Investigations on the use of circular RNA for effective inhibition of protein biosynthesis in plants and fungi

Abstract

As single-stranded molecules, circRNAs have gathered increasing attention in recent years due to their distinct covalently closed circular structure. In mammalian cells, circRNAs are recognized for their diverse functional roles, contributing to the intricate regulation of gene expression and protein function. Emerging evidence also suggests that circRNAs play special roles in plants, influencing plant development, providing resistance against biotic stress, and enhancing tolerance to abiotic stress. Our study aimed at addressing a relevant agricultural question: is it possible to apply exogenous circRNAs to regulate endogenous plant proteins or genes in a sequence-specific manner? We conducted experiments using GFP-expressing Arabidopsis protoplasts and found that treatment with a designer 50-nt GFP antisense circRNA (circRNAGFP) resulted in a dose dependent reduction in the cellular accumulation of the reporter protein. Interestingly, the corresponding sequence-identical linear form of the circRNA (linRNAGFP) had no apparent effect on protein reduction. Importantly, the inhibitory activity of circRNAGFP persisted even in Arabidopsis ARGONAUTE and DICER-like mutants with impaired RNAi pathways. The results prompt consideration of the potential for circRNAs to influence the regulation of endogenous plant genes, suggesting a potential application as a bioherbicide in the future. Expanding our investigation, we extended the application of antisense circRNAGFP to pathogenic fungi, specifically Botrytis cinerea, to explore its impact on reporter GFP accumulation in this fungus and explore the possibility of using circRNA as a fungicide. In sum, my study contributes to the emerging understanding of the versatile roles of antisense circRNAs on both fungal and plant systems, shedding light on their potential applications in agriculture for targeted gene regulation and crop protection.