Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors.

RRS1-R confers broad-spectrum resistance to several strains of the causal agent of bacterial wilt, Ralstonia solanacearum. Although genetically defined as recessive, this R gene encodes a protein whose structure combines the TIR-NBS-LRR domains found in several R proteins and a WRKY motif characteristic of some plant transcriptional factors and behaves as a dominant gene in transgenic susceptible plants. Here we show that PopP2, a R. solanacearum type III effector, which belongs to the YopJ͞AvrRxv protein family, is the avirulence protein recognized by RRS1-R. Furthermore, an interaction between PopP2 and both RRS1-R and RRS1-S, present in the resistant Nd-1 and susceptible Col-5 Arabidopsis thaliana ecotypes, respectively, was detected by using the yeast split-ubiquitin two-hybrid system. This interaction, which required the full-length R protein, was not observed between the RRS1 proteins and PopP1, another member of the YopJ͞AvrRxv family present in strain GMI1000 and that confers avirulence in Petunia. We further demonstrate that both the Avr protein and the RRS1 proteins colocalize in the nucleus and that the nuclear localization of the RRS1 proteins are dependent on the presence of PopP2. P lants rely on an innate immune response for their survival after pathogen attack. Specific recognitions between pathogen Avr and plant R proteins are crucial for the onset of the resistance response and determine the issue of many plantpathogen interactions by triggering plant defense. Disease results from the inactivation or absence of one or both partners (1). It has been postulated that R gene products are receptors for pathogen-encoded Avr components (2). Despite the cloning of numerous R and Avr genes, only two plant R proteins, Pto and Pi-ta, were shown to interact physically with their pathogen Avr counterparts, Avr-Pto and Avr-Pita, respectively, by using the yeast two-hybrid system (3-5). The hypothesis that R proteins are part of protein complexes was recently substantiated by the identification of multiprotein complexes including R and Avr proteins (6-9). Additionally, whereas Avr bacterial proteins expressed in plants carrying the cognate R protein generally induce a cell death program (10), termed the hypersensitive response, closely linked to resistance, they can also cause disease-like symptoms when expressed in plants lacking the appropriate R protein. Bacterial pathogenicity effectors such as Avr proteins are injected into the host cell via a type III secretion system (TTSS) (11). According to the guard model (9, 12), such effectors can associate and induce modifications of plant targets functioning as negative regulators of basal defense responses leading to disease development in plants lacking the corresponding R protein. In a resistant host, the plant target that interacts with both R and Avr proteins is guarded by the R protein, preventing its manipulation by pathogen effectors. The recent characterization of RIN4, a negative regulator of plant defense, strengthens this model (13).Most resistance p...

SummaryThe ability of Ralstonia solanacearum strain GMI1000 to cause disease on a wide range of host plants (including most Solanaceae and Arabidopsis thaliana ) depends on genes activated by the regulatory gene hrpB . HrpB controls the expression of the type III secretion system (TTSS) and pathogenicity effectors transiting through this pathway. In order to establish the complete repertoire of TTSS-dependent effectors belonging to the Hrp regulon and to start their functional analysis, we developed a rapid method for insertional mutagenesis, which was used to monitor the expression of 71 candidate genes and disrupt 56 of them. This analysis yielded a total of 48 novel hrpB -regulated genes. Using the Bordetella pertussis calmodulin-dependent adenylate cyclase reporter fusion system, we provide direct biochemical evidence that five R. solanacearum effector proteins are translocated into plant host cells through the TTSS. Among these novel TTSS effectors, RipA and RipG both belong to multigenic families, RipG defining a novel class of leucine-rich-repeats harbouring proteins. The members of these multigenic families are differentially regulated, being composed of genes expressed in either an hrpB -dependent or an hrpBindependent manner. Pathogenicity assays of the 56 mutant strains on two host plants indicate that, with two exceptions, mutations in individual effectors have no effect on virulence, a probable consequence of genetic and functional redundancy. This large repertoire of HrpB-regulated genes, which comprises > 20 probable TTSS effector genes with no counterparts in other bacterial species, represents an important step towards a full-genome understanding of R. solanacearum virulence.

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In many plant and animal bacterial pathogens, the Type III secretion system (TTSS) that directly translocates effector proteins into the eukaryotic host cells is essential for the development of disease. In all species studied, the transcription of the TTSS and most of its effector substrates is tightly regulated by a succession of consecutively activated regulators. However, the whole genetic programme driven by these regulatory cascades is still unknown, especially in bacterial plant pathogens. Here, we have characterised the programme triggered by HrpG, a host-responsive regulator of the TTSS activation cascade in the plant pathogen Ralstonia solanacearum. We show through genome-wide expression analysis that, in addition to the TTSS, HrpG controls the expression of a previously undescribed TTSS-independent pathway that includes a number of other virulence determinants and genes likely involved in adaptation to life in the host. Functional studies revealed that this second pathway co-ordinates the bacterial production of plant cell wall-degrading enzymes, exopolysaccharide, and the phytohormones ethylene and auxin. We provide experimental evidence that these activities contribute to pathogenicity. We also show that the ethylene produced by R. solanacearum is able to modulate the expression of host genes and can therefore interfere with the signalling of plant defence responses. These results provide a new, integrated view of plant bacterial pathogenicity, where a common regulator activates synchronously upon infection the TTSS, other virulence determinants and a number of adaptive functions, which act co-operatively to cause disease.

The phytopathogenic bacterium Ralstonia solanacearum encodes a family of seven type III secretion system (T3SS) effectors that contain both a leucine-rich repeat and an F-box domain. This structure is reminiscent of a class of typical eukaryotic proteins called F-box proteins. The latter, together with Skp1 and Cullin1 subunits, constitute the SCF-type E3 ubiquitin ligase complex and control specific protein ubiquitinylation. In the eukaryotic cell, depending on the nature of the polyubiquitin chain, the ubiquitintagged proteins either see their properties modified or are doomed for degradation by the 26S proteasome. This pathway is essential to many developmental processes in plants, ranging from hormone signaling and flower development to stress responses. Here, we show that these previously undescribed T3SS effectors are putative bacterial F-box proteins capable of interacting with a subset of the 19 different Arabidopsis Skp1-like proteins like bona fide Arabidopsis F-box proteins. A R. solanacearum strain in which all of the seven GALA effector genes have been deleted or mutated was no longer pathogenic on Arabidopsis and less virulent on tomato. Furthermore, we found that GALA7 is a host-specificity factor, required for disease on Medicago truncatula plants. Our results indicate that the GALA T3SS effectors are essential to R. solanacearum to control disease. Because the F-box domain is essential to the virulence function of GALA7, we hypothesize that these effectors act by hijacking their host SCF-type E3 ubiquitin ligases to interfere with their host ubiquitin͞proteasome pathway to promote disease.plant pathogen ͉ SCF complex ͉ type III secretion system ͉ virulence

The use of a modified procedure for the isolation of covalently closed circular DNA of high molecular weight, followed by agarose gel electrophoresis of the crude extracts, provides a simple screening method for detecting plasmids with molecular weights of more than 250 x lo6 from Agrobacterium tumefaciens, Pseudomonas putida and Rhizobium species. This method was used for a survey of plasmids in 25 symbiotically effective strains of Rhizobium meliloti from various geographical origins. Of these, 22 strains were found to carry at least one large plasmid. By electron microscopy and measurement of electrophoretic mobility in gels, the molecular weights of most of the plasmids were estimated to range from 90x lo6 to 200x lo6.

This paper describes the identification of a new class of extracellular bacterial proteins, typified by PopA1 and its derivative PopA3, which act as specific hypersensitive response (HR) elicitors. These two heat‐stable proteins, with HR‐like elicitor activities on tobacco (non‐host plant) but without activity on tomato (host plant), have been characterized from the supernatant of the plant pathogenic bacterium Pseudomonas solanacearum strain GMI1000. These two proteins induced the same pattern of response on Petunia, as a function of the genotypes tested. popA, the structural gene for PopA1, maps outside of the hrp gene cluster but belongs to the hrp regulon. The amino acid sequence of PopA1 does not show homology to any characterized proteins. Its secretion is dependent on hrp genes and is followed by stepwise removal of the 93 amino‐terminal amino acids, producing the protein PopA3. Petunia lines responsive to PopA3 and its precursors were resistant to infection by strain GMI1000, whereas non‐responsive lines were sensitive, suggesting that popA could be an avirulence gene. A popA mutant remained fully pathogenic on sensitive plants, indicating that this gene is not essential for pathogenicity. While lacking PopA1, this mutant, which remained avirulent on tobacco and on resistant Petunia lines, still produced additional extracellular necrogenic compounds. On the basis of both their structural features and the biological properties of the popA mutant, PopA1 and PopA3 clearly differ from hairpins characterized in other plant pathogenic bacteria.