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1.
Grapevine (Vitis spp.) is a widespread fruit tree hosting many viral entities that interact with the plant modifying its responses to the environment. The production of virus-free plants is becoming increasingly crucial for the use of grapevine as a model species in different studies. Using high-throughput RNA sequencing, the viromes of seven mother plants grown in a germplasm collection vineyard were sequenced. In addition to the viruses and viroids already detected in grapevine, we identified 13 putative new mycoviruses. The different spread among grapevine tissues collected in vineyard, greenhouse and in vitro conditions suggested a clear distinction between viruses/viroids and mycoviruses that can successfully be exploited for their identification. Mycoviruses were absent in in vitro cultures, while plant viruses and viroids were particularly accumulated in these plantlets. Somatic embryogenesis applied to the seven mother plants was effective in the elimination of the complete virome, including mycoviruses. However, different sanitization efficiencies for viroids and grapevine pinot gris virus were observed among genotypes. The absence of mycoviruses in in vitro plantlets, associated with the absence of all viral entities in somaclones, suggested that this regeneration technique is also effective to eradicate endophytic/epiphytic fungi, resulting in gnotobiotic or pseudo-gnotobiotic plants.  相似文献   

2.
Nucleic acid sequence-based amplification (NASBA) according to the standard protocol failed to amplify cRNA of viroids, probably because of their GC-rich and intramolecular base-paired structure. However, NASBA in the presence of inosine 5'-triphosphate successfully amplified the cRNAs to viroids in total nucleic acid extracts from citrus plants. As sequence specificity of the cRNA to viroids was confirmed by northern analysis, the amplification and fidelity of cRNAs are sufficient for the sensitive and specific detection of viroids.  相似文献   

3.
Summary In the present study we examined the possibility that viruses, viroids or dsRNA are associated with cytoplasmic male sterile (cms) petunia. The assumption was made that if viruses or viroids were present, the treatments for elimination of viruses and viroids would produce healthy fertile plants. Male sterile plants were subjected to heat and cold treatments for 10 weeks and/ or for 5 months, after which apical meristems were isolated and cultured with the addition of antiviral factors. The mother plants, the regenerated plants and their progeny were sterile. These treatments did not affect sterility in sterile plants or the fertility of fertile plants. No dsRNA was found in cms petunia by gel electrophoresis. Thus, our data suggest that there are no viruses, viroids or dsRNA associated with cms petunia. Our data are in agreement with recent data, which suggests that the mitochondrial DNA is the site of the cytoplasmic male sterile gene in petunia.Contribution no. 2198-E, 1987 series from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel  相似文献   

4.
5.
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.  相似文献   

6.
Most viroids replicate in the nuclei of infected plant cells. Nuclear import of the incoming RNA thus represents a key control point for establishment of a systemic infection. However, little is known about the mechanisms by which viroids are transported into the nucleus. We have characterized nuclear import of infectious, fluorescein-labeled potato spindle tuber viroid (F-PSTVd) in permeabilized tobacco BY2 cells. Import was observed for F-PSTVd but not for mRNA fragments of the same size or two viroids believed to replicate in the chloroplasts. Import of F-PSTVd was inhibited by addition of a 10-fold excess of non-fluorescent PSTVd but not by similar amounts of control RNAs. Import was not inhibited by pre-incubation with GTP-γ-S or GDP-β-S, however. Disruption of microtubules and actin filaments with oryzalin or cytochalasin D did not inhibit F-PSTVd import. Taken together, our results indicate that (i) PSTVd possesses a sequence and/or structural motif for nuclear import and (ii) the import is a cytoskeleton-independent process that is mediated by a specific and saturable receptor. Insensitivity to GTP-γ-S and GDP-β-S treatment suggests that PSTVd import is not coupled to Ran GTPase cycle, which mediates nuclear transport of many proteins and nucleic acids. To our knowledge, our studies are the first to examine the mechanisms of nuclear transport of RNA in plants.  相似文献   

7.
Viroids are subviral plant pathogens at the frontier of life. They are solely composed by a single-stranded circular RNA of 246-401 nt with a compact secondary structure. Viroids replicate autonomously when inoculated into their host plants and incite, in most of them, economically important diseases. In contrast to viruses, viroids do not code for any protein and depend on host enzymes for their replication, which in some viroids occurs in the nucleus and in others in the chloroplast, through a rolling-circle mechanism with three catalytic steps. Quite remarkably, however, one of the steps, cleavage of the oligomeric head-to-tail replicative intermediates to unit-length strands, is mediated in certain viroids by hammerhead ribozymes that can be formed by their strands of both polarities. Viroids induce disease by direct interaction with host factors, the nature of which is presently unknown. Some properties of viroids, particularly the presence of ribozymes, suggest that they might have appeared very early in evolution and could represent 'living fossils' of the precellular RNA world that presumably preceded our current world based on DNA and proteins.  相似文献   

8.
Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.  相似文献   

9.
Viroids: an Ariadne's thread into the RNA labyrinth   总被引:4,自引:0,他引:4  
  相似文献   

10.
A gel electrophoretic technique for the rapid and sensitive detection of viroids and virusoids is described. Starting from plant material, a typical analysis requires less than 5 hours. Viroid concentrations as low as 800 pg/g tissue can be detected unambiguously without the use of radioactivity, organic solvents, or highly specialized laboratory equipment. The sensitivity may be further increased by introducing additional purification steps. The technique is an essential improvement of the previously published bidirectional gel electrophoretic analysis (Schumacher et al.1983, Anal. Biochem. 135, 288–295). In the new procedure gel electrophoresis is first carried out under native conditions. Before the viroid (or virusoid) bands will leave the gel, conditions are changed to provide denaturing conditions which are achieved by increasing the temperature and changing the buffer. After changing the polarity of the electric field all nucleic acids in the gel “return” in that they now migrate towards their original starting point. Under the denaturing conditions in the second electrophoresis viroids (or virusoids) unfold into the conformation of a circle without in tramolecular base pairs, which structure is unique among the nucleic acids in the gel. The denatured circular viroids migrate in the gel much slower than all other nucleic acids of comparable molecular weight and, therefore stay well separated behind the edge of the other nucleic acids. Thus, viroids can easily be detected on the stained gel as a discrete band.  相似文献   

11.
Dynamics and interactions of viroids   总被引:5,自引:0,他引:5  
Viroids are single stranded circular RNA molecules of 120,000 daltons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids. For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure. The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.  相似文献   

12.
The stone fruit genus Prunus, within the family Rosaceae, comprises more than 230 species, some of which have great importance or value as ornamental or fruit crops. Prunus are affected by numerous viruses and viroids linked to the vegetative propagation practices in many of the cultivated species. To date, 44 viruses and three viroids have been described in the 9 main cultivated Prunus species. Seven of these viruses and one viroid have been identified in Prunus hosts within the last 5 years. This work addresses recent advances and prospects in the study of viruses and viroids affecting Prunus species, mostly concerning the detection and characterisation of the agents involved, pathogenesis analysis and the search for new control tools. New sequencing technologies are quickly reshaping the way we can identify and characterise new plant viruses and isolates. Specific efforts aimed at virus identification or data mining of high‐throughput sequencing data generated for plant genomics‐oriented purposes can efficiently reveal the presence of known or novel viruses. These technologies have also been used to gain a deeper knowledge of the pathogenesis mechanisms at the gene and miRNA expression level that underlie the interactions between Prunus spp. and their main viruses and viroids. New biotechnological control tools include the transfer of resistance by grafting, the use of new sources of resistance and the development of gene silencing strategies using genetic transformation. In addition, the application of next generation sequencing and genome editing techniques will contribute to improving our knowledge of virus–host interactions and the mechanisms of resistance. This should be of great interest in the search to obtain new Prunus cultivars capable of dealing both with known viruses and viroids and with those that are yet to be discovered in the uncertain scenario of climate change.  相似文献   

13.
14.
R Hammond  D R Smith    T O Diener 《Nucleic acids research》1989,17(23):10083-10094
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.  相似文献   

15.
Plant pathogenic bacteria, phytoplasmas, viruses and viroids are difficult to control, and preventive measures are essential to minimize the losses they cause each year in different crops. In this context, rapid and accurate methods for detection and diagnosis of these plant pathogens are required to apply treatments, undertake agronomic measures or proceed with eradication practices, particularly for quarantine pathogens. In recent years, there has been an exponential increase in the number of protocols based on nucleic-acid tools being those based on PCR or RT-PCR now routinely applied worldwide. Nucleic acid extraction is still necessary in many cases and in practice inhibition problems are decreasing the theoretical sensitivity of molecular detection. For these reasons, integrated protocols that include the use of molecular techniques as screening methods, followed by confirmation by other techniques supported by different biological principles are advisable. Overall, molecular techniques based on different types of PCR amplification and very especially on real-time PCR are leading to high throughput, faster and more accurate detection methods for the most severe plant pathogens, with important benefits for agriculture. Other technologies, such as isothermal amplification, microarrays, etc. have great potential, but their practical development in plant pathology is still underway. Despite these advances, there are some unsolved problems concerning the detection of many plant pathogens due to their low titre in the plants, their uneven distribution, the existence of latent infections and the lack of validated sampling protocols. Research based on genomic advances and innovative detection methods as well as better knowledge of the pathogens' lifecycle, will facilitate their early and accurate detection, thus improving the sanitary status of cultivated plants in the near future.  相似文献   

16.
Replicating circular RNAs are independent plant pathogens known as viroids, or act to modulate the pathogenesis of plant and animal viruses as their satellite RNAs. The rate of discovery of these subviral pathogens was low over the past 40 years because the classical approaches are technical demanding and time-consuming. We previously described an approach for homology-independent discovery of replicating circular RNAs by analysing the total small RNA populations from samples of diseased tissues with a computational program known as progressive filtering of overlapping small RNAs (PFOR). However, PFOR written in PERL language is extremely slow and is unable to discover those subviral pathogens that do not trigger in vivo accumulation of extensively overlapping small RNAs. Moreover, PFOR is yet to identify a new viroid capable of initiating independent infection. Here we report the development of PFOR2 that adopted parallel programming in the C++ language and was 3 to 8 times faster than PFOR. A new computational program was further developed and incorporated into PFOR2 to allow the identification of circular RNAs by deep sequencing of long RNAs instead of small RNAs. PFOR2 analysis of the small RNA libraries from grapevine and apple plants led to the discovery of Grapevine latent viroid (GLVd) and Apple hammerhead viroid-like RNA (AHVd-like RNA), respectively. GLVd was proposed as a new species in the genus Apscaviroid, because it contained the typical structural elements found in this group of viroids and initiated independent infection in grapevine seedlings. AHVd-like RNA encoded a biologically active hammerhead ribozyme in both polarities, and was not specifically associated with any of the viruses found in apple plants. We propose that these computational algorithms have the potential to discover novel circular RNAs in plants, invertebrates and vertebrates regardless of whether they replicate and/or induce the in vivo accumulation of small RNAs.  相似文献   

17.
Abstract

Viroids are single stranded circular RNA molecules of 120 000 dal tons which are pathogens of certain higher plants and replicate autonomously in the host cell. Virusoids are similar to viroids in respect to size and circularity but do replicate only as a part of a larger plant virus. The structure and structural transitions have been investigated by thermodynamic, kinetic and hydrodynamic methods and have been compared to results from calculations of the most favorable native structures and the denaturation process. The algorithm of Zuker et al. was modified for the application to circular nucleic acids.

For viroids the calculations confirm our earlier theoretical and experimental results about the extended native structure and the highly cooperative transition into a branched structure. Virusoids, although described in the literature as viroid-like, show less base pairing, branching in the native secondary structure, and only low cooperativity during denaturation. They resemble more closely the properties of random sequences with length, G:C content, and circularity as in viroids but sequences generated by a computer. The comparison of viroids, virusoids and circular RNA of random sequences underlines the uniqueness of viroid structure.

The interactions of viroids with dye and oligonucleotide-ligands and with RNA-polymerase II from wheat germ, which enzyme replicates viroids in vitro, has been studied in order to correlate viroid structure and its ability for specific interactions. Specificity of the interactions may be interpreted on the basis of the neighbourhood of double stranded and single stranded regions. In the host cell viroids are localized in the cell nucleus; they may be detected as free nucleic acids and in high molecular weight complexes together with other RNA and proteins.  相似文献   

18.
T O Diener 《FASEB journal》1991,5(13):2808-2813
Contrary to earlier beliefs, viruses are not the smallest causative agents of infectious diseases. Single-stranded RNAs as small as 246 nucleotides exist in certain higher plants and cause more than a dozen crop diseases. These RNAs have been termed viroids. Despite their extremely limited information content, viroids replicate autonomously in susceptible cells--that is, they do not require helper functions from simultaneously replicating conventional viruses. Viroids are covalently closed circular molecules with a characteristic rodlike secondary structure in which short helical regions are interrupted by internal and bulge loops. Viroids are not translated; they are replicated by a host enzyme (or enzymes) (probably RNA polymerase II) via oligomeric RNA intermediates by a rolling circle mechanism. Viroidlike satellite RNAs resemble viroids in size and molecular structure, but are found within the capsids of specific helper viruses on which they depend for their own replication. These RNAs are of great interest to molecular biology for at least two reasons: 1) they are the smallest and simplest replicating molecules known, and 2) they may represent living fossils of precellular evolution in a hypothetical RNA world.  相似文献   

19.
20.
Summary A wide range of microorganisms (filamentous fungi, yeasts, bacteria, viruses and viroids) and micro-arthropods (mites and thrips) have been identified as contaminants in plant tissue cultures. Contaminant may be introduced with the explant, during manipulations in the laboratory or by micro-arthropod vectors. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination. Disinfection protocols have now been developed for a wide range of plant species including those infected with viruses/viroids or endophytic bacteria. They may include the selection of pathogen-free donor plants or donor plant treatments such as thermotherapy. Also microbiological quality assurance systems (e.g. Hazard Analysis Critical Control Point; HACCP procedures) have been adapted to the needs of commercial plant tissue culture laboratories. These are aimed at, preventing the introduction of pathogens, into tissue cultures at establishment and in the laboratory. In established in vitro cultures preventative strategies have proved to be essential, since it is extremely difficult to eliminate environmental bacterial and fungal contaminants using, antibiotics and fungicides. In many cases anti-microbial treatments only inhibit contaminants and low levels of contamination persist. In particular, the use of antibiotics against Gram-negative bacteria (including plant pathogenic bacteria and Agrobacterium tumefaciens vector systems used in genetic engineering) has been shown frequently to be extremely difficult or unsuccessful. Detection of latent contamination may involve the use of general and semi-selective microbial growth media or serological and PCR-based molecular techniques for specific pathogens. However, it is often difficult to detect low numbers of latent bacterial contaminants (e.g. levels present following antibiotic treatment or when acidified plant media are used). This poses a particular risk in the production of transgenic plants where the elimination or detection of Agrobacterium tumefaciens-based vector systems cannot be guaranteed with the currently available methodologies. Recent research has also shown that there is a risk of the transmission of human pathogens in plant tissue cultures.  相似文献   

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