Analysis and application of viroid-specific small RNAs generated by viroid-inducing RNA silencing

Viroids are noncoding RNA pathogens inducing severe to mild disease symptoms on agriculturally important crop plants. Viroid replication is entirely dependent on host transcription machinery, and their replication/accumulation in the infected cells can activate RNA silencing-a host defense mechanism that targets the viroid itself. RNA silencing produces in the cell large amounts of viroid-specific small RNAs of 21-24-nucleotides by cleaving (or "dicing") entire molecules of viroid RNA. However, viroid replication is resistant to the effects of RNA silencing and disrupts the normal regulation of host gene expression, finally resulting in the development of disease symptoms on infected plant. The molecular mechanisms of biological processes involving RNA silencing and underlying various aspects of viroid-host interaction, such as symptom expression, are of special interests to both basic and applied areas of viroid research. Here we present a method to create infectious viroid cDNA clones and RNA transcripts, the starting material for such analyses, using Hop stunt viroid as an example. Next we describe methods for the preparation and analysis of viroid-specific small RNAs by deep sequencing using tomato plants infected with Potato spindle tuber viroid as an example. Finally we introduce bioinformatics tools and methods necessary to process, analyze, and characterize these viroid-specific small RNAs. These bioinformatic methods provide a powerful new tool for the detection and discovery of both known and new viroid species.