Functional analysis of rpfG gene in Xanthomonas arboricola pv. juglandis

Abstract:

[Objective] The purpose of this study was to reveal the biological function of rpfG gene in walnut bacterial black spot pathogen (Xanthomonas arboricola pv. juglandis) XajDW3F3, so as to provide a target for the development of prevention and treatment agents for walnut bacterial black spot. [Methods] The rpfG gene of Xanthomonas campestris pv.campestris 8004 and Xanthomonas campestris pv.campestris PXO99^A were used as template sequences to search the genome sequence of DW3F3, a wild-type strain of Xaj. With homologous recombination technology to knock out XajrpfG, the virulence factors and stress resistance of the mutant strain were detected by biochemical methods. [Results] Homologous gene was identified in XajDW3F3 genome through sequence homology analysis. The deletion mutant of rpfG was successfully obtained. Compared with the wild type, the mutant reduced the production of biofilm and extracellular polysaccharides, the ability of biofilm formation mutant strains ΔrpfG was only about 44.58% of wild type, the yield of exopolysaccharide decreased from 8.47 mg/mL in wild type to 5.23 mg/mL in ΔrpfG; but ΔrpfG increased the motility and flocculation activity, motility experiments showed that the motility diameter of ΔrpfG increased 12.38% than the wild type. The secretion of extracellular enzymes also changed at different degrees: the ability to secrete cellulase was significantly decreased and amylase activity was increased, but the ability to secrete protease was not changed. What’s more, after the deletion of rpfG, the resistance of Xaj to adversity (NaCl, pH, SDS, CuSO₄) was decreased. [Conclusion] The results showed that rpfG is an important pathogenic gene of walnut bacterial black spot pathogen, and endows bacteria with a certain degree of resistance to adversity.