Periodic Reporting for period 3 - NoPest (Novel Pesticides for a Sustainable Agriculture)
Período documentado: 2021-11-01 hasta 2023-06-30
NoPEST, Novel PESTicides for a sustainable agriculture, is a five-years project undertaken under the framework of FET-OPEN CHALLENGING CURRENT THINKING program. It is aimed to explore new approaches to identify low-risk, environmentally friendly molecules to be used for crop protection. Modern agriculture relies on pesticides to avoid losses and produce high-quality foods. A 15 to 20-fold increase in the amount of pesticides used globally has been reported over the past decades. However, the overuse of pesticides is harmful to the environment and has led to pollution, greenhouse gas emissions, and potential impact on human health. The European Union (Directives 1107/2009/EC and 473/2002) has initiated very restrictive regulatory changes in pesticide registration based on toxicology, traceability and environmental impact. The discovery of low-risk and reliable alternatives is, therefore, mandatory.
The vision of NoPEST is to contribute to the realization of a sustainable agriculture while addressing environmental and human health concerns. The innovative nature of NoPEST relies on a novel biotechnological approach based on peptide aptamers, to solve disease issues in commercial crops. Peptide aptamers are high-affinity short molecules that specifically inhibit the activity of a target enzyme, and represent a remarkable green, low-risk, alternative to pesticides. In addition, NoPEST will develop precision farming tools to optimise application sites and timing, reducing the cost and environmental impact of field treatments. Key benefits include low or no impact on the environment, on animal and human health, as peptides consist of natural amino-acids, and the fact that this strategy can be applied to potentially any pathogen.
NoPEST aims to counteract crop oomycete infections. Oomycetes, i.e. fungus-like eukaryotic microorganisms, are severe crop pathogens, extremely harmful to various crops of agricultural interest such as potato, tomato and grapevine, and they pose a threat to food production and global plant biodiversity. Highly polluting copper-based compounds are currently used to control oomycete infections. NoPEST intends to find alternatives by perceiving these intermediate objectives (see Figure 1):
• Identification of enzymes responsible of oomycete cell wall polysaccharide biosynthesis, and essential for pathogenesis, to be used as targets for antimicrobial compounds;
• Generation of linear and cyclic peptide combinatorial libraries to be screened via the yeast two-hybrid approach, using cell wall enzymes as baits;
• Identification of peptides with the best antimicrobial properties tested via in vitro and in vivo assays;
• Optimization of active compounds through peptidomimetics and identification of non-peptide small molecules;
• Development of sensors for precision farming to optimise their application.
The vision of NoPEST is to contribute to the realization of a sustainable agriculture while addressing environmental and human health concerns. The innovative nature of NoPEST relies on a novel biotechnological approach based on peptide aptamers, to solve disease issues in commercial crops. Peptide aptamers are high-affinity short molecules that specifically inhibit the activity of a target enzyme, and represent a remarkable green, low-risk, alternative to pesticides. In addition, NoPEST will develop precision farming tools to optimise application sites and timing, reducing the cost and environmental impact of field treatments. Key benefits include low or no impact on the environment, on animal and human health, as peptides consist of natural amino-acids, and the fact that this strategy can be applied to potentially any pathogen.
NoPEST aims to counteract crop oomycete infections. Oomycetes, i.e. fungus-like eukaryotic microorganisms, are severe crop pathogens, extremely harmful to various crops of agricultural interest such as potato, tomato and grapevine, and they pose a threat to food production and global plant biodiversity. Highly polluting copper-based compounds are currently used to control oomycete infections. NoPEST intends to find alternatives by perceiving these intermediate objectives (see Figure 1):
• Identification of enzymes responsible of oomycete cell wall polysaccharide biosynthesis, and essential for pathogenesis, to be used as targets for antimicrobial compounds;
• Generation of linear and cyclic peptide combinatorial libraries to be screened via the yeast two-hybrid approach, using cell wall enzymes as baits;
• Identification of peptides with the best antimicrobial properties tested via in vitro and in vivo assays;
• Optimization of active compounds through peptidomimetics and identification of non-peptide small molecules;
• Development of sensors for precision farming to optimise their application.
The 1st year of the project, January-December 2019, was dedicated to the activities planned in Workpackages (WP) 1, 2, 3, 4, 5 and 9. The main results are summarized in Table 1.
WP1 aims to decipher the enzymes involved in cell wall biosynthesis of different oomycets. Twenty potential targets were identified. WP1 also includes the initial establishment of the in vitro culture of P. viticola. Promising results have been obtained in the optimization of a synthetic medium.
WP2 activities were dedicated to the construction of 2 peptide yeast libraries suitable for screenings. 14 out of 20 target enzymes (see WP1) were cloned into suitable vectors for the yeast 2-hybrid screening.
WP3 is aimed to the production of linear and cyclic peptides identified in WP2. Using the Microwave assisted solid phase strategy, 5 linear peptides were synthesized.
WP4 has the objective to select non-peptide small molecules through pharmacophore-based virtual screening. Existing information on inhibitors and substrates of Chitin Synthase, an enzyme involved in oomycete cell wall biosynthesis, allowed the development of a pharmacophore model and used to screen the ZINC database. 13 compounds, predicted to inhibit Chitin Synthase, were identified and their antimicrobial properties will be assayed.
WP5 aims to design and adapt non-invasive sensing technologies for monitoring disease incidence and assessing the performance of the new molecules. New methods, for assessing and quantifying the visual symptoms caused by P. viticola grapevine leaf infection and for the early detection of P. viticola growth on inoculated grapevine leaf disks, are under development with encouraging results.
WP9 is dedicated to the coordination of the research and dissemination activities. During the first year of NoPEST, different activities were realized and are listed in Table 1.
The second period of the project, from 1st January 2020 to 31st October 2021, was dedicated to carry out further the experimental plan described in WP1-to-WP6 and WP9, resulting in 7 different deliverables (see Figure 2).
The characterization of oomycete cell wall continued within the frame of WP1 and was instrumental to identify the 20 target enzymes to be used as baits in the yeast two-hybrid assays, within the frame of WP2. Fourteen baits were used to run the yeast two-hybrid screenings, resulting in the identification of 301 unique peptides (WP2), 62 of which were synthetized (WP3) and tested for their antimicrobial properties (WP6). Currently, two cyclic peptides have been identified showing excellent antimicrobial properties against Plasmopara viticola and Phytophthora infestans. Protocols for the generation of peptidomimetics have been also developed (WP3) and pharmacophores identified on the basis of target protein structures (WP4). The latter ones will be used to screen in silico molecule databases in order to identify non-peptide small molecules with antimicrobial properties. Furthermore, sensors to detect oomycete infections on grapevine and potato leaves continued to be developed within the frame of WP5.
WP1 aims to decipher the enzymes involved in cell wall biosynthesis of different oomycets. Twenty potential targets were identified. WP1 also includes the initial establishment of the in vitro culture of P. viticola. Promising results have been obtained in the optimization of a synthetic medium.
WP2 activities were dedicated to the construction of 2 peptide yeast libraries suitable for screenings. 14 out of 20 target enzymes (see WP1) were cloned into suitable vectors for the yeast 2-hybrid screening.
WP3 is aimed to the production of linear and cyclic peptides identified in WP2. Using the Microwave assisted solid phase strategy, 5 linear peptides were synthesized.
WP4 has the objective to select non-peptide small molecules through pharmacophore-based virtual screening. Existing information on inhibitors and substrates of Chitin Synthase, an enzyme involved in oomycete cell wall biosynthesis, allowed the development of a pharmacophore model and used to screen the ZINC database. 13 compounds, predicted to inhibit Chitin Synthase, were identified and their antimicrobial properties will be assayed.
WP5 aims to design and adapt non-invasive sensing technologies for monitoring disease incidence and assessing the performance of the new molecules. New methods, for assessing and quantifying the visual symptoms caused by P. viticola grapevine leaf infection and for the early detection of P. viticola growth on inoculated grapevine leaf disks, are under development with encouraging results.
WP9 is dedicated to the coordination of the research and dissemination activities. During the first year of NoPEST, different activities were realized and are listed in Table 1.
The second period of the project, from 1st January 2020 to 31st October 2021, was dedicated to carry out further the experimental plan described in WP1-to-WP6 and WP9, resulting in 7 different deliverables (see Figure 2).
The characterization of oomycete cell wall continued within the frame of WP1 and was instrumental to identify the 20 target enzymes to be used as baits in the yeast two-hybrid assays, within the frame of WP2. Fourteen baits were used to run the yeast two-hybrid screenings, resulting in the identification of 301 unique peptides (WP2), 62 of which were synthetized (WP3) and tested for their antimicrobial properties (WP6). Currently, two cyclic peptides have been identified showing excellent antimicrobial properties against Plasmopara viticola and Phytophthora infestans. Protocols for the generation of peptidomimetics have been also developed (WP3) and pharmacophores identified on the basis of target protein structures (WP4). The latter ones will be used to screen in silico molecule databases in order to identify non-peptide small molecules with antimicrobial properties. Furthermore, sensors to detect oomycete infections on grapevine and potato leaves continued to be developed within the frame of WP5.
The final goal of NoPEST project is to provide low risk alternatives to conventional pesticides, a mandatory issue for an environmental friendly and sustainable agriculture. Competence in plant pathology, disease assays and plant disease management are in place and essential to validate new antimicrobial formulations. Expertise in precision farming and plant-phenotyping non-invasive technologies are also part of NoPEST, to assess the competitiveness of the new molecules on pesticide market. Overall, the NoPEST technology and competences meet all the requirements to open a totally new area in plant biotechnology with a clear final industrial application in the agrochemical sector. NoPEST technology, if successful, could represent the core of a new high-growth agrochemical industrial sector. In addition, the NoPEST strategy for disease control may have much broader impacts in the longer term by being applicable to other species and types of crop pathogens, e.g. viruses and bacteria. Thus, NoPEST holds a great bio-economical potential well beyond the specific important oomycete diseases, serving as a guide to foster new commercial initiatives and startups focused on the production and employment of peptide-based low-impact pesticides and precision farming tools in tomorrow’s agriculture world.