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

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

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

Viroids are non-coding single-stranded circular RNA molecules that replicate autonomously in infected host plants causing mild to lethal symptoms. Their genomes contain about 250-400 nucleotides, depending on viroid species. Members of the family Pospiviroidae, like the Potato spindle tuber viroid (PSTVd), replicate via an asymmetric rolling-circle mechanism using the host DNA-dependent RNA-Polymerase II in the nucleus, while members of Avsunviroidae are replicated in a symmetric rolling-circle mechanism probably by the nuclear-encoded polymerase in chloroplasts. Viroids induce the production of viroid-specific small RNAs (vsRNA) that can direct (post-)transcriptional gene silencing against host transcripts or genomic sequences. Here, we used deep-sequencing to analyze vsRNAs from plants infected with different PSTVd variants to elucidate the PSTVd quasipecies evolved during infection. We recovered several novel as well as previously known PSTVd variants that were obviously competent in replication and identified common strand-specific mutations. The calculated mean error rate per nucleotide position was less than [Formula: see text], quite comparable to the value of [Formula: see text] reported for a member of Avsunviroidae. The resulting error threshold allows the synthesis of longer-than-unit-length replication intermediates as required by the asymmetric rolling-circle mechanism of members of Pospiviroidae.

• Klíčová slova
• Error rate, Pospiviroid, error threshold, quasispecies, sequence network mapping, sequencing error, viroid-specific small RNA,
• MeSH
• genom virový * MeSH
• mutace MeSH
• reassortantní viry genetika   MeSH
• replikace viru MeSH
• RNA virová genetika   MeSH
• viroidy genetika   MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA virová MeSH

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

• Klíčová slova
• TBN1, superhelix, tomato multifunctional nuclease, type I nuclease,
• MeSH
• endodeoxyribonukleasy chemie   genetika   metabolismus   MeSH
• fosfáty metabolismus   MeSH
• krystalizace MeSH
• molekulární sekvence - údaje MeSH
• multienzymové komplexy chemie   genetika   metabolismus   MeSH
• rostlinné proteiny chemie   genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• Solanum lycopersicum enzymologie   genetika   MeSH
• vazebná místa fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endodeoxyribonukleasy MeSH
• fosfáty MeSH
• multienzymové komplexy MeSH
• rostlinné proteiny MeSH
• superhelical DNA endonuclease MeSH Prohlížeč

The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum.

• MeSH
• deoxyribonukleasy chemie   genetika   MeSH
• ionty chemie   MeSH
• konformace proteinů MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• molekulární sekvence - údaje MeSH
• rekombinantní proteiny chemie   genetika   MeSH
• rostlinné proteiny chemie   genetika   MeSH
• Solanum lycopersicum chemie   genetika   MeSH
• zinek chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• deoxyribonukleasy MeSH
• ionty MeSH
• rekombinantní proteiny MeSH
• rostlinné proteiny MeSH
• zinek MeSH

Anticancer drugs attacking nucleic acids of the target cells have so far been based on animal or fungal ribonucleases. Plant nucleases have been proved to exhibit decreased cytotoxic side effects. Tomato bifunctional nuclease 1 with activity against both single-stranded and double-stranded RNA and DNA was produced in tobacco leaves as recombinant protein. The enzyme crystallizes under several different crystallization conditions. The presence of Zn(2+) ions was confirmed by X-ray fluorescence. First crystallographic data were obtained.

• MeSH
• difrakce rentgenového záření MeSH
• dvouvláknová RNA metabolismus   MeSH
• endodeoxyribonukleasy chemie   MeSH
• endoribonukleasy chemie   MeSH
• jednovláknová DNA metabolismus   MeSH
• rekombinantní proteiny chemie   MeSH
• Solanum lycopersicum enzymologie   MeSH
• tabák enzymologie   MeSH
• zinek analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dvouvláknová RNA MeSH
• endodeoxyribonukleasy MeSH
• endoribonukleasy MeSH
• jednovláknová DNA MeSH
• rekombinantní proteiny MeSH
• zinek MeSH