The type 3 effector NopL of Sinorhizobium sp strain NGR234 is a mitogen-activated protein kinase substrate
The type 3 effector NopL of Sinorhizobium sp strain NGR234 is a mitogen-activated protein kinase substrate
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中华根瘤菌的 3 型效应子 NopL。
DOI：
10.1093/jxb/erw065
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发表时间：
2016-04-01
影响因子：
6.9
通讯作者：
Staehelin, Christian
中科院分区：
文献类型：
--
作者：
Ge, Ying-Ying;Xiang, Qi-Wang;Staehelin, Christian
病原菌利用3型分泌系统将3型效应子（T3 E）注入宿主细胞，从而破坏宿主防御反应。同样，共生固氮根瘤菌的T3 Es可以影响豆科植物根部的根瘤形成。以前的工作表明，中华根瘤菌（Ensifer）的NopL（外蛋白L）的菌株NGR 234是在真核细胞中的多重磷酸化，这种T3 E抑制有丝分裂原激活蛋白（MAP）激酶信号传导介导的反应在酵母（交配信息素信号）和植物细胞（发病相关的防御蛋白的表达）。在这里，我们表明，NopL是MAP激酶底物。洋葱细胞中荧光融合蛋白的显微观察和双分子荧光互补分析表明，NopL靶向细胞核，并与烟草MAP激酶SIPK（水杨酸诱导的蛋白激酶）形成复合物。体外实验表明，NopL被SIPK磷酸化。双向凝胶电泳后观察到至少9个不同的斑点，表明NopL可以被MAP激酶过度磷酸化。菜豆根瘤衰老的症状。Tendergreen）以确定在鉴定和预测的磷酸化位点（丝氨酸-脯氨酸基序）处具有丝氨酸至丙氨酸取代的不同NopL变体的共生效应物活性。具有6个或8个丝氨酸至丙氨酸取代的NopL变体是部分活性的，而具有10个或12个取代的丝氨酸残基的NopL形式是无活性的。总之，我们的研究结果提供的证据表明，NopL与MAP激酶相互作用，并揭示了丝氨酸-脯氨酸基序的重要性，在共生过程中的效应活性。
Pathogenic bacteria utilize type 3 secretion systems to inject type 3 effectors (T3Es) into host cells, thereby subverting host defense reactions. Similarly, T3Es of symbiotic nitrogen-fixing rhizobia can affect nodule formation on roots of legumes. Previous work showed that NopL (nodulation outer protein L) of Sinorhizobium(Ensifer) sp. strain NGR234 is multiply phosphorylated in eukaryotic cells and that this T3E suppresses responses mediated by mitogen-activated protein (MAP) kinase signaling in yeast (mating pheromone signaling) and plant cells (expression of pathogenesis-related defense proteins). Here, we show that NopL is a MAP kinase substrate. Microscopic observations of fluorescent fusion proteins and bimolecular fluorescence complementation analysis in onion cells indicated that NopL is targeted to the nucleus and forms a complex with SIPK (salicylic acid-induced protein kinase), a MAP kinase of tobacco. In vitro experiments demonstrated that NopL is phosphorylatyed by SIPK. At least nine distinct spots were observed after two-dimensional gel electrophoresis, indicating that NopL can be hyperphosphorylated by MAP kinases. Senescence symptoms in nodules of beans (Phaseolus vulgaris cv. Tendergreen) were analyzed to determine the symbiotic effector activity of different NopL variants with serine to alanine substitutions at identified and predicted phosphorylation sites (serine-proline motif). NopL variants with six or eight serine to alanine substitutions were partially active, whereas NopL forms with 10 or 12 substituted serine residues were inactive. In conclusion, our findings provide evidence that NopL interacts with MAP kinases and reveals the importance of serine-proline motifs for effector activity during symbiosis.