First Report of European mountain ash ringspot-associated virus in Sorbus aucuparia in Norway.

In July 2012, leaf mottle and intensive chlorotic ringspots were observed on urban, forest, or roadside mountain ash trees (Sorbus aucuparia L., rowan) of different ages in Norway during visual inspection of native broadleaf forest tree species. Symptoms resembled those caused by European mountain ash ringspot-associated virus (EMARaV), the type-member of the newly established genus Emaravirus, containing segmented ss(-)RNA and infecting woody host species (2). Leaves of nine out of 30 assessed rowan trees exhibiting characteristic symptoms were sampled in the counties of Nordland and Nord-Trøndelag (between 63.511806° and 66.304680°N latitude). Three of them were infested by the potential vector the eriophyid gall mite Phytoptus pyri. EMARaV was detected from total RNA extracts of leaves by reverse transcription-PCR using virus-specific primers amplifying 300 bp of RNA2 and 204 bp of RNA3, respectively (3). PCR fragments were directly sequenced from both ends and submitted to the EMBL database (accession nos. HG428680 to 97). Sequenced fragments comprising the partial gene encoding the glycoprotein-precursor (261 nucleotides of RNA2 omitting primer sequences) obtained from the nine sampled trees showed identities of 97 to 98% to the sequence of the reference strain of EMARaV from Hamburg, Germany (database accession AY563041). Comparison of 159 nucleotides of the 3' untranslated region (3' UTR) of viral RNA3 of the nine investigated rowans in Norway exhibited higher sequence diversity on nucleotide level (up to 50 nucleotide exchanges, or 31%) as previously reported from EMARaV variants from other European countries (4). When subjected to BLASTn search through GenBank, only three partial RNA3 sequences generated in this study showed sequence identities of 96% to the reference isolate (accession DQ831831). The other six sequences revealed only 68 to 73% identity to RNA3 sequences of EMARaV variants from GenBank. This led to formation of a separate cluster in phylogenetic analysis of partial RNA3 sequences of the six EMARaV variants from Norway when compared to previously characterized strains from the Czech Republic (n = 2), Finland (n = 17), Germany (n = 1), Great Britain (n = 5), Russia (n = 3), and Sweden (n = 10). From three Norwegian samples clustering separately in the tree based on the partial 3' UTR of RNA3, the partial vRNA1 was amplified by RT-PCR using a generic primer set Motif-A-sense/Motif-C-antisense (1). Sequence analyses of these PCR fragments confirmed the viruses as members of the Emaravirus genus which were most closely related to EMARaV (data not shown). This is the first report of EMARaV in Norway infecting Sorbus aucuparia, a valuable native plant of northern Europe. The data obtained suggest a higher genetic variability of the EMARaV population in mountain ash trees in Norway than in other locations in Central and Northern Europe. However, whether the EMARaV variants identified in this study represent new strains of the virus have to be investigated in the future. References: (1) T. Elbeaino et al. J. Virol. Meth. 188:37, 2013. (2) N. Mielke-Ehret. and H. P. Mühlbach. Viruses 4:1515, 2012. (3) N. Mielke et al. For. Pathol. 38:371, 2008. (4) S. von Bargen et al. For. Pathol. 43: 429, 2013.

Double-stranded RNA pattern and partial sequence data indicate plant virus infection associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.).

Double-stranded RNA (dsRNA) has been extracted from tissue of European mountain ash trees (Sorbus aucuparia L.) showing typical ringspot and mottling symptoms on leaves and a gradual decay in general. A characteristic dsRNA pattern was found in leaf samples of symptomatic mountain ash trees from various stands in Germany. Bands of dsRNA molecules of approximately 7 kb, 2.3 kb, 1.5 kb, and 1.3 kb, respectively, were repeatedly detected. By random primed reverse transcription cDNA was synthesised from dsRNA and amplified by degenerate oligonucleotide primed PCR. After TA cloning, the cDNA clones obtained were screened with an enhanced-chemiluminescence-labelled dsRNA probe. Positive clones were further analysed by using them as hybridisation probes in Northern blots of total plant RNA and in Southern hybridisation with genomic DNA from Sorbus aucuparia leaves. From cDNA clones that were found to be specific for dsRNA in Northern analysis, primers were deduced for 5'-RACE analyses and further cloning. Finally, a cDNA fragment of 3,737 bp was obtained, which showed homology to viral proteins, particularly to the RNA-dependent RNA polymerase of members of the family Bunyaviridae, but without high similarity to a known genus. The dsRNA pattern and the sequence information strongly indicate a virus associated with the mountain ash ringspot disease. The putative virus remains still unidentified.

Control of mechanical viroid transmission by the disinfection of tables and tools.

Viroids are of practical importance as the cause of several economically significant infectious diseases. Potato spindle tuber viroid (PSTVd) causes severe yield losses in several crops, because the pathogen spreads fast within the culture. Viroids are small molecules, a few hundred nucleotides long, with a high degree of secondary structure. They do not code for any polypeptides and replicate independently of any associated plant virus. Viroids are readily transmitted by contaminated tools and tables. Furthermore PSTVd is transmitted through the pollen and true seed and can remain its infectious activity in seed for long periods. Vector transmission of PSTVd was reported to occur at low frequency. However, the mechanical transmission is the predominant factor and in this case we discuss the efficient disinfection of tools and tables as a main prophylactic trail to avoid viroid transmission. In previous studies we have tested the efficiency of several disinfectants to eliminate virus contamination. This paper demonstrates the efficient disinfection of MENNO-Florades (Menno-Chemie-GmbH, Norderstedt, Germany. A selection of different concentrations of the disinfectant and various times of incubation were applied in regard to practical use. The tests were confirmed by biological assays using suitable indicator plants, tissue print hybridization, gel electrophoresis and by nucleic acid hybridization. It was shown that PSTVd was eliminated when using the determined combination: 2% of the disinfectant while incubating for one minute or alternative 3%, 30 seconds. The possibility of viroid inactivation by a chemical method of disinfection--while plants are not affected--opens a new perspective to control viroid transmission via tools and tables.

Use of plant cell cultures in biotechnology.

Plant cell cultures are being widely used in scientific studies on the physiology, biochemistry and molecular biology of primary and secondary metabolism, developmental regulation and cellular responses to pathogens and stress. In this chapter the significance of plant cell cultures in biotechnology is discussed with special emphasis on commercial production of secondary metabolites and pharmaceuticals, the potential of genetically transformed cell cultures, photosynthetically active cell cultures, production of somatic embryos, and novel assay systems based on the use of plant cells. Future aspects of biotechnical applications with respect to the potentials and limitations of these approaches are assessed, particularly in comparison with the productivity of lower eucaryotes.

Detection and tissue distribution of potato spindle tuber viroid in infected tomato plants by tissue print hybridization.

Potato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developing cv "Rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "Goldkugel" 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

Detection and tissue distribution of potato spindle tuberviroid in infected tomato plants by tissue print hybridization.

Potato spindle tuber viroid (PSTVd) was detected in two cultivars of tomato (Lycopersicon esculentum Mill.) by tissue print hybridization of cross-sections of stem and rhachis, using a 35S-labeled PSTVd RNA probe. PSTVd was detectable in the viroid-sensitive and symptom-developping cv "Rutgers" 2 weeks p.i., and in the viroid-tolerant and practically symptomless cv "Goldkugel" 3 weeks p.i. In both tomato cultivars, PSTVd accumulated in the upper parts of the plants newly grown after inoculation. It was predominantly found in association with the ring formed by the vascular tissue. The final accumulation of PSTVd as well as its spatial distribution were similar in the sensitive and in the tolerant tomato cultivar, as estimated from the tissue print autoradiographs. Thus, tissue print hybridization provides a rapid and sensitive means for viroid diagnosis and for the assessment of tissue-specific localization of the viroid RNA.

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.).

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae.

Circadian oscillations of Lhc mRNAs in a photoautotrophic cell culture of Lycopersicon peruvianum.

Fourteen genes encoding proteins of the light harvesting complex (Lhc) are expressed in a photoautotrophic cell culture from the wild species of tomato (Lycopersicon peruvianum). For two genes, Lhca2 (cab7) and Lhcb2(*)1 (cab4), a rhythmic oscillation of the transcript accumulation is observed under light/dark and constant dark conditions indicating that gene expression is controlled by a circadian clock in the tomato cell culture. The circadian expression of the Lhc genes remains present after application of 2,2'-dipyridyl. However, the amplitude of Lhc mRNA oscillations and the photosynthetic capacity (Fmax/Fo) decrease significantly. The transcript accumulations of psbA, rbcS and rbcL are less or not at all affected by 2,2'-dipyridyl.

Isolation of viroid-RNA-binding proteins from an expression library with nonradioactive-labeled RNA probes.

The detection and isolation of cDNAs of tomato proteins that are able to bind to viroid RNA molecules are described. They were found by screening of a lambda gt11 cDNA expression library using a modification of the previously established ligand-blotting procedure to detect DNA- and RNA-binding proteins. The essentials of our modifications are the use of (i) digoxigenin-labeled viroid RNA, (ii) low concentration of the labeled probes and (iii) an expression library that allows the direct isolation of cDNA clones. The analysis of various isolated clones showed that this method is reliable for RNA-ligand screening and North-Western blotting. Applied to viroid RNA, these experimental tools provide the precondition for further studies on the specificity of the isolated proteins.

In vivo desaturation of cis-delta 9-monounsaturated to cis-delta 9,12-diunsaturated alkenylether glycerolipids.

Plants convert lipid-bound cis-n-9 monoenoic to polyenoic fatty acid residues without involvement of corresponding CoA-thioesters. To provide additional evidence for this type of lipid-linked desaturation we incubated sn-1-O- and 2-O-(cis-9)octadecenylglycerol isomers with photoautotrophic cell cultures from tomato. After 14 days the fractions of phosphatidylcholine and monogalactosyldiacylglycerol were isolated and the incorporated glycerol ether backbones released by treatment with LiAlH4 (reduction of ester bonds) and short acid hydrolysis (cleavage of enol ether bonds). High performance liquid chromatography and mass spectroscopy of the products in appropriately derivatized form showed that the (cis-9)octadecenyl group in the sn-1 position of the phospholipid was nearly completely desaturated to a (cis-9,12)octadecadienyl residue having the same double bond arrangement as linoleic acid. In the galactolipid fraction the desaturation had progressed to octadecatrienyl residues. Similarly, the octadecenyl residue in the sn-2 position of the phospholipid was nearly completely desaturated to an octadecadienyl group. These results are unambiguous proof for lipid-linked desaturation by both microsomal and plastidial desaturase systems of plants.

Photosynthetically active suspension cultures of potato spindle tuber viroid infected tomato cells as tools for studying viroid - host cell interaction.

Photosynthetically active callus and cell suspension cultures were established from uninfected Lycopersicon peruvianum plants and from uninfected and potato spindle tuber viroid (PSTVd) infected plants of Lycopersicon esculentum cv. Rutgers. Viroid infection was maintained in photoheterotrophic culture on media containing 3% sucrose, but during continuous photo-mixotrophic culture in low sucrose media (1% sucrose), the level of PSTVd accumulation decreased. Photoautotrophic cell suspensions could be established with uninfected, but not with viroid infected tomato cells. As compared to uninfected cells, PSTVd infected cells grew slowly, were morphologically different in size and shape, and formed tight cell aggregates. Electronmicroscopy showed that starch accumulation in chloroplasts, deformation of the chloroplast envelope and irregular plasmalemmasomes at the cell membrane were associated with PSTVd infection.

Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells.

A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (-) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (-)RNAs are likewise processed but with a much lower efficiency. Viroid-processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.

Synthesis of (+) and (-) RNA molecules of potato spindle tuber viroid (PSTV) in isolated nuclei and its impairment by transcription inhibitors.

Transcription studies with highly purified potato cell nuclei in combination with a 'transcription-hybridization analysis' unequivocally demonstrate that the nucleus is the subcellular site where the entire process of PSTV replication takes place. Inhibition experiments with actinomycin D and alpha-amanitin furthermore suggest that the nuclear DNA-dependent RNA polymerases I and II are involved in the synthesis of PSTV (+) and (-) RNA, respectively.

Oligomeric forms of potato spindle tuber viroid (PSTV) and of its complementary RNA are present in nuclei isolated from viroid-infected potato cells.

Different oligomeric forms of PSTV are detected in nuclei isolated from PSTV-infected potato cells by means of molecular hybridization, using as probes synthetic oligodeoxyribonucleotides with sequence specificity for (+)PSTV and for (-)PSTV. In addition to several species of longer-than-unit-length (-)PSTV molecules, two oligomeric forms of (+)PSTV are detected, which correspond in size to RNA strands of approximately two and three times viroid unit-length. They must be considered as the precursors of the circular and linear (+)PSTV monomers accumulating in the cell nucleus.

Contitions for optimal growth of a PSTV-infected potato cell suspension and detection of viroid-complementary longer-than-unit-length RNA in these cells.

A suspension culture from potato spindle tuber viroid (PSTV)-infected cells of the wild type potato (Solanum demissum) has been established, which is a suitable model system for studying PSTV replicationin vivo. The conditions for rapid growth of these cells and for permanent extensive viroid biosynthesis within them are described. Biosynthesis of PSTV in the potato cells was demonstrated by(32)P-incorporation into nucleic acids and their subsequent electrophoretic analysis on polyacrylamide gels. Under optimum culture conditions the amount of(32)P-orthophosphate incorporation into PSTV reached 10% of that incorporated into the 2 M LiCl-soluble cellular RNA. (+)PSTV and its complementary form, i.e. (-)PSTV were identified after their electrophoretic separation on polyacrylamide and agarose gels by molecular hybridization. This analysis revealed the presence of six high molecular weight(-)PSTV species, which are possibly multimers of the unit length(+)PSTV molecule consisting of 359 nucleotides.

Continuous replication of potato spindle tuber viroid (PSTV) in permanent cell cultures of potato and tomato.

The continuous replication of potato spindle tuber viroid (PSTV) in callus cultures from PSTV-infected wild-type potato (Solanum demissum L.) and tomato (Lycopersicon peruvianum L. Mill) plants and in cell suspensions derived from potato protoplasts (Solanum tuberosum L.) inoculated in vitro is described. The persistence of PSTV replication in these cell lines through at least 14 subculture passages, which corresponds to a continuous replication over a period of more than one year, was demonstrated by infectivity assay and by polyacrylamide-gel electrophoresis of isolated nucleic acids. This continuous synthesis de novo of PSTV was substantiated by the incorporation of [3H]uridine and of [32P]orthophosphate into viroid RNA.

Response to chilling of tomato mesophyll protoplasts.

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.

Different regeneration potential of mesophyll protoplasts from cultivated and a wild species of tomato.

Mesophyll protoplasts were isolated from leaves of three cultivars of Lycopersicon esculentum (L.) Mill., namely "Hilda 72", "Rutgers" and "Rentita", and from the wild tomato species Lycopersicon peruvianum (L.) Mill. Protoplasts from L. peruvianum divided and grew actively in a liquid medium according to Zapata et al. (1977), whereas protoplasts from the tomato cultivars "Hilda 72" and "Rutgers" showed comparable rates for cell division only, when the content of major elements in this medium was reduced to one half of the original concentration and when mannitol as osmoticum was replaced by glucose. In "Rentita" protoplasts no cell division could be observed in about 15 different modifications of the five basic culture media tested. The morphogenetic potential of these tomato cells was assessed by comparing the root and shoot formation of protoplasts and of leaf explants. L. peruvianum exhibited the highest potential. Calli derived from protoplasts regenerated roots on Murastrige-Skoog agar containing 1 µM benzylaminopurine (BAP) plus 10 µM indole-3-acetic acid (IAA) and 0.1 µM BAP plus 1 µM IAA. Shoot formation occurred in the combinations of 10 µM BAP with 0.1, 1.0, and 10 µM IAA. Plantlets regenerated from the L. peruvianum calli could be grown in soil. No shoots or roots were regenerated from calli of "Hilda 72" and "Rutgers" protoplasts in all combinations of BAP and IAA tested in the range from 0.1 µM to 100 µM, thus indicating the rather low morphogenetic potential of these protoplasts as compared to protoplasts from L. peruvianum leaves.