Periodic Reporting for period 1 - SIGSbyBEVs (Spray-induced gene silencing using bacterial extracellular vesicles for dsRNA delivery to manage fungal diseases in agricultural and forest systems)
Periodo di rendicontazione: 2022-09-01 al 2024-10-31
The SIGSbyBEVs project was developed in response to the urgent need for sustainable solutions to manage fungal diseases affecting agricultural and forest ecosystems. These pathogens cause significant economic losses, threaten global food security, and compromise forest health by reducing the yield of staple crops, fruits, and vegetables while also risking biodiversity. Traditional chemical fungicides, often banned in forestry, are becoming less viable in agriculture due to increasing restrictions under European Green Deal policies and the emergence of resistant fungal strains.
The project aimed to develop an innovative, environmentally friendly technology based on bacterial extracellular vesicles (BEVs) to deliver double-stranded RNA (dsRNA) capable of triggering RNA interference (RNAi) in fungal pathogens. This RNAi-based strategy provides a biodegradable alternative to fungicides, targeting the molecular pathways essential for fungal growth without harming non-target organisms.
The research was structured around two key objectives. The first focused on identifying and designing effective dsRNA molecules to suppress fungal pathogens Fusarium circinatum (causal agent of pine pitch canker disease) and Fusarium oxysporum f. sp. lycopersici (Fusarium wilt disease in tomato) while minimizing potential off-target effects. The second aimed at improving the stability and uptake efficiency of these dsRNA molecules by encapsulating them within BEVs derived from bacterial strains.
Through interdisciplinary collaborations with research institutions and industry partners, SIGSbyBEVs successfully advanced RNAi-based solutions for plant disease management. The project demonstrated that BEV-encapsulated dsRNAs remain stable on plant surfaces for extended periods and are efficiently taken up by fungal pathogens, leading to reduced pathogen growth and disease symptoms.
The impacts of SIGSbyBEVs are multifaceted. Scientifically, it fills critical knowledge gaps in RNAi delivery systems and fungal dsRNA processing. Economically, it offers farmers and forest managers an innovative, low-impact tool to protect plants while preserving yield. Socially, it promotes safer agricultural practices and better food quality by reducing fungicide residues. Strategically, the project aligns with European policy objectives under the Green Deal and Farm to Fork Strategy, positioning RNAi technologies as viable alternatives for sustainable crops and forest protection.
The project aimed to develop an innovative, environmentally friendly technology based on bacterial extracellular vesicles (BEVs) to deliver double-stranded RNA (dsRNA) capable of triggering RNA interference (RNAi) in fungal pathogens. This RNAi-based strategy provides a biodegradable alternative to fungicides, targeting the molecular pathways essential for fungal growth without harming non-target organisms.
The research was structured around two key objectives. The first focused on identifying and designing effective dsRNA molecules to suppress fungal pathogens Fusarium circinatum (causal agent of pine pitch canker disease) and Fusarium oxysporum f. sp. lycopersici (Fusarium wilt disease in tomato) while minimizing potential off-target effects. The second aimed at improving the stability and uptake efficiency of these dsRNA molecules by encapsulating them within BEVs derived from bacterial strains.
Through interdisciplinary collaborations with research institutions and industry partners, SIGSbyBEVs successfully advanced RNAi-based solutions for plant disease management. The project demonstrated that BEV-encapsulated dsRNAs remain stable on plant surfaces for extended periods and are efficiently taken up by fungal pathogens, leading to reduced pathogen growth and disease symptoms.
The impacts of SIGSbyBEVs are multifaceted. Scientifically, it fills critical knowledge gaps in RNAi delivery systems and fungal dsRNA processing. Economically, it offers farmers and forest managers an innovative, low-impact tool to protect plants while preserving yield. Socially, it promotes safer agricultural practices and better food quality by reducing fungicide residues. Strategically, the project aligns with European policy objectives under the Green Deal and Farm to Fork Strategy, positioning RNAi technologies as viable alternatives for sustainable crops and forest protection.
The SIGSbyBEVs project was structured around three primary work packages (WPs), each addressing key components for the development and validation of RNAi-based plant disease management strategies using BEVs
WP1: Identification and Design of Effective dsRNA Molecules.
This WP focused on identifying target genes in the fungal pathogens Fusarium circinatum and Fusarium oxysporum f. sp. lycopersici for RNAi. Through bioinformatic analyses and experimental validation, 11 essential target genes were identified for each pathogen, grouped into three functional pathways related to vesicle trafficking, cell wall synthesis, and signal transduction. Chimeric dsRNA molecules targeting multiple genes were designed to enhance silencing efficiency and minimize functional redundancy.
WP2: BEV Production and dsRNA Encapsulation.
Two bacterial strains, Escherichia coli and Bacillus subtilis, were optimized for BEV production. A novel polyethylene glycol (PEG)-based protocol was developed for BEV purification without ultracentrifugation, yielding vesicles of appropriate purity, size, and charge for dsRNA encapsulation. Multiple encapsulation methods, including electroporation, were tested, achieving up to 90% encapsulation efficiency while maintaining dsRNA integrity and protecting it from nuclease degradation.
WP3: Efficacy Testing of BEV-dsRNA Formulations.
In vitro assays demonstrated that BEV-encapsulated dsRNA reduced fungal growth by up to 80%, with significant gene expression suppression confirmed by RT-qPCR. In planta experiments showed that BEV-encapsulated dsRNA delayed disease progression by approximately two weeks in Pinus radiata seedlings infected with Fusarium circinatum, outperforming naked dsRNA treatments. However, due to the limited lab-scale production of BEV-dsRNA complex, experiments with Fusarium oxysporum f. sp. lycopersici in tomato required adaptation to hydroponic systems, yielding variable but promising results.
WP1: Identification and Design of Effective dsRNA Molecules.
This WP focused on identifying target genes in the fungal pathogens Fusarium circinatum and Fusarium oxysporum f. sp. lycopersici for RNAi. Through bioinformatic analyses and experimental validation, 11 essential target genes were identified for each pathogen, grouped into three functional pathways related to vesicle trafficking, cell wall synthesis, and signal transduction. Chimeric dsRNA molecules targeting multiple genes were designed to enhance silencing efficiency and minimize functional redundancy.
WP2: BEV Production and dsRNA Encapsulation.
Two bacterial strains, Escherichia coli and Bacillus subtilis, were optimized for BEV production. A novel polyethylene glycol (PEG)-based protocol was developed for BEV purification without ultracentrifugation, yielding vesicles of appropriate purity, size, and charge for dsRNA encapsulation. Multiple encapsulation methods, including electroporation, were tested, achieving up to 90% encapsulation efficiency while maintaining dsRNA integrity and protecting it from nuclease degradation.
WP3: Efficacy Testing of BEV-dsRNA Formulations.
In vitro assays demonstrated that BEV-encapsulated dsRNA reduced fungal growth by up to 80%, with significant gene expression suppression confirmed by RT-qPCR. In planta experiments showed that BEV-encapsulated dsRNA delayed disease progression by approximately two weeks in Pinus radiata seedlings infected with Fusarium circinatum, outperforming naked dsRNA treatments. However, due to the limited lab-scale production of BEV-dsRNA complex, experiments with Fusarium oxysporum f. sp. lycopersici in tomato required adaptation to hydroponic systems, yielding variable but promising results.
1. The SIGSbyBEVs project has delivered several groundbreaking results that advance current knowledge and technological capabilities in the field of plant disease management through RNAi-based approaches.
2. A key scientific achievement was the development of a novel system for encapsulating dsRNA within BEVs to protect plants from fungal pathogens. This approach addresses critical challenges in RNAi technology, including dsRNA stability, delivery, and fungal uptake efficiency. Unlike previous methods reliant on chemical carriers, the use of BEVs offers a biodegradable, low-impact alternative capable of protecting dsRNA from enzymatic degradation and enabling effective delivery to target pathogens.
3. The project identified 11 essential target genes for both Fusarium circinatum and Fusarium oxysporum f. sp. lycopersici, grouped into pathways related to vesicle trafficking, cell wall synthesis, and signal transduction. The design of chimeric dsRNAs targeting multiple genes within these pathways demonstrated significant suppression of fungal growth and pathogenicity, marking an innovative approach to overcoming the functional redundancy typically seen in RNAi applications.
4. On the technological front, the project developed a scalable, cost-effective protocol for BEV production without ultracentrifugation, using polyethylene glycol (PEG)-based purification. This method yielded vesicles of appropriate purity, size, and charge for agricultural applications, setting the stage for large-scale production.
5. The in planta experiments showed that BEV-encapsulated dsRNAs delayed disease progression in Pinus radiata seedlings infected with Fusarium circinatum by four weeks, confirming the technology's efficacy and extended action. These findings underscore the potential for BEVs to serve as delivery systems in future commercial formulations.
To ensure further success, several key steps have been identified:
• Further research: Investigation into optimizing BEV production at scale and improving dsRNA encapsulation efficiency using advanced methods like high-frequency sonication.
• Demonstration and commercial viability: Continued collaboration with industry partners to evaluate the practical feasibility of large-scale field applications.
• Regulatory support: Engaging with regulatory bodies to establish a supportive framework for the adoption of RNAi-based biopesticides in agriculture.
• IPR protection: Ongoing discussions with the University of Valladolid's patent office to evaluate potential intellectual property protection for the BEV-based RNA delivery process.
2. A key scientific achievement was the development of a novel system for encapsulating dsRNA within BEVs to protect plants from fungal pathogens. This approach addresses critical challenges in RNAi technology, including dsRNA stability, delivery, and fungal uptake efficiency. Unlike previous methods reliant on chemical carriers, the use of BEVs offers a biodegradable, low-impact alternative capable of protecting dsRNA from enzymatic degradation and enabling effective delivery to target pathogens.
3. The project identified 11 essential target genes for both Fusarium circinatum and Fusarium oxysporum f. sp. lycopersici, grouped into pathways related to vesicle trafficking, cell wall synthesis, and signal transduction. The design of chimeric dsRNAs targeting multiple genes within these pathways demonstrated significant suppression of fungal growth and pathogenicity, marking an innovative approach to overcoming the functional redundancy typically seen in RNAi applications.
4. On the technological front, the project developed a scalable, cost-effective protocol for BEV production without ultracentrifugation, using polyethylene glycol (PEG)-based purification. This method yielded vesicles of appropriate purity, size, and charge for agricultural applications, setting the stage for large-scale production.
5. The in planta experiments showed that BEV-encapsulated dsRNAs delayed disease progression in Pinus radiata seedlings infected with Fusarium circinatum by four weeks, confirming the technology's efficacy and extended action. These findings underscore the potential for BEVs to serve as delivery systems in future commercial formulations.
To ensure further success, several key steps have been identified:
• Further research: Investigation into optimizing BEV production at scale and improving dsRNA encapsulation efficiency using advanced methods like high-frequency sonication.
• Demonstration and commercial viability: Continued collaboration with industry partners to evaluate the practical feasibility of large-scale field applications.
• Regulatory support: Engaging with regulatory bodies to establish a supportive framework for the adoption of RNAi-based biopesticides in agriculture.
• IPR protection: Ongoing discussions with the University of Valladolid's patent office to evaluate potential intellectual property protection for the BEV-based RNA delivery process.