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Tomato brown rugose fruit virus (ToBRFV) overcomes Tm‐22‐mediated resistance, posing a serious threat to global tomato production. We previously identified that key residues in the movement protein (MP)‐H67, N125, K129, A134, I147 and I168 are essential for ToBRFV evasion of Tm‐22 resistance. However, the underlying mechanisms remain unclear. In Tm‐22‐transgenic Nicotiana benthamiana plants at later infection stages, MP mutations H67C, N125A, K129Q and A134N abolished ToBRFV‐GFP cell‐to‐cell and systemic movement, whereas I168N abolished ToBRFV‐GFP systemic but not cell‐to‐cell movement, and I147M partially impaired both. Correspondingly, the MPH67C, MPN125A, MPK129Q and MPA134N mutants elicited strong Tm‐22‐mediated hypersensitive response (HR), MPI168N elicited mild HR and MPI147M elicited none. Confocal microscopy revealed I147M reduced MP localisation at plasmodesmata in Tm‐22‐transgenic N. benthamiana leaves. The interaction with Tm‐22 was weak for wild‐type MP and I147M (slightly enhanced), but moderately or strongly enhanced for I168N and the other four mutants. Wild‐type MP and MPI147M failed to induce Tm‐22 self‐association, MPI168N only weakly induced it, whereas the other four markedly induced it. Collectively, these findings demonstrate that ToBRFV evades Tm‐22‐mediated resistance by attenuating MP–Tm‐22 interaction strength, thereby avoiding Tm‐22 self‐association and downstream immune activation. Tomato brown rugose fruit virus evades Tm‐22‐mediated resistance by reducing affinity between movement protein and Tm‐22, thus failing to trigger Tm‐22 self‐association and activation of immune signalling, while maintaining MP function to support viral systemic infection.

Tomato brown rugose fruit virus (ToBRFV) overcomes Tm‐2 2 ‐mediated resistance, posing a serious threat to global tomato production. We previously identified that key residues in the movement protein (MP)‐H67, N125, K129, A134, I147 and I168 are essential for ToBRFV evasion of Tm‐2 2 resistance. However, the underlying mechanisms remain unclear. In Tm‐2 2 ‐transgenic Nicotiana benthamiana plants at later infection stages, MP mutations H67C, N125A, K129Q and A134N abolished ToBRFV‐GFP cell‐to‐cell and systemic movement, whereas I168N abolished ToBRFV‐GFP systemic but not cell‐to‐cell movement, and I147M partially impaired both. Correspondingly, the MP H67C , MP N125A , MP K129Q and MP A134N mutants elicited strong Tm‐2 2 ‐mediated hypersensitive response (HR), MP I168N elicited mild HR and MP I147M elicited none. Confocal microscopy revealed I147M reduced MP localisation at plasmodesmata in Tm‐2 2 ‐transgenic N. benthamiana leaves. The interaction with Tm‐2 2 was weak for wild‐type MP and I147M (slightly enhanced), but moderately or strongly enhanced for I168N and the other four mutants. Wild‐type MP and MP I147M failed to induce Tm‐2 2 self‐association, MP I168N only weakly induced it, whereas the other four markedly induced it. Collectively, these findings demonstrate that ToBRFV evades Tm‐2 2 ‐mediated resistance by attenuating MP–Tm‐2 2 interaction strength, thereby avoiding Tm‐2 2 self‐association and downstream immune activation.

Tomato mottle mosaic virus (ToMMV) poses a threat to production and quality of tomato fruits. The Tm-2 2 gene confers resistance to some tobamoviruses by recognizing viral movement proteins. However, Tm-2 2 -mediated resistance against ToMMV is not well known. Here, we found that ToMMV could infect wild-type but not Tm-2 2 transgenic Nicotiana benthamiana plants and could also infect tomato cultivar Moneymaker but not resistant cultivar Jili with homozygous Tm-2 2 . Chimeric viral ToMMV ToBRFV−MP with swapped ToMMV MP to MP of tomato brown rugose fruit virus could systemically infect Tm-2 2 transgenic N. benthamiana and tomato cultivars Jili plants. Further, transient expression of ToMMV MP in the leaves of Tm-2 2 transgenic N. benthamiana plants induced hypersensitive response-associated cell death, suggesting that the MP of ToMMV was the avirulent factor for the Tm-2 2 resistance gene. ToMMV could infect Tm-2 2 -containing cultivar Jinpeng 1 but not Chaobei. Sequence analysis revealed that cultivars Chaobei and Jinpeng 1 were heterozygous, where Chaobei consists of Tm-2 2 and Tm-2 genes, while Jinpeng 1 consists of Tm-2 2 and tm-2 genes. Transient co-expression assays showed that both Tm-2 2 and Tm-2 but not tm-2 could recognize ToMMV MP and induce hypersensitivity response-associated cell death in N. benthamiana leaves, suggesting that homozygous tomato harboring Tm-2 2 and heterozygous tomato containing Tm-2 2 and Tm-2 may exhibit more durable resistance to ToMMV than heterozygous tomato carrying Tm-2 2 and tm-2 . Further, Tm-2 2 transgenic N. benthamiana and tomato cultivar Jili plants with silenced Tm-2 2 gene were susceptible to ToMMV. Also, silencing type-I J-domain MIP1 gene compromised Tm-2 2 -mediated resistance to ToMMV in Tm-2 2 transgenic N. benthamiana and tomato cultivar Jili. Moreover, we found that viral RNA could accumulate in the systemic leaves of Tm-2 2 transgenic N. benthamiana plants and tomato cultivar Jili at 35°C, but not at 20, 25, or 30°C. Altogether, our findings reveal that the Tm-2 2 confers resistance to ToMMV by recognizing MP, and the resistance is regulated by the allele combinations, accumulation levels of Tm-2 2 , MIP1 , and the temperature.

Abstract Tomato mottle mosaic virus (ToMMV) poses a threat to production and quality of tomato fruits. The Tm-2 2 gene confers resistance to some tobamoviruses by recognizing viral movement proteins. However, Tm-2 2 -mediated resistance against ToMMV is not well known. Here, we found that ToMMV could infect wild-type but not Tm-2 2 transgenic Nicotiana benthamiana plants and could also infect tomato cultivar Moneymaker but not resistant cultivar Jili with homozygous Tm-2 2 . Chimeric viral ToMMVToBRFV−MP with swapped ToMMV MP to MP of tomato brown rugose fruit virus could systemically infect Tm-2 2 transgenic N. benthamiana and tomato cultivars Jili plants. Further, transient expression of ToMMV MP in the leaves of Tm-2 2 transgenic N. benthamiana plants induced hypersensitive response-associated cell death, suggesting that the MP of ToMMV was the avirulent factor for the Tm-2 2 resistance gene. ToMMV could infect Tm-2 2 -containing cultivar Jinpeng 1 but not Chaobei. Sequence analysis revealed that cultivars Chaobei and Jinpeng 1 were heterozygous, where Chaobei consists of Tm-2 2 and Tm-2 genes, while Jinpeng 1 consists of Tm-2 2 and tm-2 genes. Transient co-expression assays showed that both Tm-2 2 and Tm-2 but not tm-2 could recognize ToMMV MP and induce hypersensitivity response-associated cell death in N. benthamiana leaves, suggesting that homozygous tomato harboring Tm-2 2 and heterozygous tomato containing Tm-2 2 and Tm-2 may exhibit more durable resistance to ToMMV than heterozygous tomato carrying Tm-2 2 and tm-2. Further, Tm-2 2 transgenic N. benthamiana and tomato cultivar Jili plants with silenced Tm-2 2 gene were susceptible to ToMMV. Also, silencing type-I J-domain MIP1 gene compromised Tm-2 2 -mediated resistance to ToMMV in Tm-2 2 transgenic N. benthamiana and tomato cultivar Jili. Moreover, we found that viral RNA could accumulate in the systemic leaves of Tm-2 2 transgenic N. benthamiana plants and tomato cultivar Jili at 35°C, but not at 20, 25, or 30°C. Altogether, our findings reveal that the Tm-2 2 confers resistance to ToMMV by recognizing MP, and the resistance is regulated by the allele combinations, accumulation levels of Tm-2 2 , MIP1, and the temperature.

Vectors of pcDNA3.1-hTERT and pcDNA 3. 1-SV40 T were established. After linearization, they were cotransfected to mammary epithelial cells of Holstein cow, in order to research on the role of hTERT and SV40 T in immortalized mammary epithelial cells in vitro. Both PT-PCR and immunohistochemical as- says of cells were carried out. Results showed that the expression of hTERT and SV40 T could effectively prolong the culture time in vitro of mammary epithelial cells, and enhance the cell passage number. The obtained cell line could be expressed normally, indicating that the in vitro cultured mammary epithelial cells expressing both hTERT and SV40 T could effectively prolong cell llfe without affecting the characteristics of mammary cells.