Functional Analysis Of The CNMP Receptor And DNA Binding Domains Of Transcriptional Regulator Clpxoo In Xanthomonas Oryzae PV. Oryzae

Bacterial blight of rice caused by Xanthomoans oryzae pv. oryzae (Xoo) is one of the most serious diseases of rice in the world. A series of signaling networks regulate bacterial infection to the host plant during the interaction of Xoo and rice. However, until recently, little was known about the function of the signaling networks to virulence in Xoo. The nucleotide signaling molecule cyclic diguanosine monophosphate (c-di-GMP) has recently emerged as a widely conserved second messenger implicated in the regulation of various biological functions of bacteria. Our lab has done some basic work on understanding the c-di-GMP signaling pathway and its functions in Xoo. Previous work indicated transcriptional regulator Clpxoo, a putative c-di-GMP receptor, regulated extracellular polysaccharide (EPS) production, biofilm formation, cell precipitation, sensitivity to H₂O₂ and pathogenicity on rice, etc. However, Clpxoo's functional mechanism remains unknown. This study was focused on analysis of functional domains of Clpxoo: the N-terminal cNMP-binding domian and C-terminal DNA-binding domain (HTH). The following experiments have been carried out:(1) Clpxoo protein was expressed using the expression vector pET28a(+), and then was purified through Ni-NTA affinity chromatography. Purified Clpxoo protein was obtained.(2) The transcripts of rpoNxoo encoding alternative sigma factorσ⁵⁴, and engXCAxoo gene encoding endoglucanase inâ–³clpxoo were analysed by quantitative reverse transcriptase PCR (qRT-PCR). The results indicated rpoNxoo and engXCAxoo were positively regulated by Clpxoo. Conserved Clpxoo binding sequences in the promoter region of rpoNxoo and engXCAxoo were detected by bioinformatics tools, and two consensus Clp-binding sites were located at -187 and -318 bp relative to translation initiation site (TIS), respectively. They were cloned with specific primers and labeled with biotion. However, there was no binding effect detected between these promoters and Clpxoo protein using electrophoretic mobility shift assay (EMSA). The result indicated there was no direct interaction between Clpxoo and these promoters, suggesting the expression of rpoNxoo and engXCAxoo were indirectly, but not directly regulated by Clpxoo.(3) The interaction between Clpxoo and c-di-GMP was tested by isothermal titration calorimeter (ITC). The result indicated there was weak interaction between Clpxoo and c-di-GMP, implying Clpxoo might be a c-di-GMP effector.(4) PXO₀1019, PXO₀3877 and PXO₀4753, which are related to c-di-GMP signaling pathway in Xoo, were cloned into different expression vectors. Plasmids to express recombinant proteins were obtained. They will be used for purification of these proteins and further characterization of their functions.In sumary, this study prelimarily characterized Clpxoo as an effector of the second messager c-di-GMP, to regulate the expression of downstream genes.