Duration: 1. 3. 2016 - 28. 2. 2019
The authors acknowledge the project J4-7636 was financially supported by the Slovenian Research Agency.
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.