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Projects / Programmes source: ARIS

Spatiotemporal analysis of hypersensitive response to Potato virus Y in potato

Research activity

Code Science Field Subfield
4.06.05  Biotechnical sciences  Biotechnology  Plant biotechnology 

Code Science Field
B191  Biomedical sciences  Plant biochemistry 

Code Science Field
4.04  Agricultural and Veterinary Sciences  Agricultural biotechnology 
Keywords
potato, hypersensitive resistance, PVY, Potyviridae, spatiotemporal regulation, transcriptomics, redox state, statistical and mathematical modelling
Evaluation (rules)
source: COBISS
Researchers (14)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  32561  PhD Luka Ausec  Plant production  Researcher  2016 - 2019  63 
2.  19116  PhD Špela Baebler  Biotechnology  Researcher  2016 - 2019  313 
3.  34130  PhD Anna Coll Rius  Biochemistry and molecular biology  Researcher  2016 - 2019  163 
4.  12688  PhD Kristina Gruden  Biotechnology  Head  2016 - 2019  985 
5.  35356  PhD Barbara Jenko Bizjan  Medical sciences  Researcher  2018 - 2019  73 
6.  20070  PhD Aleš Kladnik  Biology  Researcher  2016 - 2019  174 
7.  37409  PhD Maja Križnik  Biotechnology  Junior researcher  2016 - 2019  42 
8.  36323  PhD Tjaša Lukan  Biology  Researcher  2016 - 2019  125 
9.  18467  PhD Maruša Pompe Novak  Biotechnology  Researcher  2016 - 2019  291 
10.  05229  PhD Maja Ravnikar  Biotechnology  Researcher  2016 - 2019  1,369 
11.  30840  PhD Amrita Roy Choudhury  Biochemistry and molecular biology  Researcher  2018 - 2019  44 
12.  29635  Katja Stare  Biology  Technical associate  2017 - 2019  86 
13.  29630  PhD Miha Štajdohar  Computer science and informatics  Researcher  2016 - 2017  21 
14.  09864  PhD Magda Tušek Žnidarič  Biology  Technical associate  2016 - 2019  414 
Organisations (3)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0105  National Institute of Biology  Ljubljana  5055784  13,239 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  66,240 
3.  3417  GENIALIS, informacijske tehnologije d.o.o. (Slovene)  Ljubljana  6323430000  136 
Abstract
Understanding the interaction between plant and pathogens is crucial to ensure long lasting and environmentally friendly system for plant protection. Potato is one of the most important crop and Potato virus Y (PVY) is one of its most detrimental pathogens, causing crop losses worldwide. Potato cv. Rywal reacts to the virus by hypersensitive response (HR), where spot necroses that limit the virus at the site of virus entry are formed. NahG-Rywal plants, compromised in salicylic acid accumulation, develop necrotic lesions that do not limit viral spread and show delayed defence response. The aim of the proposed research project is to bring new perspective into understanding of plant HR response towards viruses, more specifically, to identify the processes that restrict the virus spread and separate them from processes leading to the formation of lesions. To achieve this we will create an experimental system enabling spatiotemporal analysis of plant responses coupled with ‘omics’ analysis. We will further apply innovative data analysis approaches and mathematical modelling to identify key components/novel features of the plant defence signalling network downstream of R-proteins.   We will analyse and compare the transcriptome and miRNA responses in both potato genotypes at two time points following the inoculation with PVY: 1) early viral multiplication: the site of virus entry before lesion development will be visualised using GFP-tagged virus prepared in our lab and 2) early lesion development, when the necrotic tissue is macroscopically visible. In the collected small sections, containing few 10s of cells of the infection foci and surrounding tissue, targeted (qPCR) and non targeted (RNA-seq) analyses will be performed. As changes in redox potential seem crucial based on our preliminary experiments, we will transform investigated genotype(s) with redox-sensing constructs which will enable in-vivo spatial and temporal monitoring of redox processes within cells. This data will be complemented with staining based H2O2 monitoring. Morphological features of cell death in both genotypes will be investigated using light microscopy for detection of changes in membrane permeability and callose deposition; and changes on the ultrastructural level will be monitored using electron microscopy.   Data management structure that will allow for efficient analysis, accessibility of data to all partners, communication with public databases and reusability of data in long-term will be set in accordance with the FairDom concept. It will also be able to implement newly developed data analysis pipelines, based on integration of prior knowledge and specifically adapted for spatiotemporal studies (statistical modelling) and low amounts of starting material. We will further integrate concepts of spatial statistics, multivariate statistics and network analysis to identify key components of the plant defence signalling network which will, together with prior knowledge based modelling provide insights into understanding of defence signalling. In parallel we will develop/improve RNA/miRNA experimental data visualization options.   The project team is composed of excellent scientists from different research organisations, from Slovenia (NIB, BF, Genialis) and abroad (PAS, AU, CIPF), to cope with the interdisciplinary nature of the project and provide infrastructure needed for execution of the project. Project results will be disseminated trough high ranking scientific papers, at scientific meetings and locally in local media and a workshop.   The project findings will ultimately contribute to improvements in plant breeding, e.g. in breeding environment-resilient potato, which is currently one of the bottlenecks in optimizing this important crop’s yields. Moreover, the proposed experimental approach and knowledge can be transferable to other pathosystems and the technology developed also to other biotech applications.
Significance for science
Complex biological processes, like signalling or metabolic networks, can only be understood if studying all components of the system simultaneously. In recent decades, new techniques in molecular biology have advanced tremendously allowing even analysis of single cells as well as couple of cells. Plant immunity signalling dynamics has so far been studied mostly on homogenised leaf materials while in fact virus-infected leaf tissues comprise of a heterogeneous mixture of cells at different stages of defence response depending on the distance from the site of viral entry.   The originality of our approach lies in combining the use of fluorescently labelled virus clone, organelle-specific redox signalling sensors, different microscopy techniques and ‘omics’ analyses for analysis of small tissue sections to follow response of potato to viral infection in detailed spatiotemporal manner. A completely new research direction will be opened with analysis of the role of miRNAs in regulation of plant resistance to pathogens. It is generally accepted that gradients of miRNAs are important contributors in plant development but were so far not implicated in spatial regulation in plant defence. We will also develop novel data analysis pipelines, based on integration of prior knowledge, and specifically adapt them for spatiotemporal studies (statistical modelling) and low amounts of starting material.   We will additionally support generation of novel biological hypothesis with mathematical modelling. We foresee identification of novel players of potato defence as well as novel characteristics of immune signalling network. Therefore, the knowledge gained within the proposed project will represent a breakthrough in the concept of understanding plant-virus interactions, understanding the molecular basis determining the outcome of interaction downstream of resistance genes. The developed experimental approaches and knowledge can be transferable to other pathosystems and the technology developed also to other biotech applications.
Significance for the country
Potato is currently the third most important food crop world-wide. It produces high amounts of non-allergic vegetable proteins per hectare and contains many vitamins and health promoting compounds and has thus an increasing significance in the developing world as food crop. Yet its production is currently not optimal due to the high input costs during cultivation needed to achieve appropriate yield and susceptibility to biotic and abiotic factors. It is grown in all EU countries forming caa 1/3 of the world production. In Slovenia, potato is traditionally the most important crop.   PVY is, besides Phytophtora infestans, a major potato pathogen causing severe crop loss (10% to 80% depending on the cultivar) in Slovenia as well as in other areas worldwide. There is no agricultural management strategy available to cure such viral diseases. The most important control strategies are breeding of resistant or tolerant cultivars, and use of virus-free seeds in combination with insect-vector control strategies. In the 80s, the PVY epidemics completely eliminated sensitive, but at that time leading, potato cultivars and terminated Slovenian seed potato production.   The project group has recently established an initiative "Sustainable agriculture varieties for the future " together with Agricultural institute of Slovenia, Jožef Stefan Institute and Slovenian breeding, agrochemical and innovative technologies companies which will re-establish a value chain for sustainable potato production and contribute to food self-supply in Slovenia. In this way the project will link the scientific findings with the end-users. The initiative was recognised as an important one in the perspective of the Slovenian Smart Specialization within the priority Sustainable food production. Within this project we will develop a scientific basis for precision breeding of stress resilient cultivars. Also, better understanding of plant – pathogen interaction is a prerequisite for design of improved agricultural practices.   An additional aspect of the proposed research is development of new technologies for unrevealing complex biological pathways. The methodological principles established within the project can be easily adapted to investigations of other biological systems, for example the ones important for interested industrial partners in pharmacy or biotech. The project partners have established a tight cooperation with many pharmaceutical and biotech companies (Lek, Krka, Bia, BiaSeparations, Omega, BioSistemika) in the fields of development of molecular biology methods and their implementation in the industrial environment. Therefore, the experimental approaches and data analysis workflows/pipelines developed within the project can be transferred to other applications.
Most important scientific results Interim report, final report
Most important socioeconomically and culturally relevant results Interim report, final report
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