A Genomic Approach to Identify Regulatory Nodes in the Transcriptional Network of Systemic Acquired Resistance in Plants

Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. PLoS Pathog 2(11): e123. doi:10.1371/journal.ppat.0020123 A Genomic Approach to Identify Regulatory Nodes in the Transcriptional Network of Systemic Acquired Resistance in Plants Dong Wang 0 1 2 Nita Amornsiripanitch 0 1 2 Xinnian Dong 0 1 2 0 Current address: Department of Biological Sciences, Stanford University , Stanford, California , United States 1 Editor: Jeffrey Dangl, University of North Carolina , United States of America 2 Developmental, Cell and Molecular Biology Group, Department of Biology, Duke University , Durham, North Carolina , United States of America Many biological processes are controlled by intricate networks of transcriptional regulators. With the development of microarray technology, transcriptional changes can be examined at the whole-genome level. However, such analysis often lacks information on the hierarchical relationship between components of a given system. Systemic acquired resistance (SAR) is an inducible plant defense response involving a cascade of transcriptional events induced by salicylic acid through the transcription cofactor NPR1. To identify additional regulatory nodes in the SAR network, we performed microarray analysis on Arabidopsis plants expressing the NPR1-GR (glucocorticoid receptor) fusion protein. Since nuclear translocation of NPR1-GR requires dexamethasone, we were able to control NPR1-dependent transcription and identify direct transcriptional targets of NPR1. We show that NPR1 directly upregulates the expression of eight WRKY transcription factor genes. This large family of 74 transcription factors has been implicated in various defense responses, but no specific WRKY factor has been placed in the SAR network. Identification of NPR1regulated WRKY factors allowed us to perform in-depth genetic analysis on a small number of WRKY factors and test well-defined phenotypes of single and double mutants associated with NPR1. Among these WRKY factors we found both positive and negative regulators of SAR. This genomics-directed approach unambiguously positioned five WRKY factors in the complex transcriptional regulatory network of SAR. Our work not only discovered new transcription regulatory components in the signaling network of SAR but also demonstrated that functional studies of large gene families have to take into consideration sequence similarity as well as the expression patterns of the candidates. - Systemic acquired resistance (SAR) is an inducible plant defense response against pathogens. In Arabidopsis, the onset of SAR is preceded by an accumulation of the signaling molecule salicylic acid (SA). SA induces nuclear translocation of the transcription cofactor NPR1 to activate many genes required for disease resistance [1]. NPR1 also negatively feedback-regulates SA synthesis to mitigate its cytotoxic effect [2]. It is known that NPR1 controls the expression of antimicrobial pathogenesis-related genes (PR genes) by interacting with TGA transcription factors [35]. A microarray experiment showed that NPR1 also directly upregulates the protein secretory pathway. This is essential for SAR since disrupting this pathway diminished the secretion of PR proteins and resulted in reduced resistance [6]. NPR1 likely regulates these secretion-related genes through a novel transcription factor [6]. In addition to this unknown transcription factor and the TGAs, WRKY transcription factors have also been implicated in regulating the response against pathogen infection. Many WRKY genes are rapidly induced after treatment with elicitors associated with infection [79]. Moreover, genes induced during defense responses often contain WRKY transcription factorbinding sites, W boxes, in their promoter regions. For example, the promoter of an SA biosynthesis gene is enriched with W boxes [10]. The expression of NPR1 itself has been shown to be under the regulation of WRKY factors [11]. In a transcriptional profiling study, Maleck et al. discovered that W boxes are overrepresented in a cluster of genes sharing the induction pattern of PR-1, suggesting a role for WRKY factors in SAR [12]. Ectopic expression studies have shed some light on the functions of WRKY genes. Plants overexpressing WRKY70 have heightened resistance toward two bacterial pathogens [13]. Likewise, overexpressing WRKY18 resulted in gain of PR gene expression and resistance in a developmentally regulated manner [14]. Transiently overexpressed WRKY29, a target of a MAPK cascade activated by bacterial flagellin, also led to stronger resistance [15]. However, data from overexpression studies need to be interpreted with caution. For example, since ectopically expressing several WRKY genes all resulted in a similar range of phenotypes, it is difficult to conclude functional specificity from these studies. Typical of large gene families, phenotypic analysis of lossMany biological processes are controlled by intricate regulatory networks of gene expression. Identifying the regulatory nodes in these networks and understanding the hierarchical relationship between them are vital to our understanding of biological systems. However, this task is frequently hampered by the intrinsic complexity of these processes. Here, the authors used a controlled transcriptional profiling strategy to a plant immune response called systemic acquired resistance to study the transcriptional events one at a time. Systemic acquired resistance is activated through the induction of thousands of genes by the transcriptional regulator protein NPR1. The authors found that downstream of NPR1 are several regulatory nodes comprised of members from a large family of transcriptional factors. Disrupting these regulatory nodes compromised various functions assigned to NPR1, providing the information needed to construct a gene regulation network. of-function WRKY mutants has been hampered by functional redundancy. It has been reported that in a collection of more than 40 wrky knockout mutants, phenotypes were rarely observed [16]. This difficulty is further exacerbated by the wide range of defense responses in which WRKY factors participate. Therefore, to elucidate the function of specific WRKY genes, it is critical to identify a small number of candidates within a well-defined biological process. In this study, we used a genomics-directed approach to identify those WRKY genes whose expression is directly regulated by NPR1. The small number of candidate genes allowed more informed construction of double mutants and focused examination of the mutants on NPR1-associated phenotypes. As a result, we were able to find new regulatory nodes (i.e., WRKY factors) in the complex transcriptional regulatory network of SAR. Identification of Eight WRKY Genes as Direct Transcriptional Targets of NPR1 To dissect the transcriptional cascade leading to SAR, we performed a microarray exper (...truncated)