Functional analysis of NLP genes from Botrytis elliptica.
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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Article: Functional analysis of NLP genes from Botrytis elliptica.
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ABSTRACT: Nep-1 Like Proteins (NLPs) are best known for their cytotoxic activity in dicot plants. NLPs are taxonomically widespread among microbes with very different lifestyles. To learn more about this enigmatic protein family we analyzed more than 500 available NLP protein sequences from fungi, oomycetes, and bacteria. Phylogenetic clustering showed that, besides the previously documented two types, an additional more divergent third NLP type could be distinguished. By closely examining the three NLP types, we identified a non-cytotoxic subgroup of type 1 NLPs (designated type 1a), which have substitutions in amino acids making up a cation-binding pocket that is required for cytotoxicity. Type 2 NLPs were found to contain a putative calcium-binding motif, which was shown to be required for cytotoxicity. Members of both type 1 and type 2 NLPs were found to possess additional cysteine residues that, based on their predicted proximity, make up potential disulfide bridges that could provide additional stability to these secreted proteins. Type 1 and type 2 NLPs, although both cytotoxic to plant cells, differ in their ability to induce necrosis when artificially targeted to different cellular compartments in planta, suggesting they have different mechanisms of cytotoxicity.Molecular Plant-Microbe Interactions 07/2014; 27(10). DOI:10.1094/MPMI-04-14-0118-R · 4.46 Impact Factor