Transgenic Res, 2003 Aug, 12(4), 509 - 17 The endosperm and the embryo of Arabidopsis thaliana are independently transformed through infiltration by Agrobacterium tumefaciens; Bechtold N et al.; Several experiments had indicated that in planta transformation of Arabidopsis thaliana by Agrobacterium involves the female germ line . In order to identify the precise stage at which transformation occurs we have monitored expression of a gusA reporter gene in the two products of the double fertilization of infiltrated plants . The plantlets and the remaining endosperm of seeds were separately tested after germination . It appeared that in the majority of cases only the plantlet or the endosperm were transformed . Based on transformation with two vectors borne by two different Agrobacterium strains, the minority of 'co-transformed' plantlets and endosperm can be explained by simultaneous but independent transformation events . These results indicate that mature female gametes could be the targets of T-DNA. Transgenic Res, 2003 Aug, 12(4), 497 - 508 The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene, confers antifungal activity against Botrytis cinerea to transgenic tobacco; Carstens M et al.; The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities . In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens . Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene . Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression . Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae . Several of the F1 progeny lines were challenged with the fungal pathogen, B . cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70% . The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities. Transgenic Res, 2003 Aug, 12(4), 403 - 11 An efficient procedure to stably introduce genes into an economically important pulp tree (Eucalyptus grandis x Eucalyptus urophylla); Tournier V et al.; Regeneration problems are one of the main limitations preventing the wider application of genetic engineering strategies to the genus Eucalyptus . Seedlings from Eucalyptus grandis x Eucalyptus urophylla were selected according to their regeneration (adventitious organogenesis) and transformation capacity . After in vitro cloning, the best genotype of 250 tested was transformed via Agrobacterium tumefaciens . A cinnamyl alcohol dehydrogenase (CAD) antisense cDNA from Eucalyptus gunnii was transferred, under the control of the 35S CaMV promoter with a double enhancer sequence, into a selected genotype . According to kanamycin resistance and PCR verification, 120 transformants were generated . 58% were significantly inhibited for CAD activity, and nine exhibited the highest down-regulation, ranging from 69 to 78% (22% residual activity) . Southern blot hybridisation showed a low transgene copy number, ranging from 1 to 4, depending on the transgenic line . Northern analyses on the 5-16 and 3-23 lines (respectively one and two insertion sites) demonstrated the antisense origin of CAD gene inhibition . With respectively 26 and 22% of residual CAD activity, these two lines were considered as the most interesting and transferred to the greenhouse for further analyses. Transgenic Res, 2003 Aug, 12(4), 391 - 402 Transgene expression in the vegetative tissues of apple driven by the vascular-specific rolC and CoYMV promoters; Gittins JR et al.; The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation . Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of beta-glucuronidase (GUS) activity by fluorometric assay . The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter . These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting . The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter . In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range . The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate . This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem . Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2003, 21(2), 106 - 9 {Construction of the plant expression vectors containing the multiepitope gene of Toxoplasma gondii}; Zhou XH et al.; OBJECTIVE: To construct the plant expression vectors containing the multiepitope gene of Toxoplasma gondii (TGMG) . METHODS: 1 . TGMG was subcloned into pBAC55 vector to construct the intermediate plasmid pB35MG . The E35S/TGMG/NOS3' fragment was cleaved from pB35MG and ligated into the plant binary vector pCAMBIA2300 to construct the plant expression vector pC35MG . 2 . Tomato fruit-specific E81 . 1 promoter was introduced to pB35MG to construct pB35E1MG vector . The E35SE81 . 1/TGMG/NOS3' fragment was subcloned into pCAMBIA2300 to construct the plant expression vector pC35E1MG . 3 . Tomato fruit-specific E82.2 promoter was inserted to pB35MG to construct pBE2MG vector . The E82.2/TGMG/NOS3' fragment was subcloned into pCAMBIA2300 to construct the plant expression vector pCE2MG . The insert gene TGMG in the vectors pB35MG, pC35E1MG and pCE2MG were confirmed by sequencing . 4 . pC35MG, pC35E1MG and pCE2MG were introduced into Agrobacterium tumefaciens strain LBA4404 competent cell . RESULTS: Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments . And the sequencing results were confirmed correct . CONCLUSION: The TGMG intermediate vectors pB35MG, pB35E1MG and pBE2MG and the plant expression vectors pC35MG, pC35E1MG and pCE2MG were constructed successfully, and the three plant expression vectors were introduced into Agrobacterium tumefaciens. Biotechnol Lett, 2003 Feb, 25(4), 349 - 52 Continuous isomerization of D-fructose to D-mannose by immobilized Agrobacterium radiobacter cells; Hirose J et al.; D-Fructose was isomerized to D-mannose using immobilized Agrobacterium radiobacter that produces a thermostable mannose isomerase . The cells were immobilized by adsorption on chitosan or by glutaraldehyde crosslinking in the presence of albumin . Optimum conditions for mannose isomerase activity were 60 degrees C and pH 7.5 . Continuous reaction at 55 degrees C was achieved with immobilized cells packed in a column to produce D-mannose. Biotechnol Lett, 2003 Jan, 25(1), 67 - 72 Enhanced hydantoinase and N-carbamoylase activity on immobilisation of Agrobacterium tumefaciens; Foster IM et al.; Cell extracts of Agrobacterium tumefaciens, immobilised in calcium alginate beads, had a 7-fold increase in N-carbamoylase (N-carbamylamino acid amidohydrolase E.C . 3.5.1) activity on reaction with N-carbamylglycine . The hydantoinase (dihydropyrimidinase E.C . 3.5.2.2) and N-carbamoylase activities remained stable over 4 weeks storage at 4 degrees C relative to the non-immobilised enzymes, with the hydantoinase activity showing a 5-fold increase in activity relative to the non-immobilised hydantoinase . The pH optima of the immobilised hydantoinase and N-carbamoylase enzymes decreased to pH 7 and pH 8, respectively . The temperature optimum remained at 40 degrees C for the N-carbamoylase enzyme while the hydantoinase activity was optimal at 50 degrees C. Biotechnol Lett, 2003 May, 25(9), 739 - 44 Rhizobium radiobacter conjugation and callus-independent shoot regeneration used to introduce the cercosporin export gene cfp from Cercospora into sugar beet (Beta vulgaris L.); Kuykendall LD et al.; Leaf spot disease caused by Cercospora is responsible for crop and profitability losses in sugar beet crops in the US and worldwide . The cfp gene that encodes a protein that exports phytotoxic cercosporins from Cercospora was conjugally transferred to sugar beet using Rhizobium radiobacter (Agrobacterium tumefaciens), to improve Cercospora-induced leafspot resistance . Conditions for shoot regeneration were optimized to increase regeneration/transformation efficiencies . Low-light and room-temperature conditions were favorable to sugar beet regeneration without callus when cytokinin had been added to the tissue culture medium . Using this procedure adventitious shoots from leaf pieces were obtained in a simple, one-step regeneration procedure . T7, a cfp-transgenic clone verified by PCR with gene-specific primers, is being propagated for leaf spot disease resistance evaluation. Biotechnol Lett, 2003 Apr, 25(8), 631 - 6 Production of ajmalicine and ajmaline in hairy root cultures of Rauvolfia micrantha Hook f., a rare and endemic medicinal plant; Sudha CG et al.; Hairy roots of Rauvolfia micrantha were induced from hypocotyl explants of 2-3 weeks old aseptic seedlings using Agrobacterium rhizogenes ATCC 15834 . Hairy roots grown in half-strength Murashige & Skoog (MS) medium with 0.2 mg indole 3-butyric acid l-1 and 0.1 mg alpha-naphthaleneacetic acid l-1 produced more ajmaline (0.01 mg g-1 dry wt) and ajmalicine (0.006 mg g-1 dry wt) than roots grown in auxin-free medium . Ajmaline (0.003 mg g-1 dry wt) and ajmalicine (0.0007 mg g-1 dry wt) were also produced in normal root cultures . This is the first report of production of ajmaline and ajmalicine in hairy root cultures of Rauvolfia micrantha. Biotechnol Lett, 2003 Apr, 25(8), 593 - 7 Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum; Komaraiah P et al.; Potato (Solanum tuberosum) hairy root cultures, established by infecting potato tuber discs with Agrobacterium rhizogenes, were used as a model system for the production of antimicrobial sesquiterpenes and lipoxygenase (LOX) metabolites . Of the four sesquiterpene phytoalexins (rishitin, lubimin, phytuberin and phytuberol) detected in elicitor-treated hairy root cultures, rishitin (213 micrograms g-1 dry wt) was the most predominant followed by lubimin (171 micrograms g-1 dry wt) . The elicitors also induced LOX activity (25-fold increase) and LOX metabolites, mainly 9-hydroxyoctadecadienoic acid and 9-hydroxyoctadecatrienoic acid, in potato hairy root cultures . The combination of fungal elicitor plus cyclodextrin was the most effective elicitor treatment, followed by methyl jasmonate plus cyclodextrin in inducing sesquiterpenes and LOX metabolites. Biotechnol Lett, 2003 Apr, 25(7), 521 - 5 Development of Linum flavum hairy root cultures for production of coniferin; Lin HW et al.; Linum flavum hairy roots were initiated from leaf discs using Agrobacterium rhizogenes strains LBA9402 and TR105 though two other strains, 15834 and A4, were relatively ineffective for induction . Significant variation in coniferin accumulation was observed between hairy root lines originating from different L . flavum seedlings and/or A . rhizogenes strains . Coniferin reached 58 mg g-1 dry wt by culturing the roots in Linsmaier and Skoog (LS) medium with 2,4-dichlorophenoxyacetic acid and naphthaleneacetic acid as growth regulators. Plant Cell Rep, 2003 Sep, 22(2), 122 - 8 Epub 2003 Jul 19. The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L . Osbeck); Boscariol RL et al.; A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used . Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source . Genetic transformation was confirmed by PCR and Southern blot . The transgene expression was evaluated using a chlorophenol red assay and isoenzymes . The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar . This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides. Protein Pept Lett, 2003 Jun, 10(3), 303 - 11 Fused RolA protein enhances beta-glucoronidase activity 50-fold: implication for RolA mechanism of action; Barros LM et al.; We report that the plant oncoprotein RolA from Agrobacterium rhizogenes acts to stabilize beta-glucoronidase (Gus) when the two proteins are expressed as a fusion protein in transformed tobacco . The observed 50-fold increase of Gus activity was shown to be related to protein accumulation, with no significant changes in mRNA abundance, kinetic properties of the enzyme and thermostability . The entire RolA sequence is essential to achieve the full effect since both the N-terminal region, spanning a putative reverse signal-anchor and nuclear targeting sequences, or the contiguous C-terminal portion were shown to increase Gus activity only 10-fold . A possible interference of RolA in the protein degradation pathway regulated by auxin is discussed. Int J Med Microbiol, 2003 Jun, 293(2-3), 153 - 65 Topology and membrane interaction of Helicobacter pylori ComB proteins involved in natural transformation competence; Hofreuter D et al.; The human gastric pathogen Helicobacter pylori is naturally competent for genetic transformation . The H . pylori comB gene duster encodes the VirB4-homologous ATPase ComB4 and the structural proteins ComB7-ComB10, which share significant sequence identity to the Agrobacterium tumefaciens virB-encoded type IV secretion system . To study the topology of the ComB7-10 proteins, we applied TnMax transposon mutagenesis by generating fusions of ComB proteins with mature beta-lactamase (BlaM) or alkaline phosphatase (PhoA) . Our data show that the putative lipoprotein ComB7 is secreted and is found membrane-attached, probably by its lipid anchor . According to our topology mapping ComB8 is a bitopic membrane protein with a short N-terminal portion in the cytoplasm and the remainder of the protein expanding into the periplasmic space . ComB9 was verified as a periplasmic protein, tightly attached to the membrane . The N-terminus of ComB10 is anchored in the cytoplasmic membrane and the major portion of the protein, including a putative coiled-coil domain, is located in the periplasm . Limited protease digestion and protein extraction under different salt and pH conditions confirmed the periplasmic localization and the tight membrane association of the ComB protein complex . A hypothetical model of the ComB DNA transformation pore in H . pylori is presented. J Infect, 2003 Aug, 47(2), 167 - 9 Identification of a novel alpha-proteobacterium causing bacteraemia in an immunocompetent patient; Xu J et al.; An 89-year male with pyrexia and suspected bacteremia was admitted to hospital, where a Gram-negative rod was identified from blood culture . The organism was difficult to identify phenotypically and the resulting sequencing of a 559 bp section of the 16S rRNA gene did not have a high homology score (>97.0%) with any deposited GenBank accession numbers and hence was not able to be assigned to a species within any genus . Given that the isolate was a member of the alpha subclass of the Proteobacteria but did not fall into any of the known genera with more than 93.7% homology (Brucella, Rhizobium, Ochrobactrum, Agrobacterium), we believe this isolate to represent a novel alpha-proteobacterium, which was the cause of bacteraemia in this patient. Plant Mol Biol, 2003 May, 52(2), 483 - 93 The Agrobacterium oncogene AB-6b causes a graft-transmissible enation syndrome in tobacco; Helfer A et al.; Agrobacterium 6b oncogenes induce tumours and modify plant growth in various ways . Here we show that the AB-6b gene from strain AB4 placed under 2x35S promoter control (2x35S-AB-6b) induces a complex enation syndrome in transgenic Nicotiana tabacum plants, that also occurs in a few rare cases of genetic enations . In Arabidopsis thaliana, 2x35S-AB-6b induced radially symmetrical tubes on the abaxial side of the leaves, which must therefore be considered as the Arabidopsis equivalents of enations on other plant species . Tobacco and Arabidopsis 2x35S-AB-6b leaves contained small, supernumerary densely packed cells between the spongy mesophyll and the abaxial epidermis, close to vascular strands arising at an early stage of leaf development . On tobacco, the 2x35S-AB-6b enation syndrome could be transmitted across graft junctions to growing tissues of untransformed plants, both acropetally and basipetally . We propose that the AB-6b gene encodes the synthesis of one or more enation factor(s) that are transported by the phloem and modify the growth of developing tissues. Plant Mol Biol, 2003 May, 52(2), 421 - 32 Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment; Makarevitch I et al.; A substantial literature exists characterizing transgene locus structure from plants transformed via Agrobacterium and direct DNA delivery . However, there is little comprehensive sequence analysis of transgene loci available, especially from plants transformed by direct delivery methods . The goal of this study was to completely sequence transgene loci from two oat lines transformed via microprojectile bombardment that were shown to have simple transgene loci by Southern analysis . In line 3830, transformed with a single plasmid, one major and one of two minor loci were completely sequenced . Both loci exhibited rearranged delivered DNA and flanking genomic sequences . The minor locus contained only 296 bp of two non-contiguous fragments of the delivered DNA flanked by genomic (filler) DNA that did not originate from the integration target site . Predicted recognition sites for topoisomerase II and a MAR region were observed in the transgene integration target site for this non-functional minor locus . Line 11929, co-transformed with two different plasmids, had a single relatively simple transgene locus composed of truncated and rearranged sequences from both delivered DNAs . The transgene loci in both lines exhibited multiple transgene and genomic DNA rearrangements and regions of scrambling characteristic of complex transgene loci . The similar characteristics of recombined fragments and junctions in both transgenic oat lines implicate similar mechanisms of transgene integration and rearrangement regardless of the number of co-transformed plasmids and the level of transgene locus complexity. Plant Mol Biol, 2003 May, 52(2), 387 - 400 Expression of a cauliflower tonoplast aquaporin tagged with GFP in tobacco suspension cells correlates with an increase in cell size; Reisen D et al.; In plants, vacuoles are essential organelles that undergo dynamic volume changes during cell growth due to rapid and high flow of water through tonoplast water-carrying channels composed of integral proteins (tonoplast aquaporins) . The tonoplast BobTIP26-1 from cauliflower has previously been shown to be an efficient active aquaporin in Xenopus leavis oocytes . In this study we used tobacco (Nicotiana tabacum cv . Wisconsin 38) suspension cells to examine the effect of BobTIP26-1 expression . In order to follow the intracellular localisation of the protein in real time, the gfp sequence was fused downstream to the BobTIP26-1 coding region . The fusion protein BobTIP26-1::GFP is less active than BobTIP26-1 by itself when expressed in Xenopus oocytes . Nevertheless, this fusion protein is well targeted to the tonoplast of the plant suspension cell when expressed via Agrobacterium co-cultivation . A complex tonoplast labelling is shown when young vacuolated cells are observed . The expression of the fusion protein does not affect the growth rate of the cells but increases their volume . We postulate that the increase in cell volume is triggered by the fusion protein allowing vacuolar volume increase. FEMS Microbiol Lett, 2003 Jul 15, 224(1), 101 - 6 Transformation of Mucor miehei results in plasmid deletion and phenotypic instability; Monfort A et al.; Mucor miehei transformants resistant to geneticin have been obtained by treatment of protoplasts with different plasmids and by Agrobacterium-mediated DNA transfer . All transformants exhibited a very unstable phenotype whose maintenance required continuous selective pressure . Molecular analysis of transformants showed that the plasmid DNA was extensively modified and maintained in very low amount . Our results indicate that M . miehei reluctance to transformation is due to different causes, including the coenocytic nature of its mycelium and the existence of specific mechanisms for the detection and elimination of foreign DNA. Nucleic Acids Res, 2003 Jul 15, 31(14), 4247 - 55 The Arabidopsis AtLIG4 gene is required for the repair of DNA damage, but not for the integration of Agrobacterium T-DNA; van Attikum H et al.; The joining of breaks in the chromosomal DNA backbone by ligases in processes of replication, recombination and repair plays a crucial role in the maintenance of genomic stability . Four ATP-dependent ligases, designated DNA ligases I-IV, have been identified in higher eukaryotes, and each one has distinct functions . In mammals and yeast, DNA ligase IV is exclusively involved in the repair of DNA double-strand breaks by non-homologous end joining . Recently, an Arabidopsis thaliana orthologue of the yeast and mammalian DNA ligase IV gene was found and termed AtLIG4 . Here we describe the isolation and functional characterisation of a plant line with a T-DNA insertion in the AtLIG4 gene . Plants homozygous for the T-DNA insertion did not display any growth or developmental defects and were fertile . However, mutant seedlings were hypersensitive to the DNA-damaging agents methyl methanesulfonate and X-rays, demonstrating that AtLIG4 is required for the repair of DNA damage . Recently, we showed that a yeast lig4 mutant is deficient in Agrobacterium T-DNA integration . However, using tumorigenesis and germline transformation assays, we found that the plant AtLIG4 mutant is not impaired in T-DNA integration . Thus, in contrast to yeast, DNA ligase IV is not required for T-DNA integration in plants. Glycobiology, 2003 Oct, 13(10), 693 - 706 Epub 2003 Jul 08. Topological characterization of an inner membrane (1-->3)-beta-D-glucan (curdlan) synthase from Agrobacterium sp . strain ATCC31749; Karnezis T et al.; The crdS gene of Agrobacterium sp . strain ATCC31749 encodes the curdlan synthase (CrdS) protein based on the homology of the derived CrdS protein sequence with those of beta-glycosyl transferases with repetitive action patterns (Stasinopoulos et al . {1999} Glycobiology, 9, 31-41) . Here we show that chemical (NTG) mutagenesis of crdS abolishes curdlan production and the induced mutations can be complemented by a cloned crdS amplicon, thus providing genetic confirmation that crdS is essential for curdlan production . When expressed in the native Agrobacterium or in Escherichia coli, the largely hydrophobic CrdS protein exhibited an Mr of approximately 60 kDa (compared with the predicted mass of 73,121 Da) and was located in the inner membrane of both bacteria . By analyzing reciprocal fusions between crdS and the reporter genes, lacZ and phoA, and assessing the sensitivity of CrdS in spheroplasts to proteinase K, CrdS was shown to be an integral membrane protein with seven transmembrane helices and an Nout-Cin disposition . A central large and relatively hydrophilic cytoplasmic region carries the substrate-binding and catalytic D,D,D35QxxRW motif . The amino acid sequence of this domain of CrdS was threaded onto the 3D structure of the comparable domain of the SpsA protein, a member of the family GT-2 glycosyl transferases, and enabled the identification of corresponding amino acids involved in binding UDP in CrdS . Analysis of Agrobacterium membrane preparations using blue native-PAGE provided preliminary evidence that CrdS occurs in multimeric protein complexes of approximately 420 kDa and approximately 500 kDa. Plant J, 2003 Jul, 35(2), 219 - 36 Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression; Veena et al.; Agrobacterium tumefaciens is a plant pathogen that incites crown gall tumors by transferring to and expressing a portion of a resident plasmid in plant cells . Currently, little is known about the host response to Agrobacterium infection . Using suppressive subtractive hybridization and DNA macroarrays, we identified numerous plant genes that are differentially expressed during early stages of Agrobacterium-mediated transformation . Expression profiling indicates that Agrobacterium infection induces plant genes necessary for the transformation process while simultaneously repressing host defense response genes, thus indicating successful utilization of existing host cellular machinery for genetic transformation purposes . A comparison of plant responses to different strains of Agrobacterium indicates that transfer of both T-DNA and Vir proteins modulates the expression of host genes during the transformation process. Mol Plant Microbe Interact, 2003 Jul, 16(7), 650 - 8 A luxR homolog, aviR, in Agrobacterium vitis is associated with induction of necrosis on grape and a hypersensitive response on tobacco; Zheng D et al.; A Tn5 mutant of Agrobacterium vitis F2/5 (M1154) differs from the wild-type strain in that it has lost its abilities to cause necrosis on grape and a hypersensitive-like response (HR) on tobacco . The Tn5 insertion occurred in an open reading frame (ORF) aviR that is homologous to genes encoding the LuxR family of transcriptional regulators, thereby suggesting that the HR and necrosis are regulated by a quorum-sensing system . Fewer N-acyl-homoserine lactone autoinducers were detected in extracts from M1154 compared with extracts from F2/5 and from aviR-complemented M1154 . The complemented mutant regained full ability to cause grape necrosis and HR . Eighteen ORFs located on a 36.6-kb insert in cosmid clone CPB221, which includes aviR, were sequenced and aligned with homologous genes from A . tumefaciens C58 and Sinorhizobium meliloti Rm1021 . The order of several clustered genes is conserved among the bacteria; however, rearrangements are also apparent . Reverse transcriptase-polymerase chain reaction analysis indicated that ORF2 and ORF14 may be regulated by an aviR-encoded transcriptional regulator . Single site-directed mutations in each of the ORFs, however, had no effect on expression of HR or necrosis as compared with the wild-type parent. Shokuhin Eiseigaku Zasshi, 2003 Apr, 44(2), 77 - 82 Determination of enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase by LC/MS; Okunuki H et al.; A liquid chromatography-mass spectrometry (LC/MS) method for determining the enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), an enzyme of the shikimate pathway, was developed . EPSP synthase catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S-3-P) and phosphoenolpyruvate (PEP) in microorganisms and plants . The enzymatic activity of EPSP synthase was assessed by the determination of EPSP after a 30-min incubation with S-3-P and PEP using the LC/MS system . EPSP synthase activity is given in terms of the produced EPSP (pmol/min/mg protein) . Glyphosate (N-phosphonomethyl glycine)-tolerant EPSP synthase from the Agrobacterium sp . strain CP4 (CP4-EPSP synthase) in genetically modified soybeans (GM-soybeans) was found to have an enzymatic activity of 736 EPSP pmol/min/mg protein in the presence of 3 nmol of S-3-P . In contrast, the enzyme activity of non-GM-soybeans was 21 EPSP pmol/min/mg protein . The EPSP synthase activity was markedly decreased in the non-GM-soybeans by the addition of glyphosate, but the enzyme activity of the GM-soybeans was only slightly decreased with this treatment . This LC/MS system could also be applicable to the measurement of EPSP synthase activity in different plant species and the detection of herbicide-tolerant EPSP synthase in GM foods. Genome Res, 2003 Jul, 13(7), 1675 - 85 EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora; Torto TA et al.; Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants.These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses . We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants . PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans . The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs . Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases . Validation of PexFinder was performed using proteomic analysis of secreted protein of P . infestans . To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome . This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora . Further characterization of the crn genes indicated that they are both expressed in P . infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato . Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins . In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including oomycetes, fungi, and nematodes. Tree Physiol, 2003 Aug, 23(11), 721 - 33 Silver birch (Betula pendula) plants with aux and rol genes show consistent changes in morphology, xylem structure and chemistry; Piispanen R et al.; The effects of Agrobacterium pRiA4 rol and aux genes, controlled by their endogenous promoters, on tree growth and wood anatomy and chemistry were studied in 5- and 7-year-old silver birch (Betula pendula Roth) plants . Southern hybridization confirmed the following rol and aux gene combinations: control plants (no genes transferred); plants with rolC and rolD genes; plants with rolA, rolB, rolC and rolD genes; and plants with rolA, rolB, rolC, rolD, aux1 and aux2 genes . Transgene mRNA was most abundant in phloem/cambium samples and in the developing xylem, whereas no expression was detected in leaves . Plants with rolC and rolD genes or with all the rol genes were significantly shorter and had smaller leaves and a more bushy growth habit than control plants or plants with both aux and rol genes . Morphological observations and wood chemistry analyses revealed that plants with rol genes produced less xylem and broke bud later than control plants or plants with both aux and rol genes . Tension wood was detected in both control and transgenic plants irrespective of their gene combination, probably as a result of greenhouse cultivation . Xylem fibers were shorter in transgenic plants than in control plants, and plants with all the rol genes were characterized by shorter vessels compared with the control plants and a smaller proportional area of vessels compared with the other groups . In addition, silver birch plants with all the rol genes had approximately a 3.3% lower concentration of total acid soluble carbohydrates than control plants . We conclude that the rolC and rolD genes induced the typical "rol-phenotype," and that this was emphasized by concomitant expression of the rolA and rolB genes and alleviated by the presence of aux1 and aux2 genes . We observed consistent phenotypic effects of rol and aux genes on the morphology, anatomy and cell wall chemistry of the plants. Curr Genet, 2003 Sep, 43(6), 447 - 52 Epub 2003 Jul 01. Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium-mediated DNA transfer; Campoy S et al.; The high-level pigment-producing Monascus strain IBCC1 was characterized by random amplification of polymorphic DNA as M . purpureus . This technique allowed us to distinguish between M . purpureus and M . ruber strains . Transformation of Monascus species has not been previously reported . Protoplast formation and regeneration from M . purpureus IBCC1 was optimized by modification of growth media, lytic enzyme mixture, osmotic stabilizer and regeneration media . Of the Monascus transformants, 60% were found to be mitotically stable and retained the plasmid inserted in the chromosome after repeated sporulation cycles . Additionally, an Agrobacterium-mediated DNA transfer system was developed . The transformants obtained by Agrobacterium-mediated DNA transfer remained fully stable (98%) after four sporulation rounds and showed bands of hybridization corresponding to integration of the plasmid in different sites of the genome . The green fluorescent protein marker was well expressed in the M . purpureus transformants . The development of transformation systems is a basic tool for advanced genetic manipulation of the natural pigment producers, M . purpureus and M . ruber. Nucleic Acids Res Suppl, 2001, (1), 245 - 6 Statistical analysis of functional regions in Ti and Ri sequenced plasmids in Agrobacterium; Yoshida K et al.; The purpose of this paper is to examine functional regions of the completely sequenced DNA data of Ti and Ri plasmids, based on codon usage similarity . It is shown that a successful classification of functional regions is obtained by using statistical measures of codon usage dissimilarity and correspondence analysis. Nucleic Acids Res Suppl, 2001, (1), 173 - 4 A new method for construction of Ti plasmid-less strains in Agrobacterium tumefaciens; Uraji M et al.; Agrobacterium tumefaciens harboring a Ti plasmid causes plant tumorigenesis and it's DNA transfer system have been extensively used in plant biotechnology . Because Ti plasmids are high stable in agrobacterial hosts, their curing is very difficult by conventional methods . Here, we introduced a novel curing method by using Ti plasmids incompatibility. Plant Cell Rep, 2003 Jul, 21(11), 1103 - 7 Epub 2003 Apr 23. Genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots; Shi HP et al.; An efficient transformation system for the medicinal plant Pueraria phaseoloides was established by using agropine-type Agrobacterium rhizogenes ATCC15834 . Hairy roots could be obtained directly from the cut edges of petioles of leaf explants or via callus 10 days after inoculation with the bacteria . The highest frequency of explant transformation by A . rhizogenes ATCC15834 was about 70% after infection for 30 days . Hairy roots could grow rapidly on solid, growth regulator-free Murashige and Skoog medium and had characteristics of transformed roots such as fast growth and high lateral branching . Paper electrophoresis revealed that bacteria-free hairy roots of P . phaseoloides could synthesize agropine and mannopine . The polymerase chain reaction amplification of rooting locus genes showed that left-hand transferred DNA of the root inducing plasmid of A . rhizogenes was inserted into the genome of transformed P . phaseoloides hairy roots . The content of puerarin in hairy roots reached a level of 1.190 mg/g dry weight and was 1.067 times the content in the roots of untransformed plants. Plant Cell Rep, 2003 Jul, 21(11), 1095 - 102 Epub 2003 Apr 12. Production of Hevea brasiliensis transgenic embryogenic callus lines by Agrobacterium tumefaciens: roles of calcium; Montoro P et al.; A procedure has been established for Agrobacterium tumefaciens-mediated genetic transformation of Hevea brasiliensis embryogenic friable calli . Precultivation of tissues on a CaCl(2)-free maintenance medium dramatically enhanced the transient activity of the reporter gene, gusA encoding beta-glucuronidase (GUS) . The increase was first noticed in highly active cells (undifferentiated or/and embryogenic), in tissues precultured for 2-8 weeks . Beyond 8 weeks of preculture, GUS activity increased again, but this time in tissues consisting of differentiated cells accumulating polyphenols . Out of five Agrobacterium strains cocultivated with CaCl(2)-free precultured tissues, only inoculation with EHA105pC2301 led to high transient GUS activity . Paromomycin proved more effective than kanamycin for the selection of transformed cells, as it inhibits the growth of non-transformed cells more radically . Five paromomycin-resistant callus lines were established . The presence of gusA and neomycin phosphotransferase ( nptII) genes in the plant genome was confirmed by DNA amplification, and by Southern hybridization . These results confirmed that A . tumefaciens is an effective system for mediating stable transformation of rubber tree calli with a low copy number of transgenes . Transgenic callus lines constitute a useful tool for studying genes of interest on a cellular level and for regenerating transgenic rubber trees. Plant Cell Rep, 2003 Jun, 21(10), 1010 - 19 Epub 2003 Apr 12. Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection; Hu T et al.; An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants . The system uses 4-day precultured immature embryos as explants . A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system . The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest . More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4% . The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days . Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria . Forty-six percent of the transgenic events fit a 3:1 segregation ratio . Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene . Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations . A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat . One of the lead events from this study, no . 33391, has been identified as a Roundup Ready wheat commercial candidate. Plant Cell Rep, 2003 Jun, 21(10), 993 - 8 Epub 2003 Apr 03. Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid; Liau CH et al.; The present protocol was aimed at establishing a routine transformation procedure via Agrobacterium tumefaciens for an important Oncidium orchid cultivar . An expression vector containing hptII and gusA genes driven by the cauliflower mosaic virus (CaMV) 35S promoter was successfully introduced into the Oncidium orchid genome by A . tumefaciens . Protocorm-like bodies (PLBs) derived from protocorms, were the target explants for transformation . The transformation was performed through two stages of cocultivation, the first stage occurring on antibiotic-free medium for 3 days, and the subsequent stage on medium containing 100 mg/l timentin for 1 month . Among 1,000 inoculated PLBs, 108 putatively transformed PLBs were proliferated on 5 mg/l hygromycin selection medium . A total of 28 independent transgenic orchid plants were obtained, from which six transgenic lines that were positive for beta-glucuronidase were randomly selected and confirmed by Southern, northern and western blot analyses . These results indicated that the foreign DNA was successfully integrated into the orchid genome and expressed transcriptionally and translationally in these orchid plants . The present transformation method reported is suitable for improving the Oncidium orchid through genetic engineering. Plant Cell Rep, 2003 Jun, 21(10), 988 - 92 Epub 2003 Apr 03. Improvement of rice (Oryza sativa L.) seed oil quality through introduction of a soybean microsomal omega-3 fatty acid desaturase gene; Anai T et al.; Microsomal omega-3 fatty acid desaturase is an essential enzyme in the production of the n-3 polyunsaturated fatty acid alpha-linolenic acid during the seed developing stage . We have constructed a chimeric gene consisting of a maize Ubi1-P-int and a soybean GmFAD3 cDNA, which was introduced into rice plants by Agrobacterium-mediated transformation . Ten transformants containing the chimeric gene were established and expression subsequently confirmed by Northern blotting . Furthermore, alpha-linolenic acid content of the T(1) seeds increased dramatically up to tenfold that of the control, and this phenotype was also stably inherited in the T(2) and T(3) progenies . These results demonstrate that the alpha-linolenic acid content of rice seed oil can easily be altered using the combination of a high-activity promoter and a GmFAD3 gene. Plant Cell Rep, 2003 Jun, 21(10), 981 - 7 Epub 2003 Apr 02. Horticultural characterization of Angelonia salicariifolia plants transformed with wild-type strains of Agrobacterium rhizogenes; Koike Y et al.; Genetic transformation was carried out with wild-type strains of Agrobacterium rhizogenes for introducing a dwarf trait into the Scrophulariaceous ornamental plant, angelonia (Angelonia salicariifolia) . Leaf segments of two angelonia genotypes (Ang.1 and Ang.2) were co-cultivated with mikimopine-type strains of A . rhizogenes . Adventitious roots that showed vigorous growth and increased lateral branching when cultured on half-strength Murashige and Skoog's (MS) basal salts medium lacking plant growth regulators (PGRs) after co-cultivation were selected as putatively transformed lines . All of these selected lines produced mikimopine . Adventitious shoots were efficiently induced from putatively transformed root segments on half-strength MS basal salts medium containing 1 mg l(-1) benzyladenine (BA) under continuous illumination (24-h photoperiod), and the shoots easily rooted following their transfer to half-strength MS basal salts medium lacking PGRs . The transgenic nature of regenerated plants was confirmed by Southern hybridization . Transformed plants frequently died during their acclimatization, and acclimatized plants of eight transformed lines grew very slowly for 1-5 months after transplantation to the greenhouse . Plants of two transformed lines of Ang.2 flowered 4-6 months after transplantation . These transformed plants exhibited phenotypic alterations such as dwarfness and smaller leaves . There were no apparent alterations observed in the number, shape, and size of the flowers . Pollen fertility of the transformed plants was 60-80% based on aceto-carmine staining . These results indicate the possibility of applying A . rhizogenes-mediated transformation for introducing a dwarf trait into angelonia. Plant Cell Rep, 2003 Jun, 21(10), 955 - 60 Epub 2003 Apr 12. Slow desiccation leads to high-frequency shoot recovery from transformed somatic embryos of cotton (Gossypium hirsutum L . cv . Coker 310 FR); Chaudhary B et al.; In Agrobacterium-mediated genetic transformation of cotton (Gossypium hirsutum L . cv . Coker 310FR) the frequency at which somatic embryos were converted to plantlets was significantly improved by subjecting the embryos to slow physical desiccation . We used Agrobacterium strain GV3101 containing the binary vector pGSFR with the nos-nptII gene for in vitro selection and the 35S gus-int fragment as a reporter to optimize the transformation protocol . Although the concentration of kanamycin was reduced during embryogenesis and embryo maturation, even at the lower levels somatic embryos were predominantly abnormal, showing hypertrophy and reduced or fused cotyledons or poor radicle ends . A majority of these embryos (more than 75%) were beta-glucuronidase (GUS)-positive . Embryos with an abnormal appearance showed a very poor conversion to plantlets . However, these embryos, when subjected to slow physical desiccation followed by transfer to fresh medium, regenerated single or multiple shoots from the cotyledonary end . These shoots could be grafted on wild-type seedling stocks in vitro, which, following their transfer to soil, developed normally and set seeds . Regenerated plants tested positive for the transgene by Southern analysis . An overall scheme for the high-frequency production of cotton transgenics from both normal and abnormal appearing somatic embryos is presented. Theor Appl Genet, 2003 Sep, 107(5), 831 - 6 Epub 2003 Jun 26. Pleiotropic effect of the insertion of the Agrobacterium rhizogenes rolD gene in tomato ( Lycopersicon esculentum Mill.); Bettini P et al.; The Agrobacterium rhizogenes rolD gene, coding for an ornithine cyclodeaminase involved in the biosynthesis of proline from ornithine, has been inserted in Lycopersicon esculentum cv Tondino with the aim of studying its effects on plant morphological characters including pathogen defense response . The analysis of plants transgenic for rolD did not show major morphological modifications . First generation transgenic plants however were found to flower earlier, and showed an increased number of inflorescences and higher fruit yield . Transformed plants were also analysed for parameters linked to pathogen defense response, i.e . ion leakage in the presence of the toxin produced by the fungus Fusarium oxysporum f . sp . lycopersici, and expression of the pathogenesis-related PR-1 gene . All the plants harbouring the rolD gene were shown to be more tolerant to the toxin in ion leakage experiments, with respect to the untransformed regenerated controls and the cv Tondino . PR-1 gene expression was quantitated by means of real-time PCR both at the basal level and after treatment with salicylic acid, an inducer of Systemic Acquired Resistance . In both cases the amount of PR-1 mRNA was higher in the transgenic plants . It seems therefore that the transformation of tomato plants with rolD could lead to an increased competence for defense response, as shown by toxin tolerance and increased expression of the Systemic Acquired Resistance marker gene PR-1 . The results are finally discussed in view of their possible economic relevance. Mol Microbiol, 2003 Jul, 49(2), 441 - 55 The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon; Pappas KM et al.; The replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria . In the region upstream of the octopine-type Ti plasmid repABC operon, three promoters were recently shown to be activated by the LuxR-type regulator TraR . Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number . Here we describe a fourth promoter, designated P4 . This promoter lies directly upstream of repA and is not regulated by TraR . The promoter was localized by subcloning and demonstrated to be strongly autorepressed . RepA is the major cis-acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA-mediated repression in trans . Purified RepA bound to an approximately 70-nucleotide operator site overlapping the P4 promoter and extending well downstream . Binding affinity was increased by adenosine di- and tri-phosphates and also by purified RepB . Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters . These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid. Plant Cell Rep, 2003 Aug, 22(1), 1 - 15 Epub 2003 Jun 24. Genetic transformation of conifers and its application in forest biotechnology; Tang W et al.; Genetic modification of conifers through gene transfer technology is now an important field in forest biotechnology . Two basic methodologies, particle bombardment and Agrobacterium-mediated transformation, have been used on conifers . The use of particle bombardment has produced stable transgenic plants in Picea abies, P . glauca, P . mariana, and Pinus radiata . Transgenic plants have been produced from Larix decidua, Picea abies, P . glauca, P . mariana, Pinus strobus, P . taeda, and P . radiata via Agrobacterium-mediated transformation . Agrobacterium-mediated transformation has advantages over particle bombardment such as a simpler integration pattern and a limited rearrangement in the introduced DNA . At present, genetic transformation of conifers has been directed toward improving growth rate, wood properties and quality, pest resistance, stress tolerance, and herbicide resistance, which will drive forestry to enter a new era of productivity and quality. Plant Cell Rep, 2003 Aug, 22(1), 46 - 51 Epub 2003 Jun 24. Efficient transformation of Medicago truncatula cv . Jemalong using the hypervirulent Agrobacterium tumefaciens strain AGL1; Chabaud M et al.; The efficiency of Agrobacterium tumefaciens transformation of the model legume Medicago truncatula cv . Jemalong (genotype 2HA) was evaluated for strains LBA 4404, C58pMP90, C58pGV2260 and AGL1 . Binary vectors carrying promoter- gus/ gfp reporter gene fusions and the nptII gene as selectable marker were used for plant in vitro transformation/regeneration . The highest transformation efficiency was obtained with the disarmed hypervirulent strain AGL1 (Ti plasmid TiBo542), for which the percentage of explants forming kanamycin (Km)-resistant calli was double that obtained with each of the other three strains . In addition, we were able to reduce the time necessary for plant regeneration using AGL1, with 24% of the explants generating Km-resistant transgenic plantlets within only 4-5 months of culture . Transgene expression in planta was analysed and found to be conserved in the T(1) descendents. Plant Cell Rep, 2003 Aug, 22(1), 38 - 45 Epub 2003 Jun 24. Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants (Prunus domestica L.); Gonzalez Padilla IM et al.; We describe here an improved system for routinely developing transgenic plum plants (Prunus domestica L.) through the use of Agrobacterium tumefaciens . The production of non-transformed "escapes" has been virtually eliminated, and rates of plant establishment in the greenhouse have been dramatically improved . The system is based on the regeneration of shoots from hypocotyls extracted from mature seed . The shoot regeneration medium is Murashige and Skoog (MS) salts and vitamins supplemented with 7.5 microM thidiazuron and 0.25 microM indole-butyric acid . Transferring the explants after co-cultivation to shoot regeneration medium containing 80 mg l(-1) of kanamycin and 300 mg l(-1) of Timentin reduced the total number of regenerated shoots without affecting the transformation rate . Transformation rates using the described system averaged 1.2% of the hypocotyl slices producing transgenic plants, with a range of 0-4.2% . The transgenic shoots rooted at a rate of 90% on half-strength MS salts and vitamins supplemented with 5 microM alpha-naphthaleneacetic acid and 0.01 microM kinetin . Plantlets were transferred to a greenhouse directly from culture tubes with a 90% average survival. Plasmid, 2003 Jul, 50(1), 1 - 11 Conjugal transfer of plasmid pTF-FC2 from Agrobacterium to plant cells in the absence of T-DNA borders; Dube T et al.; The ability of the plasmid pTF-FC2 to transfer genes into plants was investigated . Using this plasmid as the backbone two plasmids were constructed namely pTD1 and pDER-bar . These plasmids contained, as plant selectable markers, the nptII and the bar genes, respectively . The nptII gene was flanked by the right and left borders and the bar gene was not . Transgenic plants were obtained through the co-cultivation of tobacco leaf discs with the Agrobacterium tumefaciens strain LBA4404(pAL4404)(pDER-bar) . Molecular and genetic analysis indicated that the bar gene had been stably integrated into the plant genome and had been inherited in a Mendelian fashion . Integration was shown to be polar and unidirectional and in some cases the entire plasmid was found to have integrated into the plant genome . Interestingly, no plants were generated from tobacco leaf discs that were co-cultivated with the strain C58C1(pMP90)(pTD1). Plant Mol Biol, 2003 May, 52(1), 17 - 29 Expression of anti human IL-4 and IL-6 scFvs in transgenic tobacco plants; Ehsani P et al.; The two murine single-chain Fv (scFv) genes against human interleukin IL-4 and IL-6 cytokines were cloned in a plant expression vector (pGEJAE1) and mobilized to Agrobacterium tumefaciens . Tobacco leaf discs were co-cultured with Agrobacterium and transferred to selective media for regeneration . The tobacco in vitro plants produced scFvs against human IL-4 and IL-6 . Only 8% of transformed plants expressing anti-IL-4 scFv were obtained versus 76% of transformed plants expressing anti-IL-6 scFv . In addition, some plants producing anti-IL-4 and anti-IL-6 scFvs aged more rapidly in in vitro conditions and in greenhouse pots than did control plants . Western blot analysis showed that the transformed Nicotiana tabacum plants contained proteins with an apparent molecular mass on electrophoresis of ca . 32 kDa, corresponding to the predicted size of the scFvs . As entire plant root seemed to accumulate more scFv than did leaves, we decided to continue working with isolated roots . Anti-IL-6 scFvs were detected in cultivated roots and their culture media . Functional anti-IL-6 scFv accounted for 0.16-0.18% of total soluble proteins . The affinity of the anti-IL-6 scFv produced in plants and measured by Biacore was similar to that of scFv produced in Escherichia coli . The high levels of antibody accumulation in isolated roots and secretion into the medium demonstrate the potential for producing recombinant protein in bioreactor systems. DNA Seq, 2003 Apr, 14(2), 87 - 94 Cloning and characterization of an outer membrane protein (Lip18) from Helicobacter bizzozeronii; Zhu J et al.; A recombinant lambda-Zap II phage was selected by screening a genomic library of Helicobacter bizzozeronii (Hb) using antibodies from a naturally infected cat . DNA sequencing resulted an open reading frame containing 172 codons with a predicted molecular mass of 18 kDa (Lip18) . The amino acid sequence showed 22.1, 55.2, 56.7 and 57.1% identity to peptidoglycan-associated lipoprotein of Helicobacter pylori, Agrobacterium tumefaciens, Bartonella bacilliformis, respectively . A peptidoglycan associating alpha-helical motif (LALGQRRSVAVRDYLVS) was located in the C-terminal region . H . bizzozeronii contains a potential lipoprotein signal peptide cleavage site (Val-Val-Gly-Cys), and yields a predicted mature protein with 148 amino acids . The Lip18 was localized into the outer membrane of the bacteria . Immunoblot analysis of serum samples from a dog and cat naturally infected with Helicobacter spp was able to recognize the purified recombinant Lip18. J Biol Chem, 2003 Sep 5, 278(36), 33786 - 92 Epub 2003 Jun 24. Biliverdin binds covalently to agrobacterium phytochrome Agp1 via its ring A vinyl side chain; Lamparter T et al.; The widely distributed phytochrome photoreceptors carry a bilin chromophore, which is covalently attached to the protein during a lyase reaction . In plant phytochromes, the natural chromophore is coupled by a thioether bond between its ring A ethylidene side chain and a conserved cysteine residue within the so-called GAF domain of the protein . Many bacterial phytochromes carry biliverdin as natural chromophore, which is coupled in a different manner to the protein . In phytochrome Agp1 of Agrobacterium tumefaciens, biliverdin is covalently attached to a cysteine residue close to the N terminus (position 20) . By testing different natural and synthetic biliverdin derivatives, it was found that the ring A vinyl side chain is used for chromophore attachment . Only those bilins that have ring A vinyl side chain were covalently attached, whereas bilins with an ethylidene or ethyl side chain were bound in a noncovalent manner . Phycocyanobilin, which belongs to the latter group, was however covalently attached to a mutant in which a cysteine was introduced into the GAF domain of Agp1 (position 249) . It is proposed that the regions around positions 20 and 249 are in close contact and contribute both to the chromophore pocket . In competition experiments it was found that phycocyanobilin and biliverdin bind with similar strength to the wild type protein . However, in the V249C mutant, phycocyanobilin bound much more strongly than biliverdin . This finding could explain why during phytochrome evolution in cyanobacteria, the chromophore-binding site swapped from the N terminus into the GAF domain. J Mol Biol, 2003 Jul 4, 330(2), 277 - 86 Consensus structural features of purified bacterial TatABC complexes; Oates J et al.; The twin-arginine translocation (Tat) system transports folded proteins across bacterial plasma membranes and the chloroplast thylakoid membrane . Here, we investigate the composition and structural organization of three different purified Tat complexes from Escherichia coli, Salmonella typhimurium and Agrobacterium tumefaciens . First, we demonstrate the functional activity of these Tat systems in vivo, since expression of the tatABC operons from S.typhimurium or A.tumefaciens in an E.coli tat null mutant strain resulted in efficient Tat-dependent export of an E.coli cofactor-containing substrate, TMAO reductase . The three isolated, affinity-tagged Tat complexes comprised TatA, TatB and TatC in each case, demonstrating a strong interaction between these three subunits . Single-particle electron microscopy studies of all three complexes revealed approximately oval-shaped, asymmetric particles with maximal dimensions up to 13 nm . A common feature is a number of stain-excluding densities surrounding more or less central pools of stain, suggesting protein-lined pores or cavities . The characteristics of size variation among the particles suggest a modular form of assembly and/or the recruitment of varying numbers of TatBC/TatA units . Despite low levels of sequence homology, the combined data indicate structural and functional conservation in the Tat systems of these three bacterial species. Protein Expr Purif, 2003 Jul, 30(1), 134 - 9 Purification of industrial hydantoinase in one chromatographic step without affinity tag; Huang CY et al.; Hydantoinase is used in industry as a biocatalyst for the production of optically pure D- or L-amino acids . Previously, homogeneous hydantoinase was obtained by multi-chromatographic purification procedures . Here, we reported a process that contained only a single chromatographic step to purify a recombinant hydantoinase to homogeneity . Hydantoinase from Agrobacterium radiobacter NRRL B11291 was expressed in Escherichia coli . The recombinant enzyme was purified following heat treatments, high concentration alcohol precipitation, and chelating Sephacel chromatography . The recombinant hydantoinase did not contain any affinity tags from the plasmid . This simplified procedure provided a convenient way to obtain hydantoinase in high yield (71%) and high purity . It should be very useful for further industrial application and for the study of the structure-function of hydantoinase. J Virol Methods, 2003 Jul, 111(1), 37 - 42 A novel co-delivery system consisting of a Tomato bushy stunt virus and a defective interfering RNA for studying gene silencing; Hou H et al.; Virus induced gene silencing (VIGS) and suppression are RNA-specific defense and counter-defense circuits in plant-virus interactions . These phenomena have been investigated extensively with an Agrobacterium-mediated transient expression system . In this study, a virus-based transient expression system was developed to study these phenomena . A Tomato bushy stunt virus (TBSV) viral vector with an inactivated P19 suppressor gene, referred to as pHST2-14, was chosen to express the P1 of Tobacco etch virus (TEV) . TEV P1 is a component of a well-characterized VIGS suppressor, TEV P1/HC-Pro protein . A TBSV defective interfering RNA (DI) that contains the 3' proximal portion of a green fluorescence protein (GFP) gene, DI-P, was used as a silencing inducer of the homologous GFP gene on GFP transgenic Nicotiana benthamiana (NbGFP) plants . The TEV P1 gene was inserted into pHST2-14 to generate TBSV-P1 . Transcripts of TBSV-P1 were then mixed with DI-P transcripts and inoculated onto NbGFP plants . DI-P consistently accumulated in NbGFP plants that were inoculated with TBSV-P1 and DI-P, and efficiently induced silencing of GFP transgene . These results demonstrate that a TBSV-based co-delivery system can provide a new alternative tool to investigate gene silencing and its influence by a TBSV-expressed foreign protein . It also can be used to elucidate functions of endogenous genes in plants. Plant Cell Rep, 2003 Aug, 21(12), 1188 - 93 Epub 2003 Jun 18. Rhizosecretion of recombinant proteins from plant hairy roots; Gaume A et al.; Rhizosecretion of a target protein in the hydroponic medium provides an alternative manufacturing platform that simplifies the downstream purification procedure and increases protein yield . In order to increase the production rates of rhizosecreted proteins, we have exploited the ability of Agrobacterium rhizogenes to induce the formation of large amounts of root tissue on transgenic tobacco plants engineered to secrete a model recombinant protein, human secreted alkaline phosphatase (SEAP) . The secretion of SEAP from hairy roots induced on the stems of transgenic tobacco plants was 5-7 times higher than that from adventitious transgenic roots. Plant Cell Rep, 2003 Aug, 21(12), 1207 - 10 Epub 2003 Jun 18. Influence of Agrobacterium tumefaciens strain on the production of transgenic peas ( Pisum sativum L.); Grant JE et al.; We compared the efficiency of two Agrobacterium tumefaciens strains, AGL 1 and KYRT1, for producing transgenic pea plants . KYRT1 is a disarmed strain of Chry5 that has been shown to be highly tumourigenic on soybean . The efficacies of the strains were compared using cotyledon explants from three pea genotypes and two plasmids . The peas were sourced from field-grown plants over three Southern Hemisphere summer seasons . Overall, KYRT1 was found to be on average threefold more efficient than AGL 1 for producing transgenic plants . We suggest that KYRT1 is sensitive to cocultivation temperature as the expected increase in efficiency was not achieved at high laboratory temperatures. J Anim Sci, 2003 Jun, 81(6), 1447 - 55 Determining whether transgenic and endogenous plant DNA and transgenic protein are detectable in muscle from swine fed Roundup Ready soybean meal; Jennings JC et al.; Questions regarding the digestive fate of DNA and protein from transgenic feed have been raised in regard to human consumption and commercial trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops . Using highly sensitive, well-characterized analytical methods, pork loin samples were analyzed for the presence of fragments of transgenic and endogenous plant DNA and transgenic protein from animals fed meal prepared from conventional or glyphosate-tolerant Roundup Ready (RR) soybeans . Pigs were fed diets containing 24, 19, and 14% RR or conventional soybean meal during grower, early-finisher, and late-finisher phases of growth, respectively, and longissimus muscle samples were collected (12 per treatment) after slaughter . Total DNA was extracted from the samples and analyzed by PCR, followed by Southern blot hybridization for the presence of a 272-bp fragment of the cp4 epsps coding region (encoding the synthetic enzyme 5-enolpyruvylshikimate-3-phosphate synthase derived from Agrobacterium sp . strain CP4) and a 198-bp fragment of the endogenous soybean gene le1 (encoding soy lectin) . Using 1 microgram of input DNA per reaction, none of the extracted samples was positive for cp4 epsps or le1 at the limit of detection (LOD) for these PCR/Southern blot assays . The LOD for these assays was shown to be approximately one diploid genome equivalent of RR soybean DNA, even in the presence of 10 micrograms of pork genomic DNA . A 185-bp fragment of the porcine preprolactin (prl) gene, used as a positive control, was amplified from all samples showing that the DNA preparations were amenable to PCR amplification . Using a competitive immunoassay with an LOD of approximately 94 ng of CP4 EPSPS protein/g of pork muscle, neither the CP4 EPSPS protein nor the immunoreactive peptide fragments were detected in loin muscle homogenates from pigs fed RR soybean meal . Taken together, these results show that neither small fragments of transgenic DNA nor immunoreactive fragments of transgenic protein are detectable in loin muscle samples from pigs fed a diet containing RR soybean meal. J Appl Microbiol, 2003, 95(1), 102 - 8 Genetic transfer of the mcd gene in soil; Desaint S et al.; AIMS: To investigate the role of horizontal gene transfer of mcd (methylcarbamate-degrading) gene in high genetic diversity of carbofuran-degrading bacteria . METHODS AND RESULTS: The actuality of genetic transfer from degraders to an Agrobacterium tumefaciens strain was determined in liquid medium . The mcd gene was chosen for transfer experiments . Transconjugants were obtained irrespective of the type of the donor strain (Gram-positive or Gram-negative), size of the inoculum, or nature and concentration of the pesticide in the medium . Soil microcosms, inoculated with or without the donor and/or recipient strains were used . The size of the initial degrading population (treated or untreated soil) and the nature of the inoculated donor strains were considered . More transconjugants were isolated in the previously treated soil than in the untreated soil . Agrobacterium transconjugants were isolated even when the donor strain was not inoculated, probably as a result of gene transfer from indigenous degrading population to the recipient strain . Moreover, potential transconjugants belonging to the Pseudomonas genus were isolated . CONCLUSIONS: Our results seem to demonstrate that the mcd gene is transferable in soil among bacterial populations . SIGNIFICANCE AND IMPACTS OF THE STUDY: The transfer of the mcd gene is partly responsible for the high genetic diversity of micro-organisms able to catabolize carbofuran. Plant Physiol, 2003 Jun, 132(2), 494 - 505 Identification of Arabidopsis rat mutants; Zhu Y et al.; Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process . To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants) . To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches . These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction . Here, we present an update on the identification and characterization of these rat mutants. DNA Cell Biol, 2003 Mar, 22(3), 179 - 86 Characterization of the ftsZ gene from Ehrlichia chaffeensis, Anaplasma phagocytophilum, and Rickettsia rickettsii, and use as a differential PCR target; Lee KN et al.; Degenerate primers corresponding to highly conserved regions of previously characterized ftsZ genes were used to PCR amplify a portion of the ftsZ gene from the genomic DNA of Ehrlichia chaffeensis (ftsZ(Ech)), Anaplasma phagocytophilum (ftsZ(Ap)), and Rickettsia rickettsii (ftsZ(Rr)) . Genome walking was then used to amplify the 5' and 3' termini of the genes . The DNA sequences of the resulting amplification products yielded open reading frames coding for proteins with molecular masses of 42.0, 45.7, and 48.3 kDa for A . phagocytophilum, E . chaffeensis, and R . rickettsii, respectively . These homologs are 20 to 70 amino acids longer than the FtsZ proteins characterized in bacteria such as Escherichia coli and Bacillus subtilis, but do not possess the large extended carboxyl-termini found in the FtsZ proteins of Bartonella, Rhizobium, and Agrobacterium species . The functional domains important for FtsZ activity are conserved within the ehrlichial and rickettsial FtsZ protein sequences . The R . rickettsii FtsZ sequence is highly homologous to the FtsZ protein previously described for Rickettsia prowazekii (89% identity), and identical to the FtsZ protein of Rickettsia conorii . The percent identity observed between the A . phagocytophilum and E . chaffeensis FtsZ proteins is only 79% and is particularly low in the carboxyl-terminal region (15.8% identity) . Primers were designed to PCR amplify a portion of the variable carboxyl-terminal region of the ftsZ gene, and used to differentiate each agent based on the size of the amplicons: A . phagocytophilum, 278 bp; E . chaffeensis, 341 bp; and Rickettsia spp., 425 bp. FEMS Microbiol Lett, 2003 Jun 6, 223(1), 1 - 6 The VirE2 protein of Agrobacterium tumefaciens: the Yin and Yang of T-DNA transfer; Duckely M et al.; Agrobacterium tumefaciens has evolved a unique mechanism to solve the problem of transferring DNA across five bilayers; the inner and outer membranes of the bacterium, the plasma membrane of the plant cell and the double membrane formed by the nuclear envelope . The two first and two last seem to be mediated by, respectively, the type IV secretion system in Agrobacterium and the nuclear pore complex in the plant cell, but the mechanism by which the transferred DNA (T-DNA) crosses the plant membrane still remains a mystery . New biophysical experiments suggest that, in addition to its established role as a single-stranded DNA (ssDNA)-binding protein, the VirE2 protein forms a channel in the plant membrane allowing the passage of the T-DNA into the cell . Such a role would be reminiscent of translocator molecules secreted by the type III secretion system of pathogenic bacteria and inserting into the host eukaryotic plasma membrane . The implications for the structure of the protein, its regulation and role in vivo are discussed. Plant J, 2003 Jun, 34(6), 778 - 87 Tumour development in Arabidopsis thaliana involves the Shaker-like K+ channels AKT1 and AKT2/3; Deeken R et al.; After completion of the Arabidopsis genome-sequencing programme, crown galls induced by Agrobacterium tumefaciens may become a model system to study plant tumour development . The molecular mechanisms of nutrient supply to support tumour growth and development are still unknown . In this study, we have identified a unique profile of Shaker-like potassium channels in agrobacteria-induced Arabidopsis tumours . Comparing the gene expression pattern of rapidly growing tumours with that of non-infected tissues, we found the suppression of shoot in favour of root-specific K+ channels . Among these, the upregulation of AKT1 and AtKC1 and the suppression of AKT2/3 and GORK were most pronounced . As a consequence, K+ uptake and accumulation were elevated in the tumour (163 mm) compared to control tissues (92 mm) . Patch clamp studies on tumour protoplasts identified a population expressing the electrical properties of the AKT1 K+ channel . Furthermore, plants lacking a functional AKT1 or the AKT2/3 phloem K+ channel gene did not support tumour growth . This indicates that the delivery of potassium by AKT1 and the direction of assimilates, triggered by AKT2/3, are essential for tumour growth. Biotechnol Prog, 2003 May-Jun, 19(3), 864 - 73 Production, IMAC purification, and molecular modeling of N-carbamoyl-D-amino acid amidohydrolase C-terminally fused with a six-his peptide; Chen HM et al.; A six-His peptide was genetically engineered to the C-terminus of Agrobacterium radiobacter N-carbamoyl-D-amino acid amidohydrolase monomer to facilitate the protein purification with immobilized metal affinity chromatography (IMAC) . The fusion enzyme, named as DCaseH, was overexpressed in Escherichia coli and one-step IMAC-purified . The production study showed that DCaseH was optimally produced at 15 degrees C for 25 h by the induction of 0.05 mM IPTG . Both Co(2+)-chelated TANOL gels and Ni(2+)-chelated nitriloacetic acid agarose gels efficiently purified DCaseH, with the former yielding purer enzyme than the latter . Highly pure DCaseH was obtained in the former purification with the addition of 5 mM imidazole in the washing buffer, and the specific enzyme activity was increased more than 11-fold . Denaturing IMAC purification successfully purified DCaseH from inclusion bodies that were mostly composed of the overexpressed DCaseH, while the attempt to refold the purified enzyme by either dialysis or solid-state refolding was not achieved . The purified native enzyme was optimally active at pH 6.5 and 50 degrees C, and the presence of 10% glycerol increased the activity . The molecular modeling of dimeric DCaseH indicated that the six-His tags were freely exposed to the protein surface, resulting in the selective and effective IMAC purification of DCaseH. Plant Cell Rep, 2003 Apr, 21(8), 785 - 8 Epub 2003 Mar 21. Establishment of a highly efficient transformation system for pepper (Capsicum annuum L.); Li D et al.; Application of modern genetic manipulation has been limited in pepper ( Capsicum annuum L.) due to the lack of an efficient transformation system . Following the development of an efficient protocol for in vitro regeneration of pepper cotyledons, we investigated the key factors affecting transformation and established a highly efficient genetic transformation system using the pepper cotyledon as starting material . In this system, cotyledon explants are preconditioned for 2 days on kanamycin (km)-free DM1 medium {Murashige and Skoog (MS) salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients and a hormone combination of 1.0 mg/l indoleacetic acid (IAA) and 5.0 mg/l 6-benzyladenine (BA) solidified with 0.7% agar, pH 5.8}, followed by co-cultivation with Agrobacterium tumefaciens on DM1 for 2 days and delay selection on DM1 with 500 mg/l carbenicillin (carb) for 2 days . The explants are then placed on DM1 containing 10 mg/l AgNO(3), 50 mg/l km-sulfate and 500 mg/l carb . After 4-5 weeks, the explants with buds are transferred to EM1 medium (MS salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients, 10 mg/l AgNO(3) and a hormone combination of 1.0 mg/l IAA, 3.0 mg/l BA and 2.0 mg/l gibberellic acid, solidified with 0.7% agar, pH 5.8) with 50 mg/l kanamycin and 500 mg/l carbenicillin for the elongation of buds . After 3-6 weeks, 1- to 2-cm-long elongated shoots are excised and planted on RM1 medium (MS basal medium supplemented with a hormone combination of 0.2 mg/l NAA and 0.1 mg/l IAA, solidified with 0.8% agar, pH 5.8) with 25 mg/l km and 200 mg/l carb for rooting . We tested four genotypes of pepper, and all presented a high differentiation efficiency (81.3% on average), elongation rate (61.5%) and rooting efficiency (89.5%) . Polymerase chain reaction analysis results showed that 40.8% of the regenerated plantlets were transgenic plants. Plant Cell Rep, 2003 Apr, 21(8), 778 - 84 Epub 2003 Mar 15. Binary transformation systems based on 'shooter' mutants of Agrobacterium tumefaciens: a simple, efficient and universal gene transfer technology that permits marker gene elimination; Mihalka V et al.; A simple transformation procedure with a positive selection scheme using the expression of the isopentenyl transferase ( ipt) gene of transfer DNA (T-DNA) 'shooter' mutants of Agrobacterium tumefaciens was elaborated . After comparing several 'shooter' mutants we found that particular strains frequently produced phenotypically normal shoots after co-culturing with tobacco leaf explants . Shoots selected for normal phenotype showed apical dominance and could be rooted with the same efficiency as non-transformed shoots . When binary vectors were introduced into these strains, stably integrated binary vector T-DNA sequences were found in some regenerants, which were produced under non-selective conditions on growth-regulator-free medium . Such phenotypically normal transformants typically lacked a stably integrated ipt gene . Normal looking shoots could also be produced in tomato, muskmelon and sweet pepper. Plant Cell Rep, 2003 Apr, 21(8), 771 - 7 Epub 2003 Feb 25. A new transformation-regeneration procedure in the model legume Lotus japonicus: root explants as a source of large numbers of cells susceptible to Agrobacterium-mediated transformation; Lombari P et al.; We describe herein a simple and efficient transformation procedure for the production of transgenic Lotus japonicus plants . In this new procedure, dedifferentiated root explants, used as starting material, are the source of a large number of cells that are competent for the regeneration procedure, with a high susceptibility to Agrobacterium infection . The application of this protocol resulted in a tenfold increase in the number of transformants produced by a single plant in comparison to the widely used hypocotyl transformation procedure . Furthermore, our procedure allowed the use of intact plants stored for a long time at 4 degrees C, thus providing a potential continuous supply of explants for transformation experiments . The overall time of incubation under tissue culture conditions required to obtain a plant transferable into soil is 4 months . The transgenic nature of the transformants was demonstrated by the detection of beta-glucuronidase (GUS) activity in the primary transformants and by molecular analysis . Stable transformation was indicated by Mendelian segregation of the hygromycin selectable marker and of the gusA activity after selfing of the transgenic plants. Plant Cell Rep, 2003 Jun, 21(9), 884 - 90 Epub 2003 Mar 22. Assembly of cholera toxin B subunit full-length rotavirus NSP4 fusion protein oligomers in transgenic potato; Kim TG et al.; A CTB-NSP4(175) fusion gene encoding the entire 175-aa murine rotavirus NSP4 enterotoxin protein was transferred into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation . The CTB-NSP4(175) enterotoxin fusion gene was detected in the genomic DNA of transformed leaves by PCR DNA amplification . Synthesis and assembly of the full-length CTB-NSP4(175) fusion protein into oligomeric structures of pentamer size was detected in transformed tuber extracts by immunoblot analysis . The binding of CTB-NSP4(175 )fusion protein pentamers to intestinal epithelial cell membrane receptors was quantified by G(M1)-ganglioside enzyme-linked immunosorbent assay (G(M1)-ELISA) . The ELISA results showed that CTB-NSP4(175) fusion protein was 0.006-0.026% of the total soluble tuber protein . The synthesis of CTB-NSP4(175) monomers and their assembly into biologically active oligomers in transformed potato tubers demonstrates the feasibility of using edible plants for the synthesis of enterocyte-targeted full-length rotavirus enterotoxin antigens that retain all of their pathogenic epitopes for initiation of a maximum mucosal immune response. Plant Cell Rep, 2003 Jun, 21(9), 872 - 83 Epub 2003 Apr 03. Stable transformation of Theobroma cacao L . and influence of matrix attachment regions on GFP expression; Maximova S et al.; We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L . using cotyledonary explants from primary somatic embryos (SEs) and A . tumefaciens strain AGL1 . Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs . The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone . However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics . Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis . Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity . Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants . Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA. Plant Cell Rep, 2003 Jun, 21(9), 851 - 9 Epub 2003 Mar 22. Stable genetic transformation of Vigna mungo L . Hepper via Agrobacterium tumefaciens; Saini R et al.; Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed . We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron . The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis . These shoots were established in soil and grown to maturity to collect the seeds . Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation . Analysis of T(0) plants showed the expression and integration of uidA into the plant genome . GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay . PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern . The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics. Plant Cell Rep, 2003 Aug, 21(12), 1167 - 74 Epub 2003 May 27. Ethylene inhibitors and low kanamycin concentrations improve adventitious regeneration from apricot leaves; Burgos L et al.; An improved method for adventitious regeneration from apricot leaves is described . The use of the ethylene inhibitors silver thiosulphate (30-60 micro M) or aminoethoxyvinylglycine (0.5 micro M) increased regeneration percentages in Helena and Canino apricot cultivars and also the consistency of results from different experiments . Use of "Pure Agar" also improved regeneration from Helena leaves as compared with agargel or agarose . Regeneration rates for Canino were dependent on the medium in which the shoots were micropropagated . When different antibiotics were tested for their influence on regeneration, the combination of cefotaxime (0.13 m M) plus vancomycin (0.63 m M), which efficiently controls Agrobacterium growth, also increased regeneration percentages in Helena two-fold but did not affect regeneration in Canino . Kanamycin, an antibiotic widely used for selection of nptII transformed cells, promoted more rapid regeneration and higher regeneration rates from Helena leaves when added at low concentrations (8.6 and 17.1 micro M) . With this improved procedure, regeneration from apricot leaves has been increased more than 200% as compared with rates reported previously. Plant Cell Rep, 2003 Aug, 21(12), 1183 - 7 Epub 2003 May 28. Agrobacterium-mediated transformation of niger { Guizotia abyssinica (L . f.) Cass.} using seedling explants; Murthy HN et al.; A protocol was developed for Agrobacterium-mediated genetic transformation of niger { Guizotia abyssinica (L.f.) Cass.} using hypocotyl and cotyledon explants . Hypocotyls and cotyledons obtained from 7-day-old seedlings were co-cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm that harbored genes for beta-glucuronidase (GUS), kanamycin, and hygromycin resistance . Following co-cultivation, the hypocotyl and cotyledon explants were cultivated on MS medium containing 1 mg/l 6-benzylaminopurine (BA) for 3 days in darkness . Subsequently, hypocotyl and cotyledon explants were transferred to selective MS medium containing 1 mg/l BA, 10 mg/l hygromycin, 10 mg/l kanamycin, and 500 mg/l cefotaxime . After 6 weeks, hypocotyls and cotyledons produced multiple adventitious shoot buds, and these explants were subcultured to MS medium containing 1 mg/l BA, 30 mg/l hygromycin, and 30 mg/l kanamycin . After a further 3 weeks, the explants (along with developing shoot buds) were subcultured to MS medium containing 1 mg/l BA, 50 mg/l kanamycin, and 50 mg/l hygromycin for further selection . Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 0.1 mg/l alpha-naphthaleneacetic acid, 50 mg/l kanamycin, and 50 mg/l hygromycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis . Genomic Southern blot hybridization confirmed the incorporation of the neomycin phosphotransferase II gene into the host genome. Plant Cell Rep, 2003 Jan, 21(5), 475 - 82 Epub 2002 Dec 04. Improved Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.): assessment of macerating enzymes and sonication; Weber S et al.; Agrobacterium -mediated transformation of shoot apices of sunflower (Helianthus annuus L.) was evaluated following wounding by cell-wall-digesting enzymes and sonication . The frequency of explants with regenerated shoots expressing GUS (beta-glucuronidase) or GFP (green fluorescent protein) increased following treatment with the macerating enzymes cellulase Onozuka R-10 and pectinase Boerozym M5, whereas treatment with macerozyme R-10 had a negative effect . When a combination of cellulase (0.1%) and pectinase (0.05%) was used, the rate of explants with uniformly GUS-positive shoots increased at least twofold . The transient expression of reporter genes was also enhanced using sonication (50 MHz; 2, 4 and 6 s), but stable expression in regenerated shoots following 4 weeks of selection did not increase with this treatment . Enzyme treatment alone (0.1% cellulase and 0.05% pectinase) was superior to a combined treatment of sonication and enzymes with respect to stable transformation . Polymerase chain reaction analyses of shoots recovered by grafting from transformation experiments using GFP as the reporter gene demonstrated the stable integration of the transgene . Regenerated plants were fertile and seeds could be harvested. Plant Cell Rep, 2003 Jan, 21(5), 459 - 66 Epub 2002 Oct 31. Regeneration of transformed verbena (Verbena x hybrida) by Agrobacterium tumefaciens; Tamura M et al.; Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid . A transformation system was developed using cvs . Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2 . Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments . Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark . After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested . These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse . GFP was expressed in all of the organs including the floral parts . Stable genomic transformation was confirmed by Southern blot analysis . No morphological differences were observed between the transformed plants and their host plants. Plant Cell Rep, 2003 Jan, 21(5), 437 - 44 Epub 2002 Oct 03. Agrobacterium tumefaciens-mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.); Bettany AJ et al.; Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum) . Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone . Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media . Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil . All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS . Three GUS-negative transgenic L . multiflorum and the two F . arundinacea plants were vernalised and allowed to flower . All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed . Progeny analysis of L . multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission . However, significant differences were noted between the paternal and maternal expression of hygromycin resistance. Plant Cell Rep, 2003 Jan, 21(5), 429 - 36 Epub 2002 Sep 26. Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium; Khanna HK et al.; Immature embryo-derived calli of spring wheat (Triticum aestivum L.) cv Veery5 were transformed using Agrobacterium tumefaciens strain LBA4404 carrying either binary vector pHK22 or superbinary vector pHK21, the latter carrying an extra set of vir genes--vir B, -C and -G . In both cases, transient beta-glucuronidase ( GUS) expression ranging from 35-63% was observed 3 days after co-cultivation, but 587 calli infected with pHK22/LBA4404 failed to produce a single stably transformed plant, whereas 658 calli infected with pHK21/LBA4404 gave rise to 17 transformants carrying both the GUS and bar genes . Regeneration media supplemented with 0.1 M spermidine improved the recovery of transformants from pHK21/LBA4404-infected calli from 7% to 24.2%, resulting in an increase in the overall transformation frequency from 1.2% to 3.9% . The results suggest that two important factors that could lead to an improvement in transformation frequencies of cereals like wheat are (1) the use of superbinary vectors and (2) modification of the polyamine ratio in the regeneration medium . Stable expression and inheritance of the transgenes was confirmed by both genetic and molecular analyses . T1 progeny showed segregation of the transgenes in a typical Mendelian fashion in most of the plants . Of the transformed plants, 35% showed single-copy insertion of the transgene as shown by both Southern analysis and the segregation ratios. Plant Cell Rep, 2003 Feb, 21(6), 599 - 604 Epub 2002 Dec 13. Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants; Cardoza V et al.; An efficient protocol for the production of transgenic Brassica napus cv . Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time . Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue . Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments . Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25% . With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25% . Transgenic shoots were selected on 200 mg/l kanamycin . Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin. Plant Cell Rep, 2003 Feb, 21(6), 563 - 8 Epub 2002 Nov 22. Production of herbicide-resistant transgenic Panax ginseng through the introduction of the phosphinothricin acetyl transferase gene and successful soil transfer; Choi YE et al.; Herbicide-resistant transgenic Panax ginseng plants were produced by introducing the phosphinothricin acetyl transferase (PAT) gene that confers resistance to the herbicide Basta (bialaphos) through Agrobacterium tumefaciens co-cultivation . Embryogenic callus gathered from cotyledon explants of P . ginseng were pre-treated with 0.5 M sucrose or 0.05 M MgSO(4 )before Agrobacterium infection . This pre-treatment process markedly enhanced the transient expression of the beta-glucuronidase (GUS) gene . Embryogenic callus was initially cultured on MS medium supplemented with 400 mg/l cefotaxime for 3 weeks and subsequently subcultured five times to a medium containing 25 mg/l kanamycin and 300 mg/l cefotaxime . Somatic embryos formed on the surfaces of kanamycin-resistant callus . Upon development into the cotyledonary stage, these somatic embryos were transferred to a medium containing 50 mg/l kanamycin and 5 mg/l gibberellic acid to induce germination and strong selection . Integration of the transgene into the plants was confirmed by polymerase chain reaction and Southern analyses . Transfer of the transgenic ginseng plantlets to soil was successfully accomplished via acclimatization in autoclaved perlite . Not all of the plantlets survived in soil that had not been autoclaved because of fungal infection, particularly in the region between the roots and leaves . Transgenic plants growing in soil were observed to be strongly resistant to Basta application. Plant Cell Rep, 2003 Feb, 21(6), 555 - 62 Epub 2002 Nov 26. Additional virulence genes and sonication enhance Agrobacterium tumefaciens-mediated loblolly pine transformation; Tang W; Additional virulence (vir) genes in Agrobacterium tumefaciens and sonication were investigated for their impact on transformation efficiency in loblolly pine (Pinus taeda L.) . Mature zygotic embryos of loblolly pine were co-cultivated with disarmed A . tumefaciens strain EHA105 containing either plasmid vector pCAMBIA1301 or vector pCAMBIA1301 with an additional 15.8-kb fragment carrying extra copies of the Vir B, Vir C, and Vir G regions from the supervirulent plasmid pTOK47 . pCAMBIA1301 contains hygromycin resistance and the beta-glucuronidase (GUS) reporter gene . Expression of GUS was observed after 3-6 days of co-cultivation, with peak expression at approximately 21 days . The highest numbers of GUS-expressing areas were visible up to 21 days after co-cultivation, declining rapidly thereafter . Both transient and stable transformation efficiencies increased when the explants were sonicated before co-cultivation and/or the additional virB, virC, and virG genes were included with the pCAMBIA1301 plasmid T-DNA . Use of the plasmid with additional vir genes and sonication dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer not only in transient expression but also in the recovery of hygromycin-resistant lines . Stably transformed cultures and transgenic plants were produced from embryos transformed with A . tumefaciens EHA105 carrying pCAMBIA1301 or pCAMBIA1301+pTOK47 in the three families of loblolly pine . The presence of the introduced GUS and hygromycin phosphotransferase genes in the transgenic plants was confirmed by polymerase chain reaction and Southern hybridization analyses. Plant Cell Rep, 2003 Feb, 21(6), 549 - 54 Epub 2003 Jan 08. Agrobacterium tumefaciens-mediated transformation of eggplant (Solanum melongena L.) using root explants; Franklin G et al.; An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed . Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII) . Transgenic calli were induced in media containing 0.1 mg l(-1) thidiazuron (TDZ), 3.0 mg l(-1) N(6)-benzylaminopurine, 100 mg l(-1) kanamycin and 500 mg l(-1) cefotaxime . The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg l(-1) kanamycin sulphate . Transgenic plants were raised in pots and seeds subsequently collected from mature fruits . Histochemical GUS assay and polymerase chain reaction analysis of field-established transgenic plants and their offsprings confirmed the presence of the GUS and NPTII genes, respectively . Integration of T-DNA into the genome of putative transgenics was further confirmed by Southern blot analysis . Progeny analysis of these plants showed a pattern of classical Mendelian inheritance for both the NPTII and GUS genes. Plant Cell Rep, 2003 Mar, 21(7), 669 - 75 Epub 2003 Feb 12. Agrobacterium tumefaciens-mediated transformation of Indian mulberry, Morus indica cv . K2: a time-phased screening strategy; Bhatnagar S et al.; An efficient and reproducible protocol for the production of transgenic plants was developed for Morus indica cv . K2 by Agrobacterium tumefaciens-mediated transformation . The hypocotyls, cotyledon, leaf and leaf callus explants precultured for 5 days on regeneration medium were co-cultivated with a bacterial suspension at 10(9) cells/ml for 3 days in the dark . Infectivity of A . tumefaciens strain LBA4404 was more than that of strains GV2260 and A281, and among the various plasmids tried, pBI121 and pBI101:Act1 transformed nearly 100% of the explants followed closely by p35SGUSINT . About 90-100% of the explants tested positive in the beta-glucuronidase (GUS) histochemical assay performed after 3 days of co-cultivation . This high level of transient expression, however, decreased to 20-25% after 15 days . Gus activity was most stable in the callus explants, which emerged as the explant of choice for transformation . The transformed explants were selected on 50-75 mg/l kanamycin for 1 month, and 25-50% of the explants developed adventitious buds . On the basis of kanamycin-resistant shoots produced from the total number of explants inoculated, the transformation efficiency was 44% . After 1 month, 40% of these shoots displayed high gus activity as assessed by the GUS fluorometric assay . On a selection-free root induction medium, 80% of the shoots developed roots and 90% of the potted plantlets acclimatized to the growth room conditions . The 3-month-old regenerates showed gus and nptII(neomycin phosphotransferase II) gene activity as assayed by the GUS fluorometric assay and nptII enzyme assay, followed by PCR polymerase chain reaction (54.5%) analysis after 6-months . Transgene integration into the nuclear genome of 1-year-old regenerates was confirmed in 10 of the 18 transformants tested by Southern analysis . The transformation efficiency as defined by the number of transgenic plants produced from the total number of explants co-cultivated was 6%. Plant Cell Rep, 2003 Mar, 21(7), 659 - 68 Epub 2003 Jan 16. Factors influencing successful Agrobacterium-mediated genetic transformation of wheat; Wu H et al.; The development of a robust Agrobacterium-mediated transformation protocol for a recalcitrant species like bread wheat requires the identification and optimisation of the factors affecting T-DNA delivery and plant regeneration . We have used immature embryos from range of wheat varieties and the Agrobacterium strain AGL1 harbouring the pGreen-based plasmid pAL156, which contains a T-DNA incorporating the bar gene and a modified uidA (beta-glucuronidase) gene, to investigate and optimise major T-DNA delivery and tissue culture variables . Factors that produced significant differences in T-DNA delivery and regeneration included embryo size, duration of pre-culture, inoculation and co-cultivation, and the presence of acetosyringone and Silwet-L77 in the media . We fully describe a protocol that allowed efficient T-DNA delivery and gave rise to 44 morphologically normal, and fully fertile, stable transgenic plants in two wheat varieties . The transformation frequency ranged from 0.3% to 3.3% . Marker-gene expression and molecular analysis demonstrated that transgenes were integrated into the wheat genome and subsequently transmitted into progeny at Mendelian ratios. Plant Cell Rep, 2003 Mar, 21(7), 651 - 8 Epub 2002 Nov 05. Expression of hemagglutinin protein of Rinderpest virus in transgenic pigeon pea {Cajanus cajan (L.) Millsp.} plants; Satyavathi VV et al.; Rinderpest virus is the causative agent of a devastating, often fatal disease in wild and domestic bovids that is endemic in Africa, the Middle East and South Asia . The existing live attenuated vaccine is heat-labile, and thus there is a need for the development of new strategies for vaccination . This paper reports the development of transgenic pigeon pea { Cajanus cajun (L.) Millsp.} expressing one of the protective antigens, the hemagglutinin (H) protein of Rinderpest virus . A 2-kb fragment containing the coding region of the H protein was cloned into pBI121 and mobilized into Agrobacterium tumefaciensstrain EHA105 . Embryonic axes and cotyledonary nodes from germinated seeds of pigeon pea were used for transformation . The presence of the transgene in transgenic plants was confirmed by Southern blots, and the specific transcription of the marker gene in the plants was demonstrated by reverse transcription-polymerase chain reaction . Integration of the H gene into the pigeon pea genome was confirmed by Southern hybridization . The expression of the H protein in the transgenic lines was confirmed by Western blot analysis using a polyclonal monospecific antibody to the H protein . The highest level of expression of the hemagglutinin protein in leaves of pigeon pea was 0.49% of the total soluble protein . The transgenic plants were fertile and the transgene expressed in the progeny. Appl Environ Microbiol, 2003 Jun, 69(6), 3288 - 98 Assessment of bioavailability of soil-sorbed atrazine; Park JH et al.; Bioavailability of pesticides sorbed to soils is an important determinant of their environmental fate and impact . Mineralization of sorbed atrazine was studied in soil and clay slurries, and a desorption-biodegradation-mineralization (DBM) model was developed to quantitatively evaluate the bioavailability of sorbed atrazine . Three atrazine-degrading bacteria that utilized atrazine as a sole N source (Pseudomonas sp . strain ADP, Agrobacterium radiobacter strain J14a, and Ralstonia sp . strain M91-3) were used in the bioavailability assays . Assays involved establishing sorption equilibrium in sterile soil slurries, inoculating the system with organisms, and measuring the CO(2) production over time . Sorption and desorption isotherm analyses were performed to evaluate distribution coefficients and desorption parameters, which consisted of three desorption site fractions and desorption rate coefficients . Atrazine sorption isotherms were linear for mineral and organic soils but displayed some nonlinearity for K-saturated montmorillonite . The desorption profiles were well described by the three-site desorption model . In many instances, the mineralization of atrazine was accurately predicted by the DBM model, which accounts for the extents and rates of sorption/desorption processes and assumes biodegradation of liquid-phase, but not sorbed, atrazine . However, for the Houghton muck soil, which manifested the highest sorbed atrazine concentrations, enhanced mineralization rates, i.e., greater than those expected on the basis of aqueous-phase atrazine concentration, were observed . Even the assumption of instantaneous desorption could not account for the elevated rates . A plausible explanation for enhanced bioavailability is that bacteria access the localized regions where atrazine is sorbed and that the concentrations found support higher mineralization rates than predicted on the basis of aqueous-phase concentrations . Characteristics of high sorbed-phase concentration, chemotaxis, and attachment of cells to soil particles seem to contribute to the bioavailability of soil-sorbed atrazine. Biosci Biotechnol Biochem, 2003 Apr, 67(4), 863 - 8 Production of an allelopathic polyacetylene in hairy root cultures of goldenrod (Solidago altissima L.); Inoguchi M et al.; Hairy roots of goldenrod (Solidago altissima L.) were induced by infecting axenic plants with Agrobacterium rhizogenes strain A4 . Growth and allelopathic polyacetylene (cis-dehydromatricaria ester, cis-DME) production of two independent hairy root clones were examined in several culture media and light regimes . cis-DME contents in hairy roots were at the same level as those in normal roots . cis-DME production in root cultures was several-fold lower than that of native plants and greatly repressed by light. J Biol Chem, 2003 Aug 8, 278(32), 29971 - 8 Epub 2003 Jun 03. Identification of organelles in bacteria similar to acidocalcisomes of unicellular eukaryotes; Seufferheld M et al.; Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds . In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes . Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane . X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium . Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration . Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A . tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles . Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane . The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide . Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining . The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport . The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged. Planta, 2003 Sep, 217(5), 726 - 35 Epub 2003 May 30. Induction of parthenocarpy in tomato via specific expression of the rolB gene in the ovary; Carmi N et al.; The molecular signals for the development of the ovary into fruit following ovule fertilization are not clear . However, in many species, including tomato ( Lycopersicon esculentum Mill.), auxins and auxin transport inhibitors can substitute for fertilization as activators of fruit set, suggesting that this plant hormone plays a key role in this process . In agreement, transgenes for auxin biosynthesis expressed under ovary- or ovule-specific promoters were shown earlier to enable parthenocarpic (i.e . seedless) fruit development . In the present study, we tested an alternative approach for the induction of parthenocarpy that is based on ovary-specific expression of the Agrobacterium rhizogenes-derived gene rolB . This gene was chosen because rolB transgenic plants manifest several syndromes characteristic of auxin treatment . Tomato plants transformed with a chimeric construct containing the rolB gene fused to the ovary- and young-fruit-specific promoter TPRP-F1 developed parthenocarpic fruits . Fruit size and morphology, including jelly fill in the locules of the seedless fruits, were comparable to th