Direct-seeded rice (DSR) offers a sustainable alternative for climate-resilient agriculture, but it is severely threatened by the root-knot nematode Meloidogyne graminicola . Knockout of susceptibility (S) genes has emerged as a promising strategy to combat such obligate parasites. Among candidate S genes, heavy metal-associated (HMA) domain containing isoprenylated plant proteins (HIPPs) are central regulators of plant stress responses. Here, we show that CRISPR/Cas9-mediated knockout of the nematode-responsive S gene, OsHIPP19 , reduces susceptibility to M. graminicola in the DSR cultivar Pusa 2090. Given the variable transformation efficiency among Oryza sativa ssp. indica rice cultivars, we first standardized an immature embryo-derived callus culture method for Pusa 2090. Subsequently, we generated three loss-of-function mutant lines harboring novel alleles. In microplot trials, these lines exhibited a 38.75%‒40.08% reduction in gall counts and a 40.26%‒42.66% reduction in nematode multiplication compared with control plants. Conversely, OsHIPP19 overexpression lines conferred hypersusceptibility to M. graminicola , and reintroducing OsHIPP19 into a resistant knockout line restored susceptibility. The role of OsHIPP19 as an S gene was further confirmed by promoter-GUS reporter assays and expression analysis of defense-related marker genes. Notably, OsHIPP19 knockout did not cause yield penalties in Pusa 2090, highlighting the translational potential of this approach for engineering sustainable nematode resistance in crops.
