Molecular basis for functional diversity among microbial Nep1-like proteins

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 mu s-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. Author summary The rhizosphere and phyllosphere of terrestrial plants are home to a number of microorganisms, many of which are potentially pathogenic at certain stages during their associations with plants. The pathogens use diverse routes to penetrate physical barriers and colonize host plants with different lifestyles. Necrosis and ethylene inducing peptide 1 (Nep1)-like proteins (NLPs) are widely distributed among prokaryotic and eukaryotic organisms, like fungi, bacteria and oomycetes, and may infect a range of different crops, such as potato, tomato, soybean and tobacco, thus posing major threat to agriculture worldwide. It was shown that NLPs function as cytolytic toxins that induce plasma membrane leakage by binding to plant membrane sphingolipid receptor. Interestingly, non-toxic NLP proteins can also be secreted by several hemibiotrophic and obligate biotrophic pathogens. This study provides a structural and functional characterization of a non-cytotoxic HaNLP3 protein from Hyaloperonospora arabidopsidis that could explain how these proteins evolved a range of new functions in variety of pathogens with different lifestyles.