Mutation of a gene with PWWP domain confers salt tolerance in rice

Salinity is a major problem due to the continuous increase in the salinization of agricultural lands, particularly, paddy fields. Using a forward genetics approach, salt-insensitive TILLING line 3, sitl3, was selected from a core population induced by gamma-ray irradiation. Under salt stress, sitl3 had greater fresh weight and chlorophyll content, and lower H₂O₂ and Na⁺ contents than the wild-type. In the gene (LOC_Os07g46180) with two PWWP domains (named Oyza sativa PWWP4, OsPWWP4) of sitl3, a premature stop was caused by an SNP, and was named OsPWWP4p.Gly462* (a stop gain occurred from the 462th amino acid residue). The OsPWWP4 and substrate proteins (OsEULS2, OsEULS3, and OsEULD2) were identified using yeast two-hybrid, bimolecular fluorescence complementation, in vitro pull-down, and in vitro methyltransferase assays. Subcellular localization of OsPWWP4 and OsPWWP4p.Gly462*GFP-tagged proteins revealed they were both localized in the nucleus, while OsEULS2, OsEULS3, and OsEULD2 GFP-tagged proteins were found in the nucleus and cytosol of rice protoplasts. The expression levels of OsEULS2, OsEULS3, OsEULD2 under salt stress were higher in sitl3 than in wild-type plants. In contrast, OsPWWP4 expression was higher in the latter. Genes involved in the salt overly sensitive (SOS) pathway showed higher expression in the aerial tissues of silt3 than in the wild-type. CRISPR/Cas9-mediated OsPWWP4 knock-out transgenic plants showed salt tolerance phenotypes with low Na⁺ contents and low Na⁺/K⁺ ratios. The data suggest that sitl3 is a valuable genetic resource for understanding protein post-translational regulation-related salinity tolerance mechanisms such as methyltransferase activities, and for improving salt tolerance in rice through breeding.