Although structurally simple, viroids can trigger numerous changes in host plants and cause loss of yield in agronomically important crops. This study investigated changes in the endogenous status of phytohormones and antioxidant enzyme activity in Solanum tuberosum cv. Désirée in response to Potato spindle tuber viroid (PSTVd) infection. Phytohormone analysis showed that the content of endogenous jasmonic acid (JA) and its precursor cis-OPDA significantly increased in leaves, while the content of castasterone (CS) increased in both leaves and tubers of systemically infected plants compared to mock-inoculated control plants at 8 weeks post-inoculation. The indole-3-acetic acid content moderately increased only in tubers, while no differences in salicylic acid and abscisic acid content were observed between infected and control plants. Changes in endogenous phytohormone content were associated with upregulated expression of genes involved in the biosynthesis of JA and brassinosteroids, and the metabolism of auxins. Additionally, PSTVd infection provoked overproduction of hydrogen peroxide, which coincided with increased activity of guaiacol peroxidase in leaves and ascorbate peroxidase in potato tubers. The activity of catalase decreased in leaves, while superoxide dismutase activity remained steady regardless of the treatment and organ type. Total ascorbate and glutathione did not change significantly, although a shift towards oxidized forms was observed. Results suggest the existence of organ-specific differences in phytohormone and antioxidative responses in potato upon PSTVd infection. Possible effects of the observed changes on symptom development are discussed.

• Klíčová slova
• Ascorbate, Brassinosteroids, IAA, JA, PSTVd, Peroxidases,
• MeSH
• antioxidancia metabolismus   MeSH
• brassinosteroidy metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• cholestanoly metabolismus   MeSH
• cyklopentany metabolismus   MeSH
• hlízy rostlin metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• listy rostlin metabolismus   MeSH
• nemoci rostlin virologie   MeSH
• oxylipiny metabolismus   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Solanum tuberosum metabolismus   fyziologie   virologie   MeSH
• viroidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia MeSH
• brassinosteroidy MeSH
• castasterone MeSH Prohlížeč
• chlorofyl MeSH
• cholestanoly MeSH
• cyklopentany MeSH
• indoleacetic acid MeSH Prohlížeč
• jasmonic acid MeSH Prohlížeč
• kyseliny indoloctové MeSH
• oxylipiny MeSH
• regulátory růstu rostlin MeSH

The effects of DNA oligonucleotides complementary to potato spindle tuber viroid (PSTVd) on viroid infection were investigated. The oligonucleotides were used to form hybrids with PSTVd in the infection mixture. A 75% reduction of viroid infection was found when an oligonucleotide complementary to nucleotides 79-110 of PSTVd was hybridized with PSTVd at a molar ratio of approximately 5000:1, respectively. A total inhibition of PSTVd infection was observed using an oligonucleotide complementary to nucleotides 42-78 at the same molar excess of DNA over PSTVd, although a 200-fold molar excess was found to be sufficient for the complete blocking of infection by PSTVd. This oligonucleotide caused a significant reduction (about 83%) of viroid infection even if the hybridization was done at a low (30 degrees C) temperature. Shorter oligonucleotides containing 22 and 15 bases corresponding to position 42-62 and 63-78, respectively, exhibited a significant effect only at a high (80 degrees C) initial temperature of molecular hybridization. Heteroduplexes formed between PSTVd RNA and antisense DNA were found to be less stable in a crude nuclease extract from tomato leaves as compared with PSTVd RNA alone. RNase H was demonstrated to cleave the molecular hybrids in vitro.

• MeSH
• antisense oligonukleotidy terapeutické užití   MeSH
• deoxyribonukleasy MeSH
• DNA * MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin mikrobiologie   MeSH
• sekvence nukleotidů MeSH
• Solanum tuberosum MeSH
• viroidy účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense oligonukleotidy MeSH
• deoxyribonukleasy MeSH
• DNA * MeSH

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
• Názvy látek
• genetické markery MeSH
• lignin MeSH
• malá nekódující RNA MeSH
• messenger RNA MeSH
• RNA virová MeSH

Thermal stress of PSTVd-infected Nicotiana benthamiana led to appearance of a broad PSTVd sequence distribution, where most of mutations accumulated in the left half of the viroid's secondary structure including the "pathogenicity" domain. A similar effect had been reported for hop latent viroid [Virology 287 (2001) 349]. The pool of viroid "thermomutants" progenies was transcribed into cDNA and used for biolistic inoculation of Raphanus sativa, where the PSTVd infection was detectable by reverse transcription and polymerase chain reaction (RT-PCR). Newly generated inoculum from R. sativa was used for biolistic transfer to Arabidopsis thaliana wild-type and silencing-deficient mutants bearing one of sde1, sde2, and sde3 locuses. Irrespective to A. thaliana silencing mutants, viroid levels in Brasicaceae species infected with mutated PSTVd variants were of approximately 300 times lower than it is expected for tomato. At the same time, no systemic infection of A. thaliana was achieved with the wild-type PSTVd. In Arabidopsis, a population of PSTVd, consisting of frequent and minor variants, was present and the sequence distribution differed from that of the original viroid "thermomutants"; that is, mutations were not predominantly restricted to the left half of viroid's secondary structure. At least 65% of viroid sequences from Arabidopsis library accumulated mutations in the upper conserved central region (UCCR). In addition, mutants having changes in "hairpin II" domain (C-->A transition at position 229) and in the conserved internal loop element in the left part of viroid structure (single insertion of G at position 39) were detected. All those mutants were inoculated biolistically to tomato and promoted infection especially after prolonged period of plant cultivation (50-80 days pi) when infection reached 70-90%. However, the sequence variants were unstable and reverted to the wild type and to other sequence variants stable in tomato. Our results demonstrate that heat stress-mediated production of viroid quasi-species could be of significance for viroid adaptations.

• MeSH
• Arabidopsis virologie   MeSH
• biolistika * MeSH
• genetická variace * MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• mutageneze MeSH
• nemoci rostlin virologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• Raphanus virologie   MeSH
• reakce na tepelný šok MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• Solanum tuberosum virologie   MeSH
• tabák virologie   MeSH
• viroidy klasifikace   genetika   růst a vývoj   patogenita   MeSH
• vysoká teplota * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

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.

• Klíčová slova
• Biolistic plant inoculation, Nicotiana benthamiana, RNA decay, Solanum lycopersicum, Transgenote, mRNA target,
• 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
• Názvy látek
• malá nekódující RNA MeSH
• messenger RNA MeSH
• regulátory růstu rostlin MeSH
• RNA virová MeSH
• rostlinné proteiny MeSH
• transkripční faktory MeSH

Strong viroid-caused pathogenesis was achieved in tomato cv. Rutgers by biolistic transfer of severe or lethal potato spindle tuber viroid (PSTVd) strains, while other tomato genotypes (e.g., Moneymaker) were tolerant. With reciprocal hybrids between sensitive and tolerant genotypes, we show that plant depression dominates over tolerance. Biolistic transfer of the most pathogenic PSTVd strain AS1 to Nicotiana benthamiana, which is considered to be a symptomless PSTVd host, led to a strong pathogenesis reaction and stunting, suggesting the presence of specific viroid pathogenesis-promoting target(s) in this plant species. Total levels of small siRNA-like PSTVd-specific RNAs were enhanced in strongly symptomatic tomato and N. benthamiana plants after biolistic infection with AS1 in comparison to the mild QFA strain. This indicates association of elevated levels of viroid-specific small RNA with production of strong symptoms. In symptom-bearing tomato leaves in comparison to controls, an RNase of approximately 18 kDa was induced and the activity of a nuclease of 34 kDa was elevated by a factor of seven in the vascular system. Sequence analysis of the nuclease cDNA designated TBN1 showed high homology with plant apoptotic endonucleases. The vascular-specific pathogenesis action is supported by light microscopic observations demonstrating a certain lack of xylem tissue and an arrest of the establishment of new vascular bundles in collapsed plants.

• MeSH
• biolistika metody   MeSH
• endonukleasy genetika   metabolismus   MeSH
• genotyp MeSH
• klonování DNA MeSH
• komplementární DNA chemie   genetika   MeSH
• konformace nukleové kyseliny MeSH
• listy rostlin genetika   metabolismus   virologie   MeSH
• malá interferující RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin genetika   virologie   MeSH
• northern blotting MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• RNA virová chemie   genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• rostlinné viry genetika   patogenita   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie aminokyselin MeSH
• Solanum lycopersicum genetika   metabolismus   virologie   MeSH
• Solanum tuberosum genetika   metabolismus   virologie   MeSH
• viroidy genetika   patogenita   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endonukleasy MeSH
• komplementární DNA MeSH
• malá interferující RNA MeSH
• RNA virová MeSH
• rostlinné proteiny 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

Parameters for biolistic transfer of viroid nucleic acids using a Helios Gene Gun device were assayed. The main achievement of this method is high efficiency of inoculation with linear monomeric viroid cDNAs and RNAs. This greatly facilitates the study of mutated sequence variants, viroid libraries and mixed populations. The lower limits for efficient inoculation of monomeric cDNA fragments with the sequence of potato spindle tuber viroid (PSTVd) and native PSTVd RNA as detected 21 days p.i. are in the range of 50 ng and 200 pg per tomato plant, respectively. At a higher dose, i.e. 2 ng of native RNA per plant, biolistic transfer causes drastic stunting compared to conventional mechanical inoculation, which points to higher PSTVd titers after the biolistic transfer. Infection is readily achieved with exact length monomeric RNA transcripts having 5'-triphosphate and 3'-OH termini in amounts ranging from 2 to 20 ng per plant, suggesting no need for any supplementary modifications of ends or RNA circularization. The biolistic transfer is efficient for viroid "thermomutants", which exhibit low or no infectivity with conventional mechanical inoculation with Carborundum. The biolistic inoculation is also efficient for two other members of the Pospiviroidae family, hop stunt and hop latent viroid.

• MeSH
• biolistika * MeSH
• dvouvláknová RNA analýza   MeSH
• nemoci rostlin virologie   MeSH
• RNA virová analýza   chemie   genetika   MeSH
• Solanum lycopersicum virologie   MeSH
• Solanum tuberosum virologie   MeSH
• viroidy klasifikace   genetika   růst a vývoj   patogenita   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dvouvláknová RNA MeSH
• RNA virová MeSH

The effect of oligodeoxyribonucleotides complementary to the region of the so-called pathogenicity domain (nucleotides 42-78) of the upper RNA strand of potato spindle tuber viroid (PSTVd) (severe) on viroid infection was investigated. The oligonucleotides were allowed to form hybrids with PSTVd in the infection mixtures before inoculation. Infectivity tests were performed using intact plants and plant protoplasts. It was found that the DNA oligonucleotides caused significant reduction of viroid infection at plant and single cell levels. The 200-fold molar excess of antisense DNA over viroid RNA is usually sufficient for the complete blocking of viroid infection. The inhibitory effect is strongly sequence specific. Inhibition by corresponding antisense RNA was much less efficient than that caused by antisense DNA.

• MeSH
• antisense DNA farmakologie   MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin mikrobiologie   MeSH
• oligonukleotidy MeSH
• sekvence nukleotidů MeSH
• Solanum tuberosum mikrobiologie   MeSH
• viroidy účinky léků   genetika   MeSH
• virové nemoci prevence a kontrola   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense DNA MeSH
• oligonukleotidy MeSH