Molecular basis for functional diversity among microbial Nep1-like proteins.

Tea Lenarčič,Alessandra Magistrato,Marjetka Podobnik,Thorsten Nürnberger,Jure Borišek,Gregor Anderluh,Katja Pirc,Isabell Albert,Vesna Hodnik,Mark J Banfield

doi:10.1371/journal.ppat.1007951

Abstract

Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic microorganisms. They trigger necrosis in various eudicot plants upon binding to plant sphingolipid glycosylinositol phosphorylceramides (GIPC). Interestingly, HaNLP3 from the obligate biotroph oomycete Hyaloperonospora arabidopsidis does not induce necrosis. We determined the crystal structure of HaNLP3 and showed that it adopts the NLP fold. However, the conformations of the loops surrounding the GIPC headgroup-binding cavity differ from those of cytotoxic Pythium aphanidermatum NLPPya. Essential dynamics extracted from μs-long molecular dynamics (MD) simulations reveals a limited conformational plasticity of the GIPC-binding cavity in HaNLP3 relative to toxic NLPs. This likely precludes HaNLP3 binding to GIPCs, which is the underlying reason for the lack of toxicity. This study reveals that mutations at key protein regions cause a switch between non-toxic and toxic phenotypes within the same protein scaffold. Altogether, these data provide evidence that protein flexibility is a distinguishing trait of toxic NLPs and highlight structural determinants for a potential functional diversification of non-toxic NLPs utilized by biotrophic plant pathogens.

Highlights

Necrosis- and ethylene-inducing peptide 1-like proteins (NLPs) have been found in plantassociated prokaryotic and eukaryotic microorganisms and are widely known to induce necrosis and ethylene production in eudicot plants [1,2,3]
Non-toxic HaNLP3 protein does not bind to glycosylinositol phosphorylceramides (GIPC)
We checked whether the difference in cytotoxicity between NLP from the oomycete Pythium aphanidermatum (NLPPya) and HaNLP3 is due to inability of the latter to bind the headgroup of plant sphingolipids

Summary

Introduction

Necrosis- and ethylene-inducing peptide 1-like proteins (NLPs) have been found in plantassociated prokaryotic (bacteria) and eukaryotic (fungi, oomycetes) microorganisms and are widely known to induce necrosis and ethylene production in eudicot plants [1,2,3]. NLPs share a conserved necrosis inducing protein (NPP1) domain, typically containing the heptapeptide motif, GHRHDWE, which is crucial for toxicity. Type 1 NLPs contain a conserved disulfide bond and are found predominately in microorganisms that are associated with plants. Several NLPs produced by oomycetes lack a complete heptapeptide motif and, as a result, are non-toxic. These proteins cluster together and form group type 1a. Type 2 NLPs are distinct from type 1 NLPs due to the presence of a second conserved disulfide bond and an additional putative calcium-binding domain. Little is known about type 3 NLPs; it is predicted that they contain three disulfide bonds and a distinct overall structure [5]

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