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-2, posing a serious threat to global tomato production. Here, we employed in vitro random mutagenesis to evolve the Tm-2 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 and Tm-2 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 resisted the naturally occurring ToBRFV (ToBRFV). However, after prolonged infection, ToBRFV evolved an E132K substitution in its MP, and the newly appeared ToBRFV evaded Tm-2-mediated resistance. However, Tm-2 still recognized MP, and the double mutant Tm-2 conferred broad resistance against ToBRFV, ToBRFV, and tobacco mosaic virus in stable transgenic tomato. Our work demonstrates that artificial NLR evolution can generate broad-spectrum resistance against tobamovirus.