Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

• Klíčová slova
• Nicotiana tabacum, plant transformation, regulation of plant morphogenesis, transcription factors, transcriptome profiling, viroid pathogenesis,
• MeSH
• Citrus * metabolismus   MeSH
• Humulus * genetika   MeSH
• kůra rostlin metabolismus   MeSH
• malá nekódující RNA * MeSH
• nemoci rostlin genetika   MeSH
• tabák genetika   metabolismus   MeSH
• transkripční faktor TFIIIA genetika   metabolismus   MeSH
• viroidy * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• malá nekódující RNA * MeSH
• transkripční faktor TFIIIA MeSH

Hop (Humulus lupulus) biosynthesizes the highly economically valuable secondary metabolites, which include flavonoids, bitter acids, polyphenols and essential oils. These compounds have important pharmacological properties and are widely implicated in the brewing industry owing to bittering flavor, floral aroma and preservative activity. Our previous studies documented that ternary MYB-bHLH-WD40 (MBW) and binary WRKY1-WD40 (WW) protein complexes transcriptionally regulate the accumulation of bitter acid (BA) and prenylflavonoids (PF). In the present study, we investigated the regulatory functions of the R2R3-MYB repressor HlMYB7 transcription factor, which contains a conserved N-terminal domain along with the repressive motif EAR, in regulating the PF- and BA-biosynthetic pathway and their accumulation in hop. Constitutive expression of HlMYB7 resulted in transcriptional repression of structural genes involved in the terminal steps of biosynthesis of PF and BA, as well as stunted growth, delayed flowering, and reduced tolerance to viroid infection in hop. Furthermore, yeast two-hybrid and transient reporter assays revealed that HlMYB7 targets both PF and BA pathway genes and suppresses MBW and WW protein complexes. Heterologous expression of HlMYB7 leads to down-regulation of structural genes of flavonoid pathway in Arabidopsis thaliana, including a decrease in anthocyanin content in Nicotiana tabacum. The combined results from functional and transcriptomic analyses highlight the important role of HlMYB7 in fine-tuning and balancing the accumulation of secondary metabolites at the transcriptional level, thus offer a plausible target for metabolic engineering in hop.

• Klíčová slova
• Bitter acids, Chalcone synthase, Flavonoids, Humulus lupulus, R2R3 MYB, RNA sequencing,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• flavonoidy metabolismus   MeSH
• Humulus * genetika   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• flavonoidy MeSH
• rostlinné proteiny MeSH
• transkripční faktory MeSH

Viroids are small, non-coding, pathogenic RNAs with a significant ability of adaptation to several basic cellular processes in plants. TFIIIA-7ZF, a splicing variant of transcription factor IIIA, is involved in replication of nuclear-replicating viroids by DNA-dependent polymerase II. We overexpressed NbTFIIIA-7ZF from Nicotiana benthamiana in tobacco (Nicotiana tabacum) where it caused morphological and physiological deviations like plant stunting, splitting of leaf petioles, pistils or apexes, irregular branching of shoots, formation of double-blade leaves, deformation of main stems, and modification of glandular trichomes. Plant aging and senescence was dramatically delayed in transgenic lines. Factors potentially involved in viroid degradation and elimination in pollen were transiently depressed in transgenic leaves. This depressed "degradome" in young plants involved NtTudor S-like nuclease, dicers, argonoute 5, and pollen extracellular nuclease I showing expression in tobacco anthers and leaves. Analysis of the "degradome" in tobacco leaves transformed with either of two hop viroids confirmed modifications of the "degradome" and TFIIIA expression. Thus, the regulatory network connected to TFIIIA-7ZF could be involved in plant pathogenesis as well as in viroid adaptation to avoid its degradation. These results support the hypothesis on a significant impact of limited TFIIIA-7ZF on viroid elimination in pollen.

• Klíčová slova
• Nicotiana tabacum, nucleolytic enzymes, plant aging, plant morphology changes, plant transformation, transcription factors, viroid,
• MeSH
• malá nekódující RNA * MeSH
• pyl genetika   MeSH
• tabák genetika   MeSH
• transkripční faktor TFIIIA MeSH
• užívání tabáku MeSH
• viroidy * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá nekódující RNA * MeSH
• transkripční faktor TFIIIA MeSH

BACKGROUND: Hop (Humulus lupulus L.) bitter acids are valuable metabolites for the brewing industry. They are biosynthesized and accumulate in glandular trichomes of the female inflorescence (hop cone). The content of alpha bitter acids, such as humulones, in hop cones can differentiate aromatic from bitter hop cultivars. These contents are subject to genetic and environmental control but significantly correlate with the number and size of glandular trichomes (lupulin glands). RESULTS: We evaluated the expression levels of 37 genes involved in bitter acid biosynthesis and morphological and developmental differentiation of glandular trichomes to identify key regulatory factors involved in bitter acid content differences. For bitter acid biosynthesis genes, upregulation of humulone synthase genes, which are important for the biosynthesis of alpha bitter acids in lupulin glands, could explain the higher accumulation of alpha bitter acids in bitter hops. Several transcription factors, including HlETC1, HlMYB61 and HlMYB5 from the MYB family, as well as HlGLABRA2, HlCYCB2-4, HlZFP8 and HlYABBY1, were also more highly expressed in the bitter hop cultivars; therefore, these factors may be important for the higher density of lupulin glands also seen in the bitter hop cultivars. CONCLUSIONS: Gene expression analyses enabled us to investigate the differences between aromatic and bitter hops. This study confirmed that the bitter acid content in glandular trichomes (lupulin glands) is dependent on the last step of alpha bitter acid biosynthesis and glandular trichome density.

• Klíčová slova
• Bitter acids, Differential gene expression, Glandular trichome development, Hop, Humulus lupulus, Lupulin gland,
• MeSH
• Humulus metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• transkripční faktory MeSH

Viroids are small, non-coding, parasitic RNAs that promote developmental distortions in sensitive plants. We analyzed pollen of Nicotiana benthamiana after infection and/or ectopic transformation with cDNAs of citrus bark cracking viroid (CBCVd), apple fruit crinkle viroid (AFCVd) and potato spindle tuber viroid (PSTVd) variant AS1. These viroids were seed non-transmissible in N. benthamiana. All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced by approximately 3.6 × 103, 800 and 59 times in mature pollen of CBCVd, AFCVd and PSTVd infected N. benthamiana, respectively, in comparison to leaves. These results suggest similar elimination processes during male gametophyte development as in the Nicotiana tabacum we presented in our previous study. Mature pollen of N. benthamiana showed no apparent defects in infected plants although all three viroids induced strong pathological symptoms on leaves. While Nicotiana species have naturally bicellular mature pollen, we noted a rare occurrence of mature pollen with three nuclei in CBCVd-infected N. benthamiana. Changes in the expression of ribosomal marker proteins in AFCVd-infected pollen were detected, suggesting some changes in pollen metabolism. N. benthamiana transformed with 35S-driven viroid cDNAs showed strong symptoms including defects in pollen development. A large number of aborted pollen (34% and 62%) and a slight increase of young pollen grains (8% and 15%) were found in mature pollen of AFCVd and CBCVd transformants, respectively, in comparison to control plants (3.9% aborted pollen and 0.3% young pollen). Moreover, pollen grains with malformed nuclei or trinuclear pollen were found in CBCVd-transformed plants. Our results suggest that "forcing" overexpression of seed non-transmissible viroid led to strong pollen pathogenesis. Viroid adaptation to pollen metabolism can be assumed as an important factor for viroid transmissibility through pollen and seeds.

• Klíčová slova
• AFCVd and PSTVd parasitic RNAs, CBCVd, Humulus lupulus, Nicotiana benthamiana, male gametophyte, plant transformation, proteomic of viroid infected pollen, viroid elimination,
• Publikační typ
• časopisecké články MeSH

Long non-coding RNAs (lncRNAs) are a highly heterogeneous class of non-protein-encoding transcripts that play an essential regulatory role in diverse biological processes, including stress responses. The severe stunting disease caused by Citrus bark cracking viroid (CBCVd) poses a major threat to the production of Humulus lupulus (hop) plants. In this study, we systematically investigate the characteristics of the lncRNAs in hop and their role in CBCVd-infection using RNA-sequencing data. Following a stringent filtration criterion, a total of 3598 putative lncRNAs were identified with a high degree of certainty, of which 19% (684) of the lncRNAs were significantly differentially expressed (DE) in CBCVd-infected hop, which were predicted to be mainly involved in plant-pathogen interactions, kinase cascades, secondary metabolism and phytohormone signal transduction. Besides, several lncRNAs and CBCVd-responsive lncRNAs were identified as the precursor of microRNAs and predicted as endogenous target mimics (eTMs) for hop microRNAs involved in CBCVd-infection.

• Klíčová slova
• Citrus bark cracking viroid, Genomics, Humulus lupulus, Long non-coding RNAs, Plant defense, RNA-sequencing,
• MeSH
• Citrus * genetika   MeSH
• Humulus * genetika   MeSH
• kůra rostlin MeSH
• nemoci rostlin genetika   MeSH
• RNA dlouhá nekódující * genetika   MeSH
• stanovení celkové genové exprese MeSH
• viroidy * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA dlouhá nekódující * MeSH

The CRISPR/Cas9-based targeted genome editing has emerged as a versatile technique, widely employed in plant genome engineering, both to decipher gene function and as an alternative to classical breeding technique for traits improvement in plants. However, to date, no such platform has been developed for hop (Humulus lupulus L.), which is an economically important crop producing valuable secondary metabolites utilized in the brewing and pharmaceutical industries. Here, we present the first report on the successful establishment of efficient CRISPR/Cas9-based genome editing using the visible endogenous marker gene phytoene desaturase (PDS) involved in carotenoid biosynthesis to demonstrate successful genome editing in hop. Agrobacterium tumefaciens-mediated transformation of in vitro generated internodal explants was used for the stable integration of constructs expressing plant codon-optimized Cas9 and a pair of co-expressed guide RNAs to target the distinct genomic sites of the PDS gene of hop. Analysis of RNA-guided genome-editing events, including mutant lines screening and homozygosity assessment using the T7 endonuclease assay showed that 33.3% of transformed plants were successfully edited at the target site, displaying albino and mosaic regenerants. Intriguingly, the detected mutations were ranges of deletions (16 bp to 39 bp) which led to disruption of the exon-intron boundary, few base substitutions, and a 1 bp insertion at 3 bp upstream of the PAM region of the target site. The decrease in chlorophyll a/b, and carotenoid content in the mutant lines further confirmed the functional disruption of the HlPDS gene. Taken together, our results demonstrate that the CRISPR/Cas9 system can precisely edit the targeted genome sequences, which may revolutionize our way to overcome some of the obstacles that have plagued the traits improvement in hop.

• Klíčová slova
• CRISPR/Cas9, Genome editing, Hop, Phytoene desaturase, Transformation and T7E1 assay,
• MeSH
• Agrobacterium tumefaciens MeSH
• chlorofyl a MeSH
• chlorofyl MeSH
• CRISPR-Cas systémy * MeSH
• editace genu MeSH
• geneticky modifikované rostliny genetika   MeSH
• genom rostlinný genetika   MeSH
• Humulus enzymologie   genetika   MeSH
• mutageneze MeSH
• oxidoreduktasy genetika   MeSH
• vodící RNA, systémy CRISPR-Cas genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl a MeSH
• chlorofyl MeSH
• chlorophyll b MeSH Prohlížeč
• oxidoreduktasy MeSH
• phytoene dehydrogenase MeSH Prohlížeč
• vodící RNA, systémy CRISPR-Cas MeSH

Members of the family Pospiviroidae have single-stranded circular RNA genomes that adopt a rod-like or a quasi-rod-like conformation. These genomes contain a central conserved region that is involved in replication in the nucleus through an asymmetric RNA-RNA rolling-circle mechanism. Members of the family Pospiviroidae lack the hammerhead ribozymes that are typical of viroids classified in the family Avsunviroidae. The family Pospiviroidae includes the genera Apscaviroid, Cocadviroid, Coleviroid, Hostuviroid and Pospiviroid, with >25 species. This is a summary of the ICTV Report on the family Pospiviroidae, which is available at ictv.global/report/pospiviroidae.

• Klíčová slova
• ICTV Report, Pospiviroidae, taxonomy,
• MeSH
• genom virový MeSH
• kruhová RNA MeSH
• replikace viru * MeSH
• RNA katalytická genetika   MeSH
• RNA virová genetika   MeSH
• RNA genetika   MeSH
• viroidy klasifikace   genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• hammerhead ribozyme MeSH Prohlížeč
• kruhová RNA MeSH
• RNA katalytická MeSH
• RNA virová MeSH
• RNA MeSH

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

• Klíčová slova
• AFCVd propagation and eradication, Nicotiana tabacum, Proteome, RNA sequencing, RT qPCR, male gametophyte, viroid degradation, viroid replication,
• MeSH
• buněčná diferenciace * MeSH
• nemoci rostlin virologie   MeSH
• proteomika * MeSH
• pyl * metabolismus   virologie   MeSH
• rostlinné viry metabolismus   MeSH
• stanovení celkové genové exprese * MeSH
• tabák * metabolismus   virologie   MeSH
• viroidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

Some viroids-single-stranded, non-coding, circular RNA parasites of plants-are not transmissible through pollen to seeds and to next generation. We analyzed the cause for the elimination of apple fruit crinkle viroid (AFCVd) and citrus bark cracking viroid (CBCVd) from male gametophyte cells of Nicotiana tabacum by RNA deep sequencing and molecular methods using infected and transformed tobacco pollen tissues at different developmental stages. AFCVd was not transferable from pollen to seeds in reciprocal pollinations, due to a complete viroid eradication during the last steps of pollen development and fertilization. In pollen, the viroid replication pathway proceeds with detectable replication intermediates, but is dramatically depressed in comparison to leaves. Specific and unspecific viroid degradation with some preference for (-) chains occurred in pollen, as detected by analysis of viroid-derived small RNAs, by quantification of viroid levels and by detection of viroid degradation products forming "comets" on Northern blots. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors, such as AGO5 nuclease, DICER-like and TUDOR S-like nuclease. In addition, the functional status of pollen, as a tissue with high ribosome content, could play a role during suppression of AFCVd replication involving transcription factors IIIA and ribosomal protein L5.

• Klíčová slova
• AFCVd and CBCVd propagation and eradication, Nicotiana tabacum, TUDOR S-nuclease, male gametophyte, recombinant AGO, small RNA, strand-specific viroid RT-qPCR, viroid degradation, viroid replication,
• MeSH
• fenotyp MeSH
• interakce hostitele a patogenu MeSH
• konformace nukleové kyseliny MeSH
• nemoci rostlin virologie   MeSH
• pyl virologie   MeSH
• replikace viru MeSH
• RNA virová MeSH
• tabák virologie   MeSH
• viroidy * MeSH
• virová nálož MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA virová MeSH