Periodic Reporting for period 1 - DETOXPEST (Towards increased biosafety for non-target insects - Damage-activated proteolysis to selectively enhance toxicity of pesticides)
Période du rapport: 2023-02-01 au 2025-07-31
Pesticides, particularly the toxic effects of insecticides on non-target insects such as bees, are among the main factors behind the rapid decline of insect numbers and diversity worldwide. The EU-funded DETOXPEST project proposes an innovative approach for the damage activation of pro-pesticides by plant proteases activated in the gut of feeding caterpillars. The method does not hurt non-target beneficial insects that cause no damage, including pollinators and natural enemies of pests. In this context, DETOXPEST will study the impact of fall armyworm (Spodoptera frugiperda), an invasive insect pest and a potential major threat to EU agriculture, on the model plant Arabidopsis thaliana and the crop maize.
Insects numbers and diversity have rapidly declined in the EU and worldwide. Pesticides, and particularly the toxicity of insecticides to non-target insects (e.g. bees), are one of the major drivers of insect declines. Apart from destabilizing natural ecosystems, pollinator disappearance directly threatens food security.
To help combat insect decline, I propose an innovative approach for the damage-activation of pro-pesticides (DAPP) by plant proteases that are activated in the gut of feeding caterpillars. Non-target beneficial insects that cause no damage, including pollinators and natural enemies of pests, are spared.
My team pioneers the study of proteolysis in the plant wound response, following our recent discovery that physical damage activates a class of proteases, called metacaspases. I hypothesize that i) damage-activated plant proteolysis is a largely unrecognized but potential key player in the plant wound response to insect herbivores and ii) this knowledge can be used to enhance pesticide biosafety.
We will study the impact of fall armyworm (Spodoptera frugiperda), an invasive insect pest and potential major threat to EU agriculture, on the model plant Arabidopsis thaliana and the economically important crop maize. A combination of advanced (N-terminomics) and novel (Proteome Integral Solubility Alteration) proteomics technologies will allow us to uncover unknown plant metacaspase substrates and damage-activated proteases and to assess their impact on insect herbivory. These fundamental studies will feed information into a pipeline of first-in-class DAPP development, where we will modify biological insecticides with newly-discovered protease cleavage sites. Finally, we will test toxicity against target (Spodoptera) and non-target insects.
My early-stage and fundamental research on damage-activated proteolysis can have a tremendous positive impact on the increase of insecticide selectivity to help combat the escalating problem of insect decline.
Insects numbers and diversity have rapidly declined in the EU and worldwide. Pesticides, and particularly the toxicity of insecticides to non-target insects (e.g. bees), are one of the major drivers of insect declines. Apart from destabilizing natural ecosystems, pollinator disappearance directly threatens food security.
To help combat insect decline, I propose an innovative approach for the damage-activation of pro-pesticides (DAPP) by plant proteases that are activated in the gut of feeding caterpillars. Non-target beneficial insects that cause no damage, including pollinators and natural enemies of pests, are spared.
My team pioneers the study of proteolysis in the plant wound response, following our recent discovery that physical damage activates a class of proteases, called metacaspases. I hypothesize that i) damage-activated plant proteolysis is a largely unrecognized but potential key player in the plant wound response to insect herbivores and ii) this knowledge can be used to enhance pesticide biosafety.
We will study the impact of fall armyworm (Spodoptera frugiperda), an invasive insect pest and potential major threat to EU agriculture, on the model plant Arabidopsis thaliana and the economically important crop maize. A combination of advanced (N-terminomics) and novel (Proteome Integral Solubility Alteration) proteomics technologies will allow us to uncover unknown plant metacaspase substrates and damage-activated proteases and to assess their impact on insect herbivory. These fundamental studies will feed information into a pipeline of first-in-class DAPP development, where we will modify biological insecticides with newly-discovered protease cleavage sites. Finally, we will test toxicity against target (Spodoptera) and non-target insects.
My early-stage and fundamental research on damage-activated proteolysis can have a tremendous positive impact on the increase of insecticide selectivity to help combat the escalating problem of insect decline.
Two years in, the project is making good progress across its various work plans (WPs), with significant milestones already achieved.
In WP1, we have made substantial advances en route to identifying additional metacaspase substrates. Our studies demonstrated that metacaspases are activated in distantly related species, such as Chlamydomonas and Arabidopsis, in response to wounding. We have developed all necessary genetic and proteomics tools (N-terminomics), and are ready to perform comparative studies between these species. The identification of substrates common to both species will help us pinpoint deeply conserved targets for further research and utilization for engineering of DAPPs. In WP2 we have discovered at least two new proteases that are involved in plant responses to damage. These proteases have shown potential for herbivore resistance, with ongoing investigations into their substrate cleavage patterns. In WP3, we are studying how damage-activated proteolysis impacts wound responses and herbivory. We are generating complementation lines for the identified proteases, including lines with inactive proteases to study their potential non-proteolytic roles. We are also generating genetic reporters to visualize protease activity in vivo during wounding and herbivory. WP4 focuses on developing DAPPs by leveraging knowledge on engineered (spider) toxins and cleavage sites from damage-activated proteases. We have acquired a range of biological toxins, and have established a protein production platform to produce these toxins with appropriate tags for purification. These toxins are being tested in combination with metacaspase cleavage sites, and we plan to expand this with newly discovered cleavage sites from our ongoing studies. Although WP5, which involves testing insecticides on S. frugiperda caterpillars, has not yet begun, we are on track to acquire the insects and start experiments this year. The project has encountered challenges, including delays in hiring key personnel, but we’ve managed to prioritize and adapt to keep progress steady, especially in developing engineered biological insecticides and establishing new lab facilities.
In WP1, we have made substantial advances en route to identifying additional metacaspase substrates. Our studies demonstrated that metacaspases are activated in distantly related species, such as Chlamydomonas and Arabidopsis, in response to wounding. We have developed all necessary genetic and proteomics tools (N-terminomics), and are ready to perform comparative studies between these species. The identification of substrates common to both species will help us pinpoint deeply conserved targets for further research and utilization for engineering of DAPPs. In WP2 we have discovered at least two new proteases that are involved in plant responses to damage. These proteases have shown potential for herbivore resistance, with ongoing investigations into their substrate cleavage patterns. In WP3, we are studying how damage-activated proteolysis impacts wound responses and herbivory. We are generating complementation lines for the identified proteases, including lines with inactive proteases to study their potential non-proteolytic roles. We are also generating genetic reporters to visualize protease activity in vivo during wounding and herbivory. WP4 focuses on developing DAPPs by leveraging knowledge on engineered (spider) toxins and cleavage sites from damage-activated proteases. We have acquired a range of biological toxins, and have established a protein production platform to produce these toxins with appropriate tags for purification. These toxins are being tested in combination with metacaspase cleavage sites, and we plan to expand this with newly discovered cleavage sites from our ongoing studies. Although WP5, which involves testing insecticides on S. frugiperda caterpillars, has not yet begun, we are on track to acquire the insects and start experiments this year. The project has encountered challenges, including delays in hiring key personnel, but we’ve managed to prioritize and adapt to keep progress steady, especially in developing engineered biological insecticides and establishing new lab facilities.
The DETOXPEST project has achieved significant breakthroughs, particularly in the emerging field of damage-activated proteolysis in plants. A major advancement is the identification of two new proteases, which, alongside metacaspase (as previously discovered in our work), are crucial to the plant’s wound response and regeneration processes. Though these proteases were known before, their role in responding to physical damage and herbivory was not recognized. This novel discovery lays the groundwork for deeper studies into their substrates and functions, potentially transforming how we approach plant defense mechanisms and the development of new, more targeted pesticides. One of the project’s key technological advancements is the establishment of N-terminomics pipelines that will allow unprecedented insights into protein cleavage during herbivory in plants like Arabidopsis and maize. We are particularly excited about the potential of simplified protocols in combination with enhanced proteome coverage, which will benefit the wider scientific community working on proteases in various biological systems. Additionally, we have expanded on a modular cloning system, allowing for efficient cloning and production of biological toxins in bacterial systems. This versatile system will facilitate the creation of novel DAPPs, a technology that has the potential to disrupt the pesticide industry.
Overall, the DETOXPEST project is advancing our understanding of plant defense mechanisms, enabling the development of more selective pesticides and offering new solutions to enhance agricultural sustainability and help combat the escalating problem of insect decline.
Overall, the DETOXPEST project is advancing our understanding of plant defense mechanisms, enabling the development of more selective pesticides and offering new solutions to enhance agricultural sustainability and help combat the escalating problem of insect decline.