Clone And Functional Prediction Of A Novel Blast-resistant Genes In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is one of the most important crops,which feed half of worldwide populations.However,pathogens and pests are severe stress conditions for rice plant growing.Rice blast disease induced by blast fungus is one of the most serious rice plant diseases,which can reduce rice yield by 11.0%-30.0% or even more.The most effective method to control rice blast is to develop new broad-spectrum rice varieties.Searching new resistant genes successfully and cultivating resistant varieties rapidly are common method in Rice Genetics and Breeding.Moreover,due to the frequent mutations of rice blast pathogens in the field,the newly blast resistant rice varieties might be weaken or lose their resistance to rice blast after several years of application.Therefore,the discovery of new broad-spectrum blast-resistant genes in rice is a critical research for the development of new blast-resistant rice varieties.In our previous study,we obtained a novel potential blast resistant gene in rice,OsHMA10,by genomic re-sequencing and comparative genomics analyzing of a set of rice Near-Isogenic lines(NILs).In this research,Nipponbare,the NIL Pi4 b and its parent,blast-susceptible rice variety CO39 were used as tested varieties.First,CRISPR / Cas9 and overexpression techniques were used to analyze the function of OsHMA10.Second,the techniques of subcellular localization,gene co-expression and protein interaction prediction were applied to reveal the blast-resistant mechanism of OsHMA10 gene.The main results were summarized at below.1.Knockout mutant of OsHMA10 gene was developed by CRISPR/Cas9 technology.Seven days after infected by blast fungus GUY11,the lesion area of the mutant was found to be larger than the wild type Pi4 b,indicating that the mutant became susceptible to blast fungus GUY11.Meanwhile,the overexpression mutant of OsHMA10 gene in Nipponbare was found to resistant to blast fungus GUY11 infection.The results showed that OsHMA10 was a new blast-resistant gene in rice.In the results of GO-analyzing,GTP-binding and GTPase activity were significantly enriched in terms molecular function GO terms of OsHMA10 co-expressing genes.As NBS-LRR genes participating in blast-resistant signal transduction through NBS domain binding to GTP or ATP,OsHMA10 gene had an HMA domain which could serve as an integration domain between immune recognition receptor proteins.Through the integration of Pikp-1 and HMA domain,Pikp-1 and Pikp-2 were able to identify the effector of rice blast fungus which induced hypersensitivity reaction and inhibit the invasion of rice blast fungus.HMA domain of OsHMA10 is highly similar to that of pikp-1.The protein interaction network prediction of OsHMA10 gene in rice found that the protein encoded by OsHMA10 gene interacted with the transcription factor GRAS56 and the peroxidase prx125.GRAS family members are involved in the response of rice to blast fungus infection,while peroxidase plays an important role in resisting rice blast fungus infection by regulating ROS metabolism in cells.Therefore,it could be inferred that OsHMA10 gene recognized the effector of Magnaporthe grisea through its HMA domain,co-expressed with GTP pathway genes to transmit the blast fungus infection signal,and then removed intracellular free radicals to protect rice plant cells through protein-protein interaction.2.It was found that the stalk of OsHMA10 gene Cas9 mutant was easy to break,which was induced by the lower thickness of cell wall and the lower content of cellulose.The cell wall in the mutant was thinner.The protein interaction network prediction showed that most of the co-expressed genes with OsHMA10 belonged to the cell composition(Golgi membrane and cell wall synthesis).Whilst,the prediction of the protein interaction network also found that the protein encoded by OsHMA10 genes could interact with GTP-activating enzymes AGD9 and ADP ribosylation factor(RAF)ARF,which regulated the transport of substances on the Golgi apparatus.These results indicated that OsHMA10 gene affected cellulose synthesis and cell wall thickness by regulating cellulose synthase on the Golgi apparatus and cell membranes.In conclusion,the OsHMA10 gene in rice was a novel blast-resistant gene.Its HMA domain might combine with one or more immune recognition receptor proteins then conducted the stress signal and eliminated the intracellular free radicals to resist blast fungus infection.The OsHMA10 gene could also affect the cell wall thickness by affecting the polysaccharides synthesis of non-cellulosic in the Golgi apparatus and cellulose in the cell membrane,thereby affecting the mechanical strength of rice stalks.Based on the principle that rice blast fungus invades rice,the thickening of the cell wall can resist the invasion of a variety of rice blast fungus,so the OsHMA10 gene may be a broad-spectrum disease-resistant gene,which is a direction for future research.In this study,the disease resistance of OsHMA10 and its molecular mechanism affecting cell wall synthesis were preliminarily revealed,which would provide theoretical guidance for the breeding of new blast resistant rice varieties.