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 of their polarity in the case of single-stranded DNA and regardless of their orientation in the vector DNA in the case of double-stranded DNA. The vector-inserted monomeric PSTV DNA units were also found to be infectious but of low specific infectivity which was increased when these monomers had been excised. Even two subgenomic DNA fragments, representing together the 359 nucleotides of the PSTV RNA genome, initiated the synthesis of viroid RNA progeny when co-inoculated although each fragment by itself is non-infectious. These results are discussed with respect to the infectivity previously observed with certain cloned DNAs of conventional RNA and DNA viruses.     

Oligomeric forms of potato spindle tuber viroid (PSTV) and of its complementary RNA are present in nuclei isolated from viroid-infected potato cells

Different oligomeric forms of PSTV are detected in nuclei isolated from PSTV-infected potato cells by means of molecular hybridization, using as probes synthetic oligodeoxyribonucleotides with sequence specificity for (+)PSTV and for (?)PSTV. In addition to several species of longer-than-unit-length (?)PSTV molecules, two oligomeric forms os (+)PSTV are detected, which correspond in size to RNA strands of approximately two and three times viroid unit-length. They must be considered as the precursors os the circular and linear (+)PSTV monomers accumulating in the cell nucleus.     

Continous 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 dem/ssum L.) and tomato (Lycopersicon peruvianum L. Mill) plants and in cell suspensions derived from potato protoplasts (Solanum tuberosum L.) inoculatedin vitro is described. The persistence of PSTV replication in these cell lines through at least 14 subculture passages, which corresponds to a continous replication over a period of more than one year, was demonstrated by infectivity assay and by polyacrylamide-gel electrophoresis of isolated nucleic acids. This continuous synthesis denovo of PSTV was substantiated by the incorporation of [³H]uridine and of [³²P]orthophosphate into viroid RNA.     

An immunochemical study of antigen expression in potato spindle tuber viroid (PSTV) - infected tomato leaves and calluses

A polyspecific antiserum against protein extracted from PSTV-infected tomato leaves was prepared and the IgGs were separated by affinity chromatography on a beaded cellulose adsorbent with an immobilized “healthy” antigen. The antibody not adsorbed entered into a preferential reaction with the antigen from PSTV-infected leaves as estimated by an enzyme-linked immunosorbent assay. The immunochemical reactions did not significantly exceed the control background, if antigens from tomato leaves infected with potato viruses X, Y and M were analyzed. By immunoblot technique we revealed, however, that several antigens not detected in healthy leaves appeared in the leaves infected either with PSTV or with viruses X and M. An accumulation of a major antigen having a molecular mass of about 70 kDa was observed in viroid-infected leaves only, suggesting the specificity for viroid infection. The antigen was found not to be an alkaline endoproteinase - the pathogenesis-related protein P-69. Some antigens with molecular masses approximately 38.0, 23.7 and 22 kDa, which occurred in PSTV-infected leaves and in healthy calluses, were not detectable in PSTV-infected calluses. No reaction exceeding the control level was observed using enzyme-linked immunosorbent assay for antigens from silver nitrate-treated tomato leaves, although such leaves showed symptoms similar to that caused by viroids.     

Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid — host cell interaction

Photosynthetically active callus and cell suspension cultures were established from uninfected Lycopersicon peruvianum plants and from uninfected and potato spindle tuber viroid (PSTVd) infected plants of Lycopersicon esculentum cv. Rutgers. Viroid infection was maintained in photoheterotrophic culture on media containing 3% sucrose, but during continuous photo-mixotrophic culture in low sucrose media (1% sucrose), the level of PSTVd accumulation decreased. Photoautotrophic cell suspensions could be established with uninfected, but not with viroid infected tomato cells. As compared to uninfected cells, PSTVd infected cells grew slowly, were morphologically different in size and shape, and formed tight cell aggregates. Electronmicroscopy showed that starch accumulation in chloroplasts, deformation of the chloroplast envelope and irregular plasmalemmasomes at the cell membrane were associated with PSTVd infection.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - CEVd citrus exocortis viroid - CSVd chrysanthemum stunt viroid - PSTVd potato spindle tuber viroid - TMV tobacco mosaic virus - phc photoheterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture     

Production of Potato Spindle Tuber Viroid (PSTV) by Large Scale Fermentation of PSTY-Infected Potato Cell Suspension Cultures

Berlin, J., Wray, V., Forche, E., Reng, H.–G , Schler,H, Luckinger, R. and Mhlbach, H.–P. 1985. Production ofpotato spindle tuber viroid (PSTV) by large scale fermentationof PSTV–infected potato cell suspension cultures.—J.exp. Bot 36: 1985–1995. Cell suspension cultures of Solatiumdemissum, infected with the potato spindle tuber viroid (PSTV),were scaled up to volumes of up to 800 dm³ to provide sufficientand uniform plant material for subsequent studies on viroidbiosynthesis. Here we describe the technological aspects ofproducing the required amounts of biomass and viroid. The cells,which had been maintained on a medium containing expensive coconutmilk, were first adapted to rapid growth on the less expensiveB5–medium. The physiological state of the cells was monitoredby in vivo ³¹P–NMR spectroscopy Under the chosen conditionsthe scale–up from 10 dm³ inoculum from shake flasks tothe harvest of the 800 dm³ stirred fermenter lasted 38 d andprovided 112 kg biomass. Growth characteristics and viroid productionin shake flasks and large bioreactors were rather similar. Gelelectrophoretic analysis of isolated nucleic acids using silverstaining and Northern blot hybridization revealed a PSTV–contentof approximately 700 µg PSTV per kg fresh mass of culturedcells. Key words: Solanum demissum, plant cell cultures, potato spindle tuber viroid, biomass production, fermentation, in vivo ³¹P-NMR     

Grapevine yellow speckle viroid: structural features of a new viroid group.

A single stranded circular RNA was isolated from grapevines infected with yellow speckle disease. The RNA which we have called grapevine yellow speckle viroid (GYSV), contains 367 nucleotide residues and has the potential to form the rod-like secondary structure characteristic of viroids. GYSV has 37% sequence homology with the recently described apple scar skin viroid (ASSV; 330 residues) and has some sequence homology with the viroids in the potato spindle tuber viroid (PSTV) group. The sequence of GYSV has characteristics which fit the structural domains described for the PSTV group. However, GYSV lacks the PSTV central conserved sequence. Instead, there is a conserved sequence in the central region of GYSV and ASSV which has the potential to form a stem loop configuration and a stable palindromic structure as does the central conserved region of the PSTV group. These structural features suggest there is a different central conserved region for GYSV and ASSV. The results support the viroid nature of GYSV and its inclusion into a separate viroid group which we suggest should be represented by ASSV.     

Nucleotide sequence of cDNA encoding α-luffin,a ribosome-inactivating protein from Luffa cylindrica

A cDNA library from tomato planta macho viroid (TPMV)-infected tomato was constructed. The library was screened at low stringency with a tobacco PR-R cDNA probe. An 832 bp cDNA from a mRNA present only in infected tissue was isolated. Nucleotide sequence showed high homology with the osmotin from both tobacco and tomato (NP24). This cDNA probably corresponds to the AP24 and P23 proteins previously described in tomato and induced upon fungal and viroid infection.     

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 residues base paired. In contrast to the extensive (69%) sequence homology of CSV with PSTV, only 18% of the ASBV sequence is homologous to PSTV and CSV. There are eight potential polypeptide translation products with chain lengths from 4 to 63 amino acid residues coded for by the plus (infectious) strand and four potential translation products (2 to 60 residues) coded for by the minus strand. An improved method is described for the synthesis of gamma-32p-ATP of high specific activity.     

Correlation between structure and pathogenicity of potato spindle tuber viroid (PSTV)

Sequence analysis by primer-extension at the level of their cDNA showed that the RNA genomes of various field isolates of potato spindle tuber viroid (PSTV) of different virulence differ from each other only in a few nucleotides in two distinct regions of the rod-shaped molecule. Despite insertions and deletions the chain length of 359 nucleotides is strictly conserved in all the isolates studied. Thermodynamic calculations revealed that due to the observed sequence differences the region located at the left hand part of the rod-like secondary structure of the PSTV molecule, denoted 'virulence modulating (VM) region', becomes increasingly unstable with the increasing virulence of the corresponding isolate. Based on these data we propose in molecular terms a model for the mechanism of viroid pathogenicity. It implies that the nucleotides of the VM region specify and modulate the binding- and hence the competition-potential of the PSTV RNA molecule for a still unknown host factor(s) and thus determine the virulence of PSTV.     

Global analysis of tomato gene expression during Potato spindle tuber viroid infection reveals a complex array of changes affecting hormone signaling

Viroids like Potato spindle tuber viroid (PSTVd) are the smallest known agents of infectious disease-small, highly structured, circular RNA molecules that lack detectable messenger RNA activity, yet are able to replicate autonomously in susceptible plant species. To better understand the possible role of RNA silencing in disease induction, a combination of microarray analysis and large-scale RNA sequence analysis was used to compare changes in tomato gene expression and microRNA levels associated with PSTVd infection in two tomato cultivars plus a third transformed line expressing small PSTVd small interfering RNAs in the absence of viroid replication. Changes in messenger (m)RNA levels for the sensitive cultivar 'Rutgers' were extensive, involving more than half of the approximately 10,000 genes present on the array. Chloroplast biogenesis was down-regulated in both sensitive and tolerant cultivars, and effects on mRNAs encoding enzymes involved in the biosynthesis of gibberellin and other hormones were accompanied by numerous changes affecting their respective signaling pathways. In the dwarf cultivar 'MicroTom', a marked upregulation of genes involved in response to stress and other stimuli was observed only when exogenous brassinosteroid was applied to infected plants, thereby providing the first evidence for the involvement of brassinosteroid-mediated signaling in viroid disease induction.     

Rapid Detection of Potato Spindle Tuber Viroid (PSTV) by Dot Blot Hybridization

A PSTV probe of 350 bases, prepared in our laboratory, was used to detect picogram quantities of viroids by a simplified, improved, dot blot, hybridization technique in crude potato and tomato extracts. Optimum conditions for certification of PSTV-free potato plants were established, involving formamide concentration, washing stringencies, exposure time during autoradiography and method of probe radiolabelling. Ten pg of purified PSTV in water and 50 pg of purified PSTV added to healthy plant extract were detected. Also hybridization signals could be detected from as little as 0.075 mg of infected plant tissue.     

Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences.

The Columnea latent viroid (CLV) occurs latently in certain Columnea erythrophae plants grown commercially. In potato and tomato, CLV causes potato spindle tuber viroid (PSTV)-like symptoms. Its nucleotide sequence and proposed secondary structure reveal that CLV consists of a single-stranded circular RNA of 370 nucleotides which can assume a rod-like structure with extensive base-pairing characteristic of all known viroids. The electrophoretic mobility of circular CLV under nondenaturing conditions suggests a potential tertiary structure. CLV contains extensive sequence homologies to the PSTV group of viroids but contains a central conserved region identical to that of hop stunt viroid (HSV). CLV also shares some biological properties with each of the two types of viroids. Most probably, CLV is the result of intracellular RNA recombination between an HSV-type and one or more PSTV-type viroids replicating in the same plant.     

Amino acid sequence of the ;pathogenesis-related' leaf protein p14 from viroid-infected tomato reveals a new type of structurally unfamiliar proteins

We have established the complete sequence of the 130 amino acid residues of the pathogenesis-related (PR) protein p14 accumulating in tomato leaves infected with the viroid of the spindle tuber disease of potato (PSTV) and partial sequences of the PR protein 1a which accumulates in tobacco mosaic virus-infected tobacco leaves. Both PR proteins are closely related to each other. However, no homology could be found between the sequence of p14 and any of the 3061 published protein sequences compiled in the protein sequence database at present. Thus, p14 represents not only the first completely sequenced PR plant protein but also a new type of structurally unfamiliar proteins whose biological function in the diseased plant remains to be elucidated.     

The sequence of a viroid from grapevine closely related to severe isolates of citrus exocortis viroid.

The primary structure of a grapevine viroid (GVs) isolated in Spain was determined. The sequence consisted of 369 nucleotide residues forming a circular molecule. GVs presented extensive homology with viroids of the potato spindle tuber viroid (PSTV) group, that was specially high in the case of citrus exocortis viroid (CEV) both with variants found in isolates inducing severe (92% with CEV-A) and mild (89% with CEV-DE26) symptoms on tomato. The secondary structure proposed for GVs showed that the changes in the sequence in relation to CEV-A generated modifications of the secondary structure particularly important in the left terminal (Tl), variable (V) and pathogenesis (P) viroid domains that have been postulated. Nevertheless it was noted in GVs a central core in the P domain that is conserved in the class A sequence variants characteristic of severe isolates, but not in the class B ones found in mild isolates of CEV. These observations indicate that GVs should be considered as a severe isolate of CEV from grapevine (CEV-g), a suggestion that correlates with the biological properties of CEV-g both in tomato and in Gynura aurantiaca. The presence of this central core in the P domain seems to characterize all the variants of CEV inducing severe symptoms in tomato.     

Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells

A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (−) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (−)RNAs are likewise processed but with a much lower efficiency. Viroid-processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.     

Site-specific mutagenesis of potato spindle tuber viroid cDNA:

Summary The infectivity of cloned viroid cDNAs permits investigation of structure/function relationships in these unusual pathogenic RNAs by systematic site-specific mutagenesis of the cDNAs and subsequent bioassay. We have used three different strategies to create nucleotide substitutions within premelting region 2, a region of potato spindle tuber viroid (PSTV) believed to be important in viroid replication: sodium bisulfitecatalyzed deamination of deoxycytosine residues, oligonucleotide-directed mutagenesis, and construction of chimeric viroid cDNAs from fragments of infectious PSTV and tomato apical stunt viroid cDNAs. Although their effects upon the rod-like native structure of PSTV should be minimal, C U transitions at positions 92 or 284 appeared to be lethal. When inoculation with PSTV cDNA containing a single nucleotide substitution was mediated by the Ti plasmid of Agrobacterium tumefaciens, PSTV progeny with an unaltered wild type sequence was obtained. Two factors, the high error frequency characteristic of RNA synthesis and the use of a systemic bioassay for PSTV replication, may explain such sequence reversion and emphasize the importance of an appropriate bioassay system for screening mutant viroid cDNAs.