Genome-wide transcriptome analysis of the plant pathogen Xanthomonas identifies sRNAs with putative virulence functions

Abstract

The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is an important model to elucidate the mechanisms involved in the interaction with the host. To gain insight into the transcriptome of the Xcv strain 85-10, we took a differential RNA sequencing (dRNA-seq) approach. Using a novel method to automatically generate comprehensive transcription start site (TSS) maps we report 1421 putative TSSs in the Xcv genome. Genes in Xcv exhibit a poorly conserved -10 promoter element and no consensus Shine-Dalgarno sequence. Moreover, 14% of all mRNAs are leaderless and 13% of them have unusually long 5'-UTRs. Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv. Expression of eight intergenic transcripts was controlled by HrpG and HrpX, key regulators of the Xcv type III secretion system. More detailed characterization identified sX12 as a small RNA that controls virulence of Xcv by affecting the interaction of the pathogen and its host plants. The transcriptional landscape of Xcv is unexpectedly complex, featuring abundant antisense transcripts, alternative TSSs and clade-specific small RNAs.

Figure 1.

Identification of TSSs, promoter elements and analysis of 5′-UTRs. (A) Distribution of dRNA-seq reads in the chromosomal locus of Xcv 85–10 spanning genes XCV0519 to XCV0524. Annotated CDSs and RNAcode high-scoring segments are highlighted in green and blue, respectively. Sequencing reads of library 1 (black) and library 2 (red) are shown on top for the (+)-strand and below for the (−)-strand. Predicted TSSs and corresponding classes are indicated in red. (B) Venn diagram illustrating the TSS classes. TSSs found maximal 300-bp upstream of coding sequences are classified as primary. Internal TSSs are found within and antisense TSSs on the opposite strand of genes (±100 bp). Orphan TSSs do not belong to other classes. (C) Sequence analysis identified a T/A-rich promoter element for 1205 of 1421 putative TSSs. The histogram depicts the position of the conserved sequence pattern relative to the annotated TSSs at position +1. (D) 5′-UTR length distribution. The x-axis is split into linear (0–50) and logarithmic (51–300) scales. The top of the histogram gives the percentage of leaderless (≤10 bp), short (≤50 bp) and longer UTRs (>50 bp).

Figure 2.

Expression of selected Xcv sRNAs and antisense RNAs depends on HrpG and HrpX. Total RNA isolated from exponential (exp) and stationary phase cultures (stat) of (a) Xcv strain 85–10, (b) 85–10 expressing hrpG* from pFG72-1 and (c) 85–10ΔhrpX carrying pFG72-1 was analyzed by northern blot. Arrows and filled squares indicate signals corresponding to the expected full-length RNA and processing products obtained by transcriptome sequencing, respectively. The open square indicates the expected size of full-length asX4 determined by RACE analysis. The expected size of sX4 according to the sequencing data is marked by an asterisk. 5S rRNA (lower panel) was probed as loading control.

Figure 3.

sX6 encodes a small protein. (A) Expression analysis of the sX6 transcript. Total RNA isolated from exponential (exp) and stationary phase cultures (stat) of (a) Xcv strain 85–10, (b) 85–10 expressing hrpG* from pFG72-1 and (c) 85–10ΔhrpX carrying pFG72-1 was analyzed by northern blot. The expected signal according to sequencing data is indicated by an arrow. 5S rRNA (lower panel) was probed as loading control. (B) Expression of the sX6 protein. Derivatives of Xcv strain 85–10 (wt) carrying promoterless empty vector pBRM-P (−) and sX6-c-Myc expression construct, respectively, were grown to OD₆₀₀ = 0.7. Protein extracts were analyzed by immunoblotting using c-Myc epitope-specific and GroEL-specific antibodies.

Figure 4.

sX12 is involved in virulence of Xcv. (A) sX12 is HrpX-dependently expressed. Total RNA isolated from exponential (exp) and stationary phase cultures (stat) of (a) Xcv strain 85–10, (b) Xcv expressing hrpG* from pFG72-1 and (c) a derivative deleted in hrpX and carrying pFG72-1 was analyzed by northern blot. The right panel shows a northern blot with RNA from (d) Xcv strain 85–10 and (e) an sX12 deletion mutant carrying empty vector pLAFR6, respectively, and (f) an sX12 deletion mutant ectopically expressing sX12 from psX12. The expected RNA size is indicated by an arrow. The asterisk denotes an unspecific signal. 5S rRNA (lower panel) was probed as loading control. (B) sX12 contributes to virulence and the HR. Strains used in (A) (right panel) were inoculated at a density of 1.25 × 10⁸ CFU ml⁻¹ into leaves of susceptible ECW and resistant ECW-10R pepper plants. Disease symptoms were photographed at 7 days post-inoculation (dpi). The HR was visualized by ethanol bleaching of the leaves at 2 days post-inoculation. Dashed lines indicate the inoculation site.