Transmission of Three Viroids Through Seed and Pollen of Tomato Plants

The severe strain of potato spindle tuber viroid (s-PSTV) as well as chrysanthemum stunt (CSV) and cucumber pale fruit (CPFV) viroids were found to be transmitted through seed and pollen of the tomato cvs. Rutgers and Najwcze?niejszy. Plants pollinated with a pollen infected with any of these three viroids became systematically infected. Plant, fruit and seed symptoms of viroid infection were noted on sap- and pollen-inoculated plants and the yield of these plants was reduced. Tomato cv. Rutgers plants grown from infected seeds were symptomless although all three viroids were detected in these plants by bioassay and by electrophoresis on 5% polyacrylamide gel. When DNA complementary to s-PSTV RNA was used for a direct viroid detection in seed samples by spot hybridization technique it hybridized not only with s-PSTV RNA but also with CSV RNA as well as with CPFV RNA.     

Australian grapevine viroid--evidence for extensive recombination between viroids.

Australian grapevine viroid (AGV, 369 residues) is a novel viroid with less than 50% sequence similarity with any known viroid. Nevertheless its entire sequence can be divided into regions, each with a high sequence similarity with segments from one of citrus exocortis, potato spindle tuber, apple scar skin, and grapevine yellow speckle viroids. AGV contains the entire central conserved region of the apple scar skin viroid group and is proposed as a member of this group. AGV appears to have originated from extensive RNA recombination involving other viroids. The vegetatively propagated grapevines which have been exposed to multiple viroid infections during their long history of cultivation may have allowed such recombination.     

Nucleotide sequence and secondary structure of apple scar skin viroid.

The complete nucleotide sequence of apple scar skin viroid(ASSV) has been established, and a probable secondary structure is proposed. A single-stranded circular ASSV RNA consists of 330 nucleotides and can assume the rodlike conformation with extensive base-pairing characteristic of all the known viroids. ASSV shows low sequence homologies with other viroids and lacks the central conserved region. These indicate that ASSV should be allocated to a separate viroid group. However, homologous sequences with potato spindle tuber viroid(PSTV) in ASSV occur in limited and scattered regions of both viroids. These homologous regions fall within the particular domains in the viroid domain model which has been previously proposed by Keese and Symons(Proc. Natl. Acad. Sci. USA. 82, 4582-4586, 1985).     

In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus

Viroids, small single-stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin-labelled RNA probes were used with subsequent detection by gold-labelled monoclonal anti-biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.     

Fatal Yellowing of Oil Palms: Search for Viroids and Double-Stranded RNA

Fatal yellowing is a serious disease of still unknown origin affecting oil palms in several regions of Central and South America. In this study a search for viroids and viroid-like RNAs in oil palms was performed using two-dimensional gel electrophoresis and return gel electrophoresis of nucleic acid extracts. Although RNAs showing viroid-like gel-electrophoretic properties were detected, the presence of the known viroids was excluded by hybridization experiments using probes specific for potato spindle tuber viroid (PSTVd), coconut cadang-cadang viroid (CCCVd), or Coleus blumei viroid 1 (CbVd1). By using double-stranded RNA (dsRNA) specific monoclonal antibodies, which do not react with viroid RNA, we were able to show that oil palm RNAs, migrating like viroids are double-stranded RNA species. Since the same dsRNA pattern was found in extracts from diseased as well as from healthy oil palms, the dsRNAs can neither be part of the causative agent of fatal yellowing, nor are they associated with the disease. Their possible origin is discussed. In addition to the standard electrophoretic methods, which have been used for identification of viroids and viroid-like RNAs, we describe additional control experiments to differentiate unequivocally between circular single stranded and linear dsRNA.     

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.     

Chrysanthemum stunt viroid: primary sequence and secondary structure.

The sequence of the 356 nucleotide residues of chrysanthemum stunt viroid (CSV) has been determined. Overlapping linear viroid fragments were obtained by partial ribonuclease digestion, radiolabelled in vitro at their 5'-ends, and sequenced using partial enzymic cleavage methods. Of the CSV sequence, 69% is contained in the published sequence of potato spindle tuber viroid (PSTV). Differences in the primary sequence of CSV and PSTV suggest that neither the positive nor putative negative strands of these two viroids code for functional polypeptide products. However, the two viroids can form similar secondary structures, implicating a role for viroid structure in replication.     

Segregation of a viroid complex from a graft-transmissible dwarfing agent source for grapefruit trees

A marked variation in tree size occurred in grapefruit trees budded in trifoliate orange rootstock and inoculated with a graft-transmissible dwarfing agent (GTD) from grapefruit. Etrog citron (Citrus medico) inoculated with budwood collected from two GTD sub-types displaying very mild (VM) and mild (M) dwarfing effects showed only mild leaf epinasty, whereas the severe (S) subtype induced severe epinasty and stunting typical of infection by the original GTD source 225-T. The characteristic symptoms of the three sub-types were persistent following three serial transfers to 'Etrog' indicators.
Extracts from 'Etrog' citron containing either the 225-T or its derivative S displayed a profile of five viroid bands when analysed by the sequential PAGE system for detection of circular RNAs. The profile obtained included the well characterised 371 nucleotide citrus exocortis viroid (CEV) and four additional viroids of approximately 330, 300, 295 and 275 nucleotides. Sub-types M and VM lacked the CEV band and each contained a complement of only three viroids of 330, 295, 275 and 300, 295, 275 nucleotides, respectively. These results indicate that a segregation of the viroid complex in grapefruit budwood was a major factor in the variation seen among trees inoculated from the GTD source 225-T.     

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 two Percoll density centrifugations as final steps. The nuclei were sonicated and the sonicate fractionated into fractions either highly enriched in nucleoli or in broken chromatin and ribonucleoprotein particles. The viroid content in the different samples was determined by gel electrophoresis. Depending upon the progress of the disease, viroid copy numbers between 200 and 10,000 per cell were observed in homogenized tissue, purified nuclei and in the nucleolar fraction. In chloroplasts, practically no viroids were detected. The results are discussed in the light of current hypotheses about the replication, pathogenicity and origin of viroids.     

A Comparative Study of Four Viroids of Plants

The host-ranges and the reactions of particular plant hosts to inoculation with severe (s-PSTV) and mild (m-PSTV) strains of potato spindle tuber viroid, as well as with chrysanthemum stunt viroid (CSV) and cucumber pale fruit viroid (CPFV) were quite similar. Some minor differences did not exceed the limits of differences noted for the strains of the same plant viroid. Two-directional crossprotection was noted for each viroid pair when s-PSTV, m-PSTV, CSV and CPFV were tested on chrysanthemum cv., ‘Bonnie Jean’ plants. Finally, the relative mobility of RNAs of s-PSTV, m-PSTV, CSV and CPFV on 5% polyacrylamide gel was identical, no matter what extraction method from plant material was used. We postulate that these four plant viroids may be regardedas the strains of the same plant viroid “species”. On the other hand, chrysanthemum chlorotic mottle viroid (ChCMV) appeared to be a quite different plant pathogen. This viroid infected and caused symptoms only in chrysanthemum plants, and was able to infect and induce symptoms on plants which had already been infected with any other viroid studied, and it did not protect chrysanthemum cv. ‘Bonnie Jean’ plants against any of the other viroids. We were not able to locate a ChCMV-RNA band on polyacrylamide gel.     

A novel aspect of the information content of viroids

Viroids were found to exhibit a structural periodicity characterized by repeat units of a length of 11 or 12 (potato spindle tuber viroid group and coconut cadang-cadang viroid), 60 (apple scar skin viroid) and 80 (avocado sunblotch viroid) nucleotide residues, respectively. It is suggested that structural periodicity of viroids is an indication of their protein-binding ability.     

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.     

Structure and structure formation of viroids

The structure of viroids and the mechanism of structure formation were investigated by different methods. Results from gel analysis, partial degradation pattern, electron microscopy, dye binding, hydrodynamic studies, and temperature-jump kinetics were interpreted in a common structural and mechanistic scheme. Gel analysis, electron microscopy and kinetic investigations show that viroids may assume the native as well as metastable conformations under the same conditions. The native conformation is obtained by complete renaturation, i.e. slow cooling throughout the transition range (e.g. 52 to 48 ° C for potato spindle tuber viroid (PST viroid) in 0.01 m-sodium cacodylate, pH 6.8). In contrast, metastable conformations were trapped if viroids were redissolved in the cold from their ethanol precipitate or if they were denatured and cooled quickly.The native secondary structure of the recently sequenced PST viroid (Gross et al., 1978) was optimized for the free energy of base-pairing. The scheme agrees with that proposed by Gross et al. (1978), which was derived from chemical arguments. The extended structure does not undergo tertiary structure folding under a wide range of conditions, as was concluded from electron microscopy, sedimentation measurements and binding studies of ethidium bromide and a new dye specific for A · U pairs (2-(4′-aminophenyl)-5-(4′-methylpiperazin-1″yl)-benzirnidazol).Intermediate structures during viroid denaturation were analysed on theoretical and experimental grounds. The experimental data, in combination with the model calculations, show that all of the native base-pairs of viroids are dissociated in one highly co-operative main transition, and that during the same process very stable hairpins are formed that are not present in the native structure. The formation of stable hairpins induces a new type of long range cooperativity, which is responsible in part for the high co-operativity observed experimentally. This interpretation is in good agreement with kinetic results presented elsewhere (Henco et al., 1979).In order to understand the uniqueness of viroids, the structure and the conformational transitions of circular RNA molecules of the same base composition as PST viroids but with 359 nucleotides arranged randomly, were studied theoretically. Common viroid features, such as the number of base-pairs, the high co-operativity and the formation of very stable hairpins, are found to be improbable in such random sequences. It is concluded that various viroid species, although differing in nucleotide sequence, follow common principles of structure and structure formation.     

Tissue and intra-cellular distribution of coconut cadang cadang viroid and citrus exocortis viroid determined by in situ hybridization and confocal laser scanning and transmission electron microscopy

Confocal laser scanning microscopy and transmission electron microscopy (TEM) were used in conjunction with in situ hybridization techniques to compare and contrast the subnuclear (ultrastructural) and tissue (histological) localizations, respectively, of citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Both these viroids, which are members of the same taxonomic subgroup of viroids, were found in the vascular tissues as well as in the nuclei of mesophyll cells of infected host plants. At the subnuclear level, however, CEV was distributed across the entire nucleus, in contrast to CCCV which was mostly concentrated in the nucleolus with the remainder distributed throughout the nucleoplasm.     

Nucleotide sequence of cucumber pale fruit viroid: homology to hop stunt viroid.

Double stranded cDNA of cucumber pale fruit viroid ( CPFV ) has been cloned by the method of Okayama and Berg (Mol.Cell.Biol.2,161-170 (1982] and the complete nucleotide sequence was established. The covalently closed circular molecules of single-stranded CPFV RNA consists of 303 nucleotides. The nucleotide sequence of CPFV was compared with the previously established sequence of hop stunt viroid (HSV), which consists of 297 nucleotides ( Ohno et al. Nucleic Acid Res.11,6185-6197 (1983]. CPFV differs from HSV in the nucleotide sequence at 16 positions which include 8 exchanges, 7 insertions and 1 deletion. Both viroids share about 95% sequence homology. Considering the pathogenic properties of both viroids together, it is concluded that CPFV is a cucumber isolate of HSV.