Screening And Identification Of Effectors Associated With Pathogenicity In Ustilaginoidea Virens

Ustilaginoidea virens(Cooke)Takah causes rice false smut(RFS),one of most devastating fungal diseases in rice.RFS not only causes significant losses in rice yield and quality,but also produces mycotoxins that are toxic to human and animals.Currently,many studies have been performed on the infection cycle of U.virens and mycotoxin identification.However,little is known on the functions of U.virens effectors and molecular mechanisms underlying its pathogenicity.Therefore,this study focused on screening,identification and function analysis of U.virens effectors that induce or suppress plant immunity.Firstly,13 out of 119 putative effectors were demonstrated to cause necrosis or necrosis-like phenotypes in N.benthamiana.The signal peptides of eleven proteins among them were confirmed to have the function to guide secretion using a yeast secretion system and the corresponding genes are all highly induced during infection except GUV₁975 and GUV₇139.Eight secreted proteins were proven to trigger cell death or defenses in rice protoplasts and the secretion signal of these proteins is essential for their cell death-inducing activity.Subsequently,it was revealed that the ability of GUV₇139 and GUV₆759 to trigger cell death is dependent on the predicted serine peptidase active sites(Asp143,His175 and Ser330)and ribonuclease catalytic active sites(His60,Glu79 and His113),respectively.Furthermore,we demonstrated that GUV₂141 and GUV₆759 are N-glycosylated proteins,which glycosylation has different impacts on their abilities to induce cell death.Secondly,42 out of 93 putative effectors in U.virens were proved to inhibit cell death triggered by Burkholderia glumae at different levels in N.benthamiana.Among them,19 and 2 putative effectors suppressed cell death induced by BAX and INF1,respectively,when these effectors were co-expressed with BAX and INF1 in N.benthamiana.When BAX and INF1 were expressed at 6 h after expressing effectors,31 and 24 putative effectors can suppress cell death induced by BAX and INF1,respectively.These results suggest that the majority of putative effectors in U.virens are associated with the modulation of plant immunity.Finally,the function assay of putative effector SCRE2 was performed.We verified that SCRE2 is a secreted protein,and can be delivered inside plant cells.SCRE2 is conserved in 31 U.virens isolates collected from different regions in China.The gene was highly induced during the early stage of U.virens infection.Furthermore,we found that the 68-85th amino acid residues in SCRE2 are the shortest functional region for inhibiting cell death and oxidative burst induced by BAX.More importantly,SCRE2 knockout in the P1 isolate attenuated U.virens pathogenicity in rice.Surprisingly,heterologous expression of SCRE2 can induce PR genes expression and MAPK activation,and enhance resistance to Xanthomonas oryzae pv.oryzae RS105 in rice.This phenomenon is perhaps an artifact caused by over-expression of pathogen effectors in rice.It needs to be explored and verified further.In addition,SCRE2 can target the RNase T2 family members,including RNS1-RNS5.The RNS1-RNS5 proteins can also interact with each other and form a complex network.RNS1 and RNS2 were verified to have RNase activities in rice protoplasts.Moreover,RNS2,RNS4 and RNS5 can suppress cell death triggered by BAX;RNS2,RNS3,RNS4 and RNS5 also suppress cell death triggered by INF1 in N.benthamiana.In summary,11 putative effectors were demonstrated to induce cell death in N.benthamiana.A group of 42 putative effectors inhibiting plant cell death were also identified,which provides a solid foundation for future researches on U.virens pathogenesis.We further identified a virulence effector SCRE2,and preliminarily explored its functions and interacting proteins.These researches lay a solid foundation for the follow-up studies on protein-protein interaction between SCRE2 and its targets.