Transcriptomic and molecular genetic analysis of the cell wall salvage response of Aspergillus niger to the absence of galactofuranose synthesis

Abstract

The 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.

Figure 1

The ΔugmA mutant strain has increased and constitutive CWI pathway activation. Conidia from the wild‐type and mutant strain were grown in liquid CM medium. The samples were collected at the indicated time points for Western blot preparation. The phosphorylated fractions and the total MpkA amount were detected using anti‐phospho p44/42 MAPK and anti‐p44/42 MAPK antibodies respectively. The γ‐tubulin antibody and the Coomassie Brilliant Blue stained gel were used as loading sample controls. Densitometry analysis of western blots showing the ratio of phosphorylated MpkA/MpkA expressed as the relative abundance (arbitrary units).

Figure 2

Cultivation of the wild‐type (N402) and the ΔugmA mutant in bioreactors. A. Hyphal morphology of N402 and ΔugmA mutant. Note the compact growth phenotype of the ΔugmA mutant. B. Dot blot assay to detect the presence of Galf residues on secreted glycoconjugates from A. niger during batch growth. A. niger wild‐type strain (N402) and ΔugmA mutant were grown for the times indicated from inoculation of the spores, and cell‐free medium was spotted on nitrocellulose filter paper. The blots were incubated with the anti‐Galf antibody (L10) (Heesemann et al., 2011) to detect the presence of Galf or incubated with ConA‐PO to detect mannoproteins.

Figure 3

Northern blot analysis of selected genes during batch growth in bioreactors. RNA was isolated from mycelium grown at pH 5.0 for 18 h, when about 60% of the glucose was consumed, and analysed by Northern blot. Hybridization with an actin probe was used to confirm similar loading.

Figure 4

Growth analysis of single and double mutants. Spores of single and double mutants were spotted in the centre of a MM plate and grown for four days at the indicated temperature. Synergistic growth reduction of the ΔugmA/msnA and ΔugmA/rlmA is indicative of an important role of the MsnA and RlmA transcription factors in survival of the ugmA mutant.

Figure 5

Overview of the number of genes overlapping between different cell wall stress conditions. Caspofungin (CA)‐induced genes (Meyer et al., 2007) and Aureobasidin A (AbaA)‐induced genes (Fiedler et al., 2014) were compared to ΔugmA‐induced genes (this study).

Figure 6

Overview of key transcription factors and some of their putative target genes involved in the cell wall salvage gene network of A. niger deduced from transcriptomic and phenotypic analysis. On the upper row, several cell wall stress conditions including FK506 (Calcineurin inhibitor), CFW (Calcofluor white), caspofungin (CA), ΔugmA (lack of galactofuranose biosynthesis), Aureobasidin A (AbaA) and fenpropimorph (FP). Arrows indicate (putative) signalling events between and the proteins involved in signalling based on transcriptomic or functional genomics studies. Boxed genes are the possible target genes of various transcription factors and the relation with the inducing condition. Major biological processes affected by the different treatments are depicted under the boxed genes. Despite limited overlap of the target genes induced by chemical compounds and in the ΔugmA mutant, the RlmA, MsnA and CrzA transcription factors are likely to play a key role in orchestrating the response.