Deep sequencing evidence from single grapevine plants reveals a virome dominated by mycoviruses.

We have characterized the virome in single grapevines by 454 high-throughput sequencing of double-stranded RNA recovered from the vine stem. The analysis revealed a substantial set of sequences similar to those of fungal viruses. Twenty-six putative fungal virus groups were identified from a single plant source. These represented half of all known mycoviral families including the Chrysoviridae, Hypoviridae, Narnaviridae, Partitiviridae, and Totiviridae. Three of the mycoviruses were associated with Botrytis cinerea, a common fungal pathogen of grapes. Most of the rest appeared to be undescribed. The presence of viral sequences identified by BLAST analysis was confirmed by sequencing PCR products generated from the starting material using primers designed from the genomic sequences of putative mycoviruses. To further characterize these sequences as fungal viruses, fungi from the grapevine tissue were cultured and screened with the same PCR probes. Five of the mycoviruses identified in the total grapevine extract were identified again in extracts of the fungal cultures.

Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus.

In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using Life Sciences 454 high-throughput sequencing. 67.5 megabases of sequence data were derived from reverse-transcribed cDNA fragments, and screened for sequences of viral or viroid origin. The data revealed that a vine showing decline symptoms supported a mixed infection that included seven different RNA genomes. Fragments identified as derived from viruses or viroids spanned a approximately ten thousand fold range in relative prevalence, from 48,278 fragments derived from Rupestris stem pitting-associated virus to 4 fragments from Australian grapevine viroid. 1527 fragments were identified as derived from an unknown marafivirus. Its complete genome was sequenced and characterized, and an RT-PCR test was developed to analyze its field distribution and to demonstrate its presence in leafhoppers (vector for marafiviruses) collected from diseased vines. Initial surveys detected a limited presence of the virus in grape-growing regions of California.

Bermuda Grass as a Potential Reservoir Host for Grapevine fanleaf virus.

Grapevine fanleaf virus (GFLV) was detected in samples of Bermuda grass (BG) from Iran by reverse transcription-polymerase chain reaction (RT-PCR) using two different pairs of GFLV-specific primers, and also by enzyme-linked immunosorbent assay (ELISA) using antiserum specific for a North American isolate of the virus. RT-PCR detected GFLV in both fresh and dried BG tissues and in virus preparations purified from these plants. Cloning and sequencing of the RT-PCR products confirmed that the amplified sequences were sections of the GFLV coat protein gene. Similar results were obtained when random and oligo(dT) primers were used on viral RNA templates recovered from BG to synthesize cDNA for cloning and sequencing. The virus induced few or no symptoms in BG, but could nonetheless be transmitted from BG to Chenopodium quinoa by mechanical inoculation. Some isolates induced systemic chlorotic spots and leaf deformation; others remained symptomless in this plant. Both symptomatic and symptomless C. quinoa plants were found to be infected with GFLV, giving positive ELISA and RT-PCR tests. A North American isolate of GFLV was found to be mechanically transmissible to BG as indicated by positive RT-PCR results from root samples of inoculated plants. GFLV-infected BG was widely distributed in the Fars province of Iran.

The detection of fake-bad and fake-good responding on the Millon Clinical Multiaxial Inventory III.

The purpose of this study was to examine the effectiveness of the 3 Modifying Indices of the Millon Clinical Multiaxial Inventory III (MCMI-III) in the detection of fake-bad and fake-good responding. The sample consisted of 160 psychiatric outpatients. Paired t tests were performed to examine the effects of instructional set (faking vs. standard instructions). As hypothesized, instructional set produced significant differences on Scale X, Scale Y, and Scale Z in both fake-bad and fake-good analyses. Single-scale cutoff scores were as effective as multiple-scale cutoffs. The overall rates of successful classification indicated moderate effectiveness and utility of the MCMI-III Modifying Indices in the detection of dissimulated responding. When base rates were varied to more closely approximate a general clinical population, overall classification accuracy increased, but identification of faking (positive predictive power) gradually eroded with declining base-rate estimates. At lower base rates of faking, MCMI-III standard cutoff points yielded a high number of false positives.

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal.

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.

The regions of sequence variation in caulimovirus gene VI.

The sequence of gene VI from figwort mosaic virus (FMV) clone x4 was determined and compared with that previously published for FMV clone DxS. Both clones originated from the same virus isolation, but the virus used to clone DxS was propagated extensively in a host of a different family prior to cloning whereas that used to clone x4 was not. Differences in the amino acid sequence inferred from the DNA sequences occurred in two clusters. An N-terminal conserved region preceded two regions of variation separated by a central conserved region. Variation in cauliflower mosaic virus (CaMV) gene VI sequences, all of which were derived from virus isolates from hosts from one host family, was similar to that seen in the FMV comparison, though the extent of variation was less. Alignment of gene VI domains from FMV and CaMV revealed regions of amino acid sequence identical in both viruses within the conserved regions. The similarity in the pattern of conserved and variable domains of these two viruses suggests common host-interactive functions in caulimovirus gene VI homologues, and possibly an analogy between caulimoviruses and certain animal viruses in the influence of the host on sequence variability of viral genes.

Point mutations in cauliflower mosaic virus gene VI confer host-specific symptom changes.

Mutants of cauliflower mosaic virus (CaMV) strain D4 have been characterized with regard to host-specific phenotypes that resulted from specific changes in the viral DNA sequence. Both the mutant and the wild-type viruses infect a brassicaceous host, Brassica campestris, systemically, giving indistinguishable symptoms. However, in the solanaceous host Datura stramonium, which was systemically infectible by the wild-type virus, mutants induced necrotic local lesions at 21 degrees C and above, and a veinal necrosis at lower temperatures. The mutants differed from the parental D4 strain by having single base changes in gene VI. The necrotic phenotype could be selected during serial passage of D4 in B. campestris or created by site-directed mutagenesis within the gene VI coding region. Full-length 62-kDa gene VI gene products were detected in extracts of plants infected with the mutant strains, as were two smaller proteins derived from the same coding region. The relationship of the host-specific phenotypes of the mutants to the detected gene VI-encoded proteins is discussed in the context of the natural variation found in this gene.

Characteristics of a strong promoter from figwort mosaic virus: comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter.

A segment of DNA from the genome of figwort mosaic virus (FMV) strain M3 possesses promoter activity when tested in electroporated protoplasts from, and transgenic plants of, Nicotiana tabacum cv. Xanthi nc. The 1.1 kb DNA segment, designated the '34S' promoter, is derived from a position on the FMV genome comparable to the position on the cauliflower mosaic virus (CaMV) genome containing the 35S promoter. The 34S and 35S promoters show approximately 63% nucleotide homology in the TATA, CCACT, and -18 to +1 domains, but in sequences further upstream the homology drops below 50%. Promoter activities were estimated using beta-glucuronidase and neomycin phosphotransferase II reporter gene systems. The activity of the 34S promoter segment approximates that of the 35S promoter in both protoplast transient expression assays and in stably transformed tobacco plants. Truncation of 5' sequences from the 34S promoter indicates that promoter strength depends upon DNA sequences located several hundred nucleotides upstream from the TATA box. In leaf tissue the 34S promoter is 20-fold more active than the mannopine synthase (MAS) promoter from Agrobacterium tumefaciens T-DNA. The 34S promoter lacks the root-specific and wound-stimulated expression of the MAS promoter, showing relatively uniform root, stem, leaf, and floral activities.

Gene I products of cauliflower mosaic virus detected in extracts of infected tissue.

The product of cauliflower mosaic virus (CaMV) gene I has been characterized from extracts of infected plants. Two size classes of this protein can be identified by the use of specific antiserum. The antiserum was induced against a chimeric protein produced in E. coli from a gene fusion between a fragment of the CaMV genome and the beta-galactosidase gene. A 36-kDa form of the gene I product is associated with virus particles. A 45-kDa form of this product was found only in the insoluble fraction of tissue homogenates. This insoluble fraction, the only fraction found to contain the product of viral gene VI, contains the virally induced inclusion bodies.

Region VI of cauliflower mosaic virus encodes a host range determinant.

A domain of cauliflower mosaic virus (CaMV) which controls systemic spread in two solanaceous hosts (Datura stramonium and Nicotiana bigelovii) was mapped to the first half of open reading frame 6. Whereas ordinary strains of CaMV are unable to infect solanaceous species except to replicate locally in inoculated leaves, a new CaMV strain (D4) induces chlorotic local lesions and systemically infects both D. stramonium and N. bigelovii. To determine which portion of the CaMV genome controls systemic spread of the virus in solanaceous hosts, nine recombinant genomes constructed between D4 and two ordinary strains of the virus were tested for their ability to infect solanaceous hosts. A 496-base-pair DNA segment comprising the first half of open reading frame 6 specified the type of local lesions and systemic spread of the virus in solanaceous hosts. Exchange of this segment of the genome between strains of CaMV converted a compatible host reaction to an incompatible (hypersensitive) one in response to infection. This suggests that the gene VI protein interacts with the plant to suppress hypersensitivity, the normal response of solanaceous hosts to CaMV infection.

Expression of disease symptoms in cauliflower mosaic virus genomic hybrids.

In an effort to determine if particular regions of the cauliflower mosaic virus (CaMV) genome could be associated with particular phenotypic characters, strains of CaMV differing markedly in biological properties were recombined to produce hybrids. DNA from pairs of (infectious) genomic clones was cleaved with restriction endonucleases, then mixed and ligated. Recombinants were found by screening transformants in E. coli, or by selection in vivo for infectious hybrids. Recombinants in infected turnip plants were characterized by restriction endonuclease mapping of their DNA to confirm the hybrid genotype. New hybrid strains that induced less severe disease, or conversely, more severe disease than either parent were observed. The experiments revealed that typical disease expression, consisting of leaf chlorosis and mottling, mapped to a genome segment containing open reading frame VI (ORF VI) and the full-length promoter. This basic disease symptom was found to be influenced by other regions of the genome. Insect transmissibility mapped to ORF II. The ability to develop generalized infections in solanaceous plants was tested in hybrids between CaMV CM1841 and a variant that infects Datura stramonium systemically. In this case the systemic mobilization of virus appeared to be controlled by ORF VI, suggesting that this gene may function in cell-to-cell movement of virus.

Insertional mutagenesis of the cauliflower mosaic virus genome.

A series of small insertions has been introduced into the various translational reading frames of the DNA of a "severe" strain of cauliflower mosaic virus (CaMV). A selectable gene (the kanamycin phosphotransferase gene of Tn903), flanked by a series of symmetrically arranged cloning sites taken from M13mp7, was used to prepare the site-specific mutants. In-phase insertions of 12 or 30 bp, which introduced unique SalI sites into reading regions I, III, IV, V and into the amino-proximal portion of region VI, destroyed infectivity. Insertions in the amino-distal portion of region VI, in the large intergenic region, and in region II retained infectivity. The amino-distal insertions in region VI reduced the severity of symptoms in plants. The insertion in region II destroyed aphid transmissibility. Longer DNA segments when inserted into region II or into the amino-distal portion of region VI destroyed infectivity, but similar insertions in the intergenic region were without effect on virus infection or development.

Mapping of the coat protein gene of cauliflower mosaic virus by its expression in a prokaryotic system.

Polypeptides recognized by antisera to cauliflower mosaic virus (CaMV) coat protein were found in lysates of Escherichia coli cells that carry the complete CaMV genome on plasmid vectors. Synthesis of this cross-reacting material in E. coli was detected when the complete CaMV DNA sequence was present at either of two sites and in either orientation at one site of the cloning vector, suggesting prokaryotic promoter activity for some sequence within the viral DNA. Using an immunosorbent assay to screen subclones containing various segments of the CaMV genome, open reading region IV alone was found to be sufficient for synthesis of the antigen, thereby mapping the coat protein gene to this region.

Multimeric forms of satellite of tobacco ringspot virus RNA.

An approximately 350-nucleotide residue RNA replicates in association with tobacco ringspot virus (TobRV) and becomes encapsidated in TobRV coat protein. Here we show by electrophoretic analyses that this small satellite RNA, RNA S, is the most abundant and most rapidly migrating of a series of at least ten encapsidated RNAs with RNA S sequences. A largely double-stranded RNA fraction from infected tissue, when denatured, gave a similar series of up to 12 zones that contained both RNA S sequences and sequences that hybridized to RNA S. Analysis of the mobilities suggests a weight increment between each zone corresponding approximately to the size of RNA S. Thus the more slowly migrating zones appear to contain covalent multimers of RNA S or, for tissue RNA, both multimers of RNA S and multimers of the complement of RNA S sequences. Neither terminal structure of TobRV genomic RNAs was found in the satellite RNA. RNA S lacks detectable polyadenylate or oligoadenylate. Covalently linked protein was not detected in RNA S or its more slowly migrating forms, and satellite RNA biological activity, unlike that of the TobRV RNAs, was not protease sensitive. Polynucleotide kinase catalyzed the phosphorylation of satellite RNAs, indicating free 5'-hydroxyl groups.

Genome-associated proteins of comoviruses.

Previously, a protein has been reported to be associated with the RNAs of cowpea mosaic virus. Here we present evidence for genome-associated proteins of other comoviruses: squash mosaic virus, Echtes Ackerbohnemosaik-Virus, and another strain of cowpea mosaic virus. Although the proteins and protein-oligonucleotide complexes derived from the RNAs of these viruses showed similar patterns of electrophoretic mobility, two-dimensional chromatograms of their tryptic peptides were distinct. Chromatograms of peptides from preparations of cowpea mosaic virus that were isolated from two hosts were very similar, indicating that the genome-associated protein may be virus specified. Upon digestion with ribonuclease T1, RNAs from the viruses gave rise to a distribution of large oligonucleotides that is consistent with the presence of polyriboadenylate.