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Projects / Programmes source: ARRS

Functional and structural analysis of Verticillium effector proteins and their targets in hop and model plants

Research activity

Code Science Field Subfield
4.03.05  Biotechnical sciences  Plant production  Phytomedicine 

Code Science Field
B390  Biomedical sciences  Phytotechny, horticulture, crop protection, phytopathology 

Code Science Field
4.01  Agricultural and Veterinary Sciences  Agriculture, Forestry and Fisheries 
Keywords
Verticillium, hop, Arabidopsis, N. bethamiana, effectors, interactors, structure, function, pathogenicity mechanisms, resistance breeding
Evaluation (rules)
source: COBISS
Researchers (12)
no. Code Name and surname Research area Role Period No. of publications
1.  21407  PhD Sabina Berne  Biotechnology  Principal Researcher  2017 - 2020  157 
2.  38323  Marinka Horvat  Biotechnology  Researcher  2018 - 2019 
3.  31143  Nataša Hren    Technician  2017 - 2019 
4.  16379  PhD Jernej Jakše  Plant production  Researcher  2017 - 2020  629 
5.  05994  PhD Branka Javornik  Plant production    2017 - 2020  1,282 
6.  34603  PhD Mirijam Kozorog  Biochemistry and molecular biology  Researcher  2018 - 2020  31 
7.  00385  PhD Nada Kraševec  Biotechnology  Researcher  2017 - 2020  201 
8.  30762  PhD Katja Pirc  Biochemistry and molecular biology  Researcher  2017 - 2019  46 
9.  12048  PhD Marjetka Podobnik  Biochemistry and molecular biology  Researcher  2017 - 2020  286 
10.  19184  PhD Nataša Štajner  Plant production  Researcher  2017 - 2020  290 
11.  15577  Nevenka Valič  Plant production  Technician  2017 - 2019  89 
12.  37428  PhD Helena Volk  Biotechnical sciences  Junior researcher  2017 - 2020  50 
Organisations (2)
no. Code Research organisation City Registration number No. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  20,101 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  63,768 
Abstract
Worldwide, plants are threatened by a variety of pests and plant pathogens, including fungi. It is considered that fungal disease outbreaks will become more severe with changing climate and globalization, presenting a significant and growing risk to global food production and leading to food insecurity, with profound social and economic impacts. To ensure sustainable food security and to reduce crop and yield losses for the growing world population, we need to engineer long-lasting disease resistance in crops, so new strategies and technologies are being adopted in modern plant breeding. Effectoromics, as a high-throughput functional genomics approach that uses effectors to detect and functionally characterize resistance (R) genes, is gaining attention in studies of plant resistance and is also the major topic of this project proposal. Effectors are broadly defined as pathogen secreted molecules that alter the host cell structure and function, thereby facilitating infection (toxins, hydrolytic enzymes) and/or triggering defence responses (avirulent proteins). The assortment of effector molecules is complex and constantly evolving, and also reflects fungal pathogenic lifestyle. Soilborne hemibiotrophic sordariomycete fungi of Verticillium spp. cause verticillium wilt disease and severely threaten the production of potato, tomato, lettuce, cauliflower, oil-seed rape, olives, cotton, flax, fruit trees and hop. Our efforts are aimed at increasing plant resistance to this disease, in which good knowledge of the molecular and cellular mechanisms of pathogenesis and the plant immune system is very important. The main objectives of our research are determination of V. nonalfalfae effector functions, discovery of their molecular targets in model and host plants and resolution of 3D structures of certain effectors. In order to achieve these goals, we will first verify the effectors' expression in planta and discriminate between apoplastic and cytoplasmic effectors. We will then prepare recombinant effector proteins using recombinational cloning, examine their ability to trigger plant defence responses and determine their subcellular localization. In addition, we will study in detail and determine the plant molecules and proteins that interact with these fungal effectors. Lastly, we will also biophysically characterize specific fungal effectors and attempt to resolve their 3D structure. We will employ various bioinformatics tools, classic molecular biology and microbiology techniques and numerous modern methods, such as yeast-2-hybrid system, bimolecular fluorescent complementation, confocal microscopy, surface plasmon resonance, HPLC/tandem mass spectrometry, microscale thermophoresis, differential scanning fluorimetry, protein modelling and X-ray crystallography. This multidisciplinary project will be conducted in collaboration with various research groups from Slovenia, as well as with experts from Germany and Scotland (GB). The project will significantly contribute to advancement of effector biology, molecular phytopathology and microbiology. It will offer new aspects of fundamental plant mechanisms and unknown processes involved in host defence and essential for the virulence of Verticillium spp. Experimentally resolved 3D structures of specific effectors will serve as valuable templates for ligand virtual screening to identify molecules that could be exploited for design of new antifungals by agrochemical companies.
Significance for science
The proposed project aims at obtaining knowledge of V. nonalfalfea infection strategies, molecular and cellular mechanisms of host-pathogen interactions (V. nonalfalfea – hop, arabidopsis, tobacco) and plant immune responses in order to address fungus pathogenicity and hop resistance potentials. This knowledge is essential for plant protection in sustainable crop production to develop efficient disease control strategies without harmful effects on human health, biodiversity and the environment and for the employment of effective resistant plant breeding. A number of predicted and identified V. nonalfalfea candidate secreted effector proteins (CSEPs) with unknown function or structure from our previous studies will be functionally characterized employing a medium throughput approach using model plants A. thaliana and N. benthamiana (CSEPs classification, subcellular localization, in vitro plant reaction to CSEPs infiltration) thereby contributing to the advancement of effector biology and plant pathology research. Subsequently, CSEPs targets in model and host plants will be identified (Y2H screening and affinity purification), unravelling the effector interactors and their mode of action as new aspects of fundamental plant mechanisms and unknown processes involved in host defence and essential for V. nonalfalfae virulence. Predicted and identified V. nonalfalfea CSEPs with no sequence similarity to proteins with known 3D structures, which are the key to understanding their molecular function, will be analysed by X-ray crystallography to obtain their 3D structures. The obtained new 3D structures will significantly aid structural biologists working with fungal effectors and increase knowledge on the effectors’ mode of action. Additionally, fungal effector 3D structures can be used as templates in ligand docking studies to identify molecules that will interfere with the effector function and could serve for the development of antifungal compounds applicable in agriculture. The results of the project will also provide a broader understanding of Verticillium spp. pathogenicity and will help to elucidate plant resistance to these vascular fungal pathogens. Our ambition through the project is additionally to improve our scientific excellence, demonstrated by increased scientific production (number of high ranked publications, high-quality of postgraduate training), increased research and innovation (R&I) potential (number of patent applications, development new products, processes and services, technology transfer) and by the development of cross-sector partnerships to spread the attained scientific capabilities and good practices to the immediate and wider environment.
Significance for Slovenia
The proposed project is well aligned with Slovenia’s Smart Specialisation Strategy (S4) under 2.2. Natural and traditional resources for the future, 2.2.2. Sustainable food production, 2.2.2.2 Technologies for sustainable agricultural production (livestock and plants), which focuses on secure food production and industrial use of renewable bio-resources. The project thus implements the strategic and government-defined priority of applications-oriented research in the areas of S4, which embraces high potential growth in R&I. With the successful realization of our project we will have at our disposal advanced scientific expertise, which, by the integration of fast growing life science technologies (biotechnology, synthetic biology), can produce solutions that can address problems of a devastating phytopathogen in a most sustainable manner. Our results will have an impact on hop growing production, on which we focus, although the overall approach to solution of the problem, i.e., the occurrence of new devastating plant pathogens due to agricultural technologies, globalization and climate change etc., is transferable to other plant–pathogen systems. Various stakeholders (breeders, growers, traders, governance and services) will benefit from translation of the project results. The discovery of new plant interactors implicated in Verticillium wilt resistance will enable the development of efficient marker(s) for enhanced marker assisted resistant breeding, which will provide hop growers with resistant varieties as the most effective means of controlling wilt disease. The results of the project will also support governance structures in building new guidelines and strategies for farming practice management aimed at fighting outbreaks of highly virulent strains of pathogens. Furthermore, characterization of pathogen effectors will also allow the development of new, faster and more cost efficient diagnostics methods for routine use by phytopathology services. In addition to knowledge creation and it's translation to practice, the project and the research group has the ambition of facilitating access for students to research infrastructure and to a competitive research environment, providing training in critical thinking, entrepreneurship skills and innovative research and thus educating the students needed to exploit the innovation potential of new research, including by the establishment of research-based firms (spin-offs, start-ups).
Most important scientific results Interim report, final report
Most important socioeconomically and culturally relevant results Interim report, final report
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