Functional analysis of NLP genes from Botrytis elliptica

Wageningen University, Laboratory of Phytopathology, Wageningen, The Netherlands.
Molecular Plant Pathology (Impact Factor: 4.72). 03/2007; 8(2):209-14. DOI: 10.1111/j.1364-3703.2007.00382.x
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SUMMARY We functionally analysed two Nep1-like protein (NLP) genes from Botrytis elliptica (a specialized pathogen of lily), encoding proteins homologous to the necrosis and ethylene-inducing protein (NEP1) from Fusarium oxysporum. Single gene replacement mutants were made for BeNEP1 and BeNEP2, providing the first example of transformation and successful targeted mutagenesis in this fungus. The virulence of both mutants on lily leaves was not affected. BeNEP1 and BeNEP2 were individually expressed in the yeast Pichia pastoris, and the necrosis-inducing activity was tested by infiltration of both proteins into leaves of several monocots and eudicots. Necrotic symptoms developed on the eudicots tobacco, Nicotiana benthamiana and Arabidopsis thaliana, and cell death was induced in tomato cell suspensions. No necrotic symptoms developed on leaves of the monocots rice, maize and lily. These results support the hypothesis that the necrosis-inducing activity of NLPs is limited to eudicots. We conclude that NLPs are not essential virulence factors and they do not function as host-selective toxins for B. elliptica.

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    • "Purified NLPs in low concentrations can induce callose apposition , accumulation of reactive oxygen species and ethylene, activation 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. "
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    ABSTRACT: a b s t r a c t 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.
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    • "Except for deleting the four C-terminal amino acids, truncation of the protein leads to a complete loss of necrosis-inducing activity (Fellbrich et al., 2002). Among the green plants (Kingdom Plantae), only dicotyledons show a necrotic response when exposed to NLPs, whereas none of the monocotyledons tested showed any response to the protein (Pemberton & Salmond, 2004; Gijzen & Nürnberger, 2006; Staats et al., 2007a). Before the onset of visual symptoms, dicot cells express defence-related genes, accumulate calcium, phytoalexins and active oxygen species, increase mitogenactivated protein (MAP) kinase activity and they produce ethylene through expression of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase genes (Bailey, 1995; Fellbrich et al., 2000; Jennings et al., 2001; Veit et al., 2001; Fellbrich et al., 2002; Keates et al., 2003). "
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