Downloaded from www.microbiologyresearch.org by IP: 54.146.27.20 On: Fri, 17 Feb 2017 04:07:51 Journal of General Microbiology (1989), 135, 2233-2239. Printed in Great Britain 2233 Ultrastructural Localization of a Cuticle-degrading Protease Produced by the Entornopathogenic Fungus Metarhizium anisopliae during Penetration of Host (Mmduca sexta) Cuticle By MARK S. GOETTEL,*t RAYMOND J. ST LEGER, NANCY W. RIZZO, RICHARD C. STAPLES AND DONALD W. ROBERTS Insect Pathology Resource Center, Boyce Thompson Institute, Tower Road, Cornell University, Ithaca, New York 14853, USA (Received 28 November 1988; revised 14 April 1989; accepted 24 April 1989) ~ ~~~ ~~~ ~ ~~ Gold-labelled rabbit antiserum was used to demonstrate that a cuticle-degrading protease (Pr 1) is produced by Metarhizium anisopliae during penetration of host (Manduca sexta) procuticle. The protease was secreted by infection structures (appressoria) on the cuticle surface and by the penetrant hyphae within the cuticle. Penetration of the procuticle was by a combination of enzymic degradation and mechanical pressure. Initially Prl was confined to the immediate vicinity of the fungal structures; however the enzyme diffused throughout the cuticle during later stages of pathogenesis. When hyphae were labelled during growth in culture under conditions conducive to rapid synthesis of Prl, gold particles distributed over the fungal cell wall, indicating binding of Prl to hyphae. INTRODUCTION The mode of penetration of fungal pathogens into their plant and animal hosts has been a matter of controversy for many years (Charnley, 1984; Van den Ende & Linskens, 1974). Penetration was considered to be accomplished by mechanical force if the cuticle was depressed inwards at the point of penetration or if cuticular structures appeared to be pushed aside by fungal growth. Lack of such a depression and the appearance of digestion was taken as evidence of enzymic penetration. However, evidence obtained from the plant pathogen Fusarium solani has shown that these two mechanisms are not mutually exclusive; ultrastructural studies with ferritin-conjugated antibody prepared against a plant cell wall degrading enzyme (cutinase) demonstrated the presence of the enzyme during penetration of host cuticle in conjunction with mechanical pressure (Shaykh et al., 1977). The major structural component of insect cuticle is protein, and recently it was shown that the entomopathogenic fungus Metarhizium anisopliae produces a cuticle-degrading protease with a chymoelastase specificity (Prl) on the surface of host cuticle during the infection process (St Leger et al., 1987a, b). Treating host cuticles with antisera against Prl or with specific inhibitors of Prl activity greatly reduced infection (St Leger et al., 1988a). A previous ultrastructural study of insect cuticle infected with M. anisopliae demonstrated apparent zones of histolysis around infection structures (Zacharuk, 1970 b). Using gold-labelled rabbit antisera to M. anisopliae chymoelastase Prl, we have followed the fate of this enzyme as it is synthesized by infection structures. The results suggest that penetration of the epicuticle is primarily by enzymic degradation while penetration of the procuticle involves both enzymic degradation and the mechanical separation of the lamellae. Present address : Crop Sciences Section, Research Station, Research Branch, Agriculture Canada, Lethbridge, Alberta, Canada TI J 4B1. 0001-5269 0 1989 SGM