Identification Of Type Ⅲ Translocators In Xanthomonas Oryzae Pv.Oryzae And Studying The Function Of XopN

Xoo(Xanthomonas oryzae pv.oryzae,Xoo)is a pathogenic variant of Xanthomonas,can use the T3SS(Type Ⅲ secretion system)to express the extracellular secretory protein,thus showing the toxicity of Bacterial blight on rice.Translocon is an important component that connects the type Ⅲ secretion system and the host cells,it is usually consisting of two hydrophobic proteins and one hydrophilic protein.The hydrophilic transport protein HpalXoo and the hydrophobic transport protein HrpFXoo were proved to be important components of the type Ⅲ secretion system.Therefore,the GAL4 and membrane yeast two-hybrid system was used to confirm whether there was a direct interaction between HpalXoo and HrpFXoo.The results showed that there was no direct interaction between the two proteins by yeast two-hybrid experiments.Then ligated the hpal and hrpF genes to the expression vector pHM1,respectively.and the protein expression condition was confirmed by Flag and His tag,The tag protein was tested by Western blotting.After that,overexpressed the protein Hpal and HrpF for co-immunoprecipitation,the results showed that via Hpal-Flag and HrpF-His protein were screened for 24 and 64 kinds of interaction proteins in total protein PX099A,respectively,including type Ⅲ secretion system effector XopN,and further confirmed the pathogenicity of.XopN.XopN is a kind of Xops(Xanthomonas outer proteins)protein,which belongs to rice pathovar Xoo.XopN is an effector,can be secreted by the Xoo type Ⅲ secretion system.At present,the XopN strain function is unknown in the Philippines strain PXO99A.In this study,we were knocked out XopN gene in PX099A wild strain by method of homologous exchange,and then we constructed the complementation carrier pHM IXopN,so we got PXO99AAXopN deletion mutant strain and C-XopN strain.The pathogenicity of XopN was studied by measuring the growth rate of PXO99A △XopN strain;Measuring the pathogenicity on host rice plants;and measuring HR reactions on non-host tobacco plants.The results showed that the growth rate ofXopN mutant was less than that of wild-type PX099A strain in NB medium;The strain that knocked XopN gene caused the shorter lesion length in the rice than wild type PXO99A,such as IRBB203,Asminori,Nipponbare,and the difference was significant,but this difference was different in various rice cultivars;Then complemented the XopN gene,the virulence of complementation strain and its growth rate on rice can be restored;In addition,the HR reaction of XopN mutant in tobacco compared to wild-type PXO99A was not obvious.