The Experts below are selected from a list of 120 Experts worldwide ranked by ideXlab platform
Jaroslav Matousek - One of the best experts on this subject based on the ideXlab platform.
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Mapping the Gene Expression Spectrum of Mediator Subunits in Response to Viroid Infection in Plants
International journal of molecular sciences, 2020Co-Authors: Vishnu Sukumari Nath, Sebastjan Radisek, Jernej Jakse, Jaroslav Matousek, Ajay Kumar Mishra, Ankita Shrestha, Praveen Awasthi, Tomáš Kocábek, Andrej Sečnik, Vipin HallanAbstract:The mediator (MED) represents a large, conserved, multi-subunit protein complex that regulates gene expression through interactions with RNA polymerase II and enhancer-bound transcription factors. Expanding research accomplishments suggest the predominant role of plant MED subunits in the regulation of various physiological and developmental processes, including the biotic stress response against bacterial and fungal pathogens. However, the involvement of MED subunits in virus/Viroid pathogenesis remains elusive. In this study, we investigated for the first time the gene expression modulation of selected MED subunits in response to five Viroid species (Apple fruit crinkle Viroid (AFCVd), Citrus bark cracking Viroid (CBCVd), Hop Latent Viroid (HLVd), Hop stunt Viroid (HSVd), and Potato spindle tuber Viroid (PSTVd)) in two model plant species (Nicotiana tabacum and N. benthamiana) and a commercially important Hop (Humulus lupulus) cultivar. Our results showed a differential expression pattern of MED subunits in response to a Viroid infection. The individual plant MED subunits displayed a differential and tailored expression pattern in response to different Viroid species, suggesting that the MED expression is Viroid- and plant species-dependent. The explicit evidence obtained from our results warrants further investigation into the association of the MED subunit with symptom development. Together, we provide a comprehensive portrait of MED subunit expression in response to Viroid infection and a plausible involvement of MED subunits in fine-tuning transcriptional reprogramming in response to Viroid infection, suggesting them as a potential candidate for rewiring the defense response network in plants against pathogens.
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Evaluation of Disease Severity and Global Transcriptome Response Induced by Citrus bark cracking Viroid, Hop Latent Viroid, and Their Co-Infection in Hop (Humulus lupulus L.).
International journal of molecular sciences, 2019Co-Authors: Nataša Štajner, Sebastjan Radisek, Jaroslav Matousek, Ajay Kumar Mishra, Vishnu Sukumari Nath, Jernej JakseAbstract: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
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One-step multiplex RT-PCR for simultaneous detection of four Viroids from Hop (Humulus lupulus L.)
European Journal of Plant Pathology, 2019Co-Authors: T. Guček, Jernej Jakse, Jaroslav Matousek, Sebastjan RadisekAbstract:Hop ( Humulus lupulus L.) plants are hosts to several Viroids; some of them can be highly aggressive, and their infection can manifest as complete plant dieback. Since molecular detection of multiple Viroids can be time consuming and cost inefficient, a reliable one-step multiplex RT-PCR (mRT-PCR) was developed to detect simultaneously all four Viroids infecting Hops: Hop Latent Viroid (HLVd), Hop stunt Viroid (HSVd), Apple fruit crinkle Viroid (AFCVd) and Citrus bark cracking Viroid (CBCVd). Several primer pairs were tested on different Viroid variants from Hops, citruses and grapevines, and from among them, specific primer pairs for detection of Hop Viroids were selected and confirmed in a single-tube assay. To improve mRT-PCR reliability and validate its effectiveness, nad5 and DRH1 genes were included as an internal control. The specificities of single and mRT-PCR assays for all four Viroids were comparable. The sensitivity of mRT-PCR was compared with that of dot-blot hybridization and single RT-PCR assay on biolistically infected Hop plants. The results show mRT-PCR to be more sensitive than the dot-blot and slightly less sensitive than the single RT-PCR assay. Furthermore, mRT-PCR was validated using field samples and a group of 135 Hop plants, which are used in the certification scheme for planting material propagation, and the method proved to be robust, rapid and simple. Additionally, this approach can be applicable to similar methods of systematic surveys of emerging diseases and epidemiological studies.
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Propagation and some physiological effects of Citrus bark cracking Viroid and Apple fruit crinkle Viroid in multiple infected Hop (Humulus lupulus L.).
Journal of plant physiology, 2017Co-Authors: Jaroslav Matousek, T. Guček, Sebastjan Radisek, Jernej Jakse, K. Siglová, Joseph R. J. Brass, Taro Tsushima, Ganesh Selvaraj Duraisamy, Teruo Sano, Gerhard StegerAbstract:The Hop metabolome important for the brewing industry and for medical purposes is endangered worldwide due to multiple Viroid infections affecting Hop physiology. Combinatorial biolistic Hop inoculation with Citrus bark cracking Viroid (CBCVd), Apple fruit crinkle Viroid (AFCVd), Hop Latent Viroid, and Hop stunt Viroid (HSVd) showed a low CBCVd compatibility with HSVd, while all other Viroid combinations were highly compatible. Unlike to other Viroids, single CBCVd propagation showed a significant excess of (-) over (+) strands in Hop, tomato, and Nicotiana benthamiana, but not in citruses. Inoculation of Hop with all Viroids led to multiple infections with unstable Viroid levels in individual plants in the pre- and post-dormancy periods, and to high plant mortality and morphological disorders. Hop isolates of CBCVd and AFCVd were highly stable, only minor quasispecies were detected. CBCVd caused a strong suppression of some crucial mRNAs related to the Hop prenylflavonoid biosynthesis pathway, while AFCVd-caused effects were moderate. According to mRNA degradome analysis, this suppression was not caused by a direct Viroid-specific small RNA-mediated degradation. CBCVd infection led to a strong induction of two Hop transcription factors from WRKY family and to a disbalance of WRKY/WDR1 complexes important for activation of lupulin genes.
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Chapter 26 – Other CocadViroids
Viroids and Satellites, 2017Co-Authors: Irene Lavagi, Jaroslav Matousek, Georgios VidalakisAbstract:Citrus bark cracking Viroid (CBCVd) and the Hop Latent Viroid (HLVd) are members of the genus CocadViroid of the family PospiViroidae. Both Viroids are globally distributed. CBCVd does not induce any significant disease of citrus but incites bark cracking of sensitive citrus rootstock species and has been associated with a severe disease of Hops. HLVd does not induce any acute Hop symptoms but causes physiological changes with negative yield and quality effects. Both Viroids are readily transmitted by mechanical means, detected by molecular assays, and eliminated from the germplasm using meristematic tissue culture. Comprehensive programs that include the use of Viroid-tested propagative materials and sanitary measures are required for the management of CBCVd and HLVd.
A N Adams - One of the best experts on this subject based on the ideXlab platform.
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The experimental transmission of Hop Latent Viroid and its elimination by low temperature treatment and meristem culture
Annals of Applied Biology, 1996Co-Authors: A N Adams, D J Barbara, A Morton, P. DarbyAbstract:Summary Two aspects of Hop Latent Viroid (HLVd) relevant to control were examined: the production of Viroid-free plants from infected material and transmission of HLVd in the field. Plants free from HLVd were obtained by a combination of storing infected source plants at low temperature (2–4oC in the dark) for several months followed by meristem culture using small explants. A total of 77 plants of six cultivars and male pollinator clones were grown from meristems and 28 of these were free from HLVd. Tests showed that the cutting of stems (mimicking the use of tools) was more effective than abrasion (mimicking natural plant to plant contact) for the mechanical transmission of HLVd between Hop plants. When field-grown test plants were inoculated, infection occurred more commonly in May before plants had grown large enough for significant contact between neighbouring plants than later in the season. The aphid Phorodon humuli could not be shown to transmit HLVd. These results indicate that all Hop varieties and pollinator clones can be made available to the industry free from HLVd and that the chances of infection can be reduced by avoiding early-season cultural operations that cut into Hop shoots.
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the distribution of Hop Latent Viroid within plants of humulus lupulus and attempts to obtain Viroid free plants
Annals of Applied Biology, 1993Co-Authors: A Morton, D J Barbara, A N AdamsAbstract:Summary The detectability of Hop Latent Viroid (HLVd) was investigated in field-grown Hop (Humulus lupulus L.; an herbaceous perennial in which all the aerial parts die at the onset of winter) plants, using dot-blot hybridisation. The Viroid was readily detected in all aerial tissues in the second half of the growing season but it could not be detected very early in the season. Between early- and mid-season, HLVd was first detected at the base of the new stems and then apparently spread up them as they grew but only became detectable near the tips of the shoots at mid-season, approximately at the time most elongation growth ended and flowering began. Petioles were the most convenient tissues to test, being easy to collect and, relative to leaf lamina tissue, low in inhibitors. Both dot-blot and in situ hybridisation failed to detect HLVd in shoot tips taken from plants grown at two ‘low’ temperatures (10°C and 15°C). Failure to produce any Viroid-free plants by in vitro culture from such tips suggested that they did contain Viroid but at levels too low to detect by either method. Lower temperatures and smaller explants are now being investigated as means of producing Viroid-free plants.
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The distribution of Hop Latent Viroid within plants of Humulus lupulus and attempts to obtain Viroid‐free plants
Annals of Applied Biology, 1993Co-Authors: A Morton, D J Barbara, A N AdamsAbstract:Summary The detectability of Hop Latent Viroid (HLVd) was investigated in field-grown Hop (Humulus lupulus L.; an herbaceous perennial in which all the aerial parts die at the onset of winter) plants, using dot-blot hybridisation. The Viroid was readily detected in all aerial tissues in the second half of the growing season but it could not be detected very early in the season. Between early- and mid-season, HLVd was first detected at the base of the new stems and then apparently spread up them as they grew but only became detectable near the tips of the shoots at mid-season, approximately at the time most elongation growth ended and flowering began. Petioles were the most convenient tissues to test, being easy to collect and, relative to leaf lamina tissue, low in inhibitors. Both dot-blot and in situ hybridisation failed to detect HLVd in shoot tips taken from plants grown at two ‘low’ temperatures (10°C and 15°C). Failure to produce any Viroid-free plants by in vitro culture from such tips suggested that they did contain Viroid but at levels too low to detect by either method. Lower temperatures and smaller explants are now being investigated as means of producing Viroid-free plants.
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The distribution and spread of Hop Latent Viroid within two commercial plantings of Hop (Humulus lupulus)
Annals of Applied Biology, 1992Co-Authors: A N Adams, D J Barbara, A Morton, M. S. RidoutAbstract:Summary The distribution and spread of Hop Latent Viroid (HLVd) in two adjacent plantings of the Hop cultivars Omega and Wye Challenger have been studied for three seasons. The planting of cv. Omega was heavily infected at the start of the survey and spread was rapid; the density of infection was lower in the planting of cv. Wye Challenger and spread was much slower. It is not known whether the difference in rate of spread was a varietal effect or because of the higher density of infection in the Omega planting. The distribution of known infections in 1991 suggests that plant-to-adjacent-plant spread, either by contact or on tools, does occur. However, the overall distribution of infection and the occurrence of new infections not adjacent to existing ones, suggests that this is not the only means of transmission; whether a vector is involved is not known.
Detlev Riesner - One of the best experts on this subject based on the ideXlab platform.
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Biolistic inoculation of plants with Viroid nucleic acids.
Journal of virological methods, 2004Co-Authors: Jaroslav Matousek, Lidmila Orctová, Gerhard Steger, Detlev RiesnerAbstract:Abstract 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.
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Analysis of thermal stress-mediated PSTVd variation and biolistic inoculation of progeny of Viroid "thermomutants" to tomato and Brassica species.
Virology, 2004Co-Authors: Jaroslav Matousek, Lidmila Orctová, Gerhard Steger, Josef Škopek, Michaela Moors, Petr Dědič, Detlev RiesnerAbstract:Abstract 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.
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The variability of Hop Latent Viroid as induced upon heat treatment.
Virology, 2001Co-Authors: Jaroslav Matousek, Josef Patzak, Lidmila Orctová, Jörg Schubert, Lukas Vrba, Gerhard Steger, Detlev RiesnerAbstract:Abstract We have previously shown that heat treatment of Hop plants infected by Hop Latent Viroid (HLVd) reduces Viroid levels. Here we investigate whether such heat treatment leads to the accumulation of sequence variability in HLVd. We observed a negligible level of mutated variants in HLVd under standard cultivation conditions. In contrast, the heat treatment of Hop led to HLVd degradation and, simultaneously, to a significant increase in sequence variations, as judged from temperature gradient–gel electrophoresis analysis and cDNA library screening by DNA heteroduplex analysis. Thirty-one cDNA clones (9.8%) were identified as deviating forms. Sequencing showed mostly the presence of quadruple and triple mutants, suggesting an accumulation of mutations in HLVd during successive replication cycles. Sixty-nine percent of base changes were localised in the left half and 31% in the right half of the secondary structure proposed for this Viroid. No mutations were found in the central part of the upper conserved region. A “hot spot” region was identified in a domain known as a “pathogenicity domain” in the group representative, potato spindle tuber Viroid. Most mutations are predicted to destabilise HLVd secondary structure. All mutated cDNAs, however, were infectious and evolved into complex progeny populations containing molecular variants maintained at low levels.
Gerhard Steger - One of the best experts on this subject based on the ideXlab platform.
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Propagation and some physiological effects of Citrus bark cracking Viroid and Apple fruit crinkle Viroid in multiple infected Hop (Humulus lupulus L.).
Journal of plant physiology, 2017Co-Authors: Jaroslav Matousek, T. Guček, Sebastjan Radisek, Jernej Jakse, K. Siglová, Joseph R. J. Brass, Taro Tsushima, Ganesh Selvaraj Duraisamy, Teruo Sano, Gerhard StegerAbstract:The Hop metabolome important for the brewing industry and for medical purposes is endangered worldwide due to multiple Viroid infections affecting Hop physiology. Combinatorial biolistic Hop inoculation with Citrus bark cracking Viroid (CBCVd), Apple fruit crinkle Viroid (AFCVd), Hop Latent Viroid, and Hop stunt Viroid (HSVd) showed a low CBCVd compatibility with HSVd, while all other Viroid combinations were highly compatible. Unlike to other Viroids, single CBCVd propagation showed a significant excess of (-) over (+) strands in Hop, tomato, and Nicotiana benthamiana, but not in citruses. Inoculation of Hop with all Viroids led to multiple infections with unstable Viroid levels in individual plants in the pre- and post-dormancy periods, and to high plant mortality and morphological disorders. Hop isolates of CBCVd and AFCVd were highly stable, only minor quasispecies were detected. CBCVd caused a strong suppression of some crucial mRNAs related to the Hop prenylflavonoid biosynthesis pathway, while AFCVd-caused effects were moderate. According to mRNA degradome analysis, this suppression was not caused by a direct Viroid-specific small RNA-mediated degradation. CBCVd infection led to a strong induction of two Hop transcription factors from WRKY family and to a disbalance of WRKY/WDR1 complexes important for activation of lupulin genes.
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Elimination of Hop Latent Viroid upon developmental activation of pollen nucleases.
Biological chemistry, 2008Co-Authors: Jaroslav Matousek, Lidmila Orctová, Josef Škopek, Karel Pešina, Gerhard StegerAbstract:Hop Latent Viroid (HLVd) is not transmissible through Hop generative tissues and seeds. Here we describe the process of HLVd elimination during development of Hop pollen. HLVd propagates in uninucleate Hop pollen, but is eliminated at stages following first pollen mitosis during pollen vacuolization and maturation. Only traces of HLVd were detected by RT-PCR in mature pollen after anthesis and no Viroid was detectable in in vitro germinating pollen, suggesting complete degradation of circular and linear HLVd forms. The majority of the degraded HLVd RNA in immature pollen included discrete products in the range of 230-100 nucleotides and therefore did not correspond to siRNAs. HLVd eradication from pollen correlated with developmental expression of a pollen nuclease and specific RNAses. Activity of the pollen nuclease HBN1 was maximal during the vacuolization step and decreased in mature pollen. Total RNAse activity increased continuously up to the final steps of pollen maturation. HBN1 mRNA, which is abundant at the uninucleate microspore stage, encodes a protein of 300 amino acids (34.1 kDa, isoeletric point 5.1). Sequence comparisons revealed that HBN1 is a homolog of S1-like bifunctional plant endonucleases. The developmentally activated HBN1 and pollen ribonucleases could participate in the mechanism of HLVd recognition and degradation.
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Biolistic inoculation of plants with Viroid nucleic acids.
Journal of virological methods, 2004Co-Authors: Jaroslav Matousek, Lidmila Orctová, Gerhard Steger, Detlev RiesnerAbstract:Abstract 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.
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Analysis of thermal stress-mediated PSTVd variation and biolistic inoculation of progeny of Viroid "thermomutants" to tomato and Brassica species.
Virology, 2004Co-Authors: Jaroslav Matousek, Lidmila Orctová, Gerhard Steger, Josef Škopek, Michaela Moors, Petr Dědič, Detlev RiesnerAbstract:Abstract 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.
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Plant 7SL RNA and tRNA^Tyr genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells
Plant Molecular Biology, 2002Co-Authors: Yasushi Yukawa, Jaroslav Matousek, Lukas Vrba, Gerhard Steger, Michael Grimm, Masahiro Sugiura, Hildburg BeierAbstract:RNA polymerase III-driven cassettes for the expression of antisense RNAs and ribozymes have recently attracted much attention because (1) pol III genes are transcribed abundantly in all kinds of tissues and (2) the transcripts are very stable by virtue of their small and compact size. We have designed two types of pol III-based expression vehicles. Antisense RNA sequences targeted against conserved structural elements or domains in the RNAs of potato spindle tuber Viroid, Hop Latent Viroid and potato virus S were either embedded in the anticodon region of a Nicotiana tRNA^Tyr gene or near the 3′ end of an Arabidopsis 7SL RNA gene. Both classes of chimeric genes were transcribed in vitro in a homologous plant extract. Our studies clearly revealed that the modified tRNA and 7SL RNA genes, carrying insertions of up to 90 and 120 bp, respectively, were expressed efficiently in the tobacco nuclear extract, resulting in high levels of stable chimeric transcripts. 7SL RNA (also termed SRP RNA) represents the RNA component of the signal recognition particle. This is the first report of demonstrating the employment of 7SL RNA genes as potential cassettes for the expression of antisense RNA and ribozyme sequences and might be helpful in future experiments to control their localization in specific sub-cellular compartments.
A Morton - One of the best experts on this subject based on the ideXlab platform.
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The experimental transmission of Hop Latent Viroid and its elimination by low temperature treatment and meristem culture
Annals of Applied Biology, 1996Co-Authors: A N Adams, D J Barbara, A Morton, P. DarbyAbstract:Summary Two aspects of Hop Latent Viroid (HLVd) relevant to control were examined: the production of Viroid-free plants from infected material and transmission of HLVd in the field. Plants free from HLVd were obtained by a combination of storing infected source plants at low temperature (2–4oC in the dark) for several months followed by meristem culture using small explants. A total of 77 plants of six cultivars and male pollinator clones were grown from meristems and 28 of these were free from HLVd. Tests showed that the cutting of stems (mimicking the use of tools) was more effective than abrasion (mimicking natural plant to plant contact) for the mechanical transmission of HLVd between Hop plants. When field-grown test plants were inoculated, infection occurred more commonly in May before plants had grown large enough for significant contact between neighbouring plants than later in the season. The aphid Phorodon humuli could not be shown to transmit HLVd. These results indicate that all Hop varieties and pollinator clones can be made available to the industry free from HLVd and that the chances of infection can be reduced by avoiding early-season cultural operations that cut into Hop shoots.
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the distribution of Hop Latent Viroid within plants of humulus lupulus and attempts to obtain Viroid free plants
Annals of Applied Biology, 1993Co-Authors: A Morton, D J Barbara, A N AdamsAbstract:Summary The detectability of Hop Latent Viroid (HLVd) was investigated in field-grown Hop (Humulus lupulus L.; an herbaceous perennial in which all the aerial parts die at the onset of winter) plants, using dot-blot hybridisation. The Viroid was readily detected in all aerial tissues in the second half of the growing season but it could not be detected very early in the season. Between early- and mid-season, HLVd was first detected at the base of the new stems and then apparently spread up them as they grew but only became detectable near the tips of the shoots at mid-season, approximately at the time most elongation growth ended and flowering began. Petioles were the most convenient tissues to test, being easy to collect and, relative to leaf lamina tissue, low in inhibitors. Both dot-blot and in situ hybridisation failed to detect HLVd in shoot tips taken from plants grown at two ‘low’ temperatures (10°C and 15°C). Failure to produce any Viroid-free plants by in vitro culture from such tips suggested that they did contain Viroid but at levels too low to detect by either method. Lower temperatures and smaller explants are now being investigated as means of producing Viroid-free plants.
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The distribution of Hop Latent Viroid within plants of Humulus lupulus and attempts to obtain Viroid‐free plants
Annals of Applied Biology, 1993Co-Authors: A Morton, D J Barbara, A N AdamsAbstract:Summary The detectability of Hop Latent Viroid (HLVd) was investigated in field-grown Hop (Humulus lupulus L.; an herbaceous perennial in which all the aerial parts die at the onset of winter) plants, using dot-blot hybridisation. The Viroid was readily detected in all aerial tissues in the second half of the growing season but it could not be detected very early in the season. Between early- and mid-season, HLVd was first detected at the base of the new stems and then apparently spread up them as they grew but only became detectable near the tips of the shoots at mid-season, approximately at the time most elongation growth ended and flowering began. Petioles were the most convenient tissues to test, being easy to collect and, relative to leaf lamina tissue, low in inhibitors. Both dot-blot and in situ hybridisation failed to detect HLVd in shoot tips taken from plants grown at two ‘low’ temperatures (10°C and 15°C). Failure to produce any Viroid-free plants by in vitro culture from such tips suggested that they did contain Viroid but at levels too low to detect by either method. Lower temperatures and smaller explants are now being investigated as means of producing Viroid-free plants.
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The distribution and spread of Hop Latent Viroid within two commercial plantings of Hop (Humulus lupulus)
Annals of Applied Biology, 1992Co-Authors: A N Adams, D J Barbara, A Morton, M. S. RidoutAbstract:Summary The distribution and spread of Hop Latent Viroid (HLVd) in two adjacent plantings of the Hop cultivars Omega and Wye Challenger have been studied for three seasons. The planting of cv. Omega was heavily infected at the start of the survey and spread was rapid; the density of infection was lower in the planting of cv. Wye Challenger and spread was much slower. It is not known whether the difference in rate of spread was a varietal effect or because of the higher density of infection in the Omega planting. The distribution of known infections in 1991 suggests that plant-to-adjacent-plant spread, either by contact or on tools, does occur. However, the overall distribution of infection and the occurrence of new infections not adjacent to existing ones, suggests that this is not the only means of transmission; whether a vector is involved is not known.