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

Schmidtke C, Findeiss S, Sharma CM, Kuhfuss J, Hoffmann S, Vogel J, Stadler PF, Bonas U - Nucleic Acids Res. (2011)

Bottom Line: Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Martin-Luther-Universität Halle-Wittenberg, Institute for Biology, D-06099 Halle, Germany. cornelius.schubert@genetik.uni-halle.de

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.

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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.
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gkr904-F4: 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.

Mentions: After completion of bioinformatic analyses, seven verified sRNAs turned out to correspond to cis-encoded antisense RNAs, termed asX1-7 (Table 1, Figure 2 and Supplementary Figure S3). We detected dRNA-seq reads mapping to both antisense RNA and mRNA for six of these transcripts and a few reads mapping to the CDS complementary to asX4, respectively (data not shown). The remaining 16 sRNAs mapped to intergenic regions and were termed sX1-15 and 6S (Table 1, Figures 2, 3A, 4A and Supplementary Figure S3). Intriguingly, three sRNAs (sX15, asX6, asX7) are encoded on the large plasmid, two of which (asX7 and sX15) are in antisense orientation to each other (Table 1 and Supplementary Figure S3). Most sRNA genes were constitutively expressed under the conditions tested, and appeared to accumulate in stationary growth phase either due to higher transcription rates or increased stability, e.g. sX14 and 6S (Figure 2). Interestingly, expression/accumulation of five intergenic sRNAs and three antisense RNAs was affected by the key regulators of hrp gene expression, HrpG and HrpX, suggesting a role of these sRNAs or their targets in the interaction of Xcv with the plant. HrpX-dependent induction of sRNA expression was observed for asX4, sX5, sX8 (Figure 2) and sX12 (see below), whereas sX11 appeared to be HrpG/HrpX-dependently repressed (Supplementary Figure S3). In case of sX4 (Figure 2) and the antisense RNAs asX1 and asX5 (Supplementary Figure S3) the sRNA stability appeared to depend on HrpG and HrpX as well as on the growth phase.Figure 3.


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

Schmidtke C, Findeiss S, Sharma CM, Kuhfuss J, Hoffmann S, Vogel J, Stadler PF, Bonas U - Nucleic Acids Res. (2011)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300014&req=5

gkr904-F4: 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.
Mentions: After completion of bioinformatic analyses, seven verified sRNAs turned out to correspond to cis-encoded antisense RNAs, termed asX1-7 (Table 1, Figure 2 and Supplementary Figure S3). We detected dRNA-seq reads mapping to both antisense RNA and mRNA for six of these transcripts and a few reads mapping to the CDS complementary to asX4, respectively (data not shown). The remaining 16 sRNAs mapped to intergenic regions and were termed sX1-15 and 6S (Table 1, Figures 2, 3A, 4A and Supplementary Figure S3). Intriguingly, three sRNAs (sX15, asX6, asX7) are encoded on the large plasmid, two of which (asX7 and sX15) are in antisense orientation to each other (Table 1 and Supplementary Figure S3). Most sRNA genes were constitutively expressed under the conditions tested, and appeared to accumulate in stationary growth phase either due to higher transcription rates or increased stability, e.g. sX14 and 6S (Figure 2). Interestingly, expression/accumulation of five intergenic sRNAs and three antisense RNAs was affected by the key regulators of hrp gene expression, HrpG and HrpX, suggesting a role of these sRNAs or their targets in the interaction of Xcv with the plant. HrpX-dependent induction of sRNA expression was observed for asX4, sX5, sX8 (Figure 2) and sX12 (see below), whereas sX11 appeared to be HrpG/HrpX-dependently repressed (Supplementary Figure S3). In case of sX4 (Figure 2) and the antisense RNAs asX1 and asX5 (Supplementary Figure S3) the sRNA stability appeared to depend on HrpG and HrpX as well as on the growth phase.Figure 3.

Bottom Line: Northern blot analyses confirmed 16 intergenic small RNAs and seven cis-encoded antisense RNAs in Xcv.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Martin-Luther-Universität Halle-Wittenberg, Institute for Biology, D-06099 Halle, Germany. cornelius.schubert@genetik.uni-halle.de

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.

Show MeSH
Related in: MedlinePlus