Overexpression of a kinase-deficient form of the EDR1 gene enhances powdery mildew resistance and ethylene-induced senescence in Arabidopsis.
Overexpression of a kinase-deficient form of the EDR1 gene enhances powdery mildew resistance and ethylene-induced senescence in Arabidopsis.
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DOI：
10.1046/j.1365-313x.2002.01482.x
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发表时间：
2002-12
期刊：
影响因子：
--
通讯作者：
Dingzhong Tang;R. Innes
中科院分区：
文献类型：
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作者：
Dingzhong Tang;R. Innes
拟南芥的EDR1基因编码一种Raf样的丝裂原活化蛋白激酶(MAPKK)，并作为抗病负调控因子发挥作用。然而，植物和动物蛋白激酶的系统发育分析表明，植物类Raf蛋白与动物混合谱系蛋白激酶(MLK)的亲缘关系比Raf类蛋白激酶更密切，而且与这两类动物蛋白激酶的亲缘关系都非常不同，这使得底物专一性的推断受到质疑。因此，我们在体外检测了重组EDR1蛋白的激酶活性。EDR1激酶结构域具有自身磷酸化活性，并能磷酸化共同的MAP激酶底物髓磷脂碱性蛋白。EDR1的激动域也能磷酸化一种缺乏激酶的EDR1蛋白，这表明EDR1的自动磷酸化可以通过分子间机制发生。在野生型拟南芥中过表达的全长EDR1基因(35S：：dnEDR1)导致了显性的负显性表型，赋予了对白粉病的抗性，并促进了乙烯诱导的衰老。RNA-Gel印迹分析表明，35S：：dnEDR1转基因植株在转基因植株中得到了高效转录。然而，Western印迹分析表明，在这些品系中都没有检测到野生型和突变型EDR1蛋白，这表明显性负表型可能是由翻译抑制机制而不是蛋白质水平效应引起的。同源dnEDR1结构的过表达可能为控制作物白粉病提供一种新的策略。
The EDR1 gene of Arabidopsis has previously been reported to encode a Raf-like mitogen-activated protein kinase kinase (MAPKK) kinase, and to function as a negative regulator of disease resistance. A phylogenetic analysis of plant and animal protein kinases revealed, however, that plant Raf-like kinases are more closely related to animal mixed lineage kinases (MLKs) than Raf-like kinases, and are deeply divergent from both classes of animal kinases, making inferences of substrate specificity questionable. We, therefore, assayed the kinase activity of recombinant EDR1 protein in vitro. The EDR1 kinase domain displayed autophosphorylation activity and phosphorylated the common MAP kinase substrate myelin basic protein. The EDR1 kinase domain also phosphorylated a kinase-deficient EDR1 protein, indicating that EDR1 autophosphorylation can occur via an intermolecular mechanism. Overexpression of a kinase-deficient full-length EDR1 gene (35S::dnEDR1) in wild-type Arabidopsis plants caused a dominant negative phenotype, conferring resistance to powdery mildew (Erysiphe cichoracearum) and enhancing ethylene-induced senescence. RNA-gel blot analyses showed that the 35S::dnEDR1 transgene was highly transcribed in transgenic plants. Western blot analysis, however, revealed that neither the wild-type nor mutant EDR1 protein could be detected in these lines, indicating that the dominant negative phenotype may be caused by a translational inhibition mechanism rather than by a protein level effect. Overexpression of orthologous dnEDR1 constructs may provide a novel strategy for controlling powdery mildew disease in crops.