A Study On The Defense Signaling Of Blast Resistance Genes PID3 And Pi9 In Rice(Oryza Sativa)

Resistance(R)genes are important components in plant innate immune system and thus considered as the crucial genetic sources for crop breeding.Most of the R genes encode nucleotide-binding,leucine-ric hrepeat domain-containing(NLR)proteins,which can perceive pathogenderived effectors and then trigger strong immune responses to limit the growth of the invading pathogens.The molecular basis of R gene-triggered immunity is one of the most significant areas in plant biology research.However,the signaling pathways for R genes have not been well understood.Rice(Oryza sativa)is one of the most important staple crops around the world.Rice blast disease,caused by the fungus Magnaporthe oryzae,is one of the most devastating diseases of rice.To date,more than 20 blast R genes have been successfully cloned in rice,most of which encode CCNLR(CNL)proteins.In fact,the rice-M.oryzae pathosystem has become an advantageous model for studying defense signaling of R genes in plant.A previous research showed that the R gene,PID3-mediated rice blast resistance requires both OsRac1,a known small GTPase,and RAI1(Rac immunity1),a b HLH(basic helix-loop-helix)transcription factor.Besides,OsRac1 as well as Os SPK1,a guanine nucleotide exchange factor(GEF)for OsRac1,has been reported to contribute to the immune responses mediated by two other R genes,Pit and Pia.In this study,to explore a conservative signaling pathway for rice R genes,we examined the roles of Os SPK1,OsRac1 and RAI1 in rice blast resistance conferred by two R genes PID3 and Pi9,which are different in resistance spectrum.We also initially investigated the molecular mechanism of RAI1 expression regulation.Our main results are as follows.1)We first generated Os SPK1 knock-down mutants in the PID3-TL background and confirmed that Os SPK1 is also genetically required for blast resistance mediated by PID3.In rice protoplasts,PID3-OsRac1-induced cell death was found partially rescued by the GEF inhibitor,suggesting that Os SPK1 contributes to OsRac1 functioning downstream of PID3 in the transient assay.And yeast two-hybridization(Y2H)assay further showed a physical interaction between PID3 and Os SPK1.These findings together with the previous research suggest that Os SPK1,OsRac1 and RAI1 constitute a major signaling pathway for PID3-mediated resistance against rice blast fungi.2)To examine whether other R genes also use the above signaling routine to convey defense signals,we chose Pi9,another R gene known to have a broad spectrum of blast resistance but differ greatly in amino acid sequences from PID3 or Pit.We also created Os SPK1 and OsRac1 knockdown lines in the Pi9-TL background,respectively,and showed that the blast resistance conferred by Pi9 is also genetically dependent on both Os SPK1 and OsRac1.Besides,two separate assays by RNA interference and chimeric repressor mutation confirmed the importance of RAI1 to Pi9-mediated resistance.And immunoblotting analysis showed that compared with those in the blast susceptible variety ‘TP309',RAI1 proteins in Pi9-TL accumulated more quickly in the early infection stage and kept at higher levels in the late stage,demonstrating that Pi9 functions possibly through regulating RAI1 levels.Furthermore,Y2 H and bimolecular fluorescence complementation assays confirmed that RAI1 interacts with Pi9 in the nucleus.Autoactivation assay in yeast showed that the N-terminal amino acids are critical for transactivation activity of RAI1,which was further confirmed by dual-luciferase reporter assay system.Overall,Os SPK1,OsRac1 and RAI1 play roles both in PID3-and Pi9-mediated resistance,which prompts us to believe that these three modules may serve as a quite conservative downstream signaling pathway for R genes conferring blast resistance in rice.3)Given its transactivation activity and regulatory roles in resistance induced by R genes,we wonder how RAI1 is regulated in vivo.We successfully identified OsRPT2a(regulatory particle AAA+ ATPase 2a)as an interactor of RAI1 in a Y2 H screen of rice c DNA library.Os RPT2 a is a subunit of the 19 S regulatory particle of 26 S proteasome in rice.Y2 H assay confirmed that the C-terminal region is important for Os RPT2 a binding to RAI1.Os RPT2 a was also proved to possess ATP hydrolysis activity in vitro.And a statistic analysis based on rice protoplasts showed that Os RPT2 a predominantly distributes in an aggregation manner inside the lumen of tonoplast and endoplasmic reticulum,and moderately in both the nucleus and cytoplasm.Besides,the subcellular localization of Os RPT2 a is partially dependent on its second conserved glycine residue at the N-terminus.Loss of Os RPT2 a function led to reduced resistance against rice blast fungi,suggesting the positive role of Os RPT2 a in plant immunity.Furthermore,co-expression experiments with rice protoplasts revealed that RAI1 levels were significantly suppressed in presence of Os RPT2 a.However,this was partially rescued by an Os RPT2 a variant losing the ATPase activity or proteasome inhibitor,which suggests that Os RPT2 a leads to the protein degradation of RAI1 in an ATPase and proteasome-dependent manner.We thus considered that Os RPT2 a mediates RAI1's protein degradation and plays roles in plant immunity.Taken together,we found a conservative signaling pathway for rice blast R genes and also initially explore the molecular basis of RAI1 expression regulation.These findings contribute to further understanding the defense mechanism mediated by R genes and provide guidance for disease control in production practice.