Improved recovery of cryotherapy-treated shoot tips following thermotherapy of in vitro-grown stock shoots of raspberry (Rubus idaeus L.).

Raspberry bushy dwarf virus (RBDV) can be efficiently eradicated from raspberry plants (Rubus idaeus) by a procedure combining thermotherapy and cryotherapy. However, the bottleneck of this procedure is that, following thermotherapy, cryopreserved shoot tips become chlorotic during regrowth and eventually die after several subcultures. In addition, survival of heat-treated stock shoots and recovery of cryopreserved shoot tips following thermotherapy are low. The present study focused towards improving regrowth of cryopreserved raspberry shoot tips following thermotherapy. Results showed that preconditioning stock shoots with salicylic acid (SA; 0.01-0.1 mM) markedly increased survival of stock shoots after 4 weeks of thermotherapy. Regrowth of cryopreserved shoot tips following thermotherapy was also significantly enhanced when SA (0.05-0.1 mM) was used for preconditioning stock shoots. Addition of either Fe-ethylenediaminetetracetic acid (Fe-EDTA, 50 mg per L) or Fe-ethylenediaminedi(o)hydroxyphenylacetic acid (Fe-EDDHA, 50 mg per L) to post-culture medium strongly promoted regrowth and totally prevented chlorosis of shoots regenerated from cryopreserved shoot tips following thermotherapy. Using the parameters optimized in the present study, about 80 percent survival of heat-treated stock shoots and about 33 percent regrowth of cryopreserved shoot tips following thermotherapy were obtained. Morphology of plants regenerated from cryopreserved shoot tips following thermotherapy was identical to that of control plants, based on observations of leaf shape and size, internode length and plant height. Optimization of the thermotherapy procedure followed by cryotherapy will facilitate the wider application of this technique to eliminate viruses which can invade meristems.

RT-PCR-RFLP for genetic diversity analysis of Tunisian Grapevine fanleaf virus isolates in their natural host plants.

Genetic diversity was characterized in 20 isolates of Grapevine fanleaf virus (GFLV) recovered from naturally infected grapevine plants (Vitis vinifera) in the North of Tunisia. Viral RNAs were isolated by oligoprobe capture, and a 605 bp fragment containing a part of the viral coat protein gene was amplified by RT-PCR. Sequence variation among isolates was characterized by restriction fragment length polymorphism (RFLP) analysis and confirmed by sequencing. The GFLV infections are found as a complex mixture of closely related genomes. In further studies, RFLP analyses of virus isolates using AluI showed that GFLV populations in Tunisian vineyards consist of two restrictotypes corresponding to distinct sub-populations Sp1 and Sp2. The relative field distribution of these sub-populations showed that Sp2 was more abundant. Individual genomes were recovered by cloning the RT-PCR products. The sequences were found to vary from each other by as much as 11%. Cloning from mixed infections showed that Sp2 are also predominant.

Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes.

Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear "viral compartment." We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

Simultaneous RT/PCR detection and differentiation of arabis mosaic and grapevine fanleaf nepoviruses in grapevines with a single pair of primers.

The movement protein genes from several isolates of ArMV and GFLV of different geographical origins were amplified by RT/PCR using degenerate primers, cloned and sequenced. A single pair of degenerate primers was designed from these sequences to allow the simultaneous amplification of parts of the movement protein genes of ArMV and GFLV. Their use in an immunocapture-RT/PCR for the detection of ArMV or GFLV in infected grapevines proved to be ten times more sensitive than the corresponding ArMV or GFLV ELISA tests. A Sph1 restriction site found in the sequences corresponding to the amplified products from the GFLV isolates, but not in the amplified products from the ArMV isolates, allowed the differentiation between ArMV and GFLV in the infected grapevines by a Sph1 restriction digestion of the amplified products.

Complete nucleotide sequence and genome organization of Grapevine fleck virus.

The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3'-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5' and 3' ends, respectively. ORF 1 potentially encodes a 215.4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24.3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3' end of the viral genome and encode proline-rich proteins of 31.4 kDa (p31) and 15.9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus.

Quasispecies nature of the genome of Grapevine fanleaf virus.

Genetic diversity was characterized in 14 isolates of Grapevine fanleaf virus (GFLV) recovered from grapevine (Vitis vinifera). Virions were collected by immunocapture, and a 1557 bp fragment containing part of the viral coat protein gene and part of the untranslated region to its 3' side was amplified by RT-PCR. Sequence variation among isolates was characterized by restriction fragment length polymorphism (RFLP) analysis and by sequencing. The AvaII-generated RFLP patterns from the various isolates were highly variable. The isolates were passaged in Chenopodium quinoa. The RFLP patterns altered with passage through the alternate host, but the variation stabilized after a number of passages. Individual genomes were recovered by cloning. The subcloned sequences were found to vary from each other by as much as 13%, and the encoded amino acid sequences by as much as 9%. The data suggest that the GFLV genome consists of quasispecies populations.

Complete nucleotide sequences of the RNAs 2 of German isolates of grapevine fanleaf and Arabis mosaic nepoviruses.

The RNAs 2 of an Arabis mosaic virus (ArMV) and a grapevine fanleaf virus (GFLV) isolate, originating from South West of Germany near Neustadt an der Weinstrasse (NW), were sequenced. They are 3820 and 3775 nucleotides long respectively, and both contain one open reading frame encoding a polypeptide of 1110 amino acids. Their 5' non-coding regions contain conserved and repeated sequences, which are able to form stem-loop structures. Nucleotide sequence comparisons between the full-length RNAs 2 revealed homology levels of 84 and 82% between the ArMV-NW and the ArMV-L and -U, respectively, 90% between GFLV-NW and GFLV-F13, and 72% between ArMV-NW and GFLV-NW. Amino acid sequence comparisons showed that the greatest difference was found between the 2A proteins of the different ArMV isolates, the 2A protein of the ArMV-NW showing more similarity to the 2A protein of GFLV-NW than to those of ArMV-L2 or -U2.

Apricot latent virus: a new species in the genus Foveavirus.

Extraction of viral double-stranded RNA from peach leaves infected with Apricot latent virus (ALV) followed by molecular cloning of synthesized cDNA and its sequencing, suggested that ALV is a new virus, whose coat protein (CP) coding region contains Apple stem pitting virus (ASPV)-related sequences. The sequenced portion of the ALV genome (1,444 nt) includes the putative CP gene and the 3' non-translated region. The 5' portion of this fragment (1-651 nt) is highly distinct whereas the 3' portion is 77% identical to the corresponding region of ASPV. Molecular hybridization experiments using a cRNA probe to ASPV with ALV-infected leaf tissue extracts also revealed that the genome of ALV contains nucleotide sequences related to that of ASPV. Western blots of tissue extracts indicated that ALV coat protein reacted with polyclonal antiserum against ASPV; however, the ALV CP differs in size from that of ASPV. ALV was graft-transmitted to several Prunus rootstocks. Based on the available sequence data, serological observations and bioassays we propose that ALV is a new species in the genus Foveavirus, typified by ASPV. ALV-specific PCR-primers and viral-specific cRNA probes developed in this investigation may be useful for detecting the virus and for studying its epidemiology and geographical distribution.

Immunodetection and subcellular localization of the proteins encoded by ORF 3 of grapevine viruses A and B.

Polyclonal antisera were raised to Escherichia coli-expressed ORF3 products (putative movement proteins) of Grapevine virus A (GVA) and Grapevine virus B (GVB) (genus Vitivirus), and used for their immunodetection in infected plants. Western blot analysis of subfractionated cellular compartments showed that the distribution of both proteins was comparable to that of plant virus movement proteins, as they were transiently present in a crude membrane fraction and accumulated in a cell wall-enriched fraction. The GVA ORF3-encoded protein, but not the comparable GVB protein, was also present in large amount in a cytoplasmic soluble fraction. Intracellular immunogold labelling localized these proteins in the cell wall and plasmodesmata of infected cells and, especially for GVA, in association with cytoplasmic virus aggregates.

Improved RNA extraction and one-tube RT-PCR assay for simultaneous detection of control plant RNA plus several viruses in plant extracts.

A procedure was developed for simultaneous detection of plant RNA viruses and of plant RNA, as a control. RT-PCR amplification with primers designed for the detection of the plant mRNAs encoding malate dehydrogenase (MDH) and the large subunit of ribulose bisphosphate carboxylase oxygenase (RubiscoL) was used for the development of a plant extraction procedure that consistently yields extracts that can be amplified. The control amplification was used successfully on extracts from cane, leaf and/or bud tissues from grapevine, apple, raspberry, strawberry, peach, apricot, plum and wheat. Multiplex RT-PCR conditions were established for the simultaneous detection in grapevine extracts of either arabis mosaic virus, rupestris stem pitting associated virus and malate dehydrogenase mRNA, or grapevine virus A, grapevine virus B, grapevine leafroll associated virus-3, and RubiscoL mRNA.

Intracellular distribution, cell-to-cell trafficking and tubule-inducing activity of the 50 kDa movement protein of Apple chlorotic leaf spot virus fused to green fluorescent protein.

The 50 kDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescent protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis and tubule formation on the surface of protoplasts were analysed. The 50KP-GFP fluorescence was distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from the cells that produced it into neighbouring cells in both young and mature leaves and targetted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from the initial cells that produced it than when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. 50KP-GFP was able to complement local spread of 50KP-deficient virus when expressed transiently in leaf epidermis of C. quinoa. Expression of 50KP-GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal region (aa 287-457) was not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus or tubule formation on protoplasts. In contrast, deletions in the N-terminal region resulted in the complete disruption of all these activities.

Grapevine fleck virus-like viruses in Vitis.

Two sets of degenerate primers for the specific amplification of 572-575 nt and 386 nt segments of the methyltransferase and RNA- dependent RNA polymerase cistrons of members of the genera Tymovirus and Marafivirus and of the unassigned virus Grapevine fleck virus (GFkV) were designed on the basis of available sequences. These primers were used for amplifying and subsequent cloning and sequencing part of the open reading frame 1 of the genome of GFkV, Grapevine asteroid mosaic-associated virus (GAMaV) and of another previously unreported virus, for which the name Grapevine red globe virus (GRGV) is proposed. Computer-assisted analysis of the amplified genome portions showed that the three grapevine viruses are phylogenetically related with one another and with sequenced tymoviruses and marafiviruses. The relationships with tymoviruses was confirmed by the type of ultrastructural modifications induced in the host cells. RdRp-specific degenerate primers were successfully used for the aspecific detection of the three viruses in crude grapevine sap extracts. Specific virus identification was obtained with RT-PCR using antisense virus-specific primers.

Sequencing of peach latent mosaic viroid variants from nine North American peach cultivars shows that this RNA folds into a complex secondary structure.

We sequenced 34 new peach latent mosaic viroid (PLMVd) variants isolated from nine different peach cultivars. This study provides the widest view of PLMVd diversity reported to date and includes the original characterization of North American variants, which cannot be differentiated from European sequences. PLMVd appears as a species in which each isolate is a complex mixture of RNAs. Analysis of base-pair covariations supports the hypothesis that PLMVd folds into a complex branched structure with the potential of including three new pseudoknots. The resulting "globular-like" structure is in contrast to the rod-like one adopted by most other viroids.

Partial sequence identification of grapevine-leafroll-associated virus-1 and development of a highly sensitive IC-RT-PCR detection method.

Using immunocapture reverse transcription PCR (IC-RT-PCR) a specific PCR product from GLRaV-1 infected vine samples was amplified with the help of degenerate primers deduced from the conserved HSP70 region of closteroviruses. 511 basepairs of the 5'end of GLRaV-1 HSP70 gene were identified. Within this region, putative GLRaV-1 specific primers were designed and an IC-RT-PCR detection procedure was developed which is about 125 times more sensitive than the established ELISA method. No PCR product was amplified in GLRaV-2,-3 and -4 infected plants which indicates the specificity of the primers. This procedure may serve as an alternative method for GLRaV-1 detection where the sensitivity of ELISA is insufficient.

Nucleotide sequence and organization of ten open reading frames in the genome of grapevine leafroll-associated virus 1 and identification of three subgenomic RNAs.

The genome of Grapevine leafroll-associated virus 1 (GLRaV-1) was cloned and the sequence of 12394 nts determined. It contains 10 major open reading frames (ORFs) and a 3'-non-coding region lacking a poly(A) tract. The first ORF (ORF 1a) encodes a putative RNA helicase at the C-terminal portion of an apparently larger protein. The downstream ORF, 1b, overlaps ORF 1a and lacks an initiation codon. This ORF encodes an RNA-dependent RNA polymerase of M(r) 59276. ORF 2 encodes a small hydrophobic protein of M(r) 6736, and ORF 3 encodes a homologue of the HSP70 family of heat shock proteins and has an M(r) of 59500. ORF 4 encodes a protein with an M(r) of 54648 that shows similarity to the corresponding proteins of other closteroviruses. ORF 5 encodes the viral coat protein (CP) with an M(r) of 35416. The identity of this ORF as the CP gene was confirmed by expression in Escherichia coli and testing with the viral antibody. ORFs 6 and 7 code for two CP-related products with M(r) of 55805 and 50164, respectively. ORFs 8 and 9 encode proteins of M(r) 21558 and 23771 with unknown functions. Using DNA probes to different regions of the GLRaV-1 sequence, three major 3'-coterminal subgenomic RNA species were identified and mapped on the GLRaV-1 genome. Phylogenetic analyses of the individual genes of GLRaV-1 demonstrated a closer relationship between GLRaV-1 and GLRaV-3 than with other closteroviruses.

Nucleotide sequence and genome organization of apple latent spherical virus: a new virus classified into the family Comoviridae.

A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1.41 and 1.43 g/cm(3) in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3' poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa ('23K'; ORF1) and 235 kDa ('235K'; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa ('108K'). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the COMOVIRIDAE: The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages.

The complete nucleotide sequence of apple mosaic virus (ApMV) RNA 1 and RNA 2: ApMV is more closely related to alfalfa mosaic virus than to other ilarviruses.

The complete nucleotide sequences of apple mosaic virus RNA 1 and 2 have been characterized. Apple mosaic virus RNA 1 is 3476 nucleotides in length and encodes a single large open reading frame (ORF), whereas apple mosaic virus RNA 2 is 2979 nucleotides in length and also encodes a single ORF. The amino acid sequences encoded by RNA 1 and 2 show similarity to all of the other ilarviruses for which sequence data are available, but both are more closely related to alfalfa mosaic virus (AMV) than to other ilarviruses. Points of similarity include the absence of ORF 2b, present on the RNA 2 of all previously characterized ilarviruses. The close relationship to AMV also occurs in the movement protein, encoded by RNA 3, but not with the coat protein. These data suggest that the present taxonomy should be revised, and that AMV should be considered an aphid-transmissible ilarvirus.

A strategy for rapid cDNA cloning from double-stranded RNA templates isolated from plants infected with RNA viruses by using Taq DNA polymerase.

A fast and efficient cDNA cloning procedure for plant RNA viruses was developed. In this procedure, double-stranded RNA (dsRNA) was used as a template source. Standard cDNA synthesis reagents and random hexamers were then used for making cDNAs. Taq DNA polymerase was used to add additional (A) at the ends of cDNAs, a TA cloning kit to ligate the cDNAs to vectors, and an electroporator to transform the DNAs to E. coli cells. dsRNAs were extracted from grapevine tissues infected with four different viruses and used for cloning. These viruses included grapevine rupestris stem pitting associated virus, grapevine leafroll associated virus 5, and two uncharacterized grapevine viruses, one each closely related to marafivirus and vitivirus groups. Selected cDNA clones were sequenced and PCR primers were developed for RT-PCR detection of these viruses in host plants.

A genetic map of cucumber composed of RAPDs, RFLPs, AFLPs, and loci conditioning resistance to papaya ringspot and zucchini yellow mosaic viruses.

The watermelon strain of papaya ringspot virus (PRSV-W) and zucchini yellow mosaic virus (ZYMV) are potyviruses that cause significant disease losses in cucumber. Resistances have been identified primarily in exotic germplasm that require transfer to elite cultivated backgrounds. To select more efficiently for virus resistances, we identified molecular markers tightly linked to PRSV-W and ZYMV resistances in cucumber. We generated F6 recombinant inbred lines (RILs) from a cross between Cucumis sativus L. 'Straight 8' and a line from 'Taichung Mou Gua', TMG1 (susceptible and resistant, respectively, to both viruses), and studied the segregations of amplified fragment length polymorphism (AFLP) markers, randomly amplified polymorphic DNAs (RAPDs), restriction fragment length polymorphisms (RFLPs), and resistances to PRSV-W and ZYMV. A 353-point map of cucumber was generated, delineating 12 linkage groups at LOD 3.5. Linkage arrangements among RFLPs were consistent with previously published maps; however linkages among RAPDs in our map did not agree with a previously published map. Resistances to PRSV-W and ZYMV were tightly linked (2.2 cM) and mapped to the end of one linkage group. One AFLP cosegregated with resistance to ZYMV.

Localization of Plum pox virus in stem and petiole tissues of apricot cultivars by immuno-tissue printing.

Localization of Plum pox virus (PPV) in stem and petiole tissues of four susceptible and four resistant apricot cultivars has been studied. Consecutive 1-mm spaced transverse sections were taken from the tissues and were printed onto nitrocellulose membrane in duplicate. For virus-specific detection, one series of prints was probed with an antibody to PPV coat and the density of stains was evaluated by light microscopy. Another series of prints was treated with a substrate but not with the antibody to reveal non-specific staining due to endogenous peroxidases. The virus was currently detected in all inoculated susceptible cultivars but only in one inoculated resistant cultivar (Harcot). In the stem tissues, the virus was localized in the pith and in the xylem. In the petiole tissues, it was localized in the epidermis and in cortical and medullae parenchyma. Non-specific staining was observed only in the stem sclerenchyma and in the petiole phloem.