| Xanthomonas campestris pv.campestris(Xcc)is a Gram-negative bacteria,the causative agent of black rot of crucifers,can infect all cultivated brassicas and thus results in poor yield and quality of these crops throughout the world.Moreover,as Xcc can infect the model plant Arabidopsis thaliana,it has become an important model organism for studying plant-microbe interactions.Helix-turn-helix(HTH)is an regulatory motif found in most of activator and repressor proteins in prokaryotes.The HTH motif consists of approximately 20 amino acids,which contains two ? helixs and a junction corner.It plays an important role in the regulation of gene expression and exists in all LysR-type transcription regulators.Xcc also encodes different types of proteins with HTH domains.The roles of VrhR,VrhA and VrhB transcriptional regulators in the interaction between Xcc and plants were studied via gene mutation and virulence analysis.The influence of these regulators(VrhA,VrhB and VrhR)on the pathogenicity of Xcc and the possible pathogenic factors as well as pathways it might regulate were analyzed.The main results of this study are as follows:(1)Bioinformatics analysis showed that VrhR contains only one HTH motif and vrhR locates between two other LysR transcriptional regulators(vrhA and vrhB)in Xcc genome,but transcribes in an opposite direction.However,promoter-GUS and RT-PCR analysis indicated that vrhR can be transcribed in two directions and both vrhR DNA double-strands can be transcribed and may encode two genes,vrhR and vrhC;(2)RT-PCR analysis showed that vrhA,vrhC and vrhB were located in the same operon;(3)The vrhR,vrhA,vrhB and vrhB/vrhR/vrhA(vrhA-vrhR-vrhB triple mutation)mutants were constructed and the effects of these genes on Xcc virulence were analyzed.Results showed that vrhR mutant had increased pathogenicity compared with the wild type Xcc 8004,but vrhA,vrhB,vrhB/vrhR/vrhA and Xcc 8004 had no difference in virulence on Brassica oleracea.Meanwhile,some virulence factors such as extracellular enzyme and motility were analyzed and data showed that there were no difference in extracellular enzyme(protease,cellulase and amylase)activities and motility between these mutants and Xcc 8004,suggesting that VrhR may regulate bacterial virulence by affecting other virulence factors;(4)To uncover the mechanism of VrhR in regulating Xcc virulence,we performed RNA sequencing(RNA-seq)and found that vrhR mutation affects the expression of a large number of genes involved in different pathways.Notably,the up-regulated genes are mostly associated with transporter and extracellular enzyme action,the down-regulated genes are mainly related to the oxidation-reduction reaction.We also confirmed these results by promoter-GUS analysis.However,the roles of these genes in Xcc virulence need further studies.In summary,we have studied three LysR-type transcription regulators(VrhR,VrhA,VrhB)which located at a gene cluster and their regulatory role in virulence.VrhR played a negative regulation during the interaction between pathogens and plants,but VrhA and VrhB did not.Notably,all of these regulators may not regulate bacterial motility and extracellular enzyme activities but affect other pathogenic factors.A large number of genes differentially expressed in vrhR mutant and the wild type strain were identified by RNA-seq and promoter-GUS analysis.Our results highlight that VrhR is vital for Xcc pathogenicity but the regulatory pathways of VrhR should be further studied in funture. |
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