[show abstract][hide abstract] ABSTRACT: Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco () expressing a hairpin RNA (hpRNA) of (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.
PLoS ONE 01/2013; 8(2):e57736. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of virological methods 11/2012; · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: To date, several viroid species have been shown to infect grapevine, including Hop stunt viroid (HpSVd), Citrus exocortis viroid (CEVd), Australian grapevine viroid (AGVd), Grapevine yellow speckle viroid-1 (GYSVd-1), Grapevine yellow speckle viroid-2 (GYSVd-2) and a tentative new species, Grapevine yellow speckle viroid-3 (GYSVd-3). Here, we identified and analyzed the distribution, genetic diversity, and molecular properties of viroids infecting grapevine cultivated in China and Japan, including old grapevines. The analysis showed that all the five known viroids and a tentative species GYSVd-3 infecting grapevine exist in China, and three of them (HpSVd, GYSVd-1 and GYSVd-3) exist in Japan. The contrast in diversity of viroid species in old grapevines from China and Japan may reflect different history of viticulture between the two countries. In general, the species of viroids infecting grapevine in China, as well as those in Iran and Australia, were more diverse than in the other countries. The population structure of viroids infecting grapevine in China and Japan showed species-dependency; i.e., HpSVd shared similar population structures in both countries, but GYSVd-1, GYSVd-2, and AGVd showed regional disparity even within the same country, although the role of sequence diversity in the biology of viroids infecting grapevine, such as the pathogenicity and evolution, still needs further study.
Virus Research 08/2012; 169(1):237-45. · 2.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: When Diener discovered Potato spindle tuber viroid in 1971 (Diener, Virology 45:411-428, 1971), only a limited number of techniques were available for plant virus detection and purification. Biological assays using indicator hosts showing characteristic symptoms of infection and able to support high levels of viroid replication played a critical role in viroid detection and characterization. Polyacrylamide gel electrophoresis (PAGE) was the first molecular technique to be used for the rapid (2-3 days) identification of viroid-infected plants. Because it is the only diagnostic method that is sequence-independent, PAGE under denaturing conditions continues to play a key role in the identification of new viroids. Starting in the early 1980s, dot blot hybridization began to replace PAGE for routine viroid diagnosis. The first diagnostic protocols based on reverse transcription-polymerase chain reaction (RT-PCR) appeared approximately 10 years later, and much effort has subsequently been devoted to simplifying the sample preparation procedure and identifying group-specific primer pairs. This chapter describes four simple, easy-to-follow protocols-two involving PAGE and two others based on enzymatic amplification of viroid cDNAs-that currently play key roles in viroid discovery and characterization.
Methods in molecular biology (Clifton, N.J.) 01/2012; 894:253-71.
[show abstract][hide abstract] ABSTRACT: To monitor the richness in microbial inhabitants in the phyllosphere of apple trees cultivated under various cultural and environmental conditions, we developed an oligo-DNA macroarray for major pathogenic and non-pathogenic fungi and bacteria inhabiting the phyllosphere of apple trees.
First, we isolated culturable fungi and bacteria from apple orchards by an agar-plate culture method, and detected 32 fungal and 34 bacterial species. Alternaria, Aureobasidium, Cladosporium, Rhodotorula, Cystofilobasidium, and Epicoccum genera were predominant among the fungi, and Bacillus, Pseudomonas, Sphingomonas, Methylobacterium, and Pantoea genera were predominant among the bacteria. Based on the data, we selected 29 major non-pathogenic and 12 phytopathogenic fungi and bacteria as the targets of macroarray. Forty-one species-specific 40-base pair long oligo-DNA sequences were selected from the nucleotide sequences of rDNA-internal transcribed spacer region for fungi and 16S rDNA for bacteria. The oligo-DNAs were fixed on nylon membrane and hybridized with digoxigenin-labeled cRNA probes prepared for each species. All arrays except those for Alternaria, Bacillus, and their related species, were specifically hybridized. The array was sensitive enough to detect 10(3) CFU for Aureobasidium pullulans and Bacillus cereus. Nucleotide sequencing of 100 each of independent fungal rDNA-ITS and bacterial 16S-rDNA sequences from apple tree was in agreement with the macroarray data obtained using the same sample. Finally, we analyzed the richness in the microbial inhabitants in the samples collected from apple trees in four orchards. Major apple pathogens that cause scab, Alternaria blotch, and Marssonina blotch were detected along with several non-phytopathogenic fungal and bacterial inhabitants.
The macroarray technique presented here is a strong tool to monitor the major microbial species and the community structures in the phyllosphere of apple trees and identify key species antagonistic, supportive or co-operative to specific pathogens in the orchard managed under different environmental conditions.
PLoS ONE 01/2012; 7(3):e34249. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of virological methods 03/2011; 174(1-2):123-7. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: To better understand the biogenesis of viroid-specific small RNAs and their possible role in disease induction, we have examined the accumulation of these small RNAs in potato spindle tuber viroid (PSTVd)-infected tomato plants. Large-scale sequence analysis of viroid-specific small RNAs revealed active production from the upper portion of the pathogenicity and central domains, two regions previously thought to be underrepresented. Profiles of small RNA populations derived from PSTVd antigenomic RNA were more variable, with differences between infected Rutgers (severe symptoms) and Moneymaker (mild symptoms) plants pointing to possible cultivar-specific differences in small RNA synthesis and/or stability. Using microarray analysis, we monitored the effects of PSTVd infection on the expression levels of >100 tomato genes containing potential binding sites for PSTVd small RNAs. Of 18 such genes down-regulated early in infection, two genes involved in gibberellin or jasmonic acid biosynthesis contain binding sites for PSTVd small RNAs in their respective ORFs.
[show abstract][hide abstract] ABSTRACT: A new variant of Potato spindle tuber viroid (PSTVd) was detected for the first time from dahlia grown in Japan. The dahlia isolate of PSTVd formed a quasi-species and
a major sequence variant consisting of 361 nucleotides in length, including five substitutions, three insertions, and one
deletion, when compared to the intermediate strain from potato. In bioassays with the new isolate, Rutgers tomato developed
mild stunting and leaf curling.
Potato spindle tuber viroid
Journal of General Plant Pathology 01/2011; 77(4):253-256. · 0.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Viroids are autonomously replicating, small single-stranded circular RNA pathogens that do not code for proteins and may cause diseases in infected, susceptible plants. They have the ability to induce both RNA-mediated transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS), or RNA silencing, in infected plants. Their induced RNA silencing has also been demonstrated in a wheat germ extract system. A possible role of RNA silencing in viroid pathogenicity and evolution has been discussed. It is suggested that RNA silencing can be employed to engineer plants for viroid resistance and attempts to produce these plants have been also discussed.
[show abstract][hide abstract] ABSTRACT: 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.
Archives of Virology 06/2009; 154(6):993-7. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hop stunt was a mysterious disorder that first emerged in the 1940s in commercial hops in Japan. To investigate the origin of this disorder, we infected hops with natural Hop stunt viroid (HpSVd) isolates derived from four host species (hop, grapevine, plum and citrus), which except for hop represent possible sources of the ancestral viroid. These plants were maintained for 15 years, then analyzed the HpSVd variants present. Here we show that the variant originally found in cultivated grapevines gave rise to various combinations of mutations at positions 25, 26, 54, 193, and 281. However, upon prolonged infection, these variants underwent convergent evolution resulting in a limited number of adapted mutants. Some of them showed nucleotide sequences identical to those currently responsible for hop stunt epidemics in commercial hops in Japan, China, and the United States. Therefore, these results indicate that we have successfully reproduced the original process by which a natural HpSVd variant naturally introduced into cultivated hops was able to mutate into the HpSVd variants that are currently present in commercial hops. Furthermore, and importantly, we have identified cultivated grapevines as a symptomless reservoir in which HSVd can evolve and be transmitted to hop crops to cause epidemics.
PLoS ONE 01/2009; 4(12):e8386. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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).
Archives of Virology 01/2009; 154(2):315-20. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Apple fruit crinkle viroid (AFCVd) infects apples and hops. To analyze the genetic diversity of AFCVd, nine apple and six hop isolates were collected from several locations in Japan. In total, 76 independent cDNA clones were used for sequencing and phylogenetic analyses. Two major population clusters were identified. The first consisted of all four hop isolates from Akita and some from Yamagata. The second cluster consisted of some Yamagata hop and all apple isolates. On the basis of the polymorphism found in the nucleotide insertion between positions 142/143 of the AFCVd genome and the history of hop cultivation in the region, it appears likely that one of the AFCVd populations that pre-existed in the Yamagata hops served as a "founder" for the Akita hop cluster. In this scenario, a genetic bottleneck caused by vegetative propagation played an important role in the shaping of viroid populations in a cultivated crop.
[show abstract][hide abstract] ABSTRACT: We have successfully applied a hybridization selection method using biotinylated-viroid transcripts and streptavidin-coupled
magnetic beads, followed by re-amplification, restriction enzyme digestion and concatemerization, to clone viroid small RNAs.
The cloning efficiency with the PSTVd-specific small RNAs increased more than 100-fold over that with random cloning of unselected
small RNAs. The method can be used to analyze small RNA biogenesis, especially to find hotspots, in various host–pathogen
Journal of General Plant Pathology 01/2008; 74(3):203-207. · 0.89 Impact Factor
[show abstract][hide abstract] ABSTRACT: Like many plant RNA viruses, infection by potato spindle tuber viroid (PSTVd) is known to lead to RNA silencing and a marked reduction in visible disease. To examine the relationship between RNA silencing and this recovery phenomenon in greater detail, we have carried out time-course analyses of viroid-specific small RNA accumulation using several viroid-host combinations. These analyses revealed the presence of two size classes of viroid-specific small RNAs in infected plants, and sequence analysis subsequently demonstrated the presence of a previously undescribed cluster of small RNAs derived primarily from negative-strand PSTVd RNA. Although the clustering patterns were similar, the size distribution of PSTVd small RNAs isolated from symptomatic leaf tissue became more heterogeneous with time. The process by which viroid-specific small RNAs are generated appears to be more complicated than previously believed, possibly involving multiple DICER-LIKE activities, viroid RNA substrates and subcellular compartments.
Journal of General Virology 01/2008; 88(Pt 12):3452-7. · 3.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Amino acid sequences of apple chlorotic leaf spot virus (ACLSV) coat protein (CP) were compared between 12 isolates from apple, plum and cherry, and 109 cDNA clones that were amplified directly from infected apple tissues. Phylogenetic analysis based on the amino acid sequences of CP showed that the isolates and cDNA clones were separated into two major clusters in which the combinations of the five amino acids at positions 40, 59, 75, 130 and 184 (Ala(40)-Val(59)-Phe(75)-Ser(130)-Met(184) or Ser(40)-Leu(59)-Tyr(75)-Thr(130)-Leu(184)) were highly conserved within each cluster. Site-directed mutagenesis using an infectious cDNA clone of ACLSV indicated that the combinations of two amino acids (Ala(40) and Phe(75) or Ser(40) and Tyr(75)) are necessary for infectivity to Chenopodium quinoa plants by mechanical inoculation. Moreover, an agroinoculation assay indicated that the substitution of a single amino acid (Ala(40) to Ser(40) or Phe(75) to Tyr(75)) resulted in extreme reduction in the accumulation of viral genomic RNA, double-stranded RNAs and viral proteins (movement protein and CP) in infiltrated tissues, suggesting that the combinations of the two amino acids at positions 40 and 75 are important for effective replication in host plant cells.
Journal of General Virology 10/2007; 88(Pt 9):2611-8. · 3.13 Impact Factor