# Heterotrimeric G-proteins and cAMP regulate gene expression during growth on cellulose in Neurospora crassa

## TL;DR

The study shows that G-proteins and cAMP signaling in Neurospora crassa are crucial for regulating genes involved in breaking down cellulose.

## Contribution

The paper identifies the role of G-proteins and cAMP in upstream regulation of cellulase genes in Neurospora crassa.

## Key findings

- G-proteins and cAMP signaling strongly impact transcriptional control of cellulase activity.
- The Δcr-1 mutant showed the most misregulated genes during growth on cellulose.
- Overexpression of clr-2 restored cellulase activity in mutant strains.

## Abstract

Aspects of transcriptional regulation of plant cell wall-degrading enzyme (PCWDE) genes have been characterized in the filamentous fungus Neurospora crassa. However, the upstream signaling pathways that regulate PCWDE expression are not well understood. We have previously reported roles for heterotrimeric G-proteins and adenylyl cyclase in the degradation of cellulose to glucose in N. crassa. Here, we performed mRNA-seq to identify patterns of gene expression after transfer from glucose to cellulose medium in wild type, the Gα mutants Δgna-1 and Δgna-3, and the adenylyl cyclase mutant Δcr-1. In wild type, 3719 genes were regulated at least twofold during growth on cellulose vs glucose. Analysis of transcriptomics data for the strains after transfer from glucose to cellulose demonstrated that the Δcr-1 mutant had the most misregulated genes, with 2,232, followed by Δgna-3 with 1,182 and Δgna-1 with 648 genes. Metabolic genes were the most prevalent differentially expressed genes in the mutants. Expression of PCWDEs, including most of the cellulases, was downregulated in the three mutants, with Δcr-1 displaying the greatest deficiency. Furthermore, several transcription factors essential for cellulase expression were misregulated in the mutants. The primary factors clr-1 and clr-2 were downregulated in Δgna-3 and Δcr-1 strains, and clr-2 was reduced in Δgna-1 mutants. Overexpression of clr-2 restored cellulase activity and increased the expression of two major cellulase genes in all three mutants. Taken together, our results demonstrate that heterotrimeric G-proteins and cAMP signaling strongly impact transcriptional control of cellulase activity, culminating in the expression of the transcription factor clr-2 in N. crassa.

Filamentous fungi are important organisms for degradation of plant biomass. Both nonpathogens and plant pathogens secrete plant cell wall degrading enzymes to release simple sugars from the plant cell wall to use as carbon sources for growth. Much is known about the transcription factors that control production of plant cell wall-degrading enzymes by fungi. However, mechanistic details for how different lignocellulosic compounds are sensed by these organisms and the resultant cellular responses that operate upstream of cellulase-regulating transcription factors are lacking. Our research helps bridge this gap by identifying the role of G-protein subunits and cAMP in the regulation of gene expression during growth on cellulose. Understanding the environmental sensing and signal transduction pathways that regulate cellulase gene expression will have applications to agricultural losses due to plant pathogens, carbon recycling in the environment, and production of biofuels.