Dynamic Transcriptomic and Phosphoproteomic Analysis During Cell Wall Stress in Aspergillus nidulans

Cynthia Chelius,Walker Huso,Samantha Reese,Alexander Doan,Stephen Lincoln,Kelsi Lawson,Bao Tran,Raj Purohit,Trevor Glaros,Ranjan Srivastava,Steven D Harris,Mark R Marten

doi:10.1074/mcp.ra119.001769

Abstract

The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a β-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture. From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 min, respectively. To differentiate statistically-significant dynamic behavior from noise, a multivariate adaptive regression splines (MARS) model was applied to both data sets. Over 1800 genes were dynamically expressed and over 700 phosphorylation sites had changing phosphorylation levels upon micafungin exposure. Twelve kinases had altered phosphorylation and phenotypic profiling of all non-essential kinase deletion mutants revealed putative connections between PrkA, Hk-8-4, and Stk19 and the CWIS pathway. Our collective data implicate actin regulation, endocytosis, and septum formation as critical cellular processes responding to activation of the CWIS pathway, and connections between CWIS and calcium, HOG, and SIN signaling pathways.

Highlights

multivariate adaptive regression splines (MARS) model identifies significantly dynamic gene expression and phosphorylation. Septation formation increases during cell-wall stress
Our findings reveal that the coordinated response of the calcium, high osmolarity-glycerol (HOG), and Septation Initiation Network (SIN) pathways are involved in the response to cell wall stress
These kinases are not members of the conserved cell-wall integrity signaling (CWIS) BckA-MkkA-MpkA mitogen activated protein kinase (MAPK) module [11] of which only the MAPKKK, BckA (S719, S1003) and MAPKK, MkkA (S169) were identified from phosphoproteomics. Both PrkA and Kin1 have been discussed above in regards to their roles in Arp2/3 regulation. As many of these kinases have not been characterized in A. nidulans, here we have identified their putative function in the CWIS network for the first time by combining phenotype and phosphoproteomic data

Summary

Introduction

MARS model identifies significantly dynamic gene expression and phosphorylation. Septation formation increases during cell-wall stress (via micafungin exposure). The fungal cell-wall integrity signaling (CWIS) pathway regulates cellular response to environmental stress to enable wall repair and resumption of normal growth. This complex, interconnected, pathway has been only partially characterized in filamentous fungi. To better understand the dynamic cellular response to wall perturbation, a ␤-glucan synthase inhibitor (micafungin) was added to a growing A. nidulans shake-flask culture From this flask, transcriptomic and phosphoproteomic data were acquired over 10 and 120 min, respectively. In the model fungus A. nidulans, the CWIS pathway is composed of cell surface receptors which sense cell wall stress [16] These sensors activate the guanine nucleotide exchange factor (GEF), Rom, which acts on the GTPase RhoA to trigger activation of PkcA (16 –19). MpkA, the final kinase in this cascade, activates the transcription factor RlmA which is responsible for controlling expression of ␣-1,3glucan synthase genes [11]

Results

Discussion

Conclusion