Objective To investigate the interaction mechanisms between Pik1-H4 and cell division cycle proteins (OsCdc48 ) and clarify their roles in rice blast disease resistance, thus providing new insights into the mechanism of rice resistance mediated by NLR proteins.
Method Firstly, the interactions between Pik1-H4 and OsCdc48 were verified using yeast two-hybrid and luciferase complementation assays. Then, the expression patterns of OsCdc48 after infection of Magnaporthe oryzae and its tissue-specific expression were analyzed by RT-qPCR. Further analysis, including the determination of OsCdc48’s sequence conservation, protein domains, construction of phylogenetic trees, protein three-dimensional structure prediction and its subcellular localization, and creation of OsCdc48 mutant strains using CRISPR/Cas9 technology were performed. The resistance identification of transgenic mutant strains to rice blast disease and expression analysis of pathogenesis-related genes were also conducted.
Result The interactions between Pik1-H4 and OsCdc48 were confirmed, and OsCdc48 was significantly induced by blast fungus infection. OsCdc48 is expressed in all tissues and was localized to the nucleus and cytoplasm. The sequence of OsCdc48 was conserved across different species, with the closest phylogenetic relationship to maize and sorghum, and it might form homologous hexamers. The OsCdc48 loss-of-function mutant ko-oscdc48 up-regulated the expression of disease-related genes and enhanced the resistance to rice blast.
Conclusion This study confirms the interaction between OsCdc48 and Pik1-H4, with OsCdc48 expression significantly induced by rice blast disease. Knocking out OsCdc48 negatively regulates rice resistance to blast disease by enhancing the expression of disease-related genes. This lays the theoretical basis for further elucidating the mechanism of OsCdc48 and NLR protein Pik1-H4 in regulating rice blast disease resistance and its application in disease-resistant breeding.
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