Summary
Ralstonia solanacearum is a soil‐borne bacterium causing the widespread disease known as bacterial wilt. Ralstonia solanacearum is also the causal agent of Moko disease of banana and brown ...rot of potato. Since the last R. solanacearum pathogen profile was published 10 years ago, studies concerning this plant pathogen have taken a genomic and post‐genomic direction. This was pioneered by the first sequenced and annotated genome for a major plant bacterial pathogen and followed by many more genomes in subsequent years. All molecular features studied now have a genomic flavour. In the future, this will help in connecting the classical field of pathology and diversity studies with the gene content of specific strains. In this review, we summarize the recent research on this bacterial pathogen, including strain classification, host range, pathogenicity determinants, regulation of virulence genes, type III effector repertoire, effector‐triggered immunity, plant signalling in response to R. solanacearum, as well as a review of different new pathosystems.
Taxonomy
Bacteria; Proteobacteria; β subdivision; Ralstonia group; genus Ralstonia.
Disease symptoms
Ralstonia solanacearum is the agent of bacterial wilt of plants, characterized by a sudden wilt of the whole plant. Typically, stem cross‐sections will ooze a slimy bacterial exudate. In the case of Moko disease of banana and brown rot of potato, there is also visible bacterial colonization of banana fruit and potato tuber.
Disease control
As a soil‐borne pathogen, infected fields can rarely be reused, even after rotation with nonhost plants. The disease is controlled by the use of resistant and tolerant plant cultivars. The prevention of spread of the disease has been achieved, in some instances, by the application of strict prophylactic sanitation practices.
Useful websites
Stock centre: International Centre for Microbial Resources—French Collection for Plant‐associated Bacteria CIRM‐CFBP, IRHS UMR 1345 INRA‐ACO‐UA, 42 rue Georges Morel, 49070 Beaucouzé Cedex, France, http://www.angers‐nantes.inra.fr/cfbp/. Ralstonia Genome browser: https://iant.toulouse.inra.fr/R.solanacearum. GMI1000 insertion mutant library: https://iant.toulouse.inra.fr/R.solanacearumGMI1000/GenomicResources. MaGe Genome Browser: https://www.genoscope.cns.fr/agc/microscope/mage/viewer.php?
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
The Ralstonia solanacearum species complex includes thousands of strains pathogenic to an unusually wide range of plant species. These globally dispersed and heterogeneous strains cause bacterial ...wilt diseases, which have major socio-economic impacts. Pathogenicity is an ancestral trait in R. solanacearum and strains with high genetic variation can be subdivided into four phylotypes, correlating to isolates from Asia (phylotype I), the Americas (phylotype IIA and IIB), Africa (phylotype III) and Indonesia (phylotype IV). Comparison of genome sequences strains representative of this phylogenetic diversity can help determine which traits allow this bacterium to be such a pathogen of so many different plant species and how the bacteria survive in many different habitats.
The genomes of three tomato bacterial wilt pathogens, CFBP2957 (phy. IIA), CMR15 (phy. III) and PSI07 (phy. IV) were sequenced and manually annotated. These genomes were compared with those of three previously sequenced R. solanacearum strains: GMI1000 (tomato, phy. I), IPO1609 (potato, phy. IIB), and Molk2 (banana, phy. IIB). The major genomic features (size, G+C content, number of genes) were conserved across all of the six sequenced strains. Despite relatively high genetic distances (calculated from average nucleotide identity) and many genomic rearrangements, more than 60% of the genes of the megaplasmid and 70% of those on the chromosome are syntenic. The three new genomic sequences revealed the presence of several previously unknown traits, probably acquired by horizontal transfers, within the genomes of R. solanacearum, including a type IV secretion system, a rhi-type anti-mitotic toxin and two small plasmids. Genes involved in virulence appear to be evolving at a faster rate than the genome as a whole.
Comparative analysis of genome sequences and gene content confirmed the differentiation of R. solanacearum species complex strains into four phylotypes. Genetic distances between strains, in conjunction with CGH analysis of a larger set of strains, revealed differences great enough to consider reclassification of the R. solanacearum species complex into three species. The data are still too fragmentary to link genomic classification and phenotypes, but these new genome sequences identify a pan-genome more representative of the diversity in the R. solanancearum species complex.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ralstonia solanacearum, the causal agent of a lethal bacterial wilt plant disease, infects an unusually wide range of hosts. These hosts can further be split into plants where R. solanacearum is ...known to cause disease (original hosts) and those where this bacterium can grow asymptomatically (distant hosts). Moreover, this pathogen is able to adapt to many plants as supported by field observations reporting emergence of strains with enlarged pathogenic properties. To investigate the genetic bases of host adaptation, we conducted evolution experiments by serial passages of a single clone of the pathogen on three original and two distant hosts over 300 bacterial generations and then analyzed the whole-genome of nine evolved clones. Phenotypic analysis of the evolved clones showed that the pathogen can increase its fitness on both original and distant hosts although the magnitude of fitness increase was greater on distant hosts. Only few genomic modifications were detected in evolved clones compared with the ancestor but parallel evolutionary changes in two genes were observed in independent evolved populations. Independent mutations in the regulatory gene efpR were selected for in three populations evolved on beans, a distant host. Reverse genetic approaches confirmed that these mutations were associated with fitness gain on bean plants. This work provides a first step toward understanding the within-host evolutionary dynamics of R. solanacearum during infection and identifying bacterial genes subjected to in planta selection. The discovery of EfpR as a determinant conditioning host adaptation of the pathogen illustrates how experimental evolution coupled with whole-genome sequencing is a potent tool to identify novel molecular players involved in central life-history traits.
Summary
An evolution experiment with the bacterial plant pathogen Ralstonia solanacearum revealed that several adaptive mutations conferring enhanced fitness in plants arose in the efpR gene encoding ...a regulator of virulence and metabolic functions. In this study, we found that an efpR mutant systematically displays colonies with two morphotypes: the type S (‘smooth’, similar to the wild type) and the type EV (‘efpR variant’). We demonstrated that the efpH gene, a homologue of efpR, plays a key role in the control of phenotypic heterogeneity, the ΔefpR‐ΔefpH double mutant being stably locked into the EV type. Using mixed infection assays, we demonstrated that the type EV is metabolically more proficient than the type S and displays fitness gain in specific environments, whereas the type S has a better fitness into the plant environment. We provide evidence that this efpR‐dependent phenotypic heterogeneity is a general feature of strains of the R. solanacearum species complex and could occur in natural conditions. This study highlights the potential role of phenotypic heterogeneity in this plant pathogen as an adaptive trait to changing environments.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Type III effectors from phytopathogenic bacteria exhibit a high degree of functional redundancy, hampering the evaluation of their precise contribution to pathogenicity. This is illustrated by the ...GALA type III effectors from Ralstonia solanacearum, which have been shown to be collectively, but not individually, required for disease on Arabidopsis thaliana and tomato. We investigated evolution, redundancy and diversification of this family in order to understand the individual contribution of the GALA effectors to pathogenicity. From sequences available, we reconstructed GALA phylogeny and performed selection studies. We then focused on the GALAs from the reference strain GMI1000 to examine their ability to suppress plant defense responses and contribution to pathogenicity on three different host plants: A. thaliana, tomato (Lycopersicum esculentum) and eggplant (Solanurn melongena). The GALA family is well conserved within R. solanacearum species. Patterns of selection detected on some GALA family members, together with experimental results, show that GALAs underwent functional diversification. We conclude that functional divergence of the GALA family likely accounts for its remarkable conservation during R. solanacearum evolution and could contribute to R. solanacearum's adaptation on several host plants.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NMLJ, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
The global regulator PhcA controls numerous traits associated to virulence and bacterial proliferation in strains of the plant pathogen Ralstonia solanacearum species complex. Here, we conducted a ...genome-wide RNA sequencing study of the GMI1000 wild-type strain and a derived phcA mutant grown in complete medium. The PhcA regulon we identified is the largest regulon described to date in the R. solanacearum species complex with 1581 regulated genes, representing about 30% of the bacterial genome. Among these genes, 166 transcription regulators were identified including known regulators controlling major cellular functions such as the Type 3 secretion system and 27 novel regulators that were not identified in previous transcriptomic studies. This study highlights that PhcA controls other functions beside pathogenicity stricto sensu which participate to the global cell homeostasis (metabolism, energy storage). We then compared the PhcA regulon identified in complete medium to the recently published PhcA regulon obtained in planta. This comparison of the set of GMI1000 genes subjected to PhcA regulation in both conditions revealed 383 common genes. Among them, 326 (85%) had a similar PhcA dependent regulation pattern in complete medium and in planta, and 57 (15%) displayed an opposite regulation pattern. A large majority of the genes repressed by PhcA in complete medium but activated in planta belong to the HrpG-HrpB regulon, which represents a set of key genes required for R. solanacearum pathogenesis. This latter class of genes appears to be specifically induced by PhcA in the plant environment whereas PhcA represses their expression in complete medium. The large set of direct and indirect targets identified in this study will contribute to enrich our knowledge of the intricate regulatory network coordinating the expression of virulence and metabolic functions in the model plant pathogen R. solanacearum.
•The PhcA regulon of R. solanacearum in complete medium (CM) is analysed.•30% of the whole bacterial genes are regulated by PhcA.•PhcA controls functions involved in pathogenicity and global cell homeostasis.•Most of PhcA-regulated genes have similar regulation pattern in CM and in planta.•Genes regulated by HrpG-HrpB are induced by PhcA in planta but repressed in CM.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
In this chapter, we describe different methods for phenotyping strains or mutants of the bacterial wilt agent, Ralstonia solanacearum, on four different host plants: Arabidopsis thaliana, tomato ...(Solanum lycopersicum), tobacco (Nicotiana benthamiana), or Medicago truncatula. Methods for preparation of high volume or low volume inocula are first described. Then, we describe the procedures for inoculation of plants by soil drenching, stem injection or leaf infiltration, and scoring of the wilting symptoms development. Two methods for measurement of bacterial multiplication in planta are also proposed: (1) counting the bacterial colonies upon serial dilution plating and (2) determining the bacterial concentration using a qPCR approach. In this chapter, we also describe a competitive index assay to compare the fitness of two strains coinoculated in the same plant. Lastly, specific protocols describe in vitro and hydroponic inoculation procedures to follow disease development and bacterial multiplication in both the roots and aerial parts of the plant.
All the strains grouped under the species
represent a species complex responsible for many diseases on agricultural crops throughout the world. The strains have different lifestyles and host range. ...Here, we investigated whether specific metabolic pathways contribute to strain diversification. To this end, we carried out systematic comparisons on 11 strains representing the diversity of the species complex. We reconstructed the metabolic network of each strain from its genome sequence and looked for the metabolic pathways differentiating the different reconstructed networks and, by extension, the different strains. Finally, we conducted an experimental validation by determining the metabolic profile of each strain with the Biolog technology. Results revealed that the metabolism is conserved between strains, with a core metabolism composed of 82% of the pan-reactome. The three species composing the species complex could be distinguished according to the presence/absence of some metabolic pathways, in particular, one involving salicylic acid degradation. Phenotypic assays revealed that the trophic preferences on organic acids and several amino acids such as glutamine, glutamate, aspartate, and asparagine are conserved between strains. Finally, we generated mutants lacking the quorum-sensing-dependent regulator PhcA in four diverse strains, and we showed that the
dependent trade-off between growth and production of virulence factors is conserved across the
species complex. IMPORTANCE
is one of the most important threats to plant health worldwide, causing disease on a very large range of agricultural crops such as tomato or potato. Behind the
name are hundreds of strains with different host range and lifestyle, classified into three species. Studying the differences between strains allows to better apprehend the biology of the pathogens and the specificity of some strains. None of the published genomic comparative studies have focused on the metabolism of the strains so far. We developed a new bioinformatic pipeline to build high-quality metabolic networks and used a combination of metabolic modeling and high-throughput phenotypic Biolog microplates to look for the metabolic differences between 11 strains across the three species. Our study revealed that genes encoding enzymes are overall conserved, with few variations between strains. However, more variations were observed when considering substrate usage. These variations probably result from regulation rather than the presence or absence of enzymes in the genome.
Experimental evolution of the plant pathogen Ralstonia solanacearum, where bacteria were maintained on plant lineages for more than 300 generations, revealed that several independent single mutations ...in the efpR gene from populations propagated on beans were associated with fitness gain on bean. In the present work, novel allelic efpR variants were isolated from populations propagated on other plant species, thus suggesting that mutations in efpR were not solely associated to a fitness gain on bean, but also on additional hosts. A transcriptomic profiling and phenotypic characterization of the efpR deleted mutant showed that EfpR acts as a global catabolic repressor, directly or indirectly down-regulating the expression of multiple metabolic pathways. EfpR also controls virulence traits such as exopolysaccharide production, swimming and twitching motilities and deletion of efpR leads to reduced virulence on tomato plants after soil drenching inoculation. We studied the impact of the single mutations that occurred in efpR during experimental evolution and found that these allelic mutants displayed phenotypic characteristics similar to the deletion mutant, although not behaving as complete loss-of-function mutants. These adaptive mutations therefore strongly affected the function of efpR, leading to an expanded metabolic versatility that should benefit to the evolved clones. Altogether, these results indicated that EfpR is a novel central player of the R. solanacearum virulence regulatory network. Independent mutations therefore appeared during experimental evolution in the evolved clones, on a crucial node of this network, to favor adaptation to host vascular tissues through regulatory and metabolic rewiring.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Ralstonia solanacearum, the causal agent of bacterial wilt, is a soil bacterium which can naturally infect a wide range of host plants through the root system. Pathogenicity relies on a type III ...secretion system which delivers a large set of approximately 75 type III effectors (T3E) into plant cells. On several plants, pathogenicity assays based on quantification of wilting symptoms failed to detect a significant contribution of R. solanacearum T3E in this process, thus revealing the collective effect of T3E in pathogenesis. We developed a mixed infection-based method with R. solanacearum to monitor bacterial fitness in plant leaf tissues as a virulence assay. This accurate and sensitive assay provides evidence that growth defects can be detected for T3E mutants: we identified 12 genes contributing to bacterial fitness in eggplant leaves and 3 of them were also implicated in bacterial fitness on two other hosts, tomato and bean. Contribution to fitness of several T3E appears to be host specific, and we show that some known avirulence determinants such as popP2 or avrA do provide competitive advantages on some susceptible host plants. In addition, this assay revealed that the efe gene, which directs the production of ethylene by bacteria in plant tissues, and hdfB, involved in the biosynthesis of the secondary metabolite 3-hydroxy-oxindole, are also required for optimal growth in plant leaf tissues.
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