Function Characterization Of Non-TAL Effector XopY From Xanthomonas Oryzae Pv.oryzae PXO86 Strain

Xanthomonas oryzae pv.oryzae(Xoo)mainly relies on T3SS(type III secretion system)to secrete transcription activator-like(TAL)effectors and non-transcription activator-like(non-TAL)effectors into plants cell.These effectors make the plant environment conducive to the propagation and spread of pathogenic bacteria by interfering with the plant immune function,stressing the transfer of nutrients and so on,which in succession induces blight disease of rice.Non-TAL effector proteins are ubiquitous in Xoo,however,their functions and mechanisms have not been elucidated well.At present,only a few numbers of effectors,such as XopR,XopAE,XopZ,XopK etc,have been studied.According to existing studies,XopY is conservatively present in X.oryzae,but it displays diversity in sequences and functions.Deletion of XopY has been shown not to affect the virulence of Xoo Philippines strain PXO99A in rice IR24.The XopY from Japanese strain MAFF3110018 can suppress Chitin-induced activation of MAPK kinase by inhibiting OsCERKl-mediated phosphorylation of OsRLCK185,which affects the immune response of rice.XopY in the X.oryzae pv.oryzicola strain RS105 displays the function of inducing the hypersensitive response(HR)of Nicotiana benthamiana.In our study,the constructed phylogenetic tree of XopY revealed that Xoc strains clustred into a small branch,which were related to some African strains.The variations of XopY among Xoo strains were large.Even strains in the same region,such as different Philippines strains or African strains,are scattered in different branches.In order to understand the function of XopY in PXO86,we carried out the following studies:The PXO86ΔXopY mutant was generated based on homologous recombination double exchange strategy.To examined its pathogenicity,we inoculated with multiple rice varieties,and found that the virulence was significantly reduced compared to wild type strain on rice varieties IRBB1,IRBB3 and IRBB11.This defect was restored to wild type by the complementation of Xop Y into the mutant on rice IRBB11,which indicated that Xop Y was required for the full virulence of PXO86.The growth rate,the expansion ability in susceptible rice,the expression of susceptible gene OsSWEET14 and the effect on T3SS transport function of PXO86ΔXopY and PXO86 were further determined.We found that the absence of XopY did not impact the growth in rich medium and the ability to expand in rice,but it influenced the multiplication of the pathogen in rice.The absence of XopY did not affect the T3SS transporting function of the strain,however,the expression of OsSWEET14 was significantly suppressed after inoculation of mutant strain in the rice IRBB11.Then we tested the effect of XopY on PXO86-induced HR,reactive oxygen species(ROS)burst and cell membrane damage on non-host plant N.benthamiana,and also studied the function of XopY when it was transiently expressed.The result revealed that XopY was involved in the positive regulation of PXO86-induced cell death and ROS burst.The degree of membrane damage of XopY-deficient mutant was significantly reduced.Transiently expression of XopY protein could not stimulate the allergic reaction,but XopY had the ability to induce ROS generation,and significantly inhibited the pathogenicity of Phytophthora parasitica on N.benthamiana.These results indicate that XopY can induce basic immune responses in plants.To sum up,during the infection of PXO86 in rice,XopY may directly or indirectly affect the expression of the susceptible gene OsSWEET14,which regulates propagation of pathogenic bacteria by affecting the sugar conversion and thus participate in the full virulence function of PXO86.At the same time,XopY can induce basic immune in non-hosts.