Arabidopsis thaliana serves as an appealing model for viroid research, though prior infection trials have largely failed. Previous studies have shown that mature circular RNAs of certain viroids can be synthesised in A. thaliana via transgenic methods. Here, we confirm this by introducing a transgene encoding the dimeric cDNA of hop stunt viroid (HSVd) genome and explore the potential of HSVd-expressing transgenic A. thaliana in viroid research. Mature HSVd circular genome RNA was detected in transgenic plants but accumulated to relatively low levels. Small RNA (sRNA) sequencing revealed minimal production of HSVd-derived sRNAs, suggesting inefficient replication. This finding highlights the importance of double-stranded replication intermediates as the primary source of viroid sRNAs. Moreover, the low replication efficiency increases the likelihood of identifying viroid-binding host factors involved in early molecular interactions using transgenic A. thaliana. Transcriptome analysis indicated that HSVd expression significantly altered the expression of thousands of A. thaliana genes, with enrichment in metabolic pathways, biosynthesis, plant hormone signalling, plant-pathogen interactions and MAPK signalling pathways. Interestingly, these pathways align with those observed in cucumber systemically infected with HSVd, suggesting that transgenic A. thaliana mimics systemic viroid infections and offers a promising model for studying viroid-host interactions. Thus, despite the challenges of establishing systemic infection, HSVd-expressing transgenic A. thaliana represents a valuable tool for advancing viroid research.

Keywords: Arabidopsis thaliana; RNA silencing; replication; viroids; viroid–host interactions.

© 2025 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.