Molecular Biosensing of the Aspergillus fumigatus Cell Wall Integrity Pathway Using the (p)agsA::luc (Luciferase) Reporter System.

In this protocol, we describe a robust luciferase-based biosensor assay to monitor the activity of the cell wall integrity (CWI) pathway in Aspergillus fumigatus in real time. The method relies on the stable integration of a markerless, synthetic reporter cassette (p)agsA::luc at the aft4 Safe Haven (Sh) genomic locus using CRISPR-Cas9. This cassette comprises a modified A. niger agsA promoter containing three tandem RlmA-binding sites, which drives the expression of the luciferase gene. Upon exposure to cell wall stress, the endogenous transcription factor RlmA activates the reporter, generating a luminescent signal proportional to promoter activity. The protocol includes the construction of aft4 locus-specific CRISPR-Cas9 plasmids, A. fumigatus transformation and candidate selection, as well as the setup of the luminescence bioassay in white 96-well microplates. This system enables highly sensitive, nondestructive, and time-resolved quantification of CWI pathway activation during early fungal growth or biofilm under various genetic or chemical perturbations. Moreover, it supports comparative studies across wild-type and mutant strains, offering a powerful platform for dissecting stress response signaling and identifying antifungal compounds that target the CWI pathway.