RING-Type E3 Ligase OsRFPH2-6 Ubiquitinates Nuclear Factor Y Subunit OsNF-YA1 to Suppress Rice Immunity

RING-type E3 ubiquitin ligases play crucial roles in plant immunity by ubiquitinating their downstream substrates for degradation. We previously revealed, using CRISR/Cas9-mediated knockout mutant lines, that the RING-H2 E3 ubiquitin ligase OsRFPH2-6, transcriptionally regulated by the NAC transcription factor ONAC083, negatively modulates rice blast resistance. In this study, we further confirmed the negative role of OsRFPH2-6 in rice immunity through overexpression lines and investigated its underlying regulatory mechanism. OsRFPH2-6 possesses E3 ubiquitin ligase activity, which depends on each cysteine residue within its RING domain. OsRFPH2-6 interacts with and ubiquitinates OsNF-YA1, a subunit of nuclear factor Y transcription factor, leading to its degradation. Knockout of OsNF-YA1 attenuates rice blast resistance and weakens chitin-induced immune responses. Transcriptome analysis revealed that OsNF-YA1 regulates a set of defence-associated genes. Notably, some negative immune regulators, like ONAC083, MNAC3 and OsSGR, were upregulated, whereas several positive immune regulators, such as genes encoding for phytoalexin biosynthesis and phenylpropanoid pathway enzymes, hydrolytic enzymes and pathogenesis-related proteins, including OsPAL6, OsATL15, OsCPS4, OsWRKY6, OsPR10a, and chi11, were downregulated in osnf-ya1 mutant plants. These findings suggest that OsNF-YA1 positively regulates rice immunity by modulating the transcription of a set of immune-associated genes. This study extends the previously established ONAC083-OsRFPH2-6 regulatory module in rice immunity by integrating OsNF-YA1 as a downstream substrate of OsRFPH2-6, offering possibilities to improve rice disease resistance through targeted genome editing of the ONAC083-OsRFPH2-6-OsNF-YA1 module.