CRISPR/Cas9-mediated mutagenesis of the susceptibility gene OsHPP04 in rice confers enhanced resistance to rice root-knot nematode

Abstract

CRISPR crops carrying a mutation in susceptibility (<i>S</i>) genes provide an effective strategy for controlling plant disease, because they could be 'transgene-free' and commonly have more broad-spectrum and durable type of resistance. Despite their importance, CRISPR/Cas9-mediated editing of <i>S</i> genes for engineering resistance to plant-parasitic nematode (PPN) disease has not been reported. In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of the <i>S</i> gene rice copper metallochaperone heavy metal-associated plant protein 04 (<i>OsHPP04</i>), and successfully obtained genetically stable homozygous rice mutants with or without transgenic elements. These mutants confer enhanced resistance to the rice root-knot nematode (<i>Meloidogyne graminicola</i>), a major plant pathogenic nematode in rice agriculture. Moreover, the plant immune responses triggered by flg22, including reactive oxygen species burst, defence-related genes expression and callose deposition, were enhanced in the 'transgene-free' homozygous mutants. Analysis of rice growth and agronomic traits of two independent mutants showed that there are no obvious differences between wild-type plants and mutants. These findings suggest that <i>OsHPP04</i> may be an <i>S</i> gene as a negative regulator of host immunity and genetic modification of <i>S</i> genes through the CRISPR/Cas9 technology can be used as a powerful tool to generate PPN resistant plant varieties.