?,the Molecular Mechanism Of The Autoimmunity Mediated By Coiled-Coil Domain Of Rice Blast Protein Pi36 ?,the Regulation Between Arabidopsis Resistance Protein RPW8.1 And Ethylene Signaling

Part One:The molecular mechanism of the autoimmunity mediated by coiled-coil domain of rice blast protein Pi36Plants have evolved structurally conserved resistance (R) proteins containing a N-terminal coiled-coil (CC) domain, a central nucleotide-binding (NB) domain and C-terminal leucine-rich repeats (LRRs). The CC domains of some CC-NB-LRR proteins can trigger autoimmunity in the model plant Arabidopsis. The majority of the rice blast R genes encode CC-NB-LRR type proteins. However, whether any CC domains of these proteins can trigger autoimmunity is unclear. Here, we successfully amplified the fragments encoding the CC domains of eight rice blast R genes, including Pi36, RGA4, RGA5, Pikm-1, Pikm-2, Pish, Pita and Pb1. Transient expressing assay demonstrated that only the CC domain alone of Pi36 is sufficient to trigger cell death in Nicotiana benthamiana and in rice protoplasts. Yeast-two-hybridization and bi-molecular fluorescent complementary assay demonstrated that self-association of the Pi36 CC domain is required for activation of cell death. Subsequently, ectopic expression of Pi36 CC domain from the promoter of Resistance to Powdery Mildew8.2 (RPW8.2) led to enhanced resistance to powdery mildew in Arabidopsis and from the promoter of Probenazole-Induced Protein1 (PBZ1) resulted in enhanced resistance to the rice blast fungus Magnaporthe oryzae in rice. Through yeast two hybrid screening, we isolated a Pi36 CC domain interactor OsEIL2, a ethylene signaling pathway transcription factor, and confirm by BiFC. We generated OsEIL2 overexpression and knockdown rice transgenic lines, disease assay demonstrated that OsEIL2 positively regulate the resistance to rice blast.Taken together, our data demonstrate that the CC domain of the rice blast resistance protein Pi36 can trigger autoimmunity and its ectopic expression confers disease resistance in Arabidopsis and rice, Pi36 CC domain interact with OsEIL2 which positively regulate resistance to rice blast. Therefore, this work demonstrates a potential way to engineer disease-resistant rice by expressing a single CC domain of certain rice resistance protein from pathogen inducible promoters.Part Two:The regulation between Arabidopsis resistance protein RPW8.1 and ethylene signalingArabidopsis broad spectrum resistance protein RPW8.1 is an unique R protein and just contain a transmembrane domain and a CCR domain. Previous study show the Arabidopsis transgenic line R1Y4 that ectopic expression of the yellow fluorescent protein (YFP)-tagged RPW8.1 from the native promoter leads to unique cell death lesions and enhances resistance to virulent fungal and oomycete pathogens that cause powdery mildew and downy mildew diseases, respectively. However, whether the molecular mechanisms of PCD and resistance mediated by RPW8.1 is still unclear. Through yeast two hybrid screening, we isolated an RPW8.1 interactor AC04, an ace oxidase, and confirm by BiFC. We data demonstrate that RPW8.1 influence ethylene biosynthesis and activate ethylene signal response. Then, we get P1Y4 x EIN3 OE? P1Y4 x ein3/eill ? P1Y4 x aco2/4 through hybridization and demonstrate that EIN3/EIL1 negatively regulate RPW8.1-mediated cell death and disease resistance.Taken together, our data demonstrate that RPW8.1 interact with AC04 and influence ethylene biosynthesis and activate ethylene signal response. Meanwhile, ethylene signaling negatively regulate RPW8.1-mediated cell death and disease resistance. Therefore, this work demonstrates a feedback loop between RPW8.1 and ET signaling.