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Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls ofC. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly.

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Infection of the chestnut blight fungus Cryphonectria parasitica with Cryphonectria hypovirus 1 (CHV1) causes disruption of virulence, pigmentation, and sporulation. Transcriptional downregulation of key developmentally regulated fungal genes occurs during infection, but vegetative growth is unaffected. Previous studies showed that CHV1 utilizes trans -Golgi network (TGN) secretory vesicles for replication. In this study, the fungal cell surface hydrophobin cryparin was chosen as a marker to follow secretion in virally infected and noninfected strains. Subcellular fractionation, cryparin-green fluorescent protein (GFP) fusion, and Western blot studies confirmed that vesicles containing cryparin copurify with the same fractions previously shown to contain elements of the viral replication complex and the TGN resident endoprotease Kex2. This vesicle fraction accumulated to a much greater concentration in the CHV1-infected strains than in noninfected strains. Pulse-chase analysis showed that the rates and amount of cryparin being secreted by the CHV1 containing strains was much lower than in noninfected strains, and the dwell time of cryparin within the cell after labeling was significantly greater in the CHV1-infected strains than in the noninfected ones. These results suggest that the virus perturbs a specific late TGN secretory pathway resulting in buildup of a key protein important for fungal development.