Title

Phosphoproteomic and transcriptomic-driven study of the cell wall integrity signaling (CWIS) pathway in the model fungus, Aspergillus nidulans.

Abstract

Filamentous fungi are industrially relevant and medically significant. In both cases the cell wall plays a critical role in protecting fungi from physical and environmental stress.  To increase our general understanding of cell-wall repair, we focused on the model species Aspergillus nidulans studying the regulation of biosynthetic proteins responsible for wall maintenance and repair via the cell wall integrity signaling pathway (CWIS). The CWIS contains a mitogen-activated protein kinase (MAPK) cascade which becomes activated upon physical or environmental stress.  MpkA, the final kinase in this cascade, controls expression of some key wall biosynthesis genes (α-1,3-glucan synthases). However, the transcription of many other wall related genes (e.g., β-1-3-glucan and chitin synthase genes) is regulated in an unknown, MpkA-independent, manner. 

To better understand the various cellular roles MpkA plays during steady-state growth, we compared global changes in protein phosphorylation and gene expression between an mpkA deletion mutant (ΔmpkA) and its isogenic parent. We found strong evidence suggesting MpkA is involved in maintaining cell-wall strength, branching regulation, and response to iron starvation.  

Next, we sought to characterize the CWIS pathway and its downstream effectors. Because cellular signaling is a dynamic process, we used a multi-omic approach employing quantitative, label-free, mass spectrometry (short timeframe; 10 min) to assess protein phosphorylation and RNA-sequencing (long timeframe; 120 min) to assess gene-expression levels.  To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets.  Overall, we identified 794 phosphorylation sites and 1800 genes dynamically phosphorylated and expressed, respectively, upon cell wall perturbation.  Several of these phosphorylation sites belong to kinases, and 12 of these kinases were tested for their involvement in CWIS using deletion strains to determine cell-wall strength and micafungin sensitivity. Combining -omic data with characterization studies reveals putative, new connections between the CWIS pathway and calcium signaling, the high osmolarity-glycerol (HOG) pathway, and the septation initiation network (SIN). This multi-omic experiment was replicated in the ΔmpkA strain to develop hypotheses about which signaling events are MpkA-independent. This work demonstrates the capacity of a multi-omics approach to study signaling networks.