Tomato brown rugose fruit virus(ToBRFV)overcomes all known tomato resistance genes,including the durable Tm-2^(2),posing a serious threat to global tomato production.Here,we employed in vitro random mutagenesis to evo...Tomato brown rugose fruit virus(ToBRFV)overcomes all known tomato resistance genes,including the durable Tm-2^(2),posing a serious threat to global tomato production.Here,we employed in vitro random mutagenesis to evolve the Tm-2^(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^(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 MPK132,and the double mutant Tm-2^(2-S723Y/N744D)conferred broad resistance against ToBRFV^(E132),ToBRF^(VK132),and tobacco mosaic virus in stable transgenic tomato.Our work demonstrates that artificial NLR evolution can generate broad-spectrum resistance against tobamovirus.展开更多
基金supported by the National Natural Science Foundation of China(32130086,32430085)the National Key Research and Development Program of China(2024ZD0407702,2022YFD1400800)。
文摘Tomato brown rugose fruit virus(ToBRFV)overcomes all known tomato resistance genes,including the durable Tm-2^(2),posing a serious threat to global tomato production.Here,we employed in vitro random mutagenesis to evolve the Tm-2^(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^(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 MPK132,and the double mutant Tm-2^(2-S723Y/N744D)conferred broad resistance against ToBRFV^(E132),ToBRF^(VK132),and tobacco mosaic virus in stable transgenic tomato.Our work demonstrates that artificial NLR evolution can generate broad-spectrum resistance against tobamovirus.
