Wang Junzhu, Shen Tianxin, Song Mengjie, Fan Jiayi, Li Yiqing, Chin Toto Zen, Yu Yidan, Huang Fan, Yang Xiuling, Li Changbao, Zhou Xueping, Li Chuanyou, Hong Yiguo, Liu Yule
MOE Key Laboratory of Bioinformatics and Center for Plant Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Tsinghua-Peking Center for Life Sciences, Beijing, 100084, China.
Sci China Life Sci. 2025 Sep 5. doi: 10.1007/s11427-025-3026-7.
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.
番茄褐色皱果病毒（ToBRFV）克服了所有已知的番茄抗性基因，包括持久的Tm-2基因，对全球番茄生产构成严重威胁。在此，我们采用体外随机诱变技术来进化Tm-2富含亮氨酸重复序列（LRR）结构域，并筛选了约8000个变体，以寻找能够识别ToBRFV运动蛋白（MP）并触发过敏细胞死亡的功能获得型突变体。我们鉴定出了五个这样的突变体。其中，Tm-2和Tm-2与ToBRFV MP共表达时诱导强烈、特异性的细胞死亡，同时保留对烟草花叶病毒MP的识别。表达Tm-2的转基因本氏烟草和番茄植株对天然存在的ToBRFV（ToBRFV）具有抗性。然而，长时间感染后，ToBRFV在其MP中发生了E132K替换，新出现的ToBRFV逃避了Tm-2介导的抗性。然而，Tm-2仍然能够识别MP，并且双突变体Tm-2在稳定转基因番茄中对ToBRFV、ToBRFV和烟草花叶病毒具有广泛抗性。我们的工作表明，人工NLR进化可以产生对烟草花叶病毒的广谱抗性。
