Molecular identification and functional analysis of Clpxoo, a homologue to the nucleotide receptor protein in Xanthomonas oryzae pv. Oryzae

Abstract:

[Objective] To better understand the mechanisms of cyclic di-GMP signaling in Xanthomonas oryzae pv. oryzae (Xoo), the casual pathogen of bacterial blight of rice, molecular identification of Clpxoo. [Methods] A putative signal receptor protein was performed through gene cloning, sequencing and deletion analysis. [Results] Sequence analysis showed Clpxoo was a homologue of Crp and Vfr, the cAMP receptor proteins in Escherichia coli and Pseudomonas aeruginosa respectively, which had the cNMP-binding domains (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. We constructed △clpxoo through a double crossover recombination and validated by PCR assay, △clpxoo displayed the reduced 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 introducing clpxoo. Moreover, we observed no significant changes in production of extracellular cellulase and xylanase in vitro, biofilm formation and induction of hypersensitive response (HR) on non-host tobacco in △clpxoo compared to PXO99A. [Conclusion] Therefore, Clpxoo played a role as one of the global regulator in regulation of flagellar motility, EPS production and H2O2 resistance.