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 consecutive plant passages. Although the severe parental sequence S23 was found to be genetically stable, all five other parental sequences analyzed, irrespective of their pathogenicity, led to the appearance of complex populations. Divergence of the progeny was observed at the sequence level, but also, more surprisingly, at the level of the pathogenicity of individual progeny molecules. In two cases, the parental sequence was retained in the progeny population. In the other cases, it was completely out-competed and eliminated, sometimes in as little as one plant passage. Although it has been observed previously that artificially mutated PSTVd molecules may revert rapidly to the wild-type sequence, this study presents direct evidence for the rapid evolution of naturally occurring PSTVd sequence variants.     

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.     

Molecular properties of potato spindle tuber viroid (PSTVd) isolates of the Russian Research Institute of Phytopathology

The complete nucleotide sequences of more than 100 isolates of Potato spindle tuber viroid (PSTVd) collected from locations in the territory of Russia and the former USSR have been determined. These sequences represent 43 individual sequence variants, each containing 1–10 mutations with respect to the “intermediate” or type strain of PSTVd (GenBank Acc. No. V01465). Isolates containing 2–5 mutations were the most common, and 24 sequence variants are described here for the first time. Twenty one isolates contained a mutation found only in Russian and Ukrainian isolates of PSTVd up till now; i.e., replacement of the adenine at position 121 with cytosine (A121C). Many of these isolates contained two mutations—deletion of one of three adenine residues occupying positions 118–120 plus replacement of the adenine at position 121 with either uracil or cytosine (?A120, A121U/C). Both combinations of mutations were phenotypically neutral, i.e. symptom expression in Rutgers tomato was unaffected. Phylogenetic analysis of the sequences of different PSTVd isolates presented in work together with sequences of other naturally-occurring isolates obtained from Internet databases suggesting that known PSTVd isolates may be divided into four groups: (i) a group of isolates from potato and ornamentals where the type strain of PSTVd (PSTVd.018) may be considered to represent the ancestral sequence, (ii) a second group of isolates from potato and ornamentals where PSTVd.123 play the same role as PSTVd.018 for the first group, and iii) a group of potato isolates where PSTVd.125 is a possible ancestral sequence. The fourth and most divergent group of PSTVd isolates differs significantly from these first three groups. The majority of isolates in this group originate from New Zealand and Australia and infect different solanaceous hosts (tomato, pepper, cape gooseberry, potato, and others).     

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.     

Biological and molecular analysis of the pathogenic variant C3 of potato spindle tuber viroid (PSTVd) evolved during adaptation to chamomile (Matricaria chamomilla)

Abstract Viroid-caused pathogenesis is a specific process dependent on viroid and host genotype(s), and may involve viroid-specific small RNAs (vsRNAs). We describe a new PSTVd variant C3, evolved through sequence adaptation to the host chamomile (Matricaria chamomilla) after biolistic inoculation with PSTVd-KF440-2, which causes extraordinary strong ('lethal') symptoms. The deletion of a single adenine A in the oligoA stretch of the pathogenicity (P) domain appears characteristic of PSTVd-C3. The pathogenicity and the vsRNA pool of PSTVd-C3 were compared to those of lethal variant PSTVd-AS1, from which PSTVd-C3 differs by five mutations located in the P domain. Both lethal viroid variants showed higher stability and lower variation in analyzed vsRNA pools than the mild PSTVd-QFA. PSTVd-C3 and -AS1 caused similar symptoms on chamomile, tomato, and Nicotiana benthamiana, and exhibited similar but species-specific distributions of selected vsRNAs as quantified using TaqMan probes. Both lethal PSTVd variants block biosynthesis of lignin in roots of cultured chamomile and tomato. Four 'expression markers' (TCP3, CIPK, VSF-1, and VPE) were selected from a tomato EST library to quantify their expression upon viroid infection; these markers were strongly downregulated in tomato leaf blades infected by PSTVd-C3- and -AS1 but not by PSTVd-QFA.     

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.     

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 interaction. In view of the nucleotide composition we call this structural element an ‘RY motif’. The RY motif located close to the terminal right hairpin loop of the PSTVd secondary structure has an ~5-fold stronger binding affinity than the more centrally located RY motif. Simultaneous sequence alterations in both RY motifs abolished the specific binding to Virp1. Mutations in any of the two RY motifs resulted in non-infectious viroid RNA, with the exception of one case, where reversion to sequence wild type took place. In contrast, the simultaneous exchange of two nucleotides within the terminal right hairpin loop of PSTVd had only moderate influence on the binding to Virp1. This variant was infectious and sequence changes were maintained in the progeny. The relevance of the phylogenetic conservation of the RY motif, and sequence elements therein, amongst various genera of the family Pospiviroidae is discussed.     

Nuclear targeting by fragmentation of the potato spindle tuber viroid genome

Transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a Potato virus X-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide Potato spindle tuber viroid (PSTVd) RNA genome. To further delimit the putative nuclear-targeting signal, PSTVd subgenomic fragments were embedded within the intron, and recombinant reporter RNAs were inoculated onto Nicotiana benthamiana plants. Appearance of green fluorescence in leaf tissue inoculated with PSTVd-fragment-containing constructs indicated shuttling of the RNA into the nucleus by fragments as short as 80 nucleotides in length. Plant-to-plant variation in the timing of intron removal and subsequent GFP fluorescence was observed; however, earliest and most abundant GFP expression was obtained with constructs containing the conserved hairpin I palindrome structure and embedded upper central conserved region. Our results suggest that this conserved sequence and/or the stem-loop structure it forms is sufficient for import of PSTVd into the 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.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02-0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02–0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

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.     

Sequence analysis of five new field isolates demonstrates that the chain length of potato spindle tuber viroid (PSTVd) is not strictly conserved but as variable as in other viroids

The sequence analysis of five new field isolates of potato spindle tuber viroid (PSTVd) of different virulence revealed that the lengthof their RNA chain is not strictly conserved to 359 nucleotides (nts), as one could have inferred from the previously sequenced PSTVd strains. It was now found that the chain length is strain-specific like in the case of practically all other viroids, and that it may vary, so far, between 356 and 360 nts. Taking our previously sequenced and least virulent mild strain PSTVd KF6-M as standard, the new mild strains PSTVd WA-M and PSTVd F-M differ from it by one or two nts. The new intermediate-severe strains PSTVd F-IS and PSTV-F 88-IS differ from the standard mild strain by eight and nine nts, respectively, whereas the new severe-lethal strain PSTVd F-SL differes in seven nts. Most of these mutations are located within the virulence-modulating (VM) region and within the variable region (VR), and only in two strains a single mutation is found in the right terminal domain.