苹果CCR-NB-LRR抗病基因MdRNL2辅助MdNL1抵御斑点落叶病的分子机制研究

苹果对斑点落叶病的抗性受抗病（R）基因调控，但具体调控机制尚不明确。本课题组前期发现CCR-NB-LRR类抗病基因MdRNL2参与苹果抗斑点落叶病，本研究筛选获得一个与其互作的NB-LRR类抗病蛋白命名为MdNL1。为进一步研究MdRNL2/MdNL1的抗病机制，首先利用体外、体内蛋白互作实验以及分段删减蛋白互作实验确认MdRNL2与MdNL1的互作关系以及互作结构域；其次在抗病品种叶片中瞬时沉默MdNL1、感病品种中瞬时过表达MdNL1或通过不同组合共注射苹果叶片后接菌并观察叶片抗性变化，从而证明MdNL1与MdRNL2共同参与苹果抗斑点落叶病途径；然后进一步探索MdNL1与MdRNL2互作后激活的下游抗病基因，并通过瞬时沉默或过表达等手段确认其抗病功能；最后，MdRNL2、MdNL1与下游抗病基因苹果转基因验证其是否参与抗病途径，从而明确苹果抗病品种抗苹果斑点落叶病分子机制。

Alternaria leaf spot was regulated by resistance (R) gene, but the specific regulatory mechanism was still unclear. Previously, we have identified a CCR-NB-LRR proteins MdRNL2 to confer ALT1 resistance in apple. And in this study, protein–protein interaction and LC-MS analyses showed that MdRNL2 interacts with MdNL1 (an NB-LRR protein). Firstly, Y2H,BiFC and pull-down assays indicated that MdRNL2 physically interacts with MdNL1; then suppressing MdNL1 expression in resistant apple (Malus × domestica cv., HF-1), and overexpressing MdNL1 expression in susceptible apple (Malus × domestica cv., NGR196), suggested that MdNL1 and MdRNL2 contribute to ALT1 resistance. Furthermore, investigating the downstream resistant gene of MdNL1 and MdRNL2 and through the suppressing its expression and overexpressing its expression to indicate the function of downstream resistant gene; Finally, MdRNL2、MdNL1 and the downstream resistant gene are made transgenic lines, proposed a mechanism that pairs of MdRNL2 and MdNL1 are both necessary to hamper ALT1 infection, which is correlated with upregulation of the downstream resistant gene and contributed to ALT1 resistance in apple. Thus, the pathway explain resistance molecular mechanism of the Alternaria leaf spot in resistant apple.