植物抗病蛋白Tm-22定位在细胞质膜上，是一个N端为CC（coiled-coil）结构域的NLR（nucleotide-binding, leucine-rich repeat receptor）蛋白，其在识别烟草花叶病毒属（Tobamovirus）病毒的运动蛋白（MP）后激活，引发ETI（effector-triggered immunity）。本文对Tm-22的抗病分子机制进行了研究，发现Tm-22在激活之后会自我互作，可能以二聚或多聚的状态发挥功能。进一步研究证明CC结构域是Tm-22的信号发射器，人为增强CC的自我互作，会增强CC诱导细胞死亡的能力。此外，本研究发现NB-ARC结构域通过不同核苷酸结合状态和自我互作，来调控CC结构域的功能。 同时，实验发现封闭CC结构域的N端会破坏CC的信号，这是我们拓展Tm-22抗性的理论依据。在此基础上，本研究开发了利用Tm-22抗马铃薯Y病毒属（Potyvirus）病毒的方法。Potyvirus属的病毒普遍编码一个NIa蛋白酶，我们将ToMV MP连接到Tm-22蛋白的N端，并在中间连接处加上NIa的识别切割位点，此时由于N端被阻滞，Tm-22处于一种功能抑制状态，当potyvirus来袭时，病毒编码的NIa将MP切离主体蛋白，Tm-22的N端被释放，而且自由活动的MP正好能被附近的Tm-22所识别，造成Tm-22激活引发ETI。在本生烟草上瞬时共表达该改造版Tm-22与NIa或者病毒本身时，会产生明显的细胞死亡现象，而相应的转基因植物获得了对PVY和TuMV病毒的部分抗性。此外，本研究还开发了另外的抗potyvirus病毒系统，将自激活形式Tm-22突变体的N端连接标签蛋白，中间连接处设计NIa的识别切割位点，这个设计在瞬时表达时也呈现了抗性。
Tm-22 is a plasma membrane-localized coiled-coil (CC)-type nucleotide-binding, leucine-rich repeat receptor (NLR). It induces effector-triggered immunity (ETI) after recognizing the viral movement protein (MP) of tobamovirus, thus conferring resistance against tobamovirus. In this study, we aim at exploring the molecular resistance mechanism of Tm-22. In the first section, we found that Tm-22 self-associates upon activation. So active Tm-22 may form a dimer or oligomer for downstream signaling. Then we demonstated that the CC domain of Tm-22 is the signaling domain for triggering cell death, and its function requires self-association, moreover，stronger self-asscociation helps CC signaling. Furthermore, we found that the NB-ARC (nucleotide-binding domain shared by Apaf‐1, R and CED4) domain regulates the activation of CC domain through its nucleotide-binding and self-association. Meanwhile, we found that blocking the N terminus of CC domain destroys its signaling, which lays a foundation for our further research on expanding Tm-22 resistance. We developed a method for conferring resistance against potyvirus, which usually encodes a protease NIa. We fused ToMV MP to the N terminus of Tm-22 with a linker containing NIa cleavage site. Becase the N terminus is blocked by MP, Tm-22 remains inactive. When the potyvirus NIa cleaves the linker between MP and Tm-22, Tm-22 with a free N terminus recognizes MP and is activated for ETI signaling. Transient co-expression of this engineered Tm-22 with NIa or potyvirus induced cell death in Nicotiana benthamiana, and transgenic plants conferred patial resistance against PVY and TuMV. Moreover, we designed other methods against potyvirus by fusing a tag to the N terminus of the auto-active version of Tm-22 with a linker containing NIa cleavage site, which functioned well in the transient expression system.
