NANOPARTICLE-BASED SEPARATION OF PLANT EXTRACELLULAR VESICLES FOR SILENCING VIRULENCE GENES

The long-term goal of this proposed project is to provide agriculture in the USA with environmentally friendly approaches for better pest and disease control, crop protection, and agricultural production. The goal will be achieved through (1) design and synthesis of nanoparticles incorporating chitosan, biotin-streptavidin complex, and lactadherin C1C2 domain for the purification of plant extracellular vesicles (EVs) and (2) study of the efficacy of preventing pathogen infection using purified plant EVs tethered with nanoparticles. Accomplishment of this goal would greatly contribute to the rural economy, the welfare of farmers, and our society in general.Objectives: Control of pests and pathogenic diseases has traditionally relied on pesticide/fungicide sprays. These tools not only threaten the human health and ecosystem but also generate uncontrollable multidrug-resistant pathogenic strains. Thus, alternative approaches to manage pests and pathogenic bacteria in a sustainable and eco-friendly manner are needed to maintain high crop production. Using natural compounds instead of synthetic compounds for pest and disease control is much more desirable because they are environmentally friendly. Silencing virulence genes of pests and pathogens is an approach which utilizes naturally occurring small RNAs carried by plant secreted EVs. It has been demonstrated that plant-originated EVs during plant-microbe interaction play prominent roles in plant immune responses. Multiple methods have been developed for isolating EVs from fluids. However, up-to-date, the more widely accepted method for isolating plant EVs is differential ultracentrifugation because the immune-affinity capture approach, depending on suitable EV biomarkers, has not been commercially available for the isolation of EVs from plants. Although time-consuming and labor-intensive, differential ultracentrifugation has remained as the most widely used method for EV purification. To meet the increasing use of EVs, there is an urgent need of more efficient and reliable methods for isolating large amounts of highly pure EVs.For this seed grant proposal, PD will initiate a proof-of-concept investigation using biodegradable chitosan nanoparticles to incorporate the C1C2 domain of lactadherin (lactC1C2) via biotin-streptavidin binding to replace the time-consuming differential ultracentrifugation for the isolation of phosphatidylserine-exposing plant EVs. The successful outcome of this proposed study will lay the framework for PD to pursue future AFRI grants. To accomplish the goals set by this seed grant project, specific studies will be conducted with the following objectives.Objective 1: To construct expression vectors containing lactC1C2-streptavidin fusion gene for bacterial transformation, protein expression and purificationObjective 2: To incorporate lactC1C2-streptavidin-bound chitosan nanoparticles with EVs secreted from Arabidopsis thaliana leaves infected with fungal pathogen Botrytis cinereaObjective 3: To examine the efficacy of EV-nanoparticle hybrids on silencing virulence genes of B. cinerea