Chloroplastic Protein OsTHF1 Affects NLR Protein Pid3-mediated Rice Blast Resistance

Rice blast is a worldwide rice disease which poses a threat to the world food security.The abuse of pesticides can control rice blast to some extent,but it also brings resource and environment problems.The research on the pathogenic mechanism of rice blast and the disease resistance mechanism of rice will provide the theoretical basis for the breeding of varieties resistant to rice blast.Pid3 is a typical NLR gene,the gene and its allele showed wide resistance to the species of rice blast fungus in the rice area of southwest China,suggesting its application value in rice molecular breeding.Therefore,it is of both theoretical and practical value to explore the working mechanism of Pid3.In a previous study,Yeast two hybridization was used to screen a cDNA library constructed from rice leaves inoculated with rice blast to search for proteins that interact with Pid3 during disease resistance.Using the CC(1-157aa)domain of Pid3 as the decoy protein,we screened a nucleo-encoded chloroplast protein OsTHF1,OsTHF1 is selected for the study of this thesis,Based on the interaction with Pid3-CC,the role of OsTHF1 in Pid3-mediated rice blast resistance was preliminarily identified by inoculation of corresponding transgenic materials.The main results are described as follows:1.Based on subcellular localization experiments,it was found that rice OsTHF1 protein localizes to the chloroplast of rice,and the specific expression of OsTHF1 protein in the chloroplast was further determined by western blot.2.Based on Y2 H,BiFC and LUC experiments,the interaction between OsTHF1 and Pid3-CC was determined.OsTHF1 interacted with Pid3-CC through its CC domain.BiFC experiments found that the interaction position of OsTHF1 and Pid3-CC were no longer in the nucleus or chloroplast.3.It was found that Pid3-CC could induce cell death by agrobacterium-mediated transient expression assay and cell survival assay using rice protoplast.However,co-transduction of Pid3-CC and OsTHF1 showed that OsTHF1 suppresses cell death induced by Pid3-CC.4.RT-qPCR detected the transcriptional level changes of OsTHF1 in Pid3-OX /TP309 strains within a few days after inoculation,and found that the expression of OsTHF1 was induced.5.OsTHF1-RNAi/Pid3-OX transgenic lines were constructed,RT-qPCR and western blot were used to identify whether the lines construct were successful.Through a series of pathological experiments,it was found that OsTHF1-knockdown lines impaired Pid3-mediated resistance to rice blast,indicating that OsTHF1 is required for Pid3-mediated resistance to rice blast.6.By DAB staining,it was found that there were differences in the accumulation of ROS Around appressorium of rice blast,and TP309 susceptible varieties produced almost no ROS,while the ROS of the transgenic OsTHF1-RNAi/Pid3-OX lines were significantly impaired compared with that of Pid3-OX strains,suggesting that OsTHF1 may be involved in Pid3-mediated resistance by regulating ROS production.The above results proved that OsTHF1 physically interacts with Pid3-CC,and OsTHF1 is involved in Pid3-mediated rice blast resistance and positively regulates the resistance.