eIFiso4G editing confers high and broad-spectrum resistance against rice yellow mottle virus

Abstract Rice yellow mottle virus (RYMV) is one of the most devastating viral pathogens affecting rice cultivation in Africa. The eukaryotic translation initiation factor 4G isoform eIFiso4G1 plays a pivotal role in rice susceptibility to the virus. Naturally occuring resistance alleles impair infection but are predominantly found in the African cultivated species Oryza glaberrima, whose use in breeding programs is limited by interspecific sterility barriers with the widely grown O. sativa. Here, we used CRISPR-Cas9 to generate knockout (KO) mutations in eIFiso4G1 as well as insertion-deletion (Indel) variants within the region involved in interaction with the viral protein VPg. KO mutations conferred high resistance to RYMV. No resistance-breaking events were observed, suggesting that this resistance may be more durable than that conferred by naturally occuring alleles. However, complete gene KO slightly affected plant growth. Lines carrying Indel mutations in the VPg-interacting region displayed variable resistance levels, with some variants conferring high resistance without compromising plant development. Structural modeling of the eIFiso4G1 variants and their complexes with VPg provided mechanistic insights into how specific Indels modulate the resistance phenotype. Competing Interest Statement The authors have declared no competing interest. Footnotes This revised version of the manuscript addresses reviewers concerns. In addition to correcting typos and reformulating text to improve accuracy and readability, the main revisions include: a modified title: a clearer rationale for generating mutants in the RYMV1 gene, now better explained in the Introduction ;the addition of RT-qPCR results (Sup. Fig. 1) ; an evaluation of grain filling rate and germination rate in plants and progenies derived from crosses between KO mutant lines, to explore the absence of double mutants (Sup. Table 3); a new supplemental table (Sup. Table 6) summarizing phenotypic results for the different mutants ; an expanded comparison in the Discussion between our variants and the natural rymv1-3 allele.