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Projects / Programmes source: ARIS

Receptors for toxins of plant pathogens

Research activity

Code Science Field Subfield
1.05.00  Natural sciences and mathematics  Biochemistry and molecular biology   

Code Science Field
P004  Natural sciences and mathematics  Biochemistry, Metabolism 

Code Science Field
1.06  Natural Sciences  Biological sciences 
Keywords
plant pathogens, NLP proteins, actinoporins, plant sphingolipids
Evaluation (rules)
source: COBISS
Researchers (16)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  15686  PhD Gregor Anderluh  Biochemistry and molecular biology  Head  2016 - 2018  966 
2.  16104  PhD Apolonija Bedina Zavec  Biotechnology  Researcher  2016 - 2018  152 
3.  24290  PhD Matej Butala  Biochemistry and molecular biology  Researcher  2016 - 2018  236 
4.  15284  PhD Stanislav Gobec  Pharmacy  Researcher  2016 - 2018  827 
5.  32587  PhD Marko Jukič  Pharmacy  Researcher  2016  170 
6.  33908  PhD Urban Košak  Pharmacy  Researcher  2017 - 2018  57 
7.  00385  PhD Nada Kraševec  Biotechnology  Researcher  2016 - 2018  210 
8.  17276  Jelka Lenarčič    Technical associate  2016 - 2018 
9.  36425  PhD Tea Lenarčič  Biochemistry and molecular biology  Junior researcher  2016 - 2018  43 
10.  06994  PhD Peter Maček  Biochemistry and molecular biology  Researcher  2016 - 2018  523 
11.  33683  Nina Orehar    Technical associate  2016 - 2018 
12.  30762  PhD Katja Pirc  Biochemistry and molecular biology  Researcher  2016 - 2018  46 
13.  12048  PhD Marjetka Podobnik  Biochemistry and molecular biology  Researcher  2016 - 2018  315 
14.  15328  PhD Kristina Sepčić  Biochemistry and molecular biology  Researcher  2016 - 2018  723 
15.  21684  Tea Tomšič    Technical associate  2016 - 2018 
16.  06905  PhD Tom Turk  Biochemistry and molecular biology  Researcher  2016 - 2018  618 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  20,869 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  66,599 
3.  0787  University of Ljubljana, Faculty of Pharmacy  Ljubljana  1626973  17,536 
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 proposal 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 was proposed 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.
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 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. We expect to find specific plant lipid receptor for NLPs and describe interactions with various sugars as the headgroups of these lipids at the molecular level. To our understanding such interactions have not yet been described before for pathogenic elicitors and plant sphingolipids, therefore, molecular description of these interactions will represent the groundwork for functioning of other similar elicitor proteins. Furthermore, the identification of the plant plasma membrane sphingolipids will also allow generation of model lipid systems for studying the pore-forming mechanism of NLP proteins. We expect that we will be able to prepare sphingolipids in quantities that would enable successful studies and, therefore, open doors for molecular dissection of NLPs cytotoxic mechanism. From the broader perspective, molecular details of sphingolipid recognition will be important to understand the evolution of structure and function of sphingolipid-binding module that is derived from the common ancestor that precedes both NLPs and actinoporins protein families. Such binding modules may be used by evolutionary distant organisms for attacking the target animal or plant cells. The molecular details of sphingolipid recognition will also allow designing of binding modules for different lipids in synthetic biology approaches. Finally, the project will also provide a solid basis for the future development of specific inhibitors of this family of proteins, with an aim to prevent interactions of pathogens with plants. Because of its wide distribution and evolutionary conservation this protein family is particularly amenable for such approaches. This, however, is a long-term outcome that will require basic knowledge of events associated with NLPs toxicity at the molecular level.
Significance for the country
The project proposal is responsive and aligned with Slovenian and EU scientific development goals and is highly intersectorial. The proposed project follows the research priorities of the Horizon 2020 regarding the safe food in food production. The project is also aligned with the activities within ARRS programme group Molecular Interactions (P1-0391), where the majority of the activities will be performed. The second part of the proposed research is aiming at clarifying molecular mechanism of NLPs action and this basic information is at this point missing in order to design approaches that will target this important protein family. This is with no doubt of a great importance since microbes that possess NLP proteins cause enormous economic loss worldwide. Furthermore, in the world of 21th century the major challenge will be to provide sufficient food for the increasing human population. Part of the solution how to produce sufficient amount of the safe food of a high quality will certainly be a development of effective suppressors of pathogenic plant microbes. Sustainable crop production is threatened also with periodic outbursts of diseases with epidemic dimensions as it was already experienced in the past. Oomycete Phytophthora infestans, for instance, is the agent of epidemic potato disease late blight that caused severe famines in 19th century with the most pronounced Irish famine. Development of diverse and highly selective suppressors of pathogenic microbes will enable the human population to be prepared for such catastrophic events. For this reason it is important to study different mechanisms of pathogenesis and develop suppressors for different stages of microbe invasion. The surface plasmon resonance and microscale thermophoresis represent state-of-the-art approaches for measurements of molecular interactions involving proteins and lipids or membranes that ask for relatively low sample quantities. We will further develop the protocols for measurements employing these two approaches and make them available to other laboratories in Slovenia and abroad. In particular, the local pharmaceutical industry, with which the principal investigator has already established a strong collaboration, will be interested in using them.
Most important scientific results Interim report, final report
Most important socioeconomically and culturally relevant results Interim report, final report
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