Sequence and Gene Expression Analysis of Recently Identified NLP from Plasmopara viticola.

Lars Askani,René Fuchs,Stefan Schumacher

doi:10.3390/microorganisms9071453

Lars Askani, René Fuchs + Show 1 more

Open Access

https://doi.org/10.3390/microorganisms9071453

Copy DOI

Abstract

Grapevine downy mildew, evoked by the obligate biotrophic oomycete Plasmopara viticola, is one of the most challenging diseases in viticulture. P. viticola establishes an infection by circumvention of plant immunity, which is achieved by the secretion of effector molecules. One family of potential effectors are the necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP). NLP are most abundant in plant pathogenic microorganisms and exist in cytotoxic and non-cyctotoxic forms. Cytotoxic NLP often act as virulence factors and are synthesized in necrotrophic or hemibiotrophic pathogens during the transition from biotrophic to necrotrophic growth. In addition to these cytotoxic NLP, many non-cytotoxic NLP have been identified; their function in biotrophic pathogens is still unknown. In 2020, eight different NLP coding genes were identified in P. viticola and named PvNLP1 to PvNLP8 (Plasmopara viticola NLP 1–8). In the present study, PvNLP4 to PvNLP8 were characterized by using qPCR analysis and transient expression in the model plant Nicotiana benthamiana. Gene expression analysis showed high PvNLP expression during the early stages of infection. Necrosis-inducing activity of PvNLP was not observed in the nonhost N. benthamiana.

Highlights

Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP) have been identified in almost 500 different species of bacteria, fungi and oomycetes [1]
The disulfide bridge between these cysteine residues as well as the cation-binding pocket are essential for the necrosis-inducing ability of these NLP [4,5]
This Ca2+ -binding pocket is necessary to induce necrosis, while the second disulfide bridge has no influence on the cytotoxicity of these proteins [3]

Summary

Introduction

Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLP) have been identified in almost 500 different species of bacteria, fungi and oomycetes [1]. Type 2 NLP, previously observed from fungi and bacteria, were just recently discovered in oomycetes [1] These NLP share four conserved cysteine residues and, besides the cation-binding pocket, share a predicted. A third class of NLP, classified as type 3, consists of proteins which are less conserved the other types but normally share three conserved disulfide bridges and have so far only been identified in ascomycete fungi and bacteria [3] Another conserved region in NLP is a 24 amino acid peptide (nlp24) which is recognized as a pathogen-associated molecular pattern (PAMP) in several plants and induces common pattern-triggered immunity (PTI) responses [6,7]. In order to fill this gap, further characterization of PvNLP4–PvNLP8 is presented here

Materials and Methods

Sequence Analysis of PvNLP4–PvNLP8

Gene Expression Analysis

Necrosis-Inducing Activity