CRISPR-Cas9-Mediated Knockout of MLO3 Confers Enhanced Resistance to Reniform Nematode in Upland Cotton

Upland cotton (<i>Gossypium hirsutum</i> L.) is a major global commodity crop whose production is threatened by the reniform nematode (<i>Rotylenchulus reniformis</i> Linford and Oliveira), a plant-parasitic pest that causes substantial yield losses. Host-plant resistance offers a sustainable management strategy, but currently available resistant cotton cultivars provide only partial protection and often require supplemental control methods. In this study, Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated 9 (Cas9) gene editing was used to generate targeted knockouts of <i>Mildew Resistance Locus O</i> (<i>GhiMLO3</i>) in cotton and assess its role in resistance to <i>R. reniformis</i>. Four independent knockout lines (A1, D3, E1, and P3) were developed, confirmed by sequencing, and evaluated for nematode resistance under controlled greenhouse conditions. Nematode reproduction was significantly reduced on lines D3 and E1, with lower egg counts and fewer vermiform life stages compared with the control genotypes, Coker 312 (WT), Delta Pearl, and Jin668. The edited lines also showed characteristic mesophyll cell-death phenotypes, suggesting potential pleiotropic effects associated with <i>MLO</i>-mediated resistance. Sequence analysis confirmed multiple homozygous and heterozygous mutations in <i>MLO3</i> alleles from both the A and D subgenomes, with D3 and E1 lines displaying the strongest resistance profiles. These findings demonstrate that <i>MLO3</i> gene editing is a promising approach for improving <i>R. reniformis</i> resistance in cotton.