Agrobacterium

Can J Microbiol, 2004 Mar, 50(3), 167 - 74
Isolation of a strain of Agrobacterium tumefaciens (Rhizobium radiobacter) utilizing methylene urea (ureaformaldehyde) as nitrogen source; Koivunen ME et al.; Methylene ureas (MU) are slow-release nitrogen fertilizers degraded in soil by microbial enzymatic activity . Improved utilization of MU in agricultural production requires more knowledge about the organisms and enzymes responsible for its degradation . A Gram-negative, MU-degrading organism was isolated from a soil in Sacramento Valley, California . The bacterium was identified as Agrobacterium tumefaciens (recently also known as Rhizobium radiobacter) using both genotypic and phenotypic characterization . The pathogenic nature of the organism was confirmed by a bioassay on carrot disks . The MU-hydrolyzing enzyme (MUase) was intracellular and was induced by using MU as a sole source of nitrogen . The bacterial growth was optimized in NH4Cl, urea, or peptone, whereas the production and specific activity of MUase were maximized with either NH4Cl or urea as a nitrogen source . The result has a practical significance, demonstrating a potential to select for this plant pathogen in soils fertilized with MU.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 6852 - 7 Epub 2004 Apr 21.
In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium; Marillonnet S et al.; We have developed an efficient, versatile, and user-friendly viral engineering and expression system that is based on in planta assembly of functional viral vectors from separate pro-vector modules . With this new system, instead of supplying a plant cell with a complete viral vector as a mature viral particle, an RNA or a linear DNA molecule, we use agrobacteria to deliver various modules that are assembled inside the cell with the help of a site-specific recombinase . The resulting DNA is transcribed, and undesired elements such as recombination sites are spliced out, generating a fully functional RNA replicon . The proposed protocol allows us, by simply treating a plant with a mixture of two or more agrobacteria carrying specific prefabricated modules, to rapidly and inexpensively assemble and test multiple vector/gene combinations, without the need to perform the various engineering steps normally required with alternative protocols . The process described here is very fast (expression requires 3-4 days); it provides very high protein yield (up to 80% of total soluble protein); more than before, it is carried out using in vivo manipulations; it is based on prefabricated genetic modules that can be developed/upgraded independently; and it is inherently scalable.

Planta Med, 2004 Apr, 70(4), 347 - 52
Studies on the effects of fpf1 gene on Artemisia annua flowering time and on the linkage between flowering and artemisinin biosynthesis; Wang H et al.; The flowering promoting factor1 ( fpf1) from Arabidopsis thaliana was transferred into Artemisia annua L . via Agrobacterium tumefaciens . The fpf1 gene was firstly inserted in the binary vector pBI121 under the control of CaMV 35S promoter to construct the plant expression vector pBIfpf1, then leaf explants of A . annua were infected with A . tumefaciens LBA4404 containing pBIfpf1, and induced shoots . Transgenic plants were obtained through the selection with kanamycin . PCR, PCR-Southern and Southern blot analyses confirmed that the foreign fpf1 gene had been integrated into the A . annua genome . RT-PCR and RT-PCR-Southern analyses suggested that the foreign fpf1 gene had expressed at the transcriptional level . Under short-day conditions, the flowering time of fpf1 transgenic plants was about 20 days earlier than the non-transformed plants; however, no significant differences were detected in artemisinin content between the flowering transgenic plants and the non-flowering non-transgenic plants . These results showed that flowering is not a necessary factor for increasing the artemisinin content, furthermore, there may be no direct linkage between flowering and artemisinin biosynthesis.

Plant Biol (Stuttg), 2004 Jan-Feb, 6(1), 100 - 3
Vectors for RNAi technology in poplar; Meyer S et al.; The potential of double-stranded RNA interference (RNAi) technology was studied for down-regulation of gene expression in poplar . A set of vectors was constructed generating RNAs capable of duplex formation of sequences specific for the beta-glucuronidase (GUS) reporter gene system . These gene cassettes are driven by the CaMV-35S promoter . To address the question of gene silencing, we tested the functionality of these vectors, both in transient assays by transforming protoplasts with the RNAi constructs, and in stably transformed GUSexpressing poplar plants . Agrobacterium-mediated transformation of those GUS-expressing plants with a GUS-specific RNAi construct showed a strong down-regulation of the reporter gene . From these results we conclude that RNAi is also functional in poplar.

Plant Biol (Stuttg), 2004 Jan-Feb, 6(1), 65 - 73
Fluorescent proteins in poplar: a useful tool to study promoter function and protein localization; Nowak K et al.; The jellyfish (Aequorea victoria) green fluorescent protein (GFP) and its variants (CFP {cyan} and YFP {yellow}) were successfully used as a vital marker system for the transformation of hybrid poplar (Populus tremula x P . alba) . Our results show that, in this woody plant, fluorescent proteins can be expressed: (i) transiently in protoplasts after PEG-mediated transformation, as well as in leaf cells after particle bombardment, and (ii) stably in callus cells and plants after Agrobacterium-mediated transformation . For these studies, we constructed vectors permitting easy recloning of any promoter fragments of interest . Confocal laser scanning microscopy was used both for visualization and differentiation between the different colours of the GFP variants and autofluorescence of chlorophyll and lignified xylem vessels . Peroxisomes were chosen as target organelles for GFP translocation via the peroxisomal targeting sequence PTS1 because this allowed us to concentrate the fluorochrome in the small volume of a few peroxisomes, giving a bright fluorescence over background noise.

Plant Biol (Stuttg), 2004 Jan-Feb, 6(1), 5 - 11
T-DNA and transposon tagging in aspen; Fladung M et al.; We have investigated the somatic activity of the maize Activator (Ac) element in haploid and diploid aspen with the objective of developing an efficient transposon-based system for gene isolation in the model tree species Populus . It was shown that Ac is reinserted, frequently into or near coding regions in aspen, and therefore can be used for gene tagging studies . A number of phenotypic variants were also found following transformation of constructs harbouring the rolC gene . Comparative analyses of T-DNA flanking regions of variants and wild type lines indicate that T-DNA insertion has occurred in or near coding regions . However, the frequency of T-DNA insertion into genes is about one half of the frequency of Ac insertion hitting coding sequences . The results obtained give a proof-of-concept for transposon tagging in a tree system . Given the long generation cycles in tree species, gene tagging strategies are practical only to obtain dominant gain-of-function mutants that do not require selfing or test crossing . In order to obtain recessive loss-of-function mutants, we have regenerated haploid lines from immature pollen . These lines were successfully transformed with a construct containing the rolC transgene from Agrobacterium rhizogenes and Ac element from maize . The results indicate that Ac is also active in haploid aspen and hence can be used in general for gene tagging in trees.

Theor Appl Genet, 2004 Aug, 109(3), 534 - 42 Epub 2004 Apr 14.
Analysis of T-DNA- Xa21 loci and bacterial blight resistance effects of the transgene Xa21 in transgenic rice; Zhai W et al.; The genetic loci and phenotypic effects of the transgene Xa21, a bacterial blight (BB) resistance gene cloned from rice, were investigated in transgenic rice produced through an Agrobacterium-mediated transformation system . The flanking sequences of integrated T-DNAs were isolated from Xa21 transgenic rice lines using thermal asymmetric interlaced PCR . Based on the analysis of 24 T-DNA- Xa21 flanking sequences, T-DNA loci in rice could be classified into three types: the typical T-DNA integration with the definite left and right borders, the T-DNA integration linked with the adjacent vector backbone sequences and the T-DNA integration involved in a complicated recombination in the flanking sequences . The T-DNA integration in rice was similar to that in dicotyledonous genomes but was significantly different from the integration produced through direct DNA transformation approaches . All three types of integrated transgene Xa21 could be stably inherited and expressed the BB resistance through derived generations in their respective transgenic lines . The flanking sequences of the typical T-DNA integration consisted of actual rice genomic DNA and could be used as probes to locate the transgene on the rice genetic map . A total of 15 different rice T-DNA flanking sequences were identified . They displayed restriction fragment length polymorphisms (RFLPs) between two rice varieties, ZYQ8 and JX17, and were mapped on rice chromosomes 1, 3, 4, 5, 7, 9, 10, 11 and 12, respectively, by using a double haploid population derived from a cross between ZYQ8 and JX17 . The blast search and homology comparison of the rice T-DNA flanking sequences with the rice chromosome-anchored sequence database confirmed the RFLP mapping results . On the basis of genetic mapping of the T-DNA- Xa21 loci, the BB resistance effects of the transgene Xa21 at different chromosome locations were investigated using homozygous transgenic lines with only one copy of the transgene . Among the transgenic lines, no obvious position effects of the transgene Xa21 were observed . In addition, the BB resistance levels of the Xa21 transgenic plants with different transgene copy numbers and on different genetic backgrounds were also investigated . It was observed that genetic background (or genome) effects were more obvious than dosage effects and position effects on the BB resistance level of the transgenic plants .

Plant J, 2004 May, 38(3), 512 - 25
Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation; Cecchetti V et al.; The effect of auxin on stamen and pistil development in tobacco flowers was investigated by means of the localized expression of rolB (root loci B), an Agrobacterium oncogene that increases auxin sensitivity in a cell-autonomous fashion . When rolB is driven by the promoter of the meiosis-specific Arabidopsis gene DMC1 (disrupted meiotic cDNA 1), expression occurs earlier in male than in female developing organs, resulting in a delay in anther dehiscence with respect to normal timing of pistil development . As a consequence of this developmental uncoupling, self-pollination is prevented in pDMC1:rolB plants . Histological analysis of pDMC1:GFP plants indicates that in tobacco, this promoter is active not only in meiocytes but also in somatic tissues of the anther . In contrast, simultaneous expression of rolB in anther and pistil somatic tissues, achieved by expressing a construct containing rolB under the control of the promoter of the petunia gene FBP7 (floral binding protein 7), results in a concomitant delay of both anther dehiscence and pistil development without affecting self-pollination of the plants . Analysis of plants harboring the pFBP7:GUS construct shows that in tobacco, this promoter is active not only in the ovules, as described for petunia, but also in pistil and anther somatic tissues involved in the dehiscence program . The delay in anther dehiscence and pistil development could be phenocopied by exogenous application of auxin . Jasmonic acid (JA) could not rescue the delay in anther dehiscence . These results suggest that auxin plays a key role in the timing of anther dehiscence, the dehiscence program is controlled by the somatic tissues of the anther, and auxin also regulates pistil development.

Curr Opin Biotechnol, 2004 Apr, 15(2), 132 - 8
A tale of two integrations, transgene and T-DNA: gene targeting by homologous recombination in rice; Iida S et al.; The first successful and reproducible gene targeting by homologous recombination, without the concomitant occurrence of ectopic events, has been reported . This will be a powerful approach for the characterization of gene function in rice, an important crop and a model for other cereal species . Models have been proposed to explain gene replacement by homologous recombination, including a possible model for Agrobacterium-mediated gene targeting using a strong positive-negative selection.

Curr Opin Biotechnol, 2004 Apr, 15(2), 126 - 31
Transgene integration in plants: poking or patching holes in promiscuous genomes?
Somers DA, Makarevitch I.
Transgene integration in plants transformed by either Agrobacterium or direct DNA delivery methods occurs through illegitimate recombination (IR) . The precise mechanism(s) for IR-mediated transgene integration and the role of host double-strand break repair enzymes remain unknown . A recent wealth of sequenced transgene loci and investigations aimed at genetically dissecting transgene integration mechanism(s) have provided new insights into the process.

Mol Ecol, 2004 May, 13(5), 981 - 95
Solanum nigrum: a model ecological expression system and its tools; Schmidt DD et al.; Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function . If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems that offer both molecular tools and interesting natural histories are needed . Here, we present Solanum nigrum L., a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system . To facilitate manipulative ecological studies with S . nigrum, we describe: (i) an Agrobacterium-based transformation system and illustrate its utility with an example of the antisense expression of RuBPCase, as verified by Southern gel blot analysis and real-time quantitative PCR; (ii) a 789-oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore-induced volatile organic compounds) defences; and (iv) growth and fitness-estimates for plants grown under field conditions . Using these tools, we demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release . Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field-grown plants . Hence, proteinase inhibitors and jasmonate-signalling are targets for manipulative studies.

Plant J, 2004 Apr, 38(2), 260 - 75
Nuclear localization and interaction of RolB with plant 14-3-3 proteins correlates with induction of adventitious roots by the oncogene rolB; Moriuchi H et al.; The rooting-locus gene B (rolB) on the T-DNA of the root-inducing (Ri) plasmid in Agrobacterium rhizogenes is responsible for the induction of transformed adventitious roots, although the root induction mechanism is unknown . We report here that the RolB protein of pRi1724 (1724RolB) is associated with Nicotianatabacum14-3-3-like protein omegaII (Nt14-3-3 omegaII) in tobacco bright yellow (BY)-2 cells . Nt14-3-3 omegaII directly interacts with 1724RolB protein . Green fluorescent protein (GFP)-fused 1724RolB is localized to the nucleus . GFP-fused mutant 1724RolB proteins having a deletion or amino acid substitution are unable to interact with Nt14-3-3 omegaII and also show impaired nuclear localization . Moreover, these 1724RolB mutants show decreased capacity for adventitious root induction . These results suggest that adventitious root induction by 1724RolB protein correlates with its interaction with Nt14-3-3 omegaII and the nuclear localization of 1724RolB protein.

Eukaryot Cell, 2004 Apr, 3(2), 420 - 9
Cryptococcus neoformans virulence gene discovery through insertional mutagenesis; Idnurm A et al.; Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes . Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance . We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO) . The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized . For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress . Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested . This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis . One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel . Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor . The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology . Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Mikrobiologiia, 2004 Jan-Feb, 73(1), 118 - 25
{Employment of associative bacteria for the inoculation of barley plants cultivated in soil contaminated with lead and cadmium}; Belimov AA et al.; In laboratory experiments, the rhizobacteria Azospirillum lipoferum 137, Arthrobacter mysorens 7, Agrobacterium radiobacter 10, and Flavobacterium sp . L30 were found to have a relatively high resistance to the toxic heavy metals lead and cadmium (except that strain L30 was found to be sensitive to Cd) . When introduced by means of seed bacterization, the heavy metal-resistant strains actively colonized the rhizosphere of barley plants cultivated in uncontaminated and contaminated soils . In both pot and field experiments, seed bacterization improved the growth of barley plants and the uptake of nutrient elements from soil contaminated with Pb and Cd . The bacterization also prevented the accumulation of Pb and Cd in barley plants, thereby mitigating the toxic effect of these heavy metals on the plants.

J Exp Bot, 2004 May, 55(399), 983 - 92 Epub 2004 Apr 08.
RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula; Limpens E et al.; RNA interference (RNAi) is a powerful reverse genetic tool to study gene function . The data presented here show that Agrobacterium rhizogenes-mediated RNAi is a fast and effective tool to study genes involved in root biology . The Arabidopsis gene KOJAK, involved in root hair development, was efficiently knocked down . A . rhizogenes-mediated root transformation is a fast method to generate adventitious, genetically transformed roots . In order to select for co-transformed roots a binary vector was developed that enables selection based on DsRED1 expression, with the additional benefit that chimaeric roots can be discriminated . The identification of chimaeric roots provided the opportunity to examine the extent of systemic spread of the silencing signal in the composite plants of both Arabidopsis and Medicago truncatula . It is shown that RNA silencing does not spread systemically to non-co-transformed (lateral) roots and only inefficiently to the non-transgenic shoot . Furthermore, evidence is presented which shows that RNAi is cell autonomous in the root epidermis.

J Clin Microbiol, 2004 Apr, 42(4), 1420 - 7
Study of genotypes and virB4 secretion gene of Bartonella henselae strains from patients with clinically defined cat scratch disease; Woestyn S et al.; Bartonella henselae is the causative agent of cat scratch disease (CSD), which usually presents as a self-limiting lymphadenopathy . Occasionally, the bacteria will spread and be responsible for tissue and visceral involvement . Two B . henselae genotypes (genotypes I and II) have been described to be responsible for uncomplicated CSD on the basis of 16S rRNA sequence analysis . A type IV secretion system (T4SS) similar to the virulence-associated VirB system of Agrobacterium tumefaciens was recently identified in the B . henselae Houston-1 genotype I strain . We studied the correlations of the B . henselae genotypes with the clinical presentations and with the presence of T4SS . Isolates originated from CSD patients whose lymph nodes were prospectively analyzed . B . henselae genotype I was identified in 13 of 42 patients (30%) . Among these, two teenage twins presented with hepatosplenic CSD and one immunocompetent adult presented with osteomyelitis . Genotype II was detected in 28 of 42 patients (67%), all of whom presented with uncomplicated CSD . The last patient was infected with both genotypes . T4SS was studied by PCR amplification of the virB4 gene . Amplification of virB4 codons 146 to 256, 273 to 357, and 480 to 537 enabled us to detect 66, 90, and 100% of the B . henselae isolates, respectively . Sequence analysis revealed sequence variations that correlated with genotype distribution . Our studies suggest that B . henselae genotype I strains harbor virB4 genes that are different from those harbored by genotype II strains and that genotype I strains might be more pathogenic.

Mol Genet Genomics, 2004 May, 271(4), 499 - 510 Epub 2004 Apr 06.
Development of a system for integrative and stable transformation of the zygomycete Rhizopus oryzae by Agrobacterium-mediated DNA transfer; Michielse CB et al.; Two transformation systems, based on the use of CaCl(2)/PEG and Agrobacterium tumefaciens, respectively, were developed for the zygomycete Rhizopus oryzae . Irrespective of the selection marker used, a pyr4 marker derived from R . niveus or a dominant amdS(+) marker from Aspergillus nidulans, and irrespective of the configuration of the transforming DNA (linear or circular), the transformants obtained with the CaCl(2)/PEG transformation method were found to carry multiple copies of tandemly linked vector molecules, which failed to integrate into the genomic DNA . Furthermore, these transformants displayed low mitotic stability . In contrast, transformants obtained by Agrobacterium-mediated transformation were mitotically stable, even under non-selective conditions . Detailed analysis of these transformants revealed that the transforming DNA had integrated into the genome of R . oryzae at a single locus in independently obtained transformants . In addition, truncation of the transforming DNA was observed, resulting in the integration of the R . niveus pyr4 marker gene, but not the second gene located on the transferred DNA . Modification of the transforming DNA, resulting in partial resistance to restriction enzyme digestion, was observed in transformants obtained with the CaCl(2)/PEG transformation method, suggesting that a specific genome defence mechanism may exist in R . oryzae . It is likely that the unique mechanism used by A . tumefaciens to deliver its transferred DNA to its hosts facilitates bypass of the host defence mechanisms, thus allowing the DNA to integrate into the chromosomal genome.

Folia Microbiol (Praha), 2003, 48(6), 794 - 8
Production of N-acylhomoserine lactone signal molecules by gram-negative soil-borne and plant-associated bacteria; Veselova M et al.; Quorum-sensing control mediated by N-acylhomoserine lactone (AHL) signal molecules has been established as a key feature in the regulation of various metabolic traits in many bacteria . Approximately 300 strains representing 6 genera and 18 species of soil-borne and plant-associated Gram-negative bacteria isolated in various regions of the former USSR using two reporter systems were screened for AHL production . The production was observed in 17.5% of the screened bacterial strains . Positive response was detected in all of the 14 tested strains of Erwinia herbicola, in 41 of the 239 strains of Pseudomonas species; in all 5 strains of Xanthomonas ampelina, X . campestris pv . malvacearum, pv . translucens, pv . vesicatoria and in one strain of Pantoea stewartii . AHL assay of 41 strains of X . maltophilia (syn . Stenotrophomonas maltophilia) isolated from soils with Chromobacterium violaceum reporter has revealed no strains synthesizing these signal molecules; 26 strains analyzed with Agrobacterium tumefaciens reporter showed the same result.

Mol Cells, 2004 Feb 29, 17(1), 81 - 5
Pathogen resistance of transgenic rice plants expressing mitogen-activated protein kinase 1, MK1, from Capsicum annuum; Lee DE et al.; Mitogen-activated protein kinase 1 (MK1) from Capsicum annuum was previously cloned and characterized . MK1 is highly conserved in plants and its amino acid sequence is 92% identical to wound-inducible protein kinase (WIPK) from tobacco . In C . annuum, MK1 is transcriptionally activated by wounding . In the present work, the MK1 gene was introduced into the rice genome by Agrobacterium-mediated transformation . Seven independent transgenic rice plants were isolated and characterized . All seven lines had a single-copy insertion of the MK1 transgene . MK1 mRNA and protein were detected in the transgenic rice, but not in wild type rice . In unwounded transgenic rice plants, the level of jasmonic acid was 3-fold higher than in the wild type, whereas in wounded leaves the level of jasmonic acid was the same in transgenic and wild type plants . Expression of the wound-inducible pathogenesis-related gene PR1a was also higher in transgenic than in wild type plants . Either overexpression of PR1a or increased PR1b and PR10 expression in transgenic rice plants may confer increased resistance to rice blast.

J Biol Chem, 2004 Jun 11, 279(24), 25359 - 63 Epub 2004 Mar 30.
Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA; Abu-Arish A et al.; Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer . A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2 . A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity . An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex . Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods . We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn . The inner face of the protein units contains a continuous wall and an inward protruding shelf . These structures appear to accommodate the DNA binding . Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors . Coexisting with the helices, we also found VirE2 tetrameric ring structures . A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.

Int Arch Allergy Immunol, 2004 May, 134(1), 1 - 9 Epub 2004 Mar 25.
A model system to study the environment-dependent expression of the Bet v 1a gene encoding the major birch pollen allergen; Tashpulatov AS et al.; BACKGROUND: The major birch pollen allergen Bet v 1 (or Bet v 1a) is one of the main causes of seasonal type I allergies . Various environmental factors such as light, temperature and air pollution may influence the activity of the Bet v 1a gene . The creation of a model system to evaluate the role of environmental factors affecting the Bet v 1a gene expression would be highly desirable . We suggest the use of transgenic tobacco plants carrying a Bet v 1a promoter-reporter gene fusion as such a system . METHODS: The promoter of the Bet v 1a gene was isolated with the use of the Universal Genome Walker kit (BD Biosciences Clontech, USA) . Web Software was used to search for putative cis-regulatory elements within the promoter . Transgenic tobacco plants harboring the promoter-beta-glucuronidase (GUS) reporter gene fusion were obtained via Agrobacterium tumefaciens-mediated transformation . Promoter activity was examined with histochemical and quantitative assays . RESULTS: Structural analysis predicted elements responsible for pollen-specific, light-, stress- and hormone-mediated induction within the Bet v 1a promoter . The evaluation of GUS activity in transgenic tobacco plants showed that the Bet v 1a promoter is pollen-specific . Moreover, the Bet v 1a promoter is considered to be the strongest isolated pollen-specific promoter reported to date . It was shown that temperature and abscisic acid positively regulate the activity of the Bet v 1a promoter during pollen development, providing evidence for environment-dependent regulation of the Bet v 1a gene . CONCLUSIONS: A model system to study the effect of environmental factors on the expression of the Bet v 1a gene encoding the major birch allergen in pollen was generated . Additionally, we suggest that this system could be used to search for factors that inhibit the activity of the gene in pollen in order to reduce the potential allergenicity of birch trees .

Plant Physiol, 2004 Apr, 134(4), 1742 - 51 Epub 2004 Mar 29.
Gene and enhancer trap tagging of vascular-expressed genes in poplar trees; Groover A et al.; We report a gene discovery system for poplar trees based on gene and enhancer traps . Gene and enhancer trap vectors carrying the beta-glucuronidase (GUS) reporter gene were inserted into the poplar genome via Agrobacterium tumefaciens transformation, where they reveal the expression pattern of genes at or near the insertion sites . Because GUS expression phenotypes are dominant and are scored in primary transformants, this system does not require rounds of sexual recombination, a typical barrier to developmental genetic studies in trees . Gene and enhancer trap lines defining genes expressed during primary and secondary vascular development were identified and characterized . Collectively, the vascular gene expression patterns revealed that approximately 40% of genes expressed in leaves were expressed exclusively in the veins, indicating that a large set of genes is required for vascular development and function . Also, significant overlap was found between the sets of genes responsible for development and function of secondary vascular tissues of stems and primary vascular tissues in other organs of the plant, likely reflecting the common evolutionary origin of these tissues . Chromosomal DNA flanking insertion sites was amplified by thermal asymmetric interlaced PCR and sequenced and used to identify insertion sites by reference to the nascent Populus trichocarpa genome sequence . Extension of the system was demonstrated through isolation of full-length cDNAs for five genes of interest, including a new class of vascular-expressed gene tagged by enhancer trap line cET-1-pop1-145 . Poplar gene and enhancer traps provide a new resource that allows plant biologists to directly reference the poplar genome sequence and identify novel genes of interest in forest biology.

Fungal Genet Biol, 2004 May, 41(5), 571 - 8
Role of bacterial virulence proteins in Agrobacterium-mediated transformation of Aspergillus awamori; Michielse CB et al.; The Agrobacterium-mediated transformation of Aspergillus awamori was optimized using defined co-cultivation conditions, which resulted in a reproducible and efficient transformation system . Optimal co-cultivation conditions were used to study the role of Agrobacterium tumefaciens virulence proteins in T-DNA transfer . This study revealed that inactivation of either of the regulatory proteins (VirA, VirG), any of the transport pore proteins (VirB), proteins involved in generation of the T-strand (VirD, VirC) or T-strand protection and targeting (VirE2) abolishes or severely reduces the formation of transformants . The results indicate that the Agrobacterium-mediated transformation of A . awamori requires an intact T-DNA machinery for efficient transformation; however, the plant host range factors, like VirE3, VirH, and VirF, are not important.

Proteomics, 2004 Apr, 4(4), 1061 - 73
Two-dimensional reference map of Agrobacterium tumefaciens proteins; Rosen R et al.; Proteomics based on two-dimensional (2-D) gel electrophoresis of proteins followed by spot identification with mass spectrometry is a commonly used method for physiological studies . Physiological proteomics requires 2-D reference maps, on which most of the main proteins are identified . We present a reference map for the bacterial plant pathogen Agrobacterium tumefaciens proteins, which contains more than 300 entries with an isoelectric point (pI) between 4 and 7 . The quantitative study of the proteins in the analytical window of the master gel demonstrated unique features, in comparison with other bacteria . In addition, a theoretical analysis of several protein parameters was performed and compared with the experimental results . A comparison of the theoretical molecular weight (MW) of the proteins and their theoretical pI with their vertical and horizontal migration distances, respectively, pointed out the existence of several proteins that strongly diverted from the graph trend-line . These proteins were clearly subjected to post-translational modifications, which changed their pI and/or MW . Additional support for post-translational modifications comes from the identification of multiple spots of the same gene products . Post-translational modifications appear to be more common than expected, at least for soluble proteins, as more than 10% of the proteins were associated with multiple spots.

Plant Cell Rep, 2004 Jul, 22(12), 925 - 30 Epub 2004 Mar 27.
Expression of the hepatitis B surface S and preS2 antigens in tubers of Solanum tuberosum; Joung YH et al.; In an attempt to develop an edible vaccine, we transformed a recombinant hepatitis B virus (HBV) gene encoding the middle protein of HBV that contains the surface S and preS2 antigen into potato by Agrobacterium-mediated transformation . The HBV gene was under control of either the CaMV 35S promoter, the double 35S promoter with the AlMV 5' non-translated leader sequence, or the tuber-specific patatin promoter . HBV mRNA levels were higher with the 35S promoter than with the double 35S and patatin promoters; however, the levels of the S and preS2 antigen in the transformed tubers were higher with the patatin promoter than with the CaMV 35S and double promoters . The levels of preS2 antigen produced are the highest reported to date . Transgenic potato tubers were fed to mice, and the mice showed an immune response against the HBV S antigen.

Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 5164 - 9 Epub 2004 Mar 26.
Expression of plant protein phosphatase 2C interferes with nuclear import of the Agrobacterium T-complex protein VirD2; Tao Y et al.; Agrobacterium tumefaciens transfers DNA to plant cells as a single-stranded DNA molecule (the T-strand) covalently linked to VirD2 protein . VirD2 contains nuclear localization signal sequences that presumably help direct the T-strand to the plant nucleus . We identified a tomato cDNA clone, DIG3, that encodes a protein that interacts with the C-terminal region of VirD2 . DIG3 encodes an enzymatically active type 2C serine/threonine protein phosphatase . Overexpression of DIG3 in tobacco BY-2 protoplasts inhibited nuclear import of a beta-glucuronidase-VirD2 nuclear localization signal fusion protein . Thus, DIG3 may be involved in nuclear import of the VirD2 protein and, consequently, the VirD2/transferred DNA complex.

Curr Genet, 2004 Jun, 45(6), 399 - 403 Epub 2004 Mar 26.
The Aspergillus nidulans amdS gene as a marker for the identification of multicopy T-DNA integration events in Agrobacterium-mediated transformation of Aspergillus awamori; Michielse CB et al.; The Aspergillus nidulans amdS selection marker was used for the identification of multicopy T-DNA insertions in Agrobacterium-mediated transformation of Asp . awamori . The selection of transformants on agar plates containing acetamide as sole nitrogen source and hygromycin resulted in a six-fold decrease in the transformation frequency, compared with the transformation frequency obtained after hygromycin selection alone . However, it was found that 47% of the transformants obtained after hygromycin and acetamide double selection contained multiple T-DNA integrations . Furthermore, it was found that the multicopy transformants could easily be identified based on their growth rate on agar plates containing acetamide medium . Based on these data, it can be concluded that the amdS marker can also be used as a selection marker in Agrobacterium-mediated transformation of Asp . awamori and that it is a very useful marker to identify those transformants containing multiple T-DNA integrations.

J Biol Chem, 2004 Jun 4, 279(23), 24291 - 6 Epub 2004 Mar 24.
Crystal structure of the quorum-sensing protein TraM and its interaction with the transcriptional regulator TraR; Vannini A et al.; Transfer of the tumor-inducing plasmid in Agrobacterium tumefaciens is controlled by a quorum-sensing system whose main components are the transcriptional regulator TraR and its autoinducer . This system allows bacteria to synchronize infection of the host plant when a "quorum" of cells has been reached . TraM is an A . tumefaciens protein involved in the regulation of this system because it binds to TraR and prevents it from binding DNA . As a first step to understanding the molecular basis for the regulation of TraR by TraM, we have determined the crystal structure of TraM at 1.65 A resolution . This protein is packed as a dimer, with each monomer consisting mainly of two antiparallel alpha helices . Monomers are tightly associated, with a large hydrophobic area buried upon dimerization . Secondly, we characterized the TraR-TraM complex in vitro . TraM (11.4 kDa, monomer molecular mass) binds tightly TraR (27 kDa, monomer molecular mass) forming a stable oligomeric complex that likely accounts for two TraR and two TraM dimers.

J Nat Prod, 2004 Mar, 67(3), 348 - 51
Effect of phenolic glycosides on Agrobacterium tumefaciens virH gene induction and plant transformation; Joubert P et al.; O-Aryl-d-glucoside (4-7) and d-xyloside (8-10) derivatives were synthesized and tested on Agrobacterium virH gene induction and plant transformation . alpha- or beta-Glycosides enhanced vir activity at concentrations above 250 micromicro . The highest vir activity was observed with beta-glucoside derivative 4 at 10 mM . A marked difference between phenol glucoside derivative 4 and the corresponding free phenol on the growth of transformants was observed . The regenerated transgenic tissues, after transformation on medium containing acetosyringyl beta-glucoside 4, grew at twice the rate of those on medium containing only free acetosyringone (AS) . Compound 4 was less toxic for tobacco explants compared to the corresponding free phenol . However, the xyloside derivatives tested (8-10) were less effective for gene induction compared with corresponding free phenols.

J Plant Res, 2004 Jun, 117(3), 191 - 8 Epub 2004 Mar 23.
Partial purification of an enzyme hydrolyzing indole-3-acetamide from rice cells; Arai Y et al.; The activity of indole-3-acetamide (IAM) hydrolase from rice cells was enriched ca . 628-fold by gel filtration and anion exchange column chromatography . The molecular masses of the IAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively . The enzyme exhibited maximum activity at pH 6.0-6.5 . The enzyme was stable against heat treatments between 4 and 50 degrees C and works optimally at 52 degrees C . The activity remained constant at 4 degrees C for at least 143 days . The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to IAM and its homologue, 1-naphthalene-acetamide, but not indole-3-acetonitrile . Km values of the enzyme were 0.96 mM and 0.55 mM for IAM and Et-IAA, respectively . Although the molecular mass of the enzyme was very similar to that of IAM hydrolase of Agrobacterium tumefaciens involved in tumor formation, the biochemical properties of the enzyme including its high Km value were considerably different from those of the A . tumefaciens enzyme . Based on these enzyme properties, we will discuss whether the amidohydrolase is involved in auxin biosynthesis in rice cells.

Plant Cell Rep, 2004 Jul, 22(12), 910 - 8 Epub 2004 Mar 23.
High frequency Agrobacterium-mediated transformation and plant regeneration via direct shoot formation from leaf explants in Beta vulgaris and Beta maritima; Hisano H et al.; We have developed a new procedure for Agrobacterium-mediated transformation of plants in the genus Beta using shoot-base as the material for Agrobacterium infection . The frequency of regeneration from shoot bases was analyzed in seven accessions of sugarbeet ( Beta vulgaris) and two accessions of B . maritima to select materials suitable for obtaining transformed plants . The frequency of transformation of the chosen accessions using Agrobacterium strain LBA4404 and selection on 150-mg/l kanamycin was found to be higher than that in previously published methods . Genomic DNA analysis and beta-glucuronidase reporter assays showed that the transgene was inherited and expressed in subsequent generations . In our method, shoot bases are prepared by a simple procedure, and transformation does not involve the callus phase, thus minimizing the occurrence of somaclonal variations.

J Gen Virol, 2004 Apr, 85(Pt 4), 993 - 9
Human influenza virus NS1 protein enhances viral pathogenicity and acts as an RNA silencing suppressor in plants; Delgadillo MO et al.; RNA silencing has a well-established function as an antiviral defence mechanism in plants and insects . Using an Agrobacterium-mediated transient assay, we report here that NS1 protein from human influenza A virus suppresses RNA silencing in plants in a manner similar to P1/HC-Pro protein of Tobacco etch potyvirus, a well-characterized plant virus silencing suppressor . Moreover, we have shown that NS1 protein expression strongly enhances the symptoms of Potato virus X in three different plant hosts, suggesting that NS1 protein could be inhibiting defence mechanisms activated in the plant on infection . These data provide further evidence that an RNA silencing pathway could also be activated as a defence response in mammals.

Infect Immun, 2004 Apr, 72(4), 2263 - 71
Molecular cloning and characterization of cgt, the Brucella abortus cyclic beta-1,2-glucan transporter gene, and its role in virulence; Roset MS et al.; The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants . Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A . tumefaciens, and nodule occupancy in S . meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm . Nucleotide sequencing revealed that B . abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S . meliloti NdvA and A . tumefaciens ChvA, respectively . Additionally, B . abortus cgt, like S . meliloti ndvA and A . tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter . Characterization of Cgt was carried out by the construction of null mutants in B . abortus 2308 and S19 backgrounds . Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells . These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.

Virus Res, 2004 Mar, 100(1), 41 - 50
Pathogen-derived resistance in potato to Potato virus Y-aspects of stability and biosafety under field conditions; Schubert J et al.; Plants of three different potato cultivars/lines were transformed via Agrobacterium tumefaciens with a truncated NIb gene of a necrotic strain of Potato virus Y (PVY(N)) which had been C-terminally fused to enhanced blue-fluorescing protein . Resistance of the resulting transgenic clones was evaluated under glass house conditions using an NTN-strain of PVY . Four clones with the highest levels of resistance were chosen for further experiments . Their type of resistance was either recovery or extreme resistance . These clones and their resistance types were also characterised at the molecular level . Mechanisms other than post-transcriptional gene silencing seemed to be involved in the resistance which was not dependent on sequence homology between transgene and challenging virus . Stability of resistance was tested under field conditions . The plants usually became infected with PVY . Tubers of the clone with extreme resistance did not recover from infection whereas those from clones with the recovery type did . No influence of transgenic potatoes was apparent on aphid population numbers in test plots . Recombination events could not be detected at the RNA level between transgene and challenging virus.

Biochemistry, 2004 Mar 30, 43(12), 3659 - 69
The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1; Lamparter T et al.; Phytochromes are widely distributed biliprotein photoreceptors . Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction . Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain . For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20 . This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven . We therefore performed mass spectrometry studies on proteolytic holopeptide fragments . For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag . Following proteolysis, the chromophore co-purified with a ca . 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus . Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site . We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis . The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome . We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.

Plant Cell Rep, 2004 Feb, 22(7), 478 - 82 Epub 2003 Sep 30.
Refined glufosinate selection in Agrobacterium-mediated transformation of soybean {Glycine max (L.) Merrill}; Zeng P et al.; Modern genetic analysis and manipulation of soybean ( Glycine max) depend heavily on an efficient and dependable transformation process, especially in public genotypes from which expressed sequence tag (EST), bacterial artificial chromosome and microarray data have been derived . Williams 82 is the subject of EST and functional genomics analyses . However, it has not previously been transformed successfully using either somatic embryogenesis-based or cotyledonary-node transformation methods, the two predominant soybean transformation systems . An advance has recently been made in using antioxidants to enhance Agrobacterium infection of soybean . Nonetheless, an undesirable effect of using these antioxidants is the compromised recovery of transgenic soybean when combined with the use of the herbicide glufosinate as a selective agent . Therefore, we optimized both Agrobacterium infection and glufosinate selection in the presence of L-cysteine for Williams 82 . We have recovered transgenic lines of this genotype with an enhanced transformation efficiency using this herbicide selection system.

FEMS Microbiol Lett, 2004 Mar 19, 232(2), 217 - 23
The role of a bifunctional catalase-peroxidase KatA in protection of Agrobacterium tumefaciens from menadione toxicity; Prapagdee B et al.; Agrobacterium tumefaciens is an aerobic plant pathogenic bacterium that is exposed to reactive oxygen species produced either as by-products of aerobic metabolism or by the defense systems of host plants . The physiological function of the bifunctional catalase-peroxidase (KatA) in the protection of A . tumefaciens from reactive oxygen species other than H(2)O(2) was evaluated in the katA mutant (PB102) . Unexpectedly, PB102 was highly sensitive to the superoxide generator menadione . The expression of katA from a plasmid vector complemented the menadione-hypersensitive phenotype . A . tumefaciens possesses an additional catalase gene, a monofunctional catalase encoded by catE . Neither inactivation nor high-level expression of the catE gene altered the menadione resistance level . Moreover, heterologous expression of the catalase-peroxidase-encoding gene katG from Burkholderia pseudomallei, but not the monofunctional catalase gene katE from Xanthomonas campestris could restore normal levels of menadione resistance to PB102 . A recent observation suggests that the menadione resistance phenotype involves increased activities of organic peroxide-metabolizing enzymes . Heterologous expression of X . campestris alkyl hydroperoxide reductase from a plasmid vector failed to complement the menadione-sensitive phenotype of PB102 . The level of menadione resistance shows a direct correlation with the level of peroxidase activity of KatA . This is a novel role for KatA and suggests that resistance to menadione toxicity is mediated by a new, and as yet unknown, mechanism in A . tumefaciens.

J Bacteriol, 2004 Apr, 186(7), 2038 - 45
Agrobacterium-mediated transformation of Aspergillus awamori in the absence of full-length VirD2, VirC2, or VirE2 leads to insertion of aberrant T-DNA structures; Michielse CB et al.; Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori . The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses . The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends . From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively . Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.

Biochimie, 2004 Feb, 86(2), 77 - 81
Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58; Martinez-Rodriguez S et al.; A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 (AthyuA2) has been cloned and expressed in Escherichia coli BL21 . The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27000 Da in SDS-gel electrophoresis . Size exclusion chromatography analysis determined a molecular mass of approximately 100000 Da, suggesting that the native enzyme is a tetramer . The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 degrees C, respectively, with L-5-ethylhydantoin as substrate . Enzyme activity was strongly inhibited by Cu(2+) and Hg(2+) . No effect on enzyme activity was detected with any other divalent cations, EDTA or DTT, suggesting that it is not a metalloenzyme . Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.

Annu Rev Phytopathol, 1997, 35, 45 - 66
The impact of Ti-plasmid-derived gene vectors on the study of the mechanism of action of phytohormones; Walden R et al.; The molecular basis of tumor formation on dicotyledonous plants by Agrobacterium relies on the transfer to the plant cell of a unique segment of bacterial DNA, the T-DNA . The T-DNA contains genes that are active in the plant cell and encode hormone biosynthetic enzymes, or proteins that deregulate the cell's response to phytohormones . Study of this process has yielded not only knowledge of how alterations in phytohormone homeostasis can affect plant cell growth, but also has provided the essential tools to study phytohormone signaling in transgenic plants . Furthermore, T-DNA insertion into the plant genome forms the basis of gene tagging, a versatile method for isolating genes involved in phytohormone signal transduction and action.

Annu Rev Plant Physiol Plant Mol Biol, 2000 Jun, 51, 223 - 256
AGROBACTERIUM AND PLANT GENES INVOLVED IN T-DNA TRANSFER AND INTEGRATION; Gelvin SB; The phytopathogenic bacterium Agrobacterium tumefaciens genetically transforms plants by transferring a portion of the resident Ti-plasmid, the T-DNA, to the plant . Accompanying the T-DNA into the plant cell is a number of virulence (Vir) proteins . These proteins may aid in T-DNA transfer, nuclear targeting, and integration into the plant genome . Other virulence proteins on the bacterial surface form a pilus through which the T-DNA and the transferred proteins may translocate . Although the roles of these virulence proteins within the bacterium are relatively well understood, less is known about their roles in the plant cell . In addition, the role of plant-encoded proteins in the transformation process is virtually unknown . In this article, I review what is currently known about the functions of virulence and plant proteins in several aspects of the Agrobacterium transformation process.

Electrophoresis, 2004 Mar, 25(6), 922 - 30
Rapid analysis of genetically modified organisms by in-house developed capillary electrophoresis chip and laser-induced fluorescence system; Obeid PJ et al.; A microfabricated, inexpensive, reusable glass capillary electrophoresis chip and a laser-induced fluorescence system were developed in-house for the rapid DNA-based analysis of genetically modified organisms (GMOs) . The 35S promoter sequence of cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens were both detected since they are present in most genetically modified organisms . The detection of genetically modified soybean in the presence of unaltered soybean was chosen as a model . Lectin, a plant-specific gene, was also detected for confirmation of the integrity of extracted DNA . The chip was composed of two glass plates, each 25 x 76 mm, thermally bonded together to form a closed structure . Photomasks with cross-topology were prepared rapidly by using polymeric material instead of chrome plates . The widths of the injection and separation channels were 30 and 70 microm, respectively, the effective separation length 4.5 cm . The glass slide was etched to a depth of 30 microm for both the injection and separation channel . The cost of the chip was less than 1 $ and required 2 days for photomask preparation and microfabrication . The separation and detection of polymerase chain reaction-amplified NOS, 35S, and lectin sequences (180, 195, and 181 bp, respectively) was completed in less than 60 s . As low as 0.1% GMO content was detectable by the proposed system after 35 and 40 amplification cycles for 35S and NOS, respectively, using 25 ng of extracted DNA as starting material . This corresponds to only 20 genome copies of genetically modified soybean.

Biocell, 2003 Dec, 27(3), 311 - 8
Agrobacterium rhizogenes vs auxinic induction for in vitro rhizogenesis of Prosopis chilensis and Nothofagus alpina; Caro LA et al.; The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep . et Endl . Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media . Microshoots of P . chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP) . After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA . For N . alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP . After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A . rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA . Rooting in P . chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots . In N . alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.

Mol Plant Microbe Interact, 2004 Mar, 17(3), 272 - 82
A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions; O'Connell R et al.; The ability of a Colletotrichum sp., originally isolated from Brassica campestris, to infect Arabidopsis thaliana was examined . Sequence analysis of the internal transcribed spacer (ITS)1, 5.8S RNA gene and ITS2 regions of ribosomal (r)DNA showed the pathogen to be Colletotrichum destructivum . The host range was broad, including many cruciferous plants and some legumes . At 25 degrees C, all A . thaliana accessions tested were susceptible to the Brassica isolates of C . destructivum; however, at 15 degrees C, the accession Ws-2 showed a temperature-dependant resistance, in which single epidermal cells underwent a rapid hypersensitive response . Legume isolates of C . destructivum were unable to infect A . thaliana and induced deposition of callose papillae at sites of attempted penetration . In compatible interactions, C . destructivum showed a two-stage, hemibiotrophic infection process . The initial biotrophic phase was associated with large, intracellular primary hyphae and was confined to one epidermal cell; whereas, in the subsequent necrotrophic phase, narrow secondary hyphae extensively colonized the tissue and conidia were produced in acervuli . An efficient transformation system was established for C . destructivum, using Agrobacterium-mediated transfer of DNA . The ability to genetically manipulate both partners in the interaction is an important advantage, and the Arabidopsis-Colletotrichum pathosystem should provide a valuable new model for dissecting plant-fungal interactions.

J Bacteriol, 2004 Mar, 186(6), 1802 - 10
Characterization of glycerol trinitrate reductase (NerA) and the catalytic role of active-site residues; Marshall SJ et al.; Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli . Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement . Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca . 10(-7) M and with restoration of activity . The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics . A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site . The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN . The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form . The mutation Y183F produced active protein with the same k(cat) as that of wild-type enzyme but with altered K(m) values for GTN and NADH, indicating a role for this residue in substrate binding . Correlation of the ratio of K(m)(GTN) to K(m)(NAD(P)H), with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.

J Agric Food Chem, 2004 Mar 10, 52(5), 1375 - 84
The composition of grain and forage from glyphosate tolerant wheat MON 71800 is equivalent to that of conventional wheat (Triticum aestivum L.); Obert JC et al.; Glyphosate tolerant wheat MON 71800, simply referred to as MON 71800, contains a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) protein from Agrobacterium sp . strain CP4 (CP4 EPSPS) that has a reduced affinity for glyphosate as compared to the endogenous plant EPSPS enzyme . The purpose of this work was to evaluate the compositional equivalence of MON 71800 to its nontransgenic parent as well as to conventional wheat varieties . The compositional assessment evaluated the levels of proximates, amino acids, fatty acids, minerals, vitamins, secondary metabolites, and antinutrients in wheat forage and grain grown during two field seasons across a total of eight sites in the United States and Canada . These data demonstrated that with respect to these important nutritional components, the forage and grain from MON 71800 were equivalent to those of its nontransgenic parent and commercial wheat varieties . These data, together with the previously established safety of the CP4 EPSPS protein, support the conclusion that glyphosate tolerant wheat MON 71800 is as safe and nutritious as commercial wheat varieties.

J Invertebr Pathol, 2004 Jan, 85(1), 18 - 24
Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker; Fang W et al.; Beauveria bassiana has been investigated for use in the biological control of several insects in agricultural practice . To understand the molecular basis of virulence and host specificity and to improve the entomopathogenicity of B . bassiana, we have developed a simple, highly efficient and reliable Agrobacterium-mediated transformation method for B . bassiana using a phosphinothricin acetyltransferase (bar) gene as a selectable marker . Most transformants contained single copies of T-DNA and the T-DNA inserts were stably inherited after five generations . With this highly efficient transformation method for B . bassiana, we also obtained two putative T-DNA-tagged mutants that may have altered growth habits or virulence . Thus, the described protocol could provide a useful tool to manipulate the genetic make-up and to tag genes that may be important for virulence or growth and development of B . bassiana.

Plant Cell Rep, 2004 Jul, 22(12), 894 - 902 Epub 2004 Feb 25.
Transgenic Pinus radiata from Agrobacterium tumefaciens-mediated transformation of cotyledons; Grant JE et al.; A method for Agrobacterium tumefaciens-mediated transformation of Pinus radiata cotyledon explants was developed using commercially available open-pollinated seed . Pinus radiata is the most widely planted commercial conifer species in the Southern Hemisphere . Reports on transformation of this species have relied on particle bombardment of embryogenic callus derived from immature embryos . The main drawback to the method is the small number of genotypes that are amenable to transformation and regeneration . Since more than 80% of genotypes of radiata pine can be regenerated using cotyledons from mature seed, cotyledon explants were cocultivated with A . tumefaciens strain AGL1 containing a plasmid coding for the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (GUS) gene (uidA) . Transformed shoots were selected using either geneticin or kanamycin . Critical factors for successful transformation were survival of the cotyledons after cocultivation and selection parameters . Of the 105 putative transformants that were recovered from selection media, 70% were positive for integration of the nptII gene when analysed by PCR . GUS histochemical assay for uidA expression was unreliable because of reaction inhibition by unidentified compounds in the pine needles . Further, only 4 of the 26 independent transformants characterised by PCR and Southern analysis contained an intact copy of both genes . The remaining 22 transformants appeared to have a truncated or rearranged copy of the T-DNA . It is possible that the truncation/rearrangements are due to the Cauliflower mosaic virus (CaMV) 35S promoter . Analysis of the T-DNA junction sites and sequencing of the introduced DNA will help elucidate the nature of T-DNA insertion so that genetic modification of radiata pine can be targeted effectively.

Plant Cell Rep, 2004 Jun, 22(11), 822 - 7 Epub 2004 Feb 25.
Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters; Lee MH et al.; Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834 . Root explants produced transformed roots at a higher frequency (76.5+/-3.5%) as compared to stem (32.7+/-4.8%) or cotyledon (16.2+/-5.7%) . Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium . The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared . The frequency of adventitious shoot formation from transformed roots was much higher (88.5+/-9.8%) than that of non-transformed roots (31.7 +/-9.5%) on hormone-free medium . Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots . The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization . Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants . Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants . The differences observed may reflect the modification of morphological root characters by introduction of rol genes .

Mol Microbiol, 2004 Mar, 51(5), 1361 - 74
The RhaS activator controls the Erwinia chrysanthemi 3937 genes rhiN, rhiT and rhiE involved in rhamnogalacturonan catabolism; Hugouvieux-Cotte-Pattat N; Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls . The linear regions of pectin are composed of an acidic sugar, D-galacturonic acid . The ramified regions of pectin also include neutral sugars, and are rich in L-rhamnose residues . E . chrysanthemi is able to degrade these polysaccharides, polygalacturonate and rhamnogalacturonate . In E . chrysanthemi, the production of pectinases acting on linear regions is induced in the presence of polygalacturonate by a mechanism involving the repressor KdgR . The induction of the two adjacent E . chrysanthemi genes, designated rhiT and rhiN, is maximal after the simultaneous addition of both polygalacturonate and L-rhamnose . The rhiT product is homologous to the oligogalacturonide transporter TogT of E . chrysanthemi . The rhiN product is homologous to various proteins of unknown function, including a protein encoded by the plant-inducible locus picA of Agrobacterium tumefaciens . Both rhiT and rhiN are highly induced during plant infection . Various data suggest that RhiT and RhiN are involved in rhamnogalacturonate catabolism . RhiN is able to degrade the oligomers liberated by the rhamnogalacturonate lyase RhiE . The induction of the rhiTN operon in the presence of polygalacturonate results from control by the repressor KdgR . The additional induction of these genes by rhamnose is directly mediated by RhaS, a protein homologous to the activator of rhamnose catabolism in Escherichia coli . The virulence of an E . chrysanthemi rhaS mutant towards different host plants was clearly reduced . In this phytopathogenic bacterial species, RhaS positively regulates the transcription of the rhaBAD operon, involved in rhamnose catabolism, of the rhiE gene and of the rhiTN operon . The regulator RhaS plays a larger role in E . chrysanthemi than in other enterobacteria . Indeed, the RhaS control is not restricted to the catabolism of rhamnose but is extended to the degradation of plant polysaccharides that contain this sugar.

Arch Insect Biochem Physiol, 2004 Mar, 55(3), 103 - 13
Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato; Brunelle F et al.; We reported earlier the importance of digestive cathepsin D-like activity for initiating dietary protein hydrolysis in Colorado potato beetle, Leptinotarsa decemlineata Say {Brunelle et al . (1999) Arch . Insect Biochem . Physiol . 42:88-98} . We assessed here whether transgenic lines of potato (Solanum tuberosum L.) expressing a cathepsin D inhibitor (CDI) from tomato would show resistance to the beetle, or if the insect would compensate for the loss of cathepsin D activity after ingesting the recombinant inhibitor . Transgenic potato lines expressing tomato CDI were developed by Agrobacterium tumefaciens genetic transformation, and selected based on their relative amount of CDI . After confirming the absence of detectable visible effects of CDI on the plant's phenotype, diet assays with control and transgenic lines were carried out to assess the impact of the inhibitor on growth and development of the insect . Leaf consumption, relative growth rate, molting incidence, and digestive protease activity were monitored at 12-h intervals over 132 h for 3rd-instar larvae provided with transgenic potato foliage . Leaf consumption and relative growth rate were slightly reduced during the first 12 h for larvae fed CDI, but no significant differences were observed thereafter . In contrast, time for molting to the 4th larval stage was significantly longer for larvae fed modified plants, with developmental delays of approximately 10 h (0.5 day) compared to control larvae . Recombinant CDI also had an impact on the insect's digestive physiology, readily inducing overproduction of digestive proteases (rubiscases), followed by a gradual decrease of total and pepstatin-sensitive activity . Overall, these observations show the ability of Colorado potato beetle to compensate for the loss of cathepsin D activity by modulating its digestive protease complement in response to aspartate-type inhibitors in the diet . From a practical viewpoint, these data stress the importance of devising improved strategies for the effective inhibition of insect digestive proteinases in vivo, based on the use of hybrid inhibitors active against different protease classes .

Plant Cell, 2004 Mar, 16(3), 755 - 68 Epub 2004 Feb 18.
The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells; Dodds PN et al.; The Linum usitatissimum (flax) L gene alleles, which encode nucleotide binding site-Leu rich repeat class intracellular receptor proteins, confer resistance against the Melampsora lini (flax rust) fungus . At least 11 different L resistance specificities are known, and the corresponding avirulence genes in M . lini map to eight independent loci, some of which are complex and encode multiple specificities . We identified an M . lini cDNA marker that cosegregates in an F2 rust family with a complex locus determining avirulence on the L5, L6, and L7 resistance genes . Two related avirulence gene candidates, designated AvrL567-A and AvrL567-B, were identified in a genomic DNA contig from the avirulence allele, whereas the corresponding virulence allele contained a single copy of a related gene, AvrL567-C . Agrobacterium tumefaciens-mediated transient expression of the mature AvrL567-A or AvrL567-B (but not AvrL567-C) proteins as intracellular products in L . usitatissimum and Nicotiana tabacum (tobacco) induced a hypersensitive response-like necrosis that was dependent on coexpression of the L5, L6, or L7 resistance gene . An F1 seedling lethal or stunted growth phenotype also was observed when transgenic L . usitatissimum plants expressing AvrL567-A or AvrL567-B (but not AvrL567-C) were crossed to resistant lines containing L5, L6, or L7 . The AvrL567 genes are expressed in rust haustoria and encode 127 amino acid secreted proteins . Intracellular recognition of these rust avirulence proteins implies that they are delivered into host cells across the plant membrane . Differences in the three AvrL567 protein sequences result from diversifying selection, which is consistent with a coevolutionary arms race.

J Bacteriol, 2004 Mar, 186(5), 1430 - 7
TeiR, a LuxR-type transcription factor required for testosterone degradation in Comamonas testosteroni; Pruneda-Paz JL et al.; We have identified a new steroid-inducible gene (designated teiR {testosterone-inducible regulator}) in Comamonas testosteroni that is required for testosterone degradation . Nucleotide sequence analysis of teiR predicts a 391-amino-acid protein which shows homology between residues 327 and 380 (C-terminal domain) to the LuxR helix-turn-helix DNA binding domain and between residues 192 and 227 to the PAS sensor domain . This domain distribution resembles that described for TraR, a specific transcriptional regulator involved in quorum sensing in Agrobacterium tumefaciens . Analysis of the gene expression indicated that teiR is tightly controlled at the transcriptional level by the presence of testosterone in the culture medium . A teiR-disrupted mutant strain was completely unable to use testosterone as the sole carbon and energy source . In addition, the expression of several steroid-inducible genes was abolished in this mutant . Northern blot assays revealed that teiR is required for full expression of sip48-beta-HSD gene mRNA (encoding a steroid-inducible protein of 48 kDa and 3beta-17beta-hydroxysteroid dehydrogenase) and also of other steroid degradation genes, including those encoding 3alpha-hydroxysteroid dehydrogenase, Delta(5)-3-ketoisomerase, 3-oxo-steroid Delta(1)-dehydrogenase, and 3-oxo-steroid Delta(4)-(5alpha)-dehydrogenase enzymes . Moreover, when teiR was provided to the teiR-disrupted strain in trans, the transcription level of these genes was restored . These results indicate that TeiR positively regulates the transcription of genes involved in the initial enzymatic steps of steroid degradation in C . testosteroni.

J Bacteriol, 2004 Mar, 186(5), 1415 - 22
VirB1 orthologs from Brucella suis and pKM101 complement defects of the lytic transglycosylase required for efficient type IV secretion from Agrobacterium tumefaciens; Hoppner C et al.; Type IV secretion systems mediate conjugative plasmid transfer as well as the translocation of virulence factors from various gram-negative pathogens to eukaryotic host cells . The translocation apparatus consists of 9 to 12 components, and the components from different organisms are believed to have similar functions . However, orthologs to proteins of the prototypical type IV system, VirB of Agrobacterium tumefaciens, typically share only 15 to 30% identical amino acids, and functional complementation between components of different type IV secretion systems has not been achieved . We here report a heterologous complementation in the case of A . tumefaciens virB1 defects with its orthologs from Brucella suis (VirB1s) and the IncN plasmid pKM101 (TraL) . In contrast, expression of the genes encoding the VirB1 orthologs from the IncF plasmid (open reading frame 169) and from the Helicobacter pylori cag pathogenicity island (HP0523) did not complement VirB1 functions . The complementation of VirB1 activity was assessed by T-pilus formation, by tumor formation on wounded plants, by IncQ plasmid transfer, and by IncQ plasmid recipient assay . Replacement of the key active-site Glu residue by Ala abolished the complementation by VirB1 from B . suis and by TraL, demonstrating that heterologous complementation requires an intact lytic transglycosylase active site . In contrast, the VirB1 active-site mutant from A . tumefaciens retained considerable residual activity in various activity assays, implying that this protein exerts additional effects during the type IV secretion process.

Tree Physiol, 1993 Jun, 12(4), 411 - 8
Agrobacterium rhizogenes-mediated transformation to improve rooting ability of eucalypts; MacRae S et al.; In an attempt to improve rooting in in vitro propagated Eucalyptus clones, root-inducing genes on the Ri plasmids of three Agrobacterium rhizogenes strains were inserted into the Eucalyptus genome . Root development was monitored in vitro and after the plantlets had hardened off . Only transformed roots grew as root cultures in hormone-free liquid medium . The potential use of this procedure for improving rooting of clonal material is discussed.

Tree Physiol, 1994 Jan, 14(1), 27 - 35
Enhancement of the endogenous cytokinin concentration in poplar by transformation with Agrobacterium T-DNA gene ipt; Von Schwartzenberg K et al.; The agrobacterial isopentenyltransferase (ipt) gene, under the control of its native promoter, was transferred to poplar (Populus tremula x P . alba) by an Agrobacterium co-cultivation method . The ipt-transformed stem explants developed calli that regenerated many buds in the absence of exogenous cytokinins . Microcuttings of the ipt transformants exhibited frequently branching shoots with short internodes that were unable to root . In this material, the concentrations of zeatin, zeatin riboside and isopentenyladenosine, determined by an indirect enzyme-linked immunosorbent assay (ELISA), were 4.8- . 17.1- and 14.6-fold higher, respectively, than in non-transformed shoots . Results are discussed with regard to cytokinin metabolism.

Plant Cell Rep, 2004 Jun, 22(11), 828 - 31 Epub 2004 Feb 13.
Plant regeneration from hairy-root cultures transformed by infection with Agrobacterium rhizogenes in Catharanthus roseus; Choi PS et al.; Hypocotyl explants of Catharanthus roseus produced hairy roots when cultured on Murashige and Skoog (MS) basal medium after infection by Agrobacterium rhizogenes . Explants gave rise to adventitious shoots at a frequency of up to 80% when cultured on MS medium supplemented with 31.1 microM 6-benzyladenine and 5.4 microM alpha-naphthaleneacetic acid . There was a significant difference in the frequency of adventitious shoot formation for each hairy-root line derived from a different cultivar . Plants derived from hairy roots exhibited prolific rooting and had shortened internodes . Approximately half of the plants had wrinkled leaves and an abundant root mass with extensive lateral branching, but otherwise appeared morphologically normal . Plants with hairy roots that were derived from the cultivar Cooler Apricot developed flowers with petals that were white in the proximal region, whereas the wild-type flower petals are red . PCR and Southern blot analyses revealed that plants derived from hairy roots retained the Ri TL-DNA .

Plant Cell Rep, 2004 Jun, 22(11), 816 - 21 Epub 2004 Feb 13.
Modification of senescence in ryegrass transformed with IPT under the control of a monocot senescence-enhanced promoter; Li Q et al.; We report here the genetic modification of ryegrass senescence . Embryogenic cell suspensions of Lolium multiflorum were transformed by microprojectile bombardment with plasmid constructs containing 1.98 kb of the 5' flanking sequence of SEE1 (a maize cysteine protease gene showing enhanced expression during senescence) fused either to the Agrobacterium tumefaciens cytokinin biosynthesis gene IPT (designated PSEE1::IPT) or to the beta-glucuronidase reporter gene UIDA (PSEE1::UIDA) . Plants were regenerated under selection for the HPH hygromycin resistance gene in the vector . PSEE1::UIDA transformants confirmed that the SEE1 flanking sequence functioned as a senescence-enhanced promoter in ryegrass . The IPT transgene was detected in 28 regenerants (PSEE1::IPT) from five independent transformation events . PSEE1::IPT leaves displayed a stay-green phenotype . Some PSEE1::IPT lines developed spontaneous lesions .

Anal Bioanal Chem, 2004 Apr, 378(7), 1748 - 53 Epub 2004 Feb 13.
Detection of transgenes in soybean via a polymerase chain reaction and a simple bioluminometric assay based on a universal aequorin-labeled oligonucleotide probe; Glynou K et al.; The recombinant photoprotein aequorin was used as a reporter in highly sensitive and automatable hybridization assays for the analysis of transgenic sequences in genetically modified organisms (GMO) . The terminator of the nopaline synthase gene (NOS) from Agrobacterium tumefaciens and the 35S promoter sequence were detected in genetically modified soybean . The endogenous, soybean-specific, lectin gene was also detected for confirmation of the integrity of extracted DNA . A universal detection reagent was produced through conjugation of aequorin to the oligonucleotide (dA)(30) . Biotinylated (through PCR) products for the three target sequences were captured onto streptavidin-coated wells, and one strand was removed by NaOH treatment . The immobilized single-stranded DNAs were then hybridized with oligonucleotide probes consisting of a target-specific segment and a poly(dT) tail . This allowed the subsequent determination of all hybrids through the use of the (dA)(30)-aequorin conjugate as a universal reagent . The bound aequorin was measured by adding Ca(2+) and integrating the light emission for 3 s . As low as 2 pM (100 amol per well) of amplified DNA was detectable for all three targets, with a signal-to-background ratio of about 2 . The analytical range extended up to 2000 pM . As low as 0.05% GMO content in soybean can be detected with a signal-to-background ratio of 8.2 . The overall repeatability of the proposed assay, including DNA extraction, PCR, and hybridization assay, ranged from 7.5-19.8% . The use of a (dA)(30)-aequorin conjugate renders the assay configuration general for any target DNA, provided that the specific probe carries a poly(dT) tail.

Methods, 2004 Mar, 32(3), 235 - 40
Over-expression and production of plant allergens by molecular farming strategies; Obermeyer G et al.; Recombinant allergens have become a valuable tool for diagnosis and may also be used for therapy in the near future . To supply the required large amounts of functional recombinant proteins on a cost-effective basis, the production of allergens in plants by molecular farming is an alternative to microbial expression systems . Especially as post-translational modifications of the allergens, e.g., phosphorylation and glycosylation, may be important for recognition by the human immune system, the plant-based production of recombinant allergens enables the correct folding, glycosylation, and other modifications of the recombinant allergen . An introduction to the methods for plant transformation via the tumor-inducing bacterium, Agrobacterium tumefaciens, is given in this paper.

Methods, 2004 Mar, 32(3), 227 - 34
Plant virus expression systems for transient production of recombinant allergens in Nicotiana benthamiana; Wagner B et al.; In recent years, several studies have demonstrated the use of autonomously replicating plant viruses as vehicles to express a variety of therapeutic molecules of pharmaceutical interest . Plant virus vectors for expression of heterologous proteins in plants represent an attractive biotechnological tool to complement the conventional production of recombinant proteins in bacterial, fungal, or mammalian cells . Virus vectors are advantageous when high levels of gene expression are desired within a short time, although the instability of the foreign genes in the viral genome may present problems . Similar levels of foreign protein production in transgenic plants often are unattainable, in some cases because of the toxicity of the foreign protein . Now virus-based vectors are for the first time investigated as a means of producing recombinant allergens in plants . Several plant virus vectors have been developed for the expression of foreign proteins . Here, we describe the utilization of tobacco mosaic virus- and potato virus X-based vectors for the transient expression of plant allergens in Nicotiana benthamiana plants . One approach involves the inoculation of tobacco plants with infectious RNA transcribed in vitro from a cDNA copy of the recombinant viral genome . Another approach utilizes the transfection of whole plants from wounds inoculated with Agrobacterium tumefaciens containing cDNA copies of recombinant plus-sense RNA viruses.

Plant Cell Rep, 2004 May, 22(10), 765 - 73 Epub 2004 Feb 10.
A quick and efficient system for antibiotic-free expression of heterologous genes in tobacco roots; Komarnytsky S et al.; Requirement for antibiotic-resistance selection markers and difficulty in identifying transgenes with the highest expression levels remain the major obstacles for rapid production of recombinant proteins in plants . An alternative approach to producing transgenic plants free of antibiotic-resistance markers is the phenotypic-based selection with root-proliferation genes (rol genes) of Agrobacterium rhizogenes . By using Agrobacterium tumefaciens harboring the pRYG transformation vector with a cluster of rol genes linked to a heterologous gene of interest, we have developed a rapid transformation tool using hairy root formation as a selection marker . The expression of beta-glucuronidase in newly induced transgenic tobacco roots could be detected as early as 12 days after inoculation . Higher levels of transgene expression in the roots correlated positively with the rates of root elongation on hormone-free medium and thus could be used for positive selection . When tobacco plants were transformed with pRYG harboring the expression cassette for secreted alkaline phosphatase (SEAP), the release of SEAP from roots of the fully regenerated transgenic plants could be quantified at rates as high as 28 microg/g root dry weight per day .

Plant Cell Rep, 2004 May, 22(10), 759 - 64 Epub 2004 Feb 10.
High-frequency transformation of Lobelia erinus L . by Agrobacterium-mediated gene transfer; Tsugawa H et al.; A highly efficient transformation procedure was developed for Lobelia erinus . Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a beta-glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers . The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots . Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83% . In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A . tumefaciens . Transgenic plants flowered 3-4 months after transformation . Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants . The transformation frequency at the stage of whole plants was very high--45% per inoculated disc .

Plant Cell Rep, 2004 Jun, 22(11), 832 - 8 Epub 2004 Feb 06.
Regeneration of herbicide-tolerant black locust transgenic plants by SAAT; Zaragoza C et al.; A protocol based on SAAT (sonication-assisted Agrobacterium-mediated transformation) has been developed to obtain herbicide-resistant transgenic black locust (Robinia pseudoacacia L.) plants . Cotyledon explants were co-cultivated with Agrobacterium AGL1 strain carrying the pTAB16 plasmid (bar and gusA genes) . The effects of bacterial concentration (OD550 of 0.3, 0.6, 0.8) and method of infection (sonication vs immersion) on bacterial delivery were determined by assaying cotyledons for transient beta-glucuronidase expression 3 days after infection . SAAT increases transient expression efficiency especially at an OD550 of 0.6 . After determining bacterial concentration and infection method, other factors affecting transformation efficiency, such as explant preconditioning and period of time before applying selection, were tested . From these experiments, the preferred protocol for black locust cotyledon transformation should include sonication of preconditioned cotyledons in AGL1 suspension, coculture for 3 days with 100 microM acetosyringone and transfer to selection medium with 4 mg/l phosphinothricin and 150 mg/l timentin . Of the initial explants, 2% produced at least one transgenic shoot . Genetic transformation was confirmed by Southern hybridization, chlorophenol red assay and herbicide tolerance of the regenerated plants .

Cell Microbiol, 2004 Mar, 6(3), 213 - 24
Integration of environmental and host-derived signals with quorum sensing during plant-microbe interactions; Newton JA et al.; Many plant-associated microbes use secreted autoinducer molecules, including N-acylhomoserine lactones (AHLs), to regulate diverse behaviours in association with their population density (quorum sensing) . Often, these responses are affected by environmental conditions, including the presence of other AHL-producing bacterial species . In addition, plant-derived metabolites, including products that arise as a direct result of the bacterial infection, may profoundly influence AHL-regulated behaviours . These plant products can interact directly and indirectly with the quorum-sensing network and can profoundly affect the quorum-sensing behaviour . Local conditions on a microscopic scale may affect signal molecule longevity, stability and accumulation, and this could be used to give information in addition to cell density . Furthermore, in many Gram-negative bacteria, AHL signalling is subservient to an additional two-component signalling system dependent upon homologues of GacS and GacA . The signal(s) to which GacS responds are not known, but recent research suggests that a self-produced ligand may be being detected . This review will focus on two well-studied examples of AHL-regulated plant-associated behaviour, Erwinia carotovora and Agrobacterium tumefaciens, to illustrate the complexity of such signalling networks.

Mol Microbiol, 2004 Feb, 51(4), 1103 - 15
Signal quenching, detoxification and mineralization of vir gene-inducing phenolics by the VirH2 protein of Agrobacterium tumefaciens; Brencic A et al.; Plant tumorigenesis by Agrobacterium tumefaciens requires approximately 20 Vir proteins, transcription of which is induced by a family of phenolic compounds released from plant wound sites . One Vir protein, VirH2, plays a role in the metabolism of at least one phenolic inducer inasmuch as it converts ferulic acid, a potent vir gene inducer, to the non-inducer caffeate by O-demethylation of a methoxyl group . Here, we tested VirH2-dependent O-demethylation of 16 other vir-inducing phenolics, and detected this activity for each compound . However, O-demethylation rates differed enormously, with the strongest vir gene inducers such as acetosyringone being demethylated extremely slowly . Compounds containing two methoxyl groups were demethylated at both positions . In general, phenolic inducers were more toxic than their demethylated counterparts . A virH2 mutant was more sensitive than the wild type to growth inhibition by virtually all phenolic inducers tested, indicating that VirH2 detoxifies these compounds . VirH2 also played a role in mineralization of some phenolics . It converted vanillate to protocatechuate, which was then mineralized via the beta-ketoadipate pathway . Vanillyl alcohol and vanillin were also mineralized after being oxidized to vanillate . All three compounds served as sole sources of carbon, whereas the remaining 13 compounds did not.

Biotechnol Prog, 2004 Jan-Feb, 20(1), 51 - 6
Cloning and functional expression of the dps gene encoding decaprenyl diphosphate synthase from Agrobacterium tumefaciens; Lee JK et al.; A newly isolated gene from Agrobacterium tumefaciens (A . tumefaciens), which encoded a decaprenyl diphosphate synthase, was cloned in Escherichia coli (E . coli), and its nucleotide sequence was determined . DNA sequence analysis revealed an open reading frame of 1077 bp capable of encoding a 358-amino-acid protein with a calculated isoelectric point of pH 5.16 and a molecular mass of 38 960 Da . The primary structure of the enzyme shared significant homology with prenyl diphosphate synthases from various sources . The deduced amino acid sequence included oligopeptide DDxxD aspartate-rich domains conserved in the majority of prenyl diphosphate synthases . High levels of the active enzyme were expressed in the soluble fraction and were readily purified to homogeneity by Ni-NTA chromatography . E . coli JM109 harboring the dps gene produced ubiquinone-10 in addition to endogenous ubiquinone-8, while E . coli JM109 harboring the dps gene mutated on the DDxxD domain lost the ability to produce ubiquinone-10, which suggests that the A . tumefaciens dps gene is functionally expressed in E . coli and that it encodes a decaprenyl diphosphate synthase.

Plant J, 2004 Feb, 37(4), 554 - 65
Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast; Jamir Y et al.; The Pseudomonas syringae pv . tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants . Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA . We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis . Mixed inoculum and Agrobacterium-mediated transient expression experiments confirmed that suppressor action occurred within plant cells . These suppressors, with the exception of AvrPpiB1Pto, inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a . DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor . Additionally, HopPtoG was identified as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant . Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner . The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P . syringae pathogenesis.

Plant J, 2004 Feb, 37(4), 635 - 44
Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures; Menges M et al.; We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised . Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines . Frozen cultures show 90% viability and rapid re-growth after recovery . We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation . The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation . Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.

Curr Microbiol, 2003 Dec, 47(6), 462 - 6
Isolation and characterization of bacteria capable of degrading phenol and reducing nitrate under low-oxygen conditions; Baek SH et al.; Nitrate-reducing bacteria capable of degrading phenol were isolated from natural and contaminated environments under low-oxygen conditions with nitrate-containing media, using phenol as a sole carbon and energy source . A total of 27 bacteria able to degrade phenol and reduce nitrate under low-oxygen conditions were isolated from all of the inoculum samples, regardless of previous phenol contamination . For all of these bacteria, oxygen was an essential requirement for phenol degradation . Nitrate reduction by 19 of the strains was insensitive to 10 mM sodium azide, and these strains were placed into the alpha- and beta-subclasses of Proteobacteria and two were Gram-positive bacteria . To date, the order of Rhizobiales has hardly been reported to have an ability to degrade aromatic compounds . Interestingly, our study showed that all isolates that were placed into the alpha-subclass of Proteobacteria are in the order of Rhizobiales . Furthermore, the genus Agrobacterium was isolated from most inoculum samples and one genus of Gram-positive bacteria, Staphylococcus, was also isolated . In the case of the remaining eight strains, nitrate reduction was inhibited by 10 mM sodium azide . Of these strains, seven were placed into the gamma-subclass of Proteobacteria.

Plant Mol Biol, 2003 Sep, 53(1-2), 247 - 59
An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics; Rosso MG et al.; The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics . Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies . T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation . To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed . DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders . After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line . Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions . Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes . Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus . In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed . Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.

FEBS Lett, 2004 Jan 30, 558(1-3), 45 - 51
Mutational analysis of GstI protein, a glutamine synthetase translational inhibitor of Rhizobium leguminosarum; Napolitani C et al.; The small GstI protein (63 amino acids) of Rhizobium leguminosarum inhibits the expression of the glnII (glutamine synthetase II) gene, thus reducing the bacterial ability to assimilate ammonium . In order to identify the residues essential for its inhibitory activity, all the 53 non-alanine amino acid residues of GstI were individually mutated into alanine . Based on their capacity to inhibit glnII expression (in two genetic backgrounds) three groups of mutants were identified . The first group displayed an inhibitory activity similar to the wild-type; the second and the third ones showed partial and total loss of inhibitory activity, respectively . Several mutations of the latter group concerned residues conserved in two related sequences from Sinorhizobium meliloti and Agrobacterium tumefaciens . Additionally, we performed experiments to exclude a GstI-mediated mechanism of glutamine synthetase II inhibition/degradation . Finally, the protein was over expressed in Escherichia coli, purified and characterised.

Arch Microbiol, 2004 Apr, 181(4), 331 - 6 Epub 2004 Jan 30.
Screening for highly active beta-lactam antibiotics against Agrobacterium tumefaciens; Ogawa Y et al.; Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 beta-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation . The antibacterial activities of beta-lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404 . Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l(-1)) . Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404 . The production of beta-lactamase was observed only in strain EHA101.

Curr Genet, 2004 Apr, 45(4), 249 - 55 Epub 2004 Jan 29.
Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans; Gardiner DM et al.; A vector system was constructed that is designed to decrease the number of transformants required to be screened when looking for gene disruption events in filamentous fungi . This vector was used to mutate two genes, an ATP-binding cassette transporter ( LmABCt4) and a two-component histidine kinase gene ( LmHK1) in the ascomycete Leptosphaeria maculans . The system uses the thymidine kinase gene from the herpes simplex virus as a negative selectable marker . Thymidine kinase expression is regulated by the TrpC regulatory elements from Aspergillus nidulans and should be applicable to other ascomycetous fungi . When thymidine kinase is expressed in the presence of particular thymidine analogues, these analogues are converted to toxic compounds which kill the cell . We also report the transformation of L . maculans using Agrobacterium tumefaciens-mediated DNA delivery.

Plant Cell Rep, 2004 Mar, 22(8), 576 - 83 Epub 2004 Jan 29.
Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea; Tewari-Singh N et al.; A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea ( Cicer arietinum L.) . Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT) . Approximately 15% of the putative transgenic shoots of vars . P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 microM thidiazuron (TDZ) and 2 mM lysine and 2 m M threonine . These shoots were subsequently grown on MSB5 medium supplemented with 2 micro M TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection . A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed . Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation . Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l(-1 )PPT . Increasing the concentrations of PPT to 15 mg l(-1) reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively . Selected putatively transformed shoots of all three varieties were rooted and grown to maturity . Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines . The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.

Ann Bot (Lond), 2004 Mar, 93(3), 303 - 10 Epub 2004 Jan 28.
An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco; Kis M et al.; BACKGROUND AND AIMS: Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting . This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence . METHODS: Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed . The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both . These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation . To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques . KEY RESULTS: Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast . The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect . Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels . CONCLUSIONS: There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system . This leads us to question the postulated targeting roles of these peptide extensions . The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency . Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic activity . These will be valuable for exploring the role of these enzymes in stress amelioration and plant development.

Curr Genet, 2004 Apr, 45(4), 242 - 8 Epub 2004 Jan 24.
A one-step method to convert vectors into binary vectors suited for Agrobacterium-mediated transformation; Takken FL et al.; Bacterial artificial chromosomes (BACs) are widely used for the construction of physical maps, positional-cloning and whole-genome sequencing strategies . Unfortunately, their use for functional genomics is limited, as currently there is no efficient method to use BACs directly for complementation . We describe a novel strategy for one-step conversion of any BAC into a binary BAC (BIBAC) . Using Agrobacterium tumefaciens, these BIBACs can be efficiently transformed to virtually all organisms, including plants, fungi, yeasts and human cells . As the strategy is based on in vivo recombineering and does not depend on restriction sites, it is applicable to any vector . To show the feasibility of the method five BACs, containing 0-75 kb of fungal DNA, were converted into BIBACs . These were subsequently transformed to the plant pathogenic fungus Fusarium oxysporum f.sp . lycopersici and to Aspergillus awamori, a filamentous fungus often used for large-scale protein production . Molecular characterisation of the transformants showed that the BIBACs were efficiently transferred to the fungi and stably integrated into their genomes.

Plant Cell Rep, 2004 Apr, 22(9), 698 - 704 Epub 2004 Jan 27.
Agrobacterium rhizogenes-mediated DNA transfer to Aesculus hippocastanum L . and the regeneration of transformed plants; Zdravkovic-Korac S et al.; Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS . Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers . Seventy-one putative transformed hairy root lines from independent transformation events were established . Regeneration was induced in MS liquid medium supplemented with 30 microM 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 microM BA . Following elongation on MS medium supplemented with 1 microM BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment . Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization . The copy number of transgenes was estimated to be from two to four.

Int J Syst Evol Microbiol . 2004 Jan;54(Pt 1):149.
Renaming of Agrobacterium larrymoorei Bouzar and Jones 2001 as Rhizobium larrymoorei (Bouzar and Jones 2001) comb . nov; Young JM; In conformity with the nomenclature of the genus Rhizobium published by Young et al . (Int J Syst Evol Microbiol 51, 89-103, 2001), it is proposed that Agrobacterium larrymoorei be named as Rhizobium larrymoorei comb . nov.

Environ Sci Technol, 2004 Jan 1, 38(1), 104 - 11
Bacterial populations associated with the oxidation and reduction of arsenic in an unsaturated soil; Macur RE et al.; Microbial populations responsible for the oxidation and reduction of As were examined in unsaturated (aerobic) soil columns treated with 75 microM arsenite {As(III)} or 250 microM arsenate {As(V)} . Arsenite {As(III)} was rapidly oxidized to As(V) via microbial activity, whereas no apparent reduction of As(V) was observed in the column experiments . Eight aerobic heterotrophic bacteria with varying As redox phenotypes were isolated from the same columns . Three isolates, identified as Agrobacterium tumefaciens-, Pseudomonas fluorescens-, and Variovorax paradoxus-like organisms (based on 16S sequence), were As(III) oxidizers, and all were detected in community DNA fingerprints generated by PCR coupled with denaturing gradient gel electrophoresis . The five other isolates were identified (16S gene sequence) as A . tumefaciens, Flavobacterium sp., Microbacterium sp., and two Arthrobacter sp . -like organisms and were shown to rapidly reduce As(V) under aerobic conditions . Although the two A . tumefaciens-like isolates exhibited opposite As redox activity,their 16S rDNA sequences (approximately 1400 bp) were 100% identical, and both were shown to contain putative arsC genes . Our results support the hypothesis that bacteria capable of either oxidizing As(III) or reducing As(V) coexist and are ubiquitous in soil environments, suggesting that the relative abundance and metabolic activity of specific microbial populations plays an important role in the speciation of inorganic As in soil pore waters.

Chromosoma, 2004 Feb, 112(5), 255 - 66 Epub 2004 Jan 23.
Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants; Docquier S et al.; We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM) . Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species . The number and distribution of CBs were investigated . To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B" and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy . Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced . AtRSp31 was detected in quiescent nuclei and in germinating cells . This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM . Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression . When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells . Treatments with alpha-amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize . Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP . The distribution of the fusion protein was identical to that of endogenous atRSp31 . Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.

Planta, 2004 Mar, 218(5), 890 - 3 Epub 2004 Jan 23.
Hairy root induction of Papaver somniferum var . album, a difficult-to-transform plant, by A rhizogenes LBA 9402; Le Flem-Bonhomme V et al.; Two strains of Agrobacterium rhizogenes (15834, LBA 9402) and one Agrobacterium tumefaciens strain {GV 3101 (PMP90RK, p35SGUS-2)} and four culture media were tested and compared for their ability to induce hairy root formation on wounded Papaver somniferum L . hypocotyls . Five weeks after the infection with A . rhizogenes LBA 9402, hairy roots appeared on 80% of the hypocotyls maintained in the hormone-free liquid medium . Six hairy-root cultures were established . Transformation was confirmed by polymerase chain reaction analysis . One clone was analysed for its alkaloid production . The total alkaloid content was higher in the transformed roots (0.46+/-0.06% DW) than in the untransformed roots (0.32+/-0.05% DW) . The transformed roots accumulated three times more codeine (0.18+/-0.02% DW) than intact roots (0.05+/-0% DW) . Moreover, morphine (0.255+/-0.03% DW) and sanguinarine (0.014+/-0% DW) were found in the liquid culture medium.

Plant Cell Rep, 2004 Apr, 22(9), 653 - 9 Epub 2004 Jan 23.
A large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.); Terada R et al.; A large-scale transformation procedure handling an adequate number of stable transformants with highly efficient positive-negative selection is a necessary prerequisite to successful gene targeting by homologous recombination, as the integration of a transgene by somatic homologous recombination in higher plants has been reported to be 10(-3) to 10(-5) compared with random integration by non-homologous end joining . We established an efficient and large-scale Agrobacterium-mediated rice transformation protocol that generated around 10(3) stable transformants routinely from 150 seeds and a strong positive-negative selection procedure that resulted in survivors at 10(-2) using the gene for diphtheria toxin A fragment as a negative marker . The established transformation procedure provides a basis for efficient gene targeting in rice.

Plant Cell Rep, 2004 Apr, 22(9), 691 - 7 Epub 2004 Jan 23.
Improvement of cotton fiber quality by transforming the acsA and acsB genes into Gossypium hirsutum L . by means of vacuum infiltration; Li X et al.; A novel method for the genetic transformation of cotton pollen by means of vacuum infiltration and Agrobacterium-mediated transformation is reported . The acsA and acsB genes, which are involved in cellulose synthesis in Acetobacter xylinum, were transferred into pollen grains of brown cotton with the aim of improving its fiber quality by incorporating useful prokaryotic features into the colored cotton plants . Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by vector pCAMBIA1301, which contains a reporter gene beta-glucuronidase (GUS), a selectable marker gene, hpt, for hygromycin resistance and the genes of interest, acsA and acsB . The integration and expression of acsA, acsB and GUS in the genome of transgenic plants were analyzed with Southern blot hybridization, PCR, histochemical GUS assay and Northern blot hybridization . We found that following pollination on the cotton stigma transformed pollen retained its capability of double-fertilization and that normal cotton seeds were produced in the cotton ovary . Of 1,039 seeds from 312 bolls pollinated with transformed pollen grains, 17 were able to germinate and grow into seedlings for more than 3 weeks in a nutrient medium containing 50 mg/l hygromycin; eight of these were transgenic plants integrated with acsA and acsB, yielding a 0.77% transformation rate . Fiber strength and length from the most positive transformants was 15% greater than those of the control (non-transformed), a significant difference, as was cellulose content between the transformed and control plants . Our study suggests that transformation through vacuum infiltration and Agrobacterium mediated transformation can be an efficient way to introduce foreign genes into the cotton pollen grain and that cotton fiber quality can be improved with the incorporation of the prokaryotic genes acsA and acsB.

Acta Biochim Pol, 2003, 50(4), 1273 - 81
Characterisation of Mesorhizobium huakuii cyclic beta-glucan; Choma A et al.; Periplasmic and extracellular glucans of Mesorhizobium huakuii were isolated and characterized by compositional and MALDI-TOF analyses, as well as 1H and 13C NMR spectroscopy . It was shown that M . huakuii produces a cyclic beta-glucan composed entirely of nonbranched glucose chains and unmodified by nonsugar substituents . The degree of polymerisation of the cyclic oligosaccharides was estimated to be in the range from 17 to 28 . The most abundant glucan molecules contained 22 glucose residues . Glucose residues within the glucan were connected by beta-(1,2) glycosidic linkages . The cyclic glucan produced by M . huakuii is quite similar to the periplasmic beta-(1,2) glucans synthesized by Agrobacterium and Sinorhizobium genera . The synthesis of beta-glucan in M . huakuii is osmoregulated and this glucan could function as an osmoprotectant in free living cells.

Planta Med, 2003 Nov, 69(11), 1018 - 23
Transformation of ipecac (Cephaelis ipecacuanha) with Agrobacterium rhizogenes; Yoshimatsu K et al.; Transformed root cultures of ipecac (Cephaelis ipecacuanha A . Richard), one of the recalcitrant woody plant species for Agrobacterium-mediated transformation, were established by co-culturing of in vitro petiole segments with Agrobacterium rhizogenes ATCC 15 834 . Southern blot analysis of the established roots revealed that only the TL-DNA was integrated into the plant genome without incorporation of the TR-DNA . The transformed roots grew slowly on phytohormone-free solid medium and adventitious shoots were regenerated after over 6 months of culture on HF, half-strength Murashige and Skoog (1/2 MS) medium in the dark . The individually separated transformed shoots developed into plantlets on phytohormone-free solid medium at 25 degrees C under 16 h/day light, and the plants demonstrated wider leaves, shorter internodes and vigorous root growth compared to non-transformed plants . Effects of basal media and auxins on the growth and the ipecac alkaloid production of the transformed roots were investigated either under light or in the dark . The roots cultured in the dark grew well in Gamborg B5 (B5) liquid medium containing 0.5 mg/L IBA and yielded 112 mg/L of cephaeline and 14 mg/L emetine after 8 weeks of culture.

Infection, 2003 Dec, 31(6), 421 - 4
Catheter-related bacteremia caused by Agrobacterium radiobacter in a cancer patient: case report and literature review; Paphitou NI et al.; Agrobacteria are a group of phytopathogenic organisms widely distributed in soil; they are now recognized as rare human pathogens affecting mostly immunocompromised hosts . We report a case of catheter-related bacteremia due to Agrobacterium radiobacter in a neutropenic patient and describe the clinical presentations, treatment strategies and outcome of Agrobacterium infections based on our experience and a literature review . The antimicrobial susceptibility patterns of these organisms appear to be quite variable and collective susceptibility data derived from this and previous reports are provided.

Mol Biotechnol, 2004 Jan, 26(1), 17 - 26
Expression of modified Cry1Ac gene of Bacillus thuringiensis in transgenic tobacco plants; Misztal LH et al.; Several mutations were introduced into the Cry1Ac toxin gene, resulting in four variants with altered sequences that were responsible for low expression of the toxin in transgenic plants . These variants were as follows: V1, with modified three A/T-rich regions, including the first signal of transcription termination; V2, with modified five putative polyadenylation signals (polyadenylation signals PAS) and the second signal of transcription termination; V3, with four initial AUUUA motifs; V4, with modification of six PASs, four AUUUA motifs, as well as the first and the second signals of transcription termination . The modified variants and the initial WT gene were cloned into the binary vector pBI121 and introduced into tobacco plants (Nicotiana tabacum) by Agrobacterium tumefaciens-mediated transformation . The presence of transgenes in the tobacco plants was confirmed by the polymerase chain reaction (PCR) method . The expression of particular variants of the Cry1Ac gene in tobacco was assayed using Western blotting with antibodies against the domain II of the Cry1Ac toxin . The average expression of WT was estimated to be 0.0025% of soluble proteins, and the expression levels of modified variants were 0.004%, 0.0098%, 0.0125%, and 0.0043% for V1, V2, V3, and V4, respectively . In this article we described the construction of a variant of the Cry1Ac gene (V3) with 12 point mutations leading to an average level of expression in transgenic plants five times higher than that observed in the case of the WT gene . Our results have shown for the first time that the modification of AUUUA sequences has a significant effect on the expression of the Cry1Ac gene in transgenic plants.

Phytochemistry, 2004 Jan, 65(2), 169 - 79
Agrobacterium tumefaciens AK-6b gene modulates phenolic compound metabolism in tobacco; Galis I et al.; The 6b gene (AK-6b) of Agrobacterium tumefaciens AKE10 can substitute for the requirement of tobacco tissues for auxin and cytokinin to maintain callus growth in the culture medium . To identify compounds that might be involved in this process we analyzed phenolic metabolites in transgenic tobacco tissues expressing the AK-6b gene . On medium containing both cytokinin and auxin (SH medium), transgenic calli accumulated higher levels of chlorogenic acid, caffeoyl putrescine, rutin and kaempferol-3-rutinoside, than did wild-type tissues . In contrast, the levels of scopolin and its aglycone, scopoletin were lower in transgenic tissues . On hormone-free medium, these phenolic compounds showed neither significant levels nor an apparent relationship with AK-6b transcript levels, except for the negatively correlated levels of scopoletin and AK-6b transcripts . Apparently, the AK-6b gene acts, in SH medium, to redirect the synthesis of scopolin in tobacco tissues towards the preferential synthesis of caffeic acid derivatives and flavonoids.

Mol Microbiol, 2004 Feb, 51(3), 765 - 76
Site-directed mutagenesis of a LuxR-type quorum-sensing transcription factor: alteration of autoinducer specificity; Chai Y et al.; TraR is a quorum-sensing transcription factor from Agrobacterium tumefaciens that regulates replication and conjugation genes of the tumour-inducing (Ti) plasmid . TraR activity requires the autoinducer pheromone N-3-oxooctanoyl-l-homoserine lactone (OOHL) . Structural studies of TraR-OOHL-DNA complexes showed that one molecule of OOHL is completely engulfed within the N-terminal domain of each TraR subunit . TraR is thought to bind OOHL via four hydrogen bonds, three of them direct and one water mediated, and by numerous hydrophobic interactions . Here, we show that all residues predicted to hydrogen bond with OOHL are essential for wild-type protein function . Mutants that failed to detect OOHL in vivo invariably failed to sequester exogenous OOHL . We showed previously that TraR is protected from cellular proteases by OOHL, and now show that mutants that failed to detect OOHL were also not protected from proteolysis by OOHL . We also describe several mutants with altered autoinducer specificity . Three mutants (T129V, T129A and T115I) detected 3-oxo-AHLs and 3-unsubstituted AHLs with equal sensitivity, indicating that these mutations perturb the water-mediated hydrogen bond to the 3-oxo moiety of OOHL . Three other mutants (A49I, A49M and Q58L) preferentially detected AHLs containing six or seven carbon atoms rather than eight . The bulkier residues in these mutations appear to have occupied a portion of the OOHL binding site, interfering with binding of the acyl chain of AHLs.

Pest Manag Sci, 2004 Jan, 60(1), 49 - 58
Biotransformation of atrazine in transgenic tobacco cell culture expressing human P450; Bode M et al.; Plant cell cultures in which the appropriate P450 cDNA is introduced are expected to metabolise certain pesticides in large quantities . Two species of human P450 (CYP1A1 and CYP1A2) were introduced into tobacco cells (Nicotiana tabacum L) by Agrobacterium-mediated transformation . The transgenic plant cell cultures were selected by combination of kanamycin-resistance, 7-ethoxycoumarin O-de-ethylase activity, PCR and Western blot analysis . For metabolism studies, 14C-labelled atrazine was used as a model substance . The metabolites de-ethylatrazine and de-isopropylatrazine were found in the control culture as well as in the transgenic culture, whereas the non-phytotoxic metabolite de-ethyl-de-isopropylatrazine was found only in the transgenic cell cultures . The results showed that both foreign enzymes CYP1A1 and CYP1A2 catalyse N-dealkylation of atrazine . However, CYP1A2 exhibited a higher conversion rate than CYP1A1 . In a time-course study the enzyme CYP1A2 catalysed predominantly N-de-ethylation followed by de-isopropylation . The extent of metabolism was considerably higher than in non-transformed cell cultures . The transgenic cell cultures can therefore be suitable tools for the production of large quantities of primary oxidised pesticide metabolites.

Plant Cell Rep, 2004 Apr, 22(9), 668 - 77 Epub 2004 Jan 15.
Penicillin derivatives induce chemical structure-dependent root development, and application for plant transformation; ur Rahman L et al.; We investigated five penicillin derivatives that are popularly used for transformation experiments with Agrobacterium rhizogenes-penicillin G, carbenicillin, ampicillin, amoxicillin and cephalexin-for their effects on the growth and morphology of Beta vulgaris, Capsicum annuum and Glehnia littoralis roots . Attention was given to the relationship between their chemical structures and functions . Ampicillin was found to stimulate root elongation but inhibit root branching, whereas carbenicillin inhibited root elongation but promoted root branching . Root cultures were also exposed to hydrolyzed products of these antibiotics-i.e . phenylmalonic acid (PM), phenylglycine and 6-aminopenicillanic acid (6-APA): PM inhibited root elongation the most, while root elongation was supported best by 6-APA . These results indicate that both the side chains and the major component of penicillin derivatives affect root development and that the nature of the side chains is responsible for the responses . Ampicillin but not carbenicillin was used in subsequent experiments described herein to eliminate bacteria and to support root growth of transformants of the recalcitrant plants.

Shi Yan Sheng Wu Xue Bao, 2003 Dec, 36(6), 459 - 64
{Genetic analysis of a rolled-leaf mutant in rice population of T-DNA insertion}; Shen GZ et al.; A rolled-leaf mutant was obtained in a T-DNA(containing bar gene and Ds element) insertion population, which consist of transgenic japonica rice Zhonghua 11 mediated by Agrobacterium tumefaciens . Through self-hybridization of three generations, one of trait-purified mutants (R1-A2) was obtained and used as parent to cross with variety Zhonghua 11 . The leaves of 36 F1 plants investigated were rolled and resistant to herbicide Basta . Among 852 F2 plants, the segregation ratio of rolled leaves to normal leaves(645:207) was consistent with 3:1 . All rolled-leaf plants were resistant to herbicide Basta, and all normal leaf plants were sensitive to herbicide Basta . These results showed that the trait of rolled-leaf is co-segregated with Basta resistance . The total DNA of 45 rolled-leaf plants and 30 normal leaf plants in F2 population were amplified to test the presence of T-DNA by Ds primers . The results showed that the positive band were amplified in all rolled-leaf plants, but not in every normal leaf plant . In F1B1 progenies, all plants which derived from backcross parent R1-A2 were rolled leaves; while variety Zhonghua 11 was used as backcross parent, the segregation ratio of rolled-leaf to normal leaf was consistent with 1:1 . Taking these data together, it indicated that the rolled-leaf mutant was co-segregation with T-DNA and controlled by single dominant gene.

Shi Yan Sheng Wu Xue Bao, 2003 Dec, 36(6), 428 - 34
{Double-antisense ACC oxidase and ACC synthase fusion gene introduced into tomato by Agrobacterium-mediated transformation and analysis the ethylene production of transgenic plants}; Xiong AS et al.; The tomato fruit-specific promoter 2A11 was amplified from tomato genomic DNA using PCR techniques . Total RNA was isolated from ripen fruit of tomato, then ACC oxidase gene and ACC synthase gene were obtained using reverse-transcription polymerase chain reaction . The fusion encoding ACC oxidase and ACC synthase gene was obtained through ACC oxidase gene and ACC synthase gene ligation . The fusion gene was then inserted into a plant binary vector pYPX145 in an inverted orientation . Finally, the binary plant expression vector pOSACC was constructed in which the double-antisense fusion gene was controlled by fruit-specific 2A11 promoter . By using hypocotyls and cotyledon petioles as explants, the unit of double-antisense fusion gene was successfully introduced into tomato (Lycopersicon esculentum Mill) cultivar "Hezuo 903" by Agrobacterium tumefaciens-mediated transformation . 105 transgenic plants were obtained through 200 mg/L kanamycin selection and GUS assay . Two lines of DR-1 and DR-2 were obtained through selecting the characteristics of prolonged shelf life and agriculture . The transgenic plants showed the characteristics of prolonged shelf life over 50 d . The amount of ethylene released from DR-1 and DR-2 fruits were reduced significantly to about 9.5% of that released by non-transformed controls.

Shi Yan Sheng Wu Xue Bao, 2003 Dec, 36(6), 407 - 13
{Culture of hairy roots in Pueraria phaseoloides and its puerarin production}; Shi HP et al.; An efficient transformation system for genetic transformation of medicinal plant, Pueraria phaseoloides was developed, by using agropine-type Agrobacterium rhizogenes ATCC15834 . Hairy roots could be obtained via callus from the cut edges of leaf explants of P . phaseoloides 20 days after inoculation with agrobacterium . 35 days after infection, the percentage of rooted leaf explants was about 85% . Hairy roots could have an autonomous growth on solid or liquid growth regulator-free MS medium but grew more rapidly and formed no callus during culture in liquid medium . The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes of Ri plasmid from A . rhizogenes . To investigate the physiological difference between solid and liquid culture, the mitochondrial membrane potential in hairy roots cultured for 15 days in solid and liquid medium were also detected by the method of fluorescence labeling of 3,3'-dipropylthiadicarbocyanide iodide . The results showed mitochondrial membrane potential of hairy roots in liquid medium was 11.8 times higher than that on solid medium . The content of puerarin in hairy roots reached a level of 1.190 mg/g.dry.wt and was 2.5 times as much as that in the roots of P . phaseoloides seedlings and was also 1.067 times as much as that in the crude drug of several year-old Pueraria roots . Our experiments have laid a foundation for large-scale production of puerarin in P . phaseoloides hairy roots.

Proc Natl Acad Sci U S A, 2004 Jan 27, 101(4), 986 - 91 Epub 2004 Jan 13.
A functional cellulose synthase from ascidian epidermis; Matthysse AG et al.; Among animals, urochordates (e.g., ascidians) are unique in their ability to biosynthesize cellulose . In ascidians cellulose is synthesized in the epidermis and incorporated into a protective coat know as the tunic . A putative cellulose synthase-like gene was first identified in the genome sequences of the ascidian Ciona intestinalis . We describe here a cellulose synthase gene from the ascidian Ciona savignyi that is expressed in the epidermis . The predicted C . savignyi cellulose synthase amino acid sequence showed conserved features found in all cellulose synthases, including plants, but was most similar to cellulose synthases from bacteria, fungi, and Dictyostelium discoidium . However, unlike other known cellulose synthases, the predicted C . savignyi polypeptide has a degenerate cellulase-like region near the carboxyl-terminal end . An expression construct carrying the C . savignyi cDNA was found to restore cellulose biosynthesis to a cellulose synthase (CelA) minus mutant of Agrobacterium tumefaciens, showing that the predicted protein has cellulose synthase activity . The lack of cellulose biosynthesis in all other groups of metazoans and the similarity of the C . savignyi cellulose synthase to enzymes from cellulose-producing organisms support the hypothesis that the urochordates acquired the cellulose biosynthetic pathway by horizontal transfer.

Mol Plant Microbe Interact, 2004 Jan, 17(1), 55 - 61
Rme1 is necessary for Mi-1-mediated resistance and acts early in the resistance pathway; Martinez de Ilarduya O et al.; The tomato gene Mi-1 confers resistance to root-knot nematodes (Meloidogyne spp.), potato aphid, and whitefly . Using genetic screens, we have isolated a mutant, rme1 (resistance to Meloidogyne spp.), compromised in resistance to M . javanica and potato aphid . Here, we show that the rme1 mutant is also compromised in resistance to M . incognita, M . arenaria, and whitefly . In addition, using an Agrobacterium-mediated transient assay in leaves to express constitutive gain-of-function mutant Pto(L205D), we demonstrated that the rme1 mutation is not compromised in Pto-mediated hypersensitive response . Moreover, the mutation in rme1 does not result in increased virulence of pathogenic Pseudomonas syringae or Mi-1-virulent M . incognita . Using a chimeric Mi-1 construct, Mi-DS4, which confers constitutive cell death phenotype and A . rhizogenes root transformation, we showed that the Mi-1-mediated cell death pathway is intact in this mutant . Our results indicate that Rme1 is required for Mi-1-mediated resistance and acts either at the same step in the signal transduction pathway as Mi-1 or upstream of Mi-1.

Plant Cell Rep, 2004 Mar, 22(8), 584 - 93 Epub 2004 Jan 09.
Antifungal activity of stilbenes in in vitro bioassays and in transgenic Populus expressing a gene encoding pinosylvin synthase; Seppanen SK et al.; The effect of two stilbene compounds, pinosylvin and resveratrol, on the growth of several fungi was evaluated in plate tests . Wood decay tests were carried out with birch and aspen samples impregnated with the two stilbenes . In plate experiments, resveratrol had an enhancing effect on growth at concentrations where pinosylvin was already enough to prevent the growth of most fungi studied . Pinosylvin impregnated at 0.2% (w/w) concentration significantly reduced the decay caused by all fungi except Phellinus tremulae . In contrast, a resveratrol content of 0.8%, did not protect the wood from decay . A pinosylvin-synthase-encoding gene from Pinus sylvestris was transferred into aspen ( Populus tremula) and two hybrid aspen clones ( Populus tremulax tremuloides) by Agrobacterium tumefaciens-mediated transformation . Transgenic plants accumulated pinosylvin synthase-specific mRNA and showed stilbene synthase enzyme activity in vitro . Transgenic aspen line H4 showed increased resistance to Phellinus tremulae, while two hybrid aspen transformants decayed faster than the control trees . However, we were unable to detect the accumulation of stilbenes in the transgenic plantlets.

Transgenic Res, 2003 Dec, 12(6), 715 - 22
Transgenic tobacco expressing Pinellia ternata agglutinin confers enhanced resistance to aphids; Yao J et al.; Tobacco leaf discs were transformed with a plasmid, pBIPTA, containing the selectable marker neomycin phosphotransferase gene (nptII) and Pinellia ternata agglutinin gene (pta) via Agrobacterium tumefaciens-mediated transformation . Thirty-two independent transgenic tobacco plants were regenerated . PCR and Southern blot analyses confirmed that the pta gene had integrated into the plant genome and northern blot analysis revealed transgene expression at various levels in transgenic plants . Genetic analysis confirmed Mendelian segregation of the transgene in T1 progeny . Insect bioassays showed that transgenic plants expressing PTA inhibited significantly the growth of peach potato aphid (Myzus persicae Sulzer) . This is the first report that transgenic plants expressing pta confer enhanced resistance to aphids . Our study indicates that the pta gene can be used as a supplement to the snowdrop (Galanthus nivalis) lectin gene (gna) in the control of aphids, a sap-sucking insect pest causing significant yield losses of crops.

Transgenic Res, 2003 Dec, 12(6), 671 - 81
Transgenic tobacco and apple plants expressing biotin-binding proteins are resistant to two cosmopolitan insect pests, potato tuber moth and lightbrown apple moth, respectively; Markwick NP et al.; Tobacco (Nicotiana tabacum cv . Samsun) and apple (Malus x domestica cv . Royal Gala) plants expressing avidin or strepavidin were produced using Agrobacterium tumefaciens-mediated transformation . ELISA assays showed that avidin expression ranged from 3.1 to 4.6 microM in tobacco and from 1.9 to 11.2 microM in apple and streptavidin expression ranged from 11.4 to 24.5 microM in tobacco and from 0.4 to 14.6 microM in apple . Expressed at these levels, both biotin-binding proteins conferred a high level of insect resistance on transformed tobacco plants to larval potato tuber moth (PTM), Phthorimaea operculella (Zeller) (fam . Gelechiidae) and on apple plants to larvae of the lightbrown apple moth (LBAM) Epiphyas postvittana (Walker) (fam . Tortricidae) . More than 90% of PTM larvae died on tobacco plants expressing either avidin or streptavidin genes within 9 days of inoculation . Mortality of LBAM larvae was significantly higher (P < 0.05) on three avidin-expressing (89.6, 84.9 and 80.1%) and two streptavidin-expressing (90 and 82.5%) apple plant lines than on non-transformed control plants (14.1%) after 21 days . Weight of LBAM larvae was also significantly reduced by feeding on all apple shoots expressing avidin and on apple shoots expressing streptavidin at levels of 3.8 microM and above.

Appl Environ Microbiol, 2004 Jan, 70(1), 404 - 12
Growth of Escherichia coli coexpressing phosphotriesterase and glycerophosphodiester phosphodiesterase, using paraoxon as the sole phosphorus source; McLoughlin SY et al.; Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies . The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation . A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries . To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source . The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate . The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source . Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source . The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase . We isolated and characterized the genes encoding the phosphohydrolase operon . The operon was identified from a shotgun clone that enabled E . coli to grow when dimethyl phosphate is the sole phosphorus source . E . coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source . By constructing a short degradative pathway, we have enabled E . coli to use phosphotriesters as a sole source of phosphorus.

J Bacteriol, 2004 Jan, 186(2), 535 - 42
An evolutionary hot spot: the pNGR234b replicon of Rhizobium sp . strain NGR234; Streit WR et al.; Rhizobium sp . strain NGR234 has an exceptionally broad host range and is able to nodulate more than 112 genera of legumes . Since the overall organization of the NGR234 genome is strikingly similar to that of the narrow-host-range symbiont Rhizobium meliloti strain 1021 (also known as Sinorhizobium meliloti), the obvious question is why are the spectra of hosts so different? Study of the early symbiotic genes of both bacteria (carried by the SymA plasmids) did not provide obvious answers . Yet, both rhizobia also possess second megaplasmids that bear, among many other genes, those that are involved in the synthesis of extracellular polysaccharides (EPSs) . EPSs are involved in fine-tuning symbiotic interactions and thus may help answer the broad- versus narrow-host-range question . Accordingly, we sequenced two fragments (total, 594 kb) that encode 575 open reading frames (ORFs) . Comparisons revealed 19 conserved gene clusters with high similarity to R . meliloti, suggesting that a minimum of 28% (158 ORFs) of the genetic information may have been acquired from a common ancestor . The largest conserved cluster carried the exo and exs genes and contained 31 ORFs . In addition, nine highly conserved regions with high similarity to Agrobacterium tumefaciens C58, Bradyrhizobium japonicum USDA110, and Mesorhizobium loti strain MAFF303099, as well as two conserved clusters that are highly homologous to similar regions in the plant pathogen Erwinia carotovora, were identified . Altogether, these findings suggest that >/==" BORDER="0">40% of the pNGR234b genes are not strain specific and were probably acquired from a wide variety of other microbes . The presence of 26 ORFs coding for transposases and site-specific integrases supports this contention . Surprisingly, several genes involved in the degradation of aromatic carbon sources and genes coding for a type IV pilus were also found.

J Bacteriol, 2004 Jan, 186(2), 445 - 53
The HWE histidine kinases, a new family of bacterial two-component sensor kinases with potentially diverse roles in environmental signaling; Karniol B et al.; Two-component signal transduction pathways play a major role in the response of bacteria to external cues . These pathways are initiated by large collection of histidine kinases (HKs) containing a sensor domain that perceives the environmental signal followed by an HK domain that triggers a histidine-aspartate phosphorelay . Previous phylogenetic analyses identified 11 major families of two-component HKs by comparing signature motifs within the HK domain . Here we describe a new family with homology to Agrobacterium tumefaciens BphP2, an HK first discovered by the presence of a phytochrome sensor domain involved in light perception . Members of this sensor HK family differ from most others by the absence of a recognizable F box and the presence of several uniquely conserved residues, including a histidine in the N box and a tryptophan-X-glutamic acid sequence in the G1 box, which we have used to define the family (HWE) . At least 81 members were identified in a variety of alpha- and gamma-proteobacteria, with a significant enrichment in the Rhizobiaceae family . Several representatives were shown to have HK activity in vitro, supporting their proposed participation in phosphorelays . One or more domains related to signal transduction were evident N-terminal to the HK domain, including chemotactic methyltransferase domains, suggesting that this family has multiple roles in environmental signaling . The discovery of the HWE family further extends the diversity within the HK superfamily and expands the importance of two-component signaling in bacteria.

Plant J, 2004 Jan, 37(2), 218 - 28
New plant growth-modifying properties of the Agrobacterium T-6b oncogene revealed by the use of a dexamethasone-inducible promoter; Gremillon L et al.; Agrobacterium 6b oncogenes induce tumours on Nicotiana glauca and enations and associated modifications in transgenic N . tabacum plants . 2x35S-AB-6b tobacco rootstocks produced a graft-transmissible factor that induced enations in wild-type scions; the nature of this enation factor remains to be identified . Here, we report on the properties of tobacco plants carrying a dexamethasone-inducible T-6b gene (dex-T-6b) . Induction with dex led to complex growth modifications, many of which have not been reported previously . Modifications were only found in growing tissues; mature tissues remained unaffected . Growth could be either stimulated or inhibited . Dex induction of young plants led to morphogenetic gradients that included enations, tubular leaves and fragmented leaf primordia . Root elongation was increased or slowed down, while radial root growth was strongly enhanced . Additional cell divisions were found in the root pericycle and vasculature . Enation factor import from mature tissues did not have the same effects on growing tissues as local T-6b synthesis: normal scions grafted on induced dex-T-6b rootstocks formed enations, whereas local dex-T-6b induction at the shoot apex led to numerous dark-green spots on the abaxial side of the leaves . In leaf patch assays, the 23-kDa T-6b protein was found to move through leaves and to enter the vascular system . This and the fact that rootstocks of spontaneous tobacco enation mutants did not modify wild-type scions contrary to 6b plants indicate that the 6b protein might be the enation factor.

Plant Cell Rep, 2004 Jan, 22(6), 359 - 64 Epub 2003 Sep 04.
Production of transgenic lily plants by Agrobacterium-mediated transformation; Hoshi Y et al.; A system for the production of transgenic plants was developed for the Oriental hybrid lily, Lilium cv . Acapulco, by Agrobacterium-mediated genetic transformation . Filament-derived calli were co-cultivated with A . tumefaciens strain EHA101/pIG121Hm, which harbored a binary vector carrying the neomycin phosphotransferase II, hygromycin phosphotransferase, and intron-containing beta-glucuronidase genes in the T-DNA region . Six hygromycin-resistant (Hyg(r)) culture lines were obtained from 200 calli by scratching them with sandpaper prior to inoculation and using NH(4)NO(3)-free medium for co-cultivation and a hygromycin-containing regeneration medium for selection . Hyg(r) culture lines regenerated shoots, which developed into plantlets following transfer to a plant growth regulator-free medium . All of these plantlets were verified to be transgenic by GUS histochemical assay and inverse PCR analysis.

Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 146 - 8 Epub 2003 Dec 18.
Crystallization and preliminary X-ray diffraction studies of the quorum-sensing regulator TraM from Agrobacterium tumefaciens; Vannini A et al.; TraM is a 11.4 kDa protein involved in the control of the conjugal transfer of Agrobacterium tumefaciens Ti plasmids by quorum-sensing . TraM was overexpressed and purified from Escherichia coli . This protein binds to the transcriptional regulator TraR, abolishing its function . Size-exclusion chromatography and dynamic light scattering show that the recombinant protein has an apparent molecular weight of 30 kDa in solution . Crystals have been obtained of both native and selenomethionine-substituted TraM by the vapour-diffusion method . Crystals diffract to 1.67 A and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 76.43, b = 47.09, c = 47.46 A and two molecules in the asymmetric unit . A two-wavelength MAD data set for the selenomethionine-substituted form has been collected to a resolution of 2.0 A . The selenium substructure (five out of six possible sites) has been solved using direct methods.

Biotechnol Lett, 2003 Nov, 25(21), 1837 - 42
Optimization of a fermentation medium using neural networks and genetic algorithms; Nagata Y et al.; Artificial neural networks and genetic algorithms are used to model and optimize a fermentation medium for the production of the enzyme hydantoinase by Agrobacterium radiobacter . Experimental data reported in the literature were used to build two neural network models . The concentrations of four medium components served as inputs to the neural network models, and hydantoinase or cell concentration served as a single output of each model . Genetic algorithms were used to optimize the input space of the neural network models to find the optimum settings for maximum enzyme and cell production . Using this procedure, two artificial intelligence techniques have been effectively integrated to create a powerful tool for process modeling and optimization.

Plant J, 2004 Jan, 37(1), 46 - 60
The tomato resistance protein Bs4 is a predicted non-nuclear TIR-NB-LRR protein that mediates defense responses to severely truncated derivatives of AvrBs4 and overexpressed AvrBs3; Schornack S et al.; The Lycopersicon esculentum Bs4 resistance (R) gene specifies recognition of Xanthomonas campestris pv . vesicatoria (Xcv) strains that express the cognate AvrBs4 avirulence protein . Bs4 was isolated by positional cloning and is predicted to encode a nucleotide-binding leucine-rich repeat (NB-LRR) protein that is homologous to tobacco N and potato Y-1 resistance proteins . Xcv infection tests demonstrate that Bs4 confers perception of AvrBs4 but not the 97% identical AvrBs3 protein . However, when delivered via Agrobacterium T-DNA transfer, both, avrBs4 and avrBs3 trigger a Bs4-dependent hypersensitive response, indicating that naturally occurring AvrBs3-homologues provide a unique experimental platform for molecular dissection of recognition specificity . Transcript studies revealed intron retention in Bs4 transcripts . Yet, an intron-deprived Bs4 derivative still mediates AvrBs4 detection, suggesting that the identified splice variants are not crucial to resistance . The L . pennellii bs4 allele, which is >98% identical to L . esculentum Bs4, has a Bs4-like exon-intron structure with exception of a splice polymorphism in intron 2 that causes truncation of the predicted bs4 protein . To test if the receptor-ligand model is a valid molecular description of Bs4-mediated AvrBs4 perception, we conducted yeast two-hybrid studies . However, a direct interaction was not observed . Defense signaling of the Bs4-governed reaction was studied in Nicotiana benthamiana by virus-induced gene silencing and showed that Bs4-mediated resistance is EDS1- and SGT1-dependent.

J Zhejiang Univ Sci, 2004 Feb, 5(2), 144 - 8
Preliminary study on a gravity-insensitive rice mutant; Jin J et al.; A gravity-insensitive mutant was isolated from rice (Oryza sativa L . cv . Zhonghua 11) transformed by Agrobacterium tumefaciens . The mutant's shoot growth (prostrate growth) was insensitive to gravity; whereas root growth displayed a normal positive gravitropism . Histological observation of root caps and leaf sheaths indicated that there was no significant difference in the number and size of amyloplasts in cells of the mutant and cells of the wild type.

Carbohydr Res, 2003 Nov 14, 338(23), 2721 - 30
Structural elucidation of a novel core oligosaccharide backbone of the lipopolysaccharide from the new bacterial species Agrobacterium larrymoorei; Molinaro A et al.; Agrobacterium larrymoorei is a Gram-negative phytopathogenic bacterium, which produces tumours on Ficus benjamina plants and differs from other Agrobacteria both genetically and biochemically . The lipopolysaccharide (LPS) plays an important role in the pathogenesis of Agrobacteria . The present paper is the first report on the molecular primary structure of the core region of an Agrobacterium LPS . The following structure of the core and lipid A carbohydrate backbone of an R-form LPS of A . larrymoorei was determined by chemical degradations and 1D and 2D NMR spectroscopy: {carbohydrate structure: see text} All sugars are alpha-D-pyranoses if not stated otherwise, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, Qui3NAcyl is 3,6-dideoxy-3-(3-hydroxy-2,3-dimethyl-5-oxoprolylamino)glucose, GlcAN and GalAN are amides of GlcA and GalA.

Syst Appl Microbiol, 2003 Nov, 26(4), 483 - 94
The variable part of the dnaK gene as an alternative marker for phylogenetic studies of rhizobia and related alpha Proteobacteria; Stepkowski T et al.; DnaK is the 70 kDa chaperone that prevents protein aggregation and supports the refolding of damaged proteins . Due to sequence conservation and its ubiquity this chaperone has been widely used in phylogenetic studies . In this study, we applied the less conserved part that encodes the so-called alpha-subdomain of the substrate-binding domain of DnaK for phylogenetic analysis of rhizobia and related non-symbiotic alpha-Proteobacteria . A single 330 bp DNA fragment was routinely amplified from DNA templates isolated from the species of the genera, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium, but also from some non-symbiotic alpha Proteobacteria such as Blastochloris, Chelatobacter and Chelatococcus . Phylogenetic analyses revealed high congruence between dnaK sequences and 16S rDNA trees, but they were not identical . In contrast, the partition homogeneity tests revealed that dnaK sequence data could be combined with other housekeeping genes such as recA, atpD or glnA . The dnaK trees exhibited good resolution in the cases of the genera Mesorhizobium, Sinorhizobium and Rhizobium, even better than usually shown by 16S rDNA phylogeny . The dnaK phylogeny supported the close phylogenetic relationship of Rhizobium galegae and Agrobacterium tumefaciens (R . radiobacter) C58, which together formed a separate branch within the fast-growing rhizobia, albeit closer to the genus Sinorhizobium . The Rhizobium and Sinorhizobium genera carried an insertion composed of two amino acids, which additionally supported the phylogenetic affinity of these two genera, as well as their distinctness from the Mesorhizobium genus . Consistently with the phylogeny shown by 16S-23S rDNA intergenic region sequences, the dnaK trees divided the genus Bradyrhizobium into three main lineages, corresponding to B . japonicum, B . elkanii, and photosynthetic Bradyrhizobium strains that infect Aeschynomene plants . Our results suggest that the 330 bp dnaK sequences could be used as an additional taxonomic marker for rhizobia and related species (alternatively to the 16S rRNA gene phylogeny).

Microbiology, 2003 Dec, 149(Pt 12), 3461 - 71
Pathways for phosphatidylcholine biosynthesis in bacteria; Martinez-Morales F et al.; Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes with important structural and signalling functions . Although many prokaryotes lack PC, it can be found in significant amounts in membranes of rather diverse bacteria . Two pathways for PC biosynthesis are known in bacteria, the methylation pathway and the phosphatidylcholine synthase (PCS) pathway . In the methylation pathway, phosphatidylethanolamine is methylated three times to yield PC, in reactions catalysed by one or several phospholipid N-methyltransferases (PMTs) . In the PCS pathway, choline is condensed directly with CDP-diacylglyceride to form PC in a reaction catalysed by PCS . Using cell-free extracts, it was demonstrated that Sinorhizobium meliloti, Agrobacterium tumefaciens, Rhizobium leguminosarum, Bradyrhizobium japonicum, Mesorhizobium loti and Legionella pneumophila have both PMT and PCS activities . In addition, Rhodobacter sphaeroides has PMT activity and Brucella melitensis, Pseudomonas aeruginosa and Borrelia burgdorferi have PCS activities . Genes from M . loti and L . pneumophila encoding a Pmt or a Pcs activity and the genes from P . aeruginosa and Borrelia burgdorferi responsible for Pcs activity have been identified . Based on these functional assignments and on genomic data, one might predict that if bacteria contain PC as a membrane lipid, they usually possess both bacterial pathways for PC biosynthesis . However, important pathogens such as Brucella melitensis, P . aeruginosa and Borrelia burgdorferi seem to be exceptional as they possess only the PCS pathway for PC formation.

Plant Cell, 2004 Jan, 16(1), 157 - 71 Epub 2003 Dec 05.
A plant caspase-like protease activated during the hypersensitive response; Chichkova NV et al.; To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV) . For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target . In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV . A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves . A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo . Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.

DNA Cell Biol, 2003 Nov, 22(11), 743 - 52
Identification and characterization of the DdlB, FtsQ and FtsA genes upstream of FtsZ in Bartonella bacilliformis and Bartonella henselae; Fiskus W et al.; Homologues of the cell division protein FtsZ were previously identified in Bartonella bacilliformis and Bartonella henselae . We report herein that ftsZ is located at the distal end of an operon that includes ddlB, ftsQ, and ftsA . These genes code for homologues of D-alanine D-alanine ligase, an enzyme involved in cell wall biosynthesis, and FtsQ, and FtsA, which are involved in cell division . The DdlB, FtsQ, and FtsA proteins from Bartonella species are most homologous to proteins in closely related species from the Order Rhizobiales, such as Brucella sp., Agrobacterium tumefaciens, and M . loti . The organization of the genes within the ddlB-ftsZ operon of B . bacilliformis and B . henselae (5'ddlB-ftsQ-ftsA-ftsZ 3') is similar to that of Mesorhizobium loti and Escherichia coli . We report the localization of three promoter regions within the ddlB-ftsA sequence of B . bacilliformis that may enhance the transcription of ftsZ mRNA . A promoter region was also identified upstream of the ddlB gene.

J Plant Physiol, 2003 Nov, 160(11), 1401 - 6
Isoenzymes of peroxidase and esterase related to morphogenesis in Mammillaria gracillis Pfeiff . tissue culture; Balen B et al.; In vitro propagated plants of the cactus Mammillaria gracillis Pfeiff . (Cactaceae) spontaneously produced callus . The habituated callus regenerated normal and hyperhydric shoots without the addition of grown regulators . Tumours were obtained by infecting cactus explants with Agrobacterium tumefaciens; the wild strain B6S3 (tumour TW) or with the rooty mutant GV3101 (tumour TR) . Both tumour lines grew vigorously, never expressing any morphogenic potential . In this study, cactus shoots, callus, normal and hyperhydric regenerants and TW and TR tumours were compared with regard to peroxidase (EC 1.11.1.7) and esterase activity, and isoenzyme patterns . Guaiacol peroxidase activity was the lowest in the cactus shoots and in the normal regenerants . Callus, hyperhydric regenerants and tumours had peroxidase activity of 6 to 7 times higher . Esterase activity was measured with 1- and 2-naphthylacetate as broad-spectrum substrates . The highest esterase activity was determined in tumours with both substrates . All tissues, except the TR tumour, had higher esterase activity for 2-compared to 1-naphtylacetate . Peroxidase and esterase isoenzyme patterns were not completely identical among the investigated tissues.

J Plant Physiol, 2003 Nov, 160(11), 1305 - 11
Transgenic tobacco plants overexpressing cotton glutathione S-transferase (GST) show enhanced resistance to methyl viologen; Yu T et al.; A GST (EC 2.5.1.18) gene (Gst-cr 1) from cotton was introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation . Transgenic tobacco plants overexpressing Gst-cr1 were normal in growth and mature compared with control, but had much higher levels of GST and GPx activities and showed an enhanced resistance to oxidative stress induced by a low concentration of methyl viologen (MV) . Six antioxidant enzymes, glutathione S-transferase, glutathione peroxidase (EC 1.11.1.9), superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), and ascorbate peroxidase (EC 1.11.1.11) were monitored in transgenic lines and non-transgenic control during MV treatments . When they were treated with 0.03 mmol/L of MV, both transgenic lines and control showed a rapid increase in the activities of GST, GPx, SOD, POD, APx, while the activity of CAT seemed to be irregular . The percent of the increase in SOD and POD activities was much higher in control than in transgenic plants . When treated with 0.05 mmol/L of MV, both control and transgenic plants were severely damaged, and the activities of the six enzymes decreased sharply.

Plant Cell Rep, 2004 Mar, 22(8), 615 - 22 Epub 2003 Dec 02.
Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination, callus and shoot formation; Zhang XH et al.; A novel acetyltransferase (Mpr1) found in Saccharomyces cerevisiae (strain Sigma1278b) has been shown to specifically detoxify a proline analog, l-azetidine-2-carboxylic acid (A2C) in yeast cells {M . Shichiri et al . (2001) J Biol Chem 276: 41998-42002} . We investigated whether the yeast MPR1 gene would function similarly in a plant system and if its expression could confer resistance to proline analogs . The MPR1 gene coding sequence driven by two different constitutive promoters, with or without the 5'- and 3'-noncoding sequence from the MPR1 gene adjacent to the conventional NOS terminator, was transformed into tobacco ( Nicotiana tabacum L . cv . Xanthi) plants via Agrobacterium tumefaciens infection . The presence of the yeast 5'- and 3'-noncoding sequences appeared to increase the likelihood of MPR1 gene expression in the transgenic plants . The kanamycin-selected transgenic plants with a high level of Mpr1 activity grew normally, and their progeny expressed acetyltransferase activity that could utilize A2C, azetidine-3-carboxylic acid and 4-hydroxy- l-proline as substrates . Resistance to A2C, but not to the other two analogs, was exhibited during leaf tissue culture and seed germination . The A2C toxicity to the wild-type plants was reversed by the addition of proline, suggesting that A2C acts as a proline analog . Our studies confirm that MPR1 can function in a similar fashion in tobacco as in yeast to detoxify the toxic proline analog A2C, so it could potentially be used as a new selectable marker for plant transformation . However, our attempts to utilize MPR1 as an efficient selectable marker gene for the A . tumefaciens-mediated transformation of tobacco were unsuccessful.

Appl Biochem Biotechnol, 2003 Nov, 111(2), 81 - 92
Degradation of ferric EDTA by Burkhol cepacia; Fang HY et al.; EDTA, the target compound of this study from the effluent of secondary biotreatment units, can be biodegraded by special microorganisms . So far, there are three species of microorganisms--Agrobacterium, Gram-negative BNCI, and DSM9103--that can degrade EDTA and are published in the literature . We have successfully isolated a bacterial strain that can degrade EDTA . It was identified as Burkhol cepacia, an aerobic species, elliptically shaped with a length of 5-15 microm . The growth medium contains 1000 mg/L of ferric-EDTA as carbon source, 750 mg/L of (NH4)2SO4 + (NH2)2CO as nitrogen source, and 1000 mg/L of KH2PO4 as phosphorus source, and mineral factors such as Fe and Mg . Incubated at pH 7.0, 30 degrees C, and 150 rpm on a shaker for 15 d, the average specific growth rate of this microbe is 0.135 d-1, which shows that the respective degradation efficiency of Fe-EDTA and Cu-EDTA is 90 and 75% individually.

Gene, 2003 Dec 11, 322, 67 - 75
Complete nucleotide sequence of the circular megaplasmid pHCG3 of Oligotropha carboxidovorans: function in the chemolithoautotrophic utilization of CO, H(2) and CO(2); Fuhrmann S et al.; Oligotropha carboxidovorans harbors the low-copy-number, circular, 133,058-bp DNA megaplasmid pHCG3, which is essential in the chemolithoautotrophic utilization of CO (carboxidotrophy), H(2) (hydrogenotrophy) and CO(2) under aerobic conditions . The complete nucleotide sequence of pHCG3 revealed 125 open reading frames . Of these, 95 were identified as putative structural genes . The plasmid carries the four gene clusters cox (14.54 kb, 12 genes), cbb (13.33 kb, 13 genes), hox (23.35 kb, 19 genes plus one ORF) and tra/trb (25.01 kb, 22 genes plus 2 ORFs), which assemble the functions required for the utilization of CO, CO(2) or H(2), and the conjugal transfer of the plasmid, respectively . The gene clusters cox, cbb and hox form a 51.2-kb chemolithoautotrophy module . The tra/trb cluster on the plasmid pHCG3 of O . carboxidovorans has a similar architecture as the Ti-plasmid of Agrobacterium tumefaciens . The tra/trb cluster is separated from the chemolithoautotrophy module by two regions (25.2 and 29.6 kb) with miscellaneous or mostly unknown functions . These regions carry a number of single genes coding for replication and stabilization of pHCG3 as well as the components of a putative system of global regulation of plasmid replication in O . carboxidovorans . An oriV encodes the replication proteins RepABC . Sequence comparisons of pHCG3-encoded genes suggest that major genetic exchange between O . carboxidovorans and the proteobacteria has occurred.

Fungal Genet Biol, 2004 Jan, 41(1), 33 - 41
Isolation and disruption of the melanin pathway polyketide synthase gene of the softwood deep stain fungus Ceratocystis resinifera; Loppnau P et al.; Ceratocystis resinifera hyphae produce a black melanin pigment causing a deep stain in softwood logs . We exploited the homology of polyketide synthases to clone PKS1, a gene responsible for dihydroxynaphthalene-melanin biosynthesis in C . resinifera . Sequence analysis indicated that PKS1 has two introns near its 5(') end and encodes a 2188-amino acid polypeptide with five functional domains: beta-ketoacyl synthase, acyl transferase, two acyl carrier proteins and a thioesterase/Claisen cyclase . A gene disruption construct designed to replace a portion of PKS1 with a hygromycin resistance cassette was transformed into C . resinifera through Agrobacterium tumefaciens-mediated transformation . PKS1 null mutants had an albino phenotype, and pigmentation was restored by the addition of scytalone, a melanin pathway intermediate . The disruption of PKS1 and restoration of pigmentation with scytalone confirmed the presence of a dihydroxynaphthalene-melanin pathway in C . resinifera . The transformation method described in this paper is the first reported for a Ceratocystis species.

Infect Immun, 2003 Dec, 71(12), 7053 - 60
Attenuated signature-tagged mutagenesis mutants of Brucella melitensis identified during the acute phase of infection in mice; Lestrate P et al.; For this study, we screened 1,152 signature-tagged mutagenesis mutants of Brucella melitensis 16M in a mouse model of infection and found 36 of them to be attenuated in vivo . Molecular characterization of transposon insertion sites showed that for four mutants, the affected genes were only present in Rhizobiaceae . Another mutant contained a disruption in a gene homologous to mosA, which is involved in rhizopine biosynthesis in some strains of Rhizobium, suggesting that this sugar may be involved in Brucella pathogenicity . A mutant was disrupted in a gene homologous to fliF, a gene potentially coding for the MS ring, a basal component of the flagellar system . Surprisingly, a mutant was affected in the rpoA gene, coding for the essential alpha-subunit of the RNA polymerase . This disruption leaves a partially functional protein, impaired for the activation of virB transcription, as demonstrated by the absence of induction of the virB promoter in the Tn5::rpoA background . The results presented here highlight the fact that the ability of Brucella to induce pathogenesis shares similarities with the molecular mechanisms used by both Rhizobium and Agrobacterium to colonize their hosts.

Gene, 2003 Dec 4, 321, 113 - 21
Construction of a genomic library of wild rice and Agrobacterium-mediated transformation of large insert DNA linked to BPH resistance locus; He RF et al.; Here we report the first genomic library of wild rice constructed on a plant-transformation-competent binary vector (BIBAC2) and transformation of the large insert DNA into rice via Agrobacterium . We selected Oryza officinalis for genomic library construction . The library consists of 55,296 clones and stored in one hundred forty-four 384-well plates . Random sampling of 140 clones indicated an average insert size of 71 Kb at a range of 15-235 Kb and 4.8% empty vectors . Four wheat chloroplast probes and four maize mitochondrial probes were hybridized separately to the library, showing that contamination with organellar DNAs is very low (0.61% and 0.04%, respectively) . The binary bacterial artificial chromosome (BIBAC) library provides 5.3 haploid genome equivalents, implying a 99.5% probability of recovering any specific sequence of interest . A stability test indicated that the large DNA inserts were stable in this BIBAC vector both in host cells of Escherichia coli and Agrobacterium . Two restriction-fragment length polymorphism (RFLP) markers R288 and C820, which co-segregate with brown planthopper (BPH) resistance gene Qbp2, were used to screen the library, and identified seven and eight positive clones, respectively . The candidate clones of target gene isolated from the library are directly used to transform cultivated rice . After screening the Agrobacterium strains and helper plasmids, and using an improved procedure of transformation, a BIBAC clone with 120 Kb O . officinalis DNA insert was successfully transferred into the rice genome via Agrobacterium-mediated transformation . The system developed here should serve as source for gene discovery, gene cloning and genome-related research in wild rice.

Biochemistry, 2003 Dec 2, 42(47), 14057 - 65
Steady-state kinetics and tryptophan fluorescence properties of halohydrin dehalogenase from Agrobacterium radiobacter . Roles of W139 and W249 in the active site and halide-induced conformational change; Tang L et al.; Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 is a homotetrameric protein containing four tryptophan residues per subunit . The fluorescence properties of the enzyme are strongly influenced by halide binding . To examine the role of the tryptophans (W139, W192, W238, and W249) in halide binding and catalysis, they were individually mutated to a phenylalanine . All mutations, except for W238F, influenced the enzymatic properties . Mutating W192 to phenylalanine inactivated the enzyme and led to dissociation into dimers and monomers . In the structure of HheC, residue W139 and residue W249 from the opposite subunit are close to the active site of the enzyme . Substitution of W139 mainly affected K(m) values with all tested substrates and reduced the enantiopreference for p-nitro-2-bromo-1-phenylethanol . Replacing W249 increased both k(cat) and K(m) values with all tested substrates except for the (S)-enantiomer of p-nitro-2-bromo-1-phenylethanol, for which k(cat) was 3-fold decreased, resulting in a 6-fold increase of the enantioselectivity . Fluorescence measurements revealed that in the ligand-free state the intrinsic protein fluorescence of mutant W139F is higher than that of the wild-type enzyme, while the fluorescence intensity of mutants W238F and W249F was lower . The fluorescence intensities of the W238F and W249F enzymes were increased when they were unfolded or when bromide was added, whereas the fluorescence of mutant W139F was not increased by unfolding or addition of bromide . These results demonstrate that the fluorescence of residues W238 and W249 is partially quenched in the folded ligand-free state, and that W139 is completely quenched and acts as an energy acceptor for the other tryptophan residues as well . Changes of the maximum fluorescence emission wavelength of the HheC variants and the results of acrylamide quenching experiments confirmed that bromide binding induces a local conformational change around the active site, resulting in residue W139 and the quencher group being separated.

Curr Genet, 2004 Feb, 45(2), 111 - 9 Epub 2003 Nov 22.
Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana; Leclerque A et al.; Agrobacterium tumefaciens was used to stably transform the entomopathogenic deuteromycete Beauveria bassiana to hygromycin B resistance by integration of the hph gene of Escherichia coli into the fungal genome . The transformation protocol was optimized to generate a library of insertion mutants of Beauveria . Transformation frequencies around 10(-4) and suppression of background growth were achieved . Over 90% of the AIM mutants investigated contained single-copy T-DNA integrations at different chromosomal locations . Integrated T-DNAs were re-isolated from ten transformants by a marker rescue approach . When the sequences flanking these T-DNAs were compared with the corresponding locations of the wild-type genome, truncations of T-DNA borders were found to be common, while none of the sites of integration had suffered deletion or rearrangement . Thus, AIM can be considered a promising tool for insertional mutagenesis studies of entomopathogenic filamentous fungi.

Biotechnol Appl Biochem . 2003 Nov 21; {Epub ahead of print}
Development of a highly efficient system for assessing recombinant gene expression in plant cell suspensions via Agrobacterium tumefaciens transformation; Fuentes AD et al.; A transient gene expression system was developed and used to characterize promoter strength, to verify suitability of bacterial gene modifications for expression in plant cells, and to express active antibody molecules . The system is based on suspension tobacco cells transformed by Agrobacterium in a transient way . Conditions as pre-culture of tobacco cells, and co-cultivation period were identified as determinants to achieve high expression levels . Under established conditions the activity strength of CaMV 35S and ToMoTV AL1 promoters were compared . A modified cry gene sequence from Bacillus thuringiensis was expressed and detected by Western blot analysis . A monoclonal antibody against Hepatitis B virus surface antigen antiHBsAg was produced in quantities allowing testing biological activity and preliminary characterization.

Farmaco, 2003 Dec, 58(12), 1351 - 4
Cynthichlorine: a bioactive alkaloid from the tunicate Cynthia savignyi; Abourriche A et al.; From ether extracts of the tunicate Cynthia savignyi, collected in Morocco, a new alkaloid-cynthichlorine-has been isolated . The structure of cynthichlorine has been characterized by extensive 2D-NMR data . Cynthichlorine possesses antifungal activity against two tomato pathogenic fungi: Botrytis cinerea and Verticillium albo atrum and antibacterial activity against Agrobacterium radiobacter, Escherichia coli and Pseudomonas aeruginosa and cytotoxicity against Artemia salina larvae.

Curr Microbiol, 2003 Oct, 47(4), 323 - 6
Atypical adaptive and cross-protective responses against peroxide killing in a bacterial plant pathogen, Agrobacterium tumefaciens; Vattanaviboon P et al.; Physiological adaptive and cross-protection responses to oxidants were investigated in Agrobacterium tumefaciens . Exposure of A . tumefaciens to sublethal concentrations of H2O2 induced adaptive protection to lethal concentrations of H2O2 . Similar treatments with organic peroxide and menadione did not produce adaptive protection to subsequent exposure to lethal concentrations of these oxidants . Pretreatment of A . tumefaciens with an inducing concentration of menadione conferred cross-protection against H2O2, but not to tert-butyl hydroperoxide (tBOOH), killing . The menadione induced cross-protection to H2O2 was due to the compound's ability to highly induce the peroxide scavenging enzyme, catalase . The levels of catalase directly correlated with the bacterium's ability to survive H2O2 treatment . Some aspects of the oxidative stress response of A . tumefaciens differ from other bacteria, and these differences may be important in plant/microbe interactions.

Planta, 2004 Mar, 218(5), 759 - 66 Epub 2003 Nov 19.
Expression of a yeast-derived invertase in companion cells results in long-distance transport of a trisaccharide in an apoplastic loader and influences sucrose transport; Zuther E et al.; Companion cell-specific expression of a cytosolic invertase from yeast ( Saccharomyces cerevisiae) was used as a tool to synthesise oligosaccharides in the sieve element/companion cell complex and study whether oligosaccharides could be transported in the phloem of an apoplastically loading species . Potato ( Solanum tuberosum L.) plants expressing the invertase under the control of the Agrobacterium tumefaciens rolC promoter produced the trisaccharide 6-kestose in leaves, which was transported via the phloem and accumulated in tubers of transgenic plants . In graft experiments with rolC invertase plants as scion and wild-type rootstocks, 6-kestose accumulated in tubers to levels comparable to sucrose . This shows that long-distance transport of oligosaccharides is possible in apoplastically loading plants, which normally transport only sucrose . The additional transport route for assimilates neither led to elevated photosynthetic activity nor to increased tuber yield . Enhanced sucrose turnover in companion cells caused large amounts of glucose and fructose to be exuded from leaf petioles, and elevated levels of sucrose were detected in phloem exudates . While the latter indicates a higher capacity for sucrose loading into the phloem due to increased metabolic activity of companion cells, the massive release of hexoses catalysed by the invertase seemed to interfere with assimilate delivery to sink organs.

Plant Cell Rep, 2004 Apr, 22(9), 678 - 83 Epub 2003 Nov 18.
Heterologous expression of Arabidopsis ERS1 causes delayed senescence in coriander; Wang Y et al.; The phytohormone ethylene is involved in many developmental processes, including leaf and flower senescence . Ethylene is perceived by plants through receptors that trigger the downstream signal transduction pathway . The mutated ethylene receptor ERS1 (ethylene response sensor) from Arabidopsis is of a dominant negative nature and confers ethylene insensitivity in Arabidopsis . To investigate if the altered ERS1 gene can affect the tissue senescence in heterologous plants, we introduced it into coriander by Agrobacterium-mediated transformation . Transgenic plants were regenerated by cocultivating hypocotyl segments with A . tumefaciens harboring binary vector pCGN1547 that carried the ERS1 gene . The presence and expression of the transgene were confirmed by genomic Southern blot and reverse transcriptase-PCR analyses . Leaf and flower senescence were delayed significantly in the transgenic plants . The ability of the mutated ERS1 gene to confer the ethylene-insensitive phenotype can be exploited for extending the shelf-life of leafy vegetables.

J Exp Bot, 2004 Jan, 55(395), 151 - 7 Epub 2003 Nov 17.
Virus-induced gene silencing of jasmonate-induced direct defences, nicotine and trypsin proteinase-inhibitors in Nicotiana attenuata; Saedler R et al.; Research into the molecular basis of plant-insect interactions is hampered by the inability to alter the expression of individual genes in plants growing under natural conditions . The ability of virus-induced gene silencing (VIGS) to silence the expression of two jasmonate-induced genes known to mediate the expression of two potent direct defences (nicotine and proteinase inhibitors) that are produced in different tissues (roots and shoots, respectively) in Nicotiana attenuata is documented here . Fragments of consensus sequences of N . attenuata's putrescine N-methyltransferase (PMT) and trypsin inhibitor (TI) genes were cloned in sense, anti-sense and inverted repeat orientations into the Tobacco Rattle Virus (TRV) to trigger post-transcriptional gene silencing by Agrobacterium-mediated inoculation in plants previously elicited with methyl jasmonate (MeJA) or left as controls . MeJA treatment elicited 2.4- and 9.8-fold increases in the concentrations of nicotine and proteinase inhibitors, respectively, and inoculation with constructs containing appropriate genes inhibited these MeJA-induced increases and halved constitutive accumulations, regardless of the orientation of the gene fragment . Root PMT transcript levels were significantly elevated in MeJA-treated plants 10 h after elicitation, but not in plants inoculated with the appropriate TRV constructs 9 d prior to MeJA treatment, demonstrating that VIGS was responsible for the inhibition of these potent direct defences . While additional research is required to minimize the effects on plant growth and the risks of using such constructs in natural settings, it is concluded that VIGS has a potential to manipulate the expression of genes important for ecological interactions.

Curr Genet, 2004 Feb, 45(2), 104 - 10 Epub 2003 Nov 14.
Cloning and targeted disruption, via Agrobacterium tumefaciens-mediated transformation, of a trypsin protease gene from the vascular wilt fungus Verticillium dahliae; Dobinson KF et al.; A gene encoding a trypsin protease was isolated from a tomato isolate of Verticillium dahliae . The gene, designated VTP1, contains two introns and is predicted to encode a protein of 256 amino acids . The gene is present in V . dahliae isolates from different host plants and in V . albo-atrum; weakly hybridizing sequences are present in V . tricorpus . VTP1 cDNA sequences were identified in a sequence tag analysis of genes expressed under growth conditions that promote microsclerotia development . Replacement of the gene, by Agrobacterium tumefaciens-mediated transformation (ATMT), with a mutant allele construct did not noticeably alter either pathogenicity or growth in culture . Searches of expressed sequence tag databases showed that, in addition to the VTP1 gene, V . dahliae contains two genes encoding subtilisin-like proteases similar to those produced by pathogenic Aspergillus spp . This is the first description of the application of ATMT to the molecular analysis of phytopathogenic Verticillium spp.

Planta, 2004 Feb, 218(4), 536 - 41 Epub 2003 Nov 14.
A partially disarmed vir helper plasmid, pKYRT1, in conjunction with 2,4-dichlorophenoxyactic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean; Ko TS et al.; Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons . KYRT1 is derived from the highly oncogenic strain Chry5 . However, pKYRT1 is not completely disarmed and still contains an entire T-right (T(R)) and a portion of T-left (T(L)) . In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean . Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues . However, none of these transformed tissues developed SEs on medium with or without 2,4-dichlorophenoxyactic acid (2,4-D) . On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D . Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis . PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of transgenic T(0) soybean plants derived by transformation using strain KYRT1 contained T(R) from pKYRT1 in addition to the uidA gene from the binary construct . None of the transgenic tissues or T(0) plants contained T(L) DNA . These results suggest that some function coded for by T(R) of pKYRT1 influences somatic embryogenesis in conjunction with exposure of the plant tissues to 2,4-D . Since the co-transformation frequency of the undesirable T-DNA sequences from the vir helper plasmid was relatively low, the partially disarmed strain KYRT1 will likely be very useful for the production of normal transgenic plants of diverse soybean cultivars.

J Appl Genet, 2003, 44(4), 449 - 58
Agrobacterium-mediated transient GUS gene expression in buffel grass (Cenchrus ciliaris L.); Batra S et al.; The study was conducted to standardize a protocol for Agrobacterium-mediated genetic transformation of buffel grass (Cenchrus ciliaris L.) . Embryogenic calli, produced from one-year-old mature seeds of buffel grass, were used as target cells for Agrobacterium-mediated transformation . A . tumefaciens strain LBA4404, harbouring pCAMBIA-1301 or pCAMBIA-2301, was used for co-cultivation with embryogenic calli from three genotypes (IG-3108, IG-9757 and IG-97101) . Co-culturing of calli with Agrobacterium for 30 minutes, followed by co-cultivation with 0.1 mM acetosyringone for 3 days was found to be optimum for maximum transformation efficiency . Presence of acetosyringone during co-cultivation was found to be necessary for transformation . Transient GUS (beta-glucuronidase) gene expression was used to monitor T-DNA delivery into the target cells . Significant genotypic variations in response to transformation were observed among the tested genotypes . A very high frequency (63.3%) of GUS gene expression was obtained following Agrobacterium-mediated gene transfer into embryogenic calli . The standardized protocol would be useful for Agrobacterium-mediated genetic transformation of buffel grass with genes of agronomic importance.

Plant Cell Rep, 2004 Apr, 22(9), 645 - 52 Epub 2003 Nov 13.
Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration; Luo H et al.; Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved . Embryogenic callus initiated from seeds (cv . Penn-A-4) was infected with an A . tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter . Plants were regenerated from 219 independent transformation events . The overall stable transformation efficiency ranged from 18% to 45% . Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation . All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements . We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.

Plant Cell Rep, 2004 Mar, 22(8), 561 - 8 Epub 2003 Nov 13.
Transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens; Akutsu M et al.; An efficient procedure is described for the transformation of the monocotyledonous Alstroemeria by Agrobacterium tumefaciens via callus regeneration . Calli derived from ovules were co-cultivated with A . tumefaciens strains EHA101 and LBA4404, which harbored the binary vector plasmids pIG121Hm and pTOK233, respectively . These plasmids contain the beta-glucuronidase gene ( gusA) as a reporter gene and the hygromycin phosphotransferase and neomycin phosphotransferase II ( nptII) genes as selective markers . Inoculated calli were first plated for 4 weeks on medium containing cefotaxime to eliminate bacteria, following which time transformed cells were selected on medium that contained 20 mg/l hygromycin . A histochemical assay for GUS activity revealed that hygromycin-based selection was completed after 8 weeks . The integration of the T-DNA of pIG121Hm and pTOK233 into the genome of the cells was confirmed by PCR analysis . Efficient shoot regeneration from the transformed calli was observed on half-strength MS medium supplemented with 0.5 mg/l naphthaleneacetic acid and 0.5 mg/l benzyladenine after about 5 months of culture . The presence of the gusA and nptII genes in the genomic DNA of regenerated plants was detected by means of PCR and PCR-Southern hybridization, and the expression of these transgenes was verified by reverse transcription-PCR.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Nov, 35(11), 1011 - 5
{Transgenic potato containing immunogenic gene of avian coronavirus and its immunogenicity in mice}; Wu JX et al.; To check the feasibility of expression of the immunogenic gene of avian coronavirus infectious bronchitis virus (IBV) in plants, the transformation of S1 gene of IBV into potato and the immunogenicity of its expression product was studied . The S1 gene of IBV-ZJ971 strain was inserted into plasmid pBI121 under the control of 35 S promoter . Agrobacterium fumefaciens EHA105 with the recombinant vector pBI121 was obtained by tri-parental mating method . So, an efficient potato transformation system mediated by Agrobacterium fumefaciens was established . The rates of calli and shoots differentiation were 100%, and more than 95% respectively, for transgenic potato with S1 gene of IBV . PCR and Southern blot analyses showed that IBV S1 gene was integrated into genomic DNA of the potato plant and most transgenic plants had two copies of S1 gene of IBV . In our experiments, 47 transgenic plantlets have been obtained . Northern blot and ELISA analyses indicated that most transgenic plants could normally transcribe and translate S1 gene of IBV, though the levels of transcription and translation were different in various transgenic plants . Immunity assay with BALB/C mice showed that expression products of transgenic potato with S1 gene of IBV were immunogenic, and ELISA antibody titer reached 1:20 to 1:40 and 1:80 to 1:160 with doses of 0.5 g and 1 g, respectively . Virus neutralization (VN) antibodies were detected by tracheal organ cultures, and the results showed that VN titers reached respectively 1:160 to 1:320 and 1:320 to 1:2048 with doses of 0.5 g and 1 g.

Plant Physiol, 2003 Nov, 133(3), 1000 - 10
Agrobacterium-mediated root transformation is inhibited by mutation of an Arabidopsis cellulose synthase-like gene; Zhu Y et al.; Agrobacterium-mediated plant genetic transformation involves a complex interaction between the bacterium and the host plant . Relatively little is known about the role plant genes and proteins play in this process . We previously identified an Arabidopsis mutant, rat4, that is resistant to Agrobacterium transformation . We show here that rat4 contains a T-DNA insertion into the 3'-untranslated region of the cellulose synthase-like gene CSLA9 . CSLA9 transcripts are greatly reduced in the rat4 mutant . Genetic complementation of rat4 with wild-type genomic copies of the CSLA9 gene restores both transformation competence and the wild-type level of CSLA9 transcripts . The CSLA9 promoter shows a distinct pattern of expression in Arabidopsis plants . In particular, the promoter is active in the elongation zone of roots, the root tissue that we previously showed is most susceptible to Agrobacterium-mediated transformation . Disruption of the CSLA9 gene in the rat4 mutant results in reduced numbers and rate of growth of lateral roots and reduced ability of the roots to bind A . tumefaciens cells under certain conditions . No major differences in the linkage structure of the non-cellulosic polysaccharides could be traced to the defective CSLA9 gene.

J Plant Physiol, 2003 Oct, 160(10), 1253 - 7
Efficient and genotype-independent Agrobacterium--mediated tomato transformation; Park SH et al.; An efficient method to transform five cultivars of tomato (Lycopersicon esculentum), Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported . A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and on 11 different media with cocultivation . Although all cultivars and explants formed callus and regenerated on the initial 7 media, cocultivation with A . tumefaciens significantly reduced the callus induction and regeneration . From these experiments, a transformation methodology using either hypocotyls or cotyledons cultured for one day on BA 1 mgL-1, NAA 0.1 mgL-1 and 3 days cocultivation with the Agrobacterium on this same medium followed by a transfer to a medium with zeatin 2 mgL-1 and IAA 0.1 mgL-1 for 4-6 weeks resulted in a greater than 20% transformation frequency for all five cultivars tested . In this transformation method, no feeder layers of tobacco, petunia or tomato suspension cultures were used, and the subculture media was minimal . Stable integration and transmission of the transgene in T1 generation plants were confirmed by Southern blot analysis . This procedure represents a simple, efficient and general means of transforming tomato.

J Plant Physiol, 2003 Oct, 160(10), 1241 - 51
Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants; Wei H et al.; GUS (uidA) reporter gene expression for two sugarcane polyubiquitin promoters, ubi4 and ubi9, was compared to expression from the maize Ubi-1 promoter in stable transgenic rice (only ubi9) and sugarcane (ubi4 and ubi9) . Ubi9 drove high-level GUS expression, comparable to the maize Ubi-1 promoter, in both callus and regenerated plants of rice transformed by Agrobacterium . This high level expression was inherited in R1 plants . Expression from ubi4 and ubi9 was quite high in sugarcane callus transformed via particle bombardment . Expression dropped to very low or undetectable levels in the resulting plants; this drop in expression resulted from PTGS . PTGS in regenerated sugarcane plants also occurred with the maize Ubi-1 promoter . In sugarcane callus, ubi4 was HS inducible, but ubi9 was not . This physiological difference corresponds to a MITE insertion that is present in the putative HSEs of ubi9 but not present in ubi4.

Appl Environ Microbiol, 2003 Nov, 69(11), 6949 - 53
Agrobacterium bioassay strain for ultrasensitive detection of N-acylhomoserine lactone-type quorum-sensing molecules: detection of autoinducers in Mesorhizobium huakuii; Zhu J et al.; An ultrasensitive bioassay system for the detection of N-acylhomoserine lactones (AHLs) was constructed in Agrobacterium tumefaciens by using the T7 expression system to overproduce the AHL receptor TraR . This strain detected many diverse AHLs, some at extremely low concentrations . We used this strain to detect for the first time AHLs made by Mesorhizobium huakuii, which symbiotically fixes nitrogen in association with the legume Astragalus sinicus, a source of green manure throughout eastern Asia.

Appl Environ Microbiol, 2003 Nov, 69(11), 6731 - 9
Plant transformation by coinoculation with a disarmed Agrobacterium tumefaciens strain and an Escherichia coli strain carrying mobilizable transgenes; Pappas KM et al.; Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens . We constructed E . coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers . Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells . However, when an E . coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A . tumefaciens strain, plant tumors arose at high frequencies . This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E . coli to the disarmed A . tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells . The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity . Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants . These techniques circumvent the need to construct A . tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.

Appl Environ Microbiol, 2003 Nov, 69(11), 6434 - 41
Glycolytic breakdown of sulfoquinovose in bacteria: a missing link in the sulfur cycle; Roy AB et al.; Sulfoquinovose (6-deoxy-6-sulfo-D-glucopyranose), formed by the hydrolysis of the plant sulfolipid, is a major component of the biological sulfur cycle . However, pathways for its catabolism are poorly delineated . We examined the hypothesis that mineralization of sulfoquinovose to inorganic sulfate is initiated by reactions of the glycolytic and/or Entner-Doudoroff pathways in bacteria . Metabolites of {U-(13)C}sulfoquinovose were identified by (13)C-nuclear magnetic resonance (NMR) in strains of Klebsiella and Agrobacterium previously isolated for their ability to utilize sulfoquinovose as a sole source of carbon and energy for growth, and cell extracts were analyzed for enzymes diagnostic for the respective pathways . Klebsiella sp . strain ABR11 grew rapidly on sulfoquinovose, with major accumulations of sulfopropandiol (2,3-dihydroxypropanesulfonate) but no detectable release of sulfate . Later, when sulfoquinovose was exhausted and growth was very slow, sulfopropandiol disappeared and inorganic sulfate and small amounts of sulfolactate (2-hydroxy-3-sulfopropionate) were formed . In Agrobacterium sp . strain ABR2, growth and sulfoquinovose disappearance were again coincident, though slower than that in Klebsiella sp . Release of sulfate was still late but was faster than that in Klebsiella sp., and no metabolites were detected by (13)C-NMR . Extracts of both strains grown on sulfoquinovose contained phosphofructokinase activities that remained unchanged when fructose 6-phosphate was replaced in the assay mixture with either glucose 6-phosphate or sulfoquinovose . The results were consistent with the operation of the Embden-Meyerhoff-Parnas (glycolysis) pathway for catabolism of sulfoquinovose . Extracts of Klebsiella but not Agrobacterium also contained an NAD(+)-dependent sulfoquinovose dehydrogenase activity, indicating that the Entner-Doudoroff pathway might also contribute to catabolism of sulfoquinovose.

Transgenic Res, 2003 Oct, 12(5), 607 - 14
Transformation of tobacco with genes encoding Helianthus tuberosus agglutinin (HTA) confers resistance to peach-potato aphid (Myzus persicae); Chang T et al.; The effects of the hta gene encoding Helianthus tuberosus agglutinin (HTA) on an insect in the order Homoptera were investigated . Homologous cDNAs of hta-a, hta-b, hta-c and hta-d with CaMV35S as promoter were introduced into tobacco via Agrobacterium tumefaciens . Southern blot results showed that the exogenous hta gene was inserted into the genome of host plants, and northern blot analysis confirmed that hta was expressed in transgenic plants . A bioassay with peach-potato aphid (Myzus persicae) demonstrated that transgenic plants had deleterious effects on the insect . The average population of aphids fed on transgenic T0 plants during an 11-day assay decreased by 70%, compared controls . In transgenic plants of T1 generation, aphid fecundity inhibitions were 53.0% (hta-b) and 64.6% (hta-c), respectively . The development of aphids was notably retarded . We conclude that hta could be a novel and promising candidate for plant transgenic engineering against homopteran insect pests.

Tree Physiol, 2003 Dec, 23(17), 1209 - 15
Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene; Li DD et al.; Valencia sweet orange (Citrus sinensis (L.) Osbeck) calluses were used as explants to develop a new transformation system for citrus mediated by Agrobacterium tumefaciens . Factors affecting Agrobacterium-mediated transformation efficiency included mode of pre-cultivation, temperature of cocultivation and presence of acetosyringone (AS) . The highest transformation efficiency was obtained with a 4-day pre-cultivation period in liquid medium . Transformation efficiency was higher when cocultivation was performed for 3 days at 19 degrees C than at 23 or 28 degrees C . Almost no resistant callus was obtained if the cocultivation medium lacked AS . The transformation procedure yielded transgenic Valencia plants containing the pTA29-barnase gene, as verified by PCR amplification and confirmed by Southern blotting . Because male sterility is a common factor leading to seedlessness in citrus cultivars with parthenocarpic characteristics, production of seedless citrus genotypes by Agrobacterium-mediated genetic transformation is a promising alternative to conventional breeding methods.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 263 - 9
Isolation and characterization of periplasmic cyclic beta-glucans of Azorhizobium caulinodans; Komaniecka I et al.; Oligoglucose molecules isolated from Azorhizobium caulinodans were characterized by compositional analysis, Smith degradation, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and (1)H and (13)C nuclear magnetic resonance analysis . A . caulinodans produced nonbranched and unsubstituted cyclic glucans composed solely of glucose, with the degree of polymerization ranging from 10 to 13 . A major fraction of the periplasmic glucans contains 11 glucose residues within rings . The glucose residues are linked by beta-(1,3) and beta-(1,6) glycosidic bonds . These molecules seem to be quite similar to the periplasmic beta-(1,3);(1,6)-glucans synthesized by the Bradyrhizobium strain and are substantially different from the cyclic beta-(1,2)-glucans produced by Agrobacterium and Sinorhizobium species . Azorhizobial cyclic glucan synthesis is not osmoregulated . The response to the osmotic stress in Azorhizobium can be regulated similarly to Brucella spp . It is probable that the biosynthesis of beta-glucans is subject to the feedback control mechanism.

Curr Genet, 2004 Feb, 45(1), 54 - 60 Epub 2003 Oct 29.
Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation; Zeilinger S; A modified Agrobacterium-mediated transformation method for the efficient disruption of two genes encoding signaling compounds of the mycoparasite Trichoderma atroviride is described, using the hph gene of Escherichia coli as selection marker . The transformation vectors contained about 1 kb of 5' and 3' non-coding regions from the tmk1 (encoding a MAP kinase) or tga3 (encoding an alpha-subunit of a heterotrimeric G protein) target loci flanking a selection marker . Transformation of fungal conidia and selection on hygromycin-containing media applying an overlay-based procedure, which overcomes the lack of formation of distinct single colonies by the fungus, led to stable clones for both disruption constructs . Southern and PCR analyses proved gene disruption by single-copy homologous integration with a frequency of approximately 60% for both genes; and the loss of tmk1 and tga3 transcript formation in the disruptants was demonstrated by RT-PCR.

Plant Cell Rep, 2003 Nov, 22(4), 274 - 81 Epub 2003 Aug 29.
Development of a novel Agrobacterium-mediated transformation method to recover transgenic Brassica napus plants; Wang WC et al.; We report here an in planta method to produce transgenic Brassica napus plants . The procedure included Agrobacterium-mediated inoculation of plants at various development stages along with a vacuum infiltration step . The flowering stage appeared to be the most receptive stage for transformation and production of transgenic plants . In some cases, the flowering stage was induced either by cold treatment or by high density planting . Molecular and genetic analysis revealed that single and multiple copy events were generated and that the transgenes were transmitted to the T1 and T2 progeny in a Mendelian fashion.

J Exp Bot, 2003 Dec, 54(393), 2643 - 53 Epub 2003 Oct 29.
The soybean sucrose binding protein gene family: genomic organization, gene copy number and tissue-specific expression of the SBP2 promoter; Contim LA et al.; The sucrose binding protein (SBP) from soybean has been implicated as an important component of the sucrose uptake system . Two SBP genomic clones, gsS641.1 and gsS641.2, which correspond to allelic forms of the GmSBP2/S64 gene, have been isolated and characterized . As a member of the seed storage protein superfamily, it has been shown that the SBP gene structure is similar to vicilin genes with intron/exon boundaries at conserved positions . Fluores cence in situ hybridization (FISH) suggested that the soybean SBP gene family is represented by at least two non-allelic genes corresponding to the previously isolated GmSBP1 and GmSBP2/S64 cDNAs . These two cDNAs share extensive sequence similarity but are located at different loci in the soybean genome . To investigate transcriptional activation of the GmSBP2 gene, 2 kb 5'-flanking sequences of gsS641.1 and gsS641.2 were fused to the beta-glucuronidase (GUS) reporter gene and to the green fluorescent protein (GFP) reporter gene and inde pendently introduced into Nicotiana tabacum by Agrobacterium tumefaciens-mediated transformation . The SBP2 promoter directed expression of both GUS and GFP reporter genes with high specificity to the phloem of leaves, stems and roots . Thus, the overall pattern of SBP-GUS or SBP-GFP expression is consistent with the involvement of SBP in sucrose translocation-dependent physiological processes.

Biotechnol Lett, 2003 Oct, 25(19), 1629 - 35
Expression and production of bioactive human interleukin-18 in transgenic tobacco plants; Zhang B et al.; The cDNA of human interleukin-18 (hIL-18) was successfully inserted into the genome of tobacco plant, Nicotiana tabacum cv . NC-89, using Agrobacterium tumefaciens-mediated transformation . Insertion and translation of hIL-18 in transformants were confirmed by PCR, ELISA, and Western blot, respectively . The transformed extracts contained the recombinant hIL-18 protein up to 0.05% of total soluble protein . Activity of the recombinant hIL-18 in plant cells was confirmed by the induction of IFN-gamma on IL-18-responsive J6-1 cells by the extracts obtained from the transformants . The expression level of hIL-18 (351 ng g(-1) tobacco tissue) obtained in the present study may be sufficient to induce responses/effects in vivo.

Microb Ecol . 2003 Oct 31; {Epub ahead of print}
Engineering Root Exudation of Lotus toward the Production of Two Novel Carbon Compounds Leads to the Selection of Distinct Microbial Populations in the Rhizosphere; Oger PM et al.; The culture of opine-producing transgenic Lotus plants induces the increase in the rhizosphere of bacterial communities that are able to utilize these molecules as sole carbon source . We used transgenic Lotus plants producing two opines, namely mannopine and nopaline, to characterize the microbial communities directly influenced by the modification of root exudation . We showed that opine-utilizers represent a large community in the rhizosphere of opine-producing transgenic Lotus . This community is composed of at least 12 different bacterial species, one third of which are able to utilize the opine mannopine and two thirds the opine nopaline . Opine utilizers are diverse, belonging to the Gram-positive and -negative bacteria . We described two novel mannopine-utilizing species, Rhizobium and Duganella spp., and five novel nopaline-utilizing species, Duganella, Afipia, Phyllobacterium, Arthrobacter, and Bosea spp . Although opine utilizers mostly belong to the alpha-Proteobacteria, Rhizobiaceae family, there is little overlap between the populations able to utilize each of the two opines produced by the plants . Noticeably, in the rhizosphere of transgenic Lotus, only the opine mannopine favors the growth of Agrobacterium tumefaciens, the bacterium from which opines have been characterized . The diversity of opine utilizers from the rhizosphere of Lotus plants is greater than that observed from any other environment . Therefore, transgenic plants with engineered exudation constitute an excellent tool to isolate and characterize specific microbial populations.

Yi Chuan Xue Bao, 2003 Jul, 30(7), 631 - 6
{Inheritance and segregation of transformants in cotton with two types of insect-resistant genes}; Wu JH et al.; A plant expression vector containing a chemeric Bt29K gene coding for the active Cry1Ac protein and the arrowhead proteinase inhibition gene API-B was introduced into an elite cotton cultivar Jihe 321 by Agrobactertium tumefaciens . Some insect-resistant cotton lines were developed . Segregation and stabilization of insect-resistant genes in six transformation lines were studied . Based on the results of kanamycin resistant test and insect bioassay using Heliethis armigera, PCR detection and Southern-blot, we found that the inheritance and segregation of Bt gene were complicated, some transformants were in accordance with Mendelian patterns of inheritance in the ratio of insect-resistant plants to non-resistant plants in Ti progeny, yet others were non-Mendelian patterns . But the inheritance and segregation of Bt gene in homozygous transformation lines were one or two pairs of major dominant genes through crossing of insect resistant homozygous lines with non-transformation cotton variety . That the insect resistance phenotype was conditioned by one or two pairs of dominant genes was ascertained in this study . There were two copies of Bt genes in two transformation lines DR248 and DR193, which was reported for the first time . The results were confirmed by Southern-blot . Through observation of segregation population of transgenic plants at different generations, we found that the exogenous Bt gene in cotton genome showed unstable in inheritance in early generations, but the gene could be stabilized through resistance screening generation by generation . The unstability of Bt gene may mean that it need time for the gene to compatibilize cotton genome.

Shi Yan Sheng Wu Xue Bao, 2003 Aug, 36(4), 289 - 94
{A simple and highly efficient Agrobacterium-mediated rice transformation system}; Li MR et al.; A simple and highly efficient rice transformation system was established based on the studying of factors influencing the Agrobacterium-mediated rice transformation . Embryogenic calli derived from mature embryos were infected and cocultivated with A . tumefaciens EHA101 harboring binary vectors: pHQ9, pHQ10, pHQT3 . The highest transformation frequency was about 100 hygromycin resistance calli per gram of calli explants, and above 85% of these calli could be regenerated into plants . The putative transgenic plants were confirmed by GUS assay and southern-blot analysis . Hygromycin resistance tests indicated that the segregation of transgene in T1 progeny corresponded to the Mendelian ratio, 3:1 . This system will benefit the functional genomic study of rice by using T-DNA insertion mutagenesis and gene targeting.

Planta, 2004 Feb, 218(4), 623 - 9 Epub 2003 Oct 23.
Inhibition of endogenous trypsin- and chymotrypsin-like activities in transgenic lettuce expressing heterogeneous proteinase inhibitor SaPIN2a; Xu ZF et al.; SaPIN2a, a proteinase inhibitor II from American black nightshade (Solanum americanum Mill.) is highly expressed in the phloem and could be involved in regulating proteolysis in the sieve elements . To further investigate the physiological role of SaPIN2a, we have produced transgenic lettuce (Lactuca sativa L.) expressing SaPIN2a from the CaMV35S promoter by Agrobacterium-mediated transformation . Stable integration of the SaPIN2a cDNA and its inheritance in transgenic lines were confirmed by Southern blot analysis and segregation analysis of the R1 progeny . SaPIN2a mRNA was detected in both the R0 and R1 transformants on northern blot analysis but the SaPIN2a protein was not detected on western blot analysis using anti-peptide antibodies against SaPIN2a . Despite an absence of significant inhibitory activity against bovine trypsin and chymotrypsin in extracts of transgenic lettuce, the endogenous trypsin-like activity in each transgenic line was almost completely inhibited, and the endogenous chymotrypsin-like activity moderately inhibited . Our finding that heterogeneously expressed SaPIN2a in transgenic lettuce inhibits plant endogenous protease activity further indicates that SaPIN2a regulates proteolysis, and could be potentially exploited for the protection of foreign protein production in transgenic plants.

Biotechnol Lett, 2003 Sep, 25(18), 1571 - 4
Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures; Kwon TH et al.; A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv . Seokwang) using Agrobacterium-mediated transformation . Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato . The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene . Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 microg l(-1) after 10 d' cultivation.

J Bacteriol, 2003 Nov, 185(21), 6481 - 5
Bacteriophage ST64B, a genetic mosaic of genes from diverse sources isolated from Salmonella enterica serovar typhimurium DT 64; Mmolawa PT et al.; The complete sequence of the double-stranded DNA (dsDNA) genome of the Salmonella enterica serovar Typhimurium ST64B bacteriophage was determined . The 40,149-bp genomic sequence of ST64B has an overall G+C content of 51.3% and is distinct from that of P22 . The genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda . Most of the putative tail genes showed sequence similarity to tail genes of Mu, a nonlambdoid phage . In addition, it is likely that these tail genes are not expressed due to insertions of fragments of genes related to virulence within some of the open reading frames . This, together with the inability of ST64B to produce plaques on a wide range of isolates, suggests that ST64B is a defective phage . In contrast to the tail genes, most of the head genes showed similarity to those of the lambdoid phages HK97 and HK022, but these head genes also have significant sequence similarities to those of several other dsDNA phages infecting diverse bacterial hosts, including Escherichia, Pseudomonas, Agrobacterium, Caulobacter, Mesorhizobium, and Streptomyces: This suggests that ST64B is a genetic mosaic that has acquired significant portions of its genome from sources outside the genus Salmonella.

World J Gastroenterol, 2003 Oct, 9(10), 2211 - 5
Expression of ORF2 partial gene of hepatitis E virus in tomatoes and immunoactivity of expression products; Ma Y et al.; AIM: To transfer hepatitis E virus (HEV) ORF2 partial gene to tomato plants, to investigate its expression in transformants and the immunoactivity of expression products, and to explore the feasibility of developing a new type of plant-derived HEV oral vaccine . METHODS: Plant binary expression vector p1301E2, carrying a fragment of HEV open reading frame-2 (named HEV-E2), was constructed by linking the fragment to a constitutive CaMV35s promoter and nos terminator, then directly introduced into Agrobacterium tumefaciens EHA105 . With leaf-disc method, tomato plants medicated by EHA105 were transformed and hygromycin-resistant plantlets were obtained in selective medium containing hygromycin . The presence and integration of foreign DNA in transgenic tomato genome were confirmed by Gus gene expression, PCR amplification and Southern dot blotting . The immunoactivity of recombinant protein extracted from transformed plants was examined by enzyme-linked immunosorbant assay (ELISA) using a monoclonal antibody specifically against HEV . ELISA was also used to estimate the recombinant protein content in leaves and fruits of the transformants . RESULTS: Seven positive lines of HEV-E2-transgenic tomato plants confirmed by PCR and Southern blotting were obtained and the immunoactivity of recombinant protein could be detected in extracts of transformants . The expression levels of recombinant protein were 61.22 ng/g fresh weight in fruits and 6.37-47.9 ng/g fresh weight in leaves of the transformants . CONCLUSION: HEV-E2 gene was correctly expressed in transgenic tomatoes and the recombinant antigen derived from them has normal immunoactivity . Transgenic tomatoes may hold a good promise for producing a new type of low-cost oral vaccine for hepatitis E virus.

Plant Physiol, 2003 Oct, 133(2), 462 - 9
A gateway cloning vector set for high-throughput functional analysis of genes in planta; Curtis MD et al.; The current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes . In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence . To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies . The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation . With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp . binary vector system that facilitates fast and reliable DNA cloning . This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly . The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the beta-glucuronidase protein so that the subcellular localization of a protein can be identified . They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments . All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.

Plant Cell Rep, 2003 Oct, 22(3), 201 - 9 Epub 2003 Jul 09.
Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell . Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene; Jayashree R et al.; Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis . Immature anther-derived calli were used to develop transgenic plants . These calli were co-cultured with A . tumefaciens harboring a plasmid vector containing the H . brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter . The beta-glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli . Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines . Transformed calli surviving on medium containing 300 mg l(-1) kanamycin showed a strong GUS-positive reaction . Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l(-1) spermine and 0.1 mg l(-1) abscisic acid . Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l(-1) gibberellic acid, 0.2 mg l(-1) kinetin (KIN) and 0.1 mg l(-1) indole-3-acetic acid . A transformation frequency of 4% was achieved . The morphology of the transgenic plants was similar to that of untransformed plants . Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants . The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.

Plant Cell Rep, 2004 Jan, 22(6), 382 - 7 Epub 2003 Oct 10.
Synthesis of an HIV-1 Tat transduction domain-rotavirus enterotoxin fusion protein in transgenic potato; Kim TG et al.; A DNA fragment encoding a 12-amino acid (aa) HIV-1 Tat transduction peptide fused to a 90-aa murine rotavirus NSP4 enterotoxin protein (Tat-NSP4(90)) was transferred to Solanum tuberosum by Agrobacterium tumefaciens-mediated transformation . The fusion gene was detected in the genomic DNA of transformed plant leaf tissues by PCR DNA amplification . The Tat-NSP4(90 )fusion protein was identified in transformed tuber extracts by immunoblot analysis using anti-NSP4(90) and anti-Tat as the primary antibodies . Enzyme-linked immunosorbent assay results showed that the Tat-NSP4(90) fusion protein made up to 0.0015% of the total soluble tuber protein . The synthesis of Tat-NSP4(90) fusion protein in transformed potato tuber tissues demonstrates the feasibility of plant cell delivery of the HIV-1 Tat transduction domain as a carrier for non-specific targeting of fused antigens to the mucosal immune system.

Plant Physiol, 2003 Nov, 133(3), 956 - 65 Epub 2003 Oct 09.
Targeted integration of T-DNA into the tobacco genome at double-stranded breaks: new insights on the mechanism of T-DNA integration; Chilton MD et al.; Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome . We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease . Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts . Short filler DNAs were also present in several junctions . When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts . Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete . This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications . Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome.

Plant Physiol, 2003 Nov, 133(3), 978 - 88 Epub 2003 Oct 09.
Recognition of the Agrobacterium tumefaciens VirE2 translocation signal by the VirB/D4 transport system does not require VirE1; Vergunst AC et al.; Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells . The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system . VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell . We analyzed whether the VirE1 chaperone is also essential for transport recognition of VirE2 by the VirB/D4 encoded type IV secretion system . In addition, we assayed whether translocation of VirF and VirE3, which also forms part of the virE operon, is affected by the absence of VirE1 . We employed the earlier developed CRAFT (Cre recombinase Reporter Assay For Translocation) assay to detect transfer of Cre::Vir fusion proteins from A . tumefaciens into plants, monitored by stable reconstitution of a kanamycin resistance marker, and into yeast, screened by loss of the URA3 gene . We show that the C-terminal 50 amino acids of VirE2 and VirE3 are sufficient to mediate Cre translocation into host cells, confirming earlier indications of a C-terminal transport signal . This transfer was independent of the presence or absence of VirE1 . Besides, the translocation efficiency of VirF is not altered in a virE1 mutant . The results unambiguously show that the VirE1 chaperone is not essential for the recognition of the VirE2 transport signal by the transport system and the subsequent translocation across the bacterial envelope into host cells.

Plant Physiol, 2003 Nov, 133(3), 989 - 99 Epub 2003 Oct 09.
Reexamining the role of the accessory plasmid pAtC58 in the virulence of Agrobacterium tumefaciens strain C58; Nair GR et al.; Isogenic strains of Agrobacterium tumefaciens carrying pTiC58, pAtC58, or both were constructed and assayed semiquantitatively and quantitatively for virulence and vir gene expression to study the effect of the large 542-kb accessory plasmid, pAtC58, on virulence . Earlier studies indicate that the att (attachment) genes of A . tumefaciens are crucial in the ability of this soil phytopathogen to infect susceptible host plants . Mutations in many att genes, notably attR and attD, rendered the strain avirulent . These genes are located on pAtC58 . Previous work also has shown that derivatives of the wild-type strain C58 cured of pAtC58 are virulent as determined by qualitative virulence assays and, hence, pAtC58 was described as nonessential for virulence . We show here that the absence of pAtC58 in pTiC58-containing strains results in reduced virulence but that disruption of the attR gene does not result in avirulence or a reduction in virulence . Our studies indicate that pAtC58 has a positive effect on vir gene induction as revealed by immunoblot analysis of Vir proteins and expression of a PvirB::lacZ fusion.

Plant Physiol, 2003 Nov, 133(3), 1011 - 23 Epub 2003 Oct 09.
Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates; Tzfira T et al.; Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome . The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure . Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process . We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB . Then, we retransformed these plants with two A . tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker . Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants . Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration . Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA . Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome.

Biotechnol Adv, 1983, 1(1), 1 - 15
Development of DNA-mediated transformation systems for plants; Pasternak JJ et al.; The genetic engineering of plants by DNA-mediated gene transfer requires that efficient transformation systems be developed . Considerable progress has been made in manipulating the Ti plasmid of Agrobacterium tumefaciens as a vehicle for delivery of foreign genes into protoplasts of dicotyle-donous plants . Part of the Ti plasmid, the T-DNA, can be incorporated into the genome of the host cell; the T-DNA can carry a foreign DNA sequence which co-integrates with it; under normal conditions, the tumorigenic-causing portion of the T-DNA can be inactivated so that transformed protoplasts can be regenerated and T-DNA with an inserted foreign gene can be stably maintained during regeneration, meiosis and gamete formation . A foreign gene has yet to be expressed in regenerated plants although a T-DNA gene for opine synthesis can function in regenerates . Developing a more ubiquitous transformation system for monocotyledons is further from fruition . Based on transformation systems for simple eukaryotic organisms, it is reasonable to expect that a DNA vector which is capable of amplifying a novel plant gene and which contains both a drug resistance marker to facilitate the selection of transformed plant protoplasts and a species-specific autonomously replicating sequence to ensure the stable maintenance of the input gene in the recipient cell can be constructed.

Biotechnol Adv, 1985, 3(1), 29 - 38
Biotechnological applications of plant cells in culture; Shargool PD; For many workers, the most exciting recent advances in the realm of plant cell biotechnology, center on results obtained from experiments concerned with the genetic engineering of plant cells . Various groups of workers have managed to introduce new genetic material into plant cells, using Ti-plasmids (or modified Ti-plasmids) from Agrobacterium tumefaciens . This genetic material has been expressed (with varying degrees of efficiency), in each case . Thus the way may possibly be coming clear to produce plant cell cultures, or whole plants with entirely new or novel properties . Other areas in which progress has been made, are in the design of media conditions to promote secondary product formation, and in ways of immobilizing plant cells and enzymes, to achieve efficient secondary product formation.

Biotechnol Adv, 1999 Dec 30, 17(8), 679 - 87
Novel biotechnological approaches in environmental remediation research; Pletsch M et al.; Two novel approaches, the use of Agrobacterium-transformed plant roots and mycelia cultures of fungi, are considered as research tools in the study of the remediation of soil, groundwater, and biowastes . Transformed roots are excellent model systems for screening higher plants that are tolerant of various inorganic and organic pollutants, and for determining the role of the root matrix in the uptake and further metabolism of contaminants . Edible and/or medicinal fungi may also be natural environmental remediators . Liquid cultures of fungal mycelia are appropriate model systems with which to commence screening and biochemical studies in this under-researched area of biotransformation.

Biotechnol Adv, 2000 Mar, 18(1), 1 - 22
Transgenic hairy roots . recent trends and applications; Giri A et al.; Agrobacterium rhizogenes causes hairy root disease in plants . The neoplastic roots produced by A . rhizogenes infection is characterized by high growth rate and genetic stability . These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants . Hairy root cultures offer promise for production of valuable secondary metabolites in many plants . The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel . Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots . Development of bioreactor models for hairy root cultures is still a recent phenomenon . It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium . Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones . This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants . In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest . In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species . The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement . Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest . This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A . rhizogenes.

Biotechnol Adv, 1995, 13(4), 653 - 71
Foreign gene delivery into monocotyledonous species; Vain P et al.; Monocotyledonous plants are generally more recalcitrant to genetic transformation than dicotyledonous species . The absence of reliable Agrobacterium-mediated transformation methods and the difficulties associated with the culture of monocotyledonous tissues in vitro are mainly responsible for this situation . Until recently, the genetic transformation of monocotyledons was essentially performed by direct transfer of DNA into regenerable protoplasts or intact cells cultured in vitro, via polyethylene glycol treatment, electroporation or particle bombardment . Since 1990, the use of particle gun technology has revolutionized the genetic engineering of monocotyledonous species, allowing transformation to be more independent of the in vitro culture requirements . Today, at least one genotype of each major monocotyledonous crop species, including cereals, can be genetically transformed.

Zhong Yao Cai, 2003 May, 26(5), 313 - 5
{Transformation of bar gene to tetraploid of Isatis indigotica}; Xu T et al.; The transgenic tetraploid of Isatis indigotica mediated by Agrobacterium tumefaciens was obtained . To transfer the plant binary expression vector pCAMBIA 3300 carrying bar gene, the Agrobacterium tumefaciens strain EHA 105 was used as engineering bacterium . The results of PCR indicated that the bar gene had been transferred into and merged with the genome of Isatis indigotica . This study will make foundation for improvement of other characters of this species with genetic engineering.

Theor Appl Genet, 2004 Feb, 108(4), 644 - 50 Epub 2003 Oct 08.
Genetic analysis of Agrobacterium tumefaciens susceptibility in Brassica oleracea; Sparrow PA et al.; The genetic control and heritability of Agrobacterium tumefaciens susceptibility was investigated using a doubled haploid (DH) mapping population of Brassica oleracea and the associated RFLP map . Preliminary studies were carried out by analysis of an 8 x 8 diallel, for which the parental lines were selected to include a range of susceptibilities to A . tumefaciens . The variation observed within the diallel was attributed to both additive and dominant gene effects, with additive gene effects being more important . A broad sense heritability value of 0.95 suggested that 95% of the observed variation was due to genetic effects, with just 5% attributed to non-genetic or environmental effects . A high narrow-sense heritability value of 0.79 suggested that 79% of this trait was controlled by additive gene effects and, therefore, the potential to introduce this trait into breeding material is high . Fifty-nine DH lines from the mapping population were screened for susceptibility towards A . tumefaciens . Variation in susceptibility was observed across the population . The results of the DH screen were entered into the mapping programme MAPQTL and a highly significant quantitative trait loci (QTL) associated with susceptibility to A . tumefaciens was identified on linkage group 09 . The use of substitution lines covering this region confirmed the location of this QTL . This work shows that susceptibility to A . tumefaciens is a heritable trait, and the transfer of susceptibility into resistant lines is demonstrated . These findings may help to overcome genotype restrictions to genetic transformation.

Appl Environ Microbiol, 2003 Oct, 69(10), 6235 - 42
Prospecting for novel biocatalysts in a soil metagenome; Voget S et al.; The metagenomes of complex microbial communities are rich sources of novel biocatalysts . We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy . A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium . Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value . Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes . Interestingly, nine of these agarase genes probably originated from gene duplications . Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119) . Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system . The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined . Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

Plant Cell Rep, 2004 Jan, 22(6), 397 - 402 Epub 2003 Oct 03.
Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes; Murray F et al.; Experiments were conducted to produce transgenic barley plants following infection of immature embryos with Agrobacterium tumefaciens . Transformed callus was obtained using hygromycin resistance as a selectable marker and either green fluorescent protein (GFP) or beta-glucuronidase (GUS) as a reporter . Significantly reduced plant transformation frequencies were obtained with the GFP gene compared to GUS . However, GFP proved to be an excellent reporter of early transformation events and was used to compare four barley cultivars for efficiency in two phases of transformation: the generation of stably transformed barley callus and the regeneration of plantlets from transformed callus . Transformed callus was generated at a high frequency (47-76%) in all four cultivars . Regeneration of transformed plantlets was also achieved for all four cultivars although the frequency was much higher for Golden Promise than for the other three genotypes, reiterating that genotype is an important determinant in the regenerative ability of barley . This study has demonstrated for the first time that Agrobacterium-mediated transformation can be used to transform the Australian cultivars Sloop and Chebec.

Syst Appl Microbiol, 2003 Sep, 26(3), 338 - 49
Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout; Buch C et al.; Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system . One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system . Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains . The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V . anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V . anguillarum indicating autoinduction . AHLs were produced by all serotypes (O1 to O10) and by non-typable strains . During infection with V . anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram . Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL . No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM . Our data show that AHLs are produced by almost all V . anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.

Plant Physiol, 2003 Nov, 133(3), 1024 - 37 Epub 2003 Oct 02.
Vascularization, high-volume solution flow, and localized roles for enzymes of sucrose metabolism during tumorigenesis by Agrobacterium tumefaciens; Wachter R et al.; Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens . Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host . Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment . CO2 release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks . However, tumor water loss contributed little to water flow to host shoots . Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance . Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors . Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization . Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase . Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport . Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment.

Planta, 2003 Dec, 218(2), 163 - 78 Epub 2003 Oct 02.
Flavonoid-related regulation of auxin accumulation in Agrobacterium tumefaciens-induced plant tumors; Schwalm K et al.; Agrobacterium tumefaciens-induced plant tumors accumulate considerable concentrations of free auxin . To determine possible mechanisms by which high auxin concentrations are maintained, we examined the pattern of auxin and flavonoid distribution in plant tumors . Tumors were induced in transformants of Trifolium repens (L.), containing the beta-glucuronidase ( GUS)-fused auxin-responsive promoter ( GH3) or chalcone synthase ( CHS2) genes, and in transformants of Arabidopsis thaliana (L.) Heynh., containing the GUS-fused synthetic auxin response element DR5 . Expression of GH3::GUS and DR5::GUS was strong in proliferating metabolically active tumors, thus suggesting high free-auxin concentrations . Immunolocalization of total auxin with indole-3-acetic acid antibodies was consistent with GH3::GUS expression indicating the highest auxin concentration in the tumor periphery . By in situ staining with diphenylboric acid 2-aminoethyl ester, by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and two-photon laser-scanning microscopy spectrometry, tumor-specific flavones, isoflavones and pterocarpans were detected, namely 7,4'-dihydroxyflavone (DHF), formononetin, and medicarpin . DHF was the dominant flavone in high free-auxin-accumulating stipules of Arabidopsis leaf primordia . Flavonoids were localized at the sites of strongest auxin-inducible CHS2::GUS expression in the tumor that was differentially modulated by auxin in the vascular tissue . CHS mRNA expression changes corresponded to the previously analyzed auxin concentration profile in tumors and roots of tumorized Ricinus plants . Application of DHF to stems, apically pretreated with alpha-naphthaleneacetic acid, inhibited GH3::GUS expression in a fashion similar to 1-N-naphthyl-phthalamic acid . Tumor, root and shoot growth was poor in inoculated tt4(85) flavonoid-deficient CHS mutants of Arabidopsis . It is concluded that CHS-dependent flavonoid aglycones are possibly endogenous regulators of the basipetal auxin flux, thereby leading to free-auxin accumulation in A . tumefaciens-induced tumors . This, in turn, triggers vigorous proliferation and vascularization of the tumor tissues and suppresses their further differentiation.

Microbiology, 2003 Oct, 149(Pt 10), 3035 - 42
Physical and gene maps of Agrobacterium biovar 2 strains and their relationship to biovar 1 chromosomes; Urbanczyk H et al.; Diverse types of genomic DNA organization have been found in Rhizobiaceae, especially among Agrobacterium species . Previous studies of Agrobacterium concentrated mainly on biovar 1 strains . Little attention has been given to biovar 2 strains . The biovar 2 genome consists of a large, circular chromosome and second megabase-sized replicon, as well as several plasmids . In this study two biovar 2 strains were analysed, A . rhizogenes (A . radiobacter) K84 and A . rhizogenes A4, by constructing physical maps of their chromosomes and mega-replicons . The maps revealed that in both strains their chromosomes consist of approximately 3.7 Mbp, while the mega-replicons are 2.6 Mbp circular DNAs . Gene mapping and comparative genomic analysis were performed based on the physical maps using Southern hybridization . It was found that rDNA, as well as analysed virulence and virulence-related genes, are present only on the chromosomes . The inter-chromosomal relationship between biovar 1 and biovar 2 strains was also analysed . Interestingly, there was a high similarity between the chromosomes of biovar 2 and the circular chromosomes of biovar 1, whereas similarity among the smaller megabase-sized replicons was restricted to each biovar . Based on these observations the possible relationship among large replicons in Agrobacterium biovars 1 and 2 is discussed.

Planta, 2003 Jul, 217(3), 349 - 55 Epub 2003 Feb 18.
Effects of Ca(2+) channel blockers and protein kinase/phosphatase inhibitors on growth and anthraquinone production in Rubia cordifolia callus cultures transformed by the rolB and rolC genes; Bulgakov VP et al.; The transformation of Rubia cordifolia L . cells by the 35S- rolB and 35S- rolC genes of Agrobacterium rhizogenes caused a growth inhibition of the resulting cultures and an induction of the biosynthesis of anthraquinone-type phytoalexins . Inhibitor studies revealed a striking difference between the rolC- and rolB-gene-transformed cultures in their sensitivity to verapamil, an L-type Ca(2+) channel blocker . The rolC culture possessed a 2-fold lowered resistance to the inhibitor than the normal culture, while the rolB culture was 4-fold more resistant to the treatment . Additionally, growth of the rolC culture was totally inhibited when the culture was grown in Ca(2+)-free medium, whereas growth of the rolB culture was reduced by less than half . We interpreted these results as evidence for a lack of calcium homeostasis in both transgenic cultures . Anthraquinone (AQ) production was not inhibited in the normal or transformed cultures by the Ca(2+) channel blockers verapamil and LaCl(3), or by diphenylene iodonium, an inhibitor of NADPH oxidase, or by the protein kinase inhibitor staurosporine . These results indicate that the induction of AQ production in non-transgenic and transgenic cultures does not proceed through the activation of the common Ca(2+)-dependent NADPH oxidase pathway that mediates signal transduction between an elicitor-receptor complex via transcriptional activation of defense genes . Okadaic acid and cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase in AQ production in transgenic cultures . Okadaic acid stimulated AQ accumulation in the non-transformed culture, whereas cantharidin had no effect . These results show that different phosphatases are involved in AQ synthesis in normal and transgenic cultures of R . cordifolia.

Plant Cell Rep, 2004 Feb, 22(7), 471 - 7 Epub 2003 Sep 27.
Studies on the immunogenic potential of plant-expressed cholera toxin B subunit; Jani D et al.; Nicotiana tabacum var . Samsun was transformed via Agrobacterium-mediated transformation with a gene encoding the cholera toxin B subunit (CTB) of Vibrio cholerae, modified to contain a sequence coding for an endoplasmic reticulum retention signal (SEKDEL), under the control of the cauliflower mosaic virus 35S promoter . Total protein from the transgenic leaf tissue was isolated and an aliquot containing 5 microg recombinant CTB was injected intradermally into Balb/c (H2K(d)) mice . CTB-specific serum IgG was detected in animals that had been administered plant-expressed or native purified CTB . A T-cell proliferation study using splenocytes and cytokine estimations in supernatants generated by in vitro stimulation of macrophages isolated from the immuno-primed animals was carried out . Inhibition of proliferation of T lymphocytes was observed in splenic T lymphocytes isolated from animals injected with either native or plant-expressed CTB . Macrophages isolated from mice immunised with native or plant-expressed CTB showed enhanced secretion of interleukin-10 but secretion of lipopolysaccharide-induced interleukin-12 and tumor necrosis factor alpha was inhibited . These studies suggest that plant-expressed protein behaved like native CTB with regards to effects on T-cell proliferation and cytokine levels, indicating the suitability of plant expression systems for the production of bacterial antigens, which could be used as edible vaccine . The transgene was found to be inherited in the progeny and was expressed to yield a pentameric form of CTB as evident by its interaction with G(M1) ganglioside.

EMBO J, 2003 Oct 1, 22(19), 4933 - 44
Structure and mechanism of a bacterial haloalcohol dehalogenase: a new variation of the short-chain dehydrogenase/reductase fold without an NAD(P)H binding site; de Jong RM et al.; Haloalcohol dehalogenases are bacterial enzymes that catalyze the cofactor-independent dehalogenation of vicinal haloalcohols such as the genotoxic environmental pollutant 1,3-dichloro-2-propanol, thereby producing an epoxide, a chloride ion and a proton . Here we present X-ray structures of the haloalcohol dehalogenase HheC from Agrobacterium radiobacter AD1, and complexes of the enzyme with an epoxide product and chloride ion, and with a bound haloalcohol substrate mimic . These structures support a catalytic mechanism in which Tyr145 of a Ser-Tyr-Arg catalytic triad deprotonates the haloalcohol hydroxyl function to generate an intramolecular nucleophile that substitutes the vicinal halogen . Haloalcohol dehalogenases are related to the widespread family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDR family), which use a similar Ser-Tyr-Lys/Arg catalytic triad to catalyze reductive or oxidative conversions of various secondary alcohols and ketones . Our results reveal the first structural details of an SDR-related enzyme that catalyzes a substitutive dehalogenation reaction rather than a redox reaction, in which a halide-binding site is found at the location of the NAD(P)H binding site . Structure-based sequence analysis reveals that the various haloalcohol dehalogenases have likely originated from at least two different NAD-binding SDR precursors.

Mol Genet Genomics, 2003 Dec, 270(4), 296 - 302 Epub 2003 Sep 26.
An improved protocol for Agrobacterium-mediated transformation of Antirrhinum majus L; Cui ML et al.; Efficient Agrobacterium -mediated transformation of Antirrhinum majus L . was achieved via indirect shoot organogenesis from hypocotyl explants of seedlings . Stable transformants were obtained by inoculating explants with A . tumefaciens strain GV2260 harboring the binary vector pBIGFP121, which contains the neomycin phosphotransferase gene ( NPT II) as a selectable marker and the gene for the Green Fluorescent Protein ( GFP) as a visual marker . Putative transformants were identified by selection for kanamycin resistance and by examining the shoots using fluorescence microscopy . PCR and Southern analyses confirmed integration of the GFP gene into the genomes of the transformants . The transformants had a morphologically normal phenotype . The transgene was shown to be inherited in a Mendelian manner . This improved method requires only a small number of seeds for explant preparation, and three changes of medium; the overall transformation efficiency achieved, based on the recovery of transformed plants after 4-5 months of culture, reached 8-9% . This success rate makes the protocol very useful for producing transgenic A . majus plants.

Appl Biochem Biotechnol, 2003 Sep, 110(3), 175 - 83
Effect of chitosan on peroxidase activity and isoenzyme profile in hairy root cultures of Armoracia lapathifolia; Flocco CG et al.; Hairy root cultures of Armoracia lapathifolia established by infection with Agrobacterium rhizogenes LBA 9402 present a level and isoenzyme pattern of peroxidases (POD) comparable to nontransformed roots . Elicitation with chitosan (10, 50, and 100 mg/L) was used in order to improve POD production . Total POD activity increased about 170% after 48 h of treatment with chitosan 100 mg/L . Elicitation effect on soluble and ionically cell-wall-bound POD fractions of A . lapathifolia hairy roots was analyzed . POD activity of the ionically cell-wall-bound protein fraction increased in the presence of chitosan in a dose-response manner . No effect on soluble POD fractions was observed, but the isoenzyme pattern analyzed by isoelectrofocusing showed an increase of an acidic isoenzyme (pI = 3.4) after the elicitation treatment . The ionically cell-wall-bound protein fraction showed only basic isoenzymes, with an increase of an isoenzyme of pI = 8.7, after the elicitation treatment.

Planta, 2003 Dec, 218(2), 226 - 32 Epub 2003 Sep 24.
Transgenic rose lines harboring an antimicrobial protein gene, Ace-AMP1, demonstrate enhanced resistance to powdery mildew ( Sphaerotheca pannosa); Li X et al.; An antimicrobial protein gene, Ace-AMP1, was introduced into Rosa hybrida cv . Carefree Beauty via Agrobacterium-mediated transformation . A total of 500 putative transgenic plants were obtained from 100 primary embryogenic calli co-cultivated with A . tumefaciens following selection on a regeneration medium containing 100 mg/l kanamycin . Polymerase chain reaction analysis of these putative transgenic lines, using primers for both Ace-AMP1 and neomycin phosphotransferase ( npt II) genes, showed that 62% of these plants were positive for both transgenes . These lines were further confirmed for stable integration of Ace-AMP1 and npt II genes by Southern blotting . Transcription of the Ace-AMP1 transgene in various transgenic rose lines was determined using Northern blotting . Transgenic rose lines inoculated with conidial spores of Sphaerotheca pannosa (Wallr.: Fr.) Lev . var . rosae showed enhanced resistance to powdery mildew using both a detached-leaf assay and an in vivo greenhouse whole-plant assay.

Plant Cell Rep, 2003 Sep, 22(2), 141 - 9 Epub 2003 Jul 09.
Transformation of apple ( Malus domestica Borkh.) with the stilbene synthase gene from grapevine ( Vitis vinifera L.) and a PGIP gene from kiwi ( Actinidia deliciosa); Szankowski I et al.; The objective of the present research was to introduce genes with antifungal potential into the commercially important apple cvs . Elstar and Holsteiner Cox in order to establish resistance against fungal diseases . The gene encoding the stilbene synthase (Vst1) from Vitis vinifera L., responsible for the synthesis of the phytoalexin resveratrol in grapevine, and the gene for a polygalacturonase-inhibiting protein (PGIP) from kiwi ( Actinidia deliciosa) were transferred into Holsteiner Cox and Elstar via Agrobacterium tumefaciens-mediated transformation . A total of nine transgenic Holsteiner Cox clones and one transgenic E clone carrying the stilbene-synthase gene as well as three transgenic Holsteiner Cox lines harbouring the polygalacturonase-inhibiting protein from Kiwi were identified via polymerase chain reaction and Southern blot analysis . High performance liquid chromatography analysis revealed the accumulation of a resveratrol-derivate, a glycoside, in transgenic Vst1 plants.

Theor Appl Genet, 2004 Jan, 108(2), 306 - 14 Epub 2003 Sep 19.
Generation and flanking sequence analysis of a rice T-DNA tagged population; Sha Y et al.; Insertional mutagenesis provides a rapid way to clone a mutated gene . Transfer DNA (T-DNA) of Agrobacterium tumefaciens has been proven to be a successful tool for gene discovery in Arabidopsis and rice ( Oryza sativa L . ssp . japonica) . Here, we report the generation of 5,200 independent T-DNA tagged rice lines . The T-DNA insertion pattern in the rice genome was investigated, and an initial database was constructed based on T-DNA flanking sequences amplified from randomly selected T-DNA tagged rice lines using Thermal Asymmetric Interlaced PCR (TAIL-PCR) . Of 361 T-DNA flanking sequences, 92 showed long T-DNA integration (T-DNA together with non-T-DNA) . Another 55 sequences showed complex integration of T-DNA into the rice genome . Besides direct integration, filler sequences and microhomology (one to several nucleotides of homology) were observed between the T-DNA right border and other portions of the vector pCAMBIA1301 in transgenic rice . Preferential insertion of T-DNA into protein-coding regions of the rice genome was detected . Insertion sites mapped onto rice chromosomes were scattered in the genome . Some phenotypic mutants were observed in the T1 generation of the T-DNA tagged plants . Our mutant population will be useful for studying T-DNA integration patterns and for analyzing gene function in rice.

Mol Cells, 2003 Aug 31, 16(1), 117 - 22
Comparing constitutive promoters using CAT activity in transgenic tobacco plants; Kang TJ et al.; The effectiveness of different promoters for use in transgenic tobacco was compared using a reporter gene expressing chloramphenicol acetyl transferase (CAT) . Plasmids with CAT gene controlled by cauliflower mosaic virus 35S (CaMV 35S), rice actin1 (Ract1) and tobacco polyubiquitin (Tubi.u4) promoters were delivered into tobacco plants by Agrobacterium-mediated transformation . The Ract1 promoter, previously shown to be a strong promoter in rice and other monocots, failed to promote strong expression in tobacco . CAT expression was greatest from the vector carrying Tubi.u4 with a 5'UTR and leader intron without a ubiquitin monomer . In transgenic plants harboring the Tubi.u4 promoter, CAT expression was approximately twice that of the CaMV 35S promoter . Our results suggest that foreign genes under the control of a ubiquitin promoter devoid of monomer will be useful for high-level gene expression in tobacco.

Mol Cells, 2003 Aug 31, 16(1), 19 - 27
Production of herbicide-tolerant zoysiagrass by Agrobacterium-mediated transformation; Toyama K et al.; Herbicide-resistant zoysiagrass (Zoysia japonica Steud.) has been developed by Agrobacterium-mediated transformation . A callus-type transformation system was established by optimizing several factors that affect the rate of transformation, including co-cultivation period and concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), CaCl2 and acetosyringone . Maximal GUS expression was observed when a Type 3 callus was co-cultivated on 2,4-D-free co-cultivation medium for 9 d . In addition, removal of calcium and addition of 50-100 mg/L acetosyringone during co-cultivation enhanced GUS expression . When this optimized protocol was applied to the transformation of the bialaphos resistance gene (bar), four plants per 700 mg of infected calluses survived on the selective medium . DNA gel-blot analysis showed that two copies of the transgene had been integrated . After application of 2 g/L bialaphos for a week the transgenic plants survived herbicide spraying, while untransformed zoysiagrasses and invading weeds died . The herbicide-tolerant zoysiagrass will permit more efficient weed control in this widely cultivated turf grass.

Methods Mol Biol, 2003, 236, 329 - 44
Vector construction for gene overexpression as a tool to elucidate gene function; Lloyd A; Gene overexpression as a means to determine plant gene function has been used almost since the first plant transformation protocols became viable . The goal of these experiments, as in classical genetic experiments, is to observe any phenotypic change associated with changing the expression of a gene of interest-in this case overexpression . Any phenotypic changes are interpreted, and the native gene's function is deduced based on the pathways or biochemistries that are altered in the transformants . Overexpression experiments may be particularly suitable in instances when genes are functionally redundant, when a plant species does not have good genetics, or when a knockout mutation is particularly deleterious . This chapter is intended as a general protocol for producing gene overexpression constructs, starting with genomic DNA, RNA, or an isolated clone, for use in plants that are transformable by Agrobacterium.

Methods Mol Biol, 2003, 236, 177 - 88
T-DNA mutagenesis in Arabidopsis; Alonso JM et al.; Insertional mutagenesis is a basic genetic tool that allows for a rapid identification of the tagged genes responsible for a particular phenotype . Transposon and Agrobacterium-mediated DNA integration are the most commonly used biological mutagens in plants . The main drawback of these technologies is the relatively low frequency of mutations, as compared to those induced by conventional chemical or physical agents, thus limiting the use of insertional mutagens to the generation of large mutant populations in few genetic backgrounds . Recent improvements in Agrobacterium-mediated transformation efficiency and an increasing repertoire of transformation vectors available to the research community is making this type of mutagen very attractive for individual laboratories interested in the studies of mutations in particular genetic backgrounds . Herein, we describe a simple yet robust Arabidopsis transformation procedure that can be used to generate large numbers of insertional mutants in Arabidopsis thaliana . Using this protocol, transformation efficiencies of up to 5% can be achieved.

Plant Physiol, 2003 Oct, 133(2), 901 - 9 Epub 2003 Sep 18.
Down-regulating alpha-galactosidase enhances freezing tolerance in transgenic petunia; Pennycooke JC et al.; Alpha-galactosidase (alpha-Gal; EC 3.2.1.22) is involved in many aspects of plant metabolism, including hydrolysis of the alpha-1,6 linkage of raffinose oligosaccharides during deacclimation . To examine the relationship between endogenous sugars and freezing stress, the expression of alpha-Gal was modified in transgenic petunia (Petunia x hybrida cv Mitchell) . The tomato (Lycopersicon esculentum) Lea-Gal gene under the control of the Figwort Mosaic Virus promoter was introduced into petunia in the sense and antisense orientations using Agrobacterium tumefaciens-mediated transformation . RNA gel blots confirmed that alpha-Gal transcripts were reduced in antisense lines compared with wild type, whereas sense plants had increased accumulation of alpha-Gal mRNAs . alpha-Gal activity followed a similar trend, with reduced activity in antisense lines and increased activity in all sense lines evaluated . Raffinose content of nonacclimated antisense plants increased 12- to 22-fold compared with wild type, and 22- to 53-fold after cold acclimation . Based upon electrolyte leakage tests, freezing tolerance of the antisense lines increased from -4 degrees C for cold-acclimated wild-type plants to -8 degrees C for the most tolerant antisense line . Down-regulating alpha-Gal in petunia results in an increase in freezing tolerance at the whole-plant level in nonacclimated and cold-acclimated plants, whereas overexpression of the alpha-Gal gene caused a decrease in endogenous raffinose and impaired freezing tolerance . These results suggest that engineering raffinose metabolism by transformation with alpha-Gal provides an additional method for improving the freezing tolerance of plants.

Plant Cell Rep, 2004 Feb, 22(7), 465 - 70 Epub 2003 Sep 17.
A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants; Leelavathi S et al.; A protocol is presented for efficient transformation and regeneration of cotton . Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos . An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus . About 83% of these plants have been confirmed to be transgenic by Southern blot analysis . An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained . The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained . In addition, multiple transformations can be performed either simultaneously or sequentially . The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.

Plant Cell Rep, 2004 Jan, 22(6), 437 - 41 Epub 2003 Sep 17.
Camptothecin and 10-hydroxycamptothecin from Camptotheca acuminata hairy roots; Lorence A et al.; Camptothecin (CPT) is an anticancer and antiviral alkaloid produced by the Chinese tree Camptotheca acuminata (Nyssaceae) and some other species belonging to the families Apocynaceae, Olacaceae, and Rubiaceae . Bark and seeds are currently used as sources for the drug . Several attempts have been made to produce CPT from cell suspensions; however, the low yields obtained limit this approach . Cultures of differentiated cell types may be an alternative source of alkaloid production . Hairy root cultures of C . acuminata were established from tissue transformed with Agrobacterium rhizogenes strains ATCC 15834 and R-1000 . Integration of the genes responsible for the hairy-root phenotype ( rol genes) into the plant genome was verified by DNA gel blot analysis . The hairy roots produce and secrete CPT as well as the more potent and less toxic natural derivative, 10-hydroxycamptothecin (HCPT), into the medium . Remarkably, the cultures were able to synthesize the alkaloids at levels equal to, and sometimes greater than, the roots in planta, i.e., 1.0 and 0.15 mg/g dry weight for CPT and the HCPT, respectively.

Plant Cell Rep, 2004 Jan, 22(6), 388 - 96 Epub 2003 Sep 12.
Expression of a magainin-type antimicrobial peptide gene (MSI-99) in tomato enhances resistance to bacterial speck disease; Alan AR et al.; MSI-99 is a synthetic analog of magainin II (MII), a small cationic peptide highly inhibitory to a wide spectrum of microbial organisms . Tomato plants were transformed to express a gene encoding the MSI-99 peptide and tested for possible enhancement of resistance to important pathogens of this crop . Thirty-six tomato transformants carrying an MSI-99 expression vector designed to target the peptide into extracellular spaces were obtained by Agrobacterium tumefaciens-mediated transformation . Expression of MSI-99 caused no obvious cytotoxic effects in these plants . In the tests with Pseudomonas syringae pv . tomato (bacterial speck pathogen) at 10(5 )CFU/ml, several MSI-99-expressing lines developed significantly fewer disease symptoms than controls . However, MSI-99-expressing lines were not significantly different from controls in their responses to the fungal pathogen Alternaria solani (early blight) and the oomycete pathogen Phytophthora infestans (late blight) . These findings are in accordance with our previous in vitro inhibition tests, which showed that the MSI-99 peptide is more inhibitory against bacteria than against fungi and oomycetes . Additional in vitro inhibition assays showed that MSI-99 loses its antimicrobial activity in the total or extracellular fluids from leaflets of non-transformed tomato plants; however, P . syringae pv . tomato could not multiply in the extracellular fluid from an MSI-99-expressing line . Our results suggest that expression strategies providing continuous high expression of MSI-99 will be necessary to achieve significant enhancement of plant disease resistance.

Int J Antimicrob Agents, 2003 Sep, 22(3), 217 - 22
Antiplasmid effect of promethazine in mixed bacterial cultures; Molnar A et al.; Promethazine has been recognised as an effective antiplasmid agent in cultures containing a single bacterial species such as Escherichia coli, Yersinia enterocolitica, Staphylococcus aureus and Agrobacterium tumefaciens . The objective of this study was to examine the effect of heterogenity of the microbial flora on plasmid elimination by promethazine in a laboratory based model system of mixed bacterial infection . F'lac plasmid elimination of E . coli K12 LE140 was studied in the presence of a numerically predominant Gram-positive species (Bacillus cereus, Staphylococcus epidermidis) by promethazine (0-120 mg/l) at 23, 37 and 39 degrees C . Growth kinetics of different bacterial species were studied at various temperatures without drug treatment in mixed bacterial cultures and it was found that the small number of added bacteria overgrew the pre-existing flora during the incubation period . We observed that bacterial-bacterial interactions modified the growth rate of individual bacterial species and gave selective advantages to some bacterial species of the microbial community . Some interactions between coexisting bacterial species enhanced the frequency of plasmid curing by promethazine in mixed cultures with S . epidermidis . In our experiments the plasmid curing action of promethazine was more effective at elevated temperature than at lower temperatures.

Indian J Exp Biol, 2002 Nov, 40(11), 1295 - 303
Regeneration from mature and immature embryos and transient gene expression via Agrobacterium-mediated transformation in emmer wheat (Triticum dicoccum Schuble); Khurana J et al.; The present study establishes a regeneration protocol and optimizes conditions for Agrobacterium-mediated transformation of the tetraploid emmer wheat, Triticum dicoccum . Regeneration from mature and immature embryos was accomplished as a two-step process involving callus induction in the presence of 2,4-D followed by regeneration on a 2,4-D free, cytokinin-containing medium (RM1) . Higher concentrations of 2,4-D (4 mg/l) though conducive for callusing (89.39% in mature embryos and 96% in immature embryos) proved detrimental for further regeneration . At lower 2,4-D (1 mg/ml) although callusing was suboptimal, (56.8% and 84% from mature and immature embryos, respectively) the regeneration response was the highest on RM1 medium (64.4% and 56.6% from mature and immature embryos, respectively) . Overall, the regeneration response of immature embryos was lower than the mature embryos by 10-12% . Due to the ease of availability of mature embryos the mature embryo-derived calli were chosen as the target tissue for Agrobacterium-mediated transformation in the two Indian varieties DDK1001 and DDK1009 . Histochemical GUS expression revealed the suitability of the mature embryo-derived calli for such investigations . Of the CaMV35S and Act1 promoters employed, the monocot promoter Act1 displayed higher GUS gene activity in the mature embryo derived calli when co-cultivated with LBA4404 (pBI101::Act1).

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1689 - 95
Classification and nomenclature of Agrobacterium and Rhizobium; Young JM et al.; Farrand et al . {Int J Syst Evol Microbiol 53 (2003), 1681-1687} have presented a critique of the proposal of Young et al . {Int J Syst Evol Microbiol 51 (2001), 89-103} to revise the nomenclature and classification of RHIZOBIUM: They argued that Young et al . (2001) are mistaken in their reclassification of all Agrobacterium species within Rhizobium, and that the resulting nomenclatural revision is 'unnecessary and unwarranted' . These objections arise because the authors appear not to understand the role of formal nomenclature, and fail to distinguish between formal and special-purpose nomenclatures (Bacteriological Code, 1990 Revision) . The arguments set out by Farrand et al . (2003) can be addressed in terms of (1) the taxonomic status of the genera Agrobacterium and Rhizobium; (2) the status of species and biovars and their nomenclature; and (3) the role of transmissible genomic elements in classification and nomenclature . Finally, an attempt is made to unravel the confusion underpinning their discussion with a consideration of the relationship between formal and special-purpose nomenclatures.

Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1681 - 7
Agrobacterium is a definable genus of the family Rhizobiaceae; Farrand SK et al.; Members of the genus Agrobacterium constitute a diverse group of organisms, all of which, when harbouring the appropriate plasmids, are capable of causing neoplastic growths on susceptible host plants . The agrobacteria, which are members of the family Rhizobiaceae, can be differentiated into at least three biovars, corresponding to species divisions based on differential biochemical and physiological tests . Recently, Young et al . {Int J Syst Evol Microbiol 51 (2003), 89-103} proposed to incorporate all members of the genus Agrobacterium into the genus RHIZOBIUM: We present evidence from classical and molecular comparisons that supports the conclusion that the biovar 1 and biovar 3 agrobacteria are sufficiently different from members of the genus Rhizobium to warrant retention of the genus AGROBACTERIUM: The biovar 2 agrobacteria cluster more closely to the genus Rhizobium, but some studies suggest that these isolates differ from species of Rhizobium with respect to their capacity to interact with plants . We conclude that there is little scientific support for the proposal to group the agrobacteria into the genus Rhizobium and consequently recommend retention of the genus AGROBACTERIUM:

J Bacteriol, 2003 Oct, 185(19), 5665 - 72
In situ activation of the quorum-sensing transcription factor TraR by cognate and noncognate acyl-homoserine lactone ligands: kinetics and consequences; Luo ZQ et al.; Conjugal transfer of Ti plasmids of Agrobacterium tumefaciens is controlled by a quorum-sensing system composed of the transcriptional activator TraR and its acyl-homoserine lactone quormone N-(3-oxo-octanoyl)-L-homoserine lactone (3-oxo-C8-HSL) . The population density dependence of quorum-sensing systems can often be circumvented by addition of the quormone to cultures at low cell numbers . However, the quorum-dependent activation of Ti plasmid conjugal transfer exhibited a lag of almost 8 h when the quormone was added to donor cells at low population densities (Piper and Farrand, J . Bacteriol . 182:1080-1088, 2000) . As measured by activation of a TraR-dependent traG::lacZ reporter fusion, TraR in cells exposed to the cognate signal for 5 min showed detectable activity, while exposure for 15 min resulted in full activity . Thus, the lag in activation is not due to some intrinsic property of TraR . Cells exposed to the agonistic analog N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) exhibited similar induction kinetics . However, activation of the reporter in cells exposed to the poorly effective alkanoyl acyl-HSL N-hexanoyl-L-homoserine lactone (C6-HSL) required the continued presence of the signal . As measured by an in vivo repressor assay, TraR activated by 3-oxo-C6-HSL or by 3-oxo-C8-HSL remained active for as long as 8 h after removal of exogenous signal . However, TraR activated by the alkanoyl quormone C6-HSL rapidly lost activity following removal of the signal . In quormone retention assays, which measure signal binding by TraR, cells grown with either of the two 3-oxo-acyl-HSL quormones retained the ligand after washing, while cells grown with C6-HSL lost the alkanoyl-HSL concomitant with the rapid loss of TraR activity . We conclude that TraR rapidly binds its quormone and that, once bound, the cognate signal and its close homologs are tightly retained . Moreover, in the absence of other regulatory factors, activated TraR remains functional after removal of the signal . On the other hand, poorly active signals are not tightly bound, and their removal by washing leads to rapid loss of TraR activity.

Plant J, 2003 Oct, 36(1), 114 - 21
Transgene-induced RNA interference: a strategy for overcoming gene redundancy in polyploids to generate loss-of-function mutations; Lawrence RJ et al.; Gene redundancy in polyploid species complicates genetic analyses by making the generation of recessive, loss-of-function alleles impractical . We show that this problem can be circumvented using RNA interference (RNAi) to achieve dominant loss of function of targeted genes . Arabidopsis suecica is an allotetraploid (amphidiploid) hybrid of A . thaliana and A . arenosa . We demonstrate that A . suecica can be genetically transformed using the floral dip method for Agrobacterium-mediated transformation . Transgenes segregate as in a diploid, indicating that chromosome pairing occurs exclusively (or almost so) among homologs and not among homeologs . Expressing a double-stranded (ds) RNA corresponding to the A . thaliana gene, decrease in DNA methylation 1 (DDM1) caused the elimination of DDM1 mRNAs and the loss of methylation at both A . thaliana- and A . arenosa-derived centromere repeats . These results indicate that a single RNAi-inducing transgene can dominantly repress multiple orthologs.

Plant Physiol, 2003 Oct, 133(2), 736 - 47 Epub 2003 Sep 11.
Development of protoporphyrinogen oxidase as an efficient selection marker for Agrobacterium tumefaciens-mediated transformation of maize; Li X et al.; In this article, we report the isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-tolerant mutants . Subsequently, an Arabidopsis double mutant (Y426M + S305L) was used to develop a selectable marker system for Agrobacterium tumefaciens-mediated transformation of maize (Zea mays) and to obtain multiple events tolerant to the PPO family of herbicides . Maize transformants were produced via butafenacil selection using a flexible light regime to increase selection pressure . Butafenacil selection per se did not change transgene copy number distribution relative to other selectable marker systems, but the most tolerant events identified in the greenhouse were more likely to contain multiple copies of the introduced mutant PPO gene . To date, more than 2,500 independent transgenic maize events have been produced using butafenacil selection . The high frequency of A . tumefaciens-mediated transformation via PPO selection enabled us to obtain single-copy transgenic maize lines tolerant to field levels of butafenacil.

Plant Physiol, 2003 Nov, 133(3), 966 - 77 Epub 2003 Sep 04.
Translation start sequences affect the efficiency of silencing of Agrobacterium tumefaciens T-DNA oncogenes; Lee H et al.; Agrobacterium tumefaciens oncogenes cause transformed plant cells to overproduce auxin and cytokinin . Two oncogenes encode enzymes that convert tryptophan to indole-3-acetic acid (auxin): iaaM (tryptophan mono-oxygenase) and iaaH (indole-3-acetamide hydrolase) . A third oncogene (ipt) encodes AMP isopentenyl transferase, which produces cytokinin (isopentenyl-AMP) . Inactivation of ipt and iaaM (or iaaH) abolishes tumorigenesis . Because adequate means do not exist to control crown gall, we created resistant plants by introducing transgenes designed to elicit posttranscriptional gene silencing (PTGS) of iaaM and ipt . Transgenes that elicit silencing trigger sequence-specific destruction of the inducing RNA and messenger RNAs with related sequences . Although PTGS has proven effective against a variety of target genes, we found that a much higher percentage of transgenic lines silenced iaaM than ipt, suggesting that transgene sequences influenced the effectiveness of PTGS . Sequences required for oncogene silencing included a translation start site . A transgene encoding a translatable sense-strand RNA from the 5' end of iaaM silenced the iaaM oncogene, but deletion of the translation start site abolished the ability of the transgene to silence iaaM . Silencing A . tumefaciens T-DNA oncogenes is a new and effective method to produce plants resistant to crown gall disease.

Plant Physiol, 2003 Sep, 133(1), 253 - 62
Does lowering glutamine synthetase activity in nodules modify nitrogen metabolism and growth of Lotus japonicus?
Harrison J, Pou de Crescenzo MA, Sene O, Hirel B.
A cDNA encoding cytosolic glutamine synthetase (GS) from Lotus japonicus was fused in the antisense orientation relative to the nodule-specific LBC3 promoter of soybean (Glycine max) and introduced into L . japonicus via transformation with Agrobacterium tumefaciens . Among the 12 independent transformed lines into which the construct was introduced, some of them showed diminished levels of GS1 mRNA and lower levels of GS activity . Three of these lines were selected and their T(1) progeny was further analyzed both for plant biomass production and carbon and nitrogen (N) metabolites content under symbiotic N-fixing conditions . Analysis of these plants revealed an increase in fresh weight in nodules, roots and shoots . The reduction in GS activity was found to correlate with an increase in amino acid content of the nodules, which was primarily due to an increase in asparagine content . Thus, this study supports the hypothesis that when GS becomes limiting, other enzymes (e.g . asparagine synthetase) that have the capacity to assimilate ammonium may be important in controlling the flux of reduced N in temperate legumes such as L . japonicus . Whether these alternative metabolic pathways are important in the control of plant biomass production still remains to be fully elucidated.

Plant Physiol, 2003 Sep, 133(1), 161 - 9
Golden Indica and Japonica rice lines amenable to deregulation; Hoa TT et al.; As an important step toward free access and, thus, impact of GoldenRice, a freedom-to-operate situation has been achieved for developing countries for the technology involved . Specifically, to carry the invention beyond its initial "proof-of-concept" status in a Japonica rice (Oryza sativa) cultivar, we report here on two transformed elite Indica varieties (IR64 and MTL250) plus one Japonica variety Taipei 309 . Indica varieties are predominantly consumed in the areas with vitamin A deficiency . To conform with regulatory constraints, we changed the vector backbone, investigated the absence of beyond-border transfer, and relied on Agrobacterium tumefaciens-mediated transformation to obtain defined integration patterns . To avoid an antibiotic selection system, we now rely exclusively on phosphomannose isomerase as the selectable marker . Single integrations were given a preference to minimize potential epigenetic effects in subsequent generations . These novel lines, now in the T(3) generation, are highly valuable because they are expected to more readily receive approval for follow-up studies such as nutritional and risk assessments and for breeding approaches leading to locally adapted variety development.

Mycol Res, 2003 Jul, 107(Pt 7), 803 - 10
Agrobacterium and PEG-mediated transformation of the phytopathogen Venturia inaequalis; Fitzgerald AM et al.; We report the development of two new transformation systems, polyethylene glycol (PEG)-mediated transformation of protoplasts and Agrobacterium tumefaciens-mediated transformation of mycelium, for the filamentous ascomycete Venturia inaequalis . New binary vectors have been created for the latter . Although transformation was initially achieved using a PEG-mediated method, this was superseded by the A . tumefaciens-mediated approach . The advantages of the latter include: ease of the protocol, no requirement for protoplasts; higher transformation efficiency; and single-site integration . A comparison between the two transformation methods is presented.

Shi Yan Sheng Wu Xue Bao, 2003 Jun, 36(3), 226 - 32
{Transformation of wml1 5' promoter region into tomato plants and studies on its transcriptional regulation role}; Wu HY et al.; According to its restriction sites, fragments of 1573 bp, 1197 bp, 896 bp and 795 bp were obtained from the 5' promoter region of wml1 and fused with the coding sequence of the GUS gene . Constructs containing these fragments were introduced into tomato plants via Agrobacterium-mediated transformation . Histochemical assay of GUS expression in transgenic tomato plants revealed that fragments of 1573 bp, 1197 bp, 896 bp were able to direct GUS expression in fruits of 15, 30, 45 days after anthesis with the expression level of GUS increasing with fruit development, but not in leaves, stems and roots . While no GUS expression was observed in tomato plants transformed by construct containing fragment of 795 bp . It was determined that the region from 857 bp to 957 bp contains the elements necessary for directing fruit-specific expression.

J Plant Physiol, 2003 Aug, 160(8), 977 - 9
Activity of a flax pectin methylesterase promoter in transgenic tobacco pollen; Lacoux J et al.; The regulatory region of the flax Lupme3 gene, which codes for a pectin methylesterase, contains two sequences (PB box) that are putative cis-active sequence elements thought to regulate transcription in pollen . The Lupme3 promoter was fused to the beta-glucuronidase (gus) reporter gene . The chimeric gene fusion was introduced into tobacco via Agrobacterium-mediated transformation . Expression of the reporter gene was monitored using a histochemical X-Gluc assay at different stages of pollen maturation and germination . The Lupme3 promoter was found to be active in germination-competent mature pollen and in pollen tube.

Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10659 - 63 Epub 2003 Sep 05.
De novo synthesis of bacterial glycogen: Agrobacterium tumefaciens glycogen synthase is involved in glucan initiation and elongation; Ugalde JE et al.; Evidence is presented indicating that initiation of glycogen synthesis in Agrobacterium tumefaciens does not require the presence of alpha(1,4)-linked glucans . Crude cell extracts incubated with ADP-glucose (Glc) were able to form alpha(1,4)-linked glucans despite the fact that cells used for extract preparation displayed a genotype that prevented synthesis of Glc-containing sugar nucleotides and thus preformation of alpha(1,4)-linked glucans and that the defined growth medium used contained glycerol as carbon source . A . tumefaciens glycogen synthase (GS) purified to homogeneity from the above-mentioned cells was able to build its own primer by transferring Glc residues from ADP-Glc to an amino acid(s) in the same protein . Primed GS then became the substrate for further GS-catalyzed glucan elongation . It was concluded that, contrary to what happens in mammalian and yeast cells in which two different proteins are required for linear alpha(1,4)-linked glucan formation (glycogenin for initiation and GS for further elongation), in A . tumefaciens and probably in all other bacteria, the same protein is involved in both glycogen initiation and elongation.

J Plant Res, 2003 Dec, 116(6), 455 - 60 Epub 2003 Sep 04.
Expression of a bacterial aroA mutant, aroA-M1, encoding 5-enolpyruvylshikimate-3-phosphate synthase for the production of glyphosate-resistant tobacco plants; Wang HY et al.; Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants . We have previously reported a strategy for engineering glyphosate-resistant class I EPSPS based on staggered-PCR technology . Selected mutant enzymes exhibited high K(i){glyphosate} and low K(m){PEP} values compared to the parental enzymes from Escherichia coli ( EcaroA) and Salmonella typhimurium ( StaroA) . One mutant, aroA-M1, was further engineered with a tobacco chloroplast leader sequence, and then placed in the binary vector pCAMBIA1300 for Agrobacterium-mediated gene transfer to tobacco ( Nicotiana tabacum cv . Xanthi) . Transgenic plants with increased resistance to glyphosate were generated.

Scand J Infect Dis, 2003, 35(6-7), 410 - 1
Endophthalmitis caused by Agrobacterium radiobacter; Pierre-Filho Pde T et al.; Infections due to Agrobacterium radiobacter are rare . This study reports 2 cases of A . radiobacter endophthalmitis . To the authors' knowledge, these are only the second and third reported cases of endophthalmitis caused by this Gram-negative rod.

J Agric Food Chem, 2003 Sep 10, 51(19), 5695 - 702
Enhancement of the primary flavor compound methional in potato by increasing the level of soluble methionine; Di R et al.; The primary flavor compound in potato, methional, is synthesized from methionine by the Strecker degradation reaction . A major problem associated with potato processing is the loss of methional . Methional or its precursor, methionine, is not added back during potato processing due to high costs of production . A novel approach to enhance the methional level in processed potato would be to increase the production of its precursor, soluble methionine (Met) . Cystathionine gamma-synthase (CGS) is a key enzyme regulating methionine biosynthesis in plants . To increase the level of soluble methionine in potato, Arabidopsis thaliana CGS cDNA was introduced under transcriptional control of the cauliflower mosaic virus 35S promoter into Russet Burbank potato by Agrobacterium-mediated transformation . Ten different transgenic potato lines (CGS1-10) were analyzed . Immunoblot analysis demonstrated that Arabidopsis CGS is expressed in the leaves, tubers, and roots of transgenic potato plants . CGS enzymatic activity was higher in the leaves and roots of the transgenic potato lines compared to the wild-type potato . Methionine levels in the leaves, roots and tubers of transgenic potato lines were enhanced as high as 6-fold compared to those in wild type potato plants . The methional level in baked tubers of field-grown transgenic potato lines was increased between 2.4- and 4.4-fold in lines CGS1, CGS2, and CGS4 . The increase observed in methional levels correlated with the soluble methionine level in the tubers from the same lines measured before processing . These results provide the first evidence that the methional level can be enhanced in processed potatoes by increasing the production of its precursor, methionine.

Mol Microbiol, 2003 Sep, 49(6), 1699 - 713
VirE2, a type IV secretion substrate, interacts with the VirD4 transfer protein at cell poles of Agrobacterium tumefaciens; Atmakuri K et al.; Agrobacterium tumefaciens transfers oncogenic DNA and effector proteins to plant cells during the course of infection . Substrate translocation across the bacterial cell envelope is mediated by a type IV secretion (TFS) system composed of the VirB proteins, as well as VirD4, a member of a large family of inner membrane proteins implicated in the coupling of DNA transfer intermediates to the secretion machine . In this study, we demonstrate with novel cytological screens - a two-hybrid (C2H) assay and bimolecular fluorescence complementation (BiFC) - and by immunoprecipitation of chemically cross-linked protein complexes that the VirE2 effector protein interacts directly with the VirD4 coupling protein at cell poles of A . tumefaciens . Analyses of truncation derivatives showed that VirE2 interacts via its C terminus with VirD4, and, further, an NH2-terminal membrane-spanning domain of VirD4 is dispensable for complex formation . VirE2 interacts with VirD4 independently of the virB-encoded transfer machine and T pilus, the putative periplasmic chaperones AcvB and VirJ, and the T-DNA transfer intermediate . Finally, VirE2 is recruited to polar-localized VirD4 as a complex with its stabilizing secretion chaperone VirE1, yet the effector-coupling protein interaction is not dependent on chaperone binding . Together, our findings establish for the first time that a protein substrate of a type IV secretion system is recruited to a member of the coupling protein superfamily.

Biochemistry (Mosc), 2003 Jul, 68(7), 795 - 801
Increase in anthraquinone content in Rubia cordifolia cells transformed by rol genes does not involve activation of the NADPH oxidase signaling pathway; Bulgakov VP et al.; It has been reported that rol plant oncogenes located in Ri-plasmids of Agrobacterium rhizogenes activated synthesis of secondary metabolites in the transformed plant cells . The activator mechanism is still unknown . In this work, we studied whether the NADPH oxidase-signaling pathway, which regulates the synthesis of defense metabolites in plants, is involved in the activator function of the rol genes . It was demonstrated that the transformation of Rubia cordifolia cells by the rolB and rolC genes caused an induction of biosynthesis of anthraquinone-type phytoalexins . Inhibition studies revealed a striking difference between the rolC and rolB transformed cultures in their sensitivity to Ca2+ channel blockers and calcium deficiency . The rolC culture displayed lowered resistance to the inhibitors compared to the non-transformed culture, while the rolB culture was more resistant to the treatment . The assumption was made that the oncogenic potential of rol genes is realized through the alteration of calcium balance in the plant cells . Anthraquinone production was not inhibited in the non-transformed and transformed cultures by Ca2+ channel blockers, as well as by diphenylene iodonium, an inhibitor of NADPH oxidase, and by the protein kinase inhibitor staurosporine . These results indicate that the induction of anthraquinone production in transgenic cultures does not involve the activation of Ca2+-dependent NADPH oxidase pathway.

Mikrobiol Z, 2003 May-Jun, 65(3), 5 - 13
{Effect of the lipopolysaccharide-protein complex of Pseudomonas syringae PV . atrofaciens on the process of tumor formation caused by Agrobacterium tumefaciens}; Hvozdiak RI et al.; It has been found that the agent of bacterial spotting of rye P . syringae pv . atrofaciens 8281 has no antagonistic action on the strains 9052 and 9054 of Agrobacterium tumefaciens--the agent of crown gall tumor . Lipopolysaccharide-protein complex of P . syringae pv . atrofaciens 8281 when added to the culture medium does not affect the growth and development of A . tumefaciens bacteria . It has been experimentally established that the lipopolysaccharide-protein complex of P . syringae pv . atrofaciens 8281, when used to treat the potato explants before inoculation of A . tumefaciens, decreases induction and development of tumours on the explants . Tumour formation inhibition depends on concentration of lipopolysaccharide-protein complex solution.

Plant J, 2003 Jan, 33(1), 161 - 75
Identification of peroxisomal targeting signal of pumpkin catalase and the binding analysis with PTS1 receptor; Kamigaki A et al.; Many peroxisomal proteins are imported into peroxisomes via recognition of the peroxisomal targeting signal (PTS1) present at the C-termini by the PTS1 receptor (Pex5p) . Catalase, a peroxisomal protein, has PTS1-like motifs around or at the C-terminus . However, it remains unclear whether catalase is imported into peroxisome via the PTS1 system . In this work, we analyzed the PTS of pumpkin catalase (Cat1) . A full or truncated pumpkin Cat1 cDNA fused at the 3' end of the green fluorescent protein (GFP) coding sequence was introduced and stably expressed in tobacco BY-2 (Nicotiana tabacum cv . Bright Yellow 2) cells or Arabidopsis thaliana by Agrobacterium-mediated transformation . The cellular localization of GFP was analyzed by fluorescence microscopy . The results showed that the C-terminal 10-amino acid region containing an SKL motif-like tripeptide (SHL) was not required for the import into peroxisomes . Surprisingly, the C-terminal 3-amino acid region was required for the import when the fusion proteins were transiently expressed by using particle gun bombardment, suggesting that the transient expression system is inadequate to analyze the targeting signal . We proposed that the C-terminal amino acid region from 13 to 11 (QKL), which corresponds with the PTS1 consensus sequence, may function as an internal PTS1 . Analysis of the binding of Cat1 to PTS1 receptor (Pex5p) by the yeast two-hybrid system revealed that Cat1 can bind with the PTS1 receptor (Pex5p), indicating that Cat1 is imported into peroxisomes by the PTS1 system.

Mol Biol (Mosk), 2003 Jul-Aug, 37(4), 654 - 62
{Construction of the synthetic genes for protein analogs of spider silk carcass spidroin 1 and their expression in tobacco plants}; Piruzian ES et al.; To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3'-fused in-frame with the reporter lichenase gene . The Tr2' weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements . The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks . On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one . Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants . The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.

Plant J, 2003 Sep, 35(5), 557 - 65
Ku80 plays a role in non-homologous recombination but is not required for T-DNA integration in Arabidopsis; Gallego ME et al.; Chromosomal breaks are repaired by homologous recombination (HR) or non-homologous end joining (NHEJ) mechanisms . The Ku70/Ku80 heterodimer binds DNA ends and plays roles in NHEJ and telomere maintenance in organisms ranging from yeast to humans . We have previously identified a ku80 mutant of the model plant Arabidopsis thaliana and shown the role of Ku80 in telomere homeostasis in plant cells . We show here that this mutant is hypersensitive to the DNA-damaging agent methyl methane sulphonate and has a reduced capacity to carry out NHEJ recombination . To understand the interplay between HR and NHEJ in plants, we measured HR in the absence of Ku80 . We find that the frequency of intrachromosomal HR is not affected by the absence of Ku80 . Previous work has clearly implicated the Ku heterodimer in Agrobacterium-mediated T-DNA transformation of yeast . Surprisingly, ku80 mutant plants show no defect in the efficiency of T-DNA transformation of plants with Agrobacterium, showing that an alternative pathway must exist in plants.

Curr Genet, 2003 Nov, 44(3), 164 - 71 Epub 2003 Aug 21.
Agrobacterium-mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences; Rolland S et al.; The Agrobacterium tumefaciens-mediated transfer of foreign DNA to the phytopathogenic fungus Botrytis cinerea was investigated . Fifteen stable transformants per 10(6) conidia were consistently produced . Monokaryons were purified in a single step and their molecular analysis demonstrated the random integration of predominantly single or tandem copies of the foreign DNA into their genome . Thermal asymmetric interlaced PCR performed directly on conidia led to the rapid identification of the genomic DNA sequences that flanked the integration sites of the transfer-DNA . Transcriptional fusions of green fluorescent protein and beta-glucuronidase-encoding genes to the promoter of the secreted proteolytic enzyme ACP1 were realised to validate the system . We provide herein observations of B . cinerea hyphae producing green fluorescent protein or beta-glucuronidase under growth conditions similar to those known to induce transcription of the acp1 gene.

Carbohydr Res, 2003 Sep 1, 338(18), 1891 - 4
Elucidation of the O-chain structure from the lipopolysaccharide of Agrobacterium tumefaciens strain C58; De Castro C et al.; A linear homopolysaccharide built of 3-alpha-L-6dTalp residues, randomly acetylated at position C-4, is described for the O-specific polysaccharide of Agrobacterium tumefaciens strain C58 . This structure, determined by spectroscopical and chemical methods, is strictly correlated to that of Rhizobium loti strain NZP2213, which differs for the degree and the position of O-acetylation.

Trends Plant Sci, 2003 Aug, 8(8), 380 - 6
Agrobacterium tumefaciens as an agent of disease; Escobar MA et al.; Twenty-six years ago it was found that the common soil bacterium Agrobacterium tumefaciens is capable of extraordinary feats of interkingdom genetic transfer . Since this discovery, A . tumefaciens has served as a model system for the study of type IV bacterial secretory systems, horizontal gene transfer and bacterial-plant signal exchange . It has also been modified for controlled genetic transformation of plants, a core technology of plant molecular biology . These areas have often overshadowed its role as a serious, widespread phytopathogen - the primary driver of the first 80 years of Agrobacterium research . Now, the diverse areas of A . tumefaciens research are again converging because new discoveries in transformation biology and the use of A . tumefaciens vectors are allowing the development of novel, effective biotechnology-based strategies for the control of crown gall disease.

Mol Genet Genomics, 2003 Nov, 270(2), 139 - 46 Epub 2003 Aug 15.
Characterization of a wound-inducible cytochrome P450 gene ( CYP72A29) that is down-regulated during crown gall tumorigenesis in potato tuber; Kato H et al.; A cDNA for a putative cytochrome P450 (CYP72A29), which is down-regulated during Agrobacterium tumefaciens -mediated tumorigenesis, was isolated from potato ( Solanum tuberosumL . May Queen) tuber by differential display . Northern analysis indicated that the CYP72A29 gene was transiently up-regulated in tuber discs after a 24-h aging period, and expression gradually increased upon further incubation (up to 7 days) . Inoculation of tuber discs with the non-pathogenic A . tumefaciens strain LBA4301 had a very similar effect, but inoculation with the wild-type strain A . tumefaciens A208 suppressed the accumulation of mRNA during further incubation . Furthermore, the accumulation of CYP72A29 mRNA was strongly suppressed by inoculation with mutant strains of A . tumefaciens that produce large amounts of indole-3-acetic acid . This down-regulation also occurred when the discs were treated with 2,4-dichlorophenoxyacetic acid or 1-naphthalenacetic acid . These results suggest that the expression of CYP72A29 is regulated by auxin . RT-PCR analysis revealed that the transcripts were most abundant in sprouts and eyes, less so in roots, and hardly detectable in leaves, flower buds and stems.

Plant Cell Rep, 2003 Nov, 22(4), 268 - 73 Epub 2003 Aug 15.
Induction of male sterile cabbage using a tapetum-specific promoter from Brassica campestris L . ssp . pekinensis; Lee YH et al.; The anther (tapetum)-specific gene BcA9 was isolated from Chinese cabbage, Brassica campestris L . ssp . pekinensis cv . Jangwon, using the Arabidopsis tapetum-specific A9 gene as a probe . The DNA and amino acid sequences of the coding region of the BcA9 gene showed high homology with A9 genes from Arabidopsis and B . napus . However, the DNA sequences of the 5' noncoding (promoter) region were different, except for the sequence from -281 to -89 . To test the specific activity of this promoter, a plant expression vector, pGR011, was constructed by fusing the BcA9 promoter and the cytotoxic diphtheria toxin A-chain (DTx-A) gene . Several transgenic plants from cabbage, B . oleracea ssp . capitata, were obtained by way of Agrobacterium-mediated transformation . Southern blot analysis indicated that the tapetum-specific BcA9 promoter and DTx-A gene were successfully integrated into the genome of the transgenic cabbage . Under the control of the BcA9 promoter, expression of the cytotoxic DTx-A gene in the tapetal cells of the transgenic plants resulted in male sterile cabbages . Microscopic examination revealed that pollen grains in anthers of the male sterile cabbages had not developed normally, but the vegetative growth and phenotype showed no difference compared to wild-type plants.

Plant Physiol, 2003 Aug, 132(4), 2205 - 17
Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase; Anoop VM et al.; Aluminum (Al) toxicity is a major constraint for crop production in acid soils, although crop cultivars vary in their tolerance to Al . We have investigated the potential role of citrate in mediating Al tolerance in Al-sensitive yeast (Saccharomyces cerevisiae; MMYO11) and canola (Brassica napus cv Westar) . Yeast disruption mutants defective in genes encoding tricarboxylic acid cycle enzymes, both upstream (citrate synthase {CS}) and downstream (aconitase {ACO} and isocitrate dehydrogenase {IDH}) of citrate, showed altered levels of Al tolerance . A triple mutant of CS (Deltacit123) showed lower levels of citrate accumulation and reduced Al tolerance, whereas Deltaaco1- and Deltaidh12-deficient mutants showed higher accumulation of citrate and increased levels of Al tolerance . Overexpression of a mitochondrial CS (CIT1) in MMYO11 resulted in a 2- to 3-fold increase in citrate levels, and the transformants showed enhanced Al tolerance . A gene for Arabidopsis mitochondrial CS was overexpressed in canola using an Agrobacterium tumefaciens-mediated system . Increased levels of CS gene expression and enhanced CS activity were observed in transgenic lines compared with the wild type . Root growth experiments revealed that transgenic lines have enhanced levels of Al tolerance . The transgenic lines showed enhanced levels of cellular shoot citrate and a 2-fold increase in citrate exudation when exposed to 150 micro M Al . Our work with yeast and transgenic canola clearly suggest that modulation of different enzymes involved in citrate synthesis and turnover (malate dehydrogenase, CS, ACO, and IDH) could be considered as potential targets of gene manipulation to understand the role of citrate metabolism in mediating Al tolerance.

Plant Physiol, 2003 Aug, 132(4), 2174 - 83
Overproduction of cytokinins in petunia flowers transformed with P(SAG12)-IPT delays corolla senescence and decreases sensitivity to ethylene; Chang H et al.; Plant senescence is regulated by a coordinated genetic program mediated in part by changes in ethylene, abscisic acid (ABA), and cytokinin content . Transgenic plants with delayed senescence are useful for studying interactions between these signaling mechanisms . Expression of ipt, a cytokinin biosynthetic gene from Agrobacterium tumefaciens, under the control of the promoter from a senescence-associated gene (SAG12) has been one approach used to delay senescence . We transformed petunia (Petunia x hybrida cv V26) with P(SAG12)-IPT . Two independently transformed lines with extended flower longevity (I-1-7-22 and I-3-18-34) were used to study the effects of elevated cytokinin content on ethylene synthesis and sensitivity and ABA accumulation in petunia corollas . Floral senescence in these lines was delayed 6 to 10 d relative to wild-type (WT) flowers . Ipt transcripts increased in abundance after pollination and were accompanied by increased cytokinin accumulation . Endogenous ethylene production was induced by pollination in both WT and IPT corollas, but this increase was delayed in IPT flowers . Flowers from IPT plants were less sensitive to exogenous ethylene and required longer treatment times to induce endogenous ethylene production, corolla senescence, and up-regulation of the senescence-related Cys protease phcp1 . Accumulation of ABA, another hormone regulating flower senescence, was significantly greater in WT corollas, confirming that floral senescence was delayed in IPT plants . These results extend our understanding of the hormone interactions that regulate flower senescence and provide a means of increasing flower longevity.

Sichuan Da Xue Xue Bao Yi Xue Ban, 2003 Jul, 34(3), 385 - 9
{Construction of plant expressive vector of human beta-defensin-2 gene}; Cai S et al.; OBJECTIVE: Several evidences suggested that transgenic plants would be a facile and economic bioreactor for large-scale production of industrial and pharmaceutical recombinant proteins . This study is made in an attempt to establish plant bioreactor for expression of recombinant hBD-2 . METHODS: Recombinant hBD-2 gene with C terminal of bi-tags of myc and 6xHis was inserted into a plant expressive vector-pCAMBIA1304, which closely located the down-stream of CaMV35S promoter . Agrobacterium tumefaciens LBA4404 was transformed with the recombinant plant expressive vector: rpCAMBIA1304/hBD-2/His . The positive clones of LBA4404 transformed by rpCAMBIA1304/hBD-2/His were selected on a culture plate containing kanamycin . The callus tissues were transfected by positive clones of LBA4404, and positive callus were examined by using the resistant selection of hygromycin gene . RESULTS: The evidences of enzyme digestion, PCR and sequence analysis confirmed that recombinant hBD-2 gene with C terminal of bi-tags of myc and 6xHis was correctly inserted into pCAMBIA1304 and was located between CaMV35S promoter and Nos terminal cordon to construct recombinant plant expressive vector: rpCAMBIA1304/hBD-2/His, thus indicating that rpCAMBIA1304/hBD-2/His has successfully transformed Agrobacterium tumefaciens LBA4404 and positive clones have been isolated . The results from resistant selection of hygromycin gene showed that rpCAMBIA1304/hBD-2/His has been transferred into the callus of wheat, and the differentiation of callus tissue under selective pressure of hygromycin is carried out continually . CONCLUSION: The above data suggest that the technique of transgenic plant is workable for the production of recombinant hBD-2.

Plant Cell Rep, 2003 Aug, 21(12), 1194 - 8 Epub 2003 May 13.
Genetic transformation of the figwort, Scrophularia buergeriana Miq., an Oriental medicinal plant; Park SU et al.; Scrophularia buergeriana Miq . (figwort) contains a diverse group of bioactive natural products and is used to treat a variety of ailments, including fever, constipation, neuritis, and laryngitis . A transformation protocol was established for S . buergeriana using Agrobacterium tumefaciens . Kanamycin-resistant plants were regenerated from leaf explants co-cultivated with A . tumefaciens strain GV3101 . The shoot regeneration medium was supplemented with 2 mg l(-1) 6-benzylaminopurine and 70 mg l(-1) putrescine to improve the efficiency of organogenesis . Detection of the neomycin phosphotransferase gene, the presence of high levels of beta-glucuronidase (GUS) transcripts and enzyme activity, and the histochemical localization of GUS confirmed the genetic transformation of S . buergeriana . This work demonstrates the potential of using A . tumefaciens to efficiently transfer foreign genes into a commercially and culturally important Oriental medicinal plant.

Mol Plant Microbe Interact, 2003 Aug, 16(8), 663 - 8
Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules; Kumagai H et al.; We investigated the efficacy of self-complementary hairpin RNA (hpRNA) expression to induce RNA silencing in the roots and nodules of model legume Lotus japonicus, using hairy root transformation mediated by Agrobacterium rhizogenes . Transgenic lines that express beta-glucuronidase (GUS) by constitutive or nodule-specific promoters were supertransformed by infection of A . rhizogenes harboring constructs for the expression of hpRNAs with sequences complementary to the GUS coding region . GUS activity in more than 60% of the hairy roots was decreased or silenced almost completely . Silencing of the GUS gene was also observed in symbiotic nodules formed on hairy roots in both early and late stages of nodule organogenesis . These results indicate that transient RNA silencing by hairy root transformation provides a powerful tool for loss-of-function analyses of genes that function in roots and root nodules.

Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 9912 - 7 Epub 2003 Aug 06.
Comparative genomics of bacterial zinc regulons: enhanced ion transport, pathogenesis, and rearrangement of ribosomal proteins; Panina EM et al.; Zinc is an important component of many proteins, but in large concentrations it is poisonous to the cell . Thus its transport is regulated by zinc repressors ZUR of proteobacteria and Gram-positive bacteria from the Bacillus group and AdcR of bacteria from the Streptococcus group . Comparative computational analysis allowed us to identify binding signals of ZUR repressors GAAATGTTATANTATAACATTTC for gamma-proteobacteria, GTAATGTAATAACATTAC for the Agrobacterium group, GATATGTTATAACATATC for the Rhododoccus group, TAAATCGTAATNATTACGATTTA for Gram-positive bacteria, and TTAACYRGTTAA of the streptococcal AdcR repressor . In addition to known transporters and their paralogs, zinc regulons were predicted to contain a candidate component of the ATP binding cassette, zinT (b1995 in Escherichia coli and yrpE in Bacillus subtilis) . Candidate AdcR-binding sites were identified upstream of genes encoding pneumococcal histidine triad (PHT) proteins from a number of pathogenic streptococci . Protein functional analysis of this family suggests that PHT proteins are involved in the invasion process . Finally, repression by zinc was predicted for genes encoding a variety of paralogs of ribosomal proteins . The original copies of all these proteins contain zinc-ribbon motifs and thus likely bind zinc, whereas these motifs are destroyed in zinc-regulated paralogs . We suggest that the induction of these paralogs in conditions of zinc starvation leads to their incorporation in a fraction of ribosomes instead of the original ribosomal proteins; the latter are then degraded with subsequent release of some zinc for the utilization by other proteins . Thus we predict a mechanism for maintaining zinc availability for essential enzymes.

Microbiology, 2003 Aug, 149(Pt 8), 1981 - 9
Novel bacteria degrading N-acylhomoserine lactones and their use as quenchers of quorum-sensing-regulated functions of plant-pathogenic bacteria; Uroz S et al.; Bacteria degrading the quorum-sensing (QS) signal molecule N-hexanoylhomoserine lactone were isolated from a tobacco rhizosphere . Twenty-five isolates degrading this homoserine lactone fell into six groups according to their genomic REP-PCR and rrs PCR-RFLP profiles . Representative strains from each group were identified as members of the genera Pseudomonas, Comamonas, Variovorax and Rhodococcus: all these isolates degraded N-acylhomoserine lactones other than the hexanoic acid derivative, albeit with different specificity and kinetics . One of these isolates, Rhodococcus erythropolis strain W2, was used to quench QS-regulated functions of other microbes . In vitro, W2 strongly interfered with violacein production by Chromobacterium violaceum, and transfer of pathogenicity in Agrobacterium tumefaciens . In planta, R . erythropolis W2 markedly reduced the pathogenicity of Pectobacterium carotovorum subsp . carotovorum in potato tubers . These series of results reveal the diversity of the QS-interfering bacteria in the rhizosphere and demonstrate the validity of targeting QS signal molecules to control pathogens with natural bacterial isolates.

J Clin Microbiol, 2003 Aug, 41(8), 3998 - 4000
Rhizobium (Agrobacterium) radiobacter identified as a cause of chronic endophthalmitis subsequent to cataract extraction; Namdari H et al.; Herein, we report a case of chronic endophthalmitis caused by a ceftazidime-resistant Rhizobium radiobacter strain in a 62-year-old male . The patient underwent an uneventful cataract extraction of the right eye a week prior to the appearance of symptoms (pain, redness, and blurring vision) which developed following a golf outing . Upon admission the patient received an emergency vitrectomy . The patient remained symptomatic, and R . radiobacter was isolated repeatedly from vitreous fluid cultures over a 5-month period . Ultimately, the infection responded to intravitreal gentamicin, oral ciprofloxacin, and removal of the lens implant.

Nucleic Acids Symp Ser, 2000, (44), 185 - 6
Different toxicity of Escherichia coli and Agrobacterium tumefaciens against tobacco BY2 cells; Fukumaru K et al.; Tobacco BY2 cells were withered away by the physical contact with intestinal bacterium Escherichia coli . In contrast, plant pathogenic bacterium Agrobacterium tumefaciens with/without tumor inducing Ti plasmid was not so toxic as E . coli . Mini-cells of E . coli decreased their toxicity.

Nucleic Acids Symp Ser, 2000, (44), 97 - 8
Toward structural and functional genomics of Agrobacterium tumefaciens: linkage map of the left region of linear chromosome; De Costa DM et al.; Genome of A . tumefaciens contains a linear and a circular chromosome . As an initial step of elucidating the structural and functional genomics of this bacterium, linkage map of the left region of its linear chromosome was constructed . Total genomic libraries of A . tumefaciens MAFF301001 were constructed in BAC vectors namely, pFOS1 and pBeloBAC11 . Upon construction of sub-libraries, minimum overlapping clones needed to cover the left region was determined . So far, four contigs have been assembled with a total of 19 overlapping clones . Detailed EcoRI physical map of contig III was constructed and it covers a 110 kb region of the Pme5 fragment of the linear chromosome . Seven end regions of the linking clones were partially sequenced but no gene existence was determined due to low homology.

Nucleic Acids Symp Ser, 2000, (44), 95 - 6
Genome structure of Ri plasmid (3) . Sequencing analysis of the vir region of pRi1724 in Japanese Agrobacterium rhizogenes; Satuti N et al.; The entire genome of the pRi1724 (217.6-kb) in the mikimopine type Agrobacterium rhizogenes strain MAFF03-01724 has been completely sequenced . The vir region covering 30.2-kb has found to be composed of 21 genes resembling virH1, virA, virB1-11, virG, virC1-2, and virD1-5 . The structural organization of the pRi1724 vir operons in this study is exactly the same as that of the previously reported vir operons of other Ri or Ti plasmids, although the size of some ORFs showed little variations among the plasmids . We also found virE3 gene in the pRi1724 (1), but different from Ti plasmids, virE1 and virE2 that are also important for the virulence do not exist in the vir region of pRi1724.

Nucleic Acids Res Suppl, 2002, (2), 91 - 2
Characterization of four ribosomal RNA operons in the genome of Agrobacterium tumefaciens MAFF301001; Bautista-Zapanta JN et al.; The four ribosomal RNA (rrn) operons rrnA, rrnB, rrnC and were identified, sequenced completely and characterized individually rrnD in the whole genome of A tumefaciens MAFF301001 . These rrn operons were located in the two topologically different chromosomal DNAs; rrnA and rrnD in the linear chromosome and rrnB and rrnC in the circular chromosome . Each operon coded for three ribosomal RNA subunit genes; 16S, 23S and 5S rRNA . The 16S-23S internal transcribed spacers (ITS) of the four rrn operons contain genes for tRNA-Ile and tRNA-Ala and tRNA-Met downstream of 5S rDNA gene while the intergenic spacer between 23S rDNA and 5S rDNA lacked tRNA genes . Sequence alignment of 23S rRNAs of A . tumefaciens MAFF301001 and C58 strains showed unrelated sequences near the 5' end region suggesting the presence of intervening sequence (IVS) . Primer extension analyses revealed that the primary transcription products for 16S and 23S rRNAs are 1497 and 2877 bases long, respectively.

Nucleic Acids Res Suppl, 2002, (2), 89 - 90
Isolation of temperature sensitive mutants and their mapping in Agrobacterium tumefaciens; Taga T et al.; The first temperature sensitive mutants of plant pathogenic bacterium Agrobacterium tumefaciens were isolated by Tn5 mutagenesis . Several genes responsible for mutants were identified and mapped on the circular and linear chromosomes in A . tumefaciens MAFF301001.

Appl Environ Microbiol, 2003 Aug, 69(8), 4989 - 93
The Ti plasmid of Agrobacterium tumefaciens harbors an attM-paralogous gene, aiiB, also encoding N-Acyl homoserine lactonase activity; Carlier A et al.; The Agrobacterium tumefaciens C58 genome contains three putative N-acyl homoserine lactone (acyl-HSL) hydrolases, which are closely related to the lactonase AiiA of BACILLUS: When expressed in Escherichia coli, two of the putative acyl-HSL hydrolases, AttM and AiiB, conferred the ability to degrade acyl-HSLs on the host . In Erwinia strain 6276, the lactonases reduced the endogenous acyl-HSL level and the bacterial virulence in planta.

Proc Natl Acad Sci U S A, 2003 Aug 19, 100(17), 10108 - 13 Epub 2003 Aug 04.
The VirD2 pilot protein of Agrobacterium-transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants; Bako L et al.; The bacterial virulence protein VirD2 plays an important role in nuclear import and chromosomal integration of Agrobacterium-transferred DNA in fungal, plant, animal, and human cells . Here we show that in nuclei of alfalfa cells, VirD2 interacts with and is phosphorylated by CAK2Ms, a conserved plant ortholog of cyclin-dependent kinase-activating kinases . CAK2Ms binds to and phosphorylates the C-terminal regulatory domain of RNA polymerase II largest subunit, which can recruit the TATA box-binding protein . VirD2 is found in tight association with the TATA box-binding protein in vivo . These results indicate that recognition of VirD2 is mediated by widely conserved nuclear factors in eukaryotes.

FEMS Microbiol Lett, 2003 Aug 8, 225(1), 167 - 72
Oxidant-inducible resistance to hydrogen peroxide killing in Agrobacterium tumefaciens requires the global peroxide sensor-regulator OxyR and KatA; Eiamphungporn W et al.; Induced adaptive and cross-protective responses to peroxide stress are important strategies used by bacteria to survive stressful environments . We have shown that exposure to low levels of peroxide (adaptive) and superoxide anions (cross-protection) induced high levels of resistance to peroxide killing in Agrobacterium tumefaciens . The mechanisms and genes involved in these processes have not been identified . Here, the roles played by peroxide (oxyR) and superoxide (soxR) global regulators and a catalase gene (katA) during these responses were investigated . H2O2-induced adaptive protection was completely abolished in both the oxyR and katA mutants . Superoxide generator (menadione)-induced cross-protection to H2O2 killing was observed in a soxR mutant, but not in either an oxyR or a katA mutant . In vivo analysis of the katA promoter, using a katA::lacZ transcriptional fusion, revealed that it could be induced by menadione in an oxyR-dependent manner . These results lead us to conclude that H2O2 and superoxide anions directly or indirectly oxidize OxyR and it is the resulting activation of katA expression that is responsible for the induced protection against lethal concentrations of H2O2.

FEMS Microbiol Lett, 2003 Aug 8, 225(1), 15 - 21
Identification of a transmissible plasmid from an Argentine Sinorhizobium meliloti strain which can be mobilised by conjugative helper functions of the European strain S . meliloti GR4; Pistorio M et al.; We describe in this work the identification and the conjugal properties of two cryptic plasmids present in the strain Sinorhizobium meliloti LPU88 isolated from an Argentine soil . One of the plasmids, pSmeLPU88b (22 kb), could be mobilised from different S . meliloti strains to other bacteria by conjugation only if the other plasmid, pSmeLPU88a (139 kb), was present . This latter plasmid, however, could not be transferred via conjugation (frequency <10(-9) transconjugants per recipient) contrasting with the conjugal system from the previously described strain GR4, where one plasmid is mobilisable and a second one (helper) is self-transmissible . Despite the differences between the two systems, the conjugative helper functions present in the cryptic plasmids of strain GR4 were active in the mobilisation of plasmid pSmeLPU88b from strain LPU88 . Contrasting with this, plasmid pSmeLPU88b was not mobilised by the helper functions of the broad-host-range plasmid RP4 . Eckhardt gel analysis showed that none of the plasmids from strain GR4 were excluded in the presence of plasmid pSmeLPU88b suggesting that they all belong to different incompatibility groups for replication . The small plasmid from strain LPU88, pSmeLPU88b, was only able to replicate in members of the Rhizobiaceae family such as Rhizobium leguminosarum, Rhizobium tropici and Agrobacterium tumefaciens, but not in Escherichia coli or Pseudomonas fluorescens . The observation suggests that most likely plasmid pSmeLPU88b was not received from a phylogenetically distant bacterium.

Plant Cell Rep, 2003 Oct, 22(3), 210 - 7 Epub 2003 Aug 01.
Regeneration and Agrobacterium-mediated transformation of hop (Humulus lupulus L.); Horlemann C et al.; An efficient procedure for direct organogenesis and regeneration of hop (Humulus lupulus L.) was established . For the first time Agrobacterium-mediated genetic transformation of hop (cv . "Tettnanger") was achieved . Shoot internodes from in vitro cultures were identified as the most suitable type of explant for regeneration . Using this type of explant, a shoot-inducing medium was developed that supported direct organogenesis of approximately 50% of the explants . Plantlets were successfully rooted and transferred to the greenhouse . Overall, in less than 6 months hop cultures propagated in vitro were regenerated to plants in the greenhouse . Agrobacterium-mediated genetic transformation was performed with the reporter gene GUS (beta-glucuronidase) . The presence and function of transgenes in plants growing in the greenhouse was verified by PCR (polymerase chain reaction) and enzyme assay for GUS activity, respectively . We have obtained 21 transgenic plants from 1,440 explants initially transformed, yielding an overall transformation efficiency of 1.5%.

Arch Microbiol, 2003 Oct, 180(4), 279 - 84 Epub 2003 Jul 31.
Agrobacterium- tumefaciens-mediated transformation of Helminthosporium turcicum, the maize leaf-blight fungus; Degefu Y et al.; Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome . Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium- tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase ( hph) and the enhanced green fluorescent protein ( EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator . Agrobacterium- tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B . The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis . Agrobacterium- tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.

Theor Appl Genet, 2003 Sep, 107(5), 958 - 64 Epub 2003 Jul 26.
BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium; Song J et al.; Development of efficient methods to transfer large DNA fragments into plants will greatly facilitate the map-based cloning of genes . The recently developed BIBAC and TAC vectors have shown potential to deliver large DNA fragments into plants via Agrobacterium-mediated transformation . Here we report that BIBAC and TAC clones containing potato genomic DNA fragments larger than 100 kb are not stable in Agrobacterium . We tested the possible factors that may cause instability, including the insert sizes of the BIBAC and TAC constructs, potato DNA fragments consisting of highly repetitive or largely single-copy DNA sequences, different Agrobacterium transformation methods and different Agrobacterium strains . The insert sizes of the potato BIBAC and TAC constructs were found to be critical to their stability in Agrobacterium . All constructs containing a potato DNA fragment larger than 100 kb were not stable in any of the four tested Agrobacterium strains, including two recA deficient strains . We developed a transposon-based technique that can be used to efficiently subclone a BAC insert into two to three BIBAC/TAC constructs to circumvent the instability problem.

Can J Microbiol, 2003 Apr, 49(4), 301 - 4
Transforming the sapstaining fungus Ophiostoma piceae with Agrobacterium tumefaciens; Tanguay P et al.; A transformation protocol mediated by Agrobacterium tumefaciens is described for the sapstaining fungus Ophiostoma piceae . We compared transformants obtained from Agrobacterium with those obtained from yeast-like cells made into spheroplasts and treated with CaCl2 . For all putative transformants analyzed, Southern hybridization confirmed that the hygromycin resistance gene had been integrated into the genomic DNA . While all transformants obtained from the treated spheroplasts had multiple copy vector insertion, 85% of the Agrobacterium-mediated transformants had single copy vector insertion.

Can J Microbiol, 2003 Apr, 49(4), 269 - 80
A phylogenetic analysis of the pSymB replicon from the Sinorhizobium meliloti genome reveals a complex evolutionary history; Wong K et al.; Microbial genomes are thought to be mosaic, making it difficult to decipher how these genomes have evolved . Whole-genome nearest-neighbor analysis was applied to the Sinorhizobium meliloti pSymB replicon to determine its origin, the degree of horizontal transfer, and the conservation of gene order . Prediction of the nearest neighbor based on contextual information, i.e., the nearest phylogenetic neighbor of adjacent genes, provided useful information for genes for which phylogenetic relationships could not be established . A large portion of pSymB genes are most closely related to genes in the Agrobacterium tumefaciens linear chromosome, including the rep and min genes . This suggests a common origin for these replicons . Genes with the nearest neighbor from the same species tend to be grouped in "patches" . Gene order within these patches is conserved, but the content of the patches is not limited to operons . These data show that 13% of pSymB genes have nearest neighbors in species that are not members of the Rhizobiaceae family (including two archaea), and that these likely represent genes that have been involved in horizontal transfer.

Science, 2003 Aug 1, 301(5633), 653 - 7
Genome-wide insertional mutagenesis of Arabidopsis thaliana; Alonso JM et al.; Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space . The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes . Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels . Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.

Fungal Genet Biol, 2003 Aug, 39(3), 264 - 75
Investigating the role of a Verticillium fungicola beta-1,6-glucanase during infection of Agaricus bisporus using targeted gene disruption; Amey RC et al.; Studies on the mycopathogen Verticillium fungicola have shown the up-regulation of beta-1,6-glucanases when grown in the presence of host cell walls and host cell wall components including chitin . These cell-wall-degrading enzymes are hypothesized to contribute to the pathogenic ability of mycopathogens . A beta-1,6-glucanase gene, VfGlu1, showing high similarity to beta-1,6-glucanase genes from Hypocrea virens, Neotyphodium sp., and Trichoderma harzianum, was isolated using degenerate PCR from V . fungicola, a serious mycopathogen of the cultivated mushroom Agaricus bisporus . Agrobacterium-mediated transformation of V . fungicola using homologous DNA from VfGlu1 resulted in homologous integration at the VfGlu1 locus in 75% of transformants, generating mutants disrupted in the VfGlu1 gene . VfGlu1 mutants displayed reduced virulence and diminished ability to utilize chitin as a carbon source, implicating VfGlu1 in the disease process . Agrobacterium-mediated transformation affords an efficient technique for the disruption of genes associated with disease symptom development in the complex V . fungicola-A . bisporus interaction.

Biotechnol Lett, 2003 Jul, 25(13), 1067 - 73
Catalytic analysis of a recombinant D-hydantoinase from Agrobacterium tumefaciens; Clemente-Jimenez JM et al.; The D-hydantoinase gene of a wild strain of Agrobacterium tumefaciens BQL9 had 99.78% nucleotide sequence identity with other available Agrobacterium genes . The resulting amino acid sequence showed two important substitutions affecting two alpha-helixes in the secondary structure of the protein . The union of Mn2+ to the protein was essential for activating the enzyme and was independent of the temperature . D-Hydantoinase only was inactivated in the presence of 70 mM EDTA and at over 40 degrees C . The enzyme showed both hydantoinase and pyrimidinase activities, but only with the D-enantiomers of the substrates . Activity was greater for substrates with apolar groups in the number 5 carbon atom of the hydantoin . The native structure of the N-terminal end of this D-hydantoinase proved to be indispensable to its enzymatic activity.

Plant J, 2003 Aug, 35(3), 418 - 27
Development of enhancer trap lines for functional analysis of the rice genome; Wu C et al.; Enhancer trapping has provided a powerful strategy for identifying novel genes and regulatory elements . In this study, we adopted an enhancer trap system, consisting of the GAL4/VP16-UAS elements with GUS as the reporter, to generate a trapping population of rice . Currently, 31 443 independent transformants were obtained from two cultivars using Agrobacterium-mediated T-DNA insertion . PCR tests and DNA blot hybridization showed that about 94% of the transformants contained T-DNA insertions . The transformants carried, on average, two copies of the T-DNA, and 42% of the transformants had single-copy insertions . Histochemical assays of approximately 1000 T0 plants revealed various patterns of the reporter gene expression, including expression in only one tissue, and simultaneously in two or more tissues . The expression pattern of the reporter gene in T1 families corresponded well with the T0 plants and segregated in a 3 : 1 Mendelian ratio in majority of the T1 families tested . The frequency of reporter gene expression in the enhancer trap lines was much higher than that in gene trap lines reported previously . Analysis of flanking sequences of T-DNA insertion sites from about 200 transformants showed that almost all the sequences had homology with the sequences in the rice genome databases . Morphologically conspicuous mutations were observed in about 7.5% of the 2679 T1 families that were field-tested, and segregation in more than one-third of the families fit the 3 : 1 ratio . It was concluded that GAL4/VP16-UAS elements provided a useful system for enhancer trap in rice.

Plant J, 2003 Aug, 35(3), 350 - 61
Absence of the Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II - effects on photosynthesis, grana stacking and fitness; Andersson J et al.; We have constructed Arabidopsis thaliana plants that are virtually devoid of the major light-harvesting complex, LHC II . This was accomplished by introducing the Lhcb2.1 coding region in the antisense orientation into the genome by Agrobacterium-mediated transformation . Lhcb1 and Lhcb2 were absent, while Lhcb3, a protein present in LHC II associated with photosystem (PS) II, was retained . Plants had a pale green appearance and showed reduced chlorophyll content and an elevated chlorophyll a/b ratio . The content of PS II reaction centres was unchanged on a leaf area basis, but there was evidence for increases in the relative levels of other light harvesting proteins, notably CP26, associated with PS II, and Lhca4, associated with PS I . Electron microscopy showed the presence of grana . Photosynthetic rates at saturating irradiance were the same in wild-type and antisense plants, but there was a 10-15% reduction in quantum yield that reflected the decrease in light absorption by the leaf . The antisense plants were not able to perform state transitions, and their capacity for non-photochemical quenching was reduced . There was no difference in growth between wild-type and antisense plants under controlled climate conditions, but the antisense plants performed worse compared to the wild type in the field, with decreases in seed production of up to 70%.

J Virol, 2003 Aug, 77(16), 9090 - 3
Expression of immunogenic S1 glycoprotein of infectious bronchitis virus in transgenic potatoes; Zhou JY et al.; The expression of infectious bronchitis virus (IBV) S1 glycoprotein in potatoes and its immunogenicity in mice and chickens were investigated . Potato plants were genetically transformed with a cDNA construct encoding the IBV S1 glycoprotein with the Agrobacterium system . Genomic DNA and mRNA analyses of the transformed plantlets confirmed the integration of the foreign cDNA into the potato genome, as well as its transcription . Mice and chickens vaccinated with the expressed IBV S1 glycoprotein produced antibodies that neutralized IBV infectivity . After three immunizations, vaccinated chickens were completely protected from virulent IBV infection . These results demonstrate that transgenic potatoes expressing IBV S1 glycoprotein can be used as a source of recombinant antigen for vaccine production.

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