Promoter Analysis and Dissection Using Reporter Genes, Comparative Genomics, and Gel Shift Assays in Phytophthora

Transcriptional regulation allows cells to execute developmental programs, maintain homeostasis, and respond to intra- and extracellular signals. Central to these processes are promoters, which in eukaryotes are sequences upstream of genes that bind transcription factors (TFs) and which recruit RNA polymerase to initiate mRNA synthesis. Valuable tools for studying promoters include reporter genes, which can be used to indicate when and where genes are activated. Moreover, functional regions within promoters (typically TF binding sites) can be identified by integrating reporter assays with promoter mutagenesis. These sites may also be revealed through comparative genomics, or by the DNA-protein binding procedure known as a gel shift or electrophoretic mobility shift assay (EMSA). The latter can also be used to test if a specific TF binds a DNA target or assess the binding kinetics or affinity of the complex. In this chapter, we describe procedures for expressing reporter genes in Phytophthora, assaying reporter activity, identifying functional sites within promoters, and testing purified TFs or proteins within nuclear extracts for DNA binding.