Steve Daubert

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.

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 β-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.

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.

Genomic and biological analysis of Grapevine leafroll-associated virus 7 reveals a possible new genus within the family Closteroviridae.

Deep sequencing analysis of an asymptomatic grapevine revealed a virome containing five RNA viruses and a viroid. Of these, Grapevine leafroll-associated virus 7 (GLRaV-7), an unassigned closterovirus, was by far the most prominently represented sequence in the analysis. Graft-inoculation of the infection to another grape variety confirmed the lack of the leafroll disease symptoms, even though GLRaV-7 could be detected in the inoculated indicator plants. A 16,496 nucleotide-long genomic sequence of this virus was determined from the deep sequencing data. Its genome architecture and the sequences encoding its nine predicted proteins were compared with those of other closteroviruses. The comparison revealed that two other viruses, Little cherry virus-1 and Cordyline virus-1 formed a well supported phylogenetic cluster with GLRaV-7.

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.

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA

Abstract Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. E . coli DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a R o t 1 2 of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a Tm of 73°C. The probe reacts with total rat nuclear RNA with a R o t 1 2 of 1.0.

Comparison of Next-Generation Sequencing Versus Biological Indexing for the Optimal Detection of Viral Pathogens in Grapevine

A bioassay is routinely used to determine the viral phytosanitary status of commercial grapevine propagation material in many countries around the world. That test is based on the symptoms developed in the field by specific indicator host plants that are graft-inoculated from the vines being tested. We compared the bioassay against next-generation sequencing (NGS) analysis of grapevine material. NGS is a laboratory procedure that catalogs the genomic sequences of the viruses and other pathogens extracted as DNA and RNA from infected vines. NGS analysis was found to be superior to the standard bioassay in detection of viruses of agronomic significance, including virus infections at low titers. NGS was also found to be superior to the bioassay in its comprehensiveness, the speed of its analysis, and for the discovery of novel, uncharacterized viruses.

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.

Development of a sensitive colorimetric-PCR assay for detection of viruses in woody plants

Diagnostic methods employing the polymerase chain reaction (PCR) provide the most sensitive means currently available for detecting viruses in woody plants. A new technique has been tested that does not rely on gel electrophoresis or molecular hybridization to detect virus-specific PCR products. This colorimetric method for detection of PCR products from woody plants was demonstrated to be at least as sensitive as gel analysis. When combined with immunocapture of virions from plant sap, colorimetric detection provides a means to apply PCR technology to a large number of samples. Here, we report on the use of this technique for detection and quantitation of a walnut isolate of cherry leafroll virus (CLRV-W), citrus tristeza virus (CTV), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and tomato ringspot virus (ToRSV) in woody and herbaceous plants. For purified virus preparations, detection limits ranged from 100 pg/ml to 100 ag/ml. We also describe the colorimetric PCR detection of CTV in pooled samples.

Sequence of the Coat Protein Gene of Bermuda Grass Etched-line Virus, and of the adjacent ‘Marafibox’ Motif

The complete nucleotide sequence of the coat protein (CP) gene of Bermuda grass etched-line virus (BELV), including 376 nucleotides (nt) of the region to its 5′ side, was determined and compared with sequences of the other viruses associated with the genus Marafivirus, substantiating the assignment of BELV to this group. The CP gene coding sequence was 585 nt in length. Inferred amino acid sequences showed homologies among marafiviral CP gene products ranging from 41% to 59%. A non-coding sequence motif characteristic of the marafiviruses lies in the region adjacent to the CP gene to the 5′ side. In contrast to various homology levels in the coding regions of the CP genes, the interspecific sequence homology in this 18 nt motif was almost perfect.