Transcriptome Analysis Of Pid3Mediated Rice Blast Resistance

Rice blast, is the most disastrous fungal disease which caused by the filamentous ascomycete Magnaporthe oryzae (M. oryzae), and resluted in drastically damage of rice production. The cloning and using of resistance (R) genes to enhance the resistance of rice is most significant both in plant immunity study and application. So far,24rice blast R genes have been cloned and characterized, and almost all of which belong to the nucleotide binding-site leucine-rich repeat (NBS-LRR) family. Pid3is a typical CC-NBS-LRR gene cloned in our lab from the indica variety DiguB by performing a genome-wide comparison of paired NBS-LRR genes and their pseudogene alleles between two rice genome-sequenced varieties,9311(indica) and Nipponbare (japonica). Keep on studing in this direction is meanningful for elucidation the mechanism of rice blasr resistance mediated by the NBS-LRR type R genes in rice.Since the whole genome sequences of rice and M. oryzae have been known already, in the prupose of elucidation of the mechanism of Pid3mediated blast resistance and the process of blast fungus infection, we could use the emerging RNA-Seq technique to analysis the mixed transcriptomes of Pid3-containning transgenic rice and blast fungus in the blast infected rice leaves at the early stage within24hours (hpi) after blast innoculatio, when the primary infection hyphae penetrate leaf epidermal cells. In rice transcriptome,2887and4969differentially expressed gene were identified in resistance and susceptible reactions respectively. These differentally expressed genes were enriched in the GO terms of response to biotic stress, photosynthesis, metabolic process, etc. And on the other hand, these genes were enriched in such pathways as biosynthesis of secondary metabolites, alpha-linolenic acid metabolism, plant-pathogen interaction, etc. Further, we discovered alpha-linolenic acid metabolism which participates in jasmonate acid (JA) synthesis was first enriched at8hpi, and diterpenoid biosynthesis which participates in phytoalexin (PA) synthesis was enriched at16hpi. These enrichments suggests that JA might regulate PA synthesis to play roles in rice blast resistance. By analyzing the expression patten of JA and PA synthesis genes, WRKY transcription factors and Pathogenesis-related (PR) genes both in Pid3resistance and susceptible reactions, we also found that Pid3has the ability to stablized the expression of these immunity related elements in resistance reaction, which mingt distinguish Pid3mediated resistance (ETI) from basic immunity (PTI) in the early stage of blast resistance ractions.For further elucidate the mechanism of NBS-LRR gene mediated rice blast resistance, another NBS-LRR gene, Pi9, was used in the same RNA-seq analysis. In comparison of Pid3and Pi9respectively mediated blast resistance transcriptoms, we found that the immunities triggered by these two R genes were similar in GO terms, such as response to biotic stress, photosynthesis, metabolic process, alpha-linolenic acid metabolism, plant-pathogen interaction, Kinase and Catalytic activity. In addition, the immunity related elements were also stable expressed in Pi9triggered blast resistance reaction. These results suggest that the resposes of the two NBS-LRR type blast resistance genes to the same blast isolate may share similar ETI reations.In the meantime, we also extracted and analysized M. oryzae transcriptome from the the mixed transcriptomes, a total of8875expressing genes were identified in all the reactions, including837genes which encode secretory proteins. GO enrichment showed that these secretory protein encoding genes were similar to those in bacterial type II secretion system, suggesting that this type of M. oryzae secretion system might play roles in the blast infection stage. Additionally,653genes including78secretory protein coding genes were found specific expressed in the rice susceptible reactions, thus they might participate in the blast pathogenity. Besides, based on above mentiond rice transcriptome analysis results, we found a Bowman-Birk type bran trypsin inhibitors (BBTI) coding gene, named as BBTI4, which was induced by blast fungus in both Pid3and Pi9transgenic plants. The BBTI4protein could be bind with and phosphorated by protein PID2encoded by another rice blast resistance gene Pid2. BBTI4could also be phosphorated by rice bacterial blight resistance protein XA21encoded by Xa21. We then conducted its functional study with BBTI4transgnic rice.