The Roles Of The Type Ⅲ Effector XopAY In The Interaction Between Xanthomonas Campestris And Plants

In order to cope with divesre enviroments,plants have evolved to have natural barriers against pathogens,such as hard cell walls,low p H values in apoplasts,self-secreted enzymes or antibacterial component.Moreover,hey have also evolved sophisticated innate immune system: induced by pathogen-related molecular characteristics(PAMPs)triggered immunity(PTI)and effector factors triggered immunity(ETI).PTI and ETI are two different levels of plant immunity,but they are closely related.For example,their receptors usually appear in the form of complexes and share some common downstream signal components 。 Plant pathogenic bacteria secrete a large number of effector proteins(Type Ⅲ Secretion Effectors,T3SEs)into plant cells through Type Ⅲ secretory system(T3SS),manipulate plant cell functions and cause plant diseases.Xanthomonas campestris pv.campestris(Xcc)is a gram-negative bacterium that can cause black rot in cruciferous plants worldwide,including cabbage and model plant Arabidopsis.With the available whole genome sequence of Xcc strains,the Arabidopsis-Xcc model system has increasingly become an effective tool for the study of plant-pathogen interactions.XopAY was predicated as a T3 SE and thus may be important to Xcc infection.In this study,we analyzed the roles of XopAY in the process of Xcc-plant interaction by genetic and biochemical analysis.Analysis showed xopAY promoter sequence contained a typical plant-inducible promoter box that can be bound by Hrp X(a key regulator for T3 SS and T3 SE genes’ expression),indicating it may be regulated by Hrp X.Furthermore,the XopAY had typical motifs that are in effector proteins,suggesting it is a predicted T3 SE.xopAY mutation significantly reduced the virulence of Xcc on cruciferous plants including cabbages and Arabidopsis,implying that XopAY is a necessary cell component for Xcc infection.We used yeast two-hybrid assays(Y2H)to screen the Arabidopsis thaliana c DNA library to identify the XopAY-interacting proteins.Two proteins,cytosolic invertase1(CINV1)and CINV2 were screened.Y2 H point-to-point analysis showed that XopAY can actually interact with CINV1/CINV2 in yeast cells.These interactions were confirmed by in vitro pull-down assays and bimolecular fluorescence complementary experiments(Bi FC),inlustrating that XopAY can interact with CINV1/CINV2 and might affect their enzymatic activities.To this end,we performed invertase activity assays.The enzyme activities showed that CINV1 and CINV2 have sucrose invertase activities and XopAY acts as an enhancer of CINV1 and CINV2 enzyme activities.