Distribution and sequence analysis of a family of type ill-dependent effectors correlate with the phylogeny of Ralstonia solanacearum strains

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.     

The awr gene family encodes a novel class of Ralstonia solanacearum type III effectors displaying virulence and avirulence activities

We present here the characterization of a new gene family, awr, found in all sequenced Ralstonia solanacearum strains and in other bacterial pathogens. We demonstrate that the five paralogues in strain GMI1000 encode type III-secreted effectors and that deletion of all awr genes severely impairs its capacity to multiply in natural host plants. Complementation studies show that the AWR (alanine-tryptophan-arginine tryad) effectors display some functional redundancy, although AWR2 is the major contributor to virulence. In contrast, the strain devoid of all awr genes (Δawr1-5) exhibits enhanced pathogenicity on Arabidopsis plants. A gain-of-function approach expressing AWR in Pseudomonas syringae pv. tomato DC3000 proves that this is likely due to effector recognition, because AWR5 and AWR4 restrict growth of this bacterium in Arabidopsis. Transient overexpression of AWR in nonhost tobacco species caused macroscopic cell death to varying extents, which, in the case of AWR5, shows characteristics of a typical hypersensitive response. Our work demonstrates that AWR, which show no similarity to any protein with known function, can specify either virulence or avirulence in the interaction of R. solanacearum with its plant hosts.     

Autoacetylation of the Ralstonia solanacearum effector PopP2 targets a lysine residue essential for RRS1-R-mediated immunity in Arabidopsis

Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as "guards". The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity.     

Regulation of the type III secretion system in phytopathogenic bacteria

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.     

Illuminating the molecular basis of gene-for-gene resistance; Arabidopsis thaliana RRS1-R and its interaction with Ralstonia solanacearum popP2

Elucidation of the molecular basis of gene-for-gene interactions between disease-resistance (R) genes and pathogen avirulence (avr) genes has been a Holy Grail of plant pathology for the past decade. Recent studies of the R-avr interaction between RRS1-R and popP2 by Laurent Deslandes et al. provide new insights and suggest a direct physical association of the encoded proteins in support of a simplistic receptor-ligand model. However, careful consideration of the experimental findings reveals that they could also be explained by molecular linker proteins that mediate formation of a PopP2 and RRS1-R uniting complex.     

Ralstonia solanacearum virulence increased following large interstrain gene transfers by natural transformation

Horizontal gene transfer (HGT) is a major driving force of evolution and is also likely to play an important role in the threatening emergence of novel pathogens, especially if it involves distantly related strains with substantially different pathogenicity. In this study, the impact of natural transformation on pathogenicity in six strains belonging to the four phylotypes of the plant-pathogenic bacterium Ralstonia solanacearum was investigated. The study focused on genomic regions that vary between donor and recipient strains and that carry genes involved in pathogenicity such as type III effectors. First, strains from R. solanacearum species complex were naturally transformed with heterologous genomic DNA. Transferred DNA regions were then determined by comparative genomic hybridization and polymerase chain reaction sequencing. We identified three transformant strains that acquired large DNA regions of up to 80 kb. In one case, strain Psi07 (phylotype IV tomato isolate) acquired 39.4 kb from GMI1000 (phylotype I tomato isolate). Investigations revealed that i) 24.4 kb of the acquired region contained 20 new genes, ii) an allelic exchange of 12 genes occurred, and iii) 27 genes (33.4 kb) formerly present in Psi07 were lost. Virulence tests with the three transformants revealed a significant increase in the aggressiveness of BCG20 over its Psi07 parent on tomato. These findings demonstrate the potential importance of HGT in the pathogenic evolution of R. solanacearum strains and open new avenues for studying pathogen emergence.     

Cloning of wild-type Pseudomonas solanacearum phcA, a gene that when mutated alters expression of multiple traits that contribute to virulence.

Pseudomonas solanacearum undergoes a spontaneous mutation that pleiotropically reduces extracellular polysaccharide (EPS) production, endoglucanase activity, and virulence and increases motility. We refer to the process that coordinately affects these traits as phenotype conversion (PC) and the resulting mutants as PC types. Previous research with the wild-type strain AW1 suggested that inactivation of a single locus could mimic phenotype conversion (T. P. Denny, F. W. Makini, and S. M. Brumbley, Mol. Plant-Microbe Interact. 1:215-223, 1988). Additional Tn5 mutagenesis of AW1 generated three more mutants (AW1-81, AW1-82, and AW1-84) that were indistinguishable from the PC type and one slightly leaky mutant (AW1-87); all four had single insertions in the same 4.0-kilobase (kb) EcoRI fragment that were responsible for the PC-like phenotype. Another insertion mutant, AW1-83, which lacks an insertion in this 4.0-kb fragment, resembled the PC type except that it was reversibly induced to produce wild-type levels of EPS when cultured adjacent to AW1. The wild-type region containing the gene that controls traits affected by phenotype conversion in AW1, designated phcA, was cloned on a 2.2-kb DNA fragment that restored all the phcA::Tn5 mutants and 11 independent spontaneous PC-type derivatives of AW1 to wild-type status. Homology with the phcA region was found in diverse wild-type strains of P. solanacearum, although restriction fragment length polymorphisms were seen. No major DNA alterations were observed in the phcA homologous region of PC types from strain AW1 or 82N. PC types from 7 of 11 conjugal strains of P. solanacearum were restored to EPS+ by phcA from AW1; however, only some PC types of strain K60 were restored, whereas others were not. We believe that a functional phcA gene is required to maintain the wild-type phenotype in P. solanacearum, and for most strains phenotype conversion results from a loss of phcA gene expression or the function of its gene product.     

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.     

Detection and functional characterization of a large genomic deletion resulting in decreased pathogenicity in Ralstonia solanacearum race 3 biovar 2 strains

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.     

RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector

Bacterial wilt, a disease impacting cultivated crops worldwide, is caused by the pathogenic bacterium Ralstonia solanacearum. PopP2 (for Pseudomonas outer protein P2) is an R. solanacearum type III effector that belongs to the YopJ/AvrRxv protein family and interacts with the Arabidopsis thaliana RESISTANT TO RALSTONIA SOLANACEARUM 1-R (RRS1-R) resistance protein. RRS1-R contains the Toll/Interleukin1 receptor–nucleotide binding site–Leu-rich repeat domains found in several cytoplasmic R proteins and a C-terminal WRKY DNA binding domain. In this study, we identified the Arabidopsis Cys protease RESPONSIVE TO DEHYDRATION19 (RD19) as being a PopP2-interacting protein whose expression is induced during infection by R. solanacearum. An Arabidopsis rd19 mutant in an RRS1-R genetic background is compromised in resistance to the bacterium, indicating that RD19 is required for RRS1-R–mediated resistance. RD19 normally localizes in mobile vacuole-associated compartments and, upon coexpression with PopP2, is specifically relocalized to the plant nucleus, where the two proteins physically interact. No direct physical interaction between RRS1-R and RD19 in the presence of PopP2 was detected in the nucleus as determined by Förster resonance energy transfer. We propose that RD19 associates with PopP2 to form a nuclear complex that is required for activation of the RRS1-R–mediated resistance response.