High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis

Abstract

The bacterium Xanthomonas campestris is an economically important pathogen of many crop species and a model for the study of bacterial phytopathogenesis. In X. campestris, a regulatory system mediated by the signal molecule DSF controls virulence to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon a system comprising the sensor RpfC and regulator RpfG. Here we have addressed the action and role of Rpf/DSF signalling in phytopathogenesis by high-resolution transcriptional analysis coupled to functional genomics. We detected transcripts for many genes that were unidentified by previous computational analysis of the genome sequence. Novel transcribed regions included intergenic transcripts predicted as coding or non-coding as well as those that were antisense to coding sequences. In total, mutation of rpfF, rpfG and rpfC led to alteration in transcript levels (more than fourfold) of approximately 480 genes. The regulatory influence of RpfF and RpfC demonstrated considerable overlap. Contrary to expectation, the regulatory influence of RpfC and RpfG had limited overlap, indicating complexities of the Rpf signalling system. Importantly, functional analysis revealed over 160 new virulence factors within the group of Rpf-regulated genes.

Fig. 1

Northern blot analysis of selected small non-coding RNAs candidates from Xcc. Schematic diagrams of the genomic context of each sRNA are given on the left. Northern hybridizations were carried out on RNA samples isolated from wild-type cells cultured in NYGB medium. The optical density at 600 nm of the cultures is indicated. The probes used in Northern analysis were complementary to the sRNA. Arrows indicate the predominant bands. Sizes of the sRNAs are given on the right. rRNAs served as control for RNA loading and RNA integrity. A summary of information on sRNAs verified is detailed in Table S4.

Fig. 2

Changes in gene expression of rpfF, rpfC, rpfG and rpfH mutants compared with the wild-type Xanthomonas campestris 8004 as measured by RNA-Seq. A and B. Venn diagrams showing the overlap of genes whose expression is (A) upregulated or (B) downregulated in different mutant backgrounds. Divergently regulated genes are not depicted in these Venn diagrams but can be found in Table S5. C. Comparison of relative fold changes between RNA-Seq and qRT-PCR results in (i) rpfF, (ii) rpfC and (iii) rpfG mutant backgrounds. All qRT-PCR results were normalized using the Cts obtained for the 16S rRNA amplifications run in the same plate. The relative levels of gene transcripts are determined from standard curves. Values given are the mean and standard deviation of triplicate measurements (three biological and three technical replicates).

Fig. 3

Reduced virulence phenotypes in Chinese radish as a consequence of mutation of genes regulated by the Rpf/DSF signalling system. A. The virulence of each mutant was tested by measurement of the lesion length after bacteria were introduced into the vascular system of Chinese radish by leaf clipping as detailed in the Experimental procedures. Mutants were assigned to classes I–V depending on the percentage lesion length when compared with the Xcc wild-type strain (Rv). B. The complementation of mutants that were in Class I. The virulence of each mutant and complemented strain was tested by measurement of the lesion length after bacteria were introduced into the vascular system of Chinese radish by leaf clipping.

Fig. 4

Regulation of expression of three sRNAs by the Rpf/DSF system and their involvement in virulence. A. Northern blots of the selected sRNAs (sRNAXcc-15, sRNAXcc-16, sRNAXcc-28) regulated by the Rpf/DSF system. RNA isolated from Xcc 8004 wild-type (i), rpfC mutant (ii) and complemented rpfC mutant [rpfC (pRPFC)] (iii) was probed for sRNAs. B. A triple deletion of sRNAXcc-15/sRNAXcc-16/sRNAXcc-28 (sR15/16/28) leads to reduction in virulence as measured in Chinese radish by leaf clipping. In contrast deletion of sRNAXcc-15, sRNAXcc-16, sRNAXcc-28 alone had no effect on virulence.

Fig. 5

Model of the DSF/Rpf signalling cascades in Xcc. A. Venn diagram depicting the overlapping and discrete regulatory actions of RpfF, RpfG and RpfC that are involved in phytopathogenesis. B. Schematic representation of regulatory pathways involving DSF, RpfC and RpfG in the virulence of Xcc. Expression of several genes including engXCA, prtA and manA are co-ordinately regulated by RpfF, which synthesizes DSF and the RpfCG two-component system, consistent with the linear pathway previously described. However RpfG controls a number of genes including XC_0638 (chemotaxis) that are not influenced by RpfF or RpfC, suggesting an interaction of RpfG with a second unknown sensor (left). DSF and RpfC also regulate expression of a number of genes including XC_1766 (transcription regulator) in a pathway independent of RpfG (centre). RpfF appears to control a number of genes independently of the other Rpf proteins, suggesting several genes including XC_0061 (hypothetical protein) are regulated by DSF but not by RpfG or RpfC. This is evidence for the occurrence of a second sensor and signal transduction system for DSF (right). All of the target genes indicated encode virulence factors that are novel, with the exception of engXCA, prtA and manA, whose role in virulence has been described previously. All predicted protein functions are given in parentheses.