Induction of the qa-y and qa-1F genes in Neurospora crassa at differing times of quinic acid exposure

Abstract

Neurospora crassa, a model filamentous fungus, contains the quinic acid (qa) gene cluster, allowing the organism to effectively metabolize quinic acid as an alternative carbon source. In the presence of quinic acid, the qa gene cluster is significantly unregulated, showing an increased expression of both regulatory proteins as well as the structural proteins required for the utilization of quinic acid as an energy source. Of these structural genes, qa-2, qa-3, and qa-4 encode for the enzymes required for the conversion of quinic acid to protocathechuic acid while qa-y encodes for a quinate permease and the function of qa-x remains unknown. The present study aims to elucidate the expression pattern of qa-y, the quinate permease-producing gene required for efficient uptake and utilization of quinic acid in N. crassa, as well as qa-1F, the activator protein of the qa gene cluster, at differing times of quinic acid exposure. Quantitative real-time PCR (RT-qPCR) using reverse transcriptase was first used to determine Histone-3 as the optimal housekeeping reference gene. This gene was then used to accurately quantify the expression of both qa-y and qa-1F at differing times of exposure to quinic acid (0min, 15min, 30min, 1hr, 2hr, and 3hr). The expression of qa-y was observed to steadily increase for the first 30 minutes, showing up to a 17-fold change in expression after only 30 minutes following introduction of quinic acid. A prompt response of the qa-1F gene was also observed. At the 15-minute time point, a 3-fold increase in expression of the qa-1F occurred, presumably to initiate activation of transcription of the genes of the qa gene cluster, including itself. When combined, these data allow for a comprehensive representation of the timing and method of regulation of the qa gene cluster in N. crassa.