Downloaded from www.microbiologyresearch.org by IP: 54.162.133.179 On: Fri, 26 Feb 2016 11:28:25 pH controls both transcription and post- translational processing of the protease BcACP1 in the phytopathogenic fungus Botrytis cinerea Ste ´ phane Rolland,3 Christophe Bruel, Christine Rascle, Vincent Girard, Genevie ` ve Billon-Grand and Nathalie Poussereau Correspondence Nathalie Poussereau nathalie.poussereau @univ-lyon1.fr Universite ´ de Lyon, Laboratoire de Ge ´ nomique Fonctionnelle des Champignons Pathoge ` nes des Plantes, UMR5240, Universite ´ Lyon 1, CNRS, Bayer CropScience, 14–20 rue Pierre Baizet, 69263 Lyon Cedex 09, France Received 18 November 2008 Revised 19 December 2008 Accepted 29 December 2008 During pathogenesis, the ascomycete Botrytis cinerea secretes a range of cell-wall-degrading enzymes such as polygalacturonases, glucanases and proteases. We report the identification of a new member of the G1 family of proteases, BcACP1, which is secreted by B. cinerea during infection. The production of BcACP1 correlates with the acidification of the plant tissue, and transcriptional analysis of the Bcacp1 gene showed that it is only expressed under acidic growth conditions. Using a transcriptional reporter system, we showed that pH regulation of Bcacp1 is not mediated by the canonical PacC transcription factor binding site. Like other G1 proteases, BcACP1 is produced as a pro-enzyme. Trapping of the zymogen form allowed investigation of its maturation process. Evidence is presented for an autocatalytic proteolysis of the enzyme that is triggered by acidic pH. Environmental pH therefore controls Bcacp1 production at both the transcriptional and post-translational level. INTRODUCTION The first barrier encountered by a phytopathogenic fungus during infection is the host cuticule and the primary plant cell wall. Successful infection depends not only on the pathogen’s ability to penetrate and colonize the host tissues, but also on its ability to degrade the plant cell wall polymers (polysaccharides and several classes of structural proteins: Carpita & Gibeaut, 1993) into nutrients required for its development. Enzymic degradation of the parietal polymers is therefore considered an important aspect of plant fungal infections. Phytopathogenic fungi secrete a wide range of cell-wall-degrading enzymes that act at different stages of the infection. Among these enzymes are pectinases, cellulases and xylanases that are involved in polysaccharide degradation (St Leger et al., 1997; Cotton et al., 2003; Olivieri et al., 2004; Brito et al., 2006). Several classes of proteases have also been described (Billon-Grand et al., 2002; Bindschedler et al., 2003; Ten Have et al., 2004), whose proposed role would be to facilitate host tissue penetration and colonization by degrading structural plant cell wall proteins, and by releasing amino acids, which represent the pathogen’s main source of nitrogen and sulfur during infection (Rauscher et al., 1995). Fungi can secrete several proteases, each characterized by an optimal pH for activity, and many produced in correlation with the environmental pH. In Sclerotinia sclerotiorum for example, secreted proteases with an acidic optimum pH can be detected when the environment has been acidified via oxalic acid production by this phytopathogenic fungus (Marciano et al., 1983; Magro et al., 1984; Poussereau et al., 2001; Cotton et al., 2003). Similarly, in the insect pathogen Metarhizium anisopliae, a secreted subtilisin with an alkaline optimum pH can be detected when the ambient pH has become alkaline following production of ammonia by the fungus (St Leger et al., 1999). In order to ensure the secretion of these proteases in their optimal environment, their production needs to be controlled in response to the environmental pH. Although the effect of ambient pH on fungal growth, physiology and differentiation is well established, the molecular responses to environmental pH are only now being elucidated. In Aspergillus nidulans, alkaline pH is perceived and transmitted via the pal signalling pathway, which activates the zinc finger tran- scription factor PacC (Penalva & Arst, 2002). PacC is a transcriptional activator of alkaline-induced genes and a transcriptional repressor of acid-induced genes (Tilburn et al., 1995). PacC and its regulatory pathway are conserved in fungi, and it has been demonstrated that this regulator participates in the virulence of some pathogenic fungi (Rollins, 2003; Caracuel et al., 2003; You & Chung, 2007; Miyara et al., 2008). Abbreviations: GUS, b-glucuronidase; p.i., post-inoculation. 3Present address: Dartmouth Medical School, Genetics Department, Remsen 7400 Building, Hanover, NH 03755, USA. Microbiology (2009), 155, 2097–2105 DOI 10.1099/mic.0.025999-0 025999 G 2009 SGM Printed in Great Britain 2097