The Crystal Structure of Necrosis- and Ethylene-Inducing Protein 2 from the Causal Agent of Cacao’s Witches’ Broom Disease Reveals Key Elements for Its Activity

Abstract

The necrosis- and ethylene-inducing peptide 1 (NEP1)-like proteins (NLPs) are proteins secreted from bacteria, fungi and oomycetes, triggering immune responses and cell death in dicotyledonous plants. Genomic-scale studies of Moniliophthora perniciosa, the fungus that causes the Witches' Broom disease in cacao, which is a serious economic concern for South and Central American crops, have identified five members of this family (termed MpNEP1-5). Here, we show by RNA-seq that MpNEP2 is virtually the only NLP expressed during the fungus infection. The quantitative real-time polymerase chain reaction results revealed that MpNEP2 has an expression pattern that positively correlates with the necrotic symptoms, with MpNEP2 reaching its highest level of expression at the advanced necrotic stage. To improve our understanding of MpNEP2's molecular mechanism of action, we determined the crystallographic structure of MpNEP2 at 1.8 Å resolution, unveiling some key structural features. The implications of a cation coordination found in the crystal structure were explored, and we show that MpNEP2, in contrast to another previously described member of the NLP family, NLP(Pya) from Pythium aphanidermatum, does not depend on an ion to accomplish its necrosis- and electrolyte leakage-promoting activities. Results of site-directed mutagenesis experiments confirmed the importance of a negatively charged cavity and an unforeseen hydrophobic β-hairpin loop for MpNEP2 activity, thus offering a platform for compound design with implications for disease control. Electron paramagnetic resonance and fluorescence assays with MpNEP2 performed in the presence of lipid vesicles of different compositions showed no sign of interaction between the protein and the lipids, implying that MpNEP2 likely requires other anchoring elements from the membrane to promote cytolysis or send death signals.

A hypothesis to reconcile the physical and chemical unfolding of proteins

“…At the structural level, two cooperative units are revealed by the mapped residues upon the pressure increase: the two most C-terminal α-helices of the protein and the main β-sheet in the protein core. The two most C-terminal α-helices form a hydrophobic bulk sandwiched with the protein manifold that is not associated with the presence of solvent-excluded cavities, as theoretically calculated (48) based on the MpNep2 crystal structure (49) (Fig. 4 A and B).…”

High-Resolution Transcript Profiling of the Atypical Biotrophic Interaction betweenTheobroma cacaoand the Fungal PathogenMoniliophthora perniciosa

“…Therefore, it is quite possible that the onset of necrosis in WBD is a physiological process of cacao caused by the metabolic disarrangement of WBD rather than a direct action of the pathogen. Consistent with this idea, M. perniciosa NEP (NECROSIS AND ETHYLENE-INDUCING PROTEIN) genes, which are involved in the necrosis of cacao tissues, are not expressed when the first signs of death are observed in infected plants (Zaparoli et al, 2011). However, as necrosis progresses, the fungus expresses its NEP2 gene, thus actively contributing to the death of the cacao tissue.…”

A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis

“…NLPs have initially been discovered as cytotoxic proteins triggering leaf necrosis and plant defenses in dicotyledonous, but not in monocotyledonous plants [27]. 3D-structural analyses of Pythium aphanidermatum or Moniliophthora perniciosa NLPs, respectively, revealed substantial fold conservation with cytolytic, pore-forming actinoporins from marine organisms, suggesting that NLPs destabilize plant plasma membranes during infection thereby facilitating host cell death [4], [28]. Indeed, cytotoxic NLPs from the necrotrophic or hemibiotrophic phytopathogens Pectobacterium carotovorum pv.…”