Unraveling the role of the AGO4 gene against rice hoja blanca virus (RHBV): From transformation to protein structure

Jhojan Smith Nanez ^1* Sandra Valdes ¹ Maribel Cruz ^1,2 Maria Camila Rebolledo ¹ Mathias Lorieux ³ Paul Chavarriaga-Aguirre ¹ Maria Fernanda Alvarez ^1*

• ¹ International Center for Tropical Agriculture (CIAT), Cali, Colombia
• ² Latin American Fund for Irrigated Rice (FLAR), Palmira, Colombia
• ³ Département Scientifique Performances des Systèmes de Production et de Transformation Tropicaux, Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), Montpellier, Languedoc-Roussillon, France

Rice hoja blanca virus (RHBV), transmitted by the insect vector Tagosodes orizicolus, poses a significant threat to rice cultivation. Here, we use CRISPR/Cas9 technology to produce specific mutations in the AGO4 gene of Oryza sativa, using the Fedearroz 2000 variety, with the aim of elucidating the participation of the gene in resistance to RHBV. We obtained 14 edited plants that presented deletions of one, two and three nucleotides in the sequence of exon 23 of the AGO4 gene. Phenotypic evaluations showed an increase in susceptibility to RHBV in the edited lines. We identified the presence of RHBV in the leaf tissue of infected plants by amplifying the nucleoprotein, NS3 and NS4 genes of the virus. Using RT-qPCR we analyzed the expression patterns of the AGO4 gene, showing that in the edited lines the expression profiles are similar to the susceptible control. Furthermore, modeling of the tertiary structure of the AGO4 protein and its mutant variant demonstrated changes in the PIWI domain and the presence of the DDH catalytic triad, confirming its role in mediating resistance to RHBV. Our study reveals the functional importance of the rice AGO4 gene in RHBV resistance.