Transcriptomic and molecular genetic analysis of the cell wall salvage response ofAspergillus nigerto the absence of galactofuranose synthesis

Abstract

SummaryThe biosynthesis of cell surface‐located galactofuranose (Galf)‐containing glycostructures such as galactomannan, N‐glycans and O‐glycans in filamentous fungi is important to secure the integrity of the cell wall. UgmA encodes an UDP‐galactopyranose mutase, which is essential for the formation of Galf. Consequently, the ΔugmA mutant lacks Galf‐containing molecules. Our previous work in Aspergillus niger work suggested that loss of function of ugmA results in activation of the cell wall integrity (CWI) pathway which is characterized by increased expression of the agsA gene, encoding an α‐glucan synthase. In this study, the transcriptional response of the ΔugmA mutant was further linked to the CWI pathway by showing the induced and constitutive phosphorylation of the CWI‐MAP kinase in the ΔugmA mutant. To identify genes involved in cell wall remodelling in response to the absence of galactofuranose biosynthesis, a genome‐wide expression analysis was performed using RNAseq. Over 400 genes were higher expressed in the ΔugmA mutant compared to the wild‐type. These include genes that encode enzymes involved in chitin (gfaB, gnsA, chsA) and α‐glucan synthesis (agsA), and in β‐glucan remodelling (bgxA, gelF and dfgC), and also include several glycosylphosphatidylinositol (GPI)‐anchored cell wall protein‐encoding genes. In silico analysis of the 1‐kb promoter regions of the up‐regulated genes in the ΔugmA mutant indicated overrepresentation of genes with RlmA, MsnA, PacC and SteA‐binding sites. The importance of these transcription factors for survival of the ΔugmA mutant was analysed by constructing the respective double mutants. The ΔugmA/ΔrlmA and ΔugmA/ΔmsnA double mutants showed strong synthetic growth defects, indicating the importance of these transcription factors to maintain cell wall integrity in the absence of Galf biosynthesis.

The FlbA-regulated predicted transcription factor Fum21 of Aspergillus niger is involved in fumonisin production

“…A. niger strain MA234.1 (transient ku70::amdS) (Park et al 2016 ) and its derivatives were grown at 30 °C using minimal medium (MM) (de Vries et al 2004 ) containing 25 mM xylose as a carbon source and either (MM-XA) or not (MM-X) containing 1.5% agar. Alternatively, strains were grown on Czapek Yeast Auto-lysate (CYA) agar (Frisvad and Samson 2004 ), Yeast Extract Sucrose (YES) agar (Frisvad and Samson 2004 ), or transformation medium (TM; MM containing 0.5% yeast extract, 0.2% casaminoacids, and 25 mM glucose, pH 6)(Kusters-van Someren et al 1991 ).…”

Updating genome annotation for the microbial cell factoryAspergillus nigerusing gene co-expression networks

“…Total RNA extraction, RNA quality control, and RNA sequencing were performed at GenomeScan (Leiden, the Netherlands). Quality analysis of raw data was done as previously described (30). In brief, ~13 million reads of 150 bp were obtained from paired-end mode for each sample.…”

A seven-membered cell wall related transglycosylase gene family in Aspergillus niger is relevant for cell wall integrity in cell wall mutants with reduced α-glucan or galactomannan

“…Moreover, we analyzed the role of Crh enzymes in the process of cell wall deficiency, by introducing a mutation in a gene required for cell wall galactomannan synthesis (Δ ugmA ) in the seven-fold crh knockout strain. A deletion of ugmA was previously shown to compromise cell wall integrity caused by a galactofuranose deficiency ( Damveld et al, 2008 ), and revealed upregulation of crhE in transcriptomic studies ( Park et al, 2016 ). Additionally, we assessed the relation between chitin to β-glucan cross-linking and α-1,3-glucan, by reducing cell wall α-glucan synthesis in both wild type and the seven-fold crh mutant.…”