Dataset Information

Dynamic expression of Ralstonia solanacearum virulence factors and metabolism-controlling genes during plant infection

ABSTRACT:

Background

Ralstonia solanacearum is the causal agent of bacterial wilt, a devastating plant disease responsible for serious economic losses especially on potato, tomato, and other solanaceous plant species in temperate countries. In R. solanacearum, gene expression analysis has been key to unravel many virulence determinants as well as their regulatory networks. However, most of these assays have been performed using either bacteria grown in minimal medium or in planta, after symptom onset, which occurs at late stages of colonization. Thus, little is known about the genetic program that coordinates virulence gene expression and metabolic adaptation along the different stages of plant infection by R. solanacearum.

Results

We performed an RNA-sequencing analysis of the transcriptome of bacteria recovered from potato apoplast and from the xylem of asymptomatic or wilted potato plants, which correspond to three different conditions (Apoplast, Early and Late xylem). Our results show dynamic expression of metabolism-controlling genes and virulence factors during parasitic growth inside the plant. Flagellar motility genes were especially up-regulated in the apoplast and twitching motility genes showed a more sustained expression in planta regardless of the condition. Xylem-induced genes included virulence genes, such as the type III secretion system (T3SS) and most of its related effectors and nitrogen utilisation genes. The upstream regulators of the T3SS were exclusively up-regulated in the apoplast, preceding the induction of their downstream targets. Finally, a large subset of genes involved in central metabolism was exclusively down-regulated in the xylem at late infection stages.

Conclusions

This is the first report describing R. solanacearum dynamic transcriptional changes within the plant during infection. Our data define four main genetic programmes that define gene pathogen physiology during plant colonisation. The described expression of virulence genes, which might reflect bacterial states in different infection stages, provides key information on the R. solanacearum potato infection process.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-021-07457-w.

SUBMITTER: de Pedro-Jove R

PROVIDER: S-EPMC7941725 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundRalstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30°C with strains that can cause disease below 20°C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18°C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS).ResultsFour hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR.ConclusionsThe putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains.

Project description:Strains of the Ralstonia solanacearum species complex in the phylotype IIB group are capable of causing Bacterial Wilt disease in potato and tomato at temperatures lower than 24°C. The capability of these strains to survive and to incite infection at temperatures colder than their normally tropical boundaries represents a threat to United States agriculture in temperate regions. In this work, we used a comparative genomics approach to identify orthologous genes linked to the lower temperature virulence phenotype. Six R. solanacearum cool virulent (CV) strains were compared to six strains non-pathogenic at low temperature (NPLT). CV strains can cause Bacterial Wilt symptoms at temperatures below 24°C, while NPLT cannot. Four R. solanacearum strains were sequenced for this work in order to complete the comparison. An orthologous genes comparison identified 44 genes present only in CV strains and 19 genes present only in NPLT strains. Gene annotation revealed a high percentage of genes compared with whole genomes in the transcriptional regulator and transport categories. A single nucleotide polymorphism (SNP) analysis identified 265 genes containing conserved non-synonymous SNPs in CV strains. Ten genes in the pathogenicity category were identified in this group. Comparisons of type 3 secretion system, type 6 secretion system (T6SS) clusters, and associated effectors did not indicate a correlation with the CV phenotype except for one T6SS VGR effector potentially associated with the CV phenotype. This is the first R. solanacearum genomic comparative analysis of multiple strains with different temperature related virulence. The candidate genes identified by this comparison are potential factors involved in virulence at low temperatures that need to be investigated. The high percentage of transcriptional regulators among the genes present only in CV strains supports the hypothesis that temperature dependent regulation of virulence genes explains the differential virulence phenotype at low temperatures. This comparison contributes to find new possible connections of temperature dependent virulence to the previously described complex regulatory system involving quorum-sensing, phenotype conversion (phcA), acyl-HSL production and responses to SA. It also added novel candidate T6SS effectors and useful detailed information about the T6SS in R. solanacearum.

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