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.
Lupulin glands localized in female hop (Humulus lupulus L.) cones are valuable source of bitter acids, essential oils and polyphenols. These compounds are used in brewing industry and are important for biomedical applications. In this study we describe the potential effect of transcription factors from WRKY family in the activation of the final steps of lupulin biosynthesis. In particular, lupulin gland-specific transcription factor HlWRKY1 that shows significant similarity to AtWRKY75, has ability to activate the set of promoters driving key genes of xanthohumol and bitter acids biosynthesis such as chalcone synthase H1, valerophenone synthase, prenyltransferase 1, 1L and 2 and O-methyltransferase-1. When combined with co-factor HlWDR1 and silencing suppressor p19, HlWRKY1 is able to enhance transient expression of gus gene driven by Omt1 and Chs_H1 promoters to significant level as compared to 35S promoter of CaMV in Nicotiana. benthamiana. Transformation of hop with dual Agrobacterium vector bearing HlWRKY1/HlWDR1 led to ectopic overexpression of these transgenes and further activation of lupulin-specific genes expression in hop leaves. It was further showed that (1) HlWRKY1 is endowed with promoter autoactivation; (2) It is regulated by post-transcriptional gene silencing (PTGS) mechanism; (3) It is stimulated by kinase co-expression. Since HlWRKY1 promotes expression of lupulin-specific HlMyb3 gene therefore it can constitute a significant component in hop lupulin regulation network. Putative involvement of HlWRKY1 in the regulation of lupulin biosynthesis may suggest the original physiological function of lupulin components in hop as flower and seed protective compounds.
- MeSH
- Humulus enzymologie genetika metabolismus MeSH
- listy rostlin enzymologie genetika MeSH
- promotorové oblasti (genetika) genetika MeSH
- regulace genové exprese u rostlin * MeSH
- rostlinné proteiny genetika metabolismus MeSH
- terpeny * MeSH
- transkripční faktory genetika metabolismus MeSH
- umlčování genů fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
Potato spindle tuber viroid (PSTVd) belongs to plant-pathogenic, circular, non-coding RNAs. Its propagation is accompanied by (mis)regulation of host genes and induction of pathogenesis symptoms including changes of leaf morphogenesis depending on the strength of viroid variant. We found strong genotype-dependent suppression of tomato morphogenesis-regulating transcription factor SANT/HTH-Myb (SlMyb) due to viroid pathogenesis. Its relative mRNA level was found to be significantly decreased in PSTVd-sensitive tomato (cvs Rutgers and Heinz 1706) due to degradation processes, but increased in PSTVd-tolerant (cv. Harzfeuer). In heterologous system of Nicotiana benthamiana, we observed a SlMyb-associated necrotic effect in agroinfiltrated leaf sectors during ectopic overexpression. Leaf sector necroses were accompanied by activation of nucleolytic enzymes but were suppressed by a strongly pathogenic PSTVd variant. Contrary to that, PSTVd's effect was inhibited by the silencing suppressor p19. It was found that in both, Solanum lycopersicum leaves and N. benthamiana leaf sectors, SlMyb mRNA degradation was significantly stronger in viroid-infected tissues. Necroses induction as well as gene silencing experiments using the SANT/HTH-Myb homologues revealed involvement of this Myb in physiological changes like distortions in flower morphogenesis and growth suppression.
- MeSH
- interakce hostitele a patogenu MeSH
- listy rostlin růst a vývoj metabolismus virologie MeSH
- malá nekódující RNA genetika metabolismus MeSH
- messenger RNA genetika metabolismus MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin genetika virologie MeSH
- regulátory růstu rostlin genetika metabolismus MeSH
- RNA virová genetika metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- sekvenční analýza RNA MeSH
- Solanum lycopersicum genetika metabolismus virologie MeSH
- tabák genetika metabolismus virologie MeSH
- transkripční faktory genetika metabolismus MeSH
- viroidy genetika patogenita fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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 TFs HlMyb2 (M2), HlbHLH2 (B2) and HlWDR1 (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) of chs_H1 in ternary combinations of B2, W1 and either M2 or the previously characterized HlMyb3 (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 native chs_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 of chs_H1-activating TFs. Comparative analyses of hop and A. thaliana TFs 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 the chs_H1 gene that depends on variable activation by combinations of R2R3Myb, bHLH and WDR TF homologues and inhibition by a Myb repressor.
Viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small RNAs (vsRNAs). We describe a new PSTVd variant C3, evolved through sequence adaptation to the host chamomile (Matricaria chamomilla) after biolistic inoculation with PSTVd-KF440-2, which causes extraordinary strong ('lethal') symptoms. The deletion of a single adenine A in the oligoA stretch of the pathogenicity (P) domain appears characteristic of PSTVd-C3. The pathogenicity and the vsRNA pool of PSTVd-C3 were compared to those of lethal variant PSTVd-AS1, from which PSTVd-C3 differs by five mutations located in the P domain. Both lethal viroid variants showed higher stability and lower variation in analyzed vsRNA pools than the mild PSTVd-QFA. PSTVd-C3 and -AS1 caused similar symptoms on chamomile, tomato, and Nicotiana benthamiana, and exhibited similar but species-specific distributions of selected vsRNAs as quantified using TaqMan probes. Both lethal PSTVd variants block biosynthesis of lignin in roots of cultured chamomile and tomato. Four 'expression markers' (TCP3, CIPK, VSF-1, and VPE) were selected from a tomato EST library to quantify their expression upon viroid infection; these markers were strongly downregulated in tomato leaf blades infected by PSTVd-C3- and -AS1 but not by PSTVd-QFA.
- MeSH
- fyziologická adaptace * MeSH
- genetické markery genetika MeSH
- interakce hostitele a patogenu MeSH
- lignin metabolismus MeSH
- malá nekódující RNA genetika MeSH
- Matricaria virologie MeSH
- messenger RNA genetika metabolismus MeSH
- molekulární evoluce * MeSH
- molekulární sekvence - údaje MeSH
- RNA virová genetika MeSH
- sekvence nukleotidů MeSH
- Solanum lycopersicum virologie MeSH
- Solanum tuberosum metabolismus virologie MeSH
- termodynamika MeSH
- viroidy genetika patogenita fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The antiproliferative and antitumor effect of leaf ribonuclease was tested in vitro on the human ML-2 tumor cell line and in vivo on athymic nude mice bearing human melanoma tumors. The antiproliferative activity of this plant ribonuclease in vitro studies was negligible. In the experiments in vivo a significant decrease of the tumor size, however was observed. From nucleases the mung bean nuclease (PhA) was studied first from nucleases. The antitumor effect of this enzyme on ML2 human tumor cell line was almost non-effective. However, significant antitumor activity was detected on human melanoma tumors in vivo. The antitumor effect of black pine pollen nuclease (PN) tested in vitro was also negligible. On the other side, in the experiments in vivo a significant decrease of the human melanoma tumor size was observed too. Recombinant plant nucleases of tomato (TBN1) and hop (HBN1) (submitted to patenting under no. PV 2008-384;Z7585) were isolated to homogeneity and examined for their antitumor effects and cytotoxicity. Although antiproliferative effects of both recombinant nucleases were not significant on the ML-2 cell culture in vitro, the nucleases were strongly cytostatic in vivo after their administration intravenously as stabilized conjugates with polyethylene glycol (PEG). Recombinant both nucleases were as effective against human melanoma tumors as previously studied pine pollen (PN) and mung bean nucleases and their effects were reached at about ten times lower concentrations compared to the use of bovine seminal RNase (BS-RNase).
- MeSH
- endonukleasy farmakologie terapeutické užití MeSH
- lidé MeSH
- melanom farmakoterapie patologie MeSH
- myši nahé MeSH
- myši MeSH
- proliferace buněk účinky léků MeSH
- ribonukleasy farmakologie terapeutické užití MeSH
- rostlinné proteiny farmakologie terapeutické užití MeSH
- xenogenní modely - testy protinádorové aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Recombinant plant nucleases R-TBN1 and R-HBN1 were isolated to homogeneity and examined for their antitumor effects and cytotoxicity. Although antiproliferative effects of both recombinant nucleases were not significant on the ML-2 cell culture in vitro, the nucleases were strongly cytostatic in vivo after their administration intravenously as stabilized conjugates with polyethylene glycol (PEG). Recombinant nucleases were as effective against melanoma tumors as previously studied pine pollen (PN) and mung bean nucleases and their effects were reached at about 10 times lower concentrations compared to the use of bovine seminal RNase (BS-RNase). Because the recombinant nucleases R-HBN1 and R-TBN1 share only 67.4% amino acid identity and showed only partial immunochemical cross-reactivity, their similar anticancerogenic effects can be mainly explained by their catalytical similarity. Both recombinant nucleases showed lower degree of aspermatogenesis compared to BS-RNAse and PN nuclease. Unlike BS-RNase, aspermatogenesis induced by both recombinant nucleases could not be prevented by the homologous antibody complexes. Owing to relatively low cytotoxicity on the one hand, and high efficiency at low protein levels on the other, recombinant plant nucleases R-HBN1 and R-TBN1 appear to be stable biochemical agents that can be targeted as potential antitumor cytostatics.
- MeSH
- endonukleasy farmakologie genetika MeSH
- glykosylace MeSH
- Humulus enzymologie MeSH
- lidé MeSH
- melanom enzymologie patologie prevence a kontrola MeSH
- myeloidní leukemie enzymologie patologie prevence a kontrola MeSH
- myši nahé MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- proliferace buněk MeSH
- protinádorové látky farmakologie MeSH
- rekombinantní proteiny farmakologie genetika MeSH
- skot MeSH
- Solanum lycopersicum enzymologie MeSH
- spermatogeneze MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Klíčová slova
- anti-c protilátky, anti-Cw protilátky,
- MeSH
- hematologické testy * metody MeSH
- lidé MeSH
- protilátky * analýza imunologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- MeSH
- hemolytické anemie prevence a kontrola MeSH
- lidé MeSH
- novorozenec MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
