Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro
Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro
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DOI：
10.1074/jbc.m109.027540
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发表时间：
2010-01-29
影响因子：
4.8
通讯作者：
Nagano, Yukio
中科院分区：
文献类型：
--
作者：
Iizasa, Ei'ichi;Mitsutomi, Masaru;Nagano, Yukio
植物通过识别几丁质诱导针对真菌病原体的免疫应答，几丁质是真菌细胞壁的组分。最近的研究表明，LysM受体样激酶1/几丁质诱导受体激酶1（LysM RLK 1/CERK 1）是拟南芥几丁质免疫应答的关键组分。然而，LysM RLK 1识别几丁质的分子机制仍然未知。在这里，我们提出的第一个证据直接绑定的LysM RLK 1几丁质。我们在酵母细胞中表达了与酵母增强型绿色荧光蛋白融合的LysM RLK 1（LysM RLK 1-yEGFP）。使用溶解的LysM RLK 1-yEGFP和几种具有相似结构的不溶性多糖的结合研究表明，LysM RLK 1-yEGFP特异性结合几丁质。随后，荧光显微镜下观察溶解的LysM RLK 1-yEGFP与几丁质珠的结合显示，结合是可饱和的，并且具有高亲和力，K-d接近82 nM。通过添加可溶性乙二醇几丁质或具有4-8个N-乙酰基葡糖胺残基的高浓度几丁质寡糖来竞争这种结合。但是，这些甲壳素寡糖的竞争力弱于乙二醇甲壳素。这些数据表明，LysM RLK 1对具有较长N-乙酰基葡糖胺残基的几丁质具有较高的亲和力。我们还发现LysM RLK 1-yEGFP在体外被自动磷酸化，几丁质不影响LysM RLK 1-yEGFP的磷酸化。我们的研究结果提供了一个新的维度几丁质激发子感知植物。
Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a K-d of similar to 82 nM. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4-8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosac-charides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was auto phosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.