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

已从烟草中鉴定出一种蛋白质(命名为TDBA12)，它具有序列特异性的DNA结合活性，并且在对烟草花叶病毒(TMV)感染和水杨酸处理后的过敏反应中显著增加。此外，已分离到两个基因，它们编码与TDBA12相同的DNA结合蛋白，它们是最近在植物中发现的新的DNA结合蛋白WRKY家族的成员。TDBA12对磷酸酶处理敏感，提示蛋白质磷酸化在其激活中起作用。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节基因。表达反义WRKY基因的转基因烟草植株在TMV侵染后发生自发性病变，并表现出加剧的黄化和坏死。这些观察结果表明，WRKY蛋白可能被激活为在植物防御反应过程中被激活的蛋白激酶级联的目标的一般模型。WRKY蛋白反过来可能调节这些反应。这些研究的总体目标是进一步确定WRKY DNA结合蛋白的特征。首先，将分离TDBA12复合体，以确定与DNA结合活性相关的亚基，并分离它们的基因。其次，将采用转基因方法来破译WRKY基因的功能。将对表达不同WRKY蛋白的反义RNA的转基因烟草植株的表型进行鉴定。第三，体外和体内实验将被用来确定TDBA12的激活是否需要特定的蛋白激酶，如果是，WRKY蛋白或它们相互作用的蛋白是否是调节磷酸化的目标。还将做出重大努力来分离调节TDBA12的DNA结合活性的蛋白激酶。这些研究将通过揭示在植物防御反应中重要的新的调控成分和机制，促进我们对植物抗病分子基础的理解。

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.