| interdependence of pathogenicity and replicability with potato spindle tuber viroid. | after the unexpected appearance of lethal symptoms on tomato plants infected with the pstvd strain intermediate di, viroids were isolated and sequenced. it was found that a new strain, named rg 1, had been generated spontaneously in our greenhouse. in a different series of plant passages two new strains, named qf a and qf b, were detected which coexisted with the wild-type strain di. strains qf a and qf b showed intermediate symptoms when inoculated separately. in order to confirm the working hy ... | 1995 | 7747485 |
| replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing. | the structure of a series of rnas extracted from avocado infected by the 247-nt avocado sunblotch viroid (asbvd) was investigated. the identification of multistranded complexes containing circular asbvd rnas of (+) and (-) polarity suggests that replication of asbvd proceeds through a symmetric pathway with two rolling circles where these two circular rnas are the templates. this is in contrast to the replication of potato spindle tuber viroid and probably of most of its related viroids, which p ... | 1994 | 7809126 |
| multimeric non-radioactive crna probes improve detection of potato spindle tuber viroid (pstvd). | experimental data showed that multimeric, complementary rna (crna) probes, labelled with non-radioactive digoxigenin (dig), improved sensitivity of detection of the potato spindle tuber viroid (pstvd) rna by 2- to 30-fold as compared with corresponding multimeric cdna probes. the degree of pstvd detectability improvement depended upon the type of alkaline phosphatase substrate (colorimetric vs. chemiluminescent) used. use of hexameric dig-labelled crna probes in combination with chemiluminescent ... | 1994 | 7822462 |
| cloning of the capsid protein gene from a blotch isolate of peanut stripe virus. | the 3' terminal 1,367 nucleotides (nts) of a blotch isolate of the potyvirus, peanut stripe virus (pstv), were cloned and sequenced. this region included the 861 nts (287 amino acids) of the pstv capsid protein (cp). the amino acid sequence of the predicted proteinase cleavage site was identified. this region shared limited homology with other potyvirus proteinase cleavage sites. the viral cp gene sequence was followed by 254 nucleotides of 3' nontranslated sequence and a poly-a tail. based on c ... | 1993 | 7916587 |
| avocado sunblotch viroid: primary sequence and proposed secondary structure. | the sequence of the 247 nucleotide residues of the single strand circular rna of avocado sunblotch viroid (asbv) was determined using partial enzymic cleavage methods on overlapping viroid fragments obtained by partial ribonuclease digestion followed by 32p-labelling in vitro at their 5'-ends. asbv is much smaller than potato spindle tuber viroid (pstv; 359 residues) and chrysanthemum stunt viroid (csv; 356 residues). a secondary structure model for asbv is proposed and contains 67% of its resid ... | 1981 | 7322921 |
| analysis of acid-extractable tomato leaf proteins after infection with a viroid, two viruses and a fungus and partial purification of the "pathogenesis-related" protein p 14. | gel electrophoretic analysis revealed marked alterations in the pattern of acid-extractable proteins from tomato leaves after infection with a viroid (pstv), two viruses [tobacco mosaic virus (tmv) and cucumber mosaic virus (cmv)], and a fungus (cladosporium fulvum) when compared to the pattern from healthy leaves. a pathogen-specific appearance of new protein bands was only found after infection with tmv (mw 17,400 and 65,000), cmv (mw 9000 and 8000) and cladosporium fulvum (mw 28,000). with th ... | 1982 | 6891893 |
| cloning and sequence analysis of the coat protein genes of an australian strain of peanut mottle and an indonesian 'blotch' strain of peanut stripe potyviruses. | we have analysed the coat protein gene sequences of two potyviruses infecting peanut. the 3' terminal 1247 nucleotides (nt) of an australian strain of peanut mottle virus (pemov-au) and the 3' terminal 1388 nt of an indonesian 'blotch' strain of peanut stripe virus (pstv-ib) were cloned and sequenced. those regions included the 861 and 864 nt encoding the respective putative coat proteins as well as the 285 and 253 nt, respectively of 3' non-coding sequences. comparison of the nucleotide sequenc ... | 1994 | 7999143 |
| gel dependence of electrophoretic mobilities of double-stranded and viroid rna and estimation of the contour length of a viroid by gel electrophoresis. | double-stranded (ds) rna normally exhibits a lower electrophoretic mobility than dsdna having the same number of base pairs. this has been attributed to its net charge density that is lower than that of b-form dna. but we show here that dsrna runs faster than corresponding dna in gels containing either > or = 2.5% agarose or > or = 8% acrylamide with high crosslinking (19:1 acrylamide:n,n'-methylenebisacrylamide). however, the relative mobility of dsrna as compared with dna, extrapolated to 0% g ... | 1994 | 7720685 |
| analysis of the population structure of three phenotypically different pstvd isolates. | phenotypically dissimilar greenhouse isolates from a polish collection of potato spindle tuber viroid (pstvd) were analysed. partially purified pstvd genomic rnas from severe, intermediate and mild isolates was reverse transcribed and the resulting cdnas enzymatically amplified. abutting-primer pcr (ab-p pcr) technology was used to obtain, in a single step, infectious full-length pstvd cdna monomers and these were sequenced. the mild isolate was found to be composed of a unique molecular variant ... | 1994 | 7998831 |
| agrobacterium-mediated inoculation of pstvd cdnas onto tomato reveals the biological effect of apparently lethal mutations. | potato spindle tuber viroid (pstvd) mutants which contain alterations in the terminal loops of the rod-like native structure have previously been reported from our laboratory. pstvd-p contains mutations at positions 2, 4, and 6 in the left terminal loop; pstvd-r+, a sequence permutation of pstvd-r, contains the same mutations at positions 177 and 178 in the right terminal loop as pstvd-r and contains in addition a 1-nucleotide g insertion at position 176. pstvd-p, pstvd-r, and pstv-r+ were nonin ... | 1994 | 7513926 |
| detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization. | in situ hybridization experiments were carried out to detect avocado sunblotch viroid (asbvd) in foliar tissue of avocado, using a digoxigenin-labelled rna probe complementary to the asbvd-rna in sections of aldehyde-fixed, lrgold-embedded leaf samples. detection of the probe was made through anti-digoxigenin antibody and protein-a colloidal gold (20 nm). seventy to 80% of the signals came from chloroplast while the cytoplasm and vacuole were labelled with ca. 10% of the gold particles. this is ... | 1994 | 7998844 |
| nucleotide sequence and genome organization of peanut stripe potyvirus. | a blotch isolate of peanut stripe virus (pstv) was cloned, sequenced and compared with other full-length potyvirus sequences. the viral genome was 10059 nucleotides (nt) in length excluding the poly(a) tail. two potential aug start codons were identified in the 5' non-translated region. analysis of in vitro translation products from transcripts containing the first 600 nt of the pstv genome indicated that the first aug (nt 134 to 136) was preferred over the second aug (nt 146 to 148) for initiat ... | 1994 | 8077957 |
| primary and secondary structure of a 360-nucleotide isolate of potato spindle tuber viroid. | full-length complementary dnas (cdna) of a mild (kf5) and a severe (s-pstvd) isolate of potato spindle tuber viroid (pstvd) were constructed. dna sequencing of four kf5 cdna clones (m3, m4, m5, and m7) revealed that kf5 is comprised of 360 nucleotides. by comparison, all three cdna clones (s2, s9, and s10) of s-pstvd possess 359 nucleotides. sequence microheterogeneity was observed among the kf5 cdna clones. clone m5 differs from mild pstvd isolate kf6 by a u-to-a transversion at position 303 fo ... | 1993 | 8435045 |
| multiple pathways of reversion in viroids for conservation of structural elements. | from site-directed mutagenesis of potato spindle tuber viroid (pstvd) it had been concluded earlier that the formation of a thermodynamically metastable structure containing hairpin ii (hp ii) is critical for infectivity. in order to differentiate between structural and sequence effects, in the present work base pairs in hp ii were exchanged by site-directed double mutations without significant alterations in the native rod-like structure of pstvd. the mutants were viable and genetically stable ... | 1993 | 8491201 |
| inhibition of potato spindle tuber viroid (pstvd) infection with dna oligonucleotides. | 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 t ... | 1993 | 8504180 |
| description of rna folding by "simulated annealing". | an algorithm is proposed which describes the thermodynamically as well as the kinetically controlled folding process of rna. the algorithm, based on a special monte carlo procedure known as "simulated annealing", takes into account the probabilities for opening and closing of single base-pairs. thus, the algorithm is able to reach structures and structure distributions near the global minimum of structure space, and is not restricted by the tendency to halt in local minima. three types of struct ... | 1996 | 8568872 |
| role of the variable domain in modulating potato spindle tuber viroid replication. | potato spindle tuber viroid (pstvd) is believed to undergo a series of specific structural transitions during replication. the variable domain of pstvd is known to contain sequences that are important for replication/accumulation as well as one of three "premelting regions" which control breakdown of the native structure in vitro. we have examined the structural and biological effects of five single and two double nucleotide substitutions within premelting region 3 in an effort to isolate temper ... | 1996 | 8623553 |
| the cdna sequence and infectious transcripts of peanut stripe virus. | a full-length cdna clone of the blotch isolate of the peanut stripe potyvirus (pstv) rna genome was constructed downstream from the bacteriophage sp6 rna polymerase promoter. the full-length pstv cdna clone (pstvsf9) was sequenced and compared to the previously published sequence of pstv-b. in vitro-synthesized pstv transcripts capped with m7gpppg were infectious in nicotiana benthamiana plants, and the progeny virus was aphid transmissible. to confirm the origin of infection, a mutant pstv (pst ... | 1996 | 8666291 |
| continuous replication of potato spindle tuber viroid (pstv) in permanent cell cultures of potato and tomato. | the continuous replication of potato spindle tuber viroid (pstv) in callus cultures from pstv-infected wild-type potato (solanum demissum l.) and tomato (lycopersicon peruvianum l. mill) plants and in cell suspensions derived from potato protoplasts (solanum tuberosum l.) inoculated in vitro is described. the persistence of pstv replication in these cell lines through at least 14 subculture passages, which corresponds to a continuous replication over a period of more than one year, was demonstra ... | 1981 | 7284577 |
| rna structural features responsible for potato spindle tuber viroid pathogenicity. | the native structure of potato spindle tuber viroid (pstvd) contains a series of short double helices and small internal loops that are organized into five structural domains. nucleotides within the pathogenicity domain are known to play a critical role in modulating pstvd symptom expression, and it has been suggested that disruption of a comparatively unstable "premelting region" within the pathogenicity domain may be required for disease induction. we have used a combination of quantitative bi ... | 1996 | 8806495 |
| a viroid from solanum pseudocapsicum closely related to the tomato apical stunt viroid. | a viroid was isolated from symptomless solanum pseudocapsicum cultivar (cv) 'new patterson' plants using the bidirectional page method for analysis of small circular rna molecules. the viroid was transmitted to tomato by mechanical inoculation. infected tomato plants developed symptoms similar to those caused by potato spindle tuber viroid (pstvd). cloning and sequencing revealed that the viroid from solanum pseudocapsicum is closely related to the tomato apical stunt viroid (tasvd). therefore, ... | 1996 | 8856021 |
| capsid protein properties of cowpea aphid-borne mosaic virus and blackeye cowpea mosaic virus confirm the existence of two major subgroups of aphid-transmitted, legume-infecting potyviruses. | a study of the capsid proteins of different legume-infecting potyviruses using specific monoclonal antibodies on immunoblots of crude extracts from infected plants revealed that cowpea aphid-borne mosaic virus (camv) and blackeye cowpea mosaic virus (bicmv) have coat protein m(r) values of 32k and 35k, respectively. immunoblot comparisons of bicmv, peanut stripe mosaic virus (pstv), bean common mosaic virus (bcmv) and azuki bean mosaic virus (azmv) revealed equal reactivity of their 35k coat pro ... | 1994 | 7996147 |
| mexican papita viroid: putative ancestor of crop viroids. | the potato spindle tuber disease was first observed early in the 20th century in the northeastern united states and shown, in 1971, to be incited by a viroid, potato spindle tuber viroid (pstvd). no wild-plant pstvd reservoirs have been identified; thus, the initial source of pstvd infecting potatoes has remained a mystery. several variants of a novel viroid, designated mexican papita viroid (mpvd), have now been isolated from solanum cardiophyllum lindl. (papita güera, cimantli) plants growing ... | 1996 | 8790341 |
| the molecular structure of iresine viroid, a new viroid species from iresine herbstii ('beefsteak plant'). | a viroid was isolated from iresine herbstii plants using the bidirectional page method for analysis of small circular rna molecules. the viroid was transmitted to viroid-free iresine herbstii plants of the cultivar 'aureoreticulata' by mechanical inoculation. infected plants did not develop any symptoms. the new viroid species has been named iresine viroid (irvd). it is a member of the potato spindle tuber viroid group and consists of 370 nucleotides, 227 g+c, 143 a+u (gc content, 61.4%). the mo ... | 1996 | 8887500 |
| cowpea aphid borne mosaic virus-morocco and south african passiflora virus are strains of the same potyvirus. | high performance liquid chromatography (hplc) profiles of tryptic peptides and partial amino acid sequence analysis have been employed to establish the taxonomic status of the moroccan isolate of cowpea aphid-borne mosaic virus (cabmv). some previous reports have suggested cabmv to be very closely related to blackeye cowpea mosaic virus (b1cmv) while other reports have concluded that this relationship is distant. in this report a tryptic digest of the coat protein of cabmv-morocco was compared w ... | 1994 | 8002788 |
| cloned single- and double-stranded dna copies of potato spindle tuber viroid (pstv) rna and co-inoculated subgenomic dna fragments are infectious. | a set of monomeric and oligomeric potato spindle tuber viroid (pstv) specific dna forms representing complete dna copies of the circular pstv rna genome were constructed and cloned in plasmid pbr322 and bacteriophage m13. both single- and double-stranded pstv dnas are capable of initiating viroid replication in mechanically inoculated tomato plants where it normally proceeds via the rna-rna pathway without dna being involved. all dimeric and higher multimeric forms were infectious irrespective o ... | 1984 | 6549294 |
| a single nucleotide substitution converts potato spindle tuber viroid (pstvd) from a noninfectious to an infectious rna for nicotiana tabacum. | mechanical inoculation of nicotiana tabacum with the pstvd isolate kf 440-2 from the host plant tomato resulted in the de novo emergence, replication, and accumulation of a new "tobacco variant," designated pstvd nt. it produces no symptoms in tobacco but, like pstv kf 440-2, severe ones in tomato. the sequence analysis of pstvd nt revealed a single nucleotide substitution from c-->u at position 259. autonomous viroid replication was also induced in tobacco by genomic integration of oligomeric c ... | 1996 | 8955038 |
| a method combining immunocapture and pcr amplification in a microtiter plate for the detection of plant viruses and subviral pathogens. | a method for the detection of rna viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (rt-pcr) and quantitation of the amplified products by fluorescence. the reverse transcription of the rna is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. the whole procedure can be carried out in a microtiter pla ... | 1993 | 8113346 |
| [design of a universal biotin-containing oligonucleotide diagnostic kit for detecting viroid plant diseases]. | a non-radioactive diagnosticum for plant viroid diseases has been designed, based on hybridization of a biotin-labeled 26-member oligonucleotide probe with the viroid rna site identical for potato spindle tuber viroid and chrysanthemum stunt viroid. the biotin label has been introduced into the synthetic oligonucleotide probe by the high-yield acylation of the oligonucleotide aminoalkylamide with the biotin imidazolide or n-hydroxysuccinimide ester. hybridization techniques have been elaborated ... | 1993 | 8250981 |
| rna-directed rna polymerase from tomato leaves. i. purification and physical properties. | an rna-directed rna polymerase (rdrp, ec 2.7.7.48) from tomato leaf tissue was purified to electrophoretic homogeneity. a terminal transferase activity that was found to cofractionate with rdrp from deae-sepharose and dna-cellulose columns was removed by chromatography on a mono q column. the highly purified rdrp exhibits a specific activity of 500 nmol x mg-1 x 30 min-1, which corresponds to a 100,000-fold enrichment of the enzyme. in buffer containing 50% glycerol, its activity decreased by ab ... | 1993 | 7685022 |
| identification and molecular properties of a 306 nucleotide viroid associated with apple dimple fruit disease. | a new viroid associated with an apple fruit disorder similar to, but more severe than, the dapple apple disease induced in some varieties by apple scar skin viroid (assvd) has been found. the new viroid, tentatively termed apple dimple fruit viroid (adfvd), is a circular rna of 306 nucleotides which adopts a quasi-rod-like conformation of minimum free energy. it contains the core nucleotides of the central conserved region (ccr) of the assvd group as well as the terminal conserved region (tcr) p ... | 1996 | 8922478 |
| mechanism of viroid pathogenesis: differential activation of the interferon-induced, double-stranded rna-activated, m(r) 68,000 protein kinase by viroid strains of varying pathogenicity. | purified potato spindle tuber viroid (pstvd) was added to an in vitro assay system containing purified interferon-induced, dsrna-activated protein kinase (p68). viroid rna activated (phosphorylated) the enzyme, although with less efficiency than did the synthetic, perfectly matched poly i-poly c. in binding experiments, rna transcripts of the intermediate strain of pstvd were shown to specifically bind to a p68-antibody complex. activation of the enzyme by a strain of pstvd that results in sever ... | 1993 | 7505621 |
| secondary structure probing of potato spindle tuber viroid (pstvd) and sequence comparison with other small pathogenic rna replicons provides evidence for central non-canonical base-pairs, large a-rich loops, and a terminal branch. | using pcr and in vitro transcription, linear (non-circular) unit-length (+)strand rna molecules of a lethal pstvd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular pstvd molecules, namely a fast migrating rod-like and a slowly migrating cruciform structure. the rod-like conformer of two end-labelled transcripts was probed with nucle ... | 1996 | 8876645 |
| antisense dna inhibits infection of potato spindle tuber viroid. | 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 ... | 1993 | 8258371 |
| viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop e conformation. | a longer-than-unit-length transcript of potato spindle tuber viroid is correctly processed in a potato nuclear extract only if the central conserved region is folded into a multi-helix junction containing at least one gnra tetraloop-hairpin. the cleavage-ligation site between g95 and g96 was mapped with s1 nuclease and primer extension. the structural motifs involved in the processing mechanism were analysed by uv crosslinking, chemical mapping, phylogenetic comparison and thermodynamic calculat ... | 1997 | 9034342 |
| classification of rna secondary structures using the techniques of cluster analysis. | an rna molecule has a lot of suboptimal secondary structures. some of these suboptimal structures are very similar and some are entirely different. in some cases, the free energy of these suboptimal structures does not differ much from that of the optimal structure. in order to characterize this relationship more clearly, we defined a metric among the secondary structures by the unweighted pair group method, using the arithmetic average and extended this metric to the sets of secondary structure ... | 1996 | 8959113 |
| rna-directed de novo methylation of genomic sequences in plants. | one monomeric and three oligomeric potato spindle tuber viroid (pstvd) cdna units were introduced into the tobacco genome via the agrobacterium-mediated leaf-disc transformation. southern analysis of the integrates revealed that only their pstvd-specific sequences become fully methylated, whereas the flanking t-dna and the genomic plant dna remain unaltered. viroid cdna methylation could only be observed after autonomous viroid rna-rna replication had taken place in these plants. these findings ... | 1994 | 8313476 |
| chemiluminescent detection of potato and pome fruit viroids by digoxigenin-labeled dot blot and tissue blot hybridization. | a chemiluminescent molecular hybridization protocol was compared to 32p autoradiography for detecting potato spindle tuber viroid (pstvd) and apple scar skin group viroids (assvd). labeled crna probes for pstvd and assvd were synthesized by sp6 rna polymerase transcription using digoxigenin-11-utp or alpha-[32p]utp. dot blot hybridization of purified viroids and sap extracts from infected plants showed that chemiluminescent detection using digoxigenin-labeled probes was as sensitive as autoradio ... | 1993 | 8366166 |
| detection and tissue distribution of potato spindle tuber viroid in infected tomato plants by tissue print hybridization. | potato spindle tuber viroid (pstvd) was detected in two cultivars of tomato (lycopersicon esculentum mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35s-labeled pstvd rna probe. pstvd was detectable in the viroid-sensitive and symptom-developing cv "rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "goldkugel" 3 weeks p.i. in both tomato cultivars, pstvd accumulated in the upper parts of the plants newly grown after inoculation. ... | 1997 | 9267443 |
| transgenic potato expressing a double-stranded rna-specific ribonuclease is resistant to potato spindle tuber viroid. | we have produced transgenic potato lines expressing the yeast-derived double-stranded rna-specific ribonuclease pac1. five lines of pac1 potato (solanum tuberosum l., cultivar russet burbank) challenged with potato spindle tuber viroid (pstvd) suppressed pstvd infection and accumulation. all of the progeny potato tubers produced by resistant plants were also free of pstvd. because the pac1 gene product digested pstvd in vitro, double-stranded regions in pstvd molecule and/or replicative intermed ... | 1997 | 9359114 |
| cell-to-cell movement of potato spindle tuber viroid. | viroids are non-translatable, autonomously replicating circular rnas that infect only plants. an important component of the viroid infection process is cell-to-cell movement; however, there is virtually no information available about the pathways and mechanisms of this process. in this study, potato spindle tuber viroid (pstvd) has been used as a model system to investigate the mechanism of viroid cell-to-cell transport. infectious rna transcripts were produced from pstvd cdna clones in vitro, l ... | 1997 | 9375403 |
| precisely full length, circularizable, complementary rna: an infectious form of potato spindle tuber viroid. | the replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. for potato spindle tuber (pstvd) and related viroids, the possible role of a circular (-)strand rna as a template for synthesis of (+)strand progeny is unclear. infected plants appear to contain only multimeric linear (-)strand rnas, and attempts to initiate infection with multimeric (-)pstvd rnas generally have failed. to examine critically the inf ... | 1998 | 9601006 |
| a mixture of synthetic oligonucleotide probes labeled with biotin for the sensitive detection of potato spindle tuber viroid. | five kinds of synthetic oligonucleotide probes labeled with biotin (bio) were designed for the detection of potato spindle tuber viroid (pstvd), and their sensitivities were compared with that of a digoxigenin (dig)- or bio-labeled cdna probe. although each oligonucleotide probe alone was less sensitive than the dig-cdna probe, sensitivity was increased by using a mixture of two or all of the five oligonucleotide probes. the sensitivity of a psmix1-5 probe, which was a mixture of five oligonucle ... | 1998 | 9626955 |
| specific rna self-cleavage in coconut cadang cadang viroid: potential for a role in rolling circle replication. | the rolling circle replication of the small, single-stranded viroid rnas requires a specific processing reaction to produce monomeric rnas that are ligated into the final circular form. for avocado sunblotch viroid, peach latent mosaic viroid, and chrysanthemum chlorotic mottle viroid, the hammerhead self-cleavage reaction is considered to provide this processing reaction. we have searched for a similar type of reaction in the 246-nt coconut cadang cadang viroid, the smallest viroid of the 24-me ... | 1998 | 9630248 |
| ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes. | a hammerhead ribozyme [r(-)] targeting the minus strand rna of potato spindle tuber viroid (pstvd) and a mutated nonfunctional ribozyme [mr(-)] were designed, cloned, and transcribed. as predicted, both monomer and dimer transcripts of the active r(-) ribozyme gene could cleave the pstvd minus strand dimer rna into three fragments of 77, 338, and 359 bases in vitro at 25 and 50 degrees c. the tandem dimer genes of r(-) and mr(-) were subcloned separately into the plant expression vector prok2. t ... | 1997 | 9144155 |
| correlation between bending of the vm region and pathogenicity of different potato spindle tuber viroid strains. | only 40 of the 359 nucleotides of potato spindle tuber viroid (pstvd) represent the virulence-modulating (vm) region. minor sequence variations in this domain distinguish mild from severe and even necrotic strains. our recent hypothesis (owens ra et al., 1996, virology 222:144-158) that these differences result in varying degrees of bending of this part of the molecule could be tested experimentally. by in vitro transcription and partial double-strand formation, three types of model rnas were pr ... | 1998 | 9769103 |
| a viroid from brunfelsia undulata closely related to the columnea latent viroid. | a viroid was isolated from symptomless brunfelsia undulata plants using the bidirectional page method for analysis of small circular rna molecules. the viroid was transmitted to tomato by mechanical inoculation. infected tomato plants developed symptoms similar to those caused by intermediate strains of potato spindle tuber viroid (pstvd). cloning and sequencing revealed that the viroid from brunfelsia undulata is closely related to the columnea latent viroid (clvd). therefore, the new viroid se ... | 1996 | 8920818 |
| sequences of the coat protein gene of five peanut stripe virus (pstv) strains from thailand and their evolutionary relationship with other bean common mosaic virus sequences. | the coat protein gene and part of the 3' non-coding region of five strains of peanut stripe virus (pstv) from thailand have been cloned and sequenced. phylogenetic comparisons of these strains, known as t1, t3, t5, t6 and t7, and related sequences showed that these strains are indeed strains of pstv. further, pstv strains appear to be related to each other according to their geographic origin. that is, the thai strains are more closely related to each other than they are to strains from the usa ... | 1998 | 9787652 |
| potyvirus aphid transmission requires helper component and homologous coat protein for maximal efficiency. | aphid transmission of potyviruses depends on the presence of specific sequence domains in two virus encoded proteins, the coat protein (cp) and helper component-proteinase (hc-pro). aphid transmissable peanut stripe virus (pstv), like most potyviruses, has an asp-ala-gly (dag) motif in the amino-terminal part of the cp. peanut mottle virus (pemov) was determined to be highly aphid transmissible but has a unique asp-ala-ala-ala (daaa) motif. to determine if the daaa motif could functionally repla ... | 1998 | 9856099 |
| evidence for heterologous encapsidation of potato spindle tuber viroid in particles of potato leafroll virus. | the aphid myzus persicae (sulz.) was shown to transmit potato spindle tuber viroid (pstvd) to potato clone dto-33 from source plants doubly infected with potato leafroll virus (plrv) and pstvd. transmission was of the persistent type and did not occur when the insects were allowed to feed on singly infected plants. only low levels of pstvd were associated with purified plrv virions, but its resistance to digestion with micrococcal nuclease indicates that the viroid rna is encapsidated within the ... | 1997 | 9191909 |
| rna movies: visualizing rna secondary structure spaces. | rna movies is a system for the visualization of rna secondary structure spaces. its input is a script consisting of primary and secondary structure information. from this script, the system fully automatically generates animated graphical structure representations. in this way, it creates the impression of an rna molecule exploring its own two-dimensional structure space. | 1999 | 10068690 |
| heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of rna-directed dna methylation. | previous analysis of potato spindle tuber viroid (pstvd) rna-infected tobacco plants has suggested that an rna-dna interaction could trigger de novo methylation of pstvd transgene sequences. using the genomic sequencing technique, the methylation pattern associated with the rna-directed dna methylation process has been characterized. three different pstvd transgene constructs all showed a similar pattern of methylation. most of the cytosines at symmetrical as well as non-symmetrical positions ap ... | 1999 | 10075993 |
| destabilization of potato spindle tuber viroid by mutations in the left terminal loop. | infectivity studies with highly infectious rna inocula generated by ribozyme cleavage were used to compare the biological properties of three apparently nonviable mutants of potato spindle tuber viroid (pstvd). one of these mutants (pstvd-p) contains three nucleotide substitutions in the left terminal loop, and mechanical inoculation of tomato seedlings with rna transcripts at levels equivalent to 10(3)-10(5) times the id50 for pstvd-intermediate failed to result in systemic infection. viable pr ... | 1997 | 9191908 |
| rapid generation of genetic heterogeneity in progenies from individual cdna clones of peach latent mosaic viroid in its natural host. | viroids, small single-stranded circular rnas endowed with autonomous replication, are unique systems to conduct evolutionary studies of complete rna genomes. the primary structure of 36 progeny variants of peach latent mosaic viroid (plmvd), evolved from inoculations of the peach indicator gf-305 with four individual plmvd cdnas differing in their pathogenicity, has been determined. most progeny variants had unique sequences, revealing that the extremely heterogeneous character of plmvd natural ... | 1999 | 10466824 |
| use of intramolecular chimeras to map molecular determinants of symptom severity of potato spindle tuber viroid (pstvd). | nucleotide sequence comparison shows that sequence variations are mostly clustered in the p (pathogenicity) and v (variable) domains of the potato spindle tuber viroid (pstvd) molecule. although these comparisons suggest the p domain as the primary determinant of pstvd symptom severity, the potential contribution of the v domain has never been analysed in detail. to investigate the relationship between the structure of these domains and pathogenicity, six intraspecific chimeric pstvd variants we ... | 1996 | 8973522 |
| the genetic stability of potato spindle tuber viroid (pstvd) molecular variants. | rna viruses propagate as a population of genetically related entities composing a quasi-species. specific representatives are the result of both a high mutation rate during replication and competition between the continuously arising sequence variants. similar to other rna pathogens, potato spindle tuber viroid (pstvd) propagates as a population of similar but nonidentical sequences. the sequence of progeny molecules derived from cloned molecular variants of pstvd were studied after one and six ... | 1997 | 8990400 |
| characterization of a tomato protein kinase gene induced by infection by potato spindle tuber viroid. | viroids--covalently closed, circular rna molecules in the size range of 250 to 450 nucleotides-are the smallest known infectious agents and cause a number of diseases of crop plants. viroids do not encode proteins and replicate within the nucleus without a helper virus. in many cases, viroid infection results in symptoms of stunting, epinasty, and vein clearing. in our study of the molecular basis of the response of tomato cv. rutgers to infection by potato spindle tuber viroid (pstvd), we have ... | 2000 | 10975647 |
| cellular basis of potato spindle tuber viroid systemic movement. | viroids are small, nontranslatable pathogenic rnas that replicate autonomously and traffic systemically in their host plants. we have used in situ hybridization to analyze the trafficking pattern of potato spindle tuber viroid (pstvd) in tomato and nicotiana benthamiana. when pstvd was inoculated onto the stem of a plant, it replicated and trafficked to sink, but not source, leaves. pstvd was absent from shoot apical meristems. in the flowers of infected plants, pstvd was present in the sepals, ... | 2001 | 11145890 |
| a novel usage of random primers for multiplex rt-pcr detection of virus and viroid in aphids, leaves, and tubers. | a multiplex reverse transcription polymerase chain reaction (m-rt-pcr) was developed for the simultaneous detection of five potato viruses and a viroid. the synthesis of cdnas used for amplification was primed by hexanucleotides (random primers, rp). an rna extraction procedure employing dnase i, is routinely used to isolate potato viruses and viroid (potato virus s, pvs; potato leafroll virus, plrv; potato virus x, pvx; potato virus a and y, pva, pvy; and potato spindle tuber viroid, pstvd) fro ... | 2001 | 11164484 |
| advantages and disadvantages of using pcr techniques to characterize transgenic plants. | the polymerase chain reaction (pcr) revolutionized molecular biology to a similar extent as the discovery of plasmids and restriction endonucleases. however, there are some limitations to the use of pcr. transgenic plants containing potato spindle tuber viroid (pstvd) cdna constructs, demonstrated to become de novo methylated upon pstvd infection, represent a good example to illustrate the advantages of pcr. pstvd is a 359 nt long autonomously replicating plant pathogenic rna where all of its en ... | 2001 | 11280933 |
| a simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and rt-pcr. | a simple, rapid method of nucleic acid extraction on a microcentrifuge tube scale for detecting viroids is presented. five distinct citrus viroids (cvds), chrysanthemum stunt viroid (csvd), hop stunt viroid (hsvd), hop latent viroid (hlvd) and potato spindle tuber viroid (pstvd) were detected in their natural host plants by hybridization using crna probes and reverse transcription-polymerase chain reaction (rt-pcr). nucleic acids (na) were liberated from tissues by incubation in a buffer contain ... | 1999 | 10029324 |
| replicating potato spindle tuber viroid rna is accompanied by short rna fragments that are characteristic of post-transcriptional gene silencing. | the low molecular weight fraction of tomato plants inoculated with potato spindle tuber viroid (pstvd) contains a population of short pstvd-specific rnas of either polarity. the main constituents were rnas of 22 and 23 nt representing different domains of the viroid genome. the occurrence of such distinct rna species indicated that the nuclear replicating pstvd rna induces post-transcriptional gene silencing. the short rnas were slightly more abundant at 30 days post-inoculation than at later st ... | 2001 | 11376158 |
| formation of metastable rna structures by sequential folding during transcription: time-resolved structural analysis of potato spindle tuber viroid (-)-stranded rna by temperature-gradient gel electrophoresis. | a model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (pstvd) was proposed earlier: a thermodynamically metastable structure containing a specific hairpin (hp ii) in the (-)-strand replication intermediate is essential for template activity during (+)-strand synthesis. we present here a detailed kinetic analysis on how pstvd (-)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slow ... | 1999 | 10199573 |
| the dimerization domain of potato spindle tuber viroid, a possible hallmark for infectious rna. | covalently closed circular (+) rna of the potato spindle tuber viroid (pstvd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or mg2+. in vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of pstvd, confirms the existence of the previ ... | 1998 | 9760245 |
| the variability of hop latent viroid as induced upon heat treatment. | 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 el ... | 2001 | 11531412 |
| transcription of potato spindle tuber viroid by rna polymerase ii starts predominantly at two specific sites. | pospiviroidae, with their main representative potato spindle tuber viroid (pstvd), are replicated via a rolling circle mechanism by the host-encoded dna-dependent rna polymerase ii (pol ii). in the first step, the (+)-strand circular viroid is transcribed into a (-)-strand oligomer intermediate. as yet it is not known whether transcription is initiated by promotors at specific start sites or is distributed non-specifically over the whole circle. an in vitro transcription extract was prepared fro ... | 2001 | 11713308 |
| genetic variability of potato spindle tuber viroid rna replicon. | the genetic continuity of the potato spindle tuber viroid (pstvd) genome was analysed after infection of tomato plants with cloned cdnas of parental strains. during the six weeks of the experiment, several new sequence variants appeared. the sequence variants detected in the progeny population induced sequence-specific disease symptoms. the pstvd genome therefore follows the pattern expected for typical pseudo-strains propagating in plants as a population of similar sequences. assessing further ... | 2001 | 11732616 |
| potato spindle tuber viroid as inducer of rna silencing in infected tomato. | potato spindle tuber viroid (pstvd), an rna plant pathogen encoding no known proteins, induces systemic symptoms on tomato plants. we report detection of small rnas of approximately 25 nucleotides with sequence specificity to pstvd in infected plants: an indication of the presence of rna silencing. rna silencing, however, did not appear to be responsible for the differing symptoms induced by a mild and a severe strain of pstvd. the unique structural and biological features of viroids make them a ... | 2001 | 11763132 |
| use of a vector based on potato virus x in a whole plant assay to demonstrate nuclear targeting of potato spindle tuber viroid. | potato spindle tuber viroid (pstvd) is a covalently closed circular rna molecule of 359 nucleotides that replicates within the nucleus of host cells. to determine how this small, highly structured rna enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (gfp) as the reporter molecule. the coding region of gfp was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato st-ls1 gene. a cdna copy of the ... | 2001 | 11369895 |
| bean common mosaic virus isolates causing different symptoms in asparagus bean in china differ greatly in the 5'-parts of their genomes. | potyvirus isolates from asparagus bean ( vigna sesquipedalis) plants in zhejiang province, china, caused either rugose and vein banding mosaic symptoms (isolate r) or severe yellowing (isolate y) in this host, but were otherwise similar in host range. both isolates were completely sequenced and shown to be isolates of bean common mosaic virus (bcmv). the complete sequences were 9992 (r) or 10062 (y) nucleotides long and shared 91.7% identical nucleotides (93.2% identical amino acids) in their ge ... | 2002 | 12111434 |
| subcellular localization of viroids in highly purified nuclei from tomato leaf tissue. | approximately 95% of the viroid rna which is present in potato spindle tuber viroid (pstv)-infected tomato plant leaf issue, is associated with the nucleolar fraction obtained from purified nuclei. viroids were released from the nucleolar fraction by increasing the ionic strength of the medium to 0.66 suggesting that viroid rna is present in these subnuclear components in a protein-nucleic acid complex. a purification procedure for nuclei from leaf tissue had to be newly developed; it involves t ... | 1983 | 11892810 |
| a novel multiplex rt-pcr probe capture hybridization (rt-pcr-elisa) for simultaneous detection of six viroids in four genera: apscaviroid, hostuviroid, pelamoviroid, and pospiviroid. | a rapid and sensitive assay was developed for the detection and identification of viroids by standard or multiplex reverse transcription-polymerase chain reaction (rt-pcr)-probe capture hybridization (rt-pcr-elisa). the assay was applied successfully for the detection and identification of the following six viroid species from infected tissues: potato spindle tuber viroid (pospiviroid), peach latent mosaic viroid (pelamoviroid), apple scar skin viroid (apscaviroid), apple dimple fruit viroid (ap ... | 2002 | 12176148 |
| movement of potato spindle tuber viroid reveals regulatory points of phloem-mediated rna traffic. | increasing evidence indicates that the phloem mediates traffic of selective rnas within a plant. how an rna enters, moves in, and exits the phloem is poorly understood. potato spindle tuber viroid (pstvd) is a pathogenic rna that does not encode proteins and is not encapsidated, and yet it replicates autonomously and traffics systemically within an infected plant. the viroid rna genome must interact directly with cellular factors to accomplish these functions and is, therefore, an excellent prob ... | 2002 | 12226494 |
| plant 7sl rna and trna(tyr) genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from nicotiana tabacum cells. | 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 p ... | 2002 | 12374302 |
| control of mechanical viroid transmission by the disinfection of tables and tools. | viroids are of practical importance as the cause of several economically significant infectious diseases. potato spindle tuber viroid (pstvd) causes severe yield losses in several crops, because the pathogen spreads fast within the culture. viroids are small molecules, a few hundred nucleotides long, with a high degree of secondary structure. they do not code for any polypeptides and replicate independently of any associated plant virus. viroids are readily transmitted by contaminated tools and ... | 2001 | 12425031 |
| potato spindle tuber viroid strains of different pathogenicity induces and suppresses expression of common and unique genes in infected tomato. | viroids are the smallest plant pathogens. these rnas do not encode proteins and are not encapsidated, and yet they can replicate autonomously, move systemically, and cause diseases in infected plants. notably, strains of a viroid with subtle differences in nucleotide sequences can cause dramatically different symptoms in infected plants. these features make viroids unique probes to investigate the role of a pathogenic rna genome in triggering host responses. we conducted a comprehensive analysis ... | 2002 | 12437296 |
| rna folding pathway functional intermediates: their prediction and analysis. | the massively parallel genetic algorithm (ga) for rna structure prediction uses the concepts of mutation, recombination, and survival of the fittest to evolve a population of thousands of possible rna structures toward a solution structure. as described below, the properties of the algorithm are ideally suited to use in the prediction of possible folding pathways and functional intermediates of rna molecules given their sequences. utilizing stem trace, an interactive visualization tool for rna s ... | 2001 | 11545583 |
| binding of ire-bp to its cognate rna sequence: sfm studies on a universal rna backbone for the analysis of rna-protein interaction. | we have used an rna consisting of the potato spindle tuber viroid (pstvd) and 240 bp of double-stranded rna derived from the gus gene as a backbone for scanning force microscope (sfm) studies on rna binding proteins. the in vitro transcribed rna forms a rod-like structure of apparent 130 nm in length with a completely base paired central part flanked by the incompletely paired viroid helix with bulges on both sides. the termini of the molecule consist of loops such that no blunt or staggered rna ... | 2001 | 11592396 |
| a dna target of 30 bp is sufficient for rna-directed dna methylation. | in higher plants, rna-dna interactions can trigger de novo methylation of genomic sequences via a process that is termed rna-directed dna methylation (rddm). in potato spindle tuber viroid (pstvd)-infected tobacco plants, this process can potentially lead to methylation of all c residues at symmetrical and nonsymmetrical sites within chromosomal inserts that consist of multimers of the 359-bp-long pstvd cdna. using pstvd cdna subfragments, we found that genomic targets with as few as 30 nt of se ... | 2000 | 10668798 |
| a mini-rna containing the tetraloop, wobble-pair and loop e motifs of the central conserved region of potato spindle tuber viroid is processed into a minicircle. | a mini-rna from potato spindle tuber viroid (pstvd) was constructed specifically for cleavage and ligation to circles in vitro. it contains the c-domain with the so-called central conserved region (ccr) of pstvd with a 17 nt duplication in the upper strand and hairpin structures at the left and rights ends of the secondary structure. the ccr was previously shown to be essential for processing of in vitro transcripts. when folded under conditions which favor formation of a kinetically controlled ... | 2003 | 12560495 |
| viroid rna systemic spread may depend on the interaction of a 71-nucleotide bulged hairpin with the host protein virp1. | viroids are noncoding circular single-stranded rnas that are propagated systemically in plants. virp1 is a protein from tomato, which is an excellent host for potato spindle tuber viroid (pstvd), and it has been isolated by virtue of its specific in vitro binding to pstvd rna. we report on the specific in vivo interaction of virp1 with full-length viroid rna as well as with subfragments in the three-hybrid system. the terminal right domain (tr) of pstvd was identified as a strong interacting par ... | 2003 | 12592008 |
| identification of neutral mutants surrounding two naturally occurring variants of potato spindle tuber viroid. | single point mutations in the pathogenicity domain of potato spindle tuber viroid (pstvd) can have a dramatic effect on disease expression, and only three substitutions are required for the spontaneous conversion of the type strain pstvd-intermediate to the rapidly replicating, highly pathogenic variant rg1 (gruner et al., virology 209, 60-69, 1995). to identify available evolutionary pathways linking these two variants, we mutagenized five positions in an infectious cdna copy of pstvd-intermedi ... | 2003 | 12604828 |
| inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid rna. | viroids are small noncoding and infectious rnas that replicate autonomously and move systemically throughout an infected plant. the rnas of the family pospiviroidae contain a central conserved region (ccr) that has long been thought to be involved in replication. here, we report that the ccr of potato spindle tuber viroid (pstvd) also plays a role in pathogenicity. a u257a change in the ccr converted the intermediate strain pstvd(int) to a lethal strain that caused severe growth stunting and pre ... | 2003 | 12782729 |
| biochemical analysis and scanning force microscopy reveal productive and nonproductive adar2 binding to rna substrates. | scanning force microscopy (sfm) can be used to image biomolecules at high resolution. here we demonstrate that single-molecule analysis by sfm complements biochemical data on rna protein binding and can provide information that cannot be obtained by the usual biochemical methods. we have used this method to study the interaction between the rna editing enzyme adar2 and rna transcripts containing selective and nonselective editing sites. the natural selectively edited r/g site from glutamate rece ... | 2003 | 12810917 |
| a bromodomain-containing protein from tomato specifically binds potato spindle tuber viroid rna in vitro and in vivo. | for the identification of rna-binding proteins that specifically interact with potato spindle tuber viroid (pstvd), we subjected a tomato cdna expression library prepared from viroid-infected leaves to an rna ligand screening procedure. we repeatedly identified cdna clones that expressed a protein of 602 amino acids. the protein contains a bromodomain and was termed viroid rna-binding protein 1 (virp1). the specificity of interaction of virp1 with viroid rna was studied by different methodologie ... | 2003 | 12915580 |
| identification of a novel structural interaction in columnea latent viroid. | pairwise sequence comparisons suggest that columnea latent viroid (clvd) may have originated from a recombination event involving potato spindle tuber viroid (pstvd) and hop stunt viroid (hsvd). to examine the role of specific structural features in determining the host range of clvd, we constructed a series of interspecific chimeras by replacing increasing portions of its terminal left and pathogenicity domains with the corresponding portions of pstvd. exchanges involving the left side of the p ... | 2003 | 12954225 |
| characterization of the rna motif responsible for the specific interaction of potato spindle tuber viroid rna (pstvd) and the tomato protein virp1. | viroids are small non-coding parasitic rnas that are able to infect their host plants systemically. this circular naked rna makes use of host proteins to accomplish its proliferation. here we analyze the specific binding of the tomato protein virp1 to the terminal right domain of potato spindle tuber viroid rna (pstvd). we find that two asymmetric internal loops within the pstvd (+) rna, each composed of the sequence elements 5'-acagg and cucuucc-5', are responsible for the specific rna-protein ... | 2003 | 14500815 |
| differential subnuclear localization of rna strands of opposite polarity derived from an autonomously replicating viroid. | the wide variety of rnas produced in the nucleus must be localized correctly to perform their functions. however, the mechanism of this localization is poorly understood. we report here the differential subnuclear localization of rna strands of opposite polarity derived from the replicating potato spindle tuber viroid (pstvd). during replication, (+)- and (-)-strand viroid rnas are produced. we found that in infected cultured cells and plants, the (-)-strand rna was localized in the nucleoplasm, ... | 2003 | 14555700 |
| resistance of human hepatitis delta virus rnas to dicer activity. | the endonuclease dicer cleaves rnas that are 100% double stranded and certain rnas with extensive but <100% pairing to release approximately 21-nucleotide (nt) fragments. circular 1,679-nt genomic and antigenomic rnas of human hepatitis delta virus (hdv) can fold into a rod-like structure with 74% pairing. however, during hdv replication in hepatocytes of human, woodchuck, and mouse origin, no approximately 21-nt rnas were detected. likewise, in vitro, purified recombinant dicer gave <0.2% cleav ... | 2003 | 14581527 |
| structural characterization of the 69 nucleotide potato spindle tuber viroid left-terminal domain by nmr and thermodynamic analysis. | the 69 nucleotide left-terminal domain (t(l)) of the potato spindle tuber rna viroid (pstvd) constitutes one of its five structural elements. due to a twofold complementary sequence repeat, two possible conformations are proposed for the t(l) secondary structure; an elongated-rod and a bifurcated form. in the present study, two t(l) mutants were designed that remove the symmetry of the sequence repeats and ensure that either the bifurcated or the elongated-rod conformation is thermodynamically f ... | 2003 | 14636600 |
| development of a real-time rt-pcr assay for the detection of potato spindle tuber viroid. | potato spindle tuber viroid (pstvd) is a quarantine pathogen in the european union and causes damaging diseases of solanaceous crops. under the eu plant health directive 2000/29/ec, countries must have the ability to detect and identify accurately and rapidly the introduction of harmful organisms in plants or plant products; furthermore, if the quarantine pathogen is found, be able to survey extensively for it. in this respect, pstvd poses an interesting technical problem, since its rna does not ... | 2004 | 14738980 |
| an rt-pcr primer pair for the detection of pospiviroid and its application in surveying ornamental plants for viroids. | a primer pair for reverse transcription-polymerase chain reaction (rt-pcr), based on the conserved sequences of the members of genus pospiviroid was designed to yield a fragment of about 200 base pairs (bp). since pospiviroids infect a large number of plants species and a few members of the genus pospiviroid have been already detected in some ornamental plants, the primer pair was evaluated for its efficacy using ornamental plants. the method of return-polyacrylamide gel electrophoresis (r-page) ... | 2004 | 14738987 |
| short interfering rnas specific for potato spindle tuber viroid are found in the cytoplasm but not in the nucleus. | short interfering (si) and micro (mi) rnas influence gene expression at post-transcriptional level. in plants, different classes of dicer-like (dcl) enzymes are responsible for the generation of these small regulatory rnas from different precursors. to characterize the cellular site of their generation and accumulation, we purified nuclei from tomato plants infected with potato spindle tuber viroid (pstvd) rna, which is known to replicate in the nucleus via double-stranded (ds) rna intermediates ... | 2004 | 14871315 |
| on the role of rna silencing in the pathogenicity and evolution of viroids and viral satellites. | viroids and most viral satellites have small, noncoding, and highly structured rna genomes. how they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. recent studies have shown that both viroids and satellites are capable of inducing rna silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral rnas. here we show that preventing rna silencing in tobacco, using a sil ... | 2004 | 14978267 |
| viroid-induced rna silencing of gfp-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencing. | viroid infection is associated with the production of short interfering rnas (sirnas), a hallmark of post-transcriptional gene silencing (ptgs). however, viroid rnas autonomously replicating in the nucleus have not been shown to trigger the degradation of homologous rna in the cytoplasm. to investigate the potential of viroids for the induction of gene silencing, non-infectious fragments of potato spindle tuber viroid (pstvd) cdna were transcriptionally fused to the 3' end of the green fluoresce ... | 2004 | 15053764 |
| sensitive detection of potato spindle tuber and temperate fruit tree viroids by reverse transcription-polymerase chain reaction-probe capture hybridization. | a rapid and sensitive assay for the specific detection of plant viroids using reverse transcription-polymerase chain reaction (rt-pcr) -probe capture hybridization (rt-pcr-enzyme-linked immunosorbent assay (elisa)) was developed. the assay was applied successfully for the detection of potato spindle tuber viroid, peach latent mosaic viroid, or apple scar skin viroid from viroid infected leaf tissue. clarified sap extract from infected leaf tissue was treated first with genereleaser polymeric mat ... | 1999 | 10471024 |
| the n-glycosidase activity of the ribosome-inactivating protein me1 targets single-stranded regions of nucleic acids independent of sequence or structural motifs. | me(1), a type i ribosome-inactivating protein (rip), belongs to a family of enzymes long believed to possess rrna n-glycosidase activity directed solely at the universally conserved residue a4324 in the sarcin/ricin loop of large eukaryotic and prokaryotic rrnas. we have investigated the effect of modifying the structure of nonribosomal rna substrates on their interaction with me(1) and other rips. me(1) was shown to depurinate a variety of partially denatured nucleic acids, randomly removing ad ... | 2004 | 15123667 |
| direct role of a viroid rna motif in mediating directional rna trafficking across a specific cellular boundary. | the plasmodesmata and phloem form a symplasmic network that mediates direct cell-cell communication and transport throughout a plant. selected endogenous rnas, viral rnas, and viroids traffic between specific cells or organs via this network. whether an rna itself has structural motifs to potentiate trafficking is not well understood. we have used mutational analysis to identify a motif that the noncoding potato spindle tuber viroid rna evolved to potentiate its efficient trafficking from the bu ... | 2004 | 15194818 |
| biolistic inoculation of plants with viroid nucleic acids. | 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 ... | 2004 | 15542139 |
| the 18s rdna sequence of synchytrium endobioticum and its utility in microarrays for the simultaneous detection of fungal and viral pathogens of potato. | resting spores extracted from wart (synchytrium endobioticum)-infected potato tubers were used for dna extraction and amplification of 18s rdna. analysis of the cloned, sequenced fragment revealed high similarity to members of the chytridiomycota. using this information, specific oligonucleotide probes were designed and arrayed onto glass slides for detection of the pathogen. viral sequence information available in the databank was retrieved, or new viral sequences were generated, and used to de ... | 2005 | 15800764 |