拟南芥的受体蛋白LORE识别LPS的分子机制研究

Lipopolysaccharide (LPS) known as endotoxin, is a major component of the Gram-negative bacteria cell wall. LPS is an important pathogen-associated molecular pattern and consists of three parts: the lipid A, core polysaccharide (core region) and the side chain O- antigens (O-specific chain). LPS can induce innate immune responses in plants, including hypersensitive response (HR), reactive oxygen species (ROS) burst, activation of calcium influx, production of nitric oxide, callose deposition, and increased expression of defense responsive genes. Recently, Ranf and colleagues isolated a LPS-insensitive mutant lore (lipooligosaccharide-specific reduced elicitation), which is defective in LPS-triggered elevation of cytosolic calcium and shows increased susceptibility to the infection of Pseudomonas syringae. LORE gene encodes a bulb-type lectin S-domain-1 receptor-like kinase (SD1-29). Although the genetic screen suggests that LORE is indispensible for sensing LPS in Arabidopsis, the biochemical regulation of LORE protein is still unclear. Therefore, we will investigate the function of various domains in LORE protein, such as the LPS binding activity of lectin domain, homodimer formation of EGF and PAN domain, and the phosphorylation ability of C-terminal kinase domain. In addition, we are going to isolate LORE protein complexes in vivo by Co-Immunoprecipitation, and perform mass spectrometry analysis to identify LORE interaction proteins (LIPs). LIPs proteins are good candidates for LORE co-receptor and downstream components of LORE-mediated LPS signal transduction. Together, our studies will broaden the understanding of LPS recognition mechanism, providing a potential way for future application in engineering new varieties with broad-spectrum disease resistance.

脂多糖(LPS)是革兰氏阴性细菌细胞壁的主要成分，又称为内毒素。在植物中，LPS是重要的病原相关分子模式，可以诱导植物的先天免疫反应。拟南芥中的LORE蛋白(lipooligosaccharide-specific reduced elicitation，LORE)是首个鉴定到的LPS受体，是包含有B-型外源凝集素和S-domain的受体类激酶，但LORE识别LPS的分子机制很不清楚。申请人拟通过遗传学，生物化学及分子生物学等手段，解析拟南芥LORE蛋白识别LPS中的分子机制。首先我们将验证LORE的结构域功能，包括其lectin结构域直接结合LPS，LORE蛋白的激酶活性，及LORE蛋白识别LPS后的磷酸化修饰位点。还将通过体内的免疫共沉淀分离LORE的复合体，筛选验证得到LORE的互作蛋白LIPs (LORE Interaction Proteins)，解析其的功能，寻找可能的共受体。

脂多糖 (Lipopolysaccharide，LPS)是革兰氏阴性细菌细胞壁的主要成份，是一种重要的病菌相关分子模式(PAPMs)，我们最新的研究发现，LPS可以诱导植物产生两次的活性氧迸发，LPS诱导产生的第一次短暂的活性氧迸发和细菌鞭毛蛋白(flg22)诱导产生的一样，但比flg22要弱很多；除了这个典型的PAMP诱导的活性氧迸发，LPS还诱导产生了第二次活性氧迸发，相比于第一次迸发，持续时间更长，强度更大。显微观察发现LPS诱导产生的第二次活性氧迸发是叶绿体产生的，并且LPS可以破坏叶绿体的结构和功能。这是一个全新的PAMPs引起的免疫反应。LPS诱导的后期活性氧迸发在各个物种中广泛存在，说明这是一个保守的反应。LPS的Lipid A组分是主要的活性中心。同时我们发现Lipid A可以诱导比中链脂肪酸3-OH-C10:0更高更持久的第二次活性氧的迸发，并且Lipid A可以诱导番茄产生后期活性氧迸发的迸发，而中链脂肪酸3-OH-C10:0不能诱导。Lipid A诱导的第二次活性氧的迸发部分依赖与中链脂肪酸3-OH-C10:0的受体LORE蛋白，所以Lipid A诱导的免疫反应并不是因为污染了游离的中链脂肪酸3-OH-C10:0。同时我们鉴定到LORE在体内的互作蛋白BIK1和NOG1-2，LORE通过磷酸化BIK1传递信号，通过小G蛋白NOG1-2调控LPS/Lipid A诱导的第二次活性氧迸发。我们的研究为寻找植物识别LPS更广谱的受体提供了研究基础，这些研究将来为农业上培育广谱的可持续的抗病品种提供重要的理论指导意义。