Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis

To understand how plant host genes are regulated during the activation of plant defence responses, we are studying a group of pathogen- and salicylic acid (SA)-induced DNA-binding proteins containing the novel WRKY domain. To identify downstream target genes of these WRKY proteins, we have searched the Arabidopsis genome and identified four closely linked genes on chromosome IV that contain an unusually large number of the W-box sequences [(T)TGAC(C/T)] recognized by WRKY proteins within a few hundred base pairs upstream of their coding regions. All four genes encode proteins characteristic of receptor-like protein kinases (RLK), each consisting of an N-terminal signal sequence, an extracellular receptor domain, a single transmembrane domain and a C-terminal cytoplasmic serine/threonine protein kinase domain. All four RLK genes were induced by treatment with SA or infection by a bacterial pathogen. Studies with one of the RLK genes (RLK4) indicated that a cluster of W-box elements in its promoter region were recognized by both purified WRKY proteins and SA-induced W-box binding activities from SA-treated Arabidopsis plants. Further analysis using the RLK4 gene promoter fused to a reporter gene in transgenic Arabidopsis indicated that the consensus WRKY protein-binding sites in the RLK4 gene promoter were important for the inducible expression of the reporter gene. These results indicate that pathogen- and SA-induced W-box binding proteins regulate not only genes encoding defence proteins with direct or indirect anti-microbial activities, but also genes encoding proteins with regulatory functions.     

Evidence for an Important Role of WRKY DNA Binding Proteins in the Regulation of NPR1 Gene Expression

The Arabidopsis NPR1 gene is a positive regulator of inducible plant disease resistance. Expression of NPR1 is induced by pathogen infection or treatment with defense-inducing compounds such as salicylic acid (SA). Transgenic plants overexpressing NPR1 exhibit enhanced resistance to a broad spectrum of microbial pathogens, whereas plants underexpressing the gene are more susceptible to pathogen infection. These results suggest that regulation of NPR1 gene expression is important for the activation of plant defense responses. In the present study, we report the identification of W-box sequences in the promoter region of the NPR1 gene that are recognized specifically by SA-induced WRKY DNA binding proteins from Arabidopsis. Mutations in these W-box sequences abolished their recognition by WRKY DNA binding proteins, rendered the promoter unable to activate a downstream reporter gene, and compromised the ability of NPR1 to complement npr1 mutants for SA-induced defense gene expression and disease resistance. These results provide strong evidence that certain WRKY genes act upstream of NPR1 and positively regulate its expression during the activation of plant defense responses. Consistent with this model, we found that SA-induced expression of a number of WRKY genes was independent of NPR1.     

Expression Profiles of Arabidopsis thaliana VQ Gene Family in Defense Related Hormones Treatments

Several VQ proteins are recently identified as WRKY factors interacting partners in Arabidopsis and involved in regulating physiological processes. Searching of genomic databases found that VQ gene family is specific to land plants and these VQ genes encode proteins characteristic of a conserved VQ motif. It consists of 34 representatives in Arabidopsis and can be divided into two groups based on the similarity of the amino acid sequences. To understand the functions of Arabidopsis VQ proteins, we examined the expression profiles of AtVQ genes in various defense related hormones treatments. qRT PCR analysis revealed that a majority of them were differently regulated in response to salicylic acid (SA), methyl jasmonate (MeJA), or 1 aminocyclopropane 1 carboxylate (ACC). And some members are induced by two of these three hormones. Moreover, four members (AtVQ3, AtVQ18, AtVQ23 and AtVQ24) are induced by SA, MeJA and ACC simultaneity. However, there is only one AtVQ gene (AtVQ27) is up regulated after spraying of abscisic acid (ABA). These results suggest that Arabidopsis VQ genes may be involved in plant defense responses.     

拟南芥VQ基因家族响应抗性相关激素表达谱分析

近年来与WRKY转录因子相互作用的VQ-motif蛋白逐渐引起广泛关注.它是一类植物特异性蛋白,目前在拟南芥(Arabidopsis thaliana)中已鉴定到34个成员,据其所编码氨基酸的序列相似性构建系统进化树,这34个成员聚集成两大簇.实时定量荧光PCR分析表明在SA(水杨酸),MeJA(茉莉酸甲酯),ACC(乙烯前体),ABA(脱落酸)处理下AtVQ基因家族中有多个成员分别受SA,JA,ET高倍诱导,部分成员受SA,JA,ET中两种激素诱导,其中AtVQ3,AtVQ18,AtVQ23和AtVQ24同时受SA,JA,ET诱导,仅AtVQ27受ABA诱导,推测拟南芥VQ家族成员在抗病方面起作用.