Symbiotic phenotypes and translocated effector proteins of the Mesorhizobium loti strain R7A VirB/D4 type IV secretion system

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

Mutants of the Agrobacterium tumefaciens virA gene exhibiting acetosyringone-independent expression of the vir regulon.

Hydroxylamine-induced mutations in the virA gene of Agrobacterium tumefaciens that do not require the plant phenolic-inducing compound acetosyringone for vir regulon induction were isolated. The isolation was based on the activation of both virB::lacZ and virE::cat fusions by mutant virA loci in the absence of acetosyringone. Three of these virA(Ais) (acetosyringone-independent signaling) mutants were characterized. All three mutants expressed a virB::lacZ fusion at high levels in the absence of acetosyringone. One virA (Ais) mutant, virA112, exhibited vir gene expression in the absence of inducing monosaccharides and acidic growth conditions, both of which are normally required for vir gene induction. The phenotype of the virA112 mutant resulted from a glycine to glutamic acid change near His-474, the site of VirA autophosphorylation.     

Dual control of Agrobacterium tumefaciens Ti plasmid virulence genes.

The virulence genes of nopaline (pTiC58) and octopine (pTiA6NC) Ti plasmids are similarly affected by the Agrobacterium tumefaciens ros mutation. Of six vir region complementation groups (virA, virB, virG, virC, virD, and virE) examined by using fusions to reporter genes, the promoters of only two (virC and virD) responded to the ros mutation. For each promoter that was affected by ros, the level of expression of its associated genes was substantially elevated in the mutant. This increase was not influenced by Ti plasmid-encoded factors, and the mutation did not interfere with the induction of pTiC58 vir genes by phenolic compounds via the VirA/VirG regulatory control mechanism. The effects of the ros mutation and acetosyringone were cumulative for all vir promoters examined. The pleiotropic characteristics of the ros mutant include the complete absence of the major acidic capsular polysaccharide.     

The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation.

The virA and virG gene products are required for the regulation of the vir regulon on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. VirA is a membrane-associated protein which is homologous to the sensor molecules of other two-component regulatory systems. We overproduced truncated VirA proteins in Escherichia coli by deleting different lengths of the 5'-coding region of the virA gene and placing these genes under lacZ control. These proteins were purified from polyacrylamide gels and renatured. The renatured proteins became radiolabeled when they were incubated with [gamma-32P]ATP but not with [gamma-32P]GTP or [alpha-32P]ATP, which suggests an ATP gamma-phosphate-specific autophosphorylation. The smallest VirA protein, which retained only the C-terminal half of the protein, gave the strongest autophosphorylation signal, which demonstrates that the C-terminal domain has the autophosphorylation site. The phosphorylated amino acid was identified as phosphohistidine, and a highly conserved histidine was found in all of the VirA homologs. When this histidine was changed to glutamine, which cannot be phosphorylated, the resulting VirA protein lost both its ability to autophosphorylate and its biological function as a vir gene regulator. Results of this study indicate that VirA autophosphorylation is required for the induction of the vir regulon and subsequent tumor induction on plants by A. tumefaciens.     

Reconstitution of acetosyringone-mediated Agrobacterium tumefaciens virulence gene expression in the heterologous host Escherichia coli

The ability to utilize Escherichia coli as a heterologous system in which to study the regulation of Agrobacterium tumefaciens virulence genes and the mechanism of transfer DNA (T-DNA) transfer would provide an important tool to our understanding and manipulation of these processes. We have previously reported that the rpoA gene encoding the alpha subunit of RNA polymerase is required for the expression of lacZ gene under the control of virB promoter (virBp::lacZ) in E. coli containing a constitutively active virG gene [virG(Con)]. Here we show that an RpoA hybrid containing the N-terminal 247 residues from E. coli and the C-terminal 89 residues from A. tumefaciens was able to significantly express virBp::lacZ in E. coli in a VirG(Con)-dependent manner. Utilization of lac promoter-driven virA and virG in combination with the A. tumefaciens rpoA construct resulted in significant inducer-mediated expression of the virBp::lacZ fusion, and the level of virBp::lacZ expression was positively correlated to the copy number of the rpoA construct. This expression was dependent on VirA, VirG, temperature, and, to a lesser extent, pH, which is similar to what is observed in A. tumefaciens. Furthermore, the effect of sugars on vir gene expression was observed only in the presence of the chvE gene, suggesting that the glucose-binding protein of E. coli, a homologue of ChvE, does not interact with the VirA molecule. We also evaluated other phenolic compounds in induction assays and observed significant expression with syringealdehyde, a low level of expression with acetovanillone, and no expression with hydroxyacetophenone, similar to what occurs in A. tumefaciens strain A348 from which the virA clone was derived. These data support the notion that VirA directly senses the phenolic inducer. However, the overall level of expression of the vir genes in E. coli is less than what is observed in A. tumefaciens, suggesting that additional gene(s) from A. tumefaciens may be required for the full expression of virulence genes in E. coli.     

Induction of a virulence response in Agrobacterium tumefaciens by exudates of Pinus pinea cotyledons

As a preliminary step in efforts to develop a successful protocol for Agrobacterium-mediated transformation of cotyledonary explants of Pinus pinea L. embryos, we tested the ability of embrionary exudates of this species to induce the expression of the virulence genes virA, virB, virC, virD, virE and virG in Agrobacterium tumefaciens containing vir: lacZ fusion constructs. The results obtained in the vir induction assay indicated the absence of bactericidal or bacteriostatic plant compounds affecting A. tumefaciens growth, and showed that cotyledonary and embrionary exudates of P. pinea are able to induce all virulence genes studied, except virG. The data suggest that A. tumefaciens can be used for gene transfer into this important forest and fruit species. This revised version was published online in June 2006 with corrections to the Cover Date.     

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

Structure activity Research of Signal Factors Inducing Agrobacterium vir Genes

Four rice ( Oryza sativa L. cv. IR72) signal factors with the functions of inducing Agrobaterium tumefaciens (Smith et Townsend) Corm vir genes were constructed. They were 5,7,4'-trihydmxy-3', 5'- dimethoxy-flavone (Fl) and its structure analogues F2, F3 and F4. These four signal factors were detected by Agrobacterium vir:: lacZ fusion gene system as to compare their functions of inducing virA, virB, virC, virD, virE and virG genes. The structure characters and stmcture-activity relationship of these signal factors with the best inducing effects to Agrobacterium exogenous gene transformation system were defined which may provide some basic information for foreign gene transformation in rice.     

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.     

Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens

The VirA/VirG two-component regulatory system of Agrobacterium tumefaciens regulates expression of the virulence (vir) genes that control the infection process leading to crown gall tumor disease on susceptible plants. VirA, a membrane-bound homodimer, initiates vir gene induction by communicating the presence of molecular signals found at the site of a plant wound through phosphorylation of VirG. Inducing signals include phenols, monosaccharides, and acidic pH. While sugars are not essential for gene induction, their presence greatly increases vir gene expression when levels of the essential phenolic signal are low. Reception of the sugar signal depends on a direct interaction between ChvE, a sugar-binding protein, and VirA. Here we show that the sugar signal received in the periplasmic region of one subunit within a VirA heterodimer can enhance the kinase function of the second subunit. However, sugar enhancement of vir gene expression was vector dependent. virA alleles expressed from pSa-derived vectors inhibited signal transduction by endogenous VirA. Inhibition was conditional, depending on the induction medium and the virA allele tested. Moreover, constitutive expression of virG overcame the inhibitory effect of some but not all virA alleles, suggesting that there may be more than one inhibitory mechanism.     

Comparative sequence analysis of the symbiosis island of Mesorhizobium loti strain R7A

The Mesorhizobium loti strain R7A symbiosis island is a 502-kb chromosomally integrated element which transfers to nonsymbiotic mesorhizobia in the environment, converting them to Lotus symbionts. It integrates into a phenylalanine tRNA gene in a process mediated by a P4-type integrase encoded at the left end of the element. We have determined the nucleotide sequence of the island and compared its deduced genetic complement with that reported for the 611-kb putative symbiosis island of M. loti strain MAFF303099. The two islands share 248 kb of DNA, with multiple deletions and insertions of up to 168 kb interrupting highly conserved colinear DNA regions in the two strains. The shared DNA regions contain all the genes likely to be required for Nod factor synthesis, nitrogen fixation, and island transfer. Transfer genes include a trb operon and a cluster of potential tra genes which are also present on the strain MAFF303099 plasmid pMLb. The island lacks plasmid replication genes, suggesting that it is a site-specific conjugative transposon. The R7A island encodes a type IV secretion system with strong similarity to the vir pilus from Agrobacterium tumefaciens that is deleted from MAFF303099, which in turn encodes a type III secretion system not found on the R7A island. The 414 genes on the R7A island also include putative regulatory genes, transport genes, and an array of metabolic genes. Most of the unique hypothetical genes on the R7A island are strain-specific and clustered, suggesting that they may represent other acquired genetic elements rather than symbiotically relevant DNA.     

The coexistence of symbiosis and pathogenicity-determining genes in Rhizobium rhizogenes strains enables them to induce nodules and tumors or hairy roots in plants

Bacteria belonging to the family Rhizobiaceae may establish beneficial or harmful relationships with plants. The legume endosymbionts contain nod and nif genes responsible for nodule formation and nitrogen fixation, respectively, whereas the pathogenic strains carry vir genes responsible for the formation of tumors or hairy roots. The symbiotic and pathogenic strains currently belong to different species of the genus Rhizobium and, until now, no strains able to establish symbiosis with legumes and also to induce tumors or hairy roots in plants have been reported. Here, we report for the first time the occurrence of two rhizobial strains (163C and ATCC11325T) belonging to Rhizobium rhizogenes able to induce hairy roots or tumors in plants and also to nodulate Phaseolus vulgaris under natural environmental conditions. Symbiotic plasmids (pSym) containing nod and nif genes and pTi- or pRi-type plasmids containing vir genes were found in these strains. The nodD and nifH genes of the strains from this study are phylogenetically related to those of Sinorhizobium strains nodulating P. vulgaris. The virA and virB4 genes from strain 163C are phylogenetically related to those of R. tumefaciens C58, whereas the same genes from strain ATCC 11325T are related to those of hairy root-inducing strains. These findings may be of high relevance for the better understanding of plant-microbe interactions and knowledge of rhizobial phylogenetic history.     

Mesorhizobium loti produces nodPQ-dependent sulfated cell surface polysaccharides

Leguminous plants and bacteria from the family Rhizobiaceae form a symbiotic relationship, which culminates in novel plant structures called root nodules. The indeterminate symbiosis that forms between Sinorhizobium meliloti and alfalfa requires biosynthesis of Nod factor, a beta-1,4-linked lipochitooligosaccharide that contains an essential 6-O-sulfate modification. S. meliloti also produces sulfated cell surface polysaccharides, such as lipopolysaccharide (LPS). The physiological function of sulfated cell surface polysaccharides is unclear, although mutants of S. meliloti with reduced LPS sulfation exhibit symbiotic abnormalities. Using a bioinformatic approach, we identified a homolog of the S. meliloti carbohydrate sulfotransferase, LpsS, in Mesorhizobium loti. M. loti participates in a determinate symbiosis with the legume Lotus japonicus. We showed that M. loti produces sulfated forms of LPS and capsular polysaccharide (KPS). To investigate the physiological function of sulfated polysaccharides in M. loti, we identified and disabled an M. loti homolog of the sulfate-activating genes, nodPQ, which resulted in undetectable amounts of sulfated cell surface polysaccharides and a cysteine auxotrophy. We concomitantly disabled an M. loti cysH homolog, which disrupted cysteine biosynthesis without reducing cell surface polysaccharide sulfation. Our experiments demonstrated that the nodPQ mutant, but not the cysH mutant, showed an altered KPS structure and a diminished ability to elicit nodules on its host legume, Lotus japonicus. Interestingly, the nodPQ mutant also exhibited a more rapid growth rate and appeared to outcompete wild-type M. loti for nodule colonization. These results suggest that sulfated cell surface polysaccharides are required for optimum nodule formation but limit growth rate and nodule colonization in M. loti.