An optimised FISH-Based approach for tissue and subcellular localisation of Apple scar skin viroid in cucumber.

Fluorescence in situ hybridisation (FISH) is a valuable technique for visualising RNA molecules in their native cellular context. Still, its application in plant tissues is often limited by tissue autofluorescence and the lack of optimised protocols. Here, we developed and validated a simple, reproducible FISH workflow to detect Apple scar skin viroid (ASSVd) in cucumber. Systematic optimisation of probe chemistry, tissue selection, and sampling stage significantly improved assay sensitivity and reproducibility. The AZDye594-labelled antisense riboprobe produced higher signal-to-background ratios and lower background fluorescence than fluorescein-labelled probes, enabling reliable detection of ASSVd in vascular-associated tissues. The optimised workflow consistently detected ASSVd in both leaves and stems. High-resolution confocal imaging further revealed predominant nuclear accumulation of ASSVd RNA in infected cells. Together, this study establishes a sensitive and accessible FISH workflow for localisation of ASSVd in cucumber and provides a practical platform for investigating the spatial distribution of viroid and other plant RNA pathogens.