Objective To investigate the interaction mechanism between Pik1-H4 and cell division cycle protein OsCdc48, clarify its role in rice blast disease resistance.

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. Next, the determination of OsCdc48’s sequence conservation, protein domains, phylogenetic relationships, protein 3D structure prediction and subcellular localization were performed. Finally, OsCdc48 mutant was created using CRISPR/Cas9 technology, the resistance identification of transgenic mutant 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 induced by rice blast fungus infection. OsCdc48 was 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 disease.

Conclusion This study lays a theoretical basis for further elucidating the mechanism of OsCdc48 and NLR protein Pik1-H4 regulating blast disease resistance and rice disease-resistant breeding.