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
Source: PubMed


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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    • "Further evidence for functional diversification within the V. dahliae NLP family comes from the identification of noncytotoxic family members. Most fungal genomes contain up to three NLP genes (Dallal et al. 2010;Garcia et al. 2007;Motteram et al. 2009;Schouten et al. 2008;Staats et al. 2007). Nevertheless, although the V. dahliae NLP family is significantly expanded, this fungus also contains only two genes that encode cytotoxic NLP. "
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    ABSTRACT: In this study, we functionally analyzed the gene family encoding necrosis- and ethylene-inducing-like proteins (NLPs) of the vascular wilt pathogen Verticillium dahliae. We show that the composition of the NLP gene family varies little among V. dahliae isolates. The cytotoxic activity of NLP family members of a tomato pathogenic V. dahliae strain was determined, demonstrating that only two of the seven NLPs induced plant cell death. The genes encoding these cytotoxic NLPs were found to be induced in V. dahliae upon colonization of tomato. Interestingly, targeted deletion of either of the two genes in V. dahliae significantly compromised virulence on tomato as well as on Arabidopsis plants, whereas deletion of only one of the two genes affected virulence on N. benthamiana. This could be attributed to differential induction of the two NLP genes in V. dahliae upon N. benthamiana colonization, revealing that the in planta induction of NLP genes varies between plant hosts. Intriguingly, one of the NLP genes appears to also affect vegetative growth and conidiospore production, as the corresponding deletion strain produced significantly less conidiospores and developed extensive aerial mycelium. In conclusion, we demonstrate that the expanded V. dahliae NLP family shows functional diversification, not only revealing differential cytotoxicity between family members, but also that the cytotoxic NLPs play a role in vegetative growth and asexual reproduction in addition to their contribution to virulence.
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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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    • "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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