Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, Lecanicillium spp: implications fo
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ORIGINAL PAPER
Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, Lecanicillium spp: implications for host specificity Natasha J. Bye Æ A. Keith Charnley
Received: 18 April 2007 / Revised: 13 June 2007 / Accepted: 4 August 2007 / Published online: 6 September 2007 Springer-Verlag 2007
Abstract The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data).
Communicated by Axel Brakhage. N. J. Bye A. K. Charnley (&) Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK e-mail: [email protected] Present Address: N. J. Bye Nutricia Ltd, Trowbridge BA14 0XQ, UK
Keywords Protease Regulation Fungus Pathogen Specificity Lecanicillium Verticillium Insect Aphid Abbreviations dH2O Suc–Ala–Ala– Pro–Phe–NA Bz–Phe–Val–Arg– NA NAG BSA IEF
Distilled water Succinyl–alanyl–alanyl–prolyl– phenylalanyl–p–nitroanilide N–benzoyl–phenylalanyl–valyl– arginyl–p–nitroanilide N-acetylglucosamine Bovine serum albumen Isoelectric focusing
Introduction Entomopathogenic fungi invade their hosts through the external skeleton (cuticle). This is a complex, composite structure with a high-protein content. Thus, particular attention has been focused on the role of proteases in the penetration process (see Charnley 2003 for a recent review). The Ascomycotina fungus Metarhizium anisopliae produces multiple isoforms of a number of families of extracellular cuticle-degrading proteases viz. subtilisins (Pr1), trypsin-like proteases (Pr2), chymotrypsins, metalloendoproteases and several classes of exo-acting enzymes (Screen and St. Leger 2000; St. Leger et al. 1993, 1994a, b). The cDNAs and genes of representatives of several classes of
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