Geminiviral-CR-gRNA expressed in cowpea efficiently edited MYMV and MYMIV genome to provide resistance against cowpea yellow mosaic disease without hampering plant growth and yield.

BACKGROUND: Cowpea is an economically important grain legume widely cultivated in Africa, Latin America, and Southeast Asia. In Southeast Asia, two of the most devastating viral diseases affecting cowpea are cowpea golden mosaic and severe leaf curl disease, both caused by Mungbean yellow mosaic India virus (MYMIV). Despite the availability of various molecular breeding strategies to manage viral infections, progress in cowpea improvement remains limited due to the lack of resistant germplasm, the absence of a reliable transformation system, and the restricted availability of efficient tools for viral gene inactivation. RESULTS: In this study, we employed CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the common region (CR) of the single-stranded DNA-A component of legume-infecting geminiviruses, using cowpea as a test system. Transgenic cowpea plants expressing Cas9 and a guide RNA (gRNA) targeting the CR of MYMV/MYMIV were evaluated for resistance to yellow mosaic disease (YMD). Agrobacterium tumefaciens strain EHA105 carrying pXSE901B-Cas9 and CR-gRNA cassettes was used to generate the transgenic plants. PCR and Southern blot analyses confirmed the integration of transgenes into the cowpea genome. Transgenic lines in the T₁ and T₂ generations were tested for YMD resistance via agroinfiltration using MYMV and MYMIV agroinfectious clones. Accumulation of AV2 and AC2 transcripts was drastically reduced in T₂ lines, which also displayed either no or minimal mosaic symptoms. Mutation analysis of the viral genome revealed frameshift mutations near the PAM region of the targeted CR sequence, with editing frequencies of 28%, 34%, 22%, and 33% in MYMV/MYMIV-infected cowpea lines #L2, #L4, #L7, and #L11, respectively. The transgenic cowpea plants exhibited a normal phenotype and did not show any yield reduction under greenhouse conditions. CONCLUSION: To the best of our knowledge, this is the first report of transgenic cowpea plants stably expressing a geminiviral common region (CR)–targeting gRNA via the CRISPR/Cas9 system, leading to efficient editing of the MYMV/MYMIV genome and conferring durable resistance to Yellow Mosaic Disease without adversely affecting plant growth or yield. These findings demonstrate the potential of CRISPR/Cas9 as a precise and robust platform for developing virus-resistant cowpea and other legume crops.