PROTEIN-LIPID INTERACTIONS IN MICROBIAL PATHOGENESIS

Members of the necrosis- and ethylene-inducing peptide 1 (NEP1)-like protein family, i.e., NLPs, are the major microbial cytolysins acting at the level of plant plasma membranes. They trigger various defense responses and cell death in eudicot plants. NLPs are produced by microbial pathogens distributed in three different kingdoms, such as fungi, bacteria, and oomycetes. These microorganisms are widespread and can infect various crops such as potato, tomato, soybean, and tobacco, causing enormous economic losses worldwide. Specific defense against microbial pathogens requires new molecular targets, and these are now urgently needed. In this project, we will provide important new information on NLPs that will enable the development of new strategies to combat the major microbial pathogens. The mechanism by which NLPs cause membrane damage is still poorly understood. In addition, there is little structural information about NLPs, which is important for a better understanding of their function as well as for the development of better strategies for ligands that might inhibit their activity. In this project, we will fill this gap and provide important new structural information about some of the NLPs of major microbial pathogens such as Phytophthora. To this end, we will develop structural approaches to study the dynamics and interactions of NLPs with their cognate ligands, as well as model membrane systems with plant sphingolipids that will be used for further research on NLPs and other plant proteins. Thus, the main objectives of this project are to (i) provide structural and functional information on important unexplored members of the NLPs superfamily; (ii) develop novel lipid-based probes and model membrane systems to study these important microbial effectors; and (iii) investigate pathways of membrane damage by NLPs from three different kingdoms of life. To achieve the goals of this project proposal, we will use state-of-the-art biochemical, biophysical, molecular, and structural biology approaches, including X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. The results of this project will provide molecular details of NLP structure, as well as data on interactions with lipids and the mechanism of membrane damage triggered by NLPs. This will elucidate the pathogenesis mechanism, reveal commonalities between NLPs from different kingdoms of life, and provide means for further development of inhibitors of NLPs activity.