P-322 - Harnessing small antisense circular RNAs for Ustilago maydis control in Zea mays

To date, RNA interference (RNAi) has been the primary mechanism explored for RNA-based crop protection. However, its application is restricted to pathogens that possess RNAi machinery, limiting its wider use. To overcome this limitation, we investigated the potential of small circular antisense RNAs (caRNAs) as a proof-of-concept approach for RNA-based plant protection beyond RNAi-dependent mechanisms. It is expected, that caRNAs can bind directly to target mRNAs and prevent translation and protein synthesis without relying on RNAi. Ustilago maydis, a fungal pathogen that infects maize and lacks an RNAi pathway, serves as an ideal model to explore the feasibility of caRNA-based control strategies. Small circular RNAs are covalently closed, single strand RNA molecules that resist exonuclease-mediated degradation, making them particularly promising for spray applications where RNA stability is a key challenge. In a detached leaf assay, drop-applied caRNAs were taken up and translocated within maize leaves, as confirmed by Northern blot analysis. An in vitro electrophoretic mobility shift assay was established to pre-screen their binding affinity to target mRNA sequences. Finally, target genes in Ustilago maydis were identified, small circular antisense RNAs were designed using in silico structure analysis, and their functionality was evaluated by infection assays in the Ustilago maydis-Zea mays pathosystem.