ABSTRACT
The type III secretion (T3S) system, an essential pathogenicity factor in most Gram-negative plant-pathogenic bacteria, injects bacterial effector proteins directly into the plant cell cytosol. Here, the type III effectors (T3Es) manipulate host cell processes to suppress defence and establish appropriate conditions for bacterial multiplication in the intercellular spaces of the plant tissue. T3E export depends on a secretion signal which is also present in 'non-effectors'. The latter are secreted extracellular components of the T3S apparatus, but are not translocated into the plant cell. How the T3S system discriminates between T3Es and non-effectors is still enigmatic. Previously, we have identified a putative translocation motif (TrM) in several T3Es from Xanthomonas campestris pv. vesicatoria (Xcv). Here, we analysed the TrM of the Xcv effector XopB in detail. Mutation studies showed that the proline/arginine-rich motif is required for efficient type III-dependent secretion and translocation of XopB and determines the dependence of XopB transport on the general T3S chaperone HpaB. Similar results were obtained for other effectors from Xcv. As the arginine residues of the TrM mediate specific binding of XopB to cardiolipin, one of the major lipid components in Xanthomonas membranes, we assume that the association of T3Es to the bacterial membrane prior to secretion supports type III-dependent export.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Secretion Systems , Conserved Sequence , Xanthomonas/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cardiolipins/metabolism , Cell Membrane/metabolism , Consensus Sequence , Models, Biological , Protein Binding , Protein Transport , Structure-Activity Relationship , Nicotiana/microbiologyABSTRACT
The genus Xanthomonas comprises a large group of plant-pathogenic bacteria. The infection and bacterial multiplication in the plant tissue depends on the type III secretion system and other virulence determinants. Recent studies revealed that bacterial virulence is also controlled at the post-transcriptional level by small non-coding RNAs (sRNAs). In this review, we highlight our current knowledge about sRNAs and RNA-binding proteins in Xanthomonas species.
Subject(s)
RNA, Small Untranslated/genetics , Xanthomonas/genetics , Gene Expression Regulation, Bacterial , Host Factor 1 Protein/metabolism , Phylogeny , Plant Diseases/microbiology , Plants/microbiology , RNA, Bacterial/chemistry , RNA, Bacterial/classification , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Small Untranslated/chemistry , RNA, Small Untranslated/classification , RNA, Small Untranslated/metabolism , RNA-Binding Proteins/metabolism , Virulence/genetics , Xanthomonas/metabolism , Xanthomonas/pathogenicityABSTRACT
Small noncoding RNAs (sRNAs) are ubiquitous posttranscriptional regulators of gene expression. Using the model plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), we investigated the highly expressed and conserved sRNA sX13 in detail. Deletion of sX13 impinged on Xcv virulence and the expression of genes encoding components and substrates of the Hrp type III secretion (T3S) system. qRT-PCR analyses revealed that sX13 promotes mRNA accumulation of HrpX, a key regulator of the T3S system, whereas the mRNA level of the master regulator HrpG was unaffected. Complementation studies suggest that sX13 acts upstream of HrpG. Microarray analyses identified 63 sX13-regulated genes, which are involved in signal transduction, motility, transcriptional and posttranscriptional regulation and virulence. Structure analyses of in vitro transcribed sX13 revealed a structure with three stable stems and three apical C-rich loops. A computational search for putative regulatory motifs revealed that sX13-repressed mRNAs predominantly harbor G-rich motifs in proximity of translation start sites. Mutation of sX13 loops differentially affected Xcv virulence and the mRNA abundance of putative targets. Using a GFP-based reporter system, we demonstrated that sX13-mediated repression of protein synthesis requires both the C-rich motifs in sX13 and G-rich motifs in potential target mRNAs. Although the RNA-binding protein Hfq was dispensable for sX13 activity, the hfq mRNA and Hfq::GFP abundance were negatively regulated by sX13. In addition, we found that G-rich motifs in sX13-repressed mRNAs can serve as translational enhancers and are located at the ribosome-binding site in 5% of all protein-coding Xcv genes. Our study revealed that sX13 represents a novel class of virulence regulators and provides insights into sRNA-mediated modulation of adaptive processes in the plant pathogen Xanthomonas.
Subject(s)
Gene Expression Regulation, Bacterial , RNA, Bacterial/metabolism , RNA, Small Untranslated/metabolism , Virulence Factors/metabolism , Xanthomonas/metabolism , Adaptation, Physiological , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Capsicum/microbiology , Chemotaxis , Mutation , Nucleic Acid Conformation , Nucleotide Motifs , Oligonucleotide Array Sequence Analysis , Plant Leaves/microbiology , Protein Biosynthesis , RNA Stability , RNA, Bacterial/chemistry , RNA, Messenger/metabolism , RNA, Small Untranslated/chemistry , Signal Transduction , Virulence , Virulence Factors/chemistry , Virulence Factors/genetics , Xanthomonas/growth & development , Xanthomonas/pathogenicityABSTRACT
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.
Subject(s)
RNA, Small Untranslated/metabolism , Virulence Factors/genetics , Xanthomonas campestris/genetics , 5' Untranslated Regions , Bacterial Proteins/genetics , Genome, Bacterial , Models, Statistical , Molecular Sequence Annotation , Phylogeny , RNA Processing, Post-Transcriptional , RNA, Small Untranslated/chemistry , RNA, Small Untranslated/genetics , Sequence Analysis, RNA , Transcription Initiation Site , Transcriptome , Virulence Factors/metabolism , Xanthomonas campestris/pathogenicityABSTRACT
The genome of Xanthomonas campestris pv. vesicatoria encodes a constitutively expressed small RNA, which we designate PtaRNA1, "Plasmid transferred anti-sense RNA". It exhibits all hallmarks of a novel RNA antitoxin that proliferates by frequent horizontal transfer. It shows an erratic phylogenetic distribution with occurrences on chromosomes in a few individual strains distributed across both beta- and gamma-proteobacteria. Moreover, a homologous gene located on plasmid pMATVIM-7 of Pseudomonas aeruginosa is found. All ptaRNA1 homologs are located anti-sense to a putative toxin, which in turn is never encountered without the small RNA. The secondary structure of PtaRNA1, furthermore, is very similar to that of the FinP anti-sense RNA found on F-like plasmids in Escherichia coli.
