Abstract
Pests and pathogens are responsible for approximately 40% of global agricultural production losses, underscoring the urgent need for sustainable crop protection strategies that increase agricultural productivity while reducing reliance on pesticides. In line with the objectives of the European Green Deal, this project aims to reduce disease impact, limit fungicide use, and advance fundamental knowledge of plant–pathogen interactions by developing resistant crop varieties through innovative RNA-based technologies. The project focuses on two economically important crops, grapevine and pear, which are threatened by major pathogens, including Botrytis cinerea (gray mold), Plasmopara viticola (downy mildew), and Stemphylium vesicarium (pear brown spot). The proposed strategy is based on Host-Induced Gene Silencing (HIGS), an RNA interference (RNAi)-based approach that exploits the plant natural gene-silencing machinery to target and suppress essential pathogen genes. RNAi is triggered by double-stranded RNA (dsRNA), which is processed into 21–24 nucleotide small RNAs that silence target genes in a sequence-specific manner. Increasing evidence indicates that these small RNAs can move bidirectionally between plants and pathogens, inducing reciprocal gene silencing through cross-kingdom RNAi. This exchange is thought to occur, at least in part, through extracellular vesicles that transport RNAi signals between interacting organisms. The project combines applied and fundamental research including the evaluation of HIGS grapevine lines expressing gene constructs against grey mold and downy mildew, the investigation of cross-kingdom RNAi mechanisms and extracellular vesicle-mediated RNA transfer during plant–pathogen interactions, the development of disease-resistant pear plants by transferring the knowledge and methodologies established in grapevine. In addition, the project explores transgrafting, an innovative application of conventional grafting in which genetically modified rootstocks constitutively produce dsRNA molecules that confer protection to non-transgenic scions. This approach offers significant regulatory and public acceptance advantages because the harvested tissues and fruits remain genetically unmodified. The expected outcomes include the development of grapevine and pear plants with enhanced resistance to major fungal diseases, a reduction in the need for chemical fungicide applications, and a deeper understanding of RNAi-mediated communication between plants and pathogens. Achieved results The project successfully achieved the main objectives originally outlined without any substantial changes in the proposed activities. It led to the generation of HIGS grapevine plants with enhanced tolerance to the fungal pathogen B. cinerea, with full validation in the cultivar Thompson Seedless and validation of the 110 Richter rootstock currently nearing completion. The study also demonstrated the occurrence of cross-kingdom RNA interference (RNAi) signal trafficking between the host plant and the target pathogen, while confirming the specificity of the targeted gene silencing. In parallel, an efficient protocol for the isolation of extracellular vesicles (EVs) from herbaceous species was developed and successfully adapted to grapevine tissues, including in vitro callus and leaves. The knowledge gained has been successfully transferred and adapted to woody plant-related explants. A major achievement was the demonstration of RNAi signal mobility in grapevine through transgrafting. Small interfering RNAs (siRNAs) produced in HIGS rootstocks were detected in grafted wild-type (WT) scions, providing clear evidence of long-distance RNAi signal movement. These mobile siRNAs conferred enhanced tolerance to B. cinerea in non-transformed WT scions, significantly reducing pathogen infection in leaves. HIGS grapevine transformed lines were also obtained hairpin-based gene (DCL1 and DCL1/2) targeting the oomycete P. viticola and were characterized and tested during one growing season. In addition, a new construct for tissue-specific expression of dsRNA was under development to obtain targeted gene expression within the cambial region. A comparative genomic analysis of 47 S. vesicarium genomes from different host plants allowed the identification of six genes exclusively present in pear-derived strains. These putative candidate target genes were tested as dsRNA in a Spray-Induced Gene Silencing (SIGS) on detached leaves to possibly be employed. The project also generated and carried out the preliminary validation of HIGS-based pear plants expressing both novel and previously developed gene constructs aimed at improving tolerance or resistance to S. vesicarium, thereby extending the applicability of the HIGS strategy to another economically important fruit crop. Target pathogens were biologically different, and especially the oomycete P. viticola, that is an obligate biotroph, required careful adaptation methods for having good quality artificial inoculum. The recalcitrance in genetic transformation and the long regeneration/selection process remain the main challenge in the obtainment of novel HIGS lines. While a well-set protocol for Thompson Seedless cultivar is defined, for other genotypes, especially for rootstocks such as 110 Richter, the protocol is still under fine-tuning, with significant advances obtained thanks to project. Although pear cultivars and rootstock often exhibit high recalcitrance to in vitro transformation and regeneration, we were able to produce three HIGS lines for Conference, with promising shoots arose in Abate cultivar. The optimization of the protocol suitable for EVs extraction from woody tissue will allow the demonstration of cross-kingdom RNAi trafficking. The newly standardized protocol for the artificial inoculation of P. viticola will be applied to HIGS plants to validate the preliminary results obtained to date. Considering the results obtained provides a strong technical foundation for future potential field-level applications. These findings highlight the feasibility of using transgrafting as a sustainable non-GM strategy to confer disease tolerance in commercial cultivars. The development of an efficient extracellular vesicle isolation protocol from model plants and its adaptation (in progress) to grapevine tissues will open new perspectives for using EV in crop protection. For future development, priority should be given to large-scale validation under field conditions to assess long-term stability, durability of tolerance, and environmental performance. Expanding multi-target strategies and testing additional rootstock–scion combinations would increase robustness and commercial relevance. In parallel, deeper analyses of off-target effects and environmental biosafety will be essential to support risk assessment processes and stakeholder confidence.
Project details
Unibo Team Leader: Elena Baraldi
Unibo involved Department/s:
Dipartimento di Scienze e Tecnologie Agro-Alimentari
Coordinator:
Università Politecnica delle Marche(Italy)
Total Unibo Contribution: Euro (EUR) 62.585,00
Project Duration in months: 24
Start Date:
12/10/2023
End Date:
28/02/2026
