Interaction Between Rice Blast Resistance Gene Pik And Its Corresponding Avirulent Gene AvrPik Based On Y2H

Rice(Oryza sativa L.)is one of the most widely consumed food crops.Rice blast,caused by the ascomycete fungus Magnaporthe oryzae,is one of the most devastating diseases in rice production.Exploitation and reasonable distribution of resistant cultivars is the most effective and environmentally friendly method to control the disease.Because it is in accordance with the classic theory “gene for gene”,the rice blast pathosystem has been a model system for pathogen-host interactions in recent years.The blast resistance(R)gene is a key factor in this system,therefore the study of it can help to reveal the molecular mechanisms of the interaction between the host plant and the fungus pathogen.The Pik locus is located on the end of the long arm of rice chromosome 11,it harbours at least 5 blast R genes(Pik,Pik-m,Pik-s,Pik-p and Pik-h),which were extremely important for blast resistance and rice breeding programmes.Except Pik-s,the remaining 4 alleles have been successfully cloned.In order to reveal the interaction molecular mechanisms of Pik gene family,some work about Pik and Pik-p was launched in this study based on yeast two-hybrid(Y2H)system.The main contents and results are as follow:1.Identification of active motifs of CC domains of Pik and Pik-p resistance proteinsInteractions between the full length and CC domain proteins of 5 alleles and a series of Avrpik mutations were detected by yeast two-hybrid system.The data showed that there was no difference on the protein activity between the full length and CC domain.Therefore,the CC domain was truncated to explore its active motifs for better understanding the molecular mechanisms.The CC domains were divided into 4 parts based on the secondary structure,CC(1-97),CC(98-130),CC(131-190)and CC(191-264).Then their activity was investigated through the Y2 H system.The data showed that the active motifs of CC domain of Pik-1 were Pik-1CC(1-97)and Pik-1CC(191-264),but Pikp-1CC(1-97),Pikp-1CC(131-190)and Pikp-1CC(191-264)were active motifs in the region of CC domain of Pikp-1.Among them,the sequence of Pik-1CC(1-97)was identical with Pikp-1CC(1-97).2.Identification of the minimal active motif of CC domainsThe motifs were truncated to detect the protein activity based on yeast two-hybrid system,thus the minimal active motif interacting with Avr Pik will be revealed.The results found that the minimal active motif of Pik-1/Pikp-1CC(1-97)was Pik-1/Pikp-1CC(1-20),then Pikp-1CC(131-138),Pik-1CC(191-200)and Pikp-1CC(191-200),respectively.3.Identification of the specific positions of CC domainsAs there were many SNP loci in CC domain between Pik-1 and Pikp-1,in order to determine whether these sites will affect the activity of resistance proteins,these sites were mutated to other side amino acids to detecte the interactions activity.There were 11 SNP loci in the motif Pikp-1CC(131-190),specific primers were designed to mutate to the corresponding sites in the motif of Pik-1CC(131-190).The data showed that the mutations of 3 sites changed their proteins activity,they were M156 K,K161E and K184 I.Then the other side mutations were done,and only I184 K caused specific interactions.In the same way,the specific positions in motifs Pik-1CC(191-264)and Pikp-1CC(191-264)were V261 A,K262N,E231 V,D252H and K263 E.4.Sub-cellular localization of active motifs and their minimal active motifs of CC domainsSub-cellular localization experiment was done to determine the location of active motifs and their minimal active motifs of CC domains.The results showed that these proteins distributed in both the cytoplasm and the nucleus evenly.Then we presumed that the interactions between active proteins and the corresponding effectors maybe exist in both the cytoplasm and the nucleus.