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

Abstract

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,N9-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.

Mitogen-Activated Protein Kinase hog1 in the Entomopathogenic FungusBeauveria bassianaRegulates Environmental Stress Responses and Virulence to Insects

“…6), suggesting that Bbhog1 may influence the expression of some genes associated with hydrophobicity or adherence. In addition, hyd2 is expressed more strongly than hyd1 both in wild-type strain Bb0062 and in a ⌬Bbhog1 mutant, which is not consistent with the data described previously (9). The difference may be due to genetic diversity of different isolates.…”

“…These proteins form an outer layer of hyphae and conidia and play a role in a broad range of processes in the growth and development of filamentous fungi. They are involved in the formation of aerial structures and in the attachment of hyphae to hydrophobic surfaces (9,56). For instance, the hydrophobin MPG1 of M. grisea directs formation of a rodlet layer on conidia, which contributes to the hydrophobicity of the surface.…”

“…Primer sequences for the eight genes used are shown in Table 1. The transcript levels of hyd1 and hyd2 were ascertained using insect cuticle media as described previously (9). Conidia harvested from plates were inoculated into 1:4-diluted Sabouraud's dextrose broth containing 1% (wt/vol) cicada cuticle at a final concentration of 5 ϫ 10 5 conidia ml Ϫ1 and were grown for 3 days at 26°C with aeration, after which the total RNA was extracted and subjected to real-time RT-PCR analysis.…”

CYP52X1, Representing New Cytochrome P450 Subfamily, Displays Fatty Acid Hydroxylase Activity and Contributes to Virulence and Growth on Insect Cuticular Substrates in Entomopathogenic Fungus Beauveria bassiana

“…Adhesion of fungal conidia, which represents the main infectious particle, to the insect cuticle is mediated in part by cell surface proteins known as hydrophobins (22,23). Once attached, the conidia germinate and produce a battery of enzymes targeting cuticular substrates.…”

Comparative characteristic of the methods of protein antigens epitope mapping

“…Існують також й бібліотеки, що відображають всю мо-лекулу, частину протеїну чи його домен [30]. Також чимало прикладів використання фаго-вого дисплею для встановлення антигенної будови протеїнів різного походження, напри-клад: α 2 -макроглобулін [39], PDZ-домени різних адаптерних протеїнів [40], вірусні протеїни (нуклеокапсидний протеїн вірусу свинячого репродуктивного та респіраторного синдрому [41], капсидний протеїн вірусу катаральної лихо-манки овець [42], глікопротеїн вірусу класичної свинячої лихоманки [43]), протеїни-антигени патогенних мікроорганізмів (гемофільної па-лички Пфейффера Haemophilus influenzae, збудника м'якого шанкру Haemophilus ducreyi, одного з етіологічних агентів парадонтиту Aggregatibacter actinomycetemcomitans, збудник пастерельозу Pasteurella multocida [44], енто-мопатогенний мікроскопічний гриб Beauveria bassiana [45]). …”