Obligate fungal biotrophs have co-evolved with their plant hosts, a direct result of an intimate interaction that protects the integrity of the plant during pathogenesis, allowing it to obtain essential nutrients. To restrict the establishment of pathogen colonization, plants have evolved complex regulatory mechanisms to control the defense response, the most extreme of which involves Resistance (R) gene-mediated programmed cell death. While it is known that de novo gene expression and subsequent protein synthesis are required for several cell death programs, the primary transcriptional targets of R gene-mediated responses are unknown. Two alternative approaches were used to identify these transcriptional targets. The first approach uses a time-course microarray experiment that contrasts wild-type and loss-of-function mutant alleles of the Mla (powdery mildew) R gene to identify transcripts that distinguish incompatibility from compatibility. Earlier expression and stronger transcriptional responses were observed in compatible plants at 20 hours after inoculation, though this reaction diminished at later time points. In contrast, incompatible interactions exhibited a time-dependent strengthening of the transcriptional response, with increases in both fold change and total number of genes differentially expressed. These results implicate MLA as a repressor of early gene expression response and provides further evidence for a link between basal and R gene-mediated resistance. The second approach uses natural variation present in a doubled-haploid population to identify the regulatory hierarchy of gene expression during the interaction of barley and stem rust. A trans-eQTL hotspot is not associated with the R gene Rpg-TTKSK, but instead an inoculation-dependent expression polymorphism in Adf3 implicates it as a candidate susceptibility gene. In contrast, co-localization of a trans-eQTL hotspot with an enhancer of R gene-mediated resistance to stem rust associates the suppression of gene expression with enhanced resistance. Lastly, Blufensin1 (Bln1) is used as a case study for functional analysis using gene expression, structural features, and phenotype. Although greater expression of Bln1 was previously associated with incompatibility, virus-induced gene silencing and transient overexpression implicates that Bln1 negatively impacts defense. Collectively, these studies suggest that our understanding of gene expression and its phenotypic consequences is more complex than previously thought.