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Phage display cDNA cloning and expression analysis of hydrophobins from the entomopathogenic fungus Beauveria (Cordyceps) bassiana

Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611, USA

Correspondence
Nemat O. Keyhani
keyhani{at}ufl.edu

Hydrophobins are small amphipathic proteins that function in a broad range of growth and developmental processes in fungi. They are involved in the formation of aerial structures, the attachment of fungal cells to surfaces, and act in signalling in response to surface cues and pathogenesis. Beauveria bassiana is an important entomopathogenic fungus used as an arthropod biological control agent. To examine the feasibility of using phage display technology to clone cDNAs encoding hydrophobins, biopanning experiments were performed using a variety of affinity resins, including N,N'-diacetylchitobiose-, fucose-, lactose-, maltose- and melibiose-coupled agarose beads. After five rounds of iterative biopanning, cDNAs corresponding to two B. bassiana (class I) hydrophobins were selectively enriched using melibiose- or lactose-coupled agarose beads. Expression analysis revealed that the hyd1 gene was expressed in all samples tested, including aerial conidia, in vitro blastospores, submerged conidia, and cells sporulating on chitin and insect cuticle, with hyd1 expression peaking in growing mycelia. In contrast, the hyd2 gene was not appreciably expressed in any of the single-cell types (aerial conidia, blastospores and submerged conidia), but was constitutively expressed in growing mycelia and when cells were sporulating on chitin and insect cuticle. MS fingerprinting of an 10 kDa protein found in boiling SDS-insoluble, trifluoroacetic acid-soluble extracts from aerial conidia identified the major component of the B. bassiana rodlet layer to be the hyd2 gene product. These results reveal the differential regulation of the isolated hydrophobins and indicate that phage display represents a novel approach to cDNA cloning of hydrophobins.

The GenBank/EMBL/DDBJ accession numbers for the hyd1 and hyd2 sequences of B. bassiana are EF452344 and EF520285, respectively.

Figures showing the sequences of hydrophobin genes used in phylogenetic analysis, atomic force micrographs and SDS-PAGE results obtained during rodlet extraction, MS results for a peptide fragment resulting from in-gel tryptic digestion, and BLAST matches of peptide fragment data are available with the online version of this paper.

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