CRISPR/Cas9-Mediated Resistance Strategies Against Tomato Leaf Curl New Delhi Virus (ToLCNDV) under Thermal Stress Conditions

Abstract

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus that has spread from the Indian subcontinent into South and Southeast Asia, the Mediterranean Basin and North Africa. Growers have few tools left. Insecticidal control of the whitefly vector is failing, and the introgressed Ty loci were selected against a different virus and break down at high temperature. CRISPR/Cas editing offers another route, because the virus replicates through a double-stranded DNA intermediate in the nucleus and can be cut directly. Proof of concept in tomato was published in 2025.

Two problems separate that proof of concept from a deployable line. The first is temperature. Almost every published geminivirus editing experiment ran at 22–25 °C, while the epidemics these constructs must survive occur at 32–38 °C. Temperature acts on the system through at least four channels that do not point the same way. SpCas9 cuts roughly five times more efficiently at 37 °C than at 22 °C. Salicylic-acid-dependent immunity collapses above 28 °C through thermosensitive transcription of CBP60g and SARD1. Virus-derived siRNA accumulation rises between 25 °C and 30 °C. Vector pressure climbs with warming. The second problem is sequence diversity, which we measured directly.

We assembled 88 complete DNA-A and 77 complete DNA-B sequences from GenBank spanning seven countries and eleven host genera, and scanned every NGG-flanked protospacer in both components. None of the 506 candidate sites matched all isolates exactly. Thirteen of 274 DNA-A protospacers reached 90% seed-plus-PAM conservation; no DNA-B protospacer did, the best reaching 83%. The non-coding intergenic and common regions, which the geminivirus editing literature recommends as the most escape-resistant targets, proved to be the least conserved parts of both components at 88.7% and 79.0% mean identity. Guide design for this virus therefore runs into a conflict between durability and coverage that has not been quantified before.

We report five DNA-A target sites above 93% exact conservation, show that no comparable DNA-B site exists in the current sequence record, and set out a three-regime thermal evaluation protocol with defined pass criteria. Constructs validated at 25 °C are tested under conditions that flatter them, and the field durability of every published geminivirus editing construct remains unknown for that reason.