Functional Primary Research Of Effectors Of Rice Seath Blight Disease Pathogen

Effecctors are a protein family that manipulate host innate immunity and strengthen parasitic infections. With the development research of phytopathogen, the research of effectors has become the priority of pathogen-host interaction study that the plant pathologists have spared no effort on. With the development of bioinformatics, the function of increasing phytopathogen effectors is being revealed. However, so far, research on effectors of rice sheath blight disease pathogen has not been reported. This paper, basead on whole genome sequenceing for assembly and prediction, carries on the study of the biological function verification and subcellular location of effectors of rice sheath blight disease pathogen to provide theoretical foundation for deep understanding of molecular mechanism of effectors and the rice host interaction.This reaserch maily foucused on the four poins behind:Firstly, clone of candidate effectors. We were based on our laboratory effort of whole genome sequenceing of Rhizoctonia solani AG1IA and predicting candidate genes coding effectors. Some primers were designed based on predicted sequences of the genes, then we got the whole gene sequence by RT-PCR technique and comfirmed the correctness of the fragment by Sanger dideoxy sequencing.Secondly, the research of programmed cell death (PCD) triggered by effcctors and host specificity (HST). To obtain a mass of crude protein of candidate effectors, we made use of heterologous expression and disrupted the bacterium by ultrasonic cell disruption system. The leaves of rice and maize were infected by the crude protein to preliminarily authenticate whether the proteins have the function of PCD and HST of effectors. Nine of these proteins programmed by the candidate genes had been identified as effectors.We used pure protein got by protein purification technology to reconfirm the PCD phenomenon.We chosed two effector proteins to lucubrate in follow-up study.Thirdly, subcellular localization of effectors in the protoplst of rice. In order to affirm the target protein of the effector in rice plant, the sequence of the gene was merged together with the sequence of yellow florecent protein. The protein were fused N-end of yellow lorecent protein to avoid affecting the location the effector itself. The fusion protein were expressed in the protoplast of rice, Confocal laser scanning microscopy indicated that the protein possibly locate in the mitochondria. Whether it really orientates in the mitochondria, further mitochondria marker gene examination is needed.Fourthly, mutation and analysis of key residues of domains of effectors. In order to confirm the functional sites of the effector, we intend to carry out sit-directed mutagenesis to hydrophobic amino acid of function domain. First, we deleted the signal peptide of the effector, cloned the remainder and implemented pathogenic identification to confirm whether the signal peptide is necessary for pathogenicity. Then, the large segment of the function domain were deleted and the remainder were cloned to confirm the protein have the function of effectors and the function domain. In our experiments the key amino acid had been predicted and the corresponding mutant had also been constructed, the expressing validation are still in progress.Our study based on the whole genome sequencing and prediction of secreted proteins and effectors of R. solani AG1IA in our laboratory, preliminary confirmed two novel effector genes, respectively called AG1IA005473and AG1IA008560(applied for patent).Acording to prediction and analysis, N-end of AG1IA005473coding protein has a signal peptide, which can guide the protein outside of the cell and C-end has a CtaG-Cox11domain locating at mitochondria or the chloroplast, which has a function of cytochrome c oxidase assembly protein. Through the usage of fluorescence confocal scanning microscope, we are initially determined that the effector is located in the mitochondria of rice. N-end of AG1IA008560coding protein also has a signal peptide and C-end has a domain of serine protease. Protein coded by AG1IA008560is initially determined located in the nucleus of rice leaves. The subsequent studies about gene knockout, complementation, affirmation of receptor in rice and signal pathway triggering rice pathogenesis are under way.In conclusion, we made it roughly clear that the protein had a function of effector and its target cell organelle provding evidence of molecular level for interaction mecnasim of rice sheath blight disease pathogens and rice. Futher studies are in progress.