9527364 Arias The goal of these studies is to better understand how the expression of protein-encoding genes of plants are regulated by environmental stimuli. A central hypothesis to be tested here is that defense gene expression in plants is modulated by a phosphorylation-dependent change in the transcriptional activity of G-box binding factors. Aside from understanding a process of general importance to all higher eukaryotes the proposed studies may ultimately lead to the development of novel and sustainable forms of crop protection. Changes in the transcription activity of plant GBF's in response to defense cues (eg., nonhost bacterial species or biotic elicitors) cDNA's of these factors will be fused to a novel DNA-binding domain and expressed in plant cells containing a reporter gene. Changes in the phosphorylation of these chimeric GBF's will be assessed by phosphopeptide mapping. Protein kinases that phosphorylate GBF's in response to defense stimuli will be biochemically purified to obtain N-terminal sequences for cloning GBF-protein kinase cDNA's. Cloned putative kinases will be tested in vivo for their ability to phosphorylate GBF and act in defense-signaling. To identify potential mediators of plant GBF-dependent transcription a far-western screening of plant cDNA expression libraries with labeled GBF as probe will be performed. Cloned proteins will by functionally characterized by standard protein binding and transfection techniques. %%% Higher plants resist microbial infection in part by synthesizing phytoalexins. The rapid and transient activation of "defense" genes that encode for enzymes in phytoalexin biosynthesis largely determines the timing and magnitude of this response. Recent evidence suggests that protein kinases couple defense signal perception to enhanced gene expression. For example, inhibitors of protein kinases block the expression of plant defense genes, whereas agents that stimulate protein kinase activities in other metazoan systems both enhance pla nt defense gene expression and result in the transient phosphorylation of a few cellular proteins. How plant gene building factors function in signal-dependent transcription is currently unknown but, like in animals, may involve changes in the phosphorylation state of the factor. ***
