Pathogen-Induced WRKY DNA-Binding Proteins as Regulators of Plant Defense Responses

已经鉴定了来自烟草植物的蛋白质（命名为TDBA12），其具有序列特异性DNA结合活性，并且在对烟草花叶病毒（TMV）感染的过敏反应期间和用水杨酸处理之后显著增加。 此外，已经分离出两个基因，它们编码与TDBA12具有相同DNA结合特异性的蛋白质，并且是最近在植物中发现的DNA结合蛋白的新WRKY家族的成员。 TDBA12对磷酸酶处理敏感，表明蛋白磷酸化在其活化中的作用。 TDBA12的潜在靶基因包括编码具有保护和防御功能的蛋白质的基因以及调控基因。 表达反义WRKY基因的转基因烟草植株在TMV感染后发生自发性病变并表现出增强的失绿和坏死。 这些观察结果表明，WRKY蛋白可能被激活的蛋白激酶级联反应，在植物防御反应过程中激活的目标的一般模型。 WRKY蛋白反过来可以调节这些反应。 这些研究的总体目标是进一步表征WRKY DNA结合蛋白。 首先，将分离TDBA12复合物以鉴定与DNA结合活性相关的亚基并分离其基因。 第二，将采用转基因方法来破译WRKY基因的功能。 表达对不同WRKY蛋白特异性的反义RNA的转基因烟草植物的表型将被表征。 第三，将使用体外和体内实验来确定TDBA12活化是否需要特定的蛋白激酶，如果需要，WRKY蛋白或其相互作用蛋白是否是调节磷酸化的靶。 一个主要的努力也将作出分离的蛋白激酶，调节DNA结合活性的TDBA12。 这些研究将通过揭示植物防御反应中重要的新的调控成分和机制，推进我们对植物抗病性分子基础的理解。

A protein (designated TDBA12) from tobacco plants has been identified that has sequence-specific DNA-binding activity, and that increases markedly during the hypersensitive response to infection with tobacco mosaic virus (TMV) and following treatment with salicylic acid. Furthermore, two genes have been isolated that encode proteins with the same DNA-binding specificity as TDBA12 and which are members of the novel WRKY family of DNA-binding proteins recently found in plants. TDBA12 is sensitive to phosphatase treatment, suggesting a role for protein phosphorylation in its activation. Potential target genes of TDBA12 include genes encoding proteins with protective and defensive functions as well as regulatory genes. Transgenic tobacco plants expressing antisense WRKY genes develop spontaneous lesions and exhibit enhanced chlorosis and necrosis after TMV infection. These observations suggest a general model in which WRKY proteins may be activated as targets of the protein kinase cascade that is activated during plant defense responses. WRKY proteins in turn may regulate these responses. The overall objective of these studies is to characterize further the WRKY DNA-binding proteins. First, the TDBA12 complex will be isolated to identify the subunits associated with DNA-binding activity and to isolate their genes. Second, transgenic approaches will be taken to decipher the functions of the WRKY genes. The phenotypes of transgenic tobacco plants expressing antisense RNAs specific for different WRKY proteins will be characterized. Third, in vitro and in vivo experiments will be used to determine if specific protein kinases are required for TDBA12 activation and, if so, whether WRKY proteins or their interacting proteins are the targets of the regulatory phosphorylation. A major effort will also be made to isolate the protein kinase that regulates the DNA-binding activity of TDBA12. These studies will advance our understanding of the molecular basis of plant disease resistance by revealing new regulatory components and mechanisms important in plant defense responses.