Identification And Functional Characterization Of The Transcriptional Regulator Clpxoo In Xanthomonas Oryzae Pv.oryzae

Xanthomoans oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight of rice, one of the most serious bacterial disease of rice. It was predicted that there was a signaling regulation network in Xoo which strictly control virulence gene expression through signal sensing, perception and transduction. However, until recently, little was known about the virulence regulation network in Xoo. As reported, cyclic diguanylate (c-di-GMP) is a bacterial second messenger of growing recognition involved in the regulation of a number of complex physiological processes. To better understand its mechanism in Xoo, our laboratory has begun extensive research on c-di-GMP signaling pathway and its functions. This research was focused on the identification and functional analysis of Clpxoo, a putative c-di-GMP nucleotide receptor protein.Firstly, clpxoo was performed through gene cloning, sequencing and deletion analysis. The transcripts of flagellar genes and genes encoding calatases were analysed by Quantitative real-time reverse transcript PCR (RT-Q-PCR). Meanwhile, the gene with conserved Clpxoo binding sequence in promoter region have been identified. Lastly, the purified Clpxoo protein was prepared for future study.Sequence analysis showed Clpxoo was a homologue of Crp and Vfr, the cAMP receptor proteins in Escherichia coli and Psudomonas aeruginosa respectively, which had the cNMP -bindnig domians (CAP-ED) at N terminal and the DNA-binding domains (HTH-CRP) at C terminal and is highly conserved in the plant-pathogenic Xanthomonas spp.△clpxoo displayed the reduced virulence, motility and extracellular polysaccharide (EPS) production, and increased sensitive to H2O2 toxicity compared with PXO99A. All these phenotype changes could be partly restored through complementation of mutants by introducting clpxoo and confirmed the former conclusion that Clp was a global transcriptional regulator. However, we observed no significant changes in production of extrcellular cellulase and xylanase in vitro, induction of hypersensitive response (HR) on non-host tobacoo in△clpxoo compared with PXO99A. Since the similar funcitons of Clpxoo to c-di-GMP, we agreed the hypothesis that Clpxoo may funciton as c-di-GMP receptor protein in Xoo.The analysis of gene transcripts indicated that fleQxoo, the gene encoding top regulator in flagellar hierarchical synthesis as well as fliCxoo,encoding flagellin ; fliAxoo, encoding RNA polymeraseσ28; fliDxoo, encoding flagellar cap protein and fliLxoo, encoding flagellar basal body-associated protein were positively regulated by Clpxoo. Moreover, computer search detected two conserved Clpxoo binding sequences in the promoter region of fleQxoo, suggested Clpxoo may directly regulate the expression of fleQxoo. Meanwhile, It was showed that Clpxoo regulated ahpCxoo and katExoo in opposite ways, which encoding alkyl hydroperocide reductase and monofunctional catalase respectively, implying Xoo may rely on a complicated compensatory mechanism to prevent the damage from H2O2.Lastly, purified Clpxoo protein has been prepared for further confirming whether Clpxoo could receive singal from c-di-GMP then directly regulate the expression of fleQxoo in the downstream.In sum, the study confirmed Clpxoo, the homologe of nucleotide receptor protien was essential for virulence, cell motility, EPS produciton and H2O2 resistance and suggested a likely regulatory hierarchy in the downstream of Clpxoo.