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RNA-seq analysis of Ralstonia solanacearum nitrosative stress response mutants

ABSTRACT: Ralstonia solanacerum causes bacterial wilt disease on many important host crops, including tomato and potato. R. solanacearum cells enter a host from soil or infested water through the roots, then multiply and spread in the water-transporting xylem vessels. Despite the low nutrient and oxygen content of xylem sap, R. solanacearum grows extremely well inside the host, using denitrification to respire in this hypoxic environment. R. solanacearum growth in planta also depends successful mitigation of oxidative stress produced by both the bacterium and the host. We found that proteins encoded by norA and hmpX that are predicted to interact with nitric oxide (NO) and protect against cellular damage caused by NO. Reducing NO toxicity is especially important for Rs that generates NO via denitrifying respiration in planta, and encounters NO produced by plants as a defense and signaling molecule. By analyzing transcriptomes of mutants lacking these genes, we found that iron, sulfur, and nitrogen metabolism genes were consistently upregulated in mutants missing these genes. Our transcriptomic analysis suggests R. solanacearum mutants lacking norA and hmpX suffer oxidative stress from an increase in cellular toxicity caused by NO that leads to damage of iron, sulfur, and nitrogen metabolic proteins. Our results suggest that NorA and HmpX, contribute to oxidative stress mitigation in denitrifying cultures.

ORGANISM(S): Ralstonia solanacearum

PROVIDER: GSE194210 | GEO | 2022/01/26

REPOSITORIES: GEO

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Project description:The plant pathogen Pseudomonas syringae pv. syringae B728a grows and survives on leaf surfaces and in the leaf apoplast of its host, bean. Global transcriptome profiling was used to understand the contribution of distinct regulators to B728a fitness and pathogenicity. We performed a transcriptome analysis of B728a and nine regulator mutants recovered from the surface and interior of leaves and exposed to various environmental stresses in culture. These mutants were nonpolar deletion mutants lacking a single target regulator gene: ahlR, aefR, gacS, retS, salA, rpoS, algU, hrpL and rpoN. RNA was collected from cells of these strains that were exposed to five treatments in vitro, namely a basal medium, sodium chloride to confer an osmotic stress, hydrogen peroxide to confer an oxidative stress, iron limitation, and nitrogen limitation, and were recovered from two bean (Phaseolus vulgaris L.) leaf sites, namely epiphytic sites after leaf surface inoculation and 72 h of growth and apoplastic sites after leaf infiltration and 48 h of growth. The results indicated that the quorum-sensing regulators AhlR and AefR influenced few genes in planta or in vitro. In contrast, GacS and a downstream regulator SalA formed a large regulatory network that included a branch that regulated diverse traits and was independent of plant-specific environmental signals, and a signal-dependent branch that positively regulated secondary metabolite genes and negatively regulated the type III secretion system. RetS functioned almost exclusively to repress secondary metabolite genes in cells in culture but not on leaves. SalA functioned as a central regulator of iron status, based on its reciprocal regulation of pyoverdine and achromobactin genes, and also sulfur uptake, suggesting a role in the iron-sulfur balance. Among the sigma factors examined, AlgU influenced many more genes than RpoS, and most AlgU-regulated genes depended on RpoN. RpoN differentially impacted many AlgU- and GacS-activated genes in cells recovered from apoplastic versus epiphytic sites, suggesting differences in environmental signals or bacterial stress status in these two habitats. Collectively, our findings illustrate a central role for GacS, SalA, RpoN and AlgU in global regulation in B728a in planta and a high level of plasticity in their response to distinct environmental signals.

Project description:Peyraud2016 - Metabolic reconstruction (iRP1476) of Ralstonia solanacearum GMI1000 This model is described in the article: A Resource Allocation Trade-Off between Virulence and Proliferation Drives Metabolic Versatility in the Plant Pathogen Ralstonia solanacearum. Peyraud R, Cottret L, Marmiesse L, Gouzy J, Genin S. PLoS Pathog. 2016 Oct; 12(10): e1005939 Abstract: Bacterial pathogenicity relies on a proficient metabolism and there is increasing evidence that metabolic adaptation to exploit host resources is a key property of infectious organisms. In many cases, colonization by the pathogen also implies an intensive multiplication and the necessity to produce a large array of virulence factors, which may represent a significant cost for the pathogen. We describe here the existence of a resource allocation trade-off mechanism in the plant pathogen R. solanacearum. We generated a genome-scale reconstruction of the metabolic network of R. solanacearum, together with a macromolecule network module accounting for the production and secretion of hundreds of virulence determinants. By using a combination of constraint-based modeling and metabolic flux analyses, we quantified the metabolic cost for production of exopolysaccharides, which are critical for disease symptom production, and other virulence factors. We demonstrated that this trade-off between virulence factor production and bacterial proliferation is controlled by the quorum-sensing-dependent regulatory protein PhcA. A phcA mutant is avirulent but has a better growth rate than the wild-type strain. Moreover, a phcA mutant has an expanded metabolic versatility, being able to metabolize 17 substrates more than the wild-type. Model predictions indicate that metabolic pathways are optimally oriented towards proliferation in a phcA mutant and we show that this enhanced metabolic versatility in phcA mutants is to a large extent a consequence of not paying the cost for virulence. This analysis allowed identifying candidate metabolic substrates having a substantial impact on bacterial growth during infection. Interestingly, the substrates supporting well both production of virulence factors and growth are those found in higher amount within the plant host. These findings also provide an explanatory basis to the well-known emergence of avirulent variants in R. solanacearum populations in planta or in stressful environments. This model is hosted on BioModels Database and identified by: MODEL1612020000. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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RNA-seq analysis of Ralstonia solanacearum nitrosative stress response mutants

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