Growth inhibition and loss of virulence in cultures of Agrobacterium tumefaciens treated with acetosyringone.

Acetosyringone, a phenolic inducer of the virulence (vir) genes of Agrobacterium tumefaciens, inhibited the growth of the nopaline-type strains T37 and C58 incubated under acidic conditions. In the course of a 6-day incubation with acetosyringone, avirulent clones were produced in different proportions by strains T37 and C58 and also by a spontaneous variant of strain C58, denominated C58F. The proportion of avirulent clones in acetosyringone-treated cultures often exceeded 50% for strains T37 and C58F and was of the order of 1% for strain C58. Control cultures not exposed to acetosyringone did not yield avirulent clones. Two other vir inducers, sinapinic acid and syringaldehyde, also inhibited growth and promoted accumulation of avirulent clones in cultures of strains C58F and T37. On the other hand, various acetosyringone analogs reported not to induce the vir genes did not act as growth inhibitors. All of the T37 and most of the C58F avirulent clones examined still carried a Ti plasmid. In all instances examined, avirulent clones still carrying a Ti plasmid were mutated in this plasmid. Mutants of strain C58F lacked the capacity to induce a virB::lacZ fusion in the presence of acetosyringone.     

Proteomic analysis of Agrobacterium tumefaciens response to the Vir gene inducer acetosyringone

Agrobacterium tumefaciens causes crown gall disease in a wide range of plants by transforming plants through the transfer and integration of its transferred DNA (T-DNA) into the host genome. In the present study, we used two-dimensional gel electrophoresis to examine the protein expression profiles of A. tumefaciens in response to the phenolic compound acetosyringone (AS), a known plant-released virulence (vir) gene inducer. Using mass spectrometry, we identified 11 proteins consisting of 9 known AS-induced Vir proteins and 2 newly discovered AS-induced proteins, an unknown protein Y4mC (Atu6162) and a small heat shock protein HspL (Atu3887). Further expression analysis revealed that the AS-induced expression of Y4mC and HspL is regulated by the VirA/VirG two-component system. This report presents the first proteomics study successfully identifying both known and new AS-induced proteins that are implicated in Agrobacterium virulence.     

A Ti-plasmid determined function is responsible for chemotaxis of Agrobacterium tumefaciens towards the plant wound product acetosyringone

Agrobacterium tumefaciens C58C¹[pTiB6S3] harbouring the octopine Ti-plasmid pTiB6S3, showed positive chemotaxis towards the phenolic plant wound exudate acetosyringone (AS). Maximal attraction was observed at 10⁻⁷ M. In contrast, A. tumefaciens C58C¹ lacking a Ti-plasmid, exhibited no chemotactic response to AS. However, chemotaxis did occur towards the plant phenolic vanillyl alcohol, but at higher concentrations (10⁻² M) and in both Ti-plasmid-containing and cured A. tumefaciens.These results indicate that at least one Ti-plasmid function is involved in the specific chemotactic response to AS, although chemotaxis per se is not Ti-plasmid-encoded. This correlates well with the specific induction of vir-operons mediated by this plant wound product [1].     

Induction of virulence response in Agrobacterium tumefaciens by tissue explants of various plant species

Forty-four plant species belonging to different taxa were tested for their ability to induce the expression of the virulence E gene in Agrobacterium tumefaciens containing virE:lacZ fusion constructs. With the exception of 6 algae, one fern and 2 monocots, tissue explants of all other plants (2 Algae, 3 Bryophytes, 2 Pteridophytes, 15 Gymnosperms, 8 Monocots and 5 Dicots) induced the expression of the virE gene as detected by the presence of -galactosidase activity in the bacteria.Abbreviations AS acetosyringone - vir virulence genes Scientific Contribution Number 1734 from the New Hampshire Agricultural Experiment Station     

Stress-induced proteins of Agrobacterium tumefaciens

The pattern of proteins produced by bacteria represents the physiological state of the organism as well as the environmental conditions encountered. Environmental stress induces the expression of several regulons encoding stress proteins. Extensive information about the proteins which constitute these regulons (or stimulons) and their control is available for very few bacteria, such as the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli (gamma-proteobacteria) and is minimal for all other bacteria. Agrobacterium tumefaciens is a Gram-negative plant pathogen of the alpha-proteobacteria, which constitutes the main tool for plant recombinant genetics. Our previous studies on the control of chaperone-coding operons indicated that A. tumefaciens has unique features and combines regulatory elements from both B. subtilis and E. coli. Therefore, we examined the patterns of proteins induced in A. tumefaciens by environmental changes using two-dimensional gel electrophoresis and dual-channel image analysis. Shifts to high temperature, oxidative and mild acid stresses stimulated the expression of 97 proteins. The results indicate that most of these stress-induced proteins (80/97) were specific to one stress stimulon. Only 10 proteins appear to belong to a general stress regulon.     

Glycine betaine allows enhanced induction of the Agrobacterium tumefaciens vir genes by acetosyringone at low pH.

We established growth conditions for efficient induction of the vir genes of Agrobacterium tumefaciens by acetosyringone. Optimal induction was attained at a pH below 5.2 in an AB minimal medium-derived high-osmotic-strength medium containing glycine betaine. This natural osmoprotectant accelerated the adaptation of the bacteria to these conditions. We established the kinetics of induction for virB, virD, virE, and virG by using lacZ fusions, and we found that the virB mutant strain could not adapt to this low-pH medium unless 1 mM CaCl2 was added. This pH control of vir gene expression was shown to act at the level of expression of virG, which was the limiting factor. This improved vir induction at a low pH correlated with an increase in a set of proteins which was analyzed by two-dimensional gel electrophoresis. The fact that high inducibility corresponded to a reduced growth rate and the demonstration that a set of proteins was associated with the inducible state suggest that vir gene induction is linked to the adaptation of the cells to an unfavorable environment. Hence, vir gene expression in A. tumefaciens is probably dependent upon a machinery which is specific to an adaptive response; the implications for plant transformation are discussed.     

virA and virG are the Ti-plasmid functions required for chemotaxis of Agrobacterium tumefaciens towards acetosyringone

Octopine and nopaline Ti-plasmids confer upon Agrobacterium tumefaciens C58C1 the ability to respond chemotactically to the vir-inducing phenolic wound exudate, acetosyringone. A. tumefaciens C58C1 containing Ti-plasmids with Tn5 insertions in virB, C, D or E exhibited marked chemotaxis towards acetosyringone. However, Ti-plasmids with mutations in virA or virG were unable to confer the responsive phenotype. Of the cosmid clones pVK219 (virAB) pVK221 (virBGC) pVK225 (virGCDE) and pVK257 (virABGC) mobilized to cured A. tumefaciens C58C1, only pVK257 bestowed acetosyringone chemotaxis. virA and virG are thus required for chemotaxis of A. tumefaciens towards acetosyringone. This suggests a multifunctional role for virA and virG: at low concentrations of acetosyringone they mediate chemotaxis and at higher concentrations they effect vir-induction.     

Plasmid required for virulence of Agrobacterium tumefaciens.

The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37 C is shown to be due to loss of a large plasmid (1.2 X 10-8 daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6 is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 X A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.     

Phosphoenolpyruvate carboxykinase is an acid-induced, chromosomally encoded virulence factor in Agrobacterium tumefaciens

The pckA gene, encoding phosphoenolpyruvate carboxykinase, catalyzes the reversible decarboxylation and phosphorylation of oxaloacetate to form phosphoenolpyruvate. Located on the circular chromosome of Agrobacterium, this locus is adjacent to the loci chvG and chvI, encoding a two-component regulatory system that has been shown to be important in virulence. Using a reporter gene fusion, studies showed that the pckA gene is induced by acidic pH but not by acetosyringone. This acid induction is regulated by the chvG-chvI regulatory system, which controls acid-inducible genes. A pckA mutant had no demonstrable PckA enzyme activity and grew on AB minimal medium with glucose but did not grow on the same medium with succinate as the sole carbon source and was more inhibited in its growth than the wild-type strain by an acidic environment. A pckA mutant was highly attenuated in tumor-inducing ability on tobacco leaf disks and was severely attenuated in vir gene expression. Although vir gene induction was completely restored when a constitutive virG gene was introduced into the mutant strain, virulence was only partially restored. These results suggest that avirulence may be due to a combination of the inhibition of this mutant in the acidic plant wound environment and the poor induction of the vir genes.     

Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens

We have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline-type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine-type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane-bound sensor of plant signal molecules.     

Characterization of an Agrobacterium tumefaciens lectin.

An Agrobacterium tumefaciens suspension induces a strong agglutination of aldehyde-fixed pig erythrocytes at pH 5.0. The agglutination is inhibited by some polysaccharides, such as fucoidin, and also when the pH is raised to 7.0. Lectins (sugar-binding proteins) associated with the bacterial cell wall of A. tumefaciens strain 84.5 were directly evidenced by spectrofluorimetry using fluoresceinylated neoglycoproteins. The specific binding of the fluorescein-labelled neoglycoprotein bearing alpha-L-fucoside residues was also optimal at pH 5.0. A lectin was purified by affinity chromatography on agarose substituted with alpha-L-fucopyranoside. Furthermore, the haemagglutination activity of this lectin was inhibited by polysaccharides isolated from poplar leaves.     

Two-dimensional reference map of Agrobacterium tumefaciens proteins

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.     

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.     

The plant cell defense and Agrobacterium tumefaciens

We previously identified changes in gene expression in Ageratum conyzoides plant cells inoculated with Agrobacterium tumefaciens by using cDNA-AFLP. Here, we show that a subset of defense-related genes is differentially regulated by an Agrobacterium attachment-deficient mutant. The expression pattern triggered by this mutant is similar to that induced by inoculation with non-pathogenic bacteria. We also observed that the expression level of the defense genes was inversely correlated with the efficiency of transformation by Agrobacterium. We propose that the plant defense system has an important role in controlling infection and transformation and that Agrobacterium may dampen some plant defense responses.