Study On The Rice Receptor Of Chitinase And Serine Protease From Magnaporthe Oryzae

The rice blast fungus, Magnaporthe oryaze, infects many economically important cereal crops, particularly rice. The interaction between M. oryzae and rice is also taken as a model for the study of fungus-plant interaction. It has been shown that the fungus could secrete chitinase and serine protease in the process of its host infection. Our previous studies indicated that chitinase and serine protease in M. oryaze are probably important pathogenic factors. However, their regulation mechanism in the process of blast fungus infection remains unclear, particularly in the regulation of pathogen-associated molecular pattern (PAMP)-induced immunity reaction. In this study, we characterized the relationship between the chitinase/serine protease in M. oryzae and their receptors in rice by the yeast two-hybrid assay, immunology and molecular genetics approach by creating the rice RNAi mutants of putative receptors.By yeast two-hybrid screening, we obtained 18 rice positive clones interacting with M. oryzae chitinase (MoChi1) and 12 rice positive clones interacting with M. oryzae serine protease (MoSP1). These clones were then sequenced and annotated by BLAST search. Interestingly, we found a common receptor, mannose-binding lectin (OsMbl1), which could interact with both MoChi1 and MoSP1.GST pull-down and Western bloting were then to confirm interaction between above candidates and MoChi1 and MoSP1. Results showed that there is interaction between the mannose-binding lectin, metallothionein-like protein (OsMtl1) and MoChi. And there was an obvious interactrion between OsMbl1, OsZfp1 and MoSP1. We then further dissected the binding properties of a protein-protein interaction domain between OsMbl1 and MoChi1 and MoSP1. First, bioinformatics analysis showed that OsMBL1 contains mannose-binding lectin domain, including three sugar-binding sites. Homology analysis showed that OsMbl1 is about 50% identical to other mannose-binding lectin from Monocotyledon, such as garlic, dendrobium and crinum. And it has conserved amino acid binding sites of mannose-binding lectin of Monocotyledon. In order to clarify the interaction domain of the smallest fragment between OsMbl1 and MoChi1 and MoSP1, we constructed the different mutants of OsMBL1, then detected the interaction by GST Pull-down assay. The results showed that the three binding sites, G…..GXXXD,GXGXXXEDE and GX[GAVIYWF] [DNEW], are independent for each other during their interaction with MoChi1. Whereas, the three binding sites are dependent for each other during their interaction with MoSP1.We also analyzed the interaction domain of another receptor OsZfp1 with MoSP1 in M. oryzae. Bioinformatics analysis indicated that the OsZFP1 gene contains a C3HC4 conserved domain, two c-terminal transmembrane helix sequence, which coding andα/β-type, non-secreted protein. Domain deletion and GST Pull-down assay demonstrated that N-terminal and C-terminal of this protein is not necessary for its interaction with the serine protease, while only C3HC4 domain is required for the interaction.The expression of OsZFP1, A11and OsMBL1 mRNA in the rice leaves which were infected by Guy11 or the knockout mutant of MoChi1 and MoSP1 were examined by the Real-Time PCR. The results showed the expression of OsMBL1 mRNA in the rice leaves infected by the knockout mutant of serine protease was down-regulated before 72 h compared with that in the Guy11-infected rice leaves,the expression of OsZFP1 was also down-regulated. However, the expression of A11 mRNA had no difference in all mutants. The expression of OsMBL1 mRNA in the rice leaves infected by the knockout mutant of MoChi1 was down-regulated compared with that in the Guy11-infected rice leaves., which is consistent with the results above.Besides above results,we also plan to analyze the biological functions of OsZFP1 an OsMBL1 through RNAi and overexpression in the rice. We have obtained RNAi and overexpression constructs of these two genes and the transgenic rice of RNAi-OsZFP1 and RNAi-OsMBL1. The lines of overexpression-OsZFP1 and overexpression -OsMBL1 were not obtained yet. Therefore, more detailed analysis of the transgenic lines generated from this study are necessary for further identification the function of these genes in rice.