The Role Of PhoP/PhoQ In Regulating The Environmental Adaptability And Pathogenicity Of Citrus Canker Pathogen

Citrus canker is a quarantine bacterial disease caused by Xac（Xanthomonas axonopodis pv.citri）,which has a great damage on citrus production in the world.At present,the pathogenic mechanism of citrus canker pathogen is still largely unclear.Pho P/Pho Q is a two-component regulatory system that plays an important role in the regulation of virulence in some pathogens,but little is known in Xac.In this study,we investigated the effect of Pho P/Pho Q two-component regulation system on the environmental adaptability and pathogenicity in Xac,and the mechanism was analyzed and clarified.The main results are as follows:1)We obtainedΔpho Q andΔpho P mutants in Xac wild-type by using the non-trace knockout method.Studies have shown that the growth rate ofΔpho P mutants are significantly slower under inappropriate environmental conditions（strongly acidic or alkaline p H,SDS,H₂O₂）compared to wild type;its motility,HR response after tobacco inoculation and the infectivity to citrus is significantly reduced;furthermore,we also found that the mutation of pho P gene can caused filamentous growth of cells.The complementary of pho P gene inΔpho P mutants could completely or partly restore various phenotypes.Different from the two-component regulatory system of Pho P/Pho Q in other pathogenic bacteria,phenotypes ofΔpho Q mutants were not significantly different from the wild type.2)Further studies by using EMSA（Electrophoretic mobility shift assay）show that Pho P protein may be involved in many important cellular processes by directly regulating Ton B-dependent receptors（Xac0144 and Xac1768）,Lys R family transcription regulator（Xac0255）,sensor histidine kinase Bae S（Xac0326）,phosphate-selective porin Opr O and Opr P（Xac3484）.Similar to other pathogens,Pho P protein has the function of self-regulation.3)Further research focused on the effects of environmental factor osmotic pressure onΔpho P mutants.Transcriptional profiling revealed that the mutation of pho P gene in NYG medium resulted in significant difference expression of 1584 genes,respectively,involving a variety of transcription regulators,Ton B-dependent receptors,and flagellar protein,virulence regulatory factors and metabolism-related genes,which 294 were down-regulated and 1290 were up-regulated.If the appropriate osmotic pressure is increased,most of the differentially expressed genes in theΔpho P mutant can be restored to the wild-type expression level.At the same time,the phenotypes of growth,motility,tobacco HR response and citrus infectivity ofΔpho P mutants were also significantly restored.4)Studies of the mechanism have shown that pho P gene may affected its pathogenicity by directly regulating XAC2416（virulence regulator）and XACfli C（flagellar protein）genes,while the appropriate osmotic pressure could enhanc its pathogenicity by increasing the expression of these two genes and increase the survival of pathogenic bacteria in plants.Interestingly,mutations of the pho P gene affect the expression and localization of cell division proteins,leading to filamentous growth of theΔpho P mutants,and increased osmotic pressure restores expression and localization of cell division proteins inΔpho P mutants.In conclusion,our study found that Pho P may be independent of Pho Q in Xac and form a new two-component regulatory system with other proteins to regulate the environmental adaptability and pathogenicity of pathogens.Based on this,we have established a possible regulation model of Pho P in Xac.The results of our study are expected to lay a theoretical foundation for the prevention and control of citrus canker disease and other bacterial diseases in the future.