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.

Keywords: Apoplast; Bacterial wilt; Dynamic gene expression; Effectors; Metabolism; RNAseq; Ralstonia solanacearum; T3SS; Virulence factors; Xylem.

Fig. 1

Transcriptomic profile of R. solanacearum UY031 in planta. a. Shared and unique DE genes across the three in planta conditions for the up-regulated (left) and down-regulated (right) genes. Each vertical bar plot represents the number of shared DE between the conditions indicated by the lines and dots in the schematic below. The horizontal bar plots on the right indicate the total DE genes per in planta condition compared to rich medium. b. For the intersection of Apoplast, Early and Late (in planta genetic programme), Early and Late (Xylem genetic programme), Apoplast and Late xylem alone, the list of genes was extracted and surveyed for enriched KEGG pathways. Dot plots of the enriched KEGG pathways for the up- (left) and down-regulated (right) genes in each environment are shown below. DE genes were identified with DEseq2 (p-adj > 0.01, log₂ FC ± 1.5) and plotted using the R package UpsetR

Fig. 2

Gene expression dynamics of R. solanacearum throughout infection. Six clusters were obtained through Mfuzz clustering of log₂-fold-change data of the apoplast, early and late xylem conditions normalised to the reference rich liquid media. Clusters include the genes with a membership higher than 70% and consistently associated to the same cluster on at least 30 out of 40 iterations. Number of genes indicated above each graph. The list of genes associated to each cluster was extracted and surveyed for enriched KEGG pathways. Dot plots of the enriched KEGG pathways in each cluster are shown next to the cluster

Fig. 3

T3E gene expression profile. Heatmaps showing the normalised transcripts per million (TPM) of the genes coding the 60 T3E described in R. solanacearum UY031 in the reference condition and in planta apoplast, early and late conditions. Only putatively functional T3E genes are included according to Peeters et al., 2013

Fig. 4

Motility gene expression profile. Heatmaps showing the normalised transcripts per million (TPM) of the genes involved in motility in the reference (rich Medium), apoplast, early and late xylem conditions. Two heatmaps are shown to differentiate the swimming (top panel) and twitching (bottom panel)

Fig. 5

T3SS regulatory cascade. Log₂ fold-change expression profile in the Apoplast, Early and Late Xylem of the genes involved in the T3SS regulatory cascade and downstream activated genes. Log₂ fold changes in transcript levels with respect to the control condition (axenic growth in rich medium) are indicated in the boxes (left to right: Apoplast, Early and Late Xylem conditions) in colour gradients according to the Colour Key

Fig. 6

Nitrogen metabolism expression profile. Log₂ fold-change expression profile in the Apoplast, Early and Late Xylem of the genes involved in the denitrification (aniA, norB), assimilatory (nasA), dissimilatory (narK1/2, narG, nirB/D) and nitrite detoxification (hmpX) pathways of nitrogen in R. solanacearum UY031. Log₂ fold changes in transcript levels with respect to the control condition (axenic growth in rich medium) are indicated in the boxes (left to right: Apoplast, Early and Late Xylem conditions) in colour gradients according to the Colour Key. O. M = Outer membrane, I.M. = Inner membrane