Viroids are small non-capsidated, single-stranded, covalently-closed circular noncoding RNA replicons of 239–401 nucleotides that exploit host factors for their replication, and some cause disease in several economically important crop plants, while others appear to be benign. The proposed mechanisms of viroid pathogenesis include direct interaction of the genomic viroid RNA with host factors and post-transcriptional or transcriptional gene silencing via viroid-derived small RNAs (vd-sRNAs) generated by the host defensive machinery. Humulus lupulus (hop) plants are hosts to several viroids among which Hop latent viroid (HLVd) and Citrus bark cracking viroid (CBCVd) are attractive model systems for the study of viroid-host interactions due to the symptomless infection of the former and severe symptoms induced by the latter in this indicator host. To better understand their interactions with hop plant, a comparative transcriptomic analysis based on RNA sequencing (RNA-seq) was performed to reveal the transcriptional alterations induced as a result of single HLVd and CBCVd infection in hop. Additionally, the effect of HLVd on the aggressiveness of CBCVd that underlies severe stunting in hop in a mixed infection was studied by transcriptomic analysis. Our analysis revealed that CBCVd infection resulted in dynamic changes in the activity of genes as compared to single HLVd infection and their mixed infection. The differentially expressed genes that are involved in defense, phytohormone signaling, photosynthesis and chloroplasts, RNA regulation, processing and binding; protein metabolism and modification; and other mechanisms were more modulated in the CBCVd infection of hop. Nevertheless, Gene Ontology (GO) classification and pathway enrichment analysis showed that the expression of genes involved in the proteolysis mechanism is more active in a mixed infection as compared to a single one, suggesting co-infecting viroids may result in interference with host factors more prominently. Collectively, our results provide a deep transcriptome of hop and insight into complex single HLVd, CBCVd, and their coinfection in hop-plant interactions

Transcription factors (TFs) play a major role in controlling gene expression by intricately regulating diverse biological processes such as growth and development, the response to external stimuli and the activation of defense responses. The systematic identification and classification of TF genes are essential to gain insight into their evolutionary history, biological roles, and regulatory networks. In this study, we performed a global mining and characterization of hop TFs and their involvement in Citrus bark cracking viroid CBCVd infection by employing a digital gene expression analysis. Our systematic analysis resulted in the identification of a total of 3,818 putative hop TFs that were classified into 99 families based on their conserved domains. A phylogenetic analysis classified the hop TFs into several subgroups based on a phylogenetic comparison with reference TF proteins from Arabidopsis thaliana providing glimpses of their evolutionary history. Members of the same subfamily and subgroup shared conserved motif compositions. The putative functions of the CBCVd-responsive hop TFs were predicted using their orthologous counterparts in A. thaliana. The analysis of the expression profiling of the CBCVd-responsive hop TFs revealed a massive differential modulation, and the expression of the selected TFs was validated using qRT-PCR. Together, the comprehensive integrated analysis in this study provides better insights into the TF regulatory networks associated with CBCVd infections in the hop, and also offers candidate TF genes for improving the resistance in hop against viroids.

Background: Lupulin glands of hop produce a specific metabolome including hop bitter acids valuable for the brewing process and prenylflavonoids with promising health-beneficial activities. The detailed analysis of the transcription factor (TF)-mediated regulation of the oligofamily of one of the key enzymes, i.e., chalcone synthase CHS_H1 that efficiently catalyzes the production of naringenin chalcone, a direct precursor of prenylflavonoids in hop, constitutes an important part of the dissection of the biosynthetic pathways leading to the accumulation of these compounds.Results: Homologues of flavonoid-regulating TFsHlMyb2(M2),HlbHLH2(B2) andHlWDR1(W1) from hop were cloned using a lupulin gland-specific cDNA library from the hop variety Osvald's 72. Using a "combinatorial" transient GUS expression system it was shown that these unique lupulin-gland-associated TFs significantly activated the promoter (P) ofchs_H1 in ternary combinations of B2, W1 and either M2 or the previously characterizedHlMyb3 (M3). The promoter activation was strongly dependent on the Myb-P binding box TCCTACC having a core sequence CCWACC positioned on its 5' end region and it seems that the complexity of the promoter plays an important role. M2B2W1-mediated activation significantly exceeded the strength of expression of nativechs_H1 gene driven by the 35S promoter of CaMV, while M3B2W1 resulted in 30% of the 35S:chs_H1 expression level, as quantified by real-time PCR. Another newly cloned hop TF,HlMyb7, containing a transcriptional repressor-like motif pdLNLD/ELxiG/S (PDLNLELRIS), was identified as an efficient inhibitor ofchs_H1-activating TFs. Comparative analyses of hop andA. thalianaTFs revealed a complex activation of Pchs_H1 and Pchs4 in combinatorial or independent manners.Conclusions: This study on the sequences and functions of various lupulin gland-specific transcription factors provides insight into the complex character of the regulation of thechs_H1 gene that depends on variable activation by combinations of R2R3Myb, bHLH and WDR TF homologues and inhibition by a Myb repressor.

Viroids are smallest known pathogen that consist of non-capsidated, single-stranded non-coding RNA replicons and they exploits host factors for their replication and propagation. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) is a serious threat, which spreads rapidly within hop gardens. In this study, we employed comprehensive transcriptome analyses to dissect host-viroid interactions and identify gene expression changes that are associated with disease development in hop. Our analysis revealed that CBCVd-infection resulted in the massive modulation of activity of over 2000 genes. Expression of genes associated with plant immune responses (protein kinase and mitogen-activated protein kinase), hypersensitive responses, phytohormone signaling pathways, photosynthesis, pigment metabolism, protein metabolism, sugar metabolism, and modification, and others were altered, which could be attributed to systemic symptom development upon CBCVd-infection in hop. In addition, genes encoding RNA-dependent RNA polymerase, pathogenesis-related protein, chitinase, as well as those related to basal defense responses were up-regulated. The expression levels of several genes identified from RNA sequencing analysis were confirmed by qRT-PCR. Our systematic comprehensive CBCVd-responsive transcriptome analysis provides a better understanding and insights into complex viroid-hop plant interaction. This information will assist further in the development of future measures for the prevention of CBCVd spread in hop fields.

Hop (Humulus lupulus L.) plants are grown primarily for the brewing industry and have been used as a traditional medicinal herb for a long time. Severe hop stunt disease caused by the recently discovered Citrus bark cracking viroid (CBCVd) is one of the most devastating diseases among other viroid infections in hop. MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in gene expression regulation. To identify miRNAs in hop and their response to CBCVd-infection, two small RNA (sRNA) libraries were prepared from healthy and CBCVd-infected hop plants and were investigated by high throughput sequencing. A total of 67 conserved and 49 novel miRNAs were identified. Among them, 36 conserved and 37 novel miRNAs were found to be differentially recovered in response to CBCVd-infection. A total of 311 potential targets was predicted for conserved and novel miRNAs based on a sequence homology search using hop transcriptome data. The majority of predicted targets significantly belonged to transcriptional factors that may regulate hop leaf, root and cone growth and development. In addition, the identified miRNAs might also play an important roles in other cellular and metabolic processes, such as signal transduction, stress response and other physiological processes, including prenylflavonoid biosynthesis pathways. Quantitative real time PCR analysis of selected targets revealed their negative correlation with their corresponding CBCVd-responsive miRNAs. Based on the results, we concluded that CBCVd-responsive miRNAs modulate several hormone pathways and transcriptional factors that play important roles in the regulation of metabolism, growth and development. These results provide a framework for further analysis of regulatory roles of sRNAs in plant defense mechanism including other hop infecting viroids in particular.