The proteome and phosphoproteome of Neurospora crassa in response to cellulose, sucrose and carbon starvation

Abstract

Improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. How fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. Here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus Neurospora crassa grown on different carbon sources, i.e. sucrose, no carbon, and cellulose, by performing isobaric tags for relative and absolute quantification (iTRAQ)-based LC–MS/MS analyses. A comparison between proteomes and transcriptomes under identical carbon conditions suggests that extensive post-transcriptional regulation occurs in N. crassa in response to exposure to cellulosic material. Several hundred amino acid residues with differential phosphorylation levels on crystalline cellulose (Avicel) or carbon-free medium vs sucrose medium were identified, including phosphorylation sites in a major transcriptional activator for cellulase genes, CLR1, as well as a cellobionic acid transporter, CBT1. Mutation of phosphorylation sites on CLR1 did not have a major effect on transactivation of cellulase production, while mutation of phosphorylation sites in CBT1 increased its transporting capacity. Our data provides rich information at both the protein and phosphorylation levels of the early cellular responses to carbon starvation and cellulosic induction and aids in a greater understanding of the underlying post-transcriptional regulatory mechanisms in filamentous fungi.

Regulation of the lignocellulolytic response in filamentous fungi

“…Although there are several highly conserved nutrient-sensing pathways that have been implicated in the lignocellulosic response, their role in the regulation of plant cell wall degrading enzyme transcription is still unclear. For example, the TOR complex is involved in nutrient sensing in eukaryotes and has been shown to be highly phosphorylated under cellulolytic conditions in N. crassa (Wullschleger et al, 2006;Xiong et al, 2014a). Putative ubiquitination and deubiquitination enzymes, such as CreB/CRE-2 and CreD, have been implicated in CCR and glycosyl hydrolase production potentially through ubiquitination of CreA itself, however, additional protein targets and the precise mechanism of action of the genes involved has yet to be identified (Boase and Kelly, 2004;Colabardini et al, 2012;Denton and Kelly, 2011;Kelly, 2001, 2002;Ries et al, 2016;Xiong et al, 2014b).…”

Chemotropism and Cell Fusion in Neurospora crassa Relies on the Formation of Distinct Protein Complexes by HAM-5 and a Novel Protein HAM-14

“…The MAPK phosphorylation site (amino acid S73) is marked by a yellow box and underlined. The 23 additional phosphorylation sites (T39, T43, T46, S47, S49, S61, S63, T65, S66, S73, T91, Y95, S345, S352, T357, S436, T496, S510, S538, S542, T550, T555, and S558) are marked in blue (Jonkers et al 2014) and green boxes (Xiong et al 2014). (B) HAM-14-GFP localizes in the cytoplasm and occasional puncta in communicating germlings (white arrow).…”

“…Eight HAM-14 phosphopeptides were detected in our phosphoproteomics experiments (Jonkers et al 2014); one phosphopeptide has a MAPK consensus site, while two additional phosphopeptides have a degenerate MAPK consensus site (pS/pT-P). From a subsequent phosphoproteomic study on N. crassa hyphal cultures (Xiong et al 2014), an additional 33 phosphopeptides were identified for HAM-14. Thus, 24 possible unique phosphosites are present in HAM-14.…”

Analysis of the Candida albicans Phosphoproteome

“…In the fungus Fusarium graminearum, 2,902 putative phosphopeptides on 1,496 different proteins were identified (23). Phosphoproteomics data sets for the fungi Cryptococcus neoformans (24), Aspergillus nidulans (25), Alternaria brassicicola (26), Botrytis cinerea (26), Neurospora crassa (27), and Schizosaccharomyces pombe (28) have also been published.…”