Rice blast is one of the main diseases that threaten rice yield and quality. OsBSK1-2 has been found to be involved in rice blast resistance regulation. Previously, OsCBSX4, a cysteine sulfide-β-Synthase, was identified by screening the proteins that interact with OsBSK1-2. The purpose of this study was to verify the interaction between OsBSK1-2 and OsCBSX4, and clarify the function and molecular mechanism of OsCBSX4 in rice blast resistance, providing a theoretical basis for rice disease resistance breeding.

Co-immunoprecipitation, bimolecular fluorescence complementation and luciferase complementation assays were used to determine the interaction between OsBSK1-2 and OsCBSX4. Then, quantitative PCR and agrobacterium-mediated transient transformation in N. benthamiana were used to detect the gene expression pattern and protein localization of OsCBSX4. Subsequently, OsCBSX4-knockingout and OsCBSX4-overexpressing plants were generated via CRISPR/Cas9 technology and Agrobacterium-mediated genetic transformation, respectively, and their resistance to rice blast was determined by inoculation with M. oryzae. Moreover, the immune responses induced by chitin and M. oryzae in the oscbsx4 mutant were analyzed using ROS burst and DAB staining assays respectively. In the end, the interaction between OsCBSX4 and OsRbohB was verified via bimolecular fluorescence complementation and split-luciferase complementation imaging assays, and the impact of the metabolite of OsCBSX4 on rice blast resistance was determined using the detached leaf inoculation method. The studies above will reveal the immune function and molecular mechanism of OsCBSX4.

The interaction between OsCBSX4 and OsBSK1-2 was verified by CoIP, LCI and BiFC assays. Compared to the wild type, the oscbsx4 mutant showed more disease lesions after inoculation with M. oryzae by the spray method, as well as a larger lesion area and greater fungal growth after inoculation with M. oryzae by the punch method, suggesting that knocking out OsCBSX4 decreased rice blast resistance. Moreover, the expression of the pathogenesis-related genes, OsPR5 and OsPR10, and the H₂O₂ accumulation induced by M. oryzae infection and the ROS burst induced by chitin treatment were reduced in the oscbsx4 mutant. Compared with the wild type, OsCBSX4 overexpressing plants showed a smaller lesion area and less fungal growth after inoculation with M. oryzae by the punch method, suggesting that overexpression of OsCBSX4 increased rice blast resistance. In addition, we found that OsCBSX4 can interact with OsRbohB, a key regulator of rice ROS production and treating rice with L-cysteine, a metabolite of OsCBSX4, does not affect rice blast resistance.

OsCBSX4 is an important component of OsBSK1-2 signaling and positively regulates rice blast resistance. OsCBSX4 may mediate ROS production by interacting with OsRbohB, thereby regulating rice immunity.