RESUMO
Characterization of viroid-derived small RNAs (vd-sRNAs) is important to understand viroid-host interactions; however, vd-sRNAs belonging to the genus Coleviroid are yet to be identified and characterized. Herein, we used coleus plants singly infected with coleus blumei viroid (CbVd)-1, -5, or -6 and doubly infected with CbVd-1 and -5 to identify and analyze their vd-sRNAs. We found sense and antisense vd-sRNAs for CbVd-1, -5 and -6, and 22-nt vd-sRNAs were the most abundant; moreover, the 5'-terminal nucleotides (nts) of CbVd-1, -5, and -6 were biased toward U and C, and sRNAs derived from these three viroids were unevenly distributed along their genomes. We also noted that CbVd-5 and -6 share a fragment that forms the right half of the rod-like secondary structure of these viroids, which implied that they generated almost the same type of vd-sRNAs. This finding indicated that vd-sRNA biogenesis is mainly determined by the primary sequence of their substrates. More importantly, we found two complementary vd-sRNAs (22 nt) that were generated from the central conserved region (CCR) of these three viroids, suggesting an important role of CCR in vd-sRNA biogenesis. In conclusion, our results provide novel insight into the biogenesis of vd-sRNAs and the biological roles of CCR.
Assuntos
Coleus/virologia , Pequeno RNA não Traduzido/genética , RNA Viral/genética , Viroides/genética , Sequência de Bases , Genoma Viral/genética , Conformação de Ácido Nucleico , Pequeno RNA não Traduzido/biossíntese , Viroides/metabolismo , Viroides/fisiologiaAssuntos
Coleus , Viroides , Canadá , Coleus/virologia , Tipagem Molecular , RNA Viral/genética , Viroides/genéticaRESUMO
Four viroid species infecting Coleus blumei, named Coleus blumei viroid 1-4 (CbVd-1-CbVd-4), and two tentative new viroid species, named CbVd-5 and CbVd-6, have been characterized, for two of which (CbVd-5 and CbVd-6, first reported in 2009), there is no established bioassay. Here, infectious clones were used as inoculums sources and the biological properties of CbVd-1, -3, -5 and -6 were assessed. When dimeric CbVd (+) RNAs synthesized in vitro were bioassayed, the first detection time for the four CbVds was different, ranging from 45 to 300 days. In addition, we confirmed that CbVd-5 and CbVd-6 can be transmitted via seeds. Molecular characterization of their progeny from slash-inoculated plants one year after inoculation demonstrated that the genetic diversity of CbVd populations may depend on the infected coleus plants and on the viroid genotype.
Assuntos
Coleus/virologia , DNA Complementar , RNA Viral/genética , RNA Viral/isolamento & purificação , Viroides/genética , Viroides/isolamento & purificação , Replicação Viral , Clonagem Molecular , Viroides/fisiologiaRESUMO
A simple, low-cost hybridization assay using a universal DIG-labeled riboprobe for the rapid detection and identification of coleus viroids is presented. An octamer of 32-nucleotide sequence derived from the central conserved region (CCR) of viroids in the genus Coleviroid was used to develop a universal cRNA probe (8-central-conserved-region probe, 8CCR probe) for coleus viroids. Dot-blot hybridization assays demonstrated that the sensitivity of this probe was similar to specific probes for each CbVd, and Northern hybridization results revealed that at least four coleus viroids could be distinguished readily and simultaneously using the 8CCR probe. Batch detection assay showed that hybridization using the 8CCR probe can identify coleus viroids rapidly and effectively. This rapid and low-cost molecular hybridization technique is an effective way to survey the occurrence of coleus viroids, and has reference for the detection of other viroids and possibly viruses.
Assuntos
Hibridização de Ácido Nucleico/métodos , Sondas de Oligonucleotídeos/genética , Viroides/genética , Viroides/isolamento & purificação , Virologia/métodos , Coleus/virologia , Sensibilidade e Especificidade , Fatores de Tempo , Virologia/economiaRESUMO
A simple and fast sap-direct RT-PCR (reverse transcription-polymerase chain reaction) for the rapid detection of 3 viroids of the genus Coleviroid is presented. The templates for cDNA synthesis were obtained directly from the sap of coleus using a pipettor, a common tool in molecular biology laboratories, and 3 coleus blumei viroids (CbVds) were detected simultaneously using a pair of universal primers designed according to sequences in the central conserved region (CCR) of CbVds. RT-PCR results demonstrated that CbVd-1, CbVd-5, and CbVd-6 can be detected accurately in viroid-infected plants but not in viroid-free plants. The results of RT-PCR, dot-blot, sequencing, and batch-detection revealed that this method can be used to identify CbVds rapidly. The method also reduces cross-contamination among different samples to a minimum. It is considered that this rapid and simple technique is an effective method for the identification and cloning of CbVds.
Assuntos
Coleus/virologia , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Viroides/isolamento & purificação , Virologia/métodos , Primers do DNA/genética , RNA Viral/genética , Viroides/genéticaRESUMO
Coleus blumei can be infected by several viroids of the genus Coleviroid. One year after detecting a mixed infection of coleus blumei viroid 1 (CbVd-1) and 5 (CbVd-5) in coleus seedlings inoculated with these two viroids, we found an additional viroid-like RNA. Sequence analysis revealed a viroid of 342 nucleotides that contains the central conserved region of coleviroids and is a chimera of the left half of CbVd-3 and the right half of CbVd-5. This new viroid, tentatively referred to as coleus blumei viroid 6 (CbVd-6), appears to have arisen from a natural recombination event or genome shuffling.
Assuntos
Coleus/virologia , Recombinação Genética , Viroides/classificação , Viroides/isolamento & purificação , Sequência de Bases , Eletroforese em Gel Bidimensional , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Viral/genética , RNA Viral/isolamento & purificação , Análise de Sequência de DNA , Viroides/genéticaRESUMO
A viroid-like RNA was detected from coleus (Coleus blumei) in China. It consisted of 274 nucleotides and had 66% sequence identity with a member of the closest known viroid species. The predicted secondary structure is rod-shaped with extensive base pairing, and it has the conserved region characteristic of the genus Coleviroid. Two terminal sequences that are highly conserved among some members of the genus were also identified. The viroid-like RNA was successfully transmitted to coleus by slash-inoculation. This viroid was identified as a new member of the genus Coleviroid, and we tentatively propose the name Coleus blumei viroid 5 (CbVd-5).
Assuntos
Coleus/virologia , Doenças das Plantas/virologia , Viroides/classificação , Viroides/patogenicidade , Sequência de Bases , China , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Viral/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Viroides/genética , VirulênciaAssuntos
Carlavirus/genética , Genoma Viral , RNA Viral/genética , Verbena/virologia , Sequência de Bases , Carlavirus/isolamento & purificação , Coleus/virologia , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Doenças das Plantas/virologia , RNA Viral/química , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Proteínas Virais/genéticaRESUMO
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) was used to determine the general presence of viroids in the test samples. Efficacy of the primer pair for members of genus Pospiviroid was demonstrated by the detection of Potato spindle tuber viroid (PSTVd) and Tomato chlorotic dwarf viroid (TCDVd) in potato, Chrysanthemum stunt viroid and Iresine viroid in Verbena and Vinca species, and Citrus exocortis viroid in Impatiens species. Specificity of the primer pair became evident, where additional viroids were detected by R-PAGE in Coleus and Magilla species, but they were not amplified by the Pospiviroid primer. This primer pair would be of benefit in indexing ornamental plants in quarantine samples or in viroid-free certification schemes, irrespective of their actual identity.