Research

Receptors for toxins of plant pathogens

Code:

J1-7515

Range:

01. January 2016 - 31. December 2018

Range:

0,10 FTE

Leader:

Stanislav Gobec

Field:

1.05 Natural sciences and mathematics/Biochemistry and molecular biology

Abstract:

Losses in crop production due to plant diseases average 20 % worldwide and severely limit production, quality, and safety of food. The main culprits, besides viruses, are fungi, bacteria and oomycetes. Practice of crop protection has been progressively reoriented towards reduction of a number of active ingredients in pesticides to those that are more selective and less toxic. The main topic of this project are the Nep1-like proteins (NLP), which are widely distributed among taxonomically non-related microorganisms like fungi, bacteria and oomycetes. These microorganisms are widespread, they infect a range of different crops like potato, tomato, soya, hop and tobacco and cause enormous economic loss worldwide. Despite their diverse phylogenetic distribution, NLPs share a high degree of sequence similarity, which is very unique in microbial world and thus makes NLPs excellent targets for antimicrobial agents. It is taken into account that NLPs function as cytolytic toxins that induce plasma membrane leakage, thus causing cytotoxicity (1). Based on known crystal structures, NLP proteins are considered to be distantly related to the well-studied pore forming toxins actinoporins. The hallmark of actinoporins is that their permeabilizing activity toward membranes is strongly enhanced in the presence of sphingomyelin. Due to resemblance between these proteins we anticipate the existence of specific receptor molecule of sphingolipid origin for NLPs in plants as well, and our research will be focused in identification of these receptor molecules. Furthermore, we also plan to dissect molecular mechanism of membrane damage induced by NLPs interaction with plant plasma membranes, which will in the future enable design of specific inhibitors for NLP proteins with potential application in commercial crop protection programs. The main objectives of our research are therefore to identify specific plant lipid receptor molecules for NLP proteins and determine molecular mechanism of action of NLPs. To address these objectives we will express recombinant NLP proteins in order to identify interacting plant lipids. Furthermore, we will study in detail NLP-receptor interactions using molecules mimicking plant lipid receptors and evaluate the effect of these molecules on the toxicity of NLP proteins in vitro and in vivo. Interactions will be measured and evaluated using a number of state-of-the-art methods like surface plasmon resonance, liquid chromatography with mass spectrometry, microscale thermophoresis, isothermal calorimetry, differential scanning fluorimetry, crystallographic structure determination and virtual screening and docking of the binder molecules to three dimensional structures of NLPs. This project will be collaboration between several research groups from Slovenia, as well as research groups from Germany and Japan. The project will reveal important details of toxicity mechanism induced by NLPs and will represent a strong basis for future development of phytopharmaceutical agents aimed at some of the most pressing plant pathogens.