草酸毒素抑制植物防卫反应的作用靶标研究

Oxalic acid (OA) is a virulence factor produced by Sclerotinia sclerotiorum. Previous studies indicated OA enhanced the infection of S. sclerotiorum on plants by suppressing the plant defense response. However, the specific target of OA in suppression of plant defense response remains unknown. Our previous study showed that, OA and its analog, malonic acid (an inhibitor of respiration, targeting the succinate dehydrogenase), can inhibit the respiration of canola significantly. Furthermore, adding exogenous OA or malonic acid can restore the virulence of OA-deficient mutant of S. sclerotiorum. Therefore, we suppose OA may be similar to malonic acid, targeting the enzymes or proteins of plant respiration pathway, and suppress the plant defense response through inhibiting plant respiration. Under the circumstances, we proposed to study the target of OA in suppression of plant defense response. The content of this project includes elucidating the target enzyme of OA in plant respiration, and determining the effects of OA to cell signaling and defense-related gene expression during the interaction between S. sclerotiorum and plants. In addition, the project will isolate and characterize OA-binding proteins in plants, and ascertain their functions. The results of this project will uncover the target of OA in plants, deepen the understanding of the underlying mechanism, and provide new insights to the genetic improvement for the resistance of canola to S. sclerotiorum.

草酸（OA）是核盘菌的一种致病因子。前人的研究表明，OA通过抑制植物的防卫反应来帮助核盘菌侵染植物。但是，对OA抑制植物防卫反应的具体作用靶标并不清楚。我们前期的研究表明：OA及其结构类似物丙二酸（呼吸作用抑制剂，作用靶标为琥珀酸脱氢酶）能显著抑制油菜的呼吸作用，外源添加OA或丙二酸均能恢复草酸缺陷型核盘菌突变体的致病力。所以，我们设想OA可能与丙二酸类似，以植物呼吸路径中某种酶或功能蛋白为靶标，通过抑制植物呼吸作用来抑制植物的防卫反应。鉴此，本项目提出研究OA抑制植物防卫反应的靶标。具体内容包括：采用代谢组学分析方法确定OA抑制植物呼吸作用的酶；分析在植物与核盘菌互作中OA对植物防卫反应信号传导和防卫基因表达等环节的影响，最后分离并鉴定与OA结合的植物蛋白，并研究其功能。项目的完成将揭示OA在植物中的作用靶标，加深对OA作用机制的认识；为油菜抗菌核病的遗传改良提供新思路。

本项目旨在探索草酸在抑制植物防卫反应过程中的作用靶标。通过测定不同浓度及pH值的草酸对不产草酸核盘菌菌株Ep-1PB菌株致病力恢复情况，明确了低pH值有利于菌株Ep-1PB致病力的恢复，并揭示其机理为低pH增大细胞膜的透性，促进草酸进入植物细胞，从而能更好地抑制植物的抗病反应，进而帮助核盘菌致病。此外，本研究构建了一种分离拟南芥中草酸结合蛋白的方法，即利用结合有草酸的凝胶珠分离植物中的草酸结合蛋白。采用这一方法，我们成功地从拟南芥中分离并鉴定出79种草酸结合蛋白。相关生物信息学分析结果表明这些蛋白参与了抗氧化压力、由RNA引起的基因沉默、细胞对光强度的应答等多种生物学进程。此外， KEGG分析发现，这些草酸结合蛋白还参与了氨基酸合成、碳代谢、三羧酸循环、光合作用的碳固定等通路。本项目首次实现对植物体内的草酸结合蛋白进行了分离鉴定，并对相关蛋白进行了生物信息学相关分析，初步明确了草酸在植物中的可能作用靶标。