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

Bacteria of the genus Xenorhabdus are symbionts of soil entomopathogenic nematodes of the genus Steinernema. This symbiotic association constitutes an insecticidal complex active against a wide range of insect pests. Unlike other Xenorhabdus species, Xenorhabdus poinarii is avirulent when injected into insects in the absence of its nematode host. We sequenced the genome of the X. poinarii strain G6 and the closely related but virulent X. doucetiae strain FRM16. G6 had a smaller genome (500–700 kb smaller) than virulent Xenorhabdus strains and lacked genes encoding potential virulence factors (hemolysins, type 5 secretion systems, enzymes involved in the synthesis of secondary metabolites, and toxin–antitoxin systems). The genomes of all the X. poinarii strains analyzed here had a similar small size. We did not observe the accumulation of pseudogenes, insertion sequences or decrease in coding density usually seen as a sign of genomic erosion driven by genetic drift in host-adapted bacteria. Instead, genome reduction of X. poinarii seems to have been mediated by the excision of genomic blocks from the flexible genome, as reported for the genomes of attenuated free pathogenic bacteria and some facultative mutualistic bacteria growing exclusively within hosts. This evolutionary pathway probably reflects the adaptation of X. poinarii to specific host.

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“…This region contains no features of GI or prophages. Its absence leads to a loss of pathogenicity (Gonzalez et al 2011). …”

Section: Discussionmentioning

confidence: 99%

Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

Ogier

Pagès

Bisch

et al. 2014

Genome Biology and Evolution

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“…Natural transformation of R . solanacearum was performed, as previously described [26], using 300 ng of the selected marker and 1 μg of unselected marker added to 50 μl of competent cells. R .…”

Section: Methodsmentioning

confidence: 99%

Enhanced in planta Fitness through Adaptive Mutations in EfpR, a Dual Regulator of Virulence and Metabolic Functions in the Plant Pathogen Ralstonia solanacearum

Perrier¹,

Peyraud²,

Rodrı́guez-Lázaro³

et al. 2016

PLoS Pathog

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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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“…The ⍀ -spectinomycin interposon (26) was cloned into the unique NotI restriction site within the hpaB open reading frame to generate plasmid pSG424. pSG424 was linearized by XbaI and used to transform R. solanacearum as previously described (27). Strain GRS474 was obtained by selecting a double recombination event using spectinomycin resistance and hpaB gene disruption was then checked by PCR.…”

Section: Methodsmentioning

confidence: 99%

“…pNP329 derivatives carrying ripD, ripH1, ripH2, ripH3, ripW, ripAB, ripAN, and ripAV-3HA tags were linearized using ScaI enzyme, and then introduced into five strains, i.e. the wild-type (GMI1000) (8), and the hrcV, hpaB, hpaD, and hpaG mutants (GMI1694, GRS474, GRS266, and GRS230) by natural transformation as previously described (27). Stable chromosomal insertions in the defined bacterial chromosome site were checked by PCR using oNP611 5Ј-GAAAGCACGCTGTTTCCGCTATTT-3Ј, oNP612 5Ј-GCGTAGTGCG-CAAGACGAACAA-3Ј and oNP613 5Ј-GGCTCAAGGAGAAGAGC-CTTCAGA-3Ј primers.…”

Section: Methodsmentioning

confidence: 99%

Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity

Lonjon

Turner

Henry

et al. 2016

Molecular & Cellular Proteomics

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Ralstonia solanacearum, the causal agent of bacterial wilt, exerts its pathogenicity through more than a hundred secreted proteins, many of them depending directly on the functionality of a type 3 secretion system. To date, only few type 3 effectors have been identified as required for bacterial pathogenicity, notably because of redundancy among the large R. solanacearum effector repertoire. In order to identify groups of effectors collectively promoting disease on susceptible hosts, we investigated the role of putative post-translational regulators in the control of type 3 secretion. A shotgun secretome analysis with label-free quantification using tandem mass spectrometry was performed on the R. solanacearum GMI1000 strain. There were 228 proteins identified, among which a large proportion of type 3 effectors, called Rip (Ralstonia injected proteins). Thanks to this proteomic approach, RipBJ was identified as a new effector specifically secreted through type 3 secretion system and translocated into plant cells. A focused Rip secretome analysis using hpa (hypersensitive response and pathogenicity associated) mutants revealed a fine secretion regulation and specific subsets of Rips with different secretion patterns. We showed that a set of Rips (RipF1, RipW, RipX, RipAB, and RipAM) are secreted in an Hpa-independent manner. We hypothesize that these Rips could be preferentially involved in the first stages of type 3 secretion. In addition, the secretion of about thirty other Rips is controlled by HpaB and HpaG. HpaB, a candidate chaperone was shown to positively control secretion of numerous Rips, whereas HpaG was shown to act as a negative regulator of secretion. To evaluate the impact of altered type 3 effectors secretion on plant pathogenesis, the hpa mutants were assayed on several host plants. HpaB was required for bacterial pathogenicity on multiple hosts whereas HpaG was found to be specifically required for full R. solanacearum pathogenicity on the legume plant Medicago truncatula. Molecular & Cellular Proteomics 15: 10.1074/ mcp.M115.051078, 598-613, 2016.The soil-borne vascular bacterium R. solanacearum is described as one of the most destructive plant pathogenic bacterium worldwide (1), mainly because of its broad host range and wide geographic distribution. Indeed, R. solanacearum attacks more than 250 plant species, distributed in more than 50 botanical families (2). R. solanacearum causes dramatic crop losses, more specifically in the tropics, notably affecting emerging countries. Durable protection strategies against this bacterium are lacking. R. solanacearum penetrates into the plant via the roots, and then colonizes the xylem vessels. The bacterium reaches the aerial parts of the plant, causes wilting symptoms leading to the death of the plant, and will eventually return to the soil, completing the cycle (3). To achieve these first colonization steps, the bacterium uses a molecular syringe called the type 3 secretion system (T3SS), 1 which deFrom the ‡INRA, Laboratoire des Interactions Plantes-...

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

Cited by 31 publications

References 46 publications

Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

Attenuated Virulence and Genomic Reductive Evolution in the Entomopathogenic Bacterial Symbiont Species, Xenorhabdus poinarii

Enhanced in planta Fitness through Adaptive Mutations in EfpR, a Dual Regulator of Virulence and Metabolic Functions in the Plant Pathogen Ralstonia solanacearum

Comparative Secretome Analysis of Ralstonia solanacearum Type 3 Secretion-Associated Mutants Reveals a Fine Control of Effector Delivery, Essential for Bacterial Pathogenicity

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