Potato (Solanum tuberosum) is the world’s most widely grown tuber crop and, in terms of fresh produce, the fourth largest food crop overall. Pests and pathogens already account for yield losses of up to 80%, a burden that is expected to worsen under climate change, making improved pest and pathogen management an urgent priority. The most economically damaging viral pathogen of potato is Potato virus Y (PVY), transmitted in a non-persistent manner by the peach-potato aphid, Myzus persicae. Current control relies on resistance breeding and vector management, but resistance is readily overcome by new viral strains, and insecticide-based vector control is becoming increasingly constrained by its impact on the environment and on non-target biodiversity.

Molecular mechanisms of plant–pathogen and plant–pest interactions have so far been studied predominantly in a two-way setting. Despite their ecological relevance, three-way interactions – in which the crop, the pathogen and the insect vector influence one another simultaneously – remain poorly characterised at the molecular level. Within EnRICH, we address this gap by investigating the three-way interaction between potato, PVY and M. persicae, with particular emphasis on non-coding RNAs (ncRNAs). These regulatory molecules, encompassing small RNAs (miRNAs, siRNAs) and long non-coding RNAs, act at the transcriptional, post-transcriptional and epigenetic level and are increasingly recognised as mediators of cross-kingdom gene regulation between interacting organisms, yet their role in plant–virus–vector systems has not been comprehensively studied.

We hypothesise that, in the co-evolutionary arms race between the plant and the aphid–virus system, specific ncRNAs have evolved to regulate signalling and metabolism both within an organism (intraspecies) and between the interacting organisms (interspecies, or cross-kingdom). We further expect that PVY-induced metabolic changes in potato will, in turn, affect aphid metabolism and fecundity.

The project pursues three specific objectives:

• identification of potato and aphid transcripts – including miRNAs and lncRNAs – that respond to PVY infection of potato;
• construction of a potato–aphid cross-species ncRNA–target transcript network; and
• characterisation of the physiological phenotypes that result from the three-way interaction.

To achieve these objectives, EnRICH combines high-throughput sequencing with network-analysis-augmented hypothesis generation across four work packages:

• WP1 – Three-way interaction transcriptional landscape profiling. A pilot experiment will establish the experimental protocol and key sampling time points, followed by a fully replicated three-way interaction experiment in which potato leaves and aphids are sampled for strand-specific short-read sequencing (sRNA-Seq and rRNA-depleted mRNA/lncRNA-Seq) as well as long-read sequencing, used to refine potato cv. Désirée genome annotation and to identify novel coding and non-coding transcripts in both potato and M. persicae.
• WP2 – Three-way interaction data analysis. Differential expression and gene set enrichment analyses will be performed for potato and aphid datasets, and the results will be integrated into a cross-species ncRNA–mRNA network built by combining newly generated predictions with existing knowledge networks, including the Plant Stress Signalling network and protein–protein interaction resources, to generate testable cross-kingdom hypotheses.
• WP3 – Experimental validation of molecular interactions and their phenotypic effects. Selected cross-kingdom ncRNA–target predictions will be functionally validated through in-vitro assays, aphid feeding bioassays and plant-based methods (target cleavage and translational repression assays, transgenic approaches), carried out in collaboration with the Agricultural Institute of Slovenia (KIS), Ghent University and the Centre for Research in Agricultural Genomics (CRAG).
• WP4 – Data, communication and valorisation. Research data will be managed according to FAIR principles throughout the project lifetime, using the pISA-tree framework and deposition in open repositories, complemented by scientific dissemination, public engagement and the evaluation of applicative and intellectual-property potential of the project’s outcomes.

The originality of EnRICH lies in its focus on non-coding RNAs as mediators of cross-kingdom communication and in the use of a data- and network-driven approach to hypothesis generation within a three-way interaction setting, sampled across multiple time points to capture the dynamics of the interaction. The project is expected to deepen fundamental understanding of plant–virus–vector interactions and to inform the development of environmentally sustainable, RNA-based crop protection strategies, with the longer-term prospect of contributing to improved crop yields and reduced reliance on chemical pesticides.

We are open for collaborations – if you are interested, please contact dr Marko Petek.