Viroid Derived Small RNAs Can Cause Change of Expression in Selected Genes of Hops

Viroids are the smallest known plant pathogens, with tiny non-protein coding RNA genomes folded into strong secondary structures. How these molecules cause severe disease symptoms is still under investigation but current data from many viroid hosts have suggested that the host defense response produces enormous amounts of viroid-derived small RNAs (vdsRNAs). In a proposed model, these vdsRNAs use host RNA silencing machinery to target host mRNAs, which eventually triggers the symptoms. Worldwide hop (Humulus lupuls) production is threatened by the occurrence of four different viroids: HLVd, HSVd, AFCVd and recently confirmed CBCVd. The last one is causing the most devastating symptoms, while HLVd infection is present in virtually all hops and is considered not harmful. An in-silico approach was therefore undertaken to search for possible targets for HLVd and CBCVd vdsRNAs. Since hop is an orphan crop, a NGS Illumina transcriptome with high coverage was constructed. The assembly of 174 M paired-end sequences resulted in a 74 Mb transcriptome, represented by 150,443 scaffolds with N50 of 984 bp. All possible HLVd and CBCVd 21, 22 and 24 bp mers were aligned against the transcriptome using RNA-target identification tools. In total, 2.041 possible targets for the action of vdsRNA were revealed and annotated. Genes of plant hormonal pathways involved in disease response were selected, together with pathogenesis related (PR) genes, for RT-qPCR analysis. Both groups of viroid infected plants (HLVd, CBCVd) showed strong up-regulation of PR genes compared to viroid free plants, while other studied genes showed either up- or down-regulation.