Projects / Programmes
The molecular mechanism of microbial NLP cytolysins membrane damage
| Code |
Science |
Field |
Subfield |
| 1.05.00 |
Natural sciences and mathematics |
Biochemistry and molecular biology |
|
| Code |
Science |
Field |
| B191 |
Biomedical sciences |
Plant biochemistry |
| Code |
Science |
Field |
| 1.06 |
Natural Sciences |
Biological sciences |
pore-forming proteins, lipid membranes, Nep1-like proteins, glycosylinositol phosphorylceramides, protein-lipid interaction, mechanism of action
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
30762 |
PhD Katja Pirc |
Biochemistry and molecular biology |
Head |
2019 - 2021 |
46 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
20,982 |
Abstract
The members of the family of necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins, i.e. NLPs, elicit diverse defence reactions and cell death in eudicot plants but not monocots. NLPs are conserved secreted effector molecules that are widely distributed among taxonomically nonrelated microorganisms like bacteria, fungi and oomycetes. These microorganisms may infect range of different crops, such as potato, tomato, soya and tobacco, and cause enormous economic loss worldwide. It was shown that NLPs function as cytolytic toxins that induce plasma membrane leakage, thus causing cytotoxicity. Based on their crystal structures, NLPs are considered to be distantly related to other pore-forming toxins of animal origin, such as actinoporins from sea anemones.
The mechanism by which NLP induce necrosis is poorly understood. Recently, we have identified glycosylinositol phosphorylceramides (GIPC), a major class of plant sphingolipids, as a target molecule for NLP binding to plant plasma membranes (Lenarčič et al., Science, 2017). GIPCs consist of a polar head group bearing variable carbohydrate moieties and inositol phosphorylceramide core. Type and number of terminal hexose groups varies significantly between plant species and plant tissues. Binding of the GIPC terminal hexose moiety induces several conformational changes within the NLP toxin that may precede membrane attachment and host cell lysis. We proposed that monocots are unaffected by NLP proteins due to their plasma membrane mostly lacking series A GIPCs, which are enriched in eudicots (Lenarčič et al., Science, 2017). We therefore explained the initial steps of NLP-plant membrane interaction and molecular basis of NLP host selectivity. However, the overall molecular mechanism by which NLP induce plant plasma membrane damage remains unknown.
The main objective of the proposed project is to unravel molecular mechanism of membrane damage of NLP proteins by using unique and newly developed model membrane systems employing GIPC lipids. This research proposal aims to provide new insights into our understanding of the cytotoxic mechanism of NLP proteins at the molecular level. Based on structural similarity of NLPs to pore-forming actinoporins, together with their ability to specifically recognize target cell membrane binding partners GIPC and sphingomyelin, respectively, we will test our central hypothesis that cytotoxic NLPs act by forming pores in lipid membranes. Plant plasma membranes are extremely complex and remain poorly characterized in terms of their composition and structure. Since NLPs represent first and only microbial protein family that elicits its cytotoxicity by disturbing plant plasma membrane integrity and due to substantial plant membrane complexity we expect to discover a novel, yet to be described membrane-damaging mechanism.
Recent identification of specific plant lipid receptor for NLPs will allow the use of a battery of biophysical approaches in order to understand the properties of lipid bilayers that contain GIPC lipids and molecular mechanism of membrane attachment and membrane damage by different members of NLPs. Therefore, the results of this research proposal will (i) provide new insights into GIPC-containing membrane structure, and will have (ii) a major contribution on our understanding of interaction between NLPs and host plants, which represent a crucial stage in the induction of necrosis and other symptoms of the disease.
Significance for science
This research proposal is aiming to clarify the cytotoxic mechanism of NLP proteins that are very important for the pathogenesis of diverse microorganisms on agriculturally most important crops. The results of this study will have a major contribution to our understanding of interaction between NLP proteins and host plants, which represent a crucial stage in the induction of necrosis and other symptoms of the diseases. Molecular details of NLP-membrane association will be important for new research directions in plant-microbe interactions, since not many families of pathogen virulence factors were shown to be involved in interactions involving plasma membrane of target plant cells.
NLPs damage only plasma membranes of eudicot but not monocot plants. We have recently proposed that differential glycosylation pattern distribution among NLP’s plant plasma membrane lipid receptors (i.e. GIPCs) of eudicot and monocot plants might explain NLP host selectivity (Lenarčič et al., Science, 2017). In this proposal we expect to decipher the mechanism of pore-forming activity of NLP proteins on the molecular level. To our understanding, molecular mechanism of membrane damaging activity of proteins upon plant plasma membrane has not yet been described before for pathogenic elicitors. Therefore, molecular description of this interaction will represent the groundwork for functioning of other related or similar elicitor proteins.
Due to their important role in the infection mechanisms of plant pathogens and their broad presence in bacteria, fungi and oomycetes, NLPs may represent an important molecular target for the development of new phytopharmaceutical products. Understanding the molecular mechanism of NLP - membrane interaction and damage and the difference between toxic and nontoxic NLP representatives will enable rational drug design directed against this proteins. This, however, is a long-term outcome that will require basic knowledge of events associated with NLPs toxicity at the molecular level.
We aim to publish the outcomes of our research in scientific journals of high impact and will also present them at scientific conferences, in forms of lectures and posters. We will also attempt to present our data to general public via publishing in social media, newspapers and other media (lectures for general public, radio, etc).
Significance for the country
This research proposal is aiming to clarify the cytotoxic mechanism of NLP proteins that are very important for the pathogenesis of diverse microorganisms on agriculturally most important crops. The results of this study will have a major contribution to our understanding of interaction between NLP proteins and host plants, which represent a crucial stage in the induction of necrosis and other symptoms of the diseases. Molecular details of NLP-membrane association will be important for new research directions in plant-microbe interactions, since not many families of pathogen virulence factors were shown to be involved in interactions involving plasma membrane of target plant cells.
NLPs damage only plasma membranes of eudicot but not monocot plants. We have recently proposed that differential glycosylation pattern distribution among NLP’s plant plasma membrane lipid receptors (i.e. GIPCs) of eudicot and monocot plants might explain NLP host selectivity (Lenarčič et al., Science, 2017). In this proposal we expect to decipher the mechanism of pore-forming activity of NLP proteins on the molecular level. To our understanding, molecular mechanism of membrane damaging activity of proteins upon plant plasma membrane has not yet been described before for pathogenic elicitors. Therefore, molecular description of this interaction will represent the groundwork for functioning of other related or similar elicitor proteins.
Due to their important role in the infection mechanisms of plant pathogens and their broad presence in bacteria, fungi and oomycetes, NLPs may represent an important molecular target for the development of new phytopharmaceutical products. Understanding the molecular mechanism of NLP - membrane interaction and damage and the difference between toxic and nontoxic NLP representatives will enable rational drug design directed against this proteins. This, however, is a long-term outcome that will require basic knowledge of events associated with NLPs toxicity at the molecular level.
We aim to publish the outcomes of our research in scientific journals of high impact and will also present them at scientific conferences, in forms of lectures and posters. We will also attempt to present our data to general public via publishing in social media, newspapers and other media (lectures for general public, radio, etc).
