Identification of potential target sites in cotton leaf Curl Multan Virus (CLCuMuV) after hybridization through candidate miRNAs in cotton to induce virus resistance.

The present study focuses on the computational identification and experimental validation of Gossypium hirsutum-derived miRNAs capable of targeting the CLCuMuV genome. The retrieved CLCuMuV genome sequence and 80 mature miRNAs were subjected to the identification of six key open reading frames (ORFs) encoding viral proteins: AC4 (100 amino acids), AC3 (134 amino acids), AC2 (150 amino acids), Rep (363 amino acids), AV2 (121 amino acids), and coat protein (256 amino acids). Using a combination of computational tools, including psRNATarget, RNA22, RNAhybrid, and miRanda, potential miRNA-mRNA interactions were predicted based on sequence complementarity and free energy calculations. The computational analysis revealed multiple cotton miRNAs manifesting increased binding potential to viral mRNAs. The potential miRNAs included ghr-miR390a, ghr-miR7486a, and ghr-miR7503 which emerged as highly promising candidates for RNA interference. Among these, ghr-miR390a witnessed the highest level of binding efficiency equipped with duplex free energy (ΔG Duplex) of - 18.70 kcal/mol with binding energy (ΔG Binding) of - 16.78 kcal/mol, indicating the maximum potential of inhibiting viral gene expression. These cotton-derived potential miRNAs target essential viral genes involved in replication, coat protein synthesis, and movement immensely critical for the survival and proliferation of CLCuMuV. Phylogenetic analysis revealed that CLCuMuV isolates share common evolutionary ancestors, suggesting potential cross-resistance mechanisms that could be exploited. To experimentally validate these computational predictions, the identified miRNAs were cloned and highly expressed in transgenic cotton lines. Real-time PCR (RT-PCR) was employed to confirm the expression level of the target miRNAs in the transgenic plants while enzyme-linked immunosorbent assay (ELISA) validated the presence of key GhDCL3 proteins in transgenic in contrast to the control. The transgenic lines expressing the ghr-miR390a encoding gene GhDCL3 witnessed a significant reduction in symptoms of CLCuMuV, and incommensurable presence of CLCuMuV infection, while control plants exhibited typical disease symptoms, including leaf curling and vein thickening. This study identifies three highly promising miRNAs ghr-miR7486a, ghr-miR390a, and ghr-miR7503 that can effectively target critical genes in the CLCuMuV genome, providing a molecular mechanism to inhibit viral replication and mitigate the impact of the disease. The successful integration of these miRNAs into cotton via genetic engineering offers a novel and sustainable approach to control CLCuMuV.