Investigating Aspergillus nidulans secretome during colonisation of cork cell walls

Abstract

Cork, the outer bark of Quercus suber, shows a unique compositional structure, a set of remarkable properties, including high recalcitrance. Cork colonisation by Ascomycota remains largely overlooked. Herein, Aspergillus nidulans secretome on cork was analysed (2DE).Proteomic data were further complemented by microscopic (SEM) and spectroscopic (ATR-FTIR) evaluation of the colonised substrate and by targeted analysis of lignin degradation compounds (UPLC-HRMS). Data showed that the fungus formed an intricate network of hyphae around the cork cell walls, which enabled polysaccharides and lignin superficial degradation, but probably not of suberin. The degradation of polysaccharides was suggested by the identification of few polysaccharide degrading enzymes (β-glucosidases and endo-1,5--L-arabinosidase).Lignin degradation, which likely evolved throughout a Fenton-like mechanism relying on the activity of alcohol oxidases, was supported by the identification of small aromatic compounds (e.g. cinnamic acid and veratrylaldehyde) and of several putative high molecular weight lignin degradation products. In addition, cork recalcitrance was corroborated by the identification of several protein species which are associated with autolysis. Finally, stringent comparative proteomics revealed that A. nidulans colonisation of cork and wood share a common set of enzymatic mechanisms. However the higher polysaccharide accessibility in cork might explain the increase of β-glucosidase in cork secretome.

The old 3-oxoadipate pathway revisited: New insights in the catabolism of aromatics in the saprophytic fungus Aspergillus nidulans

“…Mycelial and extracellular proteins were obtained and quantified as previously described (Carvalho et al, 2013;Martins et al, 2014a). Mycelial (100 mg powder mixed with 20 mg poly(vinyl)polypyrrolidone) and extracellular (concentrated using 10 kDa centrifugal devices) proteins were precipitated with cold acetone containing 10% w/v trichloroacetic acid and 60 mM dithiothreitol (DTT).…”

“…Protein isoelectric focusing, electrophoresis, staining, image acquisition and analysis followed previously described methods (Carvalho et al, 2013;Martins et al, 2014a). The gels were stained with flamingo dye (Bio-Rad) or colloidal coomassie blue for image acquisition or spot excision, respectively.…”

“…The differential protein spots were excised from gels manually, and processed using the Ettan Digester robot of the Ettan Spot Handling Workstation (GE Healthcare) (Carvalho et al, 2013;Martins et al, 2014a). Samples (0.7 lL) were then spotted on MALDI-TOF target plates (Applied Biosystems), before the deposit of 0.7 lL CHCA (7 mg mL À1 in ACN 50%, TFA 0.1%).…”

Elucidating how the saprophytic fungus Aspergillus nidulans uses the plant polyester suberin as carbon source

“…Previous studies on A. nidulans colonisation of cork revealed that suberin remained unaltered [103] probably because the outermost lignin-enriched cell wall layers hampered its degradation. Here we have shown that A. nidulans was able to utilise suberin macromolecules as sole carbon source (Figure 1b) and that the fungus also underwent sexual development (Figure 4) and boosted secondary metabolism (Table 1).…”

Physical, Chemical and Proteomic Evidence of Potato Suberin Degradation by the Plant Pathogenic Bacterium <i>Streptomyces scabiei</i>

“…Although the degradation of suberin has not been extensively examined, it has been attributed to soil fungal populations (14). When Aspergillus nidulans was grown on suberized cell walls, the degradation of the suberin aromatic domain was corroborated by the identification of some degradation products, whereas the aliphatic domain remained virtually intact (20). However, a whole-genome transcriptome analysis of A. nidulans , which was conducted in order to identify the main pathways involved in suberin degradation, proposed that initial suberin degradation involved ester hydrolysis, possibly through the actions of cutinase 1 and some lipases (21).…”