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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ABSTRACT 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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Available from: Alexander Schouten, Sep 25, 2015
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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.
    Physiological and Molecular Plant Pathology 09/2010; 74(5). DOI:10.1016/j.pmpp.2010.06.003 · 1.41 Impact Factor
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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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    ABSTRACT: Nep1-like proteins (NLPs), produced by an array of unrelated microorganisms, are phytotoxic for dicotyledonous plant cells but their mode of action has not yet been established. Two paralogous NLPs from the necrotrophic plant pathogenic fungus Botrytis cinerea were characterized, designated BcNEP1 and BcNEP2. Both proteins were produced in the heterologous host Pichia pastoris and purified to homogeneity. The localization of fluorescently labelled proteins was studied and mechanisms of cell death were investigated in protoplasts and suspension cells. Purified BcNEP1 and BcNEP2 caused necrosis in all dicotyledonous plant species tested, but not in monocotyledons. A synthetic heptapeptide comprising a sequence (GHRHDWE) that is conserved in all NLPs did not cause symptoms and was unable to interfere with necrosis induction by BcNEP1 and BcNEP2 proteins. Fluorescently labelled BcNEP1 and BcNEP2 proteins were associated with plasma membranes and the nuclear envelope, as well as in the nucleolus of responding plant cells. A strong hydrogen peroxide (H(2)O(2)) accumulation was observed in chloroplasts. The death process was characterized by TUNEL assays as apoptosis, necrosis or intermediate forms of both. BcNEP1- and BcNEP2-induced cell death execution could not be abolished by specific inhibitors. These results provide further information on mechanisms of NLP-inflicted cell death.
    New Phytologist 02/2008; 177(2):493-505. DOI:10.1111/j.1469-8137.2007.02274.x · 7.67 Impact Factor
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    ABSTRACT: Botrytis spp. associated with neck rot disease were isolated from New Zealand onions. The fungi were identified using molecular sequences of the ribosomal internal transcribed spacer (ITS) and intergenic spacer (IGS) regions, and the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene. Analyses of the sequences showed that the majority of the isolates gathered in 2005–07 were B. aclada. A new high resolution melting analysis (HRMA) assay was developed that allowed fast and simple discrimination between B. aclada and other Botrytis spp. causing onion neck rot in New Zealand. To further verify these results, Botrytis isolates from New Zealand onions, stored in the International Collection of Microorganisms from Plants (ICMP), were also examined. Only a single isolate from the ICMP collection was B. aclada while two isolates were B. byssoidea, one B. squamosa and another closely related to Botryotinia porri. Identification of the remaining Botrytis isolates was more difficult; while IGS and ITS sequences indicated a close relationship to B. allii or B. byssoidea, a previously unreported intron insertion was observed at the 3′ end of the ribosomal small subunit gene in these isolates. No evidence of heterogeneity was observed in the G3PDH gene sequences, as might have been expected of the allodiploid B. allii, but the G3PDH sequence ruled out B. byssoidea as the identity of these isolates.
    European Journal of Plant Pathology 07/2013; 136(3). DOI:10.1007/s10658-013-0182-y · 1.49 Impact Factor
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