Functional analysis and mode of action of phytotoxic Nep1-like proteins of Botrytis cinerea Yaite Cuesta Arenas, Eric R.I.C. Kalkman 1 , Alexander Schouten 2 , Mirjam Dieho, Peter Vredenbregt, Beatrice Uwumukiza, Miriam Osés Ruiz, Jan A.L. van Kan * Wageningen University, Laboratory of Phytopathology, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands article info Article history: Accepted 8 June 2010 Keywords: Gene expression Grey mould Necrotroph Phytotoxic proteins Site-directed mutagenesis Virulence factor abstract Nep1-like proteins (NLPs) induce necrosis and ethylene production in dicotyledonous plants. Botrytis cinerea contains two genes encoding NLPs, named Bcnep1 and Bcnep2. The activity of both proteins as well as the expression and function of the genes was studied. The genes are differentially expressed during pathogenesis. Mutants in either the Bcnep1 or Bcnep2 gene were equally virulent as the wild type strain. Site-directed mutant proteins were expressed in tobacco by agroinfiltration. Mutations in a conserved motif, or in either of two N-terminal cysteine residues abolished necrosis-inducing activity. The contribution of the plant to necrosis-inducing activity of B. cinerea NLPs was investigated using Arabidopsis mutants, virus-induced gene silencing and pharmacological inhibitors. The necrosis- inducing activity of B. cinerea NLPs does not seem to require cellular processes or defense signalling pathways previously identified to be involved in pathogen-induced plant cell death. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Botrytis cinerea is a necrotrophic plant pathogen with a broad host range [1]. An important determinant for virulence of B. cinerea is the production of secreted metabolites and proteins, which enable it to kill the host [2,3]. B. cinerea isolates can produce two chemically different phytotoxic metabolites (botrydial and botcinic acid) [4e8]. Besides genes involved in the biosynthesis of phyto- toxic metabolites [9,10], the B. cinerea genome contains several genes encoding phytotoxic proteins, including a protein designated SPL1 [11] and two paralogous proteins, BcNEP1 and BcNEP2, members of the Nep1-like protein (NLP) family [12,13]. NLPs are 24e26 kDa proteins that share in the central region a conserved heptapeptide motif (‘GHRHDWE’) not present in other proteins [14,15]. NLPs are divided into two groups, called type I and type II, based on the presence of either two or four conserved cysteine residues [15] and both types may occur in a single species. NLPs, first discovered in Fusarium oxysporum f.sp. erythroxyli [16], are present in bacterial, fungal and oomycete microbes, including pathogens and non-pathogens but lacking in the plant and animal kingdoms [15]. Purified NLPs in low concentrations can induce callose appo- sition, accumulation of reactive oxygen species and ethylene, acti- vation of genes involved in stress and defense responses [14,17,18] while at higher concentrations they induce cell death at the site of application [14e16]. NLPs are only phytotoxic to dicotyledonous plant cells [13,15,19]. In spite of these observations the importance of NLPs in pathogenesis remains elusive. Disruption of NLP- encoding genes in F. oxysporum f.sp. erythroxyli or Mycosphaerella graminicola did not result in reduced virulence [20,21]. The only evidence of a role of NLPs in virulence is in the soft-rotting bacteria Erwinia carotovora subsp. carotovora (syn. Pectobacterium car- otovorum) and E. carotovora subsp. atroseptica. Disruption of nip ecc and nip eca genes reduced virulence on potato [22]. Virulence of P . carotovorum mutants could partially be restored by comple- mentation with NLP-encoding genes from the oomycetes Phytoph- thora parasitica and Pythium aphanidermatum [23]. NLP-encoding genes can display differential expression during infection. In Phytophthora sojae, a hemibiotroph that causes root and stem rot on soybean, the gene encoding PsojNIP is expressed exclusively during late stages of the infection, corresponding to the transition to the necrotrophic phase [24]. Moniliophthora perniciosa, a basidiomycete that causes witches broom disease of cacao, possesses two NLP genes. Mpnep1 is expressed in the biotrophic and saprophytic phases whereas Mpnep2 is expressed only in the * Corresponding author. Tel.: þ31 317 483126; fax: þ31 317 483412. E-mail address: jan.vankan@wur.nl (J.A.L. van Kan). 1 Current address: Division of Infection & Immunity, Faculty of Biomedical & Life Sciences, Glasgow Biomedical Research Centre, University of Glasgow, 120 Univer- sity Place, Glasgow, United Kingdom. 2 Current address: INRES-Molecular Phytomedicine, University of Bonn, Nus- sallee 9, 53115 Bonn, Germany. Contents lists available at ScienceDirect Physiological and Molecular Plant Pathology journal homepage: www.elsevier.com/locate/pmpp 0885-5765/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2010.06.003 Physiological and Molecular Plant Pathology 74 (2010) 376e386