphotransferase activities, under the control of a 35S35SAMV promoter . Using a series of tissue culture media for co-cultivation, shoot initiation and rooting, we recovered transgenic plants from approximately 1.3% of the sliced embryo axes . The addition of a shoot elongation medium to the protocol improved the success rate to 3.1% but increased the time in tissue culture . Inheritance of the gus gene was followed through four generations, both through expression and Southern hybridization assays, and showed the expected Mendelian inheritance pattern. Plant Cell Rep . 2004 Oct 19; {Epub ahead of print} Different effects on ACC oxidase gene silencing triggered by RNA interference in transgenic tomato; Xiong AS et al.; RNA interference (RNAi) is a potent trigger for specific gene silencing of expression in a number of organisms and is an efficient way of shutting down gene expression . 1-Aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the oxidation of ACC to ethylene, a plant growth regulator that plays an important role in the tomato ripening process . In this research, to produce double-stranded (ds)RNA of tomato ACC oxidase, we linked the sense and antisense configurations of DNA fragments with 1,002-bp or 7-nt artificially synthesized fragments, respectively, and then placed these under the control of a modified cauliflower mosaic virus 35S promoter . The dsRNA expression unit was successfully introduced into tomato cultivar Hezuo 906 by Agrobacterium tumefaciens-mediated transformation . Molecular analysis of 183 transgenic plants revealed that the dsRNA unit was integrated into the tomato genome . With respect to the construct with the 1,002-bp linker, the severity of phenotypes indicated that 72.3% of the transformed plants had non-RNA interference, about 18.1% had semi-RNA interference, and only 9.6% had full-RNA interference . However when the construct with the 7-nt linker was used for transformation, the results were 13.0%, 18.0%, and 69.0%, respectively, indicating that the short linker was more efficient in RNAi of transgenic tomato plants . When we applied this fast way of shutting down the ACC oxidase gene, transgenic tomato plants were produced that had fruit which released traces of ethylene and had a prolonged shelf life of more than 120 days . The RNA and protein analyses indicated that there was non-RNA interference, semi-RNA interference and full-RNA interference of ACC oxidase in the transgenic tomato plants. Plant Cell, 2004 Nov, 16(11), 3148 - 67 Epub 2004 Oct 19. Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation; Hwang HH et al.; Agrobacterium tumefaciens uses a type IV secretion system (T4SS) to transfer T-DNA and virulence proteins to plants . The T4SS is composed of two major structural components: the T-pilus and a membrane-associated complex that is responsible for translocating substrates across both bacterial membranes . VirB2 protein is the major component of the T-pilus . We used the C-terminal-processed portion of VirB2 protein as a bait to screen an Arabidopsis thaliana cDNA library for proteins that interact with VirB2 in yeast . We identified three related plant proteins, VirB2-interacting protein (BTI) 1 (BTI1), BTI2, and BTI3 with unknown functions, and a membrane-associated GTPase, AtRAB8 . The three BTI proteins also interacted with VirB2 in vitro . Preincubation of Agrobacterium with GST-BTI1 protein decreased the transformation efficiency of Arabidopsis suspension cells by Agrobacterium . Transgenic BTI and AtRAB8 antisense and RNA interference Arabidopsis plants are less susceptible to transformation by Agrobacterium than are wild-type plants . The level of BTI1 protein is transiently increased immediately after Agrobacterium infection . In addition, overexpression of BTI1 protein in transgenic Arabidopsis results in plants that are hypersusceptible to Agrobacterium-mediated transformation . Confocal microscopic data indicate that GFP-BTI proteins preferentially localize to the periphery of root cells in transgenic Arabidopsis plants, suggesting that BTI proteins may contact the Agrobacterium T-pilus . We propose that the three BTI proteins and AtRAB8 are involved in the initial interaction of Agrobacterium with plant cells. Sci China C Life Sci, 2004 Aug, 47(4), 382 - 8 Effect of cDNA fragments in different length derived from potato virus Y coat protein gene on the induction of RNA-mediated virus resistance; Zhu J et al.; We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RNA-mediated rather than coat protein-mediated . In this study, we cloned cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3' end of the PVY CP gene, and the cDNA fragments were introduced into tobacco (var . NC89) plants via Agrobacterium-mediated transformation system . The results of resistance assay showed that the CP cDNA fragments of 417 bp and 603 bp could confer resistance of the transgenic plants to PVY infection, but the fragment of 202 bp in length could not . Molecular analysis revealed that the resistance was RNA-mediated, which is believed to be a result of post-transcriptional gene silencing . The results indicate that the length of cDNA fragments needed for resistance induction was located somewhere between 202 bp and 417 bp from the 3' end of PVY CP gene. Sci China C Life Sci, 2004 Aug, 47(4), 322 - 31 Distribution of T-DNA carrying a Ds element on rice chromosomes; Wang J et al.; Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agrobacterium tumefaciens mediation . Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from independent transformants and classified into six main types on the basis of the origin of filler DNA between the right border of T-DNA and flanking sequence of rice genome . Type I sequences were the most common and showed canonical integration that T-DNA right border was followed by rice genome sequence with or without filler DNA of no more than 50 bp, while type II sequences displayed a vector-genome combination that T-DNA right border was followed by a vector fragment and then connected with rice genome sequence . The location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using BLAST analysis . The 340 Ds insertions at an average interval of 0.8 megabase (Mb) constructed a basic framework of Ds starter points on whole rice chromosomes . The frequency of T-DNA (Ds) inserted into the exons of predicted genes on chromosome one was 21% . Knowledge of T-DNA (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis. Mol Microbiol, 2004 Nov, 54(3), 742 - 54 A proline tRNA(CGG) gene encompassing the attachment site of temperate phage 16-3 is functional and convertible to suppressor tRNA; Blaha B et al.; Several temperate bacteriophage utilize chromosomal sequences encoding putative tRNA genes for phage attachment . However, whether these sequences belong to genes which are functional as tRNA is generally not known . In this article, we demonstrate that the attachment site of temperate phage 16-3 (attB) nests within an active proline tRNA gene in Rhizobium meliloti 41 . A loss-of-function mutation in this tRNA gene leads to significant delay in switching from lag to exponential growth phase . We converted the putative Rhizobium gene to an active amber suppressor gene which suppressed amber mutant alleles of genes of 16-3 phage and of Escherichia coli origin in R . meliloti 41 and in Agrobacterium tumefaciens GV2260 . Upon lysogenization of R . meliloti by phage 16-3, the proline tRNA gene retained its structural and functional integrity . Aspects of the co-evolution of a temperate phage and its bacterium host is discussed . The side product of this work, i.e . construction of amber suppressor tRNA genes in Rhizobium and Agrobacterium, for the first time widens the options of genetic study. Syst Appl Microbiol, 2004 Sep, 27(5), 603 - 11 Metabolic and genomic diversity of rhizobia isolated from field standing native and exotic woody legumes in southern Ethiopia; Wolde-meskel E et al.; Eighty-seven rhizobial strains isolated from root nodules of field standing native and exotic woody legumes in southern Ethiopia were characterized using the Biolog method and AFLP fingerprinting technique . Cluster analysis of the metabolic and genomic fingerprints revealed 18 and 25 groups, respectively, demonstrating considerable diversity in rhizobial population indigenous to Ethiopian soils . While 25 strains (29%) were linked to members of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium or Sinorhizobium, the bulk of the strains formed several distinct groups in both methods and did not relate to reference species included in the study . In contrast to exotic species which formed symbiosis with strains of only one specific genomic group, indigenous host species nodulated by metabolically and genomically diverse groups . The results in this study support the view, that long-term association between the symbionts allows gradual differentiation and diversity in compatible rhizobial population resident in native soils . Lack of significant metabolic and genomic relatedness to the reference strains in our results suggested that test strains in our collection probab