Surface motility and associated surfactant production in Agrobacterium vitis

Aims:  Agrobacterium vitis is the causal agent of crown gall of grapevine. Surface motility (swarming), an important mechanism for bacterial colonization of new environments and a previously unknown behaviour of Ag. vitis , was demonstrated.
Methods:  Surface motility assays were performed on half-strength potato dextrose agar (Difco) containing 0·75% agar. To test for surfactant production, a drop-collapse test was used. Quorum-sensing (QS) negative and complemented mutants were tested for swarming activity.
Results:  Ninety-one Agrobacterium strains representing – Agrobacterium tumefaciens (17 strains), Agrobacterium rhizogenes (14 strains) and Ag. vitis (60 strains) were tested for swarming and production of surfactant. All Ag. vitis strains expressed a surface-related motility. In contrast, none of 17 strains of Ag. tumefaciens or 14 strains of Ag. rhizogenes exhibited this behaviour. Surface motility in Ag. vitis was associated with surfactant secretion; both of which are regulated by a QS system previously associated with induction of a hypersensitive response on tobacco and necrosis on grape. An aviR (belongs to luxR family) mutant was surface motility negative and did not produce surfactant. An avsI mutant (autoinducer synthase) was also surface motility negative and was complemented with an Ag. tumefaciens clone expressing avsI .
Conclusions:  Agrobacterium vitis is able to produce a characteristic swarming phenotype that is regulated by a complex QS system.
Significance and Impact of the Study:  Swarming activity is unique to Ag. vitis among Agrobacterium sp. and may be associated with the ability of the pathogen to colonize grapevines.     

Variation of 16S-23S internally transcribed spacer sequence and intervening sequence in rDNA among the three major Agrobacterium species

We analyzed 16S-23S internally transcribed spacer (ITS) and neighboring sequences among 37 strains belonging to the three major pathogenic Agrobacterium species, in order to know variation in each species and to develop a simple discrimination method. Number of ITS size variation was 9, 4, and 7 in Agrobacterium tumefaciens, Agrobacterium vitis, and Agrobacterium rhizogenes, respectively. The ITS sequence of most strains in each species was distinguishable from that of the other two species. The region surrounded by 16S rRNA gene and trn(Ala) contained information to distinguish between the ITS variants and was easy for sequencing. Intervening sequences (IVSs) in 23S rRNA gene were classified into short and long types in each species. Some long-type IVSs of A. vitis were very similar to that of A. tumefaciens, while the other long-type IVSs of A. vitis were very similar to that of A. rhizogenes. Two A. vitis strains simultaneously contained both types of IVS. Similarly, the two exceptional A. vitis strains possessed A. tumefaciens-type ITS in addition to A. vitis-type ITS. These results suggest horizontal transfer of rDNA and subsequent recombination. Among the three species, A. tumefaciens was most variable based on 16S rRNA gene, ITS and IVS sequences.     

Quorum-sensing signal production by Agrobacterium vitis strains and their tumor-inducing and tartrate-catabolic plasmids

Agrobacterium vitis strains, their tumor-inducing (pTi) and tartrate utilization (pTr) plasmid transconjugants and grapevine tumors were analyzed for the presence of N -acyl-homoserine lactones (AHLs). All wild-type A. vitis strains produced long-chain signals. PCR analysis of the A. vitis long-chain AHL synthase gene, avsI , showed the predicted amplicon. Agrobacterium tumefaciens UBAPF2 harboring various A. vitis pTi plasmids produced N -(3-oxo-octanoyl)- l -homoserine lactone encoded also by pTis of A. tumefaciens . UBAPF2 transconjugants carrying pTrs except for pTrTm4 and pTrAB3, also produced an AHL. UBAPF2 transconjugants carrying pTrAT6, pTrAB4 and pTrRr4 or pTiNi1 produced two additional AHLs not observed in the corresponding wild-type strains. We also provide evidence for in situ production of AHLs in grapevine crown gall tumors of greenhouse and field origin.     

Physical map of the vitopine Ti plasmid pTiS4.

Within the Agrobacterium vitis group the vitopine strains represent a special subclass. Vitopine bacteria carry Ti plasmids with little or no homology with the well-characterized T-DNAs of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The 262-kb Ti plasmid of the vitopine strain S4 was cloned and mapped. Homology studies with the octopine Ti plasmid pTiAch5, the nopaline Ti plasmid pTiC58, and the agropine/mannopine Ri plasmid pRiHRI identified several regions of homology. The origin of replication was localized to within 2.5 kb.     

Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.     

Construction of physical map and mapping of chromosomal virulence genes of the biovar 3 Agrobacterium (Rhizobium vitis) strain K-Ag-1

Most plant pathogenic Agrobacterium strains have been classified into three biovars, "biovar 1 (A. tumefaciens; Rhizobium radiobacter), biovar 2 (A. rhizogenes; R. rhizogenes) and biovar 3 (A. vitis; R. vitis)". The bacteria possess diverse types of genomic organization depending on the biovar. Previous genomic physical maps indicated difference in location of rDNA and chromosomally-coded virulence genes between biovar 1 and 2 genomes. In order to understand biovar 3 genome and its evolution in relation to the biovar 1, 2 and 3 genomes, we constructed physical map of a pathogenic biovar 3 strain K-Ag-1 in this study. Its genome consisted of two circular chromosomes (3.6 and 1.1 Mbp in length), and three plasmids (560, 230 and 70 kbp). Gene mapping based on the physical map showed presence of two rDNA loci in the larger chromosome and at least one rDNA locus in the smaller chromosome. Six chromosomal virulence genes, namely chvA, chvD, chvE, glgP, exoC and ros were found in the larger chromosome and not in the smaller chromosome. The location of rDNA loci is similar with that of biovar 1 genome, whereas the location of chromosomal virulence genes is similar with that of biovar 2 genome despite of the closer 16S-rRNA based phylogenetic relation of biovar 3 with biovar 1 than with biovar 2. Genomic PFGE RFLP analysis revealed that the K-Ag-1 strain, which was isolated on a kiwifruit plant in Japan, has the closest intra-species relation with two strains isolated from grapevine plants in Japan among eight biovar 3 strains examined. This datum suggests that the line of the strain is a major one in biovar 3 in Japan. Evolution of the genome of the strain is discussed based on the data.     

Agrobacterium rhizogenes GALLS protein substitutes for Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2

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

Deoxyribonucleic acid homology and taxonomy of Agrobacterium, Rhizobium, and Chromobacterium

Hybridization experiments were carried out between high molecular weight, denatured, agar-embedded deoxyribonucleic acid (DNA) and homologous, nonembedded, sheared, denatured (14)C-labeled DNA from a strain of Agrobacterium tumefaciens and Rhizobium leguminosarum (the reference strains) in the presence of sheared, nonembedded, nonlabeled DNA (competing DNA) from the same or different nomen-species of Agrobacterium, Rhizobium, Chromobacterium, and several other organisms. Percentage of DNA homology was calculated from the results. The findings are discussed in relation to previous taximetric studies, present classification schemes, and guanine-cytosine content of the DNA. Strains of A. tumefaciens, A. radiobacter, A. rubi, A. rhizogenes, R. leguminosarum, and R. meliloti exhibited a mean percentage of DNA homology greater than 50 with the two reference strains. A. tumefaciens, A. radiobacter, and A. rubi were indistinguishable on the basis of DNA homology, with strain variations for this group involving up to 30% of their base sequences. The remainder of the organisms studied fall into at least six distinct genetic groups: (i) R. (Agrobacterium) rhizogenes, which is more homologous to R. leguminosarum than to the A. tumefaciens-A. radiobacter group; (ii) R. leguminosarum; (iii) R. meliloti; (iv) R. japonicum, which has a mean DNA homology of some 38 to 45% with the reference strains; (v) Chromobacterium, which is as genetically remote from the reference strains as, for example, Pseudomonas; and (vi) A. pseudotsugae strain 180, which has a DNA homology with A. tumefaciens and R. leguminosarum of only about 10%. Since this latter homology value is similar to what was found after hybridizations between the reference strains and organisms such as Escherichia coli and Bacillus subtilis, A. pseudotsugae should definitely be removed from the genus.     

Carbohydrate Catabolism of Selected Strains in the Genus Agrobacterium

Radiorespirometric and enzyme analyses were used to reveal the glucose-catabolizing mechanisms functioning in single strains of seven presumed Agrobacterium species. The Entner-Doudoroff and pentose cycle pathways functioned in A. radiobacter, A. tumefaciens, A. rubi, and A. rhizogenes. Whereas both catabolic pathways were utilized to an almost equal degree in the A. radiobacter and A. tumefaciens strains, use of the Entner-Doudoroff pathway predominated in the A. rubi and A. rhizogenes strains. A stellulatum catabolized glucose almost solely through the Entner-Doudoroff pathway. In A. pseudotsugae and A. gypsophilae, glucose was metabolized mainly through the Emden-Meyerhof-Parnas pathway; the pentose phosphate pathway was also utilized.     

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.     

Oxazolomycin Esters,Specific Inhibitors of Plant Transformation

Oxazolomycin diacetate, dipropionate, monobutyrate and dibutyrate were derived from oxazolomycin, a product of Streptomyces sp. KBFP-2025. These esters were potent inhibitors of crown gall formation on potato tuber disks upon infection with Agrobacterium tumefaciens. They showed neither antibacterial nor phytotoxic activity, whereas oxazolomycin showed both antibacterial and phytotoxic activities. Further, they had no inhibitory activity against A. tumefaciens on the potato tuber disk. The inhibitory activity of these esters against crown gall formation seems to be due to specific inhibition of the transformation of plants with A. tumefaciens.     

Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacterium rhizogenes

Abstract Efficient transformation of strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes by electroporation with binary Ti plasmid vector is reported. This procedure yields rates of transformation of 10⁶-10³ per μg DNA, which is several orders of magnitude greater than previously published procedures for this genus, the efficiency of transformation varies with the bacterial strain used. This procedure will be useful for the construction of plant DNA libraries directly in Agrobacterium .     

Susceptibility of Paulownia elongata to Agrobacterium and production of transgenic calli and hairy roots by in vitro inoculation

Susceptibility of Paulownia elongata S.Y. Hu (princess tree) to Agrobacterium tumefaciens and A. rhizogenes was demonstrated by inoculating in vitro shoots. Shoots had a gall formation frequency of ≥83% when inoculated with any of three A. tumefaciens strains (542, A281, or C58). Timing of gall appearance and type of callus proliferation differed among A. tumefaciens strains. Rapidly proliferating callus was produced from explants that were inoculated with A. tumefaciens. Hairy roots were produced directly from wound sites on 33% of shoots inoculated with A. rhizogenes strain R1601. Rapidly growing detached roots were produced from explants that were inoculated with A. rhizogenes. Opine analyses demonstrated the expression of foreign genes in proliferating galls/hairy roots shortly after emergence from wound sites and in callus and roots after 12 weeks of in vitro culture. Southern analyses demonstrated the presence of tDNA in long-term callus and root cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of Agrobacterium tumefaciens strains isolated from grapevine tumors in China.

Thirteen strains of Agrobacterium tumefaciens isolated from grapevine tumors in northern China were surveyed. These strains varied in their host range properties, although all were tumorigenic on grapevines. Twelve of these strains belonged to Agrobacterium sp. biotype 3, and 11 strains resulted in the synthesis of the opine octopine in tumor tissue. Interestingly, one strain resulted in accumulation of arginine, a previously unrecognized opine, in tumor tissue. Although DNA in most of these strains showed homology to the previously characterized transferred DNA and vir loci, some virulent strains showed little or no homology to these loci. Thus, some of these strains represent widely divergent examples of Agrobacterium sp. The DNA in most strains exhibited little or no homology to a wide-host-range virA locus but did show strong homology to a limited-host-range virA locus. This finding further supports the idea that Agrobacterium strains associated with grapevines may have a specific virA locus.     

Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv. phaseolicola.

Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent injection at the same site of Pseudomonas syringae pv. phaseolicola. This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells. Live A. tumefaciens cells were required for the inhibitory effect. Various mutants and strains of A. tumefaciens were examined to determine the genes involved. Known chromosomal mutations generally had no effect on the ability of A. tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response. Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response. The bacteria did not need to be virulent in order to inhibit the hypersensitive response. Deletion of the vir region from pTi had no effect on the inhibition. However, the T region of the Ti plasmid was required for inhibition. Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required. These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells. The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant. An examination of the genes required in P. savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria.     

Agrobacterium rhizogenes GALLS protein contains domains for ATP binding, nuclear localization, and type IV secretion

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.     

A Plating‐PCR Technique for Detection and Quantification of the Biological Control Agent Agrobacterium vitis Strain E26 in Soil under Controlled Conditions

Agrobacterium vitis strain E26 is a promising biocontrol agent of grapevine crown gall, an economically important disease of grape worldwide. In this report, we developed a Plating‐PCR method that allows specific detection and quantification of E26 by combining classical microbiological techniques with molecular tools. Random amplified polymorphic DNA fingerprints were used to differentiate E26 from other A. vitis strains. A differentially amplified fragment from E26 was sequenced and characterized as a sequence characterized amplified region (SCAR) marker. Two primer pairs were then designed and evaluated for their specificity against E26. One of the two SCAR primer pairs, 740F/R, was further selected for specific detection of strain E26. A plating assay coupled to PCR with the SCAR primers 740F/R allowed the assessment of population dynamics of E26 in non‐sterile grape rhizosphere soil under controlled conditions.     

Elevated temperature differentially affects virulence, VirB protein accumulation, and T-pilus formation in different Agrobacterium tumefaciens and Agrobacterium vitis strains.

That gene transfer to plant cells is a temperature-sensitive process has been known for more than 50 years. Previous work indicated that this sensitivity results from the inability to assemble a functional T pilus required for T-DNA and protein transfer to recipient cells. The studies reported here extend these observations and more clearly define the molecular basis of this assembly and transfer defect. T-pilus assembly and virulence protein accumulation were monitored in Agrobacterium tumefaciens strain C58 at different temperatures ranging from 20 degrees C to growth-inhibitory 37 degrees C. Incubation at 28 degrees C but not at 26 degrees C strongly inhibited extracellular assembly of the major T-pilus component VirB2 as well as of pilus-associated protein VirB5, and the highest amounts of T pili were detected at 20 degrees C. Analysis of temperature effects on the cell-bound virulence machinery revealed three classes of virulence proteins. Whereas class I proteins (VirB2, VirB7, VirB9, and VirB10) were readily detected at 28 degrees C, class II proteins (VirB1, VirB4, VirB5, VirB6, VirB8, VirB11, VirD2, and VirE2) were only detected after cell growth below 26 degrees C. Significant levels of class III proteins (VirB3 and VirD4) were only detected at 20 degrees C and not at higher temperatures. Shift of virulence-induced agrobacteria from 20 to 28 or 37 degrees C had no immediate effect on cell-bound T pili or on stability of most virulence proteins. However, the temperature shift caused a rapid decrease in the amount of cell-bound VirB3 and VirD4, and VirB4 and VirB11 levels decreased next. To assess whether destabilization of virulence proteins constitutes a general phenomenon, levels of virulence proteins and of extracellular T pili were monitored in different A. tumefaciens and Agrobacterium vitis strains grown at 20 and 28 degrees C. Levels of many virulence proteins were strongly reduced at 28 degrees C compared to 20 degrees C, and T-pilus assembly did not occur in all strains except "temperature-resistant" Ach5 and Chry5. Virulence protein levels correlated well with bacterial virulence at elevated temperature, suggesting that degradation of a limited set of virulence proteins accounts for the temperature sensitivity of gene transfer to plants.