Engineering resistance genes against tomato brown rugose fruit virus

Tomato brown rugose fruit virus (ToBRFV) overcomes all known tomato resistance genes, including the durable Tm-2², posing a serious threat to global tomato production. Here, we employed in vitro random mutagenesis to evolve the Tm-2² leucine-rich repeat (LRR) domain and screened ∼8,000 variants for gain-of-function mutants capable of recognizing the ToBRFV movement protein (MP) and triggering hypersensitive cell death. We identified five such mutants. Among them, Tm-2^2-S723Y and Tm-2^2-N744D induced strong, specific cell death upon co-expression with ToBRFV MP, while retaining recognition of TMV MP. Transgenic Nicotiana benthamiana and tomato plants expressing Tm-2^2-S723Y resisted the naturally occurring ToBRFV (ToBRFV^E132). However, after prolonged infection, ToBRFV^E132 evolved an E132K substitution in its MP, and the newly appeared ToBRFV^K132 evaded Tm-2^2-S723Y-mediated resistance. However, Tm-2^2-N744D still recognized MP^K132, and the double mutant Tm-2^{2-S723Y/N744D} conferred broad resistance against ToBRFV^E132, ToBRFV^K132, and tobacco mosaic virus in stable transgenic tomato. Our work demonstrates that artificial NLR evolution can generate broad-spectrum resistance against tobamovirus.