Research Projects

Abstract

Verticillium wilt disease occurs worldwide and affects annual crops and perennial woody plants, affecting food and feed production. In Slovenia, Verticillium nonalfalfae causes significant damage to the production of hops (Humulus lupulus L.). In addition, the more virulent pathotype of the fungus, which appeared in 1997 and still occurs sporadically in hop fields today, is mainly responsible for the huge yield losses. Due to the extent of the damage caused by Verticillium fungi, for which no effective phytopharmaceuticals are available, it is necessary to study the mechanisms of plant defense against these pathogens intensively and to obtain resistant plants using modern biotechnological methods.

MicroRNAs (miRNAs) are small endogenous non-coding RNAs that regulate the expression of numerous genes at the post-transcriptional level by cleaving the transcripts of genes (miRNA targets), thus preventing their translation into proteins. Many targets of conserved miRNAs encode transcription factors that regulate plant growth and development. Therefore, conserved miRNAs may play a crucial role in universal regulatory mechanisms in different plant species. Moreover, studies show that miRNAs and their targets play an important role in defense responses to various biotic and abiotic stress factors. miRNAs not only directly regulate the expression of defense-related genes but also modulate signal transduction or hormone signaling.

In our previous studies, we identified miRNAs that show differential expression in the resistant and susceptible hop cultivar in the early phase of V. nonalfalfae infection and performed in silico prediction of miRNA targets. In this project, we aim to investigate the involvement of miR408, which could be involved in the defense mechanism of the resistant hop variety by regulating the expression of host genes and de novo formation of roots. Therefore, we plan to:

- elucidate the temporal expression patterns of miR408 and its target genes in hop plants during infection with V. nonalfalfae;

- confirm specific interaction between miR408 and its target transcripts;

- obtain miR408 knockouts in hop using CRISPR/Cas9,

- explore the role of miR408 in resistance to V. nonalfalfae infection.

The experimental approach of proposed project focuses on molecular plant-pathogen interactions, leveraging functional biology and transcriptomic tools, including genome editing, small RNA sequencing, and controlled inoculation experiments. The results of our study will not only contribute to the understanding of the fundamentals of miRNA functions but may also contribute to the development of new miRNA-based strategies to prevent Verticillium wilt disease in agronomically important crops.

External link to Researchers Open in new window

• University of Ljubljana, Biotechnical Faculty Open (Department of Agronomy, Chair of Genetics, Biotechnology, Statistics and Plant Breeding – Programme P4-0077 Genetics and Modern Technologies of Crops):
  - Ester Stajič (38120), Nataša Štajner (19184), Jernej Jakše (16379), Vanja Miljanić (57117), Helena Volk (37428), Taja Jeseničnik (37427), Tjaša Cesar (50811)
• Slovenian Institute for Hop Research and Brewing (IHPS; Plant protection group and Plant, soil and the environment group):
  - Sebastjan Radišek (20162), Tanja Guček (36371), Maja Dobrajc (51925), Andreja Čerenak (18828), Lucija Luskar (53760), Monika Oset Luskar (23196)

The phases of the project and their realization

WP1: Inoculation of hop plants and evaluation of the fungal colonization after Verticillium infection

Within this workpackage, the artificial inoculation of the resistant hop cultivar ‘Wye target’, which has an introgression of Verticillium wilt tolerance from US germplasm with highly virulent strain PG2 of V. nonalfalfae will be performed. The success of infection (presence of the fungus) will be determined using quantitative PCR (qPCR).

WP2: Validation of predicted miRNA targets

In WP2, samples of inoculated hop plants from WP1 will be used to determine the expression pattern of miR408 and its targets during the progression of V. nonalfalfae infection to determine the temporal miRNA and target expression patterns in the early stages of Verticillium wilt pathogenesis. The interaction between miR408 and its targets will be analyzed in selected sampling time points using the RLM-RACE method.

WP3: Preparation of different CRISPR/Cas9 vectors and their validation in hop protoplasts

The aim of WP3 is transient expression of sgRNAs for miR408 CRISPR/Cas9-based editing in hop protoplasts to test their editing efficiency and the potential impact of the induced mutations on miRNA biogenesis and function. 

WP4: Stable transformation and functional analysis in mutant hop plants

In this work package, miR408 mutant hop plants will be produced and inoculated with V. nonalfalfae to investigate the role of this miRNA in the response to infection. Our goal is to generate miR408-knockout plants of susceptible variety that phenotypically mimic the natural downregulation of this miRNA observed in resistant hop variety, allowing us to assess its functional role in pathogen response.

WP5: Project management and dissemination activities (Months 1-36)

WP5 deals with project management which will be carried out by the project leader in cooperation with WP leaders. Dissemination of the project results is intended for both the general public and the scientific community, which will be achieved through various outreach activities (e.g. websites, social media and newspaper articles) and public lectures to popularize science. The project results will be presented at national and international conferences, meetings and workshops and will be published in high-ranking open-access journals. The project has the potential to have an impact on the wider scientific community as well as relevant policy- and decision-makers. 

Project partners

• SHIRB - THE SLOVENIAN INSTITUTE OF HOP RESEARCH AND BREWING