Project phases and realization

The project was organized into the following tasks:

WP1: Integration of prior knowledge of the defense signaling network
1.1. Summarizing information from knowledge of the functioning of the Arabidopsis thaliana plant
Data from public databases and important publications in the field have been collected, and a plan for collecting manual updates of the network has been prepared on the basis of more specialized articles. The methodology for the automatic addition of new plant defense response components is ready, the methodology for facilitating the review of new links to the expert and the combination of different versions of the model is ready and in the testing phase by users.
For synthetic biology methods, the GenoCAD tool has been complemented so that it is possible to design genetic cassettes for functional analysis of genes in plants
Realization: The work is fully completed and published.

1.2. Inference of miRNA in the regulatory network 
Experimental data on the miRNA network in Arabidopsis are included in the plant defense model. We also compiled a network of links between potato small RNAs and their target mRNAs.
Realization: The work is fully completed and published.

1.3. Translation of the knowledge from the Arabidopsis plant to potatoes
A reference source was selected as the basis for translating Arabidopsis/potato genes, the translation tables were prepared, including selected POCI microarray probes. Thus, we combined the knowledge gained from the model organism and experimental data in potato, which was the basis for generating new hypotheses about the functioning of immune signaling in potato.
Realization: The work is fully completed and published.

WP2: Establishment of a methodology for obtaining spatial-temporal data
2.2. Approaches for the dissection of plant tissues and cell sorting
We tested two methods for microdissection, namely manual microdissection and laser microdissection of a live leaf. Hand-held microdissection was optimized by using lights to pieces containing ca 40 cells. Laser microdissection allows the collection of slightly smaller pieces of tissue, but is time-consuming and can lead to the creation of several artifacts of sampling in the final results. Therefore, we decided to work with small pieces for further work. From the collected tissue, we were able to analyze target transcripts and target sRNAs.
Realization: The work is fully completed.

2.3. Analysis of microscale data
We used different methods for isolating long and short RNAs. We have prepared protocols that allow simultaneous isolation of both types of RNA in quantities that should be sufficient for non-sequential sequencing analysis.

We also prepared bioinformatics files for the analysis of long and small RNA transcriptome – we have prepared reference transcriptomes for cultivar Desiree and breeding clone PW363. We have prepared it according to the principle of a hybrid assembly after two unconstrained methods, the Trinity method and fitting on known genomes with the CLC Genomics algorithms. The obtained transcriptomes were unified using the Evigene program. We developed the quantGenius tool ( for reliable and high-throughput analysis of qPCR data.

For the analysis of small RNAs we have established the set of all potato small RNAs and showed which are regulated in a viral infection in homogenized material.
Realization: The work is fully completed and published.

WP4: Generating data in a multitrophic system potato/pest/pathogen
4.1 The concept of a multitrophic system
In order to closely monitor the spread of the virus, we tested different approaches for inoculation of potato leaves. We used GFP-labeled PVY-N, which allowed us to easily monitor the course of the infection. Point infiltration of the infected plant extract proved to be unsuccessful, so we focused on mechanical point inoculation on the surface of the leaf. The method, which included mechanical damage to the leaf, was also unsuccessful. A point infection has been achieved by adapting the existing system of inoculations to a smaller surface, but we need to optimize the method in order to achieve greater efficiency of infection.
In the subsequent phase, the leaves were infected with standard mechanical inoculation with the help of carborundum. The site of the virus entry was observed both under the confocal microscope and visually through the formation of necrosis. Under the confocal microscope, the virus can only be detected after the formation of microlesion and observed with the aid of a translucent light. We analyzed the response of potato at a time when we observed the fluorescence of individual cells under the confocal microscope, at the time when we observed micro-lesions and in the time of fully developed lesions (work on the ARRS project J4-7636). Responses were not repeatable at the first time point, meaning that the virus cannot be repeatedly detected at such early stages of development in potato leaves. For such analyzes, tobacco plants would be more suitable, in which a lower degree of silencing the fluorescence emission would occur. For further analysis, we will focus on subsequent responses. We also tested a spatial response to Colorado potato beetle attack. The results showed that the immune response was initiated 10 minutes after the first bite of the larvae. Thus, we have now prepared the basis for further triangular analysis of potato with PVY and potato beetle.
Realization: The work is fully completed.