抗啶酰菌胺灰葡萄孢菌（H272R型突变体）对氯苯醚酰胺（Y13149）高敏感性及其机制

Y13149 and boscalid both belong to SDHI. Previous results of this project research group have shown that His at the position 272 was replaced by Tyr of SDH subunit B from Botrytis cinerea leading to resistance to boscalid and sensitivity to Y13149, however no site mutation at position 272 of isolate leading more sensitive to boscalid than Y13149. Site-directed mutagenesis were constructed by the gene replacement strategy, preliminary results indicated that the point mutation H272R in Sdh B confers sensitivity to Y13149.It is no cross-resistance between Y13149 and boscalid, although they shared the same mode of action. This was not an accident, but the mechanism was unknown. This project intends to explore the sensitive mechanism of the amino acid at position 272 contributing to sensitivity to Y13149: after expression and purification B05.10-SDH B and BC-3-SDH B, the proteins are analyzed by SDS-PAGE and Blue native PAGE. These results will reveal the effect of 272aa for expression and interaction of SDH B. Extraction of complex Ⅱ from the SDH B mutations are fused His tag, after SDH activity test, drug test in vitro by microplate reader and interaction between SDH and SDHIs by Blue native PAGE with western blot. This project will provide novel insights into explore the mechanism of resistant or sensitivity from protein nature function and a new thinking to compound SDHIs.

氯苯醚酰胺(Y13149)和啶酰菌胺都是琥珀酸脱氢酶抑制剂，本项目前期研究发现，灰葡萄孢菌的琥珀酸脱氢酶B亚基272位组氨酸突变成精氨酸将导致对啶酰菌胺的抗性，但对氯苯醚酰胺表现为敏感，而未发生突变的菌株对啶酰菌胺更敏感；同源重组构建的点突变材料初步证明272位的突变引起了菌株对氯苯醚酰胺的敏感性。作用机制相同的Y13149与啶酰菌胺不存在正交互抗性，这不是一种偶然现象，但是造成其差异的机制未知。本项目拟从蛋白水平上探索B亚基272位氨基酸突变引起对氯苯醚酰胺敏感的分子机制:表达纯化B05.10-SDH B和BC-3-SDH B，利用变性/非变性蛋白胶分析272位氨基酸对蛋白表达、互作的影响；构建带His标签的B05.10-SDH B和BC-3-SDH B突变材料，抽提复合体Ⅱ进行酶活、体外药效测定，非变性蛋白胶和蛋白杂交分析不同类型琥珀酸脱氢酶与药剂结合情况。这将提供抗药性研究的新视角。

氯苯醚酰胺是基于药效团连接碎片虚拟筛选策略发现的新型琥珀酸脱氢酶抑制剂，其作用靶标琥珀酸脱氢酶包含A、B、C、D 4个亚基，通过转录组测序，我们发现氯苯醚酰胺主要作用于灰葡萄孢呼吸作用的氧化磷酸化通路，可引起该通路上9个基因上调表达并显著富集；对9个基因编码的蛋白功能分析，我们推测琥珀酸脱氢酶B亚基是该药剂的重要作用靶标。室内生物活性表明氯苯醚酰胺对田间草莓灰霉病菌的抑菌活性要优于啶酰菌胺；对2018-2019年田间分离菌株的琥珀酸脱氢酶各亚基基因测序比对，发现B亚基上有3类点突变：H272R、P225F和N230I；H272R型在田间最为常见，菌株数从2018年到2019年明显上升，该类突变体对啶酰菌胺表现为抗性而对氯苯醚酰胺表现敏感，P225F和N230I突变体对两种药剂均表现抗性；分子对接模拟结果表明，氯苯醚酰胺对H272R基因型菌株具有抑菌活性源于药剂与H272R突变体仍然形成氢键和π−π互作，两者间的结合能力并未被减弱；田间灰葡萄孢存在一定数量的C亚基多点突变体（G85A、I93V、M158V 和V168I；I93V和V168I；G85A 和V168I），但与药剂抗性无关。综上，氯苯醚酰胺作用于灰葡萄孢琥珀酸脱氢酶B亚基，该亚基上发生H272R点突变不影响氯苯醚酰胺对突变菌株的抑菌活性，可用于防治田间草莓灰霉病；开发新型琥珀酸脱氢酶抑制剂时，可利用分子对接，综合评估药剂对田间已有或实验室诱导突变体的抑菌活性。