Agrobacterium

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 probably included 'unique' types, which belong to several yet undefined rhizobial species.

Planta . 2004 Oct 13; {Epub ahead of print}
An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato; Chye ML et al.; Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains . Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability . BjCHI1 expression in B . juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus . To verify this, transgenic potato ( Solanum tuberosum L . cv . Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R . solani . We also transformed potato with a cDNA encoding Hevea brasiliensis beta-1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU . In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect . Consistently, in vivo fungal bioassays with soil-borne R . solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU . Light microscopy and transmission electron microscopy revealed abundant intact R . solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants . Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.

J Bacteriol, 2004 Nov, 186(21), 7254 - 61
Osa protein constitutes a strong oncogenic suppression system that can block vir-dependent transfer of IncQ plasmids between Agrobacterium cells and the establishment of IncQ plasmids in plant cells; Lee LY et al.; The osa (oncogenic suppressive activity) gene of the IncW group plasmid pSa is sufficient to suppress tumorigenesis by Agrobacterium tumefaciens . osa confers oncogenic suppression by inhibiting VirE2 protein export . This result is similar, but not identical, to that of oncogenic suppression by the IncQ plasmid RSF1010 . We conducted a series of experiments to compare oncogenic suppression by these two systems . Agrobacterium strains harboring plasmids containing osa are more able to effect oncogenic suppression than are similar strains containing various RSF1010 derivatives . When osa is present within a donor Agrobacterium strain that also carries a derivative of RSF1010, the transfer of RSF1010 derivatives to recipient bacteria and their establishment in plants are blocked . Oncogenic suppression is still effected when the osa gene is integrated into the Agrobacterium chromosome, suggesting that it is the osa gene product that is active in suppression and that suppression does not require a protein-nucleic acid intermediate like that described for IncQ plasmids . Extracellular complementation experiments with tobacco leaf disks indicated that Osa blocks stable transfer of RSF1010 to plant cells by inhibiting transfer of VirE2, which is essential for the transfer of RSF1010 into plant cells, and not by inhibiting the actual transfer of RSF1010 itself . Our results suggest that Osa and RSF1010 cause oncogenic suppression by using different mechanisms.

Curr Microbiol, 2004 Nov, 49(5), 334 - 40
Mutants of Agrobacterium tumefaciens virG gene that activate transcription of vir promoter in Escherichia coli; Jung YC et al.; The virA and virG two-component regulatory system is essential for transcriptional activation of virulence (vir) genes in Agrobacterium tumefaciens in the presence of inducer molecules . The VirA/VirG mediated vir gene transcription depends on a specific interaction between the C-terminal domain of the alpha subunit (RpoA) of A . tumefaciens RNA polymerase (RNAP) and N-terminal domain of the VirG . However, such interaction does not occur between RNAP of E . coli and the VirG, thus vir gene activation in E . coli requires the presence of rpoA gene from A . tumefaciens . In this report, we describe VirG mutants that are capable of activating the expression of vir genes in E . coli in the absence of A . tumefaciens RpoA . The selected 45 VirG mutants exhibited a common amino acid substitution at position 56 and additional one or more substitutions at different positions; thus the amino acid at position 56 is likely to play a key role in the interaction with the RpoA of E . coli . Furthermore, two virG mutants, with amino acid substitutions of G56V/V7I/I106N and G56V/I77V, respectively, are capable of activating vir genes in E . coli in response to inducer acetosyringone in a virA-dependent manner, demonstrating that the interaction site between VirG and RpoA is separable from that of VirG and VirA . Therefore, it is possible to establish inducer-mediated vir gene expression in heterologous hosts using virG mutants that are capable of interacting with the RpoA of the respective bacterial hosts while retaining the ability to interact with the sensor VirA.

Yi Chuan Xue Bao, 2004 Aug, 31(8), 858 - 63
{Heteologous expression of Mortierella isabellina delta6 -fatty acid desaturase gene in soybean}; Li MC et al.; Gamma-linolenic acid (GLA, C18:3delta(6, 9, 12) is one of nutritionally important polyunsaturated fatty acids in human and animal diets . Vast majority oilseeds crops do not produce GLA . Delta6-fatty acid desaturase (D6D) is the rate-limiting enzyme, which catalyzes the conversion of linoleic acid (LA) and alpha-linolenic acid (ALA) to GLA and octadecatetraenoic acid (OTA) . In this study, a delta6-fatty acid desaturase gene isolated from Mortierella isabellina was transformed into some soybean cultivars Jilin 35, Jilin 47, Suinong 10, Suinong 14 and Heinong 37 successfully by agrobacterium-mediated cotylendon node transformation system . The results of PCR analysis, Southern blotting and Northern blotting with transgenic plants indicated that target gene was integrated into transgenic soybean genome and expressed at the level of RNA . Meanwhile, total fatty acids were extracted from transgenic seeds and methyl-esterified . Analysis of gas chromatograph (GC) showed that a novel peak corresponding to the standard of GLA methyl ester was detected, the highest percentage of gamma-lenolenic acid to total fatty acids in transgenic seeds was 27 . 067%, which indicated that Mortierella isabellina delta6-fatty acid desaturase gene was expressed in transgenic soybean . It is so far the first report on the expression of delta6-fatty acid desaturase gene in transgenic soybean in the world.

Plant Cell Rep . 2004 Oct 9; {Epub ahead of print}
Transgenic tall fescue containing the Agrobacterium tumefaciens ipt gene shows enhanced cold tolerance; Hu Y et al.; Embryogenic calli of Festuca arundinacea were transformed with the Agrobacterium tumefaciens isopentenyl transferase ( ipt) gene driven by a maize ubiquitin promoter . Tillering ability, levels of chlorophyll a and b, and cold tolerance were greatly increased in the transgenic turfgrass, which resulted in the plants remaining more vigorous and staying green longer under lower temperatures.

Plant Cell Rep . 2004 Oct 12; {Epub ahead of print}
Agrobacterium-mediated transformation of bottle gourd ( Lagenaria siceraria Standl.); Han JS et al.; We describe a procedure for producing transgenic bottle gourd plants by inoculating cotyledon explants with Agrobacterium tumefaciens strain AGL1 that carries the binary vector pCAMBIA3301 containing a glufosinate ammonium-resistance ( bar) gene and the beta- d-glucuronidase (GUS) reporter gene . The most effective bacterial infection was observed when cotyledon explants of 4-day-old seedlings were co-cultivated with Agrobacterium for 6-8 days on co-cultivation medium supplemented with 0.1-0.001 mg/l l-alpha-(2-aminoethoxyvinyl) glycine (AVG) . The putatively transformed shoots directly emerged at the proximal end of cotyledon explants after 2-3 weeks of culturing on selection medium containing 2 mg/l dl-phosphinothricin . These shoots were rooted after 3 weeks of culturing on half-strength MS medium containing 0.1 mg/l indole acetic acid and 1 mg/l dl-phosphinothricin . Transgenic plants were obtained at frequencies of 1.9% . Stable integration and transmission of the transgenes in T(1) generation plants were confirmed by a histochemical GUS assay, polymerase chain reaction and Southern blot analyses . Genetic segregation analysis of T(1) progenies showed that transgenes were inherited in a Mendelian fashion . To our knowledge, this study is the first to show Agrobacterium-mediated transformation in bottle gourd.

Plant Cell Rep . 2004 Oct 5; {Epub ahead of print}
Evaluation of 12 beta-lactam antibiotics for Agrobacterium-mediated transformation through in planta antibacterial activities and phytotoxicities; Ogawa Y et al.; The antibacterial activities of 12 beta-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 living in tobacco ( Nicotiana tabacum L.) leaf tissues, and their phytotoxicities to tobacco leaf tissues were evaluated . All beta-lactams at minimum bactericidal concentration (MBC) or higher showed weak bactericidal activities against agrobacteria persisting in tobacco leaf tissues . The beta-lactams evaluated were classified into two major groups according to their inhibitory effect on shoot regeneration of tobacco leaf tissues: (1) highly phytotoxic drugs, and (2) moderately phytotoxic drugs . According to these results, suitable kind and concentration of beta-lactam antibiotics were evaluated for Agrobacterium-mediated transformation.

Curr Genet . 2004 Oct 5; {Epub ahead of print}
Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium; Godio RP et al.; The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound . Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation . Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested . The promoter used was critically important in order to express heterologous genes in H . sublateritium . Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (P gpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective . Transformant clones showed a random integration pattern of plasmid DNA . Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations . All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic . The green fluorescent protein gene was expressed from the A . bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed . Transformation of H . sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.

Yi Chuan Xue Bao, 2004 May, 31(5), 518 - 24
{Endosperm-specific expression of the ferritin gene in transgenic rice (Oryza sativa L.) results in increased iron content of milling rice}; Liu QQ et al.; Iron deficiency is the most widespread micronutrient deficiency world-wild, and is estimated to affect about 30% of the world population . To increase the iron content of rice in Chinese, the 764 bp cDNA of ferritin gene was cloned from soybean (Phaseolus limensis), and constructed between the 1 353 bp rice glutelin GluB-1 promoter and NOS terminator in a binary vector pCAMIBA1301 . The constructed pYF1067 vector was introduced into Agrobacterium strain EHA105, and used for transformation of the primary callus derived from immature embryos of a high-yielding rice ( Oryza sativa L . ssp . japonica) variety Wuxiangjin 9 . Under the selection on hygromycin-containing medium, seventeen independent transgenic rice lines, total more than 80 transgenic plants, were finally regenerated, and most of these transgenic rice plants grew normally . PCR and Southern blot analysis of total DNA from primary transformants confirmed that one to three copies of the transgenes integrated into the genome of most of the transgenic plants, and they could be stably transmitted into the progeny of the transgenic rice . Northern blot analysis showed that the ferritin gene could specifically express in the endosperm of transgenic rice with high level, while no or low expression in leaves . The expression level varied among different independent transgenic rice plants . There was a significant effect of the high-expression of ferritin on the increased iron content in transgenic rice, the iron content in the milling rice of transgenic rice was up to 64% higher than that of the untransformed wild-type plant, whereas no significant alteration of the zinc level occurred between these two type rice plants.

Mol Biotechnol, 2004 Oct, 28(2), 113 - 9
A rapid and highly efficient method for transformation of sugarcane callus; Santosa DA et al.; Modern sugarcane cultivars have complex genetic characteristics and low fertility that render their genetic improvement through traditional breeding difficult . Genetic engineering methodology to introduce foreign genes provides new opportunities for the genetic improvement of sugarcane cultivars . One of prerequisites for successful insertion of a gene cassette into the plant genome is the availability of an efficient transformation protocol . An improved protocol for Agrobacterium-mediated transformation of sugarcane is described . Between 85 and 100% of calli transformed using this procedure produced new calli, and 100% of them were positive for the inserted gene . The whole procedure permitted the production of transgenic calli in a short time (1.5 mo) . The transformed calli can be cultured further for the production of the inserted gene-encoded enzyme by using cell culture, or they can be regenerated into transgenic plants . This protocol may be implemented also for the generation of transgenic plants from other species.

FEMS Microbiol Lett, 2004 Oct 15, 239(2), 241 - 8
Identification of an antibacterial compound, benzylideneacetone, from Xenorhabdus nematophila against major plant-pathogenic bacteria; Ji D et al.; An entomopathogenic bacterium, Xenorhabdus nematophila, is known to have potent antibiotic activities to maintain monoxenic condition in its insect host for effective pathogenesis and ultimately for optimal development of its nematode symbiont, Steinernema carpocapsae . In this study we assess its antibacterial activity against plant-pathogenic bacteria and identify its unknown antibiotics . The bacterial culture broth had significant antibacterial activity that increased with development of the bacteria and reached its maximum at the stationary growth phase . The antibiotic activities were significant against five plant-pathogenic bacterial strains: Agrobacterium vitis, Pectobacterium carotovorum subsp . atrosepticum, P . carotovorum subsp . carotovorum, Pseudomonas syringae pv . tabaci, and Ralstonia solanacearum . The antibacterial factors were extracted with butanol and fractionated using column chromatography with the eluents of different hydrophobic intensities . Two active antibacterial subfractions were purified, and the higher active fraction was further fractionated and identified as a single compound of benzylideneacetone (trans-4-phenyl-3-buten-2-one) . With heat stability, the synthetic compound showed equivalent antibiotic activity and spectrum to the purified compound . This study reports a new antibiotic compound synthesized by X . nematophila, which is a monoterpenoid compound and active against some Gram-negative bacteria.

Phytochemistry, 2004 Oct, 65(20), 2751 - 61
Selection of high ginsenoside producing ginseng hairy root lines using targeted metabolic analysis; Woo SS et al.; To develop an experimental system for studying ginsenoside biosynthesis, we generated thousands of ginseng (Panax ginseng C.A . Meyer) hairy roots, genetically transformed roots induced by Agrobacterium rhizogenes, and analyzed the ginsenosides in the samples . 27 putative ginsenosides were detected in ginseng hairy roots . Quantitative and qualitative variations in the seven major ginsenosides were profiled in 993 ginseng hairy root lines using LC/MS and HPLC-UV . Cluster analysis of metabolic profiling data enabled us to select hairy root lines, which varied significantly in ginsenoside production . We selected hairy root lines producing total ginsenoside contents 4-5 times higher than that of a common hairy root population, as well as lines that varied in the ratio of the protopanaxadiol to protopanaxatriol type ginsenoside . Some of the hairy root lines produce only a single ginsenoside in relatively high amounts . These metabolites represent the end product of gene expression, thus metabolic profiling can give a broad view of the biochemical status or biochemical phenotype of a hairy root line that can be directly linked to gene function.

Med Mycol, 2004 Aug, 42(4), 391 - 5
Agrobacterium tumefaciens-mediated transformation of Paracoccidioides brasiliensis; Leal CV et al.; Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, an important mycosis endemic to Latin America . As the tools to study gene function in P . brasiliensis are only in the early stage of development, there is presently no system that allows for both the delivery and integration of exogenous nucleic acids into its genome . We report in this paper the transformation of the yeast phase of P . brasiliensis (ATCC-60855) with Agrobacterium tumefaciens (GV3101) carrying the vector pAD1625 . The microorganisms were co-cultivated for 2 days and then incubated for 10 days at 35 degrees C on selective media . PCR and dot-blot targeted at a fragment of 222 bp from the hph (hygromycin phosphotransferase) gene which confers Hygr confirmed the transformation of P . brasiliensis.

Yi Chuan Xue Bao, 2004 Feb, 31(2), 183 - 8
{Transgenic cotton plants of chitinase and glucanase genes and their performance of resistance to Verticillium dahliea}; Wu JH et al.; The two disease-resistance genes chitinase and glucanase, which were respectively directed by commelina yellow mottle virus promoter (CoYMV, vascular specific) and CaMV35S promoter, were introduced into cotton genome via Agrobacterium tumefaciens . Transgenic plants were obtained from two popularly cultivated varieties Jihe321 and CRIC35 . After screening by spraying kanamycin over unfolding leaves, the kanamycin resistance (KmR) plants were tested by PCR and Southern blot . The results showed that there were one or two inserts of transgenes in cotton genome . Performance test of resistance of T3 families in field and greenhouse showed that seven lines were resistant or tolerant to Verticillium dahliea . Meanwhile, the resistance at seedling stage in greenhouse was in accordance with that at the boll-setting stage in field . Among the seven lines, D9910, D9915 and D9919 had a disease resistance index of 6.5, 9.4 and 9.5, respectively in field, which showed a high resistance level . Genetics analysis of the three lines showed a classical Mendelian pattern of one pair of genes, which meant that each of the three lines contains one copy of transgene . Southern blotting also confirmed the copy number of inserts.

Mol Microbiol, 2004 Oct, 54(2), 561 - 74
Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system; Hubber A et al.; The symbiosis island of Mesorhizobium loti strain R7A contains genes with strong similarity to the structural vir genes (virB1-11; virD4) of Agrobacterium tumefaciens that encode the type IV secretion system (T4SS) required for T-DNA transfer to plants . In contrast, M . loti strain MAFF303099 lacks these genes but contains genes not present in strain R7A that encode a type III secretion system (T3SS) . Here we show by hybridization analysis that most M . loti strains contain the VirB/D4 T4SS and not the T3SS . Strikingly, strain R7A vir gene mutants formed large nodules containing bacteroids on Leucaena leucocephala in contrast to the wild-type strain that formed only uninfected tumour-like structures . A rhcJ T3SS mutant of strain MAFF303099 also nodulated L . leucocephala, unlike the wild type . On Lotus corniculatus, the vir mutants were delayed in nodulation and were less competitive compared with the wild type . Two strain R7A genes, msi059 and msi061, were identified through their mutant phenotypes as possibly encoding translocated effector proteins . Both Msi059 and Msi061 were translocated through the A . tumefaciens VirB/D4 system into Saccharomyces cerevisiae and Arabidopsis thaliana, as shown using the Cre recombinase Reporter Assay for Translocation (CRAfT) . Taken together, these results suggest that the VirB/D4 T4SS of M . loti R7A plays an analogous symbiotic role to that of T3SS found in other rhizobia . The heterologous translocation of rhizobial proteins by the Agrobacterium VirB/D4 T4SS is the first demonstration that rhizobial effector proteins are translocated into plant cells and confirms functional conservation between the M . loti and A . tumefaciens T4SS.

Yi Chuan Xue Bao, 2004 Jan, 31(1), 97 - 101
{Saline tolerance white clover transformed with the betaine aldehyde dehyrogenase gene by Agrobacterium tumefaciens}; Chen CF et al.; The white clover has been transformed with the Betaine Aldehyde Dehydrogenase (BADH) gene cloned from Atriplex hortensis by Agrobacterium tumefaciens . The relative electronic conductivity of the transgenic plants under 1% NaCl stress for 48 hours was about 20%, less than the control plant's relative electronic conductivity (more than 40%), these showed the cell membrane of the transgenic plants has been less injured than control plants under salt stress . The other experience showed that the transgenic plant could grow well in water culture included 0.5% NaCl for more than two weeks, but the control plants could not.

EMBO J, 1987, 6(12), 3559 - 64
Localization of sequences in wheat endosperm protein genes which confer tissue-specifc expression in tobacco; Colot V et al.; The developing cereal grain accumulates large quantities of proteins which are unique to the endosperm tissue . The DNA sequences which determine their endosperm-specific expression have not yet been identified . In the absence of a suitable transformation-regeneration system for cereals, we have investigated whether chimaeric genes consisting of low mol . wt (LMW) and high mol . wt (HMW) glutenin gene upstream sequences coupled to the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene could be specifically expressed in transgenic tobacco . The fusions, made in a Ti-derived binary vector, were introduced into tobacco via Agrobacterium tumefaciens-mediated transformation and their activity assayed . Both the LMW and HMW glutenin chimaeric genes exhibited endosperm-specific CAT activity in the transformed plants . In addition, a deletion series of the LMW glutenin sequence indicated that sequences present between 326 bp and 160 bp upstream of the transcription start point are necessary to confer endosperm-specific CAT activity.

Biol Pharm Bull, 2004 Oct, 27(10), 1621 - 5
Activation of phenylpropanoid metabolism in sesame by over-expression of carrot calmodulin gene; Mitsuma S et al.; Transgenic sesame (Sesamum schinzianum ASCH.) was produced by Agrobacterium-mediated transfection of a carrot calmodulin gene, cam-4, which was specifically expressed upon the contact of carrot cells with oligogalacturonide elicitor . Coding region of cam-4 was ligated to the downstream of 35S promoter of cauliflower mosaic virus and subcloned into pMATGBO-DB3.1 . A . tumefaciens 4404 was transformed with the constructed vector, and the crown gall tissues formed in the sesame seedlings were transferred onto appropriate media to obtain the re-differentiated plants . The reverse-transcription polymerase chain reaction followed by Southern blot analysis revealed that cam-4 gene was appreciably expressed in the transgenic plants . Activities of two key enzyme regulating phenylpropanoid metabolisms, phenylalanine ammonia-lyase and caffeic acid O-methyltransferase, and the contents of phenolic compounds in the transformed sesame were markedly elevated as compared with those of the control . These results suggest that the over-expression of cam-4 gene enhances the biosynthetic activities of phenylpropane derivatives in the transformed sesame plants.

J Biol Chem, 2004 Dec 10, 279(50), 52753 - 61 Epub 2004 Dec 10.
Hexameric assembly of the bifunctional methylerythritol 2,4-cyclodiphosphate synthase and protein-protein associations in the deoxy-xylulose-dependent pathway of isoprenoid precursor biosynthesis; Gabrielsen M et al.; The bifunctional methylerythritol 4-phosphate cytidylyltransferase methylerythritol 2,4-cyclodiphosphate synthase (IspDF) is unusual in that it catalyzes nonconsecutive reactions in the 1-deoxy-D-xylulose 5-phosphate (DOXP) pathway of isoprenoid precursor biosynthesis . The crystal structure of IspDF from the bacterial pathogen Campylobacter jejuni reveals an elongated hexamer with D3 symmetry compatible with the dimeric 2C-methyl-D-erythritol-4-phosphate cytidylyltransferase and trimeric 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase monofunctional enzymes . Complex formation of IspDF with 4-diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE), the intervening enzyme activity in the pathway, has been observed in solution for the enzymes from C . jejuni and Agrobacterium tumefaciens . The monofunctional enzymes (2C-methyl-D-erythritol-4-phosphate cytidylyltransferase, IspE, and 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase) involved in the DOXP biosynthetic pathway of Escherichia coli also show physical associations . We propose that complex formation of the three enzymes at the core of the DOXP pathway can produce an assembly localizing 18 catalytic centers for the early stages of isoprenoid biosynthesis.

J Bacteriol, 2004 Oct, 186(20), 6824 - 9
Replicon-specific regulation of small heat shock genes in Agrobacterium tumefaciens; Balsiger S et al.; Four genes coding for small heat shock proteins (sHsps) were identified in the genome sequence of Agrobacterium tumefaciens, one on the circular chromosome (hspC), one on the linear chromosome (hspL), and two on the pAT plasmid (hspAT1 and hspAT2) . Induction of sHsps at elevated temperatures was revealed by immunoblot analyses . Primer extension experiments and translational lacZ fusions demonstrated that expression of the pAT-derived genes and hspL is controlled by temperature in a regulon-specific manner . While the sHsp gene on the linear chromosome turned out to be regulated by RpoH (sigma32), both copies on pAT were under the control of highly conserved ROSE (named for repression of heat shock gene expression) sequences in their 5' untranslated region . Secondary structure predictions of the corresponding mRNA strongly suggest that it represses translation at low temperatures by masking the Shine-Dalgarno sequence . The hspC gene was barely expressed (if at all) and not temperature responsive.

Microbiol Res, 2004, 159(3), 285 - 93
Rhizosheath of sinai desert plants is a potential repository for associative diazotrophs; Othman AA et al.; Among 42 plant species representing the flora of north Sinai, two possessed sand grain sheath encasing the roots . They are Panicum turgidum Forssk . and Stipagrostis scoparia (Trin.and Rupr.) deWinter . Rhizosheaths, compared to surrounding free sand, accommodated higher population density of microorganisms including associative diazotrophs . Isolates secured belonged to the species of Bacillus circulans, Paenib . macerans (Bacillus macerans), Enterobacter agglomerans, Agrobacterium radiobacter and Chryseomonas luteola (Pseudomonos luteola) . The rhizosheath potentiality in re-vegetating sand dunes and arid lands, through nitrogen fixation, plant-water relationship and root continuity for nutrient uptake, are discussed.

Mol Biotechnol, 2004 Sep, 28(1), 33 - 40
Synthesis and assembly of SIVmac Gag p27 capsid protein cholera toxin B subunit fusion protein in transgenic potato; Kim TG et al.; A deoxyribonucleic acid (DNA) fragment encoding the cholera toxin B subunit (CTB) was linked 5' to the simian immunodeficiency virus (SIVmac) Gag p27 capsid gene (CTB-Gag) . The fusion gene was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated . The CTB-Gag gene fusion was detected in transformed potato leaf genomic DNA by polymerase chain reaction-mediated DNA amplification . The results of immunoblot analysis with anti-CTB and anti-Gag antibodies verified the synthesis of biologically active CTB-Gag fusion protein in transformed leaf and tuber tissues . Synthesis and assembly of the CTB-Gag fusion protein into oligomeric structures of pentamer size was confirmed by GM1-ganglioside-enzyme-linked immunosorbent assay (GM1-ELISA) of transformed potato tuber tissue extracts . The binding of CTB-Gag fusion protein oligomers to intestinal epithelial cell membrane receptors quantified by GM1-ELISA showed that CTB-Gag fusion protein made up approx 0.016-0.022% of the total soluble tuber protein . The synthesis of CTB-Gag monomers and their assembly into biologically active CTB-Gag fusion protein oligomers in potato tuber tissues provides the opportunity for employment of the carrier and adjuvant properties of CTB for the development of edible plant-based subunit mucosal vaccines for enhanced mucosal immunity against SIV in macaques.

Plant Cell Rep, 2004 Nov, 23(5), 297 - 303 Epub 2004 Sep 29.
An efficient transformation system for chickpea (Cicer arietinum L.); Senthil G et al.; A reproducible and efficient transformation method was developed for Desi and Kabuli chickpeas (Cicer arietinum L.) using germinated seedlings as sources of explants . Slices derived from plumules were the most efficient at generating transformed shoots . The AGL1 Agrobacterium-treated explants were first incubated on thidiazuron-containing media, then selected using phosphinothricin . Resistant shoots were successfully transferred to soil either by grafting or in vitro rooting . In experiments each taking 4-9 months, a total of 41 confirmed transformed lines were created using embryo axis slices as source explants, giving a transformation frequency of 5.1% . Southern analysis and histochemical and leaf painting assays demonstrated integration and expression of the transgenes in the initial transformants and two generations of progeny.

Curr Opin Microbiol, 2004 Oct, 7(5), 505 - 12
Protein interaction networks in bacteria; Noirot P et al.; The complement of expressed cellular proteins - the proteome - is organized into functional, structured networks of protein interactions that mediate assembly of molecular machines and dynamic cellular pathways . Recent studies reveal the biological roles of protein interactions in bacteriophage T7 and Helicobacter pylori, and new methods allow to compare and to predict interaction networks in other species . Smaller scale networks provide biological insights into DNA replication and chromosome dynamics in Bacillus subtilis and Archeoglobus fulgidus, and into the assembly of multiprotein complexes such as the type IV secretion system of Agrobacterium tumefaciens, and the cell division machinery of Escherichia coli . Genome-wide interaction networks in several species are needed to obtain a biologically meaningful view of the higher order organization of the proteome in bacteria.

FEMS Microbiol Lett, 2004 Oct 1, 239(1), 41 - 9
Identification of Mycobacterium avium genes up-regulated in cultured macrophages and in mice; Danelishvili L et al.; To investigate Mycobacterium avium gene expression upon infection of macrophages, we created a M . avium-promoter library upstream of a promoter-less gene encoding the green fluorescent protein (GFP) in Mycobacterium smegmatis . Clones were evaluated for increased expression of GFP after infection of U937 macrophages . A number of M . avium genes were up-regulated more than 3-fold after 24 and 48 h following macrophage infection . M . avium genes expressed by M . smegmatis during growth in macrophages include genes encoding transport/binding proteins, synthesis, modification and degradation of macromolecules, and a great majority of genes for which no function is currently known . For some of the unknown genes, homologues were identified in bacteria such as Mycobacterium leprae, Salmonella typhimurium and Agrobacterium tumefaciens . In order to investigate if these genes were also expressed in M . avium during macrophage infection in vitro and in vivo, transcripts of selected genes were quantified using real time RT-PCR . Evaluation of most expressed genes in M . smegmatis confirmed their up-regulation in M . avium after 24 h infection of macrophages in vitro and mice.

Plant Cell Rep . 2004 Sep 22; {Epub ahead of print}
Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with a double T-DNA binary vector system; Chen S et al.; We investigated the potential of a novel double T-DNA vector for generating marker-free transgenic plants . Co-transformation methods using a double T-DNA vector or using mixture of two Agrobacterium tumefaciens strains were compared, and showed that the double T-DNA vector method could produce marker-free transgenic tobacco ( Nicotiana tabacum L.) plants more efficiently . A dual marker double T-DNA vector was then constructed by assembling the green fluorescent protein (GFP) gene mgfp5 and the neomycin phosphotransferase gene nptII into the same T-DNA . The frequency of co-transformants produced by this vector was 56.3% . Co-expression of mgfp5 and nptII was found in 28 out of 29 T(1) lines, and segregation of the reporter beta-glucuronidase gene, gusA, from mgfp5 to nptII was found in 12 out of 29 T(1) lines . Therefore, GFP could be used as a vital marker to improve the transformation efficiency and to easily monitor the segregation of marker genes, thus facilitating screening of marker-free progeny.

Plant Cell Rep . 2004 Sep 22; {Epub ahead of print}
Consistent and stable expression of the nptII, uidA and bar genes in transgenic Pinus radiata after Agrobacterium tumefaciens-mediated transformation using nurse cultures; Charity JA et al.; An Agrobacterium tumefaciens-mediated transformation protocol has been developed for embryogenic cell cultures of Pinus radiata . Transgenic lines were only produced when embryogenic tissue was placed on nurse tissue during the Agrobacterium co-cultivation and recovery stages of the procedure . Plantlets were regenerated via somatic embryogenesis from ten of the 11 transgenic lines tested and at least 20 of each line were planted in a GMO glasshouse . Expression of the nptII, uidA and bar genes in up to ten plants of each individual transgenic line was evaluated by molecular, biochemical and functional analysis . As expected, expression of the nptII gene varied among the ten lines, while within ten replicates of the same line, nptII expression appeared to be consistent, with the exception of one line, K3 . Likewise, the level of GUS activity varied among transgenic lines, but was relatively consistent in plants derived from the same tissue, except for two lines, G4 and G5 . Moreover, similar absolute values and pattern of gene expression of uidA was observed in the transgenic plants, for two consecutive years . Plantlets from eight lines survived a spray treatment with the equivalent of 2 kg/ha and 4 kg/ha of the commercial formulation Buster, whereas non-transformed controls died . Southern hybridisation analysis of embryogenic tissue and green needle tissue from putative transgenic lines demonstrated a relatively low number of gene insertions (from one to nine) of both the bar and nptII genes in the nine transgenic lines tested.

Theor Appl Genet, 2004 Nov, 109(8), 1562 - 7 Epub 2004 Nov.
Co-transformation using a negative selectable marker gene for the production of selectable marker gene-free transgenic plants; Park J et al.; A negative selectable marker gene, codA, was successfully co-transformed with a GUS reporter gene to develop selectable marker gene-free transgenic plants . The pNC binary vector contained a T-DNA harboring the codA gene next to the nptII gene, while a second binary vector, pHG, contained a GUS reporter gene . Tobacco plants ( Nicotiana tabacum cv . Samsun NN) were co-transformed via the mixture method with Agrobacterium tumefaciens LBA4404 strains harboring pNC and pHG, respectively . Seeds harvested from the co-transformants were sown on germination media containing 5-fluorocytosine (5-FC) . Analysis of the progeny by GUS staining and PCR amplification revealed that all of the 5-FC-resistant R(1) plants were codA free, and that the codA gene segregated independently of the GUS gene . Because codA-free seedlings developed normally on 5-FC-containing medium, we suggest that co-transformation with negatively selectable markers is a viable method for the production of easily distinguished, selectable marker gene-free transgenic plants.

Proc Natl Acad Sci U S A, 2004 Oct 5, 101(40), 14533 - 8 Epub 2004 Sep 24.
Evidence for landscape-level, pollen-mediated gene flow from genetically modified creeping bentgrass with CP4 EPSPS as a marker; Watrud LS et al.; Sampling methods and results of a gene flow study are described that will be of interest to plant scientists, evolutionary biologists, ecologists, and stakeholders assessing the environmental safety of transgenic crops . This study documents gene flow on a landscape level from creeping bentgrass (Agrostis stolonifera L.), one of the first wind-pollinated, perennial, and highly outcrossing transgenic crops being developed for commercial use . Most of the gene flow occurred within 2 km in the direction of prevailing winds . The maximal gene flow distances observed were 21 km and 14 km in sentinel and resident plants, respectively, that were located in primarily nonagronomic habitats . The selectable marker used in these studies was the CP4 EPSPS gene derived from Agrobacterium spp . strain CP4 that encodes 5-enol-pyruvylshikimate-3-phosphate synthase and confers resistance to glyphosate herbicide . Evidence for gene flow to 75 of 138 sentinel plants of A . stolonifera and to 29 of 69 resident Agrostis plants was based on seedling progeny survival after spraying with glyphosate in greenhouse assays and positive TraitChek, PCR, and sequencing results . Additional studies are needed to determine whether introgression will occur and whether it will affect the ecological fitness of progeny or the structure of plant communities in which transgenic progeny may become established.

Plant J, 2004 Oct, 40(2), 322 - 31
Agrodrench: a novel and effective agroinoculation method for virus-induced gene silencing in roots and diverse Solanaceous species; Ryu CM et al.; Summary Virus-induced gene silencing (VIGS) is an extremely powerful tool for plant functional genomics . We used Tobacco rattle virus (TRV)-derived VIGS vectors expressed from binary vectors within Agrobacterium to induce RNA silencing in plants . Leaf infiltration is the most common method of agroinoculation used for VIGS but this method has limitations as it is laborious for large-scale screening and some plants are difficult to infiltrate . Here we have developed a novel and simple method of agroinoculation, called 'agrodrench', where soil adjacent to the plant root is drenched with an Agrobacterium suspension carrying the TRV-derived VIGS vectors . By agrodrench we successfully silenced the expression of phytoene desaturase (PDS), a 20S proteasome subunit (PB7) or Mg-protoporphyrin chelatase (Chl H) encoding genes in Nicotiana benthamiana and in economically important crops such as tomato, pepper, tobacco, potato, and Petunia, all belonging to the Solanaceae family . An important aspect of agrodrench is that it can be used for VIGS in very young seedlings, something not possible by the leaf infiltration method, which usually requires multiple fully expanded leaves for infiltration . We also demonstrated that VIGS functioned to silence target genes in plant roots . The agrodrench method of agroinoculation was more efficient than the leaf infiltration method for VIGS in roots . Agrodrench will facilitate rapid large-scale functional analysis of cDNA libraries and can also be applied to plants that are not currently amenable to VIGS technology by conventional inoculation methods.

Carbohydr Res, 2004 Oct 4, 339(14), 2451 - 5
Elucidation of two O-chain structures from the lipopolysaccharide fraction of Agrobacterium tumefaciens F/1; De Castro C et al.; Agrobacterium tumefaciens F/1 produces two different O-chains, both are constituted of rhamnose and glucosamine: the less abundant has a linear disaccharidic repeating unit 3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1--> and the second one 4)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1--> . The two intact antigenic moieties were studied in mixture by 2D NMR . Additional supporting data were obtained by periodate degradation, the major component was cleaved selectively, leading to a glucosamine glycoside, whereas the minor one was recovered unaffected.

Infect Immun, 2004 Oct, 72(10), 5693 - 703
Release of periplasmic proteins of Brucella suis upon acidic shock involves the outer membrane protein Omp25; Boigegrain RA et al.; The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH . In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels . No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B . suis released a substantial amount of soluble proteins . Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location . Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens . The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome . We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats . In B . suis, this protein contained 9 repeats, while 12 were present in the B . melitensis orthologue . B . suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product . However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects . Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.

Mol Plant Microbe Interact, 2004 Sep, 17(9), 951 - 7
The assimilation of gamma-butyrolactone in Agrobacterium tumefaciens C58 interferes with the accumulation of the N-acyl-homoserine lactone signal; Carlier A et al.; Agrobacterium tumefaciens C58 communicates using N-acyl-homoserine lactones (acyl-HSL) and contains two lactonase-encoding genes, attM and aiiB, the products of which are capable of inactivating the acyl-HSL signal . In A . tumefaciens A6, the expression of the attKLM operon is controlled by the transcriptional repressor encoded by an adjacent gene, attJ . An attJ::Tn5 mutant does not accumulate acyl-HSL because of the constitutive expression of the lactonase AttM, the activity of which inactivates acyl-HSL . In this work, the attKLM operon of A . tumefaciens C58 was shown to be involved in an assimilative pathway of gamma-butyrolactone (GBL), gamma-hydroxybutyrate (GHB), and succinate semialdehyde (SSA), in which AttM and AttL are key enzymes for GBL and GHB assimilation . The expression of the attKLM promoter was activated in the presence of GBL, GHB, and SSA . Under these conditions, A . tumefaciens C58 did not accumulate the acyl-HSL that it naturally synthesizes, and also became able to inactivate exogenous acyl-HSL signals . Therefore, in A . tumefaciens C58, the assimilative pathway of gamma-butyrolactone interferes with the acyl-HSL signaling.

J Biosci, 2004 Sep, 29(3), 309 - 17
Do leaf surface characteristics affect Agrobacterium infection in tea {Camellia sinensis (L.) O Kuntze}?
Kumar N, Pandey S, Bhattacharya A, Ahuja PS.
The host range specificity of Agrobacterium with five tea cultivars and an unrelated species (Artemisia parviflora) having extreme surface characteristics was evaluated in the present study . The degree of Agrobacterium infection in the five cultivars of tea was affected by leaf wetness, micro-morphology and surface chemistry . Wettable leaf surfaces of TV1, Upasi-9 and Kangra jat showed higher rate (75%) of Agrobacterium infection compared to Upasi-10 and ST-449, whereas non-wettable leaves of A . parviflora showed minimum (25%) infection . This indicated that the leaves with glabrous surface having lower q (larger surface area covered by water droplet), higher phenol and wax content were more suitable for Agrobacterium infection . Caffeine fraction of tea promoted Agrobacterium infection even in leaves poor in wax (Upasi-10), whereas caffeine-free wax inhibited both Agrobacterium growth and infection . Thus, study suggests the importance of leaf surface features in influencing the Agrobacterium infection in tea leaf explants . Our study also provides a basis for the screening of a clone/cultivar of a particular species most suitable for Agrobacterium infection the first step in Agrobacterium-mediated genetic transformation.

Int J Biochem Cell Biol, 2005 Jan, 37(1), 177 - 91
Rational mutagenesis of a 40 kDa heat shock protein from Agrobacterium tumefaciens identifies amino acid residues critical to its in vivo function; Hennessy F et al.; Prokaryotic DnaJ and DnaK, homologous to the eukaryotic 40 and 70kDa heat shock proteins (Hsp40 and Hsp70) respectively, play an important role as molecular chaperones in assisted protein folding under both normal and stressed conditions . DnaJ-like proteins are defined by the presence of a 70 amino acid domain termed the J domain, similar to the initial 73 amino acids of the Escherichia coli protein DnaJ . The J domain comprises four alpha-helices and a loop region containing the invariant tripeptide of histidine, proline and aspartic acid (HPD motif) . This motif and Helix II have been shown previously to be important for the interaction with partner Hsp70s . Conserved amino acid residues present in the J domain were identified, and substitutions of these residues were performed to examine their effect on the in vivo functioning of the J domain of Agrobacterium tumefaciens DnaJ . Three conserved, charged residues, and three conserved, hydrophobic residues, in addition to the HPD motif, were shown to be important for the correct functioning of A . tumefaciens DnaJ . These included Arg26 located on Helix II, Arg63 and Asp59 located on Helix IV, Tyr7 and Leu10 located on Helix I, and Leu57 located on Helix III . This study has identified charged and hydrophobic residues on all the structural elements of the J domain that were critical to the structure and function of DnaJ, and in particular shown that Helix IV may have an important role in the structure and function of DnaJs in general.

Proteomics, 2004 Oct, 4(10), 3128 - 40
DARTs: A DNA-based in vitro polypeptide display technology; de Figueiredo P et al.; Display technologies link proteins with the genes that encode them, providing a means of selecting proteins with desired properties through the process of directed evolution . Here, we describe DNA/protein attachment and recovery tools (DARTs), a novel polypeptide display technology that utilizes the Agrobacterium tumefaciens protein VirD2 to generate DNA-protein hybrid molecules . The resulting DNA-protein hybrids are small, robust, and are not expected to be subject to the synthesis and selection biases associated with viral- and cell-based display systems . We demonstrated that these DNA-protein hybrids could be used to display a variety of peptides that bind to appropriate antibodies for immunodetection and immunopurification . Further, the DNA components of the hybrid molecules can hybridize to complementary DNA molecules in solution or on a solid substrate . Because full-length VirD2 self-associated, we constructed a truncation that did not self-associate but still exhibited DNA linking activity and efficiently displayed peptides . Finally, we purified DNA-protein hybrids using their displayed peptide epitopes and amplified their DNA components by polymerase chain reaction . We suggest that the DART polypeptide display system will be valuable for performing directed evolution and generating protein arrays.

Plant Cell Rep, 2005 Jan, 23(8), 548 - 56 Epub 2004 Sep 16.
Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation; Tsai FY et al.; A total of 35 independent transgenic tobacco plants were produced using the Agrobacterium tumefaciens-leaf segment co-cultivation method followed by selection with kanamycin for the nptII gene . The vector also carried the tobacco feedback-insensitive anthranilate synthase gene (ASA2) . Many of the lines showed increased ASA2 mRNA levels but only three contained increased free tryptophan (Trp) and many lines contained lower Trp than the untransformed control . The line with the highest Trp level (threefold that of the untransformed control) contained increased anthranilate synthase activity (AS) both in leaves and a cell suspension culture derived from the plant while the feedback insensitivity was most evident in the suspension culture . Other kinetic data also indicated that the ASA2 encoded AS alpha-subunit was more abundant in the tissue culture than in leaves . Progeny seedlings from this line were resistant to certain toxic Trp analogs, especially alpha-methyltryptophan (alphaMT) and less so to the most commonly used analog, 5-methyltryptophan . Shoots formed more readily from leaves of two of the transgenic lines than from leaves of the untransformed control on alphaMT, indicating that it might be possible to use ASA2 as a selectable marker gene and alphaMT as the selection agent.

Plant Cell Rep . 2004 Sep 15; {Epub ahead of print}
Enhanced anthocyanin synthesis in foliage plant Caladium bicolor; Li SJ et al.; A protocol was developed for Agrobacterium-mediated genetic transformation of monocotyledon foliage plant Caladium bicolor cv . Jackie Suthers using leaf disc and petiole as the explants . The explants were inoculated with Agrobacterium strain LBA4404 harboring a binary vector with the maize anthocyanin regulatory gene Lc under the control of the cauliflower mosaic virus promoter . Callus formation was induced in MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (6-BA), 0.1 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g/l sucrose and kanamycin 50 mg/l for selection . Resistant calli were induced for shoot generation in MS medium with 2 mg/l 6-BA and 0.2 mg/l alpha-naphthaleneacetic acid . As much as 10% of the explants gave rise to kanamycin-resistant shoots with our procedure . Transformed plants had enhanced anthocyanin accumulation in the roots, leaves and stems (epidermis and vascular bundles) . Integration of the transgene into the host genome was confirmed by genomic Southern blot hybridization, and RNA blot hybridization analysis indicated that the expression of the transgene correlated with anthocyanin accumulation . This investigation illustrates the utility of anthocyanin regulatory genes in the genetic manipulation of the color of foliage plants . It also supports the premise that the Lc gene can be used as a powerful non-destructive cell autonomous visual marker in a wide variety of plants, as exemplified by the perfect symmetrical half-green/half-red plant presumably derived from the symmetrical division of one transgenic and one non-transgenic precursor meristematic cell.

Plant Cell Rep . 2004 Sep 15; {Epub ahead of print}
Development of transgenic rice plants overexpressing the Arabidopsis H(+)/Ca(2+) antiporter CAX1 gene; Kim KM et al.; The gene of the Arabidopsis thaliana H(+)/Ca(2+) transporter, CAX1 (cation exchanger 1) was introduced into Japonica cultivars of rice (Ilpumbyeo) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced . The neomycin phosphotransferase II (NPTII) gene was used as a selectable marker . The activity of neomycin phosphotransferase could be successfully detected in transgenic rice callus . The introduction of the CAX1 gene was also proven by PCR using CAX1-specific oligonucleotide primers in regenerated plants . Stable integration and expression of the CAX1 gene in T(0) plants and T(1) progeny were confirmed by DNA hybridization, Northern blot analysis, and luminescent analysis.

J Biol Chem, 2004 Dec 3, 279(49), 51193 - 202 Epub 2004 Dec 3.
The H(+)-pyrophosphatase of Rhodospirillum rubrum is predominantly located in polyphosphate-rich acidocalcisomes; Seufferheld M et al.; Acidocalcisomes are acidic, calcium storage compartments with a H(+) pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, and have also been found in the bacterium Agrobacterium tumefaciens . In this work, we report that the H(+)-pyrophosphatase (H(+)-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified and thought to be localized only to chromatophore membranes, is predominantly located in acidocalcisomes . The identification of the acidocalcisomes of R . rubrum was carried out by using transmission electron microscopy, x-ray microanalysis, and immunofluorescence microscopy . Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H(+)-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs) but a lack of markers of the plasma membrane . polyP was also localized to the acidocalcisomes by using 4',6'-diamino-2-phenylindole staining and identified by using 31P NMR and biochemical methods . Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations . The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP increased when bacteria were grown in the light . Taken together, these results suggest that the H(+)-PPase of R . rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the chromatophore membranes.

Plant Cell Rep, 2004 Nov, 23(6), 386 - 90 Epub 2004 Nov.
Agrobacterium-mediated genetic transformation of Perilla frutescens; Kim KH et al.; A reproducible plant regeneration and an Agrobacterium tumefaciens-mediated genetic transformation protocol were developed for Perilla frutescens (perilla) . The largest number of adventitious shoots were induced directly without an intervening callus phase from hypocotyl explants on MS medium supplemented with 3.0 mg/l 6-benzylaminopurine (BA) . The effects of preculture and extent of cocultivation were examined by assaying beta-glucuronidase (GUS) activity in explants infected with A . tumefaciens strain EHA105 harboring the plasmid pIG121-Hm . The highest number of GUS-positive explants were obtained from hypocotyl explants cocultured for 3 days with Agrobacterium without precultivation . Transgenic perilla plants were regenerated and selected on MS basal medium supplemented with 3.0 mg/l BA, 125 mg/l kanamycin, and 500 mg/l carbenicillin . The transformants were confirmed by PCR of the neomycin phosphotransferase II gene and genomic Southern hybridization analysis of the hygromycin phosphotransferase gene . The frequency of transformation from hypocotyls was about 1.4%, and the transformants showed normal growth and sexual compatibility by producing progenies.

Transgenic Res, 2004 Jun, 13(3), 271 - 87
A novel two T-DNA binary vector allows efficient generation of marker-free transgenic plants in three elite cultivars of rice (Oryza sativa L.); Breitler JC et al.; A pilot binary vector was constructed to assess the potential of the 2 T-DNA system for generating selectable marker-free progeny plants in three elite rice cultivars (ZhongZuo321, Ariete and Khao Dawk Mali 105) known to exhibit contrasting amenabilities to transformation . The first T-DNA of the vector, delimited by Agrobacterium tumefaciens borders, contains the hygromycin phosphotransferase (hpt) selectable gene and the green fluorescent protein (gfp) reporter gene while the second T-DNA, delimited by Agrobacterium rhizogenes borders, bears the phosphinothricin acetyl transferase (bar) gene, featuring the gene of interest . 82-90% of the hygromycin-resistant primary transformants exhibited tolerance to ammonium glufosinate mediated by the bar gene suggesting very high co-transformation frequency in the three cultivars . All of the regenerated plants were analyzed by Southern blot which confirmed co-integration of the T-DNAs at frequencies consistent with those of co-expression and allowed determination of copy number for each gene as well as detection of two different vector backbone fragments extending between the two T-DNAs . Hygromycin susceptible, ammonium glufosinate tolerant phenotypes represented 14.4, 17.4 and 14.3% of the plants in T1 progenies of ZZ321, Ariete and KDML105 primary transformants, respectively . We developed a statistical model for deducing from the observed copy number of each T-DNA in T0 plants and phenotypic segregations in T1 progenies the most likely constitution and linkage of the T-DNA integration locus . Statistical analysis identified in 40 out of 42 lines a most likely linkage configuration theoretically allowing genetic separation of the two T-DNA types and out segregation of the T-DNA bearing the bar gene . Overall, though improvements of the technology would be beneficial, the 2 T-DNA system appeared to be a useful approach to generate selectable marker-free rice plants with a consistent frequency among cultivars.

Transgenic Res, 2004 Jun, 13(3), 225 - 33
Superoxide dismutase transgenes in sugarbeets confer resistance to oxidative agents and the fungus C . beticola; Tertivanidis K et al.; Sugarbeets carrying superoxide dismutase transgenes were developed in order to investigate the possibility of enhancing their resistance to oxidative stress . Binary T-DNA vectors carrying the chloroplastic and cytosolic superoxide dismutase genes from tomato, were used for Agrobacterium-mediated transformation of sugarbeet petioles . The transgenic plants were subjected to treatments known to cause oxidative stress, such as the herbicide methyl viologen and a natural photosensitizer toxin produced by the fungus Cercospora beticola, namely cercosporin . The transgenic plants exhibited increased tolerance to methyl viologen, to pure cercosporin, as well as to leaf infection with the fungus C . beticola.

Clin Diagn Lab Immunol, 2004 Sep, 11(5), 868 - 73
Occurrence and potential diagnostic applications of serological cross-reactivities between Brucella and other alpha-proteobacteria; Delpino MV et al.; Agrobacterium, Sinorhizobium, and Ochrobactrum are genera closely related to Brucella but, in contrast to the latter, are not pathogenic for humans and animals . We studied by an indirect enzyme-linked immunosorbent assay (ELISA) the reactivities of brucellosis sera against cytosolic (CYT) and membrane (MA) antigens from these nonpathogenic bacteria, and we evaluated the potential usefulness of these cross-reactions for the diagnosis of brucellosis in humans, sheep, cows, and dogs . Canine infection by Brucella canis was detected with high specificity by CYT antigen-based ELISAs (96% for Agrobacterium, 96% for Sinorhizobium, and 91% for Ochrobactrum), while sensitivity was variable (58% for Agrobacterium, 88% for Sinorhizobium, and 84% for Ochrobactrum) . In addition, it was possible to diagnose canine disease shortly after exposure to the pathogen (15 days) . Similar results for canine brucellosis were obtained with MA antigens . In contrast, normal sera from humans, sheep, and cattle reacted strongly with all the antigens (CYT and MA antigens from the three bacteria), producing high cutoff values and, consequently, low sensitivities . While for some host species the reactivity patterns of normal sera by Western blotting were similar to those produced with sera from infected individuals, the reactivity pattern of bovine sera against Sinorhizobium meliloti antigens exhibited some differential bands for the two groups of sera . These results show that crude fractions from nonpathogenic alpha-proteobacteria can be used to diagnose canine brucellosis but may need to be further separated into simpler fractions to have diagnostic usefulness in ovine, bovine, or human infection . By reducing the biosafety requirements, the use of antigens derived from these nonpathogenic bacteria would simplify the production of diagnostic kits for brucellosis, especially in settings where biosafety level-3 facilities are scarce or absent.

FEMS Microbiol Lett, 2004 Sep 15, 238(2), 273 - 9
Protein secretion in Legionella pneumophila and its relation to virulence; Lammertyn E et al.; Protein secretion is a universal process of fundamental importance for various aspects of cell physiology including the infection of a host organism by a bacterial pathogen . Many Gram-negative pathogens export virulence-associated proteins across one or two cell membranes to their place of action using a wide plethora of secretory pathways with the objective of infecting the host . For Legionella pneumophila, a facultative intracellular, human pathogen which is ubiquitously found in natural and artificial aquatic environments, two major secretory pathways known to be involved in virulence have been described . These are the PilD-dependent Lsp type II secretion pathway and the type IV secretion system encoded by the dot/icm genes . In addition, a second type IV system, with high sequence similarity to the Agrobacterium tumefaciens VirB system for conjugal transfer of oncogenic DNA, is present . Albeit dispensable for intracellular growth, this type IV system is important for efficient host cell infection at lower temperatures . Further more, evidence exists for the presence of at least one type I secretion system in L . pneumophila as well as for the presence of a twin arginine dependent translocation (Tat) pathway . This is a recently detected, signal peptide-dependent, secretion pathway complementary to the well-known Sec-dependent pathway for protein transport across the cytoplasmic membrane.

Protein Expr Purif, 2004 Oct, 37(2), 486 - 92
Expression and characterization of extracellular fungal phytase in transformed sesame hairy root cultures; Jin UH et al.; A recombinant fungal phytase was produced by cultures of sesame hairy roots transformed with Agrobacterium rhizogenes, purified and its molecular properties were characterized . Its transcription level and the phytase production were rapidly increased after 4 weeks of the cultures, suggesting that its transcription and protein synthesis might concur . Western blot analysis provided evidence that the recombinant fungal phytase was secreted into the liquid culture medium of the hairy roots . The phytase enzyme secreted was purified by three steps of ultrafiltration, DEAE-Sepharose ion exchange chromatography, and Sephadex G-100 size-exclusion chromatography . As a result, one single band signal was observed with SDS-PAGE, indicating that the purification step was reasonable . The positive signs of both the zymogram and the PAS staining on SDS-PAGE suggested that the activity of the final product phytase was active and glycosylated . The optimal reaction temperature of the phytase was between 50 and 60 degrees C and at over 60 degrees C its activity was reduced by 30-90%, depending on the temperatures applied . Pre-incubation at temperatures of 20-50 degrees C showed stable catalytic activity, while at over 50 degrees C the phytase activity was gradually decreased by 90% . The optimal pH was between 4 and 5 pH values for the recombinant fungal phytase, while for native phytase it was at pH 5.0 . Addition of iron ion inhibited the phytase activity but treatments of some cations, EDTA, and PMSF showed no effect on the activity or slightly stimulated it positively.

Plant Cell Physiol, 2004 Aug, 45(8), 1023 - 31
Expression analysis of the NgORF13 promoter during the development of tobacco genetic tumors; Udagawa M et al.; We investigated the expression pattern of the promoter of Nicotiana glauca (Ng) ORF13 in the hybrids between N . glauca and N . langsdorffii harboring the NgORF13-beta-glucuronidase (GUS) chimeric gene . The promoter of NgORF13 of N . glauca had lower activities than the promoter of RiORF13 of Agrobacterium rhizogenes agropine-type root-inducing (Ri) plasmid . However, the localization of GUS activity in the NgORF13 transgenic plants was similar to that in the RiORF13 transgenic plants . The GUS activity of NgORF13-GUS was high in genetic tumors cultured in vitro or developed spontaneously on F1 plants with aging or by wounding . The GUS activity in tumors was observed in bud primordia, vascular bundles and leaves in the buds . While the activity was lower than in tumors, NgORF13-GUS was also expressed in vascular bundles and the parenchymatous tissues in plants regenerated from tumors . Furthermore, the promoter activity of NgORF13 was induced by wounding and activated by exogenous application of methyl jasmonate . During tumorization, NgORF13 was induced at an early stage and showed expression patterns similar to both NgrolB and NgrolC whose expression were investigated by Nagata et al . (1996) Plant Cell Physiol . 37: 489-498 . It is thought that Ngrol genes might be involved in the formation of genetic tumors, and, moreover, NgORF13 might work in cooperation with NgrolB and NgrolC.

Izv Akad Nauk Ser Biol, 2004 May-Jun, (3), 310 - 8
{Genetically transformed plant roots as a model for studying specific metabolism and symbiotic contacts of the root system}; Active site engineering of the epoxide hydrolase from Agrobacterium radiobacter AD1 to enhance aerobic mineralization of cis-1 et al.; Department of Chemical Engineering, University of Connecticut, Storrs, Connecticut 06269-3222, USAChlorinated ethenes are the most prevalent ground-water pollutants, and the toxic epoxides generated during their aerobic biodegradation limit the extent of transformation . Hydrolysis of the toxic epoxide by epoxide hydrolases represents the major biological detoxification strategy; however, chlorinated epoxyethanes are not accepted by known bacterial epoxide hydrolases . Here, the epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA), which enables growth on epichlorohydrin, was tuned to accept cis-1,2-dichloroepoxyethane as a substrate by accumulating beneficial mutations from three rounds of saturation mutagenesis at three selected active site residues, Phe-108, Ile-219, and Cys-248 (no beneficial mutations were found at position Ile-111) . The EchA F108L/I219L/C248I variant coexpressed with a DNA-shuffled toluene ortho-monooxygenase, which initiates attack on the chlorinated ethene, enhanced the degradation of cis-dichloroethylene (cis-DCE) an infinite extent compared with wild-type EchA at low concentrations (6.8 microm) and up to 10-fold at high concentrations (540 microm) . EchA variants with single mutations (F108L, I219F, or C248I) enhanced cis-DCE mineralization 2.5-fold (540 microm), and EchA variants with double mutations, I219L/C248I and F108L/C248I, increased cis-DCE mineralization 4- and 7-fold, respectively (540 microm) . For complete degradation of cis-DCE to chloride ions, the apparent Vmax/Km for the Escherichia coli strain expressing recombinant the EchA F108L/I219L/C248I variant was increased over 5-fold as a result of the evolution of EchA . The EchA F108L/I219L/C248I variant also had enhanced activity for 1,2-epoxyhexane (2-fold) and the natural substrate epichlorohydrin (6-fold).

Acta Biochim Biophys Sin (Shanghai), 2004 Sep, 36(9), 644 - 8
Preparation of polyclonal antibodies of Rubisco large and small subunits and their application in the functional analysis of the genes; Ma PD et al.; Spinach Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) large (rbcL) and small (rbcS) subunits were separated by SDS-PAGE, and protein amount and purity were determined by Bradford assay . Polyclonal antibodies against rbcL and rbcS subunit were generated in female BALB/c mice and had no cross-reaction with each other . A total of 81 microg antigens were used and 0.3 ml anti-sera with titer of 1:5000 were yielded . The antibodies were also applicable to study rbcL and rbcS in tobacco plant Nicotiana benthamiana . Potato virus X vector pGR107 induced silencing of rbcS gene by Agrobacterium in Nicotiana benthamiana was performed . The expression level of rbcL and rbcS was lower in rbcS silenced plants than that in control plants as detected by the corresponding antibodies . This implied that the expression of rbcL was regulated by rbcS.

Shi Yan Sheng Wu Xue Bao, 2002 Jun, 35(2), 117 - 22
{Transformation of Bt-CpTi fusion protein gene to cabbage (Brassica oleracea var . capitata) mediated by Agrobacterium tumefaciens and particle bombardment}; Yang GD et al.; Bt-CpTI fusion protein gene was transferred to the explants of hypocotyl, cotyledon with petiole and shoot apex of cabbage (Brassica oleracea L . var . capitata) variety "Zhonggan No 8" via Agrobacterium tumefaciens and particle bombardment, and 13 kanamycin-resistant plants were obtained . PCR and Southern blotting hybridization verified that all these plants of the kanr plants of type I regenerated from hypocotyl and cotyledon with petiole mediated by A . tumefaciens were transgenic plants, 2 karr plants of type II stemed from shoot apex mediated by A . tumefaciens were "false-positive"plants and one of 2 kanr plants of type III regenerated from shoot apex via particle bombardment was non-transformed plants . It was showed that part of transgenic plants had high activity of trypsin inhibitor and strong resistance to resist common cabbage worm through the analysis of CpTI relative capacity and insect-resistant test.

Nature, 2004 Sep 2, 431(7004), 87 - 92
Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium; Tzfira T et al.; Genetic transformation of plant cells by Agrobacterium represents a unique case of trans-kingdom DNA transfer . During this process, Agrobacterium exports its transferred (T) DNA and several virulence (Vir) proteins into the host cell, within which T-DNA nuclear import is mediated by VirD2 (ref . 3) and VirE2 (ref . 4) and their host cell interactors AtKAP-alpha and VIP1 (ref . 6), whereas its integration is mediated mainly by host cell proteins . The factors involved in the uncoating of T-DNA from its cognate proteins, which occurs before integration into the host genome, are still unknown . Here, we report that VirF-one of the few known exported Vir proteins whose function in the host cell remains unknown-is involved in targeted proteolysis of VIP1 and VirE2 . We show that VirF localizes to the plant cell nucleus and interacts with VIP1, a nuclear protein . VirF, which contains an F-box motif, significantly destabilizes both VIP1 and VirE2 in yeast cells . Destabilization of VIP1 in the presence of VirF was then confirmed in planta . These results suggest that VIP1 and its cognate VirE2 are specifically targeted by the VirF-containing Skp1-Cdc53-cullin-F-box complex for proteolysis . The critical role of proteasomal degradation in Agrobacterium-mediated genetic transformation was also evident from inhibition of T-DNA expression by a proteasomal inhibitor.

Fungal Genet Biol, 2004 Oct, 41(10), 963 - 71
Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats; Fitzgerald A et al.; RNA-mediated gene silencing has been demonstrated in plants, animals, and more recently in filamentous fungi . Here, we report high frequency, RNA-mediated gene silencing in the apple scab fungus, Venturia inaequalis . The green fluorescent protein (GFP) transgene was silenced in a GFP-expressing transformant . An endogenous gene, trihydroxynaphthalene reductase (THN), involved in melanin biosynthesis, was also silenced . Silencing of these two genes resulted in obvious phenotypes in vitro . High frequency gene silencing was achieved using hairpin constructs for the GFP or the THN genes transferred by Agrobacterium (71 and 61%, respectively) . THN-silenced transformants exhibited a distinctive light brown phenotype and maintained the ability to infect apple . Of significance was the simultaneous silencing of the two genes from a single chimeric, inverted repeat hairpin construct . Silencing of both genes with this construct occurred at a frequency of 51% of all the transformants . All 125 colonies silenced for the GFP gene were also silenced for THN . As THN and GFP silenced transformants have readily detectable phenotypes, the genes have utility as markers for gene silencing . Simultaneous, multiple gene silencing, utilising such marker genes, will enable the development of high through-put screening for functional genomics . This chimeric technology will be particularly valuable when linked with silenced genes that have no obvious phenotype in vitro.

J Plant Res, 2004 Aug, 117(4), 329 - 37 Epub 2004 Jul 28.
Resurrection of an ancestral gene: functional and evolutionary analyses of the Ngrol genes transferred from Agrobacterium to Nicotiana; Aoki S; The Ng rol genes, which have high similarity in sequence to the rol genes of Agrobacterium rhizogenes, are present in the genome of untransformed plants of Nicotiana glauca . It is thought that bacterial infection resulted in the transfer of the Ng rol genes to plants early in the evolution of the genus Nicotiana, since several species in this genus contain rol-like sequences but others do not . Plants transformed with the bacterial rol genes exhibit various developmental and morphological changes . The presence of rol-like sequences in plant genomes is therefore thought to have contributed to the evolution of Nicotiana species . This paper focuses on studies of the Ng rol genes in present-day plants and during the evolution of the genus Nicotiana . The functional sequences of several Ng rol genes may have been conserved after their ancient introduction from a bacterium to the plant . Resurrection of an ancestral function of one of the Ng rol genes, as examined by physiological and evolutionary analyses, is also described . The origin of the Ng rol genes is then considered, based on results of molecular phylogenetic analyses . The effects of the horizontal transfer of the Ng rol genes and mutations in the genes are discussed on the plants of the genus Nicotiana during evolution.

Plant Cell Rep, 2004 Nov, 23(5), 311 - 8 Epub 2004 Aug 26.
Agrobacterium-mediated transformation of European chestnut embryogenic cultures; Corredoira E et al.; An innovative and efficient genetic transformation protocol for European chestnut is described in which embryogenic cultures are used as the target material . When somatic embryos at the globular or early-torpedo stages were cocultured for 4 days with Agrobacterium tumefaciens strain EHA105 harbouring the pUbiGUSINT plasmid containing marker genes, a transformation efficiency of 25% was recorded . Murashige and Skoog culture medium containing 150 mg/l of kanamycin was used as the selection medium . The addition of acetosyringone was detrimental to the transformation efficiency . Transformation was confirmed by a histochemical beta-glucuronidase (GUS ) assay, PCR and Southern blot analyses for the uidA (GUS) and nptII (neomycin phosphotransferase II) genes . At present, 93 GUS-positive chestnut embryogenic lines are being maintained in culture . Low germination rates (6.3%) were recorded for the transformed somatic embryos . The presence of the transferred genes in leaves and shoots derived from the germinated embryos was also verified by the GUS assay and PCR analysis.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 125 - 31
Diversity of 16S rDNA sequences of Rhizobium spp . implications for species determinations; Young JM et al.; Comparative analysis of 70 16S rDNA sequences representing 20 Rhizobium species (including pathogenic Agrobacterium spp.) was conducted using Maximum Likelihood to establish relationships of species using multiple sequences . There is no significant internal division of the Rhizobium clade to suggest that it represents more than one genus . Plant pathogenic (Agrobacterium) species are distributed within the genus . The analysis supported the synonymy of some species (Rhizobium gallicum and Rhizobium mongolense) and the need for comparative investigations of the tumorigenic and nodulating properties of Rhizobium tropici and Rhizobium rhizogenes . Misidentification of some sequences may conceal one or more putative novel species . Some sequences appear to be misidentified because of faulty sequencing or incomplete or inadequate analysis.

Ontogenez, 2004 May-Jun, 35(3), 220 - 8
{Saving insertional recessive lethal mutants of Arabidopsis thaliana}; Shoot regeneration from GUS-transformed tomato (Lycopersicon esculentum) hairy root; Department of Environmental Dynamics and Management, Graduate School of Biosphere Sciences, Hiroshima University, Higashi-Hiroshima, 739-8528, JapanTo study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector . The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses . The regeneration efficiency from hairy root explants was assessed . The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl(-1) 2, 4-D plus 0.25 mgl(-1) kinetin . Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl(-1) GA3 along with 0.5 mgl(-1) NAA . The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi . Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis . Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors.

Plant Physiol Biochem, 2004 Jul-Aug, 42(7-8), 671 - 9
Isoflavonoid accumulation in soybean hairy roots upon treatment with Fusarium solani; Lozovaya VV et al.; Hairy roots were initiated from two soybean {Glycine max (L.) Merr.} genotypes with different susceptibility (susceptible 'Spencer' and partially resistant 'PI567.374') to the disease sudden death syndrome (SDS) caused by the soil-borne fungal pathogen Fusarium solani f . sp . glycines (FSG) to study the role of isoflavonoids in the plant response to FSG infection . Hairy root cultures obtained by transformation with Agrobacterium rhizogenes allows normal root growth that can be visually monitored . The principal isoflavones (genistin, daidzin, glycitin and their malonyl conjugates and aglycones) and also isoflavonoid phytoalexins (coumestrol and glyceollin) were measured by HPLC in extracts of the FSG-inoculated and non-inoculated hairy roots . FSG mycelia grew more slowly on inoculated PI567.374 hairy roots than on Spencer hairy roots . The glyceollin content was higher in FSG-inoculated PI567.374 hairy roots than in Spencer hairy roots even though the glyceollin precursor, the isoflavone daidzein, was higher in Spencer . The de novo synthesis of isoflavones and glyceollin was confirmed by {(14)C}Phe incorporation into glyceollin, which was higher both in the FSG-inoculated roots and surrounding medium of the cv . PI567.374 than that of Spencer . Glyceollin was the most inhibitory to FSG growth among eight isoflavonoids tested . The levels of coumestrol, a putative phytoalexin, did not change upon FSG inoculation . The defense response was also elicited by FSG culture filtrates in hairy roots grown in liquid culture . The data obtained indicate that the ability of soybean roots to rapidly produce sufficient amounts of glyceollin in response to FSG infection might be important in providing partial resistance to this fungus .

J Mol Biol, 2004 Aug 20, 341(4), 961 - 77
Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion System; Jakubowski SJ et al.; The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) translocates DNA and protein substrates across the bacterial cell envelope . Six presumptive channel subunits of this T4SS (VirD4, VirBll, VirB6, VirB8, VirB2, and VirB9) form close contacts with the VirD2-T-strand transfer intermediate during export, as shown recently by a novel transfer DNA immunoprecipitation (TrIP) assay . Here, we characterize the contribution of the hydrophobic channel component VirB6 to substrate translocation . Results of reporter protein fusion and cysteine accessibility studies support a model for VirB6 as a polytopic membrane protein with a periplasmic N terminus, five transmembrane segments, and a cytoplasmic C terminus . TrIP studies aimed at characterizing the effects of VirB6 insertion and deletion mutations on substrate translocation identified several VirB6 functional domains: (i) a central region composed of a large periplasmic loop (P2) (residues 84 to 165) mediates the interaction of VirB6 with the exiting T-strand; (ii) a multi-membrane-spanning region carboxyl-terminal to loop P2 (residues 165 to 245) is required for substrate transfer from VirB6 to the bitopic membrane subunit VirB8; and (iii) the two terminal regions (residues 1 to 64 and 245 to 290) are required for substrate transfer to the periplasmic and outer membrane-associated VirB2 and VirB9 subunits . Our findings support a model whereby the periplasmic loop P2 comprises a portion of the secretion channel and distinct domains of VirB6 participate in channel subunit interactions required for substrate passage to the cell exterior .

Shi Yan Sheng Wu Xue Bao, 2004 Jun, 37(3), 189 - 98
An efficient method for sonication assisted Agrobacterium-mediated transformation of coat protein (CP) coding genes into papaya (Carica papaya L.); Jiang L et al.; An efficient method for the production of transgenic papaya was developed via Sonication Assisted Agrobacterium-mediated Transformation (SAAT) of somatic embryos . The plasmid pGA482G was modified to contain gene PTi-Epj-TL-PLDMV with CP coding sequence of PLDMV Japan strain and chimeric gene PTi-NP-YKT with multiple CP coding sequences from PRSV Taiwan strain, PRSV Hawaii strain and PRSV Thailand strain, respectively . Disarmed Agrobacterium tumefaciens strain LBA4404 carrying the binary plasmid pGA482G with the CP genes and nptII gene was used to transform embryo calli of papaya variety Sunset to produce transgenic papaya plants . The experiment was focused on the screening of effective transformation method . The engineered Agrobacterium grown overnight was diluted with an infection media of high osmotic pressure (1/2 MS medium contain 6% sucrose and 1% glucose, pH 5.7) and adjusted to optical density OD600nm = 0.15-0.20, embryonic calli were immerged in it for 30 min and treated with 5 s, 15 s, and 20 s sonication respectively during the infection . Results indicated that 15 s sonication treatment improved the transformation efficiency dramatically . After 15 s sonication treatment on embryo calli loaded in 15 ml sterile plastic tubes, 21 putative transgenic lines were produced from 80 pieces embryonic calli (26.3%) transformed by Agrobacterium {pGA482G/CPG} and 8 putative transgenic lines was produced from 48 pieces embryonic calli (16.7%) transferred by Agrobacterium {pGA482G/CPB}, while only a single line came out of 64 pieces embryonic calli (1.6%) transformed by Agrobacterium {pGA482G/CPG} and none from 25 pieces embryonic calli transformed by Agrobacterium {pGA482G/CPB} in the non-treatment control . Results also showed that the best concentration of selection antibiotic was 120 mg/L kanamycin . A total of 42 resistant shoots were produced from 421 pieces of original embryonic calli in 9 months . The presence of the CP genes in the transgenic plants and their integration into the papaya genome were confirmed by PCR and Southern hybridization respectively.

Shi Yan Sheng Wu Xue Bao, 2004 Jun, 37(3), 167 - 75
{Intergeneric somatic hybridization between Astragalus adsurgens Pall and Medicago sativa L.}; Jin H et al.; The protoplast fusion was induced between Astragalus adsurgens Pall and Agrobacterium rhizogenes-transformed cell line of Medicago Sativa L . by use of PEG method . Putative somatic hybrid cells were selected based on the observation that only intergeneric hybrid cells continued to divide on the DPD medium without any phytohormone while the cells and clusters derived from parental cells did not survive further . After being cultured, the somatic hybrid calli were obtained . Although both of the parental calli were not capable of regenerating , one of the hydrid cell lines recovered the differentiation ability and produced small shoots . Characterizations of the hybrid calli were carried out by examination of chromsome number, opine synthetase assay, isoenzyme and RAPD analyses.

Plant Cell Rep . 2004 Aug 20; {Epub ahead of print}
Agrobacterium-mediated in vitro transformation of wood-producing stem segments in eucalypts; Spokevicius AV et al.; The genetic manipulation of perennial woody tree species presents a range of additional challenges compared to that of annual weedy crop species . These include long generation times and reproductive cycle, the heterogeneity of plants under investigation and, when investigating wood properties, a number of physical and biochemical limitations to microscopical and molecular experimentation . The use of in vitro wood formation systems for molecular studies and Agrobacterium-mediated introduction of transgenes overcomes many of these obstacles . Using a commercially relevant Eucalyptus species as model organism, we demonstrate here that in vitro wood formation systems can be readily employed to introduce transgenes into growing wood-producing tissue, initially leading to frequent transient gene expression in a range of cell types . Stable transformation events were observed as sectors of transformed tissue derived from primary transformation events in individual cells . The usefulness of such systems for the analysis of gene function during the process of wood formation and wood quality determination, as well as for constructing developmental fate maps of cambial derivatives, is discussed.

FEMS Microbiol Lett, 2004 Aug 15, 237(2), 219 - 26
Analysis of growth phase regulated KatA and CatE and their physiological roles in determining hydrogen peroxide resistance in Agrobacterium tumefaciens; Prapagdee B et al.; Agrobacterium tumefaciens possesses two catalases, a bifunctional catalase-peroxidase, KatA and a homologue of a growth phase regulated monofunctional catalase, CatE . In stationary phase cultures and in cultures entering stationary phase, total catalase activity increased 2-fold while peroxidase activity declined . katA and catE were found to be independently regulated in a growth phase dependent manner . KatA levels were highest during exponential phase and declined as cells entered stationary phase, while CatE was detectable at early exponential phase and increased during stationary phase . Only small increases in H2O2 resistance levels were detected as cells entering stationary phase . The katA mutant was more sensitive to H2O2 than the parental strain during both exponential and stationary phase . Inactivation of catE alone did not significantly change the level of H2O2 resistance . However, the katA catE double mutant was more sensitive to H2O2 during both exponential and stationary phase than either of the single catalase mutants . The data indicated that KatA plays the primary role and CatE acts synergistically in protecting A . tumefaciens from H2O2 toxicity during all phases of growth . Catalase-peroxidase activity (KatA) was required for full H2O2 resistance . The expression patterns of the two catalases in A . tumefaciens reflect their physiological roles in the protection against H2O2 toxicity, which are different from other bacteria.

J Biotechnol, 2004 Sep 9, 112(3), 247 - 53
Expression of His-tagged Shigella IpaC in Arabidopsis; MacRae AF et al.; Although the expression of histidine (His)-tagged proteins in bacteria is routine, few His-tagged proteins have been expressed in plants, and no His-tagged proteins from bacterial pathogens have been expressed in plants, to our knowledge . Here, we demonstrate expression of the Shigella flexneri invasion plasmid antigen, IpaC, in Arabidopsis thaliana . S . flexneri is the causitive trigger for bacillary dysentery, and IpaC is essential for bacterial entry into epithelial cells . IpaC, attached to a 5' leader containing six tandem His codons, was cloned into a pBI121 vector . This clone was introduced into Agrobacterium tumefaciens and Arabidopsis plants were then transformed . T1 and T2 plant generations were obtained . Total plant proteins were extracted from T2 leaves; the Bradford assay was used to determine protein concentrations . A nickel-coated ELISA plate method, using both anti-His and anti-IpaC 1 degrees antibodies, was used to detect and quantify IpaC in transgenic Arabidopsis plants . Between 1.9 and 2.3 microg IpaC/mg total plant protein was obtained; this equals 0.2% of total protein, an amount comparable to other recombinant protein estimates in plants . Expressing His-tagged proteins from bacterial pathogens, in plants, is important because plant material could ultimately be fed or applied intranasally to animals that are "at risk" for infection by such bacterial pathogens, thus causing them to raise antibodies against the pathogens--functioning as a vaccine.

Methods Mol Biol, 2005, 286, 351 - 64
Agrobacterium persistence in plant tissues after transformation; Cubero J et al.; Agrobacterium spp . are routinely used in plant transformation to introduce genes of interest in valuable economic species . However, several agrobacteria species are also plant pathogens with ability to survive in different environments including the inner part of the plants . To avoid the release of genetic modified bacteria a successful plant transformation protocol must include the total elimination of agrobacteria by the use of antibiotics . Because sometimes these antibiotics failed in removing the bacteria entirely, confirmation of agrobacteria absence after plant transformation and regeneration is required . Different methodologies can be used for this purpose: isolation techniques followed by identification are used if detection of viable and culturable bacteria is necessary and techniques based on the polymerase chain reaction can be used to detect agrobacteria independently of their physiological state . Here we present several protocols to detect Agrobacterium in tissues of transformed plants as well as methods to identify the strains isolated . These identification methods can help to elucidate if they are the engineered bacteria used in the transformation process or just part of the natural endophytic microbiota.

Methods Mol Biol, 2005, 286, 237 - 54
Elimination of marker genes from transgenic plants using MAT vector systems; Ebinuma H et al.; We have developed an efficient system (Multi-Auto-Transformation {MAT} vectors) for the removal of marker genes and to increase the regeneration frequency of transgenic crops without using antibiotic selection, reducing their possible environmental impact . The MAT vector system is designed to use the oncogenes (ipt, iaaM/H, rol) of Agrobacterium, which control the endogenous levels of plant hormones and the cell responses to plant growth regulators, to differentiate transgenic cells, and to select marker-free transgenic plants . The oncogenes are combined with the site-specific recombination system (R/RS) . At transformation, the oncogenes regenerate transgenic plants and then are removed by the R/RS system to generate marker-free transgenic plants . The choice of a promoter for the oncogenes and the recombinase (R) gene, the state of plant materials and the tissue culture conditions greatly affect efficiency of both the regeneration of transgenic plants and the generation of marker-free plants . We have evaluated these conditions in several plant species to increase their generation efficiency . This chapter describes our transformation protocols using MAT vectors.

Methods Mol Biol, 2005, 286, 165 - 76
Regeneration of transgenic cassava from transformed embryogenic tissues; Zhang P et al.; Production of transgenic plants is gradually becoming routine in cassava biotechnology . Green cotyledons of maturing somatic embryos (somatic cotyledons for short) and friable embryogenic suspensions (FES) are the target tissues for transformation by Agrobacterium or biolistics . Putative transgenic shoots develop from transformed somatic cotyledons via shoot organogenesis or from FES via somatic embryogenesis under selection . Maturation of transgenic somatic embryos is induced by transfer to maturation medium with reduced concentrations of selective agents . Mature somatic embryos can also develop directly from FES cells under selection . Transgenic plants are regenerated by the elongation of transgenic shootlets from organogenesis experiments and by the germination of or shoot development from transgenic mature embryos cultured without selection . beta-Glucuronidase (GUS) assays and rooting tests can be used to screen for escapes from selection, which improves the regeneration rate of truly transgenic plants.

Methods Mol Biol, 2005, 286, 151 - 64
Genetic transformation of conifers utilizing somatic embryogenesis; Klimaszewska K et al.; Over the last 5 yr, the production of transgenic conifers has been greatly facilitated by the ability to transform somatic embryonal tissues (somatic embryos) via cocultivation with Agrobacterium tumefaciens . This has allowed us to develop protocols for the genetic transformation of several spruce species . Furthermore, these procedures can produce an average of 20 independent transgenic lines (translines) per gram fresh mass of embryonal tissue, providing for the first time the magnitude-of-scale required for implementing large-scale functional genomics studies in conifers . Combined with efficient regeneration of transgenic trees via somatic embryos, the potential for genetic engineering of conifers has been demonstrated by stable reporter gene expression (GUS or GFP) resulting from single insert T-DNA integration events.

Methods Mol Biol, 2005, 286, 141 - 50
Organogenesis from transformed tomato explants; Frary A et al.; Tomato was one of the first crops for which a genetic transformation system was reported involving regeneration by organogenesis from Agrobacterium-transformed explants . Since the initial reports, various factors have been studied that affect the efficiency of tomato transformation and the technique has been useful for the isolation and identification of many genes involved in plant disease resistance, morphology and development . In this method, cotyledon explants from in vitro-grown seedlings are precultured overnight on a tobacco suspension feeder layer . The explants are then inoculated with Agrobacterium and returned to the feeder layer for a 2-d period of cocultivation . After cocultivation, the explants are transferred to an MS-based selective regeneration medium containing zeatin . Regenerated shoots are then rooted on a separate selective medium . This protocol has been used with several tomato cultivars and routinely yields transformation efficiencies of 10-15%.

Methods Mol Biol, 2005, 286, 91 - 102
Floral dip: agrobacterium-mediated germ line transformation; Clough SJ; Many researchers use the flowering plant Arabidopsis thaliana to study gene function and basic plant biology . This easy-to-grow, small plant is ideal for genetic studies as it has a relatively simple genome compared to crop plants and its genetic material has been recently sequenced . Another very useful feature of Arabidopsis is that it is extremely simple to transform genetically . The ability to insert genes of interest stably into a given plant is essential to understand and verify gene function . Transformation is also a means of introducing specific traits that are difficult or impossible to introduce by conventional breeding techniques . This chapter provides detailed explanations on the floral dip protocol, a simple method to transform Arabidopsis by inoculating immature flowers with Agrobacterium tumefaciens.

Methods Mol Biol, 2005, 286, 47 - 60
Production of hairy root cultures and transgenic plants by Agrobacterium rhizogenes-mediated transformation; Christey MC et al.; Agrobacterium rhizogenes-mediated transformation results in the development of hairy roots at the site of infection . The production of hairy roots involves cocultivation of explants with A . rhizogenes and the subsequent selection of hairy roots on hormone-free medium . Hairy roots have many applications for research including secondary product production and for the study of biochemical pathways . In addition, transgenic plants regenerated from hairy roots often show an altered phenotype due to the presence of the rol genes . In this chapter we describe how to produce and grow hairy root cultures, how to regenerate shoots from these hairy roots, and how to conduct molecular analysis of these cultures.

Methods Mol Biol, 2005, 286, 35 - 46
Plant transformation: agrobacterium-mediated gene transfer; Dandekar AM et al.; Plant transformation is the process by which DNA is introduced into plant cells or tissues . The DNA can come from virtually any source . Gene transfer methodology has become part of an essential technology to manipulate plants for both scientific and commercial purposes . Transgenic plants, the products of this methodology, are useful for dissecting the mechanism(s) of plant gene regulation . This technology is also useful for identifying and evaluating agriculturally useful traits (genes) as well as for their introduction into commercially valuable crops . One of the most efficient methods for gene transfer employs Agrobacterium tumefaciens and takes advantage of the naturally evolved crown gall-inducing mechanisms of DNA transfer present in this common soil pathogen . Much has been learned about the mechanisms of this form of DNA movement and subsequent crown gall induction . This information has been applied to develop methods that result in the formation of gall-free, genetically transformed plants . This chapter describes a detailed protocol for Agrobacterium-mediated transformation of tobacco cells and their subsequent selection and regeneration into transgenic plants.

Plant Cell Rep, 2005 Jan, 23(8), 540 - 7 Epub 2004 Aug 07.
Optimising the tissue culture conditions for high efficiency transformation of indica rice; Lin YJ et al.; Establishment of high efficiency Agrobacterium-mediated transformation techniques has greatly accelerated the widespread application of transformation in japonica rice . However, transformation in indica rice remains difficult . In this study, we identify two new media for subculture and differentiation, the two major steps in the tissue culture process for transformation . These media were tested using four cultivars representing very different germplasms of indica rice . The results show that the new media significantly improved the growth rate and quality of the calli, and also increased the differentiation rate for all four cultivars tested . Use of these modified media in transformation experiments also greatly improved the transformation efficiency of all four indica cultivars.

J Virol, 2004 Sep, 78(17), 9487 - 98
Differential roles of AC2 and AC4 of cassava geminiviruses in mediating synergism and suppression of posttranscriptional gene silencing; Vanitharani R et al.; Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection . In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses . Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-{CM}) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants . Here, we report that synergism between ACMV-{CM} and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-{CM} or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-{CM}, respectively, in tobacco BY-2 protoplasts . Furthermore, transient expression of ACMV-{CM} AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by approximately 8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-{CM} DNA accumulation, also by approximately 8-fold . An Agrobacterium-based leaf infiltration assay determined that ACMV-{CM} AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation . In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host . The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.

Biol Pharm Bull, 2004 Aug, 27(8), 1261 - 5
Tropane alkaloid production and shoot regeneration in hairy and adventitious root cultures of Duboisia myoporoides-D . leichhardtii hybrid; Yoshimatsu K et al.; Co-culture conditions for Duboisia myoporoides-D . leichhardtii hybrid hairy root induction were investigated using leaf explants and Agrobacterium rhizogenes ATCC 15834 . The bacteria density and duration of co-culture greatly affected the induction rate; the highest rate of 50% was obtained when the leaf explants were co-cultured for 2 d with 10(6) bacteria . One hairy root clone that showed the fastest root growth was selected and used for comparison study with adventitious roots cultured with 0.5 mg/l indole-3-acetic acid (IAA) . The hairy roots cultured in Murashige and Skoog (MS) liquid medium grew well and yielded much more tropane alkaloids (35 mg/l scopolamine and 17 mg/l hyoscyamine) than adventitious roots cultured in 0.5 mg/l IAA after 6 weeks of culture at 25 degrees C in the dark . The hairy and adventitious roots (2.5 cm) grown in liquid media were divided into 5 parts (each 0.5 cm) along the root axis . Distribution of scopolamine and IAA was then determined by enzyme-linked immunosorbent assay (ELISA) . Inverse relationship between contents of scopolamine and IAA was observed in the hairy roots; increase of scopolamine and decrease of IAA were proportional to the distance from the root meristem . In contrast, the contents of scopolamine and IAA were relatively constant in the adventitious roots . In shoot regeneration experiments, the hairy and adventitious root segments (1 cm) were placed onto 1/2 MS solid medium containing various concentrations of IAA and BA cultured at 25 degrees C under 16 h light . In adventitious roots, the shoots regenerated on media containing 6-benzyladenine (BA) (0.5 to 5 mg/l), and 100% regeneration was observed in medium with 0.1 mg/l IAA and 2 mg/l BA . On the other hand, shoot regeneration was only observed in 33% of hairy roots cultured on medium containing 5 mg/l BA.

Appl Biochem Biotechnol, 2004 Jun, 117(3), 175 - 87
Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence; Kang TJ et al.; Escherichia coli heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant for coadministered antigens . We synthesized a gene encoding the B-subunit of LT (LTB) adapted to the coding sequence of tobacco plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its level of expression in plants . The synthetic LTB gene was cloned into a plant expression vector adjacent to the CaMV 35S promoter and was introduced into tobacco by Agrobacterium-mediated transformation . The amount of LTB protein detected in transgenic tobacco leaves was 2.2% of the total soluble plant protein, which is approx 200-fold higher than in previous reports of native LTB gene expression in transgenic plants . Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers . Copryright 2004 Humana Press Inc.

Appl Biochem Biotechnol, 2004 Jul-Sep, 118(1-3), 21 - 31
Fermentative production of curdlan; Saudagar PS et al.; Curdlan was produced by pure culture fermentation using Agrobacterium radiobacter NCIM 2443 . Three different carbon sources (glucose, sucrose, maltose) were selected for study . Sucrose was found to be the most efficient . Utilization of sugar during the course of fermentation was studied, and the data were correlated to the production of curdlan . Curdlan mimics a secondary metabolite, in that its synthesis is associated with the poststationary growth phase of nitrogen-depleted batch culture . This was inferred from the results obtained from utilization of nitrogen . Regulation of pH at 6.1 +/- 0.3 resulted in an increased yield of curdlan from 2.48 to 4.8 g/L, and the corresponding increase in succinoglucan production was from 1.78 to 2.8 g/L . An attempt was made to increase curdlan production by the addition of the uridine nucleotides UMP and UDP-glucose to the fermentation broth . It was found that UDP-glucose at 0.8 microg/mL and UMP at 0.6 microg/mL served as precursors for curdlan and succinoglucan production when added after 18 h of nitrogen depletion in the fermentation broth.

Plant Cell Rep . 2004 Aug 5; {Epub ahead of print}
Agrobacterium tumefaciens-mediated transformation of blueberry ( Vaccinium corymbosum L.); Song GQ et al.; Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 micro M for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days . Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene ( nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)(3)AmasPmas . Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 micro M AS . Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime . Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C . After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora . Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer . Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe . The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

Theor Appl Genet, 2004 Nov, 109(7), 1512 - 8 Epub 2004 Nov.
Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip; Labra M et al.; The occurrence of DNA modification is an undesired phenomenon accompanying plant cell transformation . The event has been correlated with the stress imposed by the presently utilised transformation procedures, all depending on plant differentiation from in vitro cell culture, but other causes have not been excluded . In this work, transgenic Arabidopsis thaliana plants have been produced by an approach that does not require cell dedifferentiation, being based on in planta Agrobacterium-mediated gene transfer by flower infiltration, which is followed by recovery and selection of transgenic progeny . Genomic DNA changes in transgenic and control plants have been investigated by AFLP and RAMP analysis . Results show no statistically relevant genomic modifications in transgenic plants, as compared with control untreated plants . Variations were observed in callus-derived A . thaliana plants, thus supporting the conclusion that somaclonal variation is essentially correlated with the stress imposed by the in vitro cell culture, rather than with the integration of a foreign gene.

Plant Physiol, 2004 Aug, 135(4), 2411 - 23 Epub 2004 Aug 06.
Relocalization of nuclear ALY proteins to the cytoplasm by the tomato bushy stunt virus P19 pathogenicity protein; Uhrig JF et al.; The P19 protein of tomato bushy stunt virus (TBSV) is a multifunctional pathogenicity determinant involved in suppression of posttranscriptional gene silencing, virus movement, and symptom induction . Here, we report that P19 interacts with the conserved RNA-binding domain of an as yet uncharacterized family of plant ALY proteins that, in animals, are involved in export of RNAs from the nucleus and transcriptional coactivation . We show that the four ALY proteins encoded by the Arabidopsis genome and two ALY proteins from Nicotiana benthamiana are localized to the nucleus . Moreover, and in contrast to animal ALY, all but one of the proteins are also in the nucleolus, with distinct subnuclear localizations . Infection of plants by TBSV or expression of P19 from Agrobacterium results in relocation of three of the six ALY proteins from the nucleus to the cytoplasm demonstrating specific targeting of the ALY proteins by P19 . The differential effects on subcellular localization indicate that, in plants, the various ALY proteins may have different functions . Interaction with and relocalization of ALY is prevented by mutation of P19 at residues previously shown to be important for P19 function in plants . Down-regulation of expression of two N . benthamiana ALY genes by virus-induced gene silencing did not interfere with posttranscriptional gene silencing . Targeting of ALY proteins during TBSV infection may therefore be related to functions of P19 in addition to its silencing suppression activity.

Commun Agric Appl Biol Sci, 2003, 68(2 Pt A), 277 - 83
Oligosaccharides and derivatives--integrating biocatalyst selectivity and chemical diversity; Buchholz K et al.; Several biocatalytic pathways are available to synthesize rare sugars, oligosaccharides and derivatives . As examples selective hydrolysis, transglycosylation and regioselective oxidation are presented illustrating their potential . From inulin, difructose anhydrides and inulobiose are accessible via specific hydrolases with transglycosylation activity . Glycosyltransferases of the sucrase type utilise sucrose as a versatile substrate for the selective transfer of either glucosyl- or fructosyl units to a broad range of acceptors, sugars as well as derivatives, to yield oligosaccharides of different type . Kinetic analysis and reaction engineering can provide high product yields and concentration, as will be shown for glucose, yielding isomaltooligosaccharides as an example . Several new acceptor saccharides, including sugar alcohols and acids, have been shown to give new oligosaccharides . Regioselective oxidation of disaccharides like sucrose, lactose and others with resting cells of Agrobacterium tumefaciens yield 3-keto-disaccharides . These can further be functionalized in aquous systems without protecting groups via established chemical routes, such as catalytic hydration . As an example allosucrose is thus obtained from 3-keto-sucrose with high yield and stereochemical selectivity, which in turn can easily be hydrolysed and separated from fructose to give allose . Amino acid and peptide conjugates are accessible via reductive amination and acylation, as are building blocks for polymerisation.

Protein Expr Purif, 2004 Sep, 37(1), 196 - 202
HIV-1 gp120 V3 cholera toxin B subunit fusion gene expression in transgenic potato; Kim TG et al.; A cDNA fragment encoding the V3 loop of human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp120 was fused to the cholera toxin B subunit gene (CTB-gp120) and transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation . The CTB-gp120 fusion gene was detected in genomic DNA from transformed potato leaves by PCR DNA amplification . Synthesis and assembly of the CTB-gp120 fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis . The binding of CTB-gp120 fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by GM1-ganglioside enzyme-linked immunosorbent assay (GM1-ELISA) . The ELISA results indicated that CTB-gp120 fusion protein made up 0.002-0.004% of the total soluble tuber protein . Synthesis of CTB-gp120 monomers and their assembly into biologically active oligomers in transformed potato tuber tissues demonstrates for the first time the expression of HIV-1 gp120 in plants and emphasizes the feasibility of using edible plant-based vaccination for protection against HIV-1 infection.

J Plant Res . 2004 Jul 28; {Epub ahead of print}
Resurrection of an ancestral gene: functional and evolutionary analyses of the Ng rol genes transferred from Agrobacterium to Nicotiana; Aoki S; The Ng rol genes, which have high similarity in sequence to the rol genes of Agrobacterium rhizogenes, are present in the genome of untransformed plants of Nicotiana glauca . It is thought that bacterial infection resulted in the transfer of the Ng rol genes to plants early in the evolution of the genus Nicotiana, since several species in this genus contain rol-like sequences but others do not . Plants transformed with the bacterial rol genes exhibit various developmental and morphological changes . The presence of rol-like sequences in plant genomes is therefore thought to have contributed to the evolution of Nicotiana species . This paper focuses on studies of the Ng rol genes in present-day plants and during the evolution of the genus Nicotiana . The functional sequences of several Ng rol genes may have been conserved after their ancient introduction from a bacterium to the plant . Resurrection of an ancestral function of one of the Ng rol genes, as examined by physiological and evolutionary analyses, is also described . The origin of the Ng rol genes is then considered, based on results of molecular phylogenetic analyses . The effects of the horizontal transfer of the Ng rol genes and mutations in the genes are discussed on the plants of the genus Nicotiana during evolution.

Arch Virol, 2004 Aug, 149(8), 1643 - 52 Epub 2004 Apr 05.
Analysis of an isolate of Mungbean yellow mosaic virus (MYMV) with a highly variable DNA B component; Karthikeyan AS et al.; One DNA A (KA30) and five different DNA B components (KA21, KA22, KA27, KA28 and KA34) of a geminivirus, Mungbean yellow mosaic virus-Vigna (MYMV-Vig) were cloned from a pooled sample of field-infected Vigna mungo plants from Vamban, South India . MYMV-Vig DNA A (KA30) and one of the DNA B components (KA27) exhibited 97% and 95% sequence identities, respectively, to those of MYMV reported from Thailand . However, the DNA B components KA21, KA22, KA28 and KA34 exhibited only 71 to 72% sequence identity to MYMV DNA B . Co-existence of multiple DNA B components in field-infected V . mungo was proved by Southern and PCR analyses . Each of the five DNA B components was infective together with the DNA A upon agroinoculation . Agroinoculation with mixed cultures of Agrobacterium with partial dimers of DNA A and all five DNA Bs proved that all five DNA B components can co-infect a single V . mungo plant.

Plant Cell Rep . 2004 Jul 28; {Epub ahead of print}
Transformation of Saussurea medusa for hairy roots and jaceosidin production; Zhao D et al.; Axenically grown Saussurea medusa plantlets were inoculated with four Agrobacterium rhizogenes strains, and hairy root lines were established with A . rhizogenes strain R1601 in N6 medium . PCR and Southern hybridization confirmed integration of the T-DNA fragment of the Ri plasmid from A . rhizogenes into the genome of S . medusa hairy roots . In N6 medium, maximum biomass of the hairy root cultures was achieved {8 g (dry weight) per liter; growth ratio 35-fold} after 21 days of culture . The amount of jaceosidin extracted from the hairy root cultures was 46 mg/l (production ratio of 37-fold) after 27 days of culture . The maximum jaceosidin content obtained using N6 medium was higher than that obtained with Modified White, MS or B5 medium . In N6 medium, the tip segments were more efficient for hairy root growth and jaceosidin production than the middle and basal regions of the root.

Biotechnol Bioeng, 2004 Aug 20, 87(4), 495 - 500
Potato flour viscosity improvement is associated with the expression of a wheat LMW-glutenin gene; Benmoussa M et al.; It has been previously shown that expression of a high-molecular-weight glutenin (HMW-GS) in transgenic wheat seeds resulted in the improvement of flour functional properties . In this study, potato flour viscosity was improved through a specific expression of a low-molecular-weight glutenin (LMW-GS-MB1) gene in tuber . The resulting construct was introduced into potato leaf explants (Solanum tuberosum cv Kennebec) through Agrobacterium tumefaciens-mediated gene transfer . Southern and Northern analysis of transgenic potato confirmed that the integration of LMW-GS-MB1 in genomic DNA was stable and its mRNA was abundant in transgenic line 16 tubers . Western blot analysis of line 16 extract shows a LMW-GS subunit accumulation in tuber . To demonstrate the capacity of transgenic lines to produce tubers with improved flour functional properties, transgenic lines 9 and 16 exhibiting, respectively, moderate and high expression of LMW-GS-MB1 mRNA and nontransgenic plants were transferred to field plots . The mean viscosity value of flour obtained from the field-grown tubers of transgenic line 16 exhibited a 3-fold increase in viscosity at 23 degrees C when compared to flour from nontransgenic tubers.

Plant Physiol, 2004 Aug, 135(4), 2162 - 71 Epub 2004 Jul 30.
Characterization of the Arabidopsis lysine-rich arabinogalactan-protein AtAGP17 mutant (rat1) that results in a decreased efficiency of agrobacterium transformation; Gaspar YM et al.; Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants . The Arabidopsis rat1 mutant, previously characterized as resistant to Agrobacterium tumefaciens root transformation, is due to a mutation in the gene for the Lys-rich AGP, AtAGP17 . We show that the phenotype of rat1 correlates with down-regulation of AGP17 in the root as a result of a T-DNA insertion into the promoter of AGP17 . Complementation of rat1 plants by a floral dip method with either the wild-type AGP17 gene or cDNA can restore the plant to a wild-type phenotype in several independent transformants . Based on changes in PR1 gene expression and a decrease in free salicylic acid levels upon Agrobacterium infection, we suggest mechanisms by which AGP17 allows Agrobacterium rapidly to reduce the systemic acquired resistance response during the infection process.

Annu Rev Phytopathol, 2004, 42, 439 - 64
Chemical biology of multi-host/pathogen interactions: chemical perception and metabolic complementation; Palmer AG et al.; The xenognostic mechanisms of two multi-host pathogens, the causative agent of crown gall tumors Agrobacterium tumefaciens and the parasitic plant Striga asiatica, are compared . Both organisms are general plant pathogens and require similar information prior to host commitment . Two mechanistic strategies, chemical perception and metabolic complementation, are used to ensure successful host commitment . The critical reactions at host-parasite contact are proton and electron transfer events . Such strategies may be common among multi-host pathogens.

Int J Syst Evol Microbiol, 2004 Jul, 54(Pt 4), 1271 - 5
Bradyrhizobium betae sp . nov., isolated from roots of Beta vulgaris affected by tumour-like deformations; Rivas R et al.; Some varieties of sugar beet, Beta vulgaris, cultivated in northern Spain have large deformations that resemble the tumours produced by Agrobacterium species . In an attempt to isolate the agent responsible for these deformations, several endophytic slow-growing bacterial strains were isolated, the macroscopic morphology of which resembled that of Bradyrhizobium species . These strains were not able to produce tumours in Nicotiana tabacum plants and, based on phylogenetic analysis of their 16S rRNA, they are closely related to the genus Bradyrhizobium . Phenotypic and molecular characteristics of these strains revealed that they represent a species different from all Bradyrhizobium species previously described . Sequence analysis of the 16S-23S rDNA intergenic spacer region indicated that these novel strains form a homogeneous group, related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense . DNA-DNA hybridization confirmed that these strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium betae sp . nov . is proposed . The type strain is PL7HG1T (=LMG 21987T=CECT 5829T).

Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1518 - 24
Intracellular localization of a class IV chitinase from yam; Mitsunaga T et al.; Genomic DNA encoding a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves in previous research (Biosci . Biotechnol . Biochem., 68, 1508-1517 (2004)) . But this chitinase had an additional sequence composed of eight amino acids (a C-terminal extension) at the C-terminal, compared with class IV chitianses from other plants . In order to clarify the role of this C-terminal extension in cellular localization, plants and suspension-cultured cells of Nicotiana tabacum were transformed with either the cloned yam class IV chitinase gene carrying the C-terminal extension or its truncated gene by the Agrobacterium-mediated method, and then their localization was investigated . The results suggest that the C-terminal extension of yam class IV chitinase plays a role as a targeting signal for plant vacuoles . This is the first report presenting the existence of vacuolar type class IV chitinase.

Chem Biol, 2004 Jul, 11(7), 981 - 90
Directed evolution of epoxide hydrolase from A . radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling; van Loo B et al.; The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling . An agar plate assay was used to screen the mutant libraries for activity . Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE) . Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides . The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic . Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate . Several mutations in the shuffled mutants had additive effects.

Biochem Soc Trans, 2004 Aug, 32(Pt 4), 614 - 6
Choice of a start codon in a single transcript determines DNA ligase 1 isoform production and intracellular targeting in Arabidopsis thaliana; Sunderland PA et al.; DNA ligase 1 (AtLIG1) is the only essential DNA ligase activity in Arabidopsis and is implicated in the important processes of DNA replication, repair and recombination and in transgene insertion during Agrobacterium-mediated plant transformations . The mitochondrial and nuclear forms of DNA ligase 1 in Arabidopsis are translated from a single mRNA species through the control of translation initiation from either the first (M1) or second (M2) in-frame AUG codons respectively . Translation from a third in-frame AUG codon (M3) occurs on transcripts in which M1 and M2 are mutagenized to stop codons . Wild-type AtLIG1-GFP constructs (where GFP stands for green fluorescent protein) can be targeted in planta to both the nucleus and mitochondria . AtLIG1-GFP translation from M1 specifically targets the fusion protein only to mitochondria in planta, whereas translation from M2 or M3 targets the fusion protein only to the nucleus . Interestingly, the AtLIG1-GFP fusion protein in which translation is initiated from M1 contains both an N-terminal mtPS (mitochondrial targeting presequence) and a nuclear localization signal; nonetheless, this protein is only targeted to the mitochondria . This result raises intriguing questions on the translational control mechanisms that regulate how the protein products of a single transcript are targeted to more than one cellular compartment.

Biotechnol Lett, 2004 May, 26(10), 787 - 92
Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana; Wu H et al.; VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens . Transgenic lines of Arabidopsis thaliana expressing recombinant VP2 were developed . The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression . A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciens by electroporation . Agrobacterium containing the rpE-VP2 construct was used to transform Ar . thaliana and transgenic plants were selected using bialaphos . The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR . Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants . The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein . Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.

Methods Mol Biol, 2004, 267, 329 - 50
Gene transfer and expression in plants; Lorence A et al.; Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between plant species . However, recent developments in plant molecular biology and genomics now give us access to knowledge and understanding of plant genomes and the possibility of modifying them . This chapter presents an updated overview of the two most powerful technologies for transferring genetic material (DNA) into plants: Agrobacterium-mediated transformation and microparticle bombardment (biolistics) . Some of the topics that are discussed in detail are the main variables controlling the transformation efficiency that can be achieved using each one of these approaches; the advantages and limitations of each methodology; transient versus stable transformation approaches; the potential of some in planta transformation systems; alternatives to developing transgenic plants without selection markers; the availability of diverse genetic tools generated as part of the genome sequencing of different plant species; transgene expression, gene silencing, and their association with regulatory elements; and prospects and ways to possibly overcome some transgene expression difficulties, in particular the use of matrix-attachment regions (MARs).

Methods Mol Biol, 2004, 267, 315 - 25
Three decades of fungal transformation: novel technologies; Casas-Flores S et al.; Fungi are lower eukaryotes that play important roles in many human activities, including biotechnological processes, phytopathology, and biomedical research . In addition, they are excellent models for molecular and genetic studies . An important key in the advancement of genetics and molecular biology of a given organism is the development of genetic transformation systems . This technology makes possible the analysis and manipulation of the genome of the organism of interest . Thirty years from the first report of transformation of a fungus, transformation of many other fungi has been achieved . However, the development of gene tagging systems generally applicable to a wide range of filamentous fungi has remained elusive . A widely used gene tagging strategy for filamentous fungi is restriction enzyme mediated integration (REMI) . In recent years numerous reports have been published describing the effective application of REMI . However, REMI shows certain disadvantages for some fungi . Recently a very promising alternative strategy has been reported based on the use of the soil bacterium Agrobacterium tumefaciens . Using this system a well-defined DNA segment (T-DNA) is transferred, which integrates by illegitimate recombination and is 100-1000 times more efficient than conventional methods . The T-DNA can be used as an efficient tool to generate recombinant strains where DNA is integrated as a single copy, allowing the generation of collections of gene-tagged mutants of the fungus of interest.

Biotechnol Lett, 2004 Jul, 26(14), 1147 - 52
Stable and specific expression of 4-coumarate:coenzyme A ligase gene (4CL1) driven by the xylem-specific Pto4CL1 promoter in the transgenic tobacco; Lu H et al.; The ability of 4-coumarate:coenzyme A ligase promoter from Populus tomentosa (Pto4CL1p) to drive expression of the GUS reporter gene and 4-coumarate:coenzyme A ligase gene in tobacco has been studied using transgenic plants produced by Agrobacterium-mediated transformation . Intense GUS histochemical staining was detected in the xylem of stem in transgenic tobacco plants carrying the 1140 bp Pto4CL1p promoter . To further investigate the regulation function of the tissue-specific expression promoter, Pto4CL1p, a binary vector containing Pto4CL1p promoter fused with 4CL1 gene was transferred into tobacco . The activity of the 4CL1 enzyme doubled in the stems of transgenic tobacco but did not increase in the leaves . The content of lignin was increased 25% in the stem but there was no increase in the leaves of transgenic tobacco.

Trends Genet, 2004 Aug, 20(8), 375 - 83
Agrobacterium T-DNA integration: molecules and models; Tzfira T et al.; Genetic transformation mediated by Agrobacterium involves the transfer of a DNA molecule (T-DNA) from the bacterium to the eukaryotic host cell, and its integration into the host genome . Whereas extensive work has revealed the biological mechanisms governing the production, Agrobacterium-to-plant cell transport and nuclear import of the Agrobacterium T-DNA, the integration step remains largely unexplored, although several different T-DNA integration mechanisms have been suggested . Recent genetic and functional studies have revealed the importance of host proteins involved in DNA repair and maintenance for T-DNA integration . In this article, we review our understanding of the specific function of these proteins and propose a detailed model for integration.

Microb Ecol, 2004 Jan, 47(1), 96 - 103
Engineering root exudation of Lotus toward the production of two novel carbon compounds leads to the selection of distinct microbial populations in the rhizosphere; Oger PM et al.; The culture of opine-producing transgenic Lotus plants induces the increase in the rhizosphere of bacterial communities that are able to utilize these molecules as sole carbon source . We used transgenic Lotus plants producing two opines, namely mannopine and nopaline, to characterize the microbial communities directly influenced by the modification of root exudation . We showed that opine-utilizers represent a large community in the rhizosphere of opine-producing transgenic Lotus . This community is composed of at least 12 different bacterial species, one third of which are able to utilize the opine mannopine and two thirds the opine nopaline . Opine utilizers are diverse, belonging to the Gram-positive and -negative bacteria . We described two novel mannopine-utilizing species, Rhizobium and Duganella spp., and five novel nopaline-utilizing species, Duganella, Afipia, Phyllobacterium, Arthrobacter, and Bosea spp . Although opine utilizers mostly belong to the alpha-Proteobacteria, Rhizobiaceae family, there is little overlap between the populations able to utilize each of the two opines produced by the plants . Noticeably, in the rhizosphere of transgenic Lotus, only the opine mannopine favors the growth of Agrobacterium tumefaciens, the bacterium from which opines have been characterized . The diversity of opine utilizers from the rhizosphere of Lotus plants is greater than that observed from any other environment . Therefore, transgenic plants with engineered exudation constitute an excellent tool to isolate and characterize specific microbial populations.

Indian J Exp Biol, 2003 Feb, 41(2), 149 - 53
Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens; Kanwar K et al.; Transgenic Robinia pseudoacacia plants were obtained by Agrobacterium tumefaciens mediated gene transfer . Agrobacterium strain LBA4404 harbouring a binary vector that contained the chimeric neomycin phosphotransferase II (NPTII) and beta-glucuronidase (GUS) genes was co-cultivated with hypocotyl segments of in vitro raised seedlings of Robinia . Parameters important for high efficiency regeneration and transformation rates included type of explant, pre-conditioning of explants and appropriate length of co-cultivation period with Agrobacterium . A transformation frequency 16.67% was obtained by 48 hr of pre-conditioning followed by 48 hr of co-cultivation . Transformed tissue was selected by the ability to grow on kanamycin containing medium . Successful regeneration was followed after histochemical GUS assay for the detection of transgenic tissue . This transformation procedure has the potential to expand the range of genetic variation in Robinia.

Theor Appl Genet, 2004 Nov, 109(7), 1399 - 405 Epub 2004 Nov.
Transgenic rice plants expressing the snowdrop lectin gene (gna) exhibit high-level resistance to the whitebacked planthopper (Sogatella furcifera); Nagadhara D et al.; Transgenic rice plants, expressing snowdrop lectin {Galanthus nivalis agglutinin (GNA)}, obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH) . The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter . In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses . Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T1 to T5 generations . Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH . Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants . Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants . Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants . Transgenic lines expressing GNA exhibited high-level resistance against the WBPH . As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper.

J Biol Chem, 2004 Oct 8, 279(41), 42787 - 93 Epub 2004 Jul 13.
Directed evolution of a glycosynthase from Agrobacterium sp . increases its catalytic activity dramatically and expands its substrate repertoire; Kim YW et al.; The Agrobacterium sp . beta-glucosidase (Abg) is a retaining beta-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using alpha-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars . Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay . Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively . Relative catalytic efficiencies (kcat/Km) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using alpha-D-galactopyranosyl fluoride and 4-nitrophenyl beta-D-glucopyranoside as substrates . The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates . Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site . Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate . The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (+1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.

FEBS Lett, 2004 Jul 16, 570(1-3), 30 - 6
Cryptic endotoxic nature of Bacillus thuringiensis Cry1Ab insecticidal crystal protein; Vazquez-Padron RI et al.; Cry1Ab is one of the most studied insecticidal proteins produced by Bacillus thuringiensis during sporulation . Structurally, this protoxin has been divided in two domains: the N-terminal toxin core and the C-terminal portion . Although many studies have addressed the biochemical characteristics of the active toxin that corresponds to the N-terminal portion, there are just few reports studying the importance of the C-terminal part of the protoxin . Herein, we show that Cry1Ab protoxin has a unique natural cryptic endotoxic property that is evident when their halves are expressed individually . This toxic effect of the separate protoxin domains was found against its original host B . thuringiensis, as well as to two other bacteria, Escherichia coli and Agrobacterium tumefaciens . Interestingly, either the fusion of the C-terminal portion with the insecticidal domain-III or the whole N-terminal region reduced or neutralized such a toxic effect, while a non-Cry1A peptide such as maltose binding protein did not neutralize the toxic effect . Furthermore, the C-terminal domain, in addition to being essential for crystal formation and solubility, plays a crucial role in neutralizing the toxicity caused by a separate expression of the insecticidal domain much like a dot/anti-dot system.

Protein Expr Purif, 2004 Aug, 36(2), 312 - 7
SIVmac Gag p27 capsid protein gene expression in potato; Kim TG et al.; A cDNA encoding the Simian immunodeficiency virus type (SIV(mac)) Gag capsid protein was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods . The gag gene was detected in the genomic DNA of transformed leaf tissues by PCR DNA amplification . Immunoblot analysis of transformed potato plant extracts with anti-Gag monoclonal antibody showed that biologically active Gag protein was synthesized in transformed tuber tissues . Based on ELISA results, recombinant Gag protein made up 0.006-0.014% of total soluble tuber protein . The synthesis of SIV Gag in transformed potato tubers opens the way for development of Gag-based edible plant vaccines for protection against SIV and potentially HIV-1 infection.

Plant Cell Rep, 2004 Nov, 23(6), 377 - 85 Epub 2004 Nov.
Introduction of a citrus blight-associated gene into Carrizo citrange {Citrus sinensis (L.) Osbc . x Poncirus trifoliata (L.) Raf.} by Agrobacterium-mediated transformation; Kayim M et al.; The protein p12 accumulates in leaves of trees with citrus blight (CB), a serious decline of unknown cause . The function of p12 is not known, but sequence analysis indicates it may be related to expansins . In studies to determine the function of p12, sense and antisense constructs were used to make transgenic Carrizo citrange using an Agrobacterium-mediated transformation system . Homogeneous beta-glucuronidase+ (GUS+) sense and antisense transgenic shoots were regenerated using kanamycin as a selective agent . Twenty-five sense and 45 antisense transgenic shoots were in vivo grafted onto Carrizo citrange for further analyses . In addition, 20 sense and 18 antisense shoots were rooted . The homogeneous GUS+ plants contained either the p12 sense or antisense gene (without the intron associated with the gene in untransformed citrus) as shown by PCR and Southern blotting . Northern blots showed the expected RNA in the sense and antisense plants . A protein of identical size and immunoreactivity was observed in seven of nine sense plants but not in nine antisense or non-transgenic plants . At the current stage of growth, there are no visual phenotypic differences between the transgenic and non-transgenic plants . Selected plants will be budded with sweet orange for field evaluation for resistance or susceptibility to CB and general rootstock performance.

Plant Physiol, 2004 Jul, 135(3), 1798 - 808 Epub 2004 Jul 09.
The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells; Stieger PA et al.; Plant infections by the soil bacterium Agrobacterium rhizogenes result in neoplastic disease with the formation of hairy roots at the site of infection . Expression of a set of oncogenes residing on the stably integrated T-DNA is responsible for the disease symptoms . Besides the rol (root locus) genes, which are essential for the formation of hairy roots, the open reading frame orf13 mediates cytokinin-like effects, suggesting an interaction with hormone signaling pathways . Here we show that ORF13 induced ectopic expression of KNOX (KNOTTED1-like homeobox) class transcription factors, as well as of several genes involved in cell cycle control in tomato (Lycopersicon esculentum) . ORF13 has a retinoblastoma (RB)-binding motif and interacted with maize (Zea mays) RB in vitro, whereas ORF13, bearing a point mutation in the RB-binding motif (ORF13*), did not . Increased cell divisions in the vegetative shoot apical meristem and accelerated formation of leaf primordia were observed in plants expressing orf13, whereas the expression of orf13* had no influence on cell division rates in the shoot apical meristem, suggesting a role of RB in the regulation of the cell cycle in meristematic tissues . On the other hand, ectopic expression of LeT6 was not dependent on a functional RB-binding motif . Hormone homeostasis was only altered in explants of leaves, whereas in the root no effects were observed . We suggest that ORF13 confers meristematic competence to cells infected by A . rhizogenes by inducing the expression of KNOX genes and promotes the transition of infected cells from the G1 to the S phase by binding to RB.

Mol Plant Microbe Interact, 2004 Jul, 17(7), 798 - 804
Introduction of plant and fungal genes into pea (Pisum sativum L.) hairy roots reduces their ability to produce pisatin and affects their response to a fungal pathogen; Wu Q et al.; Pisatin is an isoflavonoid phytoalexin synthesized by pea (Pisum sativum L.) . Previous studies have identified two enzymes apparently involved in the synthesis of this phytoalexin, isoflavone reductase (IFR), which catalyzes an intermediate step in pisatin biosynthesis, and (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM), an enzyme catalyzing the terminal step . To further evaluate the involvement of these enzymes in pisatin biosynthesis, sense- and antisense-oriented cDNAs of Ifr and Hmm fused to the 35s CaMV promoter, and Agrobacterium rhizogenes, were used to produce transgenic pea hairy root cultures . PDA, a gene encoding pisatin demethylating activity (pda) in the pea-pathogenic fungus Nectria haematococca, also was used in an attempt to reduce pisatin levels . Although hairy root tissue with either sense or antisense Ifr cDNA produced less pisatin, the greatest reduction occurred with sense or antisense Hmm cDNA . The reduced pisatin production in these lines was associated with reduced amounts of Hmm transcripts, HMM protein, and HMM enzyme activity . Hairy roots containing the PDA gene also produced less pisatin . To evaluate the role of pisatin in disease resistance, the virulence of N . haematococca on the transgenic roots that produced the lowest levels of pisatin was tested . Hairy roots expressing antisense Hmm were more susceptible than the control hairy roots to isolates of N . haematococca that are either virulent or nonvirulent on wild-type pea plants . This appears to be the first case of producing transgenic plant tissue with a reduced ability to produce a phytoalexin and demonstrating that such tissue is less resistant to fungal infection: these results support the hypothesis that phytoalexin production is a disease resistance mechanism.

Appl Environ Microbiol, 2004 Jul, 70(7), 4040 - 7
Initial reaction(s) in biotransformation of CL-20 is catalyzed by salicylate 1-monooxygenase from Pseudomonas sp . strain ATCC 29352; Bhushan B et al.; CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C(6)H(6)N(12)O(12)), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp . strain FA1 and Agrobacterium sp . strain JS71; however, the nature of the enzyme(s) involved in the process was not understood . In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp . strain ATCC 29352, biotransformed CL-20 at rates of 0.256 +/- 0.011 and 0.043 +/- 0.003 nmol min(-1) mg of protein(-1) under anaerobic and aerobic conditions, respectively . The disappearance of CL-20 was accompanied by the release of nitrite ions . Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion {M - H}(-) at 345 Da, corresponding to an empirical formula of C(6)H(6)N(10)O(8), produced as a result of two sequential N denitration steps on the CL- 20 molecule . We also detected two isomeric metabolites with {M - H}(-) at 381 Da corresponding to an empirical formula of C(6)H(10)N(10)O(10) . The latter was a hydrated product of the metabolite C(6)H(6)N(10)O(8) with addition of two H(2)O molecules, as confirmed by tests using (18)O-labeled water . The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion . Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase . The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical.

Appl Environ Microbiol, 2004 Jul, 70(7), 3898 - 903
Disruption of the subtilase gene, albin1, in Ophiostoma piliferum; Hoffman B et al.; Wood sapstaining fungi produce multiple proteases that break down wood protein . Three groups of subtilases have been identified in sapstaining fungi; however, it is not known if these groups have distinct physiological roles (B . Hoffman and C . Breuil, Curr . Genet . 41:168-175, 2002) . In this work we examined the role of the subtilase Albin1 from Ophiostoma piliferum . Reamplification of cDNA ends PCR was used to obtain the albin1 gene sequence . The encoded subtilase is probably extracellular and involved in nutrient acquisition . This gene was disrupted with an Agrobacterium tumefaciens-mediated transformation system . Two of the disruptants obtained had significantly lower levels of proteolytic activity, slower growth in bovine serum albumin, and significantly reduced growth on wood . Thus, albin1 plays an important role in O . piliferum's ability to acquire nitrogen from wood proteins.

BMC Biotechnol . 2004 Jul 07;4(1):13.
Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude; Horstmann V et al.; BACKGROUND: In addition to studies of plant gene function and developmental analyses, plant biotechnological use is largely dependent upon transgenic technologies . The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due to an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants . However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters . To satisfy this requirement, a set of plant expression vectors was constructed and equipped with either heterologous or endogenous promoters . RESULTS: Promoter activity was quantified using the dual-luciferase reporter assay system . The eight different heterologous promoter constructs tested exhibited expression levels spanning three orders of magnitude . Of these, the complete rice actin1 gene promoter showed the highest activity in Physcomitrella, followed by a truncated version of this promoter and three different versions of the cauliflower mosaic virus 35S promoter . In contrast, the Agrobacterium tumefaciens nopaline synthase promoter induced transcription rather weakly . Constructs including promoters commonly used in mammalian expression systems also proved to be functional in Physcomitrella . In addition, the 5' -regions of two Physcomitrella glycosyltransferases (i.e . alpha1,3-fucosyltransferase and beta1,2-xylosyltransferase) were identified and functionally characterised in comparison to the heterologous promoters . Furthermore, motifs responsible for enhancement of translation efficiency - such as the TMV omega element and a modified sequence directly prior the start codon - were tested in this model . CONCLUSION: We developed a vector set that enables gene expression studies, both in lower and higher land plants, thus providing valuable tools applicable in both basic and applied molecular research.

Biochim Biophys Acta, 2004 Jul 6, 1673(1-2), 45 - 55
Glycogenin: the primer for mammalian and yeast glycogen synthesis; Lomako J et al.; Glycogen synthesis, whether in mammalian tissue, yeast, or Agrobacterium tumefaciens or other bacteria, is initiated by autoglucosylation of a protein . Initiation in muscle, by a self-glucosylating protein, glycogenin-1, is the most thoroughly studied system, as is described here . These relatively recent findings have prompted a rekindling of interest in the intermediates lying between the primer and mature mammalian glycogen.

J Biol Chem, 2004 Sep 24, 279(39), 40844 - 51 Epub 2004 Jul 04.
Domains formed within the N-terminal region of the quorum-sensing activator TraR are required for transcriptional activation and direct interaction with RpoA from agrobacterium; Qin Y et al.; TraR, a quorum-sensing activator, induces transcription from its binding site, the tra-box, located upstream of Ti plasmid target promoters . TraR activated expression of a lacZ reporter in Escherichia coli only when RpoAAt from Agrobacterium tumefaciens was co-expressed . As assessed by gel retardation assays RpoAAt, but not RpoAEc, formed a ternary complex with TraR and a tra-box probe in vitro . TraR formed similar ternary complexes with alphaCTDAt but not with NTDAt, the C- and N-terminal segments of RpoAAt . As measured by surface plasmon resonance refractometry, TraR interacted directly with RpoAAt with an affinity about five times greater than that observed for its interaction with RpoAEc . The activator interacted with alphaCTDAt with kinetics and affinities similar to those of the full-sized -subunit . Positive control (PC) mutations at Asp-10 and Gly-123 of TraR did not affect DNA binding but greatly decreased the TraR-RpoAAt interaction . These two residues combine to form two patches on the activator, one of which may be involved in interaction with RpoA . When co-expressed, mutants of TraR with substitutions at Asp-10 complementing mutants with substitutions at Gly-123 for gene activation in an allele-specific manner . Co-expression studies with TraR and its PC mutants, and also with complementary PC alleles of TraR, coupled with three-dimensional structure are consistent with a hypothesis that both Asp-10/Gly-123 patches are required for activator function .

J Zhejiang Univ Sci, 2004 Aug, 5(8), 897 - 9
Characteristics of photosynthesis in rice plants transformed with an antisense Rubisco activase gene; Jin SH et al.; Transgenic rice plants with an antisense gene inserted via Agrobacterium tumefaciens were used to explore the impact of the reduction of Rubisco activase (RCA) on Rubisco and photosynthesis . In this study, transformants containing 15% to 35% wild type Rubisco activase were selected, which could survive in ambient CO2 concentration but grew slowly compared with wild type controls . Gas exchange measurements indicated that the rate of photosynthesis decreased significantly, while stomatal conductance and transpiration rate did not change; and that the intercellular CO2 concentration even increased . Rubisco determination showed that these plants had approximately twice as much Rubisco as the wild types, although they showed 70% lower rate of photosynthesis, which was likely an acclimation response to the reduction in Rubsico activase and/or the reduction in carbamylation.

J Microbiol Methods, 2004 Aug, 58(2), 197 - 202
Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana; dos Reis MC et al.; Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana . Conidia of B . bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator . The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors . High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium . Abortive transformants were observed for all the hph(r) vectors used . Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis . The latter analysis revealed the integration of two or more copies of the hph gene in the genome . The agro-transformation method was found to be effective for the isolation of B . bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.

J Biol Chem, 2004 Sep 3, 279(36), 37377 - 84 Epub 2004 Jun 29.
Molecular cloning, expression, and properties of an unusual aldo-keto reductase family enzyme, pyridoxal 4-dehydrogenase, that catalyzes irreversible oxidation of pyridoxal; Yokochi N et al.; Microbacterium luteolum YK-1 has pyridoxine degradation pathway I . We have cloned the structural gene for the second step enzyme, pyridoxal 4-dehydrogenase . The gene consists of 1,026-bp nucleotides and encodes 342 amino acids . The enzyme was overexpressed under cold shock conditions with a coexpression system and chaperonin GroEL/ES . The recombinant enzyme showed the same properties as the M . luteolum enzyme . The primary sequence of the enzyme was 54% identical with that of d-threo-aldose 1-dehydrogenase from Agrobacterium tumefaciens, a probable aldo-keto reductase (AKR) . Upon multiple alignment with enzymes belonging to the 14 AKR families so far reported, pyridoxal 4-dehydrogenase was found to form a new AKR superfamily (AKR15) together with A . tumefaciens d-threo-aldose 1-dehydrogenase and Pseudomonas sp . l-fucose dehydrogenase . These enzymes belong to a distinct branch from the two main ones found in the phylogenic tree of AKR proteins . The enzymes on the new branch are characterized by their inability to reduce the corresponding lactones, which are produced from pyridoxal or sugars . Furthermore, pyridoxal 4-dehydrogenase prefers NAD(+) to NADP(+) as a cofactor, although AKRs generally show higher affinities for the latter.

Plant Cell Rep, 2004 Aug, 23(1-2), 50 - 8 Epub 2004 Jun 23.
A new selection method for pepper transformation: callus-mediated shoot formation; Lee YH et al.; We used two genes, TMV-CP and PPI1 (pepper-PMMV interaction 1 transcription factor), to transform commercially important chili pepper (Capsicum annuum) inbred lines (P915, P409) by means of Agrobacterium co-culture . Eighteen independently transformed T0 plants were obtained . The most critical point in the pepper transformation protocol was the selection of shoots growing on calli--referred to as callus-mediated shoot formation (indirect shooting)--because shoots not grown from the callus (direct shooting from the wounded surface) developed into non-transformants . Selection of the correct right callus type also proved to be an important requirement for obtaining transformed peppers . Six different types of callus developed during the selection process . Shoots regenerated from two of these types, while one type regenerated significantly more shoots than the other types, suggesting that the capacity for shoot formation is callus type-specific . Although the transformation rate was low, transformation via callus-mediated shoot formation proved to be reproducible and was confirmed by Southern and Northern blot analyses . Based on the experimental data, we have succeeded in developing a new protocol for the selection and transformation of pepper and expect that it will be used in the future for pepper transformation.

Plant Cell Rep, 2004 Sep, 23(3), 148 - 54 Epub 2004 Jun 25.
Growth and terpenoid indole alkaloid production in Catharanthus roseus hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration; Batra J et al.; Hairy root cultures of Catharanthus roseus var . Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain . Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents . On the basis of growth pattern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories . However based on the integration of the Ri T(L)-DNA and T(R)-DNA genes, there were only three different categories of independent hairy root clones-C1 (rolA&B(+)/ags(+)), C2 (rolA&B(-)/ags(+)) and C3 (rolA&B(+)/ags(-)) . Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones . The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the T(L)-DNA and T(R)-DNA genes (C1) and the T(L)-DNA gene (C3) alone . Two rolA&B(-) but ags(+) clones (C2) accumulated much less or only very negligible amounts of ajmalicine . The possible role of the T(L)-DNA and T(R)-DNA genes on growth and alkaloid accumulation in these root clones is discussed.

J Biotechnol, 2004 Jul 15, 111(2), 131 - 41
Development of stem borer resistant transgenic parental lines involved in the production of hybrid rice; Ramesh S et al.; Stem borer resistant transgenic parental lines, involved in hybrid rice, were produced by Agrobacterium-mediated gene transfer method . Two pSB111 super-binary vectors containing modified cry1Ab/cry1Ac genes driven by maize ubiquitin promoter, and herbicide resistance gene bar driven by cauliflower mosaic virus 35S promoter were, used in this study . Embryogenic calli after co-cultivation with Agrobacterium were selected on the medium containing phosphinothricin . Southern blot analyses of primary transformants revealed the stable integration of bar, cry1Ab and cry1Ac coding sequences into the genomes of three parental lines with a predominant single copy integration and without any rearrangement of T-DNA . T1 progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses . Furthermore, the co-segregation of bar and cry genes in T1 progenies suggested that the transgenes are integrated at a single site in the rice genome . In different primary transformants with alien inbuilt resistance, the levels of cry proteins varied between 0.03 and 0.13% of total soluble proteins . These transgenic lines expressing insecticidal proteins afforded substantial resistance against stem borers . This is the first report of its kind dealing with the introduction of Bacillus thuringiensis (Bt) cry genes into the elite parental lines involved in the development of hybrid rice.

J Biotechnol, 2004 Jul 15, 111(2), 121 - 30
Generation of the transgenic potato expressing full-length spike protein of infectious bronchitis virus; Zhou JY et al.; To seek a new delivery system of vaccine for infectious bronchitis virus (IBV), transgenic potato expressing full-length spike (S) protein of IBV was produced and its immunogenicity in chickens was investigated . One to three copies of S gene of IBV were randomly and stably inserted into potato (Solanum tuberrosum cv . Dongnong 303) genome by Agrobacterium tumefaciens-mediated transformation . Transcription and translation of S gene for IBV were confirmed by Northern blot and Western blot analyses in transgenic plantlets . Chickens immunized orally and intramuscularly with transgenic potato tubers expressing S protein generated the detectable levels of serum neutralizing antibodies and were protected against the challenge with the virulent IBV . In vitro secretion of interleukin 2 and T lymphocyte proliferation of spleen cells from the immunized chickens varied with the dose and the manner of vaccination with S protein derived from transgenic plants . The results indicated that S protein expressed in transgenic plants might be a new source for the production of Coronaviridae IBV vaccine.

Biotechnol Lett, 2004 Jul, 26(13), 1057 - 9
Scale-up production of puerarin from hairy roots of Pueraria phaseoloides in an airlift bioreactor; Kintzios S et al.; Hairy roots of the Chinese herb, Pueraria phaseoloides, obtained from leaf explants and transformed with the Agrobacterium rhizogenes, were cultured in 2.5 l airlift bioreactors for three weeks . Puerarin accumulated at 5,570 microg g(-1) dry wt, which is near 200 times as much as in 250 ml flask cultures . In addition, puearin was exuded into the nutrient medium at final concentrations higher than in the hairy roots themselves.

Biosci Biotechnol Biochem, 2004 Jun, 68(6), 1193 - 200
Agrobacterium-mediated transformation of Javanica rice cv . Rojolele; Rachmawati D et al.; The Agrobacterium-mediated transformation system was extended to a famous Javanica rice variety, Rojolele, that is cultivated in Indonesia now . Efficient callus induction from immature and mature seeds of Rojolele did not succeed by any previous method for any rice cultivar . In this study, the callus from mature seeds of Rojolele exhibited a compact and nodular appearance on C medium after the carbon source and medium pH was modified . Scutellum-derived calli from mature seeds were co-cultivated with Agrobacterium tumefaciens strains EHA101 or LBA4404 that carried plasmid pAFT14, which contained the genes for beta-glucuronidase (gus) and hygromycin resistance (hpt) . Finally, the transformation efficiency of Rojolele variety using A . tumefaciens strain EHA101 (pAFT14) was improved to about 23%, similar to that of the Japonica rice variety Nipponbare . The seed fertility of transgenic Rojolele was more than 90% . The copy number of the transgene varied from one to three copies in the T(0) transgenic lines . Both the gus and the hpt genes were inherited and expressed in the progeny.

Cell Mol Biol Lett, 2004, 9(2), 287 - 300
Agrobacterium-mediated transformation of bangladeshi indica rices; Al-Forkan M et al.; Morphologically normal, fertile transgenic plants were obtained by co-culturing embryogenic calli of the Bangladeshi Indica rice cultivars BR26 and Binni with Agrobacterium tumefaciens strain LBA4404 carrying the super binary vector pTOK233 . Acetosyringone (100 microM) in the medium during co-culture (25-28 degrees C) and selection on hygromycin B (50 mg l(-1)) were essential for efficient transformation . Stable integration and expression of beta-glucuronidase, neomycin phosphotransferase and hygromycin phosphotransferase genes in regenerated plants were confirmed by histochemical and fluorometric assays, ELISA and Southern analysis . Two to 3 copies of T-DNA were integrated into regenerated plants; transgene expression did not correlate with gene copy number . Mendelian segregation of transgenes occurred in T1 seed progeny.

Plasmid, 2004 Jul, 52(1), 57 - 62
Integrative plasmid vector for constructing single-copy reporter systems to study gene regulation in Rhizobium meliloti and related species; Ferenczi S et al.; The integrative system of phage 16-3 of Rhizobium meliloti 41 was shown to function in several bacterial species belonging to the Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium genera . It might also function in many other bacterial species provided that both the target site (attB) and the required host factor(s) are present . Here we report on the construction of a new integrative vector that can be utilized in gene regulation studies . It provides an opportunity to create a single-copy set-up for characterizing DNA-protein interactions in vivo, in a wide range of bacteria . To demonstrate the usefulness of the vector, transcription repression by binding of the C repressor protein of phage 16-3 to wild type operators was studied . The assay system provided highly reproducible quantitative data on repression.

Plasmid, 2004 Jul, 52(1), 1 - 12
Modified RP4 and Tn5-Mob derivatives for facilitated manipulation of large plasmids in Gram-negative bacteria; Quandt J et al.; We have constructed a set of RP4 (NmS/TcS) and Tn5-Mob derivatives which have applications in experiments involving mobilization of replicons in many Gram-negative organisms . The different selection markers of the RP4 and Tn5-Mob derivatives include streptomycin, chloramphenicol, gentamicin, and spectinomycin resistance as well as mercury resistance, and a constitutively expressed lacZ gene . This choice of markers allows the use of these derivatives in bacteria which are naturally resistant to many antibiotics, and in strains which contain pre-existing resistance plasmids, transposons, or antibiotic cassette insertions . In addition, a RP4 derivative carrying the sacB gene of Bacillus subtilis was constructed . This allows the selection for the loss of RP4 after it has been used to mobilize other plasmids . A Tn5-Mob-sacB derivative with a new marker (Gm) was also developed, as were vectors which take advantage of the sacB gene to facilitate replacement of existing Tn5 inserts with other Tn5 derivatives . As an example of the use of these tools, three Rhizobium leguminosarum bv . viciae VF39 plasmids which have been shown to be involved in symbiosis were differentially tagged and mobilized (individually and in various combinations) to the plasmid-free Agrobacterium tumefaciens strain UBAPF2 . None of the resultant Agrobacterium strains was able to fix nitrogen in symbiosis with peas.

Transgenic Res, 2004 Apr, 13(2), 135 - 42
Prototype demonstration of transgenic resistance to the nematode Radopholus similis conferred on banana by a cystatin; Atkinson HJ et al.; Cavendish banana was transformed using Agrobacterium tumefaciens to express a protein engineered rice cystatin (OcIdeltaD86) of value for control of plant parasitic nematodes . Expression for each line was under control of a constitutive promoter from the maize ubiquitin gene (UBI-1), a constitutive, chimeric promoter from the octopine and mannopine synthase genes of A . tumefaciens or a promoter from a root-preferentially expressed tubulin gene Arabidopsis (TUB-1) . Lines were selected as of potential interest after 8 weeks challenge in containment if their mean R . similis/25 g roots for three sibling plants were more than 1 standard normal variate below the grand mean for all plants in c7-15 lines challenged concurrently . A total of 16 lines were selected on this basis as putative positives . Western blots confirmed that eight of these lines expressed cystatin with a mean of 0.08 +/- 0.04% tsp . All but two of 19 negatively selected lines from bioassays did not express cystatin . The mean resistance level of the confirmed positive lines was 69 +/- 17% . ELISA established the positive lines under control of UBI provided significantly higher expression levels of OcIdeltaD86 than recorded for the other two promoters . Lines of interest were confirmed as producing a transcript for OcIdeltaD86 by RT-PCR . Eight plants of one UBI promoter line expressing only 0.1 +/- 0.004% tsp as cystatin were re-challenged with R . similis and achieved a resistance of 70 +/- 10% . Subsequent repeat western blotting confirmed that this line still produced the cystatin after the trial . This is the first report of transgenic resistance against a major pest or disease of banana.

Transgenic Res, 2004 Apr, 13(2), 109 - 18
A ROS repressor-mediated binary regulation system for control of gene expression in transgenic plants; Schafer UA et al.; We describe a novel binary system to control transgene expression in plants . The system is based on the prokaryotic repressor, ROS, from Agrobacterium tumefaciens, optimized for plant codon usage and for nuclear targeting (synROS) . The ROS protein bound in vitro to double stranded DNA comprising the ROS operator sequence, as well as to single stranded ROS operator DNA sequences, in an orientation-independent manner . A synROS-GUS fusion protein was localized to the nucleus, whereas wtROS-GUS fusion remained in the cytoplasm . The ability of synROS to repress transgene expression was validated in transgenic Arabidopsis thaliana and Brassica napus . When expressed constitutively under the actin2 promoter, synROS repressed the expression of the reporter gene gusA linked to a modified CaMV35S promoter containing ROS operator sequences in the vicinity of the TATA box and downstream of the transcription initiation signal . Repression ranged from 32 to 87% in A . thaliana, and from 23 to 76% in B . napus . These results are discussed in relation to the potential application of synROS in controlling the expression of transgenes and endogenous genes in plants and other organisms.

Plant Cell Rep, 2004 Aug, 23(1-2), 64 - 70 Epub 2004 Jun 09.
The use of phenotypic markers to identify Brassica oleracea genotypes for routine high-throughput Agrobacterium-mediated transformation; Sparrow PA et al.; Doubled haploid (DH) genotypes from a genetic mapping population of Brassica oleracea were screened for ease of transformation . Candidate genotypes were selected based on prior knowledge of three phenotypic markers: susceptibility to Agrobacterium tumefaciens, shoot regeneration potential and mode of shoot regeneration . Mode of regeneration was found to be the most significant of the three factors . Transgenic plants were successfully obtained from genotypes that regenerated multiple shoots via a distinct swelling or callus phase . The absence of tissue culture blackening (associated with genotypes that formed callus) was found to be critical for transformation success . Transgenic shoots were obtained from genotypes that regenerated via an indirect callus mode, even when susceptibility to Agrobacterium was low . The most efficient genotype (DH AG1012) produced transgenic shoots at an average rate of 15% (percentage of inoculated explants giving rise to transgenic plants) . The speed and efficiency of regeneration enabled the isolation of transgenic shoots 5-6 weeks after inoculation with A . tumefaciens . This line was also self-compatible, enabling the production of seed without the need for hand-pollination . A genetically uniform DH genotype, with an associated genetic map, make DH AG1012 highly desirable as a potential model B . oleracea genotype for studying gene function . The possibility of applying the same phenotypic tissue culture markers to other Brassica species is discussed.

Plant Cell Rep, 2004 Oct, 23(4), 236 - 45 Epub 2004 Jun 09.
An efficient mannose selection protocol for tomato that has no adverse effect on the ploidy level of transgenic plants; Sigareva M et al.; A protocol for Agrobacterium-mediated transformation with mannose selection was developed for cotyledon petiole, hypocotyl and leaf explants of tomato (Lycopersicon esculentum L . Mill) . More than 400 transgenic plants from three tomato varieties were selected with 1% mannose in combination with 0.1-0.5% glucose . Average transformation frequencies ranged from 2.0 to 15.5% depending on the construct, genotype and type of tissue used for transformation . The highest transformation rate was obtained for hypocotyl explants from tomato variety SG048 . The ploidy levels of 264 independent transgenic events and 233 non-transgenic plants regenerated from tissue culture were assessed by flow cytometry . The incidence of polyploids within the total population of transgenic plants varied from 10 to 78% and was not significantly different from the non-transgenic population . The greatest variation in the proportion of polyploids was observed in plants derived from different explant types, both in transgenic and non-transgenic regenerants, across three studied genotypes . Transgenic and non-transgenic plants regenerated from leaves included the highest number of normal diploid plants (82-100%), followed by cotyledon petiole-derived plants (63-78%) . Transgenic plants produced from hypocotyls contained 22-58% diploids depending on the genotype used in transformation . Results described in this study demonstrate that, although transformation frequencies for leaf tissue are still lower under current protocols, the high percentage of diploids obtained make leaf tissue an attractive transformation target.

Mol Plant Microbe Interact, 2004 Jun, 17(6), 571 - 6
Site-directed mutagenesis and generation of chimeric viruses by homologous recombination in yeast to facilitate analysis of plant-virus interactions; Liang D et al.; A yeast homologous recombination system was used to generate mutants and chimeras in the genome of Potato leafroll virus (PLRV) . A yeast-bacteria shuttle vector was developed that allows mutants and chimeras generated in yeast to be transformed into Escherichia coli for confirmation of the mutations and transformed into Agrobacterium tumefaciens to facilitate agroinfection of plants by the mutant PLRV genomes . The advantages of the system include the high frequency of recovered mutants generated by yeast homologous recombination, the ability to generate over 20 mutants and chimeras using only two restriction endonuclease sites, the ability to introduce multiple additional sequences using three and four DNA fragments, and the mobilization of the same plasmid from yeast to E . coli, A . tumefaciens, and plants . The wild-type PLRV genome showed no loss of virulence after sequential propagation in yeast, E . coli, and A . tumefaciens . Moreover, many PLRV clones with mutations generated in the capsid protein and readthrough domain of the capsid protein replicated and moved throughout plants . This approach will facilitate the analysis of plant-virus interactions of in vivo-generated mutants for many plant viruses, especially those not transmissible mechanically to plants.

Yi Chuan Xue Bao, 2004 Mar, 31(3), 281 - 6
Expression pattern of UidA gene under the control of rice glutelin GluA-2 gene upstream sequence in transgenic rice endosperm; Chen Y et al.; In order to study the expression pattern of rice glutelin endosperm specific promoter in Chinese cultivar Zhonghua 8 (Oryza sativa L . subsp japonica), UidA gene was fused with rice glutelin GluA-2 gene 5' upstream sequence 2.3 kb and 750 bp upstream respectively and transferred into rice by Agrobacterium mediated transformation . Southern blot indicated that UidA gene was integrated into the genome of transgenic plants as single copy . Northern blot demonstrated that the expression of UidA gene and endogenous GluA-2 gene reached their highest level at 13-15 days and 11-13 days after pollination respectively, and then declined . Histochemical staining of immature transgenic rice seeds showed UidA gene was specifically expressed in endosperm and the highest level GUS expression was observed in aleurone layer . Quantitative analysis of GUS activity showed seeds GUS activity of that 2.3 kb transgenic plant was about two to three folds of those of 750 bp transgenic plant . Sequence analysis suggested that the G-box located in the -2,170 bp (from transcription start site) may be a quantitative cis-element.

Bioorg Med Chem, 2004 Jul 1, 12(13), 3485 - 95
Probing the aglycon binding site of a beta-glucosidase: a collection of C-1-modified 2,5-dideoxy-2,5-imino-D-mannitol derivatives and their structure-activity relationships as competitive inhibitors; Wrodnigg TM et al.; A range of new C-1 modified derivatives of the powerful glucosidase inhibitor 2,5-dideoxy-2,5-imino-D-mannitol has been synthesised and their biological activities probed with the beta-glucosidase from Agrobacterium sp . Ki values are compared with those of previously prepared close relatives . Findings suggest dramatic effects exerted by the aglycon binding site on substrate/inhibitor binding.

Mol Microbiol, 2004 Jun, 52(6), 1641 - 51
Quorum-sensing antiactivator TraM forms a dimer that dissociates to inhibit TraR; Chen G et al.; The quorum-sensing transcriptional activator TraR of Agrobacterium tumefaciens, which controls the replication and conjugal transfer of the tumour-inducing (Ti) virulence plasmid, is inhibited by the TraM antiactivator . The crystal structure of TraM reveals this protein to form a homodimer in which the monomer primarily consists of two long coiled alpha-helices, and one of the helices from each monomer also bundles to form the dimeric interface . The importance of dimerization is addressed by mutational studies in which disruption of the hydrophobic dimer interface leads to aggregation of TraM . Biochemical studies confirm that TraM exists as a homodimer in solution in equilibrium with the monomeric form, and also establish that the TraM-TraR complex is a heterodimer . Thus, the TraM homodimer undergoes dissociation in forming the antiactivation complex . Combined with the structure of TraR (Zhang et al., 2002, Nature 417: 971-974; Vannini et al., 2002, EMBO J 21: 4393-4401), our structural analysis suggests overlapping interactive surfaces in homodimeric TraM with those in the TraM-TraR complex and a mechanism for TraM inhibition on TraR.

Plant Cell Rep, 2004 Oct, 23(4), 218 - 23 Epub 2004 Jun 08.
Genetic transformation of selected mature cork oak (Quercus suber L.) trees; Alvarez R et al.; A transformation system for selected mature cork oak (Quercus suber L.) trees using Agrobacterium tumefaciens has been established . Embryos obtained from recurrent proliferating embryogenic masses were inoculated with A . tumefaciens strains EHA105, LBA4404 or AGL1 harbouring the plasmid pBINUbiGUSint {carrying the neomycin phosphotransferase II (nptII) and beta-glucuronidase (uidA) genes} . The highest transformation efficiency (4%) was obtained when freshly isolated explants were inoculated with A . tumefaciens strain AGL1 . Evidence of stable transgene integration was obtained by PCR for the nptII and uidA genes, Southern blotting and expression of the uidA gene . The transgenic embryos were germinated and successfully transferred to soil.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 349 - 55
Sequence-based approach to finding functional lipases from microbial genome databases; Kwoun Kim H et al.; A sequence-based approach was used to retrieve functional lipases from microbial genome databases . Many novel genes assigned as putative lipases were tested using the criteria of the typical lipase sequence rule, based on a consensus sequence of a catalytic triad (Ser, Asp, His) and oxyanion hole sequence (HG) . To obtain the lipase genes satisfying the sequence rule, PCR cloning was performed, while the lipase activities were tested using a tributyrin/tricaprylin plate and p-nitrophenyl caproate . Among nine putative lipases from four strains, five functional lipolytic proteins were obtained from Archaeoglobus fulgidus, Deinococcus radiodurans, and Agrobacterium tumefaciens . All five lipases exhibited a relatively low sequence similarity (less than 26.7%) with known lipases and turned out to belong to different lipase families . Accordingly, the current results indicate that the proposed strategic approach based on the microbial genome is an efficient and rapid method for finding novel and functional lipases.

J Mol Biol, 2004 Jun 18, 339(5), 1103 - 13
X-ray analysis of Mycobacterium smegmatis Dps and a comparative study involving other Dps and Dps-like molecules; Roy S et al.; The structure of the DNA binding protein from starved cells from Mycobacterium smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources . The dodecameric molecule can be described as a distorted icosahedron . The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers . The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M.smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first . Trimerisation is aided in the three proteins by the additional N-terminal stretches that they possess . The M.smegmatis protein has an additional C-terminal stretch compared to other related proteins . The stretch, known to be involved in DNA binding, is situated on the surface of the molecule . A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule . The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid . Regions in the vicinity of the acidic holes centred around molecular 3-fold axes, are relatively flexible . So are the DNA binding regions . The crystal structures of the protein from M.smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the arrangement of the protein molecules . However, contrary to earlier suggestions, the spaces do not need to be between layers of protein molecules . The cubic form provides an arrangement in which grooves, which could hold DNA molecules, criss-cross the crystal.

Biotechnol Prog, 2004 May-Jun, 20(3), 890 - 6
Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture; Collens JI et al.; Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression . Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system . Twelve of these auxotrophs were characterized on a nutrient matrix . Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32) . Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs . The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation . The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A . tumefaciens was achieved.

Biol Res, 2004, 37(1), 71 - 82
Replicase mediated resistance against potato leafroll virus in potato Desirée plants; Ehrenfeld N et al.; Potato leafroll virus (PLRV) is a major menace for the potato production all over the world . PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides . Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases . The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicate transgene in potato plants cv . Desiree, Plants were genetically modified to express the complete sense PLRV replicase gene . Two constructions were used, one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes . Transgenic plants were infected with PLRV in vitro, using infested aphids . In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected, although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection . In turn, those plants with the RolA promoter displayed an initial resistance that was later overcome . Different molecular mechanisms are likely to participate in the response to PLRV infection of these two types of transgenic plants.

J Plant Growth Regul . 2004 Jun 1; {Epub ahead of print}
Hormonal Control of Tumor Formation in Radish; Matveeva TV et al.; The role of phytohormones in genetic tumor formation on radish crop-roots was investigated using the collection of inbred Raphanus sativus lines as a model system . The genetic analysis showed that the trait <<tumor formation>> was recessive and monogenic in some crossings . The spectrum of main phytohormones in tumor and non-tumor radish lines has shown that at the initiation of tumor formation (30 days old plants) the amounts of main cytokinins in the lower part of plants from the tumor line were dramatically increased . The transformation of the non-tumor line by the ipt gene of Agrobacterium tumefaciens resulted in tumor formation in plants of the T(1) progeny . We propose that increasing the cytokinin/auxin ratio may lead to tumor formation on radish crop roots.

Biosci Biotechnol Biochem, 2004 May, 68(5), 1113 - 8
Effects of truncation at the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens; Ying L et al.; The function of the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens (Cbg1) was studied by analyzing the properties of mutant enzymes that have internal truncated amino acid sequences in the region . Five truncated mutants named Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-d119 having deletions of 4, 31, 62, 89, and 119 amino acid residues starting from Phe417, respectively, were expressed in Escherichia coli and purified . All the mutants exhibited beta-glucosidase activity, indicating that the non-homologous region was not essential for the activity . The truncation caused thermal instability, decrease in pK(a) of the proton donor residue (Glu616), and deficient transglycosylation activity . The thermal stability and the pK(a) of Glu616 were partially recovered with longer truncation, suggesting that the truncation perturbed the structure and that their presence in the region was not essential . The main role of the non-homologous region could be formation of a hydrophobic atmosphere at the acceptor site to make the enzyme suitable for hydrolyzing hydrophobic glucosides.

Biotechnol Lett, 2004 Apr, 26(7), 545 - 8
Solasodine glycoside production by hairy root cultures of Physalis minima Linn; Putalun W et al.; Hairy root cultures of Physalis minima L . were developed using Agrobacterium rhizogenes, strain ATCC 15834 mediated transformation and grown in half strength of Murashige and Skoog medium containing 8% (w/v) sucrose . Media supplementation with 1 mg naphthalenacetic acid l(-1) and 1 mg benzyladenine increased solasodine glycoside up to 900 g dry wt, which was 20 times higher than that in the native root.

Plant Cell Rep, 2004 Sep, 23(3), 155 - 8 Epub 2004 May 28.
Chemical nursing: phytosulfokine improves genetic transformation efficiency by promoting the proliferation of surviving cells on selective media; Matsubayashi Y et al.; The relative growth rate of plant cells in vitro is considerably affected by initial cell density . This troublesome effect has interfered with the establishment of efficient plant cell culture systems, especially when only a small number of cells are expected to survive, such as in the genetic transformation of cells under antibiotic selection . To improve the recovery of antibiotic-resistant cells, we examined the use of the peptide plant hormone phytosulfokine (PSK), which has been shown to promote cellular growth and development in vitro . The addition of PSK to selective media increased the recovery of transformed callus from Agrobacterium-infected carrot hypocotyl explants from 7% to 39%, which is more than a fivefold improvement over the control . Most calluses developed into normal plantlets with cotyledons and primary roots and, eventually, formed foliage leaves . Thus, chemical nursing using PSK shows promise as a tool for basic research in plant biology and biotechnological applications.

Plant Cell Rep, 2004 Aug, 23(1-2), 104 - 13 Epub 2004 May 28.
Use of the tobacco feedback-insensitive anthranilate synthase gene (ASA2) as a selectable marker for legume hairy root transformation; Cho HJ et al.; The feedback-insensitive anthranilate synthase ( ASA2) cDNA--isolated from a 5-methyltryptophan (5MT)-resistant tobacco cell line--driven by the CaMV 35S promoter or 606 bp of the native ASA2 promoter, was introduced into the forage legume plant Astragalus sinicus or soybean ( Glycine max), using Agrobacterium rhizogenes strains DC-AR2 or K599, respectively . Hairy roots of A . sinicus transformed with 35S-ASA2 but not 606- ASA2 could be directly selected using 20-75 micro M 5MT . ASA2 mRNA was expressed in all A . sinicus lines selected with 5MT, but nptII mRNA was expressed only in some lines even though the gene was present . Free tryptophan was increased 8- to 26-fold in A . sinicus and 3- to 6-fold in soybean (selected with kanamycin) . An HPLC method was used to measure anthranilate synthase (AS) activity since there was a fluorescent compound or compounds present in the soybean hairy root extracts . The transformed soybean hairy roots contained more feedback-resistant AS activity, showing that there is interaction of the tobacco ASA2 alpha-subunit with the soybean beta-subunit to form an active enzyme . Soybean hairy roots that express ASA2 also exhibit 5MT resistance . These results demonstrate that the tobacco feedback-insensitive ASA2 gene can be used as a selectable marker for transformation of the legume A . sinicus.

Mol Microbiol, 2004 Jun, 52(5), 1389 - 401
The quormone degradation system of Agrobacterium tumefaciens is regulated by starvation signal and stress alarmone (p)ppGpp; Zhang HB et al.; A unique signal degradation system has recently been discovered in Agrobacterium tumefaciens . Upon entering stationary phase, A . tumefaciens terminates quorum sensing-dependent Ti-plasmid conjugation by degradation of acyl homoserine lactone (AHL) quormone via the enzyme AttM (AHL-lactonase) . attM, together with attK and attL, constitute one transcriptional unit subjected to the control of a common promoter . AttJ, the other member of the signal degradation system, is an IclR-like negative transcriptional factor, which tightly represses the expression of AttM at the early stage of bacterial growth . In this study, we found that this quormone degradation system is activated by either carbon or nitrogen starvation . Quormone degradation was significantly delayed when bacterial culture was supplemented with extra carbon or nitrogen source in the nutrient-limited minimal medium before the onset of stationary phase . To identify the signalling pathway and regulatory mechanisms that mediate quormone degradation, we constructed a reporter strain A6(attKLM::lacZ) in which the promoterless lacZ was transcriptionally fused to the attKLM promoter . Transposon mutagenesis of strain A6(attKLM::lacZ) led to identification of the relA gene, which encodes the stress alarmone (p)ppGpp synthetase . Tn5 knock-out of relA abolished the stationary phase-dependent expression of attM . We concluded that the A . tumefaciens quormone degradation system is coupled to and regulated by the generic (p)ppGpp stress response machinery.

Mol Microbiol, 2004 Jun, 52(5), 1349 - 62
VirA of Agrobacterium tumefaciens is an intradimer transphosphorylase and can actively block vir gene expression in the absence of phenolic signals; Brencic A et al.; The VirA-VirG two-component system regulates the 30-gene vir regulon in response to host-released chemical signals . VirA is a homodimeric membrane-spanning histidine protein kinase . Here, we show that mutations in two essential VirA residues, His-474 and Gly-657, can be complemented by the formation of mixed heterodimers, indicating that each subunit of a VirA dimer transphosphorylates the opposite subunit . VirA contains a receiver domain that inhibits kinase activity . We use the forced heterodimer system to show that the two receiver domains of a VirA dimer act independently and that each inhibits the phosphoacceptor subdomain of the opposite subunit . We also demonstrate that merodiploid strains co-expressing constitutive VirA mutants and wild-type VirA show levels of vir gene expression far lower than haploid strains expressing just the constitutive alleles . The fact that wild-type VirA can actively block vir gene expression in the absence of phenolic signals suggests that it might have a phospho-VirG phosphatase activity . The receiver domain of VirA is essential for this activity, whereas residues H474 and G657 of the kinase domain are not required . Merodiploid strains co-expressing a constitutive VirA allele and an allele that is kinase inactive but proficient in the inhibitory activity show strongly inducible vir gene expression, indicating that the inhibitory activity is modulated by environmental signals.

Microb Ecol, 2004 Jul, 48(1), 10 - 8 Epub 2004 May 28.
A highly selectable and highly transferable Ti plasmid to study conjugal host range and Ti plasmid dissemination in complex ecosystems; Teyssier-Cuvelle S et al.; A conjugal donor system, ST2, was constructed to study the conjugal dissemination of a Ti plasmid to wild-type recipient bacteria in vitro and in situ . The system consisted of a polyauxotrophic derivative of C58 harboring a hyperconjugative and highly selectable Ti plasmid, pSTiEGK, which was constructed by inserting a multiple antibiotic resistance cassette in the traM- mcpA region of pTiC58Delta accR . ST2 transfers pSTiEGK constitutively at frequencies up to 10(-1) to plasmidless Agrobacterium recipients . The host range of pSTiEGK includes all the known genomic species of Agrobacterium, indigenous soil agrobacteria and some Rhizobium and Phyllobacterium spp . All transconjugants became pathogenic upon acquisition of the Ti plasmid and were also able to transfer pSTiEGK by conjugation . This host range was indistinguishable from that of its wild-type parent pTiC58, and therefore pSTiEGK constitute a valid proxy to study the dissemination of Ti plasmids directly in the environment . Transconjugants can be selected on a combination of four antibiotics, which efficiently prevents the growth of the indigenous microbiota present in complex environments . The transfer of pSTiEGK to members of the genus Agrobacterium was affected primarily by the plasmid content of the recipient strain (10(3)- to 10(5)-fold reduction), e.g., the presence of incompatible plasmids . As a consequence, a species should be considered permissive to Ti transfer whenever one permissive isolate is found.

J Basic Microbiol, 2004, 44(3), 203 - 14
Searching for nitrile hydratase using the Consensus-Degenerate Hybrid Oligonucleotide Primers strategy; Lourenco PM et al.; Consensus-Degenerate Oligonucleotide Hybrid Primers (CODEHOP) were designed by using the WWW-implemented strategy, based on multiple alignments of nitrile hydratase (NHase) alpha subunit, available from EMBL database . These primers were successfully tested with known NHase-producing bacterial strains such as Agrobacterium tumefaciens DSM 9674, Rhodococcus erythropolis DSM 9675, R . erythropolis 9685 and R . erythropolis 11397 and also allowed amplification from organisms not previously referenced (Variovorax sp . DSM 11402 and Agrobacterium sp . DSM 11401) . Hybrid primer utilisation was evaluated by analysing the incorporated sequences in cloned PCR fragments . Several primers from the oligonucleotide pool seemed to participate in the amplification of the correct fragment . Common garden soil was used as a source for both acetonitrile culture enrichment screening and direct DNA extraction . A R . erythropolis strain (CCMI 1005) was isolated by culture enrichment and allowed the PCR amplification of a DNA sequence (AJ548493) identical to the NHase coding sequences commonly described for that species, while the direct use of soil DNA as template led to the CODEHOP detection of two putative NHase coding sequences (AJ548498 and AJ548499), which were significantly different from any other known sequence . The phylogenetic relationship between all sequences obtained and the published NHase coding sequences was assessed by neighbour-joining analysis . The results demonstrate the use of consensus-degenerate primers in NHase detection from organisms known to express it and in the screening for new NHase family members .

Plant Mol Biol, 2004 Jan, 54(2), 245 - 59
Transgenic rice plants expressing the antifungal AFP protein from Aspergillus giganteus show enhanced resistance to the rice blast fungus Magnaporthe grisea; Coca M et al.; The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea . In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation . Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants . In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein . Transgenic rice plants showed stable integration and inheritance of the transgene . No effect on plant morphology was observed in the afp -expressing rice lines . The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M . grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active . Several of the T(2) homozygous afp lines were challenged with M . grisea in a detached leaf infection assay . Transformants exhibited resistance to rice blast at various levels . Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M . grisea.

Glycobiology, 2004 Sep, 14(9), 805 - 15 Epub 2004 May 24.
Full structural characterization of the lipid A components from the Agrobacterium tumefaciens strain C58 lipopolysaccharide fraction; Silipo A et al.; For the first time, the complete structure of the lipid A from the lipopolysaccharide of an Agrobacterium species is here reported . In particular, the structure of the lipid A from A . tumefaciens strain C58, a soil pathogen bacterium strictly related to Rhizobiaceae, was determined . The structural study, carried out by chemical analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy, revealed that lipid A fraction consisted of a mixture of species all sharing the bis-phosphorylated glucosamine disaccharide backbone that could be designated in two main structural motifs, according to the acylation pattern . The main species was a penta-acylated lipid A bearing two unsubstituted 14:0 (3-OH) fatty acids in ester linkage and two 16:0 (3-OH) in amide linkage; the one on GlcN II was O-acylated by a long chain fatty acid, 28:0 (27-OH) . This in turn was esterified by a 3-hydroxy-butyroyl residue at its hydroxy group . The second species, in lesser amounts, was identified as a tetra-acylated lipid A and lacked the 14:0 (3-OH) residue on GlcN I . Other species deriving from these two lacked a phosphate group or 3-hydroxy-butyroyl residue or otherwise carried a 26:0 (25-OH) as long chain fatty acid . The lipid A structure of phytopathogen A . tumefaciens strain C58 presents deep structural analogies with lipid A of symbiotic Rhizobium, and the hypothesis is advanced that it can be a strategy of the bacterium to escape or attenuate the plant response.

Arch Biochem Biophys, 2004 Jun 15, 426(2), 266 - 78
Controlled glycosylation of therapeutic antibodies in plants; Tekoah Y et al.; Recombinant therapeutic monoclonal antibodies (mAb) can be expressed, assembled, and glycosylated in plants . Transgenic plants, producing anti-rabies mAb and anti-colorectal cancer mAb, were obtained from Agrobacterium-mediated transformation . The heavy chain (HC) of anti-rabies mAb was fused to the Lys-Asp-Glu-Leu (KDEL) endoplasmic reticulum retention signal whereas the HC of anti-colorectal cancer mAb was not fused to the KDEL sequence . Gel release of glycans and detection by high-performance liquid chromatography (HPLC), together with computer assisted analysis and matrix-assisted laser desorption/ionization time-of-flight (MALD-TOF) mass spectrometry, revealed that the plant-derived anti-rabies mAb with KDEL contained mainly oligomannose type N-glycans while the plant-derived anti-colorectal cancer mAb carried mainly biantennary glycans with and without a pentose sugar, that is thought to be xylose . This finding indicates that the KDEL sequence can affect the N-glycosylation processing of antibody in plant cells . The plant-derived mAbs with addition of a KDEL sequence did not contain any of the known antigenic glycan epitopes that are frequently found in other plant glycans or in mammalian-derived mAbs . The altered glycosylation on both plant-derived mAbs did not affect the activities that are required for therapy . These results indicate that plant genetic engineering could provide an effective and inexpensive means to control the glycosylation of therapeutic proteins such as mAbs, by the addition of a KDEL signal as a regulatory element.

Science, 2004 May 21, 304(5674), 1170 - 3
Definition of a bacterial type IV secretion pathway for a DNA substrate; Cascales E et al.; Bacteria use conjugation systems, a subfamily of the type IV secretion systems, to transfer DNA to recipient cells . Despite 50 years of research, the architecture and mechanism of action of the channel mediating DNA transfer across the bacterial cell envelope remains obscure . By use of a sensitive, quantifiable assay termed transfer DNA immunoprecipitation (TrIP), we identify contacts between a DNA substrate (T-DNA) and 6 of 12 components of the VirB/D4 conjugation system of the phytopathogen Agrobacterium tumefaciens . Our results define the translocation pathway for a DNA substrate through a bacterial conjugation machine, specifying the contributions of each subunit of the secretory apparatus to substrate passage.

Biotechnol Appl Biochem, 2004 Jun, 39(Pt 3), 355 - 61
Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via Agrobacterium tumefaciens transformation; Fuentes A et al.; A transient gene-expression system was developed and used to characterize promoter strength, to verify suitability of bacterial gene modifications for expression in plant cells, and to express active antibody molecules . The system is based on suspension tobacco cells transformed by Agrobacterium in a transient way . Conditions such as pre-culture of tobacco cells and the co-cultivation period were identified as determinants to achieve high expression levels . Under established conditions the activity strength of CaMV (cauliflower mosaic virus) 35 S and ToMoTV (tomato mottle taino virus) AL1 promoters were compared . A modified cry gene sequence from Bacillus thuringiensis was expressed and detected by Western-blot analysis . A monoclonal antibody against anti-(hepatitis B virus surface antigen) was produced in such quantities as to allow testing of biological activity and preliminary characterization.

EMBO Rep, 2004 Jun, 5(6), 632 - 7 Epub 2004 May 21.
Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells; Pelczar P et al.; Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants . In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells . Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events . These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors . Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.

Physiol Plant, 2004 Jun, 121(2), 231 - 238
Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit; Badawi GH et al.; The role of APX (ascorbate peroxidase) in protection against oxidative stress was examined using transgenic tobacco plants . The full length cDNA, coding Arabidopsis thaliana L . APX fused downstream to the chloroplast transit sequence from A . thaliana glutathione reductase, was cloned into appropriate binary vector and mobilized into Agrobacterium tumefaciens C58C2 . Leaf discs were infected with the Agrobacterium and cultured on medium supplied with kanamycin . The incorporation of the gene in tobacco genome was confirmed by Southern dot blot hybridization . Transgenic lines were generated, and the line Chl-APX5 shown to have 3.8-fold the level of APX activity in the wild-type plants . The isolated chloroplasts from this line showed higher APX activity . During early investigation, this line showed enhanced tolerance to the active oxygen-generating paraquat and sodium sulphite . The first generation of this line, also, showed enhanced tolerance to salt, PEG and water stresses, as determined by net photosynthesis . The present data indicate that overproducing the cytosolic APX in tobacco chloroplasts reduces the toxicity of H(2)O(2).

Proteins, 2004 Jun 1, 55(4), 846 - 55
Sequence and structure of epoxide hydrolases: a systematic analysis; Barth S et al.; Epoxide hydrolases (EC 3.3.2.3) are ubiquitous enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols . More than 100 epoxide hydrolases (EH) have been identified or predicted, and 3 structures are available . Although they catalyze the same chemical reaction, sequence similarity is low . To identify conserved regions, all EHs were aligned . Phylogenetic analysis identified 12 homologous families, which were grouped into 2 major superfamilies: the microsomal EH superfamily, which includes the homologous families of Mammalian, Insect, Fungal, and Bacterial EHs, and the cytosolic EH superfamily, which includes Mammalian, Plant, and Bacterial EHs . Bacterial EHs show a high sequence diversity . Based on structure comparison of three known structures from Agrobacterium radiobacter AD1 (cytosolic EH), Aspergillus niger (microsomal EH), Mus musculus (cytosolic EH), and multisequence alignment and phylogenetic analysis of 95 EHs, the modular architecture of this enzyme family was analyzed . Although core and cap domain are highly conserved, the structural differences between the EHs are restricted to only two loops: the NC-loop connecting the core and the cap and the cap-loop, which is inserted into the cap domain . EHs were assigned to either of three clusters based on loop length . By using this classification, core and cap region of all EHs, NC-loops and cap-loops of 78% and 89% of all EHs, respectively, could be modeled . Representative models are available from the Lipase Engineering Database,

Ann Bot (Lond), 2004 Jul, 94(1), 67 - 74 Epub 2004 May 14.
Early events in Agrobacterium-mediated genetic transformation of citrus explants; Pena L et al.; BACKGROUND AND AIMS: Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells . Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species . In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out . Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied . METHODS: A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A . tumefaciens . In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry . KEY RESULTS: It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring . Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants . Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA . CONCLUSIONS: Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.

Hepatogastroenterology, 2004 May-Jun, 51(57), 891 - 4
Genes inside the cagPAI of Helicobacter pylori are not associated with gastric cancer in Japan; Nishiya D et al.; BACKGROUND/AIMS: In Japan, infection with cagA-positive H . pylori is not associated with gastric cancer unlike Western populations . Both cagE and Agrobacterium VirD4 homologue are genes inside the cagPAI . The aim of this study was to examine whether the presence of genes inside the cagPAI, cagA, cagE and Agrobacterium VirD4 homologue, is associated with gastric cancer in Japan . METHODOLOGY: Thirty-nine patients with gastric cancer and 39 subjects with only chronic gastritis were infected with H . pylori . Seventy-eight H . pylori strains were isolated from gastric biopsies and the presence of 23S rRNA, cagA, cagE, and VirD4 homologue were studied by polymerase chain reaction . RESULTS: The positivity of cagA was 97.4% in patients with gastric cancer, and 92.3% in control subjects . Thirty-six strains (92.3%) isolated from patients and 35 strains (89.7%) from control subjects had both cagE and VirD4 . All the 3 cagA-negative strains did not have both cagE and VirD4 . There were no significant differences in the positivities of cagA, cagE, and VirD4 between patients and control subjects . CONCLUSIONS: cagE and VirD4 were possessed by most Japanese strains, and thus the structure of the cagPAI of H . pylori might not be associated with the development of gastric cancer in Japan.

Protein Expr Purif, 2004 Jun, 35(2), 313 - 9
Synthesis and assembly of a cholera toxin B subunit SHIV 89.6p Tat fusion protein in transgenic potato; Kim TG et al.; A cDNA encoding the simian-human immunodeficiency virus (SHIV 89.6p) Tat regulatory element protein was fused to the c-terminus of the cholera toxin B subunit gene (ctxB-tat) and introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation methods . The fusion gene was detected in the genomic DNA of transformed potato leaf cells by PCR DNA amplification . Synthesis and assembly of the CTB-Tat fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis . The binding of CTB-Tat fusion protein pentamers to intestinal epithelial cell membrane glycolipid receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA) . Based on the ELISA results, CTB-Tat fusion protein made up about 0.005-0.007% of total soluble tuber protein or approximately 4.6mg per 100g potato tuber tissue . The synthesis and assembly of CTB-Tat monomers into biologically active oligomers in transformed potato tuber tissues demonstrates the feasibility of using viral pathogen antigens synthesized in edible plants for mucosal immunization against HIV-1 infection.

Curr Pharm Des, 2004, 10(13), 1551 - 65
Coupling factors in macromolecular type-IV secretion machineries; Gomis-Ruth FX et al.; Type IV secretion systems (T4SSs) are bacterial multiprotein organelles specialised in the transfer of (nucleo)protein complexes across cell membranes . They are essential for conjugation, bacterial-induced tumour formation in plant cells, as observed in Agrobacterium, toxin secretion, like in Bordetella and Helicobacter, cell-to-cell translocation of virulence factors, and intracellular activity of mammalian pathogens like Legionella . By enabling conjugative DNA delivery, these systems contribute to the spread of antibiotic resistance genes among bacteria . These translocons are made up by 10-15 proteins that are analogous to Vir proteins of Agrobacterium and traverse both membranes and the periplasmic space in between in Gram-negative bacteria . Their secretion substrates range from single-stranded DNA/protein complexes to multicomponent toxins and they are assisted by integral inner-membrane coupling factors, the multimeric type-IV coupling proteins (T4CPs), to connect the macromolecular complexes to be transferred with the secretory conduit . To do so, these T4CPs may be required to localise close to the secretion machinery within the donor cell . The T4CP structural prototype is the hexameric protein TrwB of Escherichia coli conjugative plasmid R388, closely related to Agrobacterium VirD4 protein . It is responsible for coupling the relaxosome with the DNA transport apparatus during cell mating . T4CP family members are related to SpoIIIE/FtsK proteins, essential for DNA pumping during sporulation and cell division . These features suggest possible mechanisms for conjugal T4CP function: as a simple coupler between two molecular machines, as a rotating device to pump DNA through the type-IV transport pore, or as a DNA injector, whereby its central channel would function as part of the transport pore.

Plant Cell Rep, 2004 Sep, 23(3), 134 - 43 Epub 2004 May 05.
Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds; Manickavasagam M et al.; Direct regeneration from explants without an intervening callus phase has several advantages, including production of true type progenies . Axillary bud explants from 6-month-old sugarcane cultivars Co92061 and Co671 were co-cultivated with Agrobacterium strains LBA4404 and EHA105 that harboured a binary vector pGA492 carrying neomycin phosphotransferase II, phosphinothricin acetyltransferase (bar) and an intron containing beta-glucuronidase (gus-intron) genes in the T-DNA region . A comparison of kanamycin, geneticin and phosphinothricin (PPT) selection showed that PPT (5.0 mg l(-1)) was the most effective selection agent for axillary bud transformation . Repeated proliferation of shoots in the selection medium eliminated chimeric transformants . Transgenic plants were generated in three different steps: (1) production of putative primary transgenic shoots in Murashige-Skoog (MS) liquid medium with 3.0 mg l(-1) 6-benzyladenine (BA) and 5.0 mg l(-1) PPT, (2) production of secondary transgenic shoots from the primary transgenic shoots by growing them in MS liquid medium with 2.0 mg l(-1) BA, 1.0 mg l(-1) kinetin (Kin), 0.5 mg l(-1) alpha-napthaleneacetic acid (NAA) and 5.0 mg l(-1) PPT for 3 weeks, followed by five more cycles of shoot proliferation and selection under same conditions, and (3) rooting of transgenic shoots on half-strength MS liquid medium with 0.5 mg l(-1) NAA and 5.0 mg l(-1) PPT . About 90% of the regenerated shoots rooted and 80% of them survived during acclimatisation in greenhouse . Transformation was confirmed by a histochemical beta-glucuronidase (GUS) assay and PCR amplification of the bar gene . Southern blot analysis indicated integration of the bar gene in two genomic locations in the majority of transformants . Transformation efficiency was influenced by the co-cultivation period, addition of the phenolic compound acetosyringone and the Agrobacterium strain . A 3-day co-cultivation with 50 micro M acetosyringone considerably increased the transformation efficiency . Agrobacterium strain EHA105 was more effective, producing twice the number of transgenic shoots than strain LBA4404 in both Co92061 and Co671 cultivars . Depending on the variety, 50-60% of the transgenic plants sprayed with BASTA (60 g l(-1) glufosinate) grew without any herbicide damage under greenhouse conditions . These results show that, with this protocol, generation and multiplication of transgenic shoots can be achieved in about 5 months with transformation efficiencies as high as 50%.

Biol Pharm Bull, 2004 May, 27(5), 657 - 60
Auxins affected ginsenoside production and growth of hairy roots in Panax hybrid; Washida D et al.; Hairy roots of interspecific hybrid ginseng (Panax ginseng x P . quinquefolium), induced by Agrobacterium rhizogenes ATCC 15834, grew well in B5 liquid media supplemented with 2.5 microM auxins (3-indole butyric acid (IBA), 1-naphtaleneacetic acid (NAA) and 3-indoleacetic acid (IAA)) . The hairy roots cultured in B5 liquid medium supplemented with 2.5 microM IBA showed best growth (6.39 g fresh weight per a flask, at week 8) . The highest content of the total ginsenosides was 1.63% as dry weight at week 8 when cultured with 2.5 microM NAA . The different auxins affected the numbers and lateral branching roots . Especially, 2.5 microM IBA promoted the lateral root formation (43.7+/-4.0 roots, at week 8), and 2.5 microM NAA promoted the lateral root growth (45.3+/-5.6 mm, at week 8) . The growth and ginsenosides production of 8-week old hairy roots cultured in B5 liquid media supplemented with IBA and NAA combinations were also investigated . Hairy roots produced higher amounts of ginsenosides in B5 liquid media supplemented with 0.5-1.0 microM IBA and NAA combinations than that cultured in B5 liquid media supplemented with only IBA and NAA . The highest yield of ginsenoside was obtained when cultured with 0.5 microM IBA and 1.0 microM IBA combination (6.38 mg per a flask, at week 8).

Plant Physiol, 2004 May, 135(1), 421 - 31 Epub 2004 May 07.
Crop improvement through modification of the plant's own genome; Rommens CM et al.; Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes . Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA . Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA . Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene . By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA . Further refinements are accomplished by employing Omega-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively . The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato . The modified plants represent the first example of genetically engineered plants that only contain native DNA.

J Appl Genet, 2004, 45(2), 127 - 44
Transgene inheritance in plants; Yin Z et al.; The patterns of transgene inheritance in plants and the possible explanations for non-Mendelian transmission are reviewed . The non-Mendelian inheritance of a transgene has been recorded with a frequency between 10% and 50% in transgenic plants produced either by Agrobacterium-mediated transformation or through particle bombardment . Different effects such as deletion, duplication, rearrangement, repeated sequence recombination as well as gene interaction have been observed for transgenic loci . The nature of the recipient genome, nature of the transgene and the interactions between them seem to contribute to the non-Mendelian segregation of transgenes.

Appl Environ Microbiol, 2004 May, 70(5), 2779 - 85
Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other alpha-Proteobacteria in cucumber and tomato crops affected by root mat; Weller SA et al.; Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi) . Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops . Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed . All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons . Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A . radiobacter strains (64%) . However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).

J Bacteriol, 2004 May, 186(10), 3065 - 77
Agrobacterium rhizogenes GALLS protein substitutes for Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2; Hodges LD et al.; Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells . The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease . DNA transfer from A . tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4 . Bacteria also secrete certain Vir proteins into plant cells via this pore . One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration . VirE2 binds incoming ssT-DNA and helps target it into the nucleus . Some strains of A . rhizogenes lack VirE2, but they still transfer T-DNA efficiently . We isolated a novel gene from A . rhizogenes that restored pathogenicity to virE2 mutant A . tumefaciens . The GALLS gene was essential for pathogenicity of A . rhizogenes . Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein . Despite their lack of similarity, GALLS substituted for VirE2.

J Biol Chem, 2004 Jul 9, 279(28), 29528 - 33 Epub 2004 Apr 28.
Protein interactions involved in nuclear import of the Agrobacterium VirE2 protein in vivo and in vitro; Citovsky V et al.; Agrobacterium, the only known organism capable of trans-kingdom DNA transfer, genetically transforms plants by transferring a segment of its DNA, T-DNA, into the nucleus of the host cell where it integrates into the plant genome . One of the central events in this genetic transformation process is nuclear import of the T-DNA molecule, which to a large degree is mediated by the bacterial virulence protein VirE2 . VirE2 is distinguished by its nuclear targeting, which occurs only in plant but not in animal cells and is facilitated by the cellular VIP1 protein . The molecular mechanism of the VIP1 function is still unclear . Here, we used in vitro assays for nuclear import and quantification of protein-protein interactions to directly demonstrate formation of ternary complexes between VirE2, VIP1, and a component of the cellular nuclear import machinery, karyopherin alpha . Our results indicate that VIP1 functions as a molecular bridge between VirE2 and karyopherin alpha, allowing VirE2 to utilize the host cell nuclear import machinery even without being directly recognized by its components.

J Med Virol, 2004 Jun, 73(2), 208 - 15
Transgenic tobacco cells producing the human monoclonal antibody to hepatitis B virus surface antigen; Yano A et al.; The recombinant human monoclonal antibody (MAb) against hepatitis B virus (HBV) surface antigen (HBsAg) was expressed in tobacco suspension cultures . The parental CL4MAb was produced by the Epstein-Barr virus (EBV) transformed human cell line TAPC301-CL4 . The CL4MAb cDNA was introduced into tobacco suspension cells by Agrobacterium mediated transformation . The monoclonal antibodies (MAbs), B294 and B303, which were derived from CL4 and subsequently produced in plant cells were selected for study . After purification on Protein A columns, B294 and B303 MAbs had anti-HBs relative affinity constants similar to the parental CL4MAb . B303 MAb interacted with cell surface HBsAgs and showed complement-dependent cytotoxicity in a manner that was similar to anti-HBs human immunoglobulins (HBIg) that are used clinically . The results of this study point to the feasibility of producing MAbs to HBsAg in plants as an alternative to HBIg .

Arch Microbiol, 2004 Jun, 181(6), 391 - 7 Epub 2004 Apr 29.
Purification and characterization of 2,6-dihydroxybenzoate decarboxylase reversibly catalyzing nonoxidative decarboxylation; Yoshida T et al.; A nonoxidative decarboxylase, 2,6-dihydroxybenzoate decarboxylase, was found in Agrobacterium tumefaciens IAM12048 . The enzyme activity was induced specifically by 2,6-dihydroxybenzoate . The purified enzyme was a homotetramer of identical 38 kDa subunits . The purified decarboxylase catalyzed the nonoxidative decarboxylation of 2,6-dihydroxybenzoate and 2,3-dihydroxybenzoate without requiring any cofactors . In the presence of KHCO(3), the enzyme also catalyzed the regioselective carboxylation of 1,3-dihydroxybenzene into 2,6-dihydroxybenzoate at a molar conversion ratio of 30%.

Mol Biol Evol, 2004 Jul, 21(7), 1294 - 307 Epub 2004 Apr 28.
Patterns of bacterial gene movement; Hao W et al.; Lateral gene transfer has emerged as an important force in bacterial evolution . A substantial number of genes can be inserted into or deleted from genomes through the process of lateral transfer . In this study, we looked for atypical occurrence of genes among related organisms to detect laterally transferred genes . We have analyzed 50 bacterial complete genomes from nine groups . For each group we use a 16s rRNA phylogeny and a comparison of protein similarity to map gene insertions/deletions onto their species phylogeny . The results reveal that there is poor correlation of genes inserted, deleted, and duplicated with evolutionary branch length . In addition, the numbers of genes inserted, deleted, or duplicated within the same branch are not always correlated with each other . Nor is there any similarity within groups . For example, in the Rhizobiales group, the ratio of insertions to deletions in the evolutionary branch leading to Agrobacterium tumefaciens str . C58 (Cereon) is 0.52, but it is 39.52 for Mesorhizobium loti . Most strikingly, the number of insertions of foreign genes is much larger in the external branches of the trees . These insertions also greatly outnumber the occurrence of deletions, and yet the genome sizes of these bacteria remain roughly constant . This indicates that many of the insertions are specific to each organism and are lost before related species can evolve . Simulations of the process of insertion and deletion, tailored to each phylogeny, support this conclusion.

Plant Cell Rep, 2004 Aug, 23(1-2), 59 - 63 Epub 2004 Apr 28.
Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots; Sriskandarajah S et al.; An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210) . This is the first report on the transformation of C . carpatica . Various factors affecting the transformation efficiency and subsequent regeneration were identified . The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots . Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots . Explants taken from 5-week-old seedlings produced only transformed callus . The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration . The cultivar "Blue Uniform" was more responsive than "White Uniform" . Both bacterial strains and plasmids were equally effective in producing transformed tissue . Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by beta-glucuronidase and ELISA analyses, respectively.

Plant Cell Physiol, 2004 Apr, 45(4), 490 - 5
Simple RNAi vectors for stable and transient suppression of gene function in rice; Miki D et al.; Since the recent sequencing of the rice genome, the functional identification of rice genes has become increasingly important . Various tagged lines have been generated; however, the number of tagged genes available is not sufficient for extensive study of gene function . To help identify the functions of genes in rice, we developed a Gateway vector, pANDA, for RNA interference of rice genes . This vector can be used for Agrobacterium transformation of rice and allows easy and fast construction of efficient RNAi vectors . In the construct, hairpin RNA derived from a given gene is transcribed from a strong maize ubiquitin promoter, and an intron is placed 5' upstream of inverted repeats to enhance RNA expression . Analysis of rice genes using this vector showed that suppression of mRNA expression was observed in more than 90% of transgenic plants examined, and short interfering RNA indicative of RNA silencing was detected in each silenced plant . A similar vector, pANDA-mini, was also developed for direct transfer into leaf cells or protoplasts . This vector can be used for transient suppression of gene function in rice . These vectors should help identify the functions of rice genes whose tagged mutants are not available at present and complement existing methods for functional genomics of rice.

Dev Dyn, 2004 May, 230(1), 12 - 22
Reorganization of specific chromosomal domains and activation of silent genes in plant cells acquiring pluripotentiality; Avivi Y et al.; The transition from leaf cells to protoplasts (plant cells devoid of cell walls) confers pluripotentiality coupled with chromatin reorganization . Here, we sought to identify remodeled chromosomal domains in Arabidopsis protoplasts by tracking DNA sequences undergoing changes in DNA methylation and by identifying up-regulated genes . We observed a reduction in DNA methylation at a pericentromeric region of chromosome 1, and up-regulation of several members of the NAC (NAM/ATAF1/CUC2) domain family, two of which are located near the telomeric region of chromosome 1 . Fluorescence in situ hybridization (FISH) analysis demonstrated that both pericentromeric and telomeric subdomains underwent chromatin decondensation . This decondensation is subdomain-specific inasmuch as centromeric repeats remained largely unchanged, whereas the 18S rDNA underwent condensation . Within the pericentromeric subdomain, VIP1, a gene encoding a b-Zip nuclear protein required for Agrobacterium infectivity, was transcriptionally activated . Overexpression of this gene in tobacco resulted in growth retardation and inhibition of differentiation and shoot formation . Altogether, our data indicate that acquisition of pluripotentiality involves changes in DNA methylation pattern and reorganization of specific chromosomal subdomains . This change leads to activation of silent genes whose products are involved in acquisition or maintenance of pluripotentiality and/or the ensuing fate of the cell .

J Plant Res, 2004 Apr, 117(2), 95 - 9 Epub 2004 Feb 13.
Obtaining transgenic plants using the bio-active beads method; Liu H et al.; Several methods of transformation are currently available for delivering exogenous DNA into animal and plant cells . In this study, a novel and efficient transformation system for DNA delivery/expression with a capacity to transport DNA of high molecular weight was developed . This system can overcome the shortcomings of traditional transformation methods such as Agrobacterium-mediated transformation, particle bombardment, and the electroporation method . The method developed in this study uses calcium alginate micro beads to immobilize DNA molecules in combination with polyethylene glycol treatment . In addition, it is simple and low-cost, and requires limited equipment . Using this method, we have successfully transformed tobacco plants, screening by kanamycin resistance . The transformed genes in the transformants were confirmed by PCR and Southern hybridization.

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