Molecular genetic analysis of vesicular transport in Aspergillus niger reveals partial conservation of the molecular mechanism of exocytosis in fungi

Abstract

The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologues of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in Saccharomyces cerevisiae, was analysed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p orthologue in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in S. cerevisiae is lethal. Similarly, deletion of the A. niger orthologue of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (encoding SecA, SecH and SsoA the A. niger orthologues of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similar to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.

FgSec2A, a guanine nucleotide exchange factor of FgRab8, is important for polarized growth, pathogenicity and deoxynivalenol production in Fusarium graminearum

“…Rabin8/Rabin3 and GRAB are two mammalian Sec2 homologues, which exhibit GEF activity toward Rab8 (Hattula et al, 2002;Yoshimura et al, 2010). SEC2 deletion is lethal in yeast (Nair et al, 1990), but AnSEC2 deletion shows a mild phenotype in Aspergillus niger (Kwon et al, 2014). Rabin8 as a GEF for Rab8 is required for ciliogenesis and epithelial lumenogenesis (Bryant et al, 2010) but can also suppress autophagosome formation independent of its GEF activity toward Rab8 (Amagai et al, 2015).…”

Functional distinction of hyphal compartments

“…The Spitzenkörper of A. niger FG7 is fluorescently labelled due to a eGFP:: SncA fusion protein 28 . Surface area, length, and fluorescence intensity of the Spitzenkörper in the apical compartment was determined before and after dissecting the 2 nd compartment.…”

“…strains N402 34 , Δ hexA 26 , and FG7 28 of A. niger were grown at 30 °C in water-saturated air at 700 lux white light (Osram Lumilux L36w/840, Osram, Munich, Germany). Spores were harvested in 10 ml 0.9% NaCl (w/v), 0.05% (v/v) Tween-20 from 7-day-old cultures grown in 9 cm Petri dishes on complete medium (CM) consisting of minimal medium (0.6% NaNO 3 , 0.15% KH 2 PO 4 , 0.05% KCl, 0.05% MgSO 4 7H 2 O, 0.2 ml −1 Vishniac solution 35 ) with 0.2% tryptone, 0.1% casamino acids, 0.1% yeast extract, 0.05% yeast ribonucleic acids, 1.5% agarose, and 25 mM maltose.…”

Endocytosis and exocytosis in hyphal growth

“…In fungi, the exocyst is composed of eight proteins, corresponding to S. cerevisiae Exo70p, Exo84p, Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, and Sec15p (TerBush et al, 1996;Guo et al, 1999a,b). Of these proteins, only Sec3 is dispensable in yeast (Haarer et al, 1996), which also holds true for Aspergillus niger and Candida albicans (Kwon et al, 2014;Li et al, 2007). In N. crassa, however, Sec-3 was essential and Sec-5 was not , and both a Magnaporthe oryzae exo70 and a sec5 deletion were viable (Giraldo et al, 2013).…”