CAS9 Mediated In-Planta Defence Strategy Against Tomato Leaf Curl New Delhi Virus (ToLCNDV) in Tomato

Tomato leaf curl New Delhi virus (ToLCNDV), a begomovirus, that causes severe leaf curling, stunting, and reduced yield in tomato plants is consistently threatening its production worldwide. CRISPR/Cas9-mediated genome editing has shown immense potential in developing disease-resistant crops. This study successfully focuses on designing a precise and efficient strategy for in planta defence against ToLCNDV. Five key targets within the viral genome, essential for its replication and pathogenicity, were selected. Five Cas9-expressing constructs, along with the ToLCNDV infectious clone, were agroinfiltrated into tomato plants. Three constructs effectively disrupted the ToLCNDV genome. These three constructs, 1T, 2T, and 4T, were shortlisted based on symptom severity level. They showed a relatively low viral titre of 0.5, 0.42, and 0.25 through quantitative real time PCR (qPCR) after 3, 6, and 9 days of post-co-infiltration, respectively. Positive control plants showed significant signs of infection like yellowing of leaves, thickening of veins, and majorly upward curling of leaves. In comparison, plants infiltrated with three Cas9 constructs had mild yellowing of leaves that recovered after approximately 21-dpi. Furthermore, we assessed the agronomic performance of Cas9-mediated tomato plants through in planta Agrobacterium-mediated transformation with three short-listed guided RNA (gRNA) constructs under greenhouse conditions. Also, qPCR analysis of Cas9 protein in 7, 14, and 21-day of intervals gave a relative expression of 0.85, 0.76 and 0.51 respectively in genetically engineered (GE) plants through in planta transformation. In conclusion, this research contributes to CRISPR-Cas9-mediated plant genome editing. Our findings substantiate the efficacy of the CRISPR/Cas9 system in achieving durable engineering of resistance against ToLCNDV with 30% transformation efficiency in tomato plant. Furthermore, this study illuminates potential avenues for extending the application of this technology to confer resistance against singular and multiple infectious viruses in diverse crop species.