Design of a real-time PCR tool to study cell wall stress in fungi

Abstract

The cell wall integrity (CWI) pathway is responsible forthe reparation and/or biosynthesis of the cell wall and is activated whenchanges on the cell surface occurred mainly under imposed stress. A new toolable to detect changes in stress-related genes would be useful to understandthe action mechanism of some filamentous fungi against antifungal compounds. Theobjective was to develop a real-time PCR (qPCR) using SYBR Green to monitor changesin the Rho1 gene expression levels inmoulds. Optimization of reaction conditions includedevaluation of different primer pairs, primer concentrations and annealing temperatures/times.The final reaction mixture contained 200nM of each primer and an annealingtemperature of 55ºC. The specificity of primers was demonstrated when amplifieda unique qPCR product with a Tm value of 86ºC. The qPCR showed a R2 value =0.9994 and amplificationefficiency of 97.5%. The method was validated by treating Aspergillus flavus and Penicilliumpolonicum with the antifungal protein PgAFP. The PgAFP-resistant P. polonicum showed an overexpression ofRho1, while the opposite trend wasdetected in A. flavus with theantifungal treatment. This qPCR assay is a valuable tool to analyseintracellular responses linked to CWI pathway activation. This provides data toin-depth understand the ability of fungi to colonise different environments andto develop new antifungals.