CASyM winter school of Systems Medicine took place between March 29th and April 1st 2017 in Ljubljana, Slovenia and is entitled »The 3rd SysBioMed hands-on tutorial: Systems Medicine Approaches in Personalized Medicine«
1Department of Agronomy, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia, 2Slovenian Institute of Hop Research and Brewing, Žalec, Slovenia, 3Institute of Plant Molecular Biology, Biology Centre ASCR v.v.i, České Budějovice, Czech Republic
In 2007, hop growers in the Savinja valley reported the appearance of severely stunted hop plants, a phenomenon that spread rapidly within hop gardens and among farms. Classical diagnostic methods were unable to detect a new pathogen; single step high-throughput parallel sequencing of total RNA and small RNAs from plants with and without symptoms was therefore employed to identify the novel pathogen. Sequences were assembled de-novo or mapped to reference genomes, resulting in the identification of a novel sequence of Citrus bark cracking viroid (CBCVd) in the stunted hop plants, never before reported on hops. Hop plants are hosts to several viroids. Two of them, hop latent viroid (HLVd) and the recently confirmed CBCVd, are interesting models to study host-viroid interactions, due to the symptomless infection of the former and the severe stunting disease caused by the latter. Although the RNA molecules are insignificant, ranging in size from 246 to 412 bp, the pathogenicity of the viroid molecules is still puzzling the scientific community, although several factors and mechanisms have been proposed for plant hosts, such as gene silencing, alteration of gene expression, and translocation of proteins. To study possible gene silencing in the viroid infected plants by viroid derived small RNAs, in-silico prediction of target transcripts for HLVd and CBCVd was performed. Prediction models revealed that 1062 and 1387 hop transcripts share nucleotide homologies with HLVd- and CBCVd-derived small RNAs, respectively, so they may be silenced in RNA interference process. Selected transcripts involved in the plant hormones metabolism, small RNA biogenesis, transcripts with high homology with viroid derived small RNAs and transcripts targeted by CBCVd-derived small RNAs showing high cellular concentration, mainly showed expression fluctuations compared to viroid free plants, with possible evidence of down-regulation of two transcripts. Additional expression profiles of 5 pathogenesis related genes confirmed high expression levels of the four pathogenesis related genes in viroid infected plants. To study the response of the hop micro RNA genes to CBCVd infection, we identified 116 miRNAs from the hop genome. Seventy-three of them were found to be differentially expressed in response to CBCVd-infection. Several potential targets (311) were predicted for miRNAs, based on a sequence homology search using hop transcriptome data. Significantly, the majority of the predicted targets belonged to transcriptional factors that may regulate hop leaf, root and cone growth and development. Quantitative real time PCR analysis of selected targets revealed their negative correlation with their corresponding CBCVd-responsive miRNAs. Our efforts to achieve better understanding of the viroid’s disease molecular mechanisms could initiate novel strategies to fight these diseases and help in the search for possible genetic resistance.
2006 - University of Ljubljana, Faculty of Medicine, Center for Functional Genomics and Bio-chips.