Identification of Pistacia-associated flexivirus 1, a putative mycovirus of the family Gammaflexiviridae, in the mastic tree (Pistacia lentiscus) transcriptome.

In this study, we identified the genome sequence of the novel virus Pistacia-associated flexivirus 1 (PAFV1), a putative member of the mycovirus family Gammaflexiviridae (the order Tymovirales), via analysis of a transcriptome dataset for the mastic tree (Pistacia lentiscus, the family Anacardiaceae). PAFV1 was predicted to have three open reading frames (ORFs): ORF1, encoding a replicase (REP) with RNA-dependent RNA polymerase activity; ORF2, a movement protein (MP); and ORF3, a hypothetical protein. The PAFV1 REP sequence showed high similarity to those of three known members of the family Gammaflexiviridae i.e., Entoleuca gammaflexivirus 1 (EnFV1), Entoleuca gammaflexivirus 2 (EnFV2), and Botrytis virus F (BVF). A genome contig of the fungus Monosporascus cannonballus also contained a sequence of an endogenous virus similar to that of PAFV1. Sequence comparison and phylogenetic analysis indicated that PAFV1, EnFV1, and the endogenous virus of M. cannonballus formed a distinct subgroup (apart from EnFV2 and BVF), and may be the founding members of a novel genus in the family Gammaflexiviridae. Notably, MP sequences of PAFV1/EnFV1 showed similarity to the MP sequences of the mycovirus group called tobamo-like mycoviruses (an unassigned taxon), implying that genomic recombination occurred between members of the family Gammaflexiviridae and tobamo-like mycoviruses. Since PAFV1 is phylogenetically related to mycoviruses, PAFV1 may also be a mycovirus that infected a fungus associated with the mastic tree sample, which is evidenced by the presence of fungal ribosomal RNA sequences in the mastic tree transcriptome. Thus, the PAFV1 genome sequence may be useful in elucidating the genome evolution of Gammaflexiviridae and tobamo-like mycoviruses. Keywords: Pistacia-associated flexivirus 1; Gammaflexiviridae; mycovirus, mastic tree.

Identification of Trichosanthes associated rhabdovirus 1, a novel member of the genus Cytorhabdovirus of the family Rhabdoviridae, in the Trichosanthes kirilowii transcriptome.

The genome sequence of a novel RNA virus, Trichosanthes associated rhabdovirus 1 (TrARV1), was identified in a transcriptome dataset isolated from a root sample of Trichosanthes kirilowii, which is a flowering plant belonging to the family Cucurbitaceae. The fruits, seeds, and root tubers of T. kirilowii have been used clinically in traditional Chinese medicine. The TrARV1 genome sequence was predicted to have six open reading frames (ORFs) encoding five canonical structural proteins of the family Rhabdoviridae (N ORF for nucleocapsid, P ORF for phosphoprotein, M ORF for matrix protein, G ORF for glycoprotein, and L ORF for polymerase), and an accessory protein. Sequence comparisons and phylogenetic analyses based on L and N proteins confirmed that TrARV1 is a novel member of the genus Cytorhabdovirus of the family Rhabdoviridae. TrARV1 is most closely related to Wuhan insect virus 5 and persimmon virus A. The putative cis-regulatory elements involved in transcription termination and polyadenylation, commonly found in the gene junction regions of rhabdoviruses, were also identified in the TrARV1 genome having the consensus sequence 3'- ACUAAAUUAUUUUGAUCUUU-5'. The genome sequence of TrARV1 may be useful to study the evolution and molecular biology of cytorhabdoviruses. Keywords: Trichosanthes associated rhabdovirus 1; Cytorhabdovirus; Rhabdoviridae; Trichosanthes kirilowii.

Zostera virus T - a novel virus of the genus Tepovirus identified in the eelgrass, Zostera muelleri.

Analysis of a transcriptome dataset obtained from tissue samples of the eelgrass Zostera muelleri, an aquatic flowering plant species of the family Zosteraceae, yielded three genome sequence contigs of a novel RNA virus. Sequence comparison and phylogenetic analysis revealed that the novel RNA virus, named Zostera virus T (ZoVT), belongs to the genus Tepovirus of the family Betaflexiviridae. The three genome contigs of ZoVT showed 88.2‒97.2% nucleotide sequence identity to each other, indicating that they descended from a common ancestor. The ZoVT genome contains three open reading frames (ORFs): ORF1 encodes a 1816 amino acid (aa) replicase (REP) with RNA-dependent RNA polymerase (RdRp) activity; ORF2, a 398 aa movement protein (MP); and ORF3, a 240 aa coat protein (CP). The phylogenetic analysis using REP sequences of ZoVT and other Betaflexiviridae viruses showed that Prunus virus T is the closest known virus to ZoVT, whereas potato virus T, the type species of the genus Tepovirus, is the second closest virus. Genome sequences of ZoVT, which is the third tepovirus species identified to date, may be useful for investigating the evolution and molecular biology of tepoviruses. Keywords: Zostera virus T; Tepovirus; Betaflexiviridae; eelgrass; Zostera muelleri.

Identification of a novel member of the family Betaflexiviridae from the hallucinogenic plant Salvia divinorum.

Betaflexiviridae is a family of plant-infecting RNA viruses with 11 recognized genera, of which genomes have diverse organization with three to six open reading frames (ORFs). A genome sequence of a novel Betaflexiviridae species, named Salvia divinorum RNA virus 1 (SdRV1), was identified in Salvia divinorum, herbal mint plant with hallucinogenic properties. The SdRV1 genome was predicted to have four ORFs encoding a replicase polyprotein (REP), a movement protein (MP), a coat protein (CP), and a putative nucleic acid-binding protein (NBP). Phylogenetic analyses based on the REP, MP, and CP sequences indicated that SdRV1 is most closely related to members of the genus Citrivirus. However, the genome organization of SdRV1 is the same as that of the genus Prunevirus. Moreover, the SdRV1 NBP had greatest sequence similarity with members of the genus Carlavirus. A complex evolutionary history involving ancestors of these three genera might have resulted in the unique phylogenetic position of SdRV1, which could be considered the founding member of a new genus in the family Betaflexiviridae. The genome sequence of SdRV1 might be useful for studies on the evolution of Betaflexiviridae. Keywords: Salvia divinorum RNA virus 1; Betaflexiviridae; Salvia divinorum.

Novel Divavirus (the family Betaflexiviridae) and Mitovirus (the family Narnaviridae) species identified in basil (Ocimum basilicum).

Transcriptome data obtained from a plant sample often contain a large number of reads that are derived from associated RNA virus genomes that were co-isolated during RNA preparation. These virus-derived reads can be assembled into a novel plant RNA genome sequence. Here, a basil (Ocimum basilicum) transcriptome dataset was analyzed to identify two new RNA viruses, which were named Ocimum basilicum RNA virus 1 (ObRV1) and Ocimum basilicum RNA virus 2 (ObRV2). A phylogenetic analysis of the ObRV1 RNA-dependent RNA polymerase (RdRp) motif indicated that ObRV1 is a novel species of the genus Divavirus of the family Betaflexiviridae. ObRV1 is the fourth divavirus species to be identified. The ObRV2 RdRp motif showed sequence similarity to viruses of the genus Mitovirus of the family Narnaviridae, which infect fungal mitochondria. Although most of the known mitoviruses do not produce a functional RdRp using the plant mitochondrial genetic code, the ObRV2 encodes a full-length RdRp using both the fungal and plant mitochondrial genetic codes.

Identification of a novel plant amalgavirus (Amalgavirus, Amalgaviridae) genome sequence in Cistus incanus.

Amalgaviridae is a family of double-stranded, monosegmented RNA viruses that are associated with plants, fungi, microsporidians, and animals. A sequence contig derived from the transcriptome of a eudicot, Cistus incanus (the family Cistaceae; commonly known as hoary rockrose), was identified as the genome sequence of a novel plant RNA virus and named Cistus incanus RNA virus 1 (CiRV1). Sequence comparison and phylogenetic analysis indicated that CiRV1 is a novel species of the genus Amalgavirus in the family Amalgaviridae. The CiRV1 genome contig has two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. An ORF1+2 fusion protein, which functions in viral RNA replication, is produced by a +1 programmed ribosomal frameshifting (PRF) mechanism. A +1 PRF motif UUU_CGU, which matches the conserved amalgavirus +1 PRF consensus sequence UUU_CGN, was found at the boundary of CiRV1 ORF1 and ORF2. Comparison of 25 amalgavirus ORF1+2 fusion proteins revealed that only three different positions within a 13-amino acid segment were recurrently used at the boundary, possibly being selected so as not to interfere with correct folding and function of the fusion protein. CiRV1 is the first virus found to be associated with the Cistus species and may be useful for studying amalgaviruses.

Lambda exonuclease-based subtractive hybridization approach to isolate differentially expressed genes from leaf cultures of Paulownia kawakamii.

Genes that are preferentially expressed in a particular developmental pathway can be isolated by subtractive hybridization (SH). We developed a PCR-based approach coupled with lambda exonuclease digestion that allows for generating single-stranded tester and driver nucleic acids suitable for SH starting from cDNA libraries. An efficient subtraction strategy was developed to overcome some of the problems in the previously described SH protocols, such as the need for large amounts of experimental tissue, RNase contamination during solution hybridization, and postsubtraction recovery of nucleic acids. We used this method to obtain cDNA corresponding to genes expressed during adventitious shoot regeneration from excised leaf cultures of the fast-growing tree Paulownia kawakamii. Over 36 cDNA clones were isolated and 1 of the differentially expressed clones codes for a leucine zipper transcription factor. This clone showed about sixfold higher level of expression in the shoot-forming tissues (tester) compared to that in the callus-forming tissues (driver) of Paulownia, suggesting that differentially expressed genes can be efficiently isolated using this simple lambda exonuclease-based subtractive hybridization method.

The expression of Brostm, a KNOTTED1-like gene, marks the cell type and timing of in vitro shoot induction in Brassica oleracea.

We studied the early events of de novo formation of adventitious shoot meristems in stem segments of Brassica oleracea. A regeneration system was used that is efficient, rapid, highly responsive to cytokinins and does not involve callus formation, thus allowing studies on a direct developmental switch of cells in the stem segment to form adventitious shoot meristem cells. Shoot meristem cells and dividing cells were marked from very early stages using in situ hybridization studies with Brostm, a Brassica homologue of the Arabidopsis SHOOTMERISTEMLESS (STM) gene, and a cyclin box-derived probe, Brocyc, respectively. We show that the process of developmental switching starts before any cell division occurs in the stem explants. This switching occurs synchronously both longitudinally and transversely in the explant, in groups of 5-7 phloem parenchyma cells subtending vascular bundles in the explant. Brostm is induced specifically in response to a cytokinin, benzyladenine, within 4 h of treatment and the transcripts persist during cell proliferation leading to shoot differentiation. We also show that during adventitious shoot formation, cells expressing Brostm are distinct from those expressing Brocyc. Lastly, our data suggest that, although developmental switching is initiated synchronously within 4 h of treatment, it requires 8 h of treatment for the establishment of organogenic determinance. The latter process is aynchronous, implying that additional factors formed later than Brostm are required to achieve maximal levels of determined cell populations to form adventitious shoots in vitro.

DOH1, a class 1 knox gene, is required for maintenance of the basic plant architecture and floral transition in orchid.

We report here the isolation and identification of an orchid homeobox gene, DOH1, from Dendrobium Madame Thong-In. Analyses of its sequence and genomic organization suggest that DOH1 may be the only class 1 knox gene in the genome. DOH1 mRNA accumulates in meristem-rich tissues, and its expression is greatly downregulated during floral transition. In situ hybridization analysis demonstrates that DOH1 is also expressed in the incipient leaf primordia and is later detected in the same region of the inflorescence apex, as in DOMADS1. Overexpression of DOH1 in orchid plants completely suppresses shoot organization and development. Transgenic orchid plants expressing antisense mRNA for DOH1 show multiple shoot apical meristem (SAM) formations and early flowering. In addition, both the sense and antisense transformants exhibit defects in leaf development. These findings suggest that DOH1 plays a key role in maintaining the basic plant architecture of orchid through control of the formation and development of the SAM and shoot structure. Investigations of DOMADS1 expression in the SAM during floral transition reveal that the precocious flowering phenotype exhibited by DOH1 antisense transformants is coupled with the early onset of DOMADS1 expression. This fact, together with the reciprocal expression of DOH1 and DOMADS1 during floral transition, indicates that downregulation of DOH1 in the SAM is required for floral transition in orchid and that DOH1 is a possible upstream regulator of DOMADS1.

Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition.

Gene expressions associated with in vitro floral transition in an orchid hybrid (Dendrobium grex Madame Thong-In) were investigated by differential display. One clone, orchid transitional growth related gene 7 (otg7), encoding a new MADS-box gene, was identified to be specifically expressed in the transitional shoot apical meristem (TSAM). Using this clone as a probe, three orchid MADS-box genes, DOMADS1, DOMADS2, and DOMADS3, were subsequently isolated from the TSAM cDNA library. Phylogenetic analyses show that DOMADS1 and DOMADS2 are new members of the AGL2 subfamily and SQUA subfamily, respectively. DOMADS3 contains the signature amino acids as with the members in the independent OSMADS1 subfamily separated from the AGL2 subfamily. All three of the DOMADS genes were expressed in the TSAM during floral transition and later in mature flowers. DOMADS1 RNA was uniformly expressed in both of the inflorescence meristem and the floral primordium and later localized in all of the floral organs. DOMADS2 showed a novel expression pattern that has not been previously characterized for any other MADS-box genes. DOMADS2 transcript was expressed early in the 6-week-old vegetative shoot apical meristem in which the obvious morphological change to floral development had yet to occur. It was expressed throughout the process of floral transition and later in the columns of mature flowers. The onset of DOMADS3 transcription was in the early TSAM at the stage before the differentiation of the first flower primordium. Later, DOMADS3 transcript was only detectable in the pedicel tissues. Our results suggest that the DOMADS genes play important roles in the process of floral transition.

Differential gene expression during floral transition in an orchid hybrid Dendrobium Madame Thong-In.

We have developed an in vitro orchid (Dendrobium grex Madame Thong-In) system as a model for investigating gene expression during floral transition. Total RNA was isolated from vegetative shoot apical meristems (VSAM) and transitional shoot apical meristems (TSAM). Using the mRNA differential display method, we identified 53 cDNA clones differentially expressed in VSAMs and 16 cDNA fragments specifically expressed in TSAMs. Northern blot analysis confirmed that, on the basis of their transcripts, 12 cDNAs decreased and 8 cDNAs increased during floral transition. Sequence analysis suggested that 5 clones are likely to be transcription factors, including one MADS-box gene of the AGL2 subfamily, one class 1 knox gene and one homolog of the Drosophila seven-up gene. Other clones showed homology with those coding for Arabidopsis SOD copper chaperone, CKI and the tobacco 21D7 protein.

Synergism in replication of cymbidium mosaic potexvirus (CymMV) and odontoglossum ringspot tobamovirus (ORSV) RNA in orchid protoplasts.

An in vitro orchid protoplast isolation method to study replication kinetics of CymMV and ORSV was developed. This method allows the isolation of viable and raphid-free petal protoplasts from an orchid hybrid, Dendrobium Sonia (Dendrobium Caesar x Dendrobium Tomie Drake). The optimum field strength for both viral RNA to achieve good efficiency of electroporation was 750 V/cm and the optimum viral RNA concentration required was 1 microgram and 4 micrograms per 2 x 10(6) protoplasts for CymMV and ORSV, respectively. Autoradiographs of Northern blots depicting the viral genomic and subgenomic RNA in the extracts, referred to as the "Replication Footprint Profiles" (RFP) of specific CymMV/ORSV virus were prepared at different time intervals. Viral RNA synthesis reached a maximum at 18 h for CymMV and 24 h for ORSV. When CymMV and ORSV viral RNA were electroporated into the protoplasts simultaneously, detection signals of both the positive and negative strand viral RNA increased as compared to the singly infected protoplasts. Thus, synergism in replication of CymMV and ORSV was observed in orchid protoplasts.

Influence of petiole and lamina on adventitious shoot initiation from leaf explants of Paulownia fortunei.

Adventitious shoot bud differentiation occurred preferentially from the petiolar cut ends of leaf explants of Paulownia fortunei cultured on Murashige and Skoog medium supplemented with 4 µMα-naphthaleneacetic acid and 20 µM benzyladenine. The details of plantlet regeneration and successful transplantation to soil have been reported earlier. We now show that besides medium supplementation with auxin and cytokinin, the presence of lamina and petiole in the explant influence shoot bud induction. Explants with the basal half of the lamina and the entire petiole were much more responsive than those with whole lamina and petiole. A dual-culture-medium technique which permitted incubation of the two ends of excised petioles under two different phytohormone regimes was devised. Our data suggest that some of the diffusible factors from the lamina may be phytohormones, and that the establishment of an endogenous phytohormone gradient in the explants may affect shoot bud differentiation in this culture system.

An efficient in vitro method for mass propagation of a woody ornamentalIxora coccinea L.

Various factors that affect culture establishment, shoot growth, proliferation and rooting ofIxora coccinea L., a woody shrub, were studied. Stem cuttings (decapitated shoot, three nodes) were the most suitable explants for multiple-shoot proliferation, and when cultured on a woody plant medium (WPM) containing 2.5 µM BA produced axillary shoots which branched repeatedly, yielding an average of 27 shoots per explant after 6 weeks in culture. Kinetin, 2-iP, zeatin and thidiazuron all induced multiple-shoot formation, but were less effective than BA. While the presence of IAA in the multiplication medium was detrimental to shoot proliferation, shoot growth was not affected by IAA. The production of large amounts of basal callus and vitrification of shoots were the major problems to be avoided in proliferating shoot cultures. Addition of TIBA to the multiplication medium markedly reduced basal callusing, while sealing the culture vessels with a fluorocarbon polymer (tetrafluoroethyleneperfluoroalkyl vinyl ether) film (Neoflon PFA film) almost completely eliminated vitrification. A reduction in the number of vitrified shoots was also achieved with AVG treatment. Following this protocol of using BA-supplemented WPM and Neoflon film, it would be possible to produce more than 100,000 plants from a single stem cutting in 1 year.

The complete sequence of a Singapore isolate of odontoglossum ringspot virus and comparison with other tobamoviruses.

The complete sequence of a Singapore isolate of odontoglossum ringspot virus (ORSV-S1) comprises 6609 nucleotides (nt) and four open reading frames (ORFs 1 to 4). The 126/183-kDa RNA-dependent RNA polymerase (RdRp), 33-kDa movement protein (MP) and 18-kDa coat protein (CP) cistrons are located at nt 63-3401/4901, 4807-5718, and 5721-6197 on the genome, respectively. The 5' UTR contains three copies of an 8-base direct repeat and (CAA)n motifs. Characteristic tRNA-like structure and three consecutive homologous regions were present in the 3' UTR. The genomic RNA and MP of ORSV-S1 are one of the longest among all members of the TOV group. Phylogenetic analysis of all four genes indicates evolutionary divergence, but within each gene there are some degrees of evolutionary convergence. The conserved amino acid sequences in the MP can be used for the classification of tobamoviruses.

High frequency adventitious shoot regeneration from excised leaves ofPaulownia spp. cultured in vitro.

High frequency, direct regeneration of shoots was induced in leaf cultures ofPaulownia tomentosa, P. fortunei x P. tomentosa andP. kawakamii. The optimum culture medium for the leaf explants derived from shoot cultures was Murashige-Skoog (MS) medium supplemented with 10 µM indole-3-acetic acid and 50 µM benzyladenine. Up to 40 shoots were obtained over a 4 month culture period from each leaf explant. Rooting occurred spontaneously in the shoots that were about 1 cm tall when subcultured on phytohormone-free MS medium. The plantlets could be transplanted successfully. Some of the transplantedP. tomentosa plantlets flowered in the greenhouse one year after transplanting. The protocol is suitable not only for rapid multiplication of the various species ofPaulownia, but also for analytical studies associated with adventitious shoot regeneration.

Delineation of an immunodominant and human T-cell lymphotropic virus (HTLV)-specific epitope within the HTLV-I transmembrane glycoprotein.

Antibody reactivity to the transmembrane region of human T-cell lymphotropic virus type I (HTLV-I) envelope, gp21, is observed in virtually all individuals infected with HTLV-I or HTLV-II. Recombinant proteins encoding selected portions of gp21 are described and used to define two immunogenic regions. The first epitope (designated GD21-I) contains amino acids 361 to 404 of the HTLV-I envelope and reacted with all of 54 sera from HTLV-I- and HTLV-II-infected individuals. The second epitope (designated BA21) expresses amino acids 397 to 430 of the HTLV-I envelope and was recognized by 33 of 54 HTLV antisera. To determine the specificity of GD21-I and BA21, sera from 17 HTLV-negative individuals with nonspecific reactivity to p21E were tested. None of these sera reacted with GD21-I, but 16 of 17 sera reacted with BA21. With virtually complete reactivity to sera from HTLV-infected individuals and no reactivity to sera from p21E-reactive uninfected individuals, GD21-I will be useful in immunoassays for the detection of HTLV infection.