Plants usually keep resistance (R) proteins in a static state under normal conditions to avoid autoimmunity and save energy for growth, but R proteins can be rapidly activated upon perceiving pathogen invasion. Pib, the first cloned blast disease R gene in rice, encoding a nucleotide-binding leucine-rich repeat (NLR) protein, mediates resistance to the blast fungal (Magnaporthe oryzae) isolates carrying the avirulence gene AvrPib. However, the molecular mechanisms about how Pib recognizes AvrPib and how it is inactivated and activated remain largely unclear. In this study, through map-based cloning and CRISPR-Cas9 gene editing, we proved that Pib contributes to the blast disease resistance of rice cultivar Yunyin (YY). Furthermore, an SH3 domain-containing protein, SH3P2, was found to associate with Pib mainly at clathrin-coated vesicles in rice cells, via direct binding with the coiled-coil (CC) domain of Pib. Interestingly, overexpression of SH3P2 in YY compromised Pib-mediated resistance to M. oryzae isolates carrying AvrPib and Pib-AvrPib recognition-induced cell death. SH3P2 competitively inhibits the self-association of the Pib CC domain in vitro, suggesting that binding of SH3P2 with Pib undermines its homodimerization. Moreover, SH3P2 can also interact with AvrPib and displays higher affinity to AvrPib than to Pib, which leads to dissociation of SH3P2 from Pib in the presence of AvrPib. Taken together, our results suggest that SH3P2 functions as a "protector" to keep Pib in a static state by direct interaction during normal growth but could be triggered off by the invasion of AvrPib-carrying M. oryzae isolates. Our study reveals a new mechanism about how an NLR protein is inactivated under normal conditions but is activated upon pathogen infection.

The lipoxygenase (LOX) gene family is widely distributed in plants, and its activity is closely associated with seed viability and stress tolerance. In this study, we cloned the rice(Oryza sativa)lipoxygenase gene OsLOX1, a key participant in the 13-lipoxygenase metabolic pathway. Our primary focus was to investigate its role in mediating responses to drought stress and seed germination in rice. Histochemical staining and qPCR analysis indicated that the expression level of OsLOX1 was relatively high in leaves and early germinating seeds. Our findings revealed that mutant lines with CRISPR/Cas9-induced knockout of OsLOX1 exhibited reduced tolerance to drought stress compared with the wild-type. This was accompanied by elevated levels of H₂O₂ and malondialdehyde, and a decrease in the expression levels of genes associated with antioxidant enzymes. Furthermore, knockout of OsLOX1 reduced the longevity of rice seeds increased H₂O₂ and MDA levels, and decreased the activities of the antioxidant enzymes superoxide dismutase and catalase, compared with the wild-type. These findings demonstrated that OsLOX1 positively regulated rice seed vigor and drought stress.