UC Riverside

Fungal pathogens pose a significant threat to global food security, as resistance to conventional fungicides becomes increasingly widespread. RNA-based antifungals have emerged as promising new technologies for combatting this microbial threat. In one specific approach, Spray-Induced Gene Silencing (SIGS), fungal gene-targeting RNAs are topically applied to plant materials where they silence important fungal genes, limiting their virulence. This practical deployment of SIGS, however, is severely limited by the instability of RNA in the environment, especially in field settings where rainfall, UV light, and high humidity can quickly degrade RNA. Further, many fungal pathogens are soilborne, and the rhizosphere is an even more inhospitable place for RNAs than plant surfaces. In order to become more feasible for agricultural use, RNA-based antifungals need to be improved for stability on both plant surfaces and in the rhizosphere. To address these issues, I worked with three different RNA delivery systems in this work. First, the lipid-based nanoparticle, artificial nanovesicles (AVs), were developed to shield RNAs from environmental degradation. These AVs effectively protected RNAs, and significantly extended the duration of RNA-mediated protection against the foliar pathogen Botrytis cinerea. Three different formulations of AVs with different lipid compositions were all found to be effective in stabilizing and delivering RNA. Next, to address the issue of soilborne fungi and decrease the costs of RNA synthesis, an innovative bacterial delivery platform was developed by engineering two plant-beneficial bacteria, Bacillus subtilis and Pseudomonas putida, to produce and excrete fungal gene-targeting RNAs via extracellular vesicles (EVs). These bacterial platforms conferred protection to tomatoes and Arabidopsis thaliana against both B. cinerea and the soil-borne pathogen Verticillium dahliae. Finally, another nanoparticle-based solution, layered double hydroxide (LDH) clay nanosheets, were investigated for their ability to stabilize and prolong the efficacy of RNA applications. A variety of BioClay formulations enhanced RNA durability and efficacy, further demonstrating the potential for nanoparticles to be optimized in their formulations to decrease production costs and toxicity while increasing antifungal effects. Further refinement of RNA targets and formulations will help optimize SIGS for field deployment, offering sustainable and effective tools for fungal disease management.