SummaryThe entomogenous filamentous fungus, Beauveria bassiana expresses two hydrophobin genes, hyd1 and hyd2, hypothesized to be involved in cell surface hydrophobicity, adhesion, virulence, and to constitute the protective spore coat structure known as the rodlet layer. Targeted gene inactivation of hyd1 resulted in seemingly 'bald' conidia that contained significantly altered surface fascicles or bundles. These cells displayed decreased spore hydrophobicity, loss of water mediated dispersal, changes in surface carbohydrate epitopes and b-1,3-glucan distribution, lowered virulence in insect bioassays, but no effect on adhesion. In contrast, Dhyd2 mutants retained distorted surface bundles, but truncated/ incomplete rodlets could be seen within the bundles. Dhyd2 conidia displayed both decreased cell surface hydrophobicity and adhesion, but the mutant was unaffected in virulence. The double Dhyd1Dhyd2 mutant was distinct from the single mutants, lacking both bundles and rodlets, and displaying additively decreased cell surface hydrophobicity, reduced cell attachment and lowered virulence than the Dhyd1 mutant. Epitope tagged constructs of the proteins were used to examine the expression and distribution of the proteins and to demonstrate the continued presence of Hyd2 in the Dhyd1 strain and vice versa. The implications of our results with respect to fascicle and rodlet assembly on the spore surface are discussed.

Broad host range entomopathogenic fungi such as Beauveria bassiana attack insect hosts via attachment to cuticular substrata and the production of enzymes for the degradation and penetration of insect cuticle. The outermost epicuticular layer consists of a complex mixture of non-polar lipids including hydrocarbons, fatty acids, and wax esters. Long chain hydrocarbons are major components of the outer waxy layer of diverse insect species, where they serve to protect against desiccation and microbial parasites, and as recognition molecules or as a platform for semiochemicals. Insect pathogenic fungi have evolved mechanisms for overcoming this barrier, likely with sets of lipid degrading enzymes with overlapping substrate specificities. Alkanes and fatty acids are substrates for a specific subset of fungal cytochrome P450 monooxygenases involved in insect hydrocarbon degradation. These enzymes activate alkanes by terminal oxidation to alcohols, which are further oxidized by alcohol and aldehyde dehydrogenases, whose products can enter β-oxidation pathways. B. bassiana contains at least 83 genes coding for cytochrome P450s (CYP), a subset of which are involved in hydrocarbon oxidation, and several of which represent new CYP subfamilies/families. Expression data indicated differential induction by alkanes and insect lipids and four CYP proteins have been partially characterized after heterologous expression in yeast. Gene knockouts revealed a phenotype for only one (cyp52X1) out of six genes examined to date. CYP52X1 oxidizes long chain fatty acids and participates in the degradation of specific epicuticular lipid components needed for breaching the insect waxy layer. Examining the hydrocarbon oxidizing CYP repertoire of pathogens involved in insect epicuticle degradation can lead to the characterization of enzymes with novel substrate specificities. Pathogen targeting may also represent an important co-evolutionary process regarding insect cuticular hydrocarbon synthesis.

The insect epicuticle or waxy layer comprises a heterogeneous mixture of lipids that include abundant levels of long-chain alkanes, alkenes, wax esters and fatty acids. This structure represents the first barrier against microbial attack and for broad-host-range insect pathogens, such as Beauveria bassiana, it is the initial interface mediating the host-pathogen interaction, since these organisms do not require any specialized mode of entry and infect target hosts via the cuticle. B. bassiana is able to grow on straight chain alkanes up to n-C 33 as a sole source of carbon and energy. The cDNA and genomic sequences, including putative regulatory elements, for eight cytochrome P450 enzymes, postulated to be involved in alkane and insect epicuticle degradation, were isolated and characterized. Expression studies using a range of alkanes as well as an insect-derived epicuticular extract from the blood-sucking bug Triatomas infestans revealed a differential expression pattern for the P450 genes examined, and suggest that B. bassiana contains a series of hydrocarbon-assimilating enzymes with overlapping specificity in order to target the surface lipids of insect hosts. Phylogenetic analysis of the translated ORFs of the sequences revealed that the enzyme which displayed the highest levels of induction on both alkanes and the insect epicuticular extract represents the founding member of a new cytochrome P450 family, with three of the other sequences assigned as the first members of new P450 subfamilies. The remaining four proteins clustered with known P450 families whose members include alkane monooxygenases. INTRODUCTIONUnlike other insect-pathogenic micro-organisms which must be ingested to initiate disease (virus, bacteria, nematodes and protozoa), entomopathogenic fungi, such as Metarhizium anisopliae and Beauveria bassiana, mostly invade target hosts by penetrating through their cuticle.The outermost insect surface or epicuticle is covered by a lipid-rich layer, usually composed of very long-chain hydrocarbons together with variable amounts of fatty alcohols, fatty acids and wax esters. The epicuticle protects the insect from desiccation, chemical and biological attack, and acts as a platform for host semiochemicals (Blomquist et al., 1987;Figueiras et al., 2009;Juarez, 1994). Entomopathogenic fungi have the ability to degrade insect cuticular lipids with hydrocarbons, the preferred components capable of supporting fungal growth (Napolitano & Juarez, 1997). The first evidence of the complete catabolism of insect-like hydrocarbons by entomopathogenic fungi was obtained from B. bassiana and M. anisopliae (Crespo et al., 2000). In addition, alkane-grown B. bassiana showed enhanced virulence, by both increasing mortality, i.e. a greater proportion of target hosts killed over time (Crespo et al., 2002), and reducing the time to kill its insect hosts (Pedrini et al., 2009). Although several aspects of the biochemistry of the interaction between entomopathogenic Abbreviations: AhR, aryl hydrocarbon receptor; ARNT, AhR ...

Background:The lipid-rich insect epicuticle mediates the initial interaction with microbial pathogens. Results: A novel cytochrome P450, CYP52X1, implicated in cuticular hydrocarbon assimilation was characterized from Beauveria bassiana. Conclusion: CPY52X1 displays fatty acid hydroxylase activity, contributes to cuticle penetration, but is dispensable for virulence once the cuticle has been breached. Significance: These results expand the enzymatic repertoire entomopathogenic fungi express in targeting insects.