Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein - Isolation and characterization of a rice Mlo homologue
Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein - Isolation and characterization of a rice Mlo homologue
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DOI：
10.1074/jbc.m108478200
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发表时间：
2002-05-31
影响因子：
4.8
通讯作者：
Cho, MJ
中科院分区：
文献类型：
--
作者：
Kim, MC;Lee, SH;Cho, MJ
Ca~(2+)的瞬时内流是触发植物防御反应的信号级联反应的早期事件。然而，防御相关的钙信号的下游成分在很大程度上是未知的。由于Ca~(2+)信号是由钙调素(CaM)等钙结合蛋白介导的，鉴定和鉴定病原菌诱导的CaM结合蛋白有助于深入了解Ca~(2+)调节防御反应的机制。在本研究中，我们从病原菌诱导的水稻悬浮细胞构建的表达文库中，通过蛋白质-蛋白质相互作用筛选获得了水稻MLO(Oryza sativa MLO；OsMlo)的编码基因。OsMlo的分子质量为62 kDa，与大麦MLO有65%的序列同源性和支架拓扑结构，MLO是一种七螺旋跨膜蛋白，其功能是广谱抗病和叶细胞死亡的负调控因子。通过凝胶覆盖实验，我们发现在大肠杆菌中产生的OsMlo以依赖于钙离子的方式与大豆CaM异构体-1(SCAM-1)结合。我们在OsMlo C末端的胞质尾部定位了一个20个氨基酸的CaM结合域(CaMBD)，这是形成钙依赖的CaM复合体的必要条件和充分条件。保守的CaMBD与CaM的特异性结合通过定点突变、凝胶迁移率改变实验和与钙/CaM依赖的酶的竞争实验得到证实。OsMlo的表达受真菌病原菌和植物防御信号分子的强烈诱导。我们认为，钙离子负载的CaM与C末端的结合可能是MLO蛋白的一个共同特征。
Transient influx of Ca2+ constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca2+ signaling are largely unknown. Because Ca2+ signals are mediated by Ca2+-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca2+ regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca2+-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca2+-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca2+/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca2+-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.