32P- and biotin-labelled in vitro transcribed cRNA probes for the detection of potato spindle tuber viroid and chrysanthemum stunt viroid

Replacing nick-translated DNA probes by in vitro transcribed complementary RNA (cRNA) probes considerably increased the sensitivity of dot-blot detection tests of potato spindle tuber viroid and chrysanthemum stunt viroid. As compared to the limit of detection of 5–10 pg of viroid obtained with ³²P-labelled DNA probes, cRNA probes allow the detection of less than 1 pg of pure viroid. When labelled with biotin by incorporation of biotin-labelled ribonucleotides, the cRNA probes have a limit of detection of approximately 5 pg of purified viroid.     

Elimination of chrysanthemum stunt viroid from an infected chrysanthemum cultivar by shoot regeneration from a leaf primordium-free shoot apical meristem dome attached to a root tip

In this research we eliminated chrysanthemum stunt viroid (CSVd) from a highly infected chrysanthemum cultivar using a newly established method. Piato is one of the most difficult cultivars in which to obtain CSVd-free plants by conventional methods. Leaf primordium-free shoot apical meristems (LP-free SAMs) of Piato plants were dissected and attached to CSVd-free chrysanthemum or cabbage root tips. As shown by nested-PCR, CSVd was not detected in some shoots regenerated on both types of root tip. The production rates of CSVd-free plants using chrysanthemum and cabbage root tips were 14% and 3%, respectively. Regeneration of plants from LP-free SAMs of chrysanthemum plants by attaching these SAMs to root tips is an efficient method of generating CSVd-free chrysanthemum plants.Communicated by K.K. Kamo     

Mutational analysis of viroid pathogenicity: tomato apical stunt viroid

A series of nucleotide substitutions within the pathogenicity domain of tomato apical stunt viroid have been evaluated for their effects upon infectivity and symptom expression. None of the 12 A----G substitutions and one C----U substitution that were examined abolished infectivity in a whole plant bioassay, and the resulting progeny were characterized by nucleotide sequence analysis of cDNAs amplified by the polymerase chain reaction. Four of the 13 substitutions gave rise to altered progeny, but the patterns of sequence changes observed were unexpectedly complex. Mutations that did not rapidly revert to the wild-type sequence are located near the right border of the pathogenicity domain, a region which shows considerable natural sequence variability. None had a detectable effect upon symptom expression. The ability to observe viroid sequence evolution in vivo may provide insight into the molecular interactions responsible for viroid host range and symptom formation.     

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–function analysis of the ribozymes of chrysanthemum chlorotic mottle viroid: a loop–loop interaction motif conserved in most natural hammerheads

Loop–loop tertiary interactions play a key role in the folding and catalytic activity of natural hammerhead ribozymes. Using a combination of NMR spectroscopy, site-directed mutagenesis and kinetic and infectivity analyses, we have examined the structure and function of loops 1 and 2 of the (+) and (–) hammerheads of chrysanthemum chlorotic mottle viroid RNA. In both hammerheads, loop 1 is a heptanucleotide hairpin loop containing an exposed U at its 5′ side and an extrahelical U at its 3′-side critical for the catalytic activity of the ribozyme in vitro and for viroid infectivity in vivo, whereas loop 2 has a key opened A at its 3′-side. These structural features promote a specific loop–loop interaction motif across the major groove. The essential features of this tertiary structure element, base pairing between the 5′ U of loop 1 and the 3′ A of loop 2, and interaction of the extrahelical pyrimidine of loop 1 with loop 2, are likely shared by a significant fraction of natural hammerheads.     

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.     

Determination of potato spindle tumor viroid and chrysanthenum stund viroid using biotinylated olideoxyribonucleotides]

A 26 base long oligodeoxyribonucleotide complementary to a common RNA sequence of potato spindle tuber viroid (PSTV) and chrysantemum stunt viroid (CSV) was synthesized. The 3'-end biotinylated one was used for the detection of PSTV and CSV RNA immobilized on nitrocellulose filters by nucleic acid hybridization. Visualization of hybridization results was performed by two ways, either by streptavidin-alkaline phosphatase conjugate or streptavidine and biotinylated alkaline phosphatase. It was possible to detect 0.65 ng of purified CSV and PSTV RNA. The suggested system of viroid diseases detection can be used by agricultural and horticultural enterprises.     

不同菊花品种抗蚜虫性鉴定

为发掘抗蚜虫种质,对32份切花菊进行了人工蚜虫接种,统计了蚜虫接种后不同品种上虫口数量和蚜量比值。结果表明:不同品种对蚜虫的抗性表现出明显差异,蚜虫接种21d时各品种上虫口数量差异甚大,为0～267头不等;根据蚜量比值可将菊花抗蚜虫性分为5级,分别为:高抗,0～0.25;中抗,0.26～0.50;抗,0.51～0.75;低抗,0.76～1.25;不抗,1.25。32份切花菊中,有14个菊花品种为不抗品种;4个品种为低抗品种;3个品种为抗虫品种;1个品种为中抗品种;10个品种为高抗品种。     

Attempts to eliminate chrysanthemum stunt from chrysanthemum by meristem-tip culture after heat-treatment

‘Mistletoe’ chrysanthemums infected with stunt were grown at 35°C for 14–37 weeks, and meristem-tips cultured from them at intervals. Of 337 meristems, seventy-two survived to plants but their development took 50% longer than did that of stunt-free meristem-tips. All plants were symptomless for at least 5 weeks after planting in 75 mm pots of soil-mix, and only three showed symptoms after 9 weeks. No more plants developed symptoms during the next 5 (winter) months, but between mid-March and late May sixty-seven did so. Only two plants were freed from stunt. ‘Mini-cuttings’ rooted from shoot tips of infected chrysanthemums grown at 35°C for 37 weeks all developed stunt symptoms within 27 weeks.     

RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct

Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (–)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.     

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, P_L and P_R, of the proposed native structure. Infectivity studies with full-length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that P_L is the pathogenicity-modulating domain. The role of the P_R domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.     

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.     

Hop stunt viroid: A polyphagous pathogenic RNA that has shed light on viroid–host interactions

Taxonomy Hop stunt viroid (HSVd) is the type species of the genus Hostuviroid (family Pospiviroidae). The other species of this genus is Dahlia latent viroid, which presents an identical central conserved region (CCR) but lacks other structural hallmarks present in Hop stunt viroid. HSVd replication occurs in the nucleus through an asymmetric rolling‐circle model as in the other members of the family Pospiviroidae, which also includes the genera Pospiviroid, Cocadviroid, Apscaviroid, and Coleoviroid.Physical properties Hop stunt viroid consists of a single‐stranded, circular RNA of 295–303 nucleotides depending on isolates and sequence variants. The most stable secondary structure is a rod‐like or quasi‐rod‐like conformation with two characteristic domains: a CCR and a terminal conserved hairpin similar to that of cocadviroids. HSVd lacks a terminal conserved region.Hosts and symptomsHSVd infects a very broad range of natural hosts and has been reported to be the causal agent of five different diseases (citrus cachexia, cucumber pale fruit, peach and plum apple apricot distortion, and hop stunt). It is distributed worldwide.TransmissionHSVd is transmitted mechanically and by seed.     

The viroid and viroid-like RNA database.

The viroid and viroid-like RNA database is a compilation of all natural sequences published in journals or available from the GenBank and EMBL nucleotide sequence libraries. Several information regarding these RNA species such as the position of their self-catalytic domains and the open reading frame of the human hepatitits delta virus are provided. The database also includes a determination of the likely ancestral sequence of most species and a prediction of the most stable secondary structures of these sequences. This online database is available on the World Wide Web (http://www.callisto.si.usherb.ca/[symbol: see text]jpperra ). It should provide an excellent reference point for further phylogenetic and structure-function studies of these RNA species.