Transcriptional Regulation And Functional Analysis Of Chemotaxis Gene Che In Xanthomonas Oryzae Pv.Oryzae

Cyclic-diguanylate（c-di-GMP） is a universal second messenger existing in bacteria, which is involved in the regulation of various cellular behaviors including the motility, biofilm formation, EPS production, and pathogenicity. As a global transcriptional regulation factor, Clpxoo can act as the receptor protein of c-di-GMP. Clpxoo includes two main important domains including c NMP and DNA-binding domain. Binding between c NMP and c-di-GMP will change Clpxoo conformational structure; meanwhile, by directly binding to DNA, Clpxoo can regulate a number of cellular functions in Xoo. In this study, we chose the chemotaxis gene（PXO₀0043） as a target gene which is regulated positively by transcriptional regulator Clpxoo. We have made the following progress:1. We firstly identified the transcription start point of che gene by using 5’RACE technique. We then constructed a GFP-based reporter system to check the promoter activity of che gene. We found that Clpxoo was required for activating the che promoter. Besides, che is also upregulated in minimum medium XOM2, but regulatory protein Hrp G and Hrp X did not affect the transcription of che significantly..2. Clpxoo variants carrying point mutations D70 A, R166 A, and E99 S, respectively, were constructed and the proteins were purified. We found that ClpxooR166 A was not stable, and became degraded after ClpxooE99 S was reduced by fivefold. In comparison, ClpxooD70 A lost its ability to bind to c-di-GMP. We concluded that D70 and E99 of Clpxoo were critical for its binding to c-di-GMP.3. We constructed the deletion mutant of chemotaxis gene（PXO₀0043, che） and also the complemented strain. Phenotype analysis showed that the che mutant was impaired in its virulence on host rice plants, suggesting it contributed to the virulence of Xoo.In conclusion, this study identified the transcriptional start point of che gene and the promoter region of che gene. We confirmed that che gene was positively controlled by Clpxoo in Xoo. Also, we confirmed that both D70 and E99 are the key residues for Clpxoo to bind to the second messenger c-di-GMP.