水稻细菌性条斑病菌三型效应蛋白AvrBs2致病机理研究

Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, is one of important phytopathogenic bacteria. The type three secreted effectors were demonstrated to function as key virulence factors in X. oryzae pv. oryzicola. To reveal molecular mechanisms underlying virulence functions of essential virulence effectors will facilitate elucidating virulence and pathogenicity of this pathogen. It has been previously shown that AvrBs2 in X. oryzae pv. oryzicola suppresses rice immunity and plays an essential role in X. oryzae pv. oryzicola virulence to rice. Structural analyses predict that AvrBs2 contains a novel phospholipase A2 domain in the N terminus besides the previously identified glycerophosphodiesterase domain in the C terminus. In addition, in-vitro purified AvrBs2 was demonstrated to disrupt the stable structure of the lipidosome formed with total rice phospholipids, indicating that AvrBs2 is an enzyme with dual catalytic domains to digest phospholipids. In this project, we will explore the effects of AvrBs2 mutation in the predicted catalytic active sites on its ability to suppress rice immunity using the transgenic plants. The catalytic activity of AvrBs2 will be also confirmed by in-vitro enzyme assays to determine the relationship of immunity suppression and the catalytic activities of AvrBs2. Secondly, the substrate of AvrBs2 will be investigated by profiling glycerophospholipids by LC-MS in the avrBs2-transgenic plants; Lastly, it will be figured out molecular mechanism of immunity suppression and infection promotion by AvrBs2 in multiple aspects including protein-protein interaction and the effect on plant immunity of the digested product of AvrBs2. This research will provide information to understand the pathogenicity mechanism of X. oryzae pv. oryzicola.

细菌性条斑病菌，简称细条病菌是水稻上重要病原。解析细条病菌关键致病效应蛋白的毒性机理将有助于揭示该病菌的致病机制。前期研究发现，细条病菌效应蛋白AvrBs2能抑制水稻免疫反应，在致病性中起关键作用。申请人通过新方法预测，AvrBs2除已鉴定的甘油磷酸二酯酶结构域外，在其N端还具有磷酸脂酶A2结构域；而且体外纯化的AvrBs2能够破坏水稻磷脂微粒体的稳定性。因此，推测其为一个具有双酶活性的磷脂降解酶。本项目将探究预测的AvrBs2酶活位点突变对抑制植物免疫能力的影响，通过体外酶活分析，对AvrBs2的生化活性进行验证，确定AvrBs2酶活与免疫抑制间的关系；其次，利用LC-MS技术分析AvrBs2转基因水稻中甘油磷酸脂的种类和含量，鉴定AvrBs2的作用底物；最后，从蛋白互作、产物的效应等多角度探究AvrBs2抑制植物免疫、促进致病的分子机理。研究结果将加深对细条病菌致病分子机制的理解。

三型分泌效应蛋白AvrBs2是稻生黄单胞菌条斑致病变种（Xanthomonas oryzae pv. oryzicola, Xoc）的关键毒力因子; 然而其毒性机制尚不清楚。对其氨基酸序列分析发现，该蛋白包含一个完整的甘油磷酸二酯酶（glycerolphosphodiesterase，GDE）结构域和两端功能未知的区域；前期实验显示AvrBs2可能与脂肪微粒体的稳定性有关。由此，我们开展了对AvrBs2的毒性机制研究。首先，我们构建了avrBs2基因的结构域缺失体和酶活丧失突变体表达质粒，互补到ΔavrBs2敲除体菌株，然后在野生型水稻中进行致病力分析。我们发现只有完整的avrBs2基因才具有毒性功能，GDE结构域或两端未知功能区中缺失任何一个部分均完全丧失毒性功能，并且GDE催化活性中心对avrBs2毒性至关重要。接着我们构建了异源表达酶活丧失点突变体avrBs2E304A, D306A 和avrBs2H319A的转基因水稻。在接种ΔavrBs2菌株后，野生型avrBs2转基因水稻对Xoc的抗病性受到抑制，而avrBs2E304A,D306A和avrBs2H319A转基因水稻对Xoc的抗病性无显著改变，这一结果进一步证实GDE酶活中心是AvrBs2毒力的关键。然而，用原核表达纯化的AvrBs2蛋白进行体外磷脂水解活性分析实验发现，AvrBs2不能降解磷脂酰胆碱、4，5-二磷酸磷脂酰肌醇和溶血磷脂酰胆碱等不同类型的磷脂类化合物，说明AvrBs2的靶标不是磷脂，而是其他化合物。有趣的是，我们发现野生型的avrBs2能够引起植物的细胞死亡，而酶活中心点突变体则不能诱导植物的细胞死亡；然而野生型和酶活丧失点突变的avrBs2对酵母均没有细胞毒性。另一方面，通过荧光素酶互补实验我们验证了AvrBs2与两个水稻蛋白（FAD依赖的氧化还原酶Os02g22260和酯酶Os07g06850）均能互作。这些结果进一步揭示，AvrBs2不能降解磷脂，而是具有依赖于GDE催化中心的新的酶活，靶向植物细胞内的某一未知化合物，引起植物的细胞死亡，从而发挥毒性功能。