A Semi-In Vivo Transcriptional Assay to Dissect Plant Defense Regulatory Modules

Plants use different regulatory modules in response to changes in their surroundings. With the transcriptomic approaches governing all research areas, an integrative, fast, and sensitive approach toward validating genes of interest becomes a critical step prior to functional studies in planta. This chapter describes a detailed method for a quantitative analysis of transcriptional readouts of defense response genes using tobacco leaves as a transient system. The method uses Luciferase reporter assays to monitor activities of defense pathway promoters. Under normal conditions, the JASMONATE ZIM-DOMAIN (JAZ) proteins repress defense genes by preventing their expression. Here, we will provide a detailed protocol on the use of a dual-luciferase system to analyze activities of various defense response promoters simultaneously. We will use two well-characterized modules from the Jasmonic acid (JA) defense pathway; the JAZ3 repressor protein and the promoters of three of JA responsive genes, MYC2, 3 and 4. This assay revealed not only differences in promoter strength but also provided quantitative insights on the JAZ3 repression of MYCs in a quantitative manner.