The plant pathogen Ralstonia solanacearum has two genes encoding for the sigma factor σ54: rpoN1, located in the chromosome and rpoN2, located in a distinct “megaplasmid” replicon. In this study, individual mutants as well as a double mutant of rpoN were created in R. solanacearum strain GMI1000 in order to determine the extent of functional overlap between these two genes. By virulence assay we observed that rpoN1 is required for virulence whereas rpoN2 is not. In addition rpoN1 controls other important functions such twitching motility, natural transformation and growth on nitrate, unlike rpoN2. The rpoN1 and rpoN2 genes have different expression pattern, the expression of rpoN1 being constitutive whereas rpoN2 expression is induced in minimal medium and in the presence of plant cells. Moreover, the expression of rpoN2 is dependent upon rpoN1. Our work therefore reveals that the two rpoN genes are not functionally redundant in R. solanacearum. A list of potential σ54 targets was identified in the R. solanacearum genome and suggests that multiple traits are under the control of these regulators. Based on these findings, we provide a model describing the functional connection between RpoN1 and the PehR pathogenicity regulator and their dual role in the control of several R. solanacearum virulence determinants.

This study provides insights into the pathogenesis of Ralstonia solanacearum, in particular with regards to strains belonging to phylotype IIB, sequevar 1 (IIB-1) and their interaction with potato, its natural host. We performed a comparative genomic analysis among IIB-1 R. solanacearum strains with different levels of virulence in order to identify candidate virulence genes. With this approach, we identified a 33.7-kb deletion in a strain showing reduced virulence on potato. This region contains a cluster of six genes putatively involved in type IV pili (Tfp) biogenesis. Functional analysis suggests that these proteins contribute to several Tfp-related functions such as twitching motility and biofilm formation. In addition, this genetic cluster was found to contribute to early bacterial wilt pathogenesis and colonization fitness of potato roots.

MetE and MetH are two distinct enzymes that catalyze a similar biochemical reaction during the last step of methionine biosynthesis, MetH being a cobalamin-dependent enzyme whereas MetE activity is cobalamin-independent. In this work, we show that the last step of methionine synthesis in the plant pathogen Ralstonia solanacearum is under the transcriptional control of the master pathogenicity regulator HrpG. This control is exerted essentially on metE expression through the intermediate regulator MetR. Expression of metE is strongly and specifically induced in the presence of plant cells in a hrpG- and metR-dependent manner. metE and metR mutants are not auxotrophic for methionine and not affected for growth inside the plant but produce significantly reduced disease symptoms on tomato whereas disruption of metH has no impact on pathogenicity. The finding that the pathogen preferentially induces metE expression rather than metH in the presence of plant cells is indicative of a probable metabolic adaptation to physiological host conditions since this induction of metE occurs in an environment in which cobalamin, the required co-factor for MetH, is absent. It also shows that MetE and MetH are not functionally redundant and are deployed during specific stages of the bacteria lifecycle, the expression of metE and metH being controlled by multiple and distinct signals.

Bacterial wilt (brown rot) disease of potato caused by Ralstonia solanacearum is one of the most important bacterial diseases and a major constraint on potato production worldwide. Through a comparative genomic analysis between R. solanacearum'race 3 biovar 2' (R3bv2) strains, we identified a 77 kb region in strain UW551 which is specifically absent in the hypoaggressive strain IPO1609. We proved that IPO1609 indeed carries a 77 kb genomic deletion and provide genetic evidence that occurrence of this deletion is responsible for almost complete loss of pathogenicity of this strain. We carried out a functional analysis of this 77 kb region in strain UW551 using a combination of gene deletion and functional complementation approaches which identified the methionine biosynthesis genes metER as having a major contribution to IPO1609 pathogenesis. Deletion of the metER genes significantly impacts pathogenicity of R3bv2 strains but does not lead to methionine auxotrophy nor reduced ability to multiply in planta. In addition, this study indicated that three type III secretion system effectors or a type VI secretion system present within the 77 kb region have no or very minor contribution to pathogenicity.

The model pathogen Ralstonia solanacearum GMI1000 is the causal agent of the bacterial wilt disease that attacks many solanaceous plants and other hosts but not tobacco (Nicotiana spp.). We found that two type III secretion system effector genes, avrA and popP1, are limiting the host range of strain GMI1000 on at least three tobacco species (N. tabacum, N. benthamiana, and N. glutinosa). Both effectors elicit the hypersensitive response (HR) on these tobacco species, although in different manners; AvrA is the major determinant recognized by N. tabacum and N. benthamiana, while PopP1 appears to be the major HR elicitor on N. glutinosa. Only the double inactivation of the avrA and popP1 genes allowed GMI1000 to wilt tobacco plants, thus showing that GMI1000 intrinsically possesses the functions necessary to wilt tobacco plants. A focused analysis on AvrA revealed that the first 58 N-terminal amino acids are sufficient to direct its injection into plant cells. We identified a hypervariable region in avrA, which contains variable numbers of tandem repeats (VNTR), each composed of 12 base pairs. We show that an 18-amino acid region in which the VNTR insertion occurs is an important domain involved in HR elicitation on N. benthamiana. avrA appears to be the target of various DNA insertions or mobile elements that probably allow R. solanacearum to evade the recognition and defense responses of tobacco.

The transcriptional activator HrpB of the bacterial wilt causing betaproteobacterium Ralstonia solanacearum represents a key regulator for pathogenicity. In particular, it drives expression of hrp genes encoding a type III secretion system (T3SS) as well as effector molecules for delivery into the host cytosol to promote disease. However, the HrpB regulon extends beyond this T3SS. We describe here an HrpB-activated operon of six genes that is responsible for the synthesis of a fluorescent isatin derivative of 149 Amu that we named HDF for HrpB-dependent factor and that we purified from culture supernatants. The structure of the labile molecule was solved by using NMR and CD spectroscopy to be (3S)-3-hydroxy-indolin-2-one and confirmed by its chemical synthesis and MS spectrometry. HDF was found to be present at 20 nM in wild-type cultures grown on minimal medium, and its synthesis increased 15-fold upon overproduction of HrpB, confirming that HrpB activates HDF synthesis. The addition of tryptophan significantly stimulated HDF biosynthesis and was shown to represent the precursor molecule for HDF synthesis. A search for the biological function of the molecule revealed that HDF induces acyl-homoserine lactone receptor-mediated reporter activity of the well studied LuxR transcriptional regulator of Vibrio fischeri. Thus, our results provide evidence that the specificity of acyl-homoserine lactone (acyl-HSL) receptors is clearly broader than previously considered. The failure to detect induction by HDF of the described endogenous quorum-sensing circuits of the pathogen points to a role in interfering with cell–cell signaling of rivalling bacteria.

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 ubiquitin-tagged 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. solanacea-rum 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 ubiquitin/proteasome pathway to promote disease.

ABSTRACT Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum.

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.

ABSTRACT In the present study, we investigated the gene distribution among strains of the highly polymorphic plant pathogenic β-proteobacterium Ralstonia solanacearum, paying particular attention to the status of known or candidate pathogenicity genes. Based on the use of comparative genomic hybridization on a pangenomic microarray for the GMI1000 reference strain, we have defined the conditions that allowed comparison of the repertoires of genes among a collection of 18 strains that are representative of the biodiversity of the R. solanacearum species. This identified a list of 2,690 core genes present in all tested strains. As a corollary, a list of 2,338 variable genes within the R. solanacearum species has been defined. The hierarchical clustering based on the distribution of variable genes is fully consistent with the phylotype classification that was previously defined from the nucleotide sequence analysis of four genes. The presence of numerous pathogenicity-related genes in the core genome indicates that R. solanacearum is an ancestral pathogen. The results establish the long coevolution of the two replicons that constitute the bacterial genome. We also demonstrate the clustering of variable genes in genomic islands. Most genomic islands are included in regions with an alternative codon usage, suggesting that they originate from acquisition of foreign genes through lateral gene transfers. Other genomic islands correspond to genes that have the same base composition as core genes, suggesting that they either might be ancestral genes lost by deletion in certain strains or might originate from horizontal gene transfers.

A 70-mer oligonucleotide-based DNA microarray covering 5,074 of the 5,120 predicted genes from Ralstonia solanacearum has been generated and used to investigate the repertoire of genes that are under the control of the transcription activator HrpB, which governs pathogenicity in this plant pathogenic bacterium. This study identified 143 hrpB up-regulated genes and 50 hrpB down-regulated genes. In addition to extending the repertoire of type III effector proteins with 26 new candidates, this work demonstrates that the hrpB regulon extends beyond type III secretion system-related functions to include a number of genes governing chemotaxy, biosynthesis or catabolism of various low-molecular-weight chemical compounds, and siderophore production and uptake. The presence of several transcripttional regulators and a cluster of genes predicted to encode the synthesis of an acylhomoserine lactone together with the absence of a consensus hrpII box in the promoter of a significant proportion of the hrpB-regulated genes suggest that, for some genes, hrpB regulation might be indirect. Altogether, the data indicate that hrpB acts as a master regulatory gene governing a physiological swing associated with the shift from saprophytic to parasitic life.

Expression of several virulence factors in the plant pathogen bacterium Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA. We provide genetic evidence that PhcA also represses the expression of hrp genes that code for the Type III protein secretion system, a major pathogenicity determinant in this bacterium. The repression of hrp genes in complete medium is relieved in a phcA mutant and two distinct signals, a quorum-sensing signal and complex nitrogen sources, appear to trigger this PhcA-dependent repression. This control of hrp gene expression by PhcA is realized at the level of the HrpG regulatory protein.

▪ Abstract Ralstonia solanacearum is a devastating plant pathogen with a global distribution and an unusually wide host range. This bacterium can also be free-living as a saprophyte in water or in the soil in the absence of host plants. The availability of the complete genome sequence from strain GMI1000 provided the basis for an integrative analysis of the molecular traits determining the adaptation of the bacterium to various environmental niches and pathogenicity toward plants. This review summarizes current knowledge and speculates on some key bacterial functions, including metabolic versatility, resistance to metals, complex and extensive systems for motility and attachment to external surfaces, and multiple protein secretion systems. Genome sequence analysis provides clues about the evolution of essential virulence genes such as those encoding the Type III secretion system and related pathogenicity effectors. It also provided insights into possible mechanisms contributing to the rapid adaptation of the bacterium to its environment in general and to its interaction with plants in particular.

In Ralstonia solanacearum, we previously have reported on the characterization of popP1 and popP2 genes. These genes encode type III-dependent pathogenicity effectors related to the large family of AvrRxv/YopJ cysteine proteases that are shared among pathogens of plants and animals. In this study, we identify a third gene, named popP3, that is inactivated in the genome sequence of strain GMI1000 by insertion of a copy of the insertion sequence ISRso13. The three popP genes are localized on two large chromosomal pathogenicity islands, with popP1 and popP2 being present on the same island. Phylogenic analysis demonstrated that the PopP2 and PopP3 proteins are clearly distinct from other effectors of this family previously characterized in plant and animal pathogens. Analysis of the distribution and allelic variations of the three genes in 30 strains representative of the biodiversity of R. solanacearum established that popP genes are distributed widely among strains from two of the three phyla previously defined on the basis of the structure of the core genome. Sequencing of the popP genes from the different strains revealed limited allelic variations at the three loci but did not show evidence of recombination between the popP genes. Limited allelic variation together with occurrence of insertion sequences within or in the close vicinity of popP genes and the presence of gene duplications in these pathogenicity islands suggest that genomic rearrangements might be a major evolutionary driving force controlling evolution of the genes encoded in these regions. The implications of these observations in terms of bacterial evolution, gene acquisition, and horizontal gene transfers are discussed.

Ralstonia solanacearum is a devastating plant pathogen with a global distribution and an unusually wide host range. This bacterium can also be free-living as a saprophyte in water or in the soil in the absence of host plants. The availability of the complete genome sequence from strain GNU 1000 provided the basis for an integrative analysis of the molecular traits determining the adaptation of the bacterium to various environmental niches and pathogenicity toward plants. This review summarizes current knowledge and speculates on some key bacterial functions, including metabolic versatility, resistance to metals, complex and extensive systems for motility and attachment to external surfaces, and multiple protein secretion systems. Genome sequence analysis provides clues about the evolution of essential virulence genes such as those encoding the Type III secretion system and related pathogenicity effectors. It also provided insights into possible mechanisms contributing to the rapid adaptation of the bacterium to its environment in general and to its interaction with plants in particular.

ABSTRACT The ability of Ralstonia solanacearum to cause disease on plants depends on its type III secretion system (TTSS) encoded by hrp genes. The expression of hrp genes and known TTSS substrates is coordinately regulated by HrpB, a member of the AraC family of transcriptional regulators. Two HrpB-regulated promoters ( hrpY and popABC ) were characterized by deletion analysis, and the HrpB-dependent activation of these promoters was found to be conferred by a 25-nucleotide DNA element, the hrp II box (TTCGn16TTCG), which is present in other hrp promoters. The hrp II box element is an imperfect plant inducible promoter box, an element which was originally found in hrp promoters of Xanthomonas campestris (S. Fenselau and U. Bonas, Mol. Plant-Microbe Interact. 8:845-854, 1995) but which was not characterized at the molecular level. Site-directed mutagenesis showed that the hrp II box is essential for hrpY promoter activation in vivo. Functional analysis of the hrp II box element identified critical parameters that are required for HrpB-dependent activity. Further mapping analyses of several other hrpB -dependent promoters also indicated that the position of the hrp II box is conserved, at −70 to −47 bp from the transcriptional start. As a first step toward identifying novel TTSS effectors, we used the hrp II box consensus sequence to search for potential HrpB-regulated promoters in the complete genome sequence of R. solanacearum strain GMI1000. Among the 114 genes identified, a subset of promoters was found to have a structural relationship with hrp promoters, thus providing a pool of candidate genes encoding TTSS effectors.