Association of Satellite RNA with Grapevine fanleaf virus in its Geographical Origin and Sequence Characteristics

The region between The Caspian Sea and Black Sea has been hypothesized as the origin of grapevine. Likewise, an extensive study on Grapevine fanleaf virus (GFLV) from this region suggests this region as a possible origin of the virus. However, as yet there is no information as to whether or not the virus variants from this region accompanied with the virus satellite RNA (satRNA) and if so, how diverse theses satRNAs can be. To answer these questions, Grapevine fanleaf virus isolates were collected from different vineyards in the northwest region of Iran for the occurrence of the satellite RNA. A total of 421 samples including Vitis vinifera, Chenopodium quinoa, C. amaranticolor, Cynodon dactylis, Medicago sativa were initially screened against GFLV by RT-PCR with the coat protein primers (Cp433, Cp912). When the GFLV-infected plants (36 samples) were screened by RT-PCR with the newly designed satRNA primers Gf750 and Gr750, three samples appeared to possess the satRNA. The satellite cDNA fragments were cloned and sequenced and when the resulting data were compared with sequences of previously-reported satRNA of GFLV and Arabis mosaic virus (ArMV), 70-98% and 69-71% similarities were found, respectively. Phylogenetic studies revealed distinctness of the satRNAs from Iran. This may suggest coevolution of the satRNA with the helper virus because GFLV isolates from this region also form a distinct branch. Among previously reported corresponding satRNA sequences to those from Slovenia and South Central Europe were the closest. This study also reports C. amaranticolor as the natural host for GFLV, which was previously reported only as an experimental host

Extraction and molecular detection of viral dsRNA from different infected plants

Extraction of viral double stranded RNA (dsRNA) from infected plants is helpful in identification of the viruses involved in infection. To date, there have been several methods developed to isolate dsRNA; however, type of the plant and virus is determinative in extraction efficiency. In this study we extracted dsRNA from different woody and herbaceous plants through a modified method which reduces the costs and time of extraction procedure. This method is based on different affinity of nucleic acids for the cellulose CF-11 in1X STE (Sodium chloride Tris EDTA) buffer containing 16 % ethanol. There is no phenol treatment or mini columns used in the isolation procedure. Extracted dsRNAs were identified by ribonuclease treatment and RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction). We have applied the procedure on five different hosts representing Amaranthaceae, Vitaceae, Fabaceae and Rosaceae infected with four different viruses representing Secoviridae and Bromoviridae.&nbsp

Molecular detection of Grapevine fanleaf Virus by the isolation of ssRNA and dsRNA from Xiphinema index

Xiphinema index is an important grapevine pathogen nematode which also vectors Grapevine fanleaf virus. The viral genes involved in transmission by the vector nematode are mapped to the C-terminal residues of RNA2-encoded polyprotein. To recognize viruliferous nematodes, there are some serological and molecular methods. In this study, we extract RNA and dsRNA of the virus, then Reverse transcription-polymerase Chain Reaction was done with virus specific primers to detect virus in its vector. The virus was detected by visualizing the desired 350 and 750 bp gene fragments in electrophoresis. This study reduces the virus detection time to only couple of hours with least imposed charges, and could be employed in transmission experiments as well.&nbsp

Serological Methods to Confirm Expression of Coat Protein Gene From an Iranian Isolate of Cucumber Mosaic Virus in Escherichia coli

Background: Cucumber mosaic virus (CMV) has isometric particles with a diameter of about 28 - 29 nm. Detection and prevention are the critical steps in the control of plant viruses. Detection in a large number of samples is still done by serological methods due to their robustness and perhaps low cost. Objectives: To this end, our aim was to express the CMV CP gene in E. coli to be used as the antigen for antibody production in the future. Materials and Methods: Coat Protein (CP) gene cDNA from an isolate (B13) of Cucumber Mosaic Virus (CMV) was subcloned from pTZ57RCMVCP to pET21a expression vector and transformed to E. coli strain Rosetta. Expression of CMV CP was successful and confirmed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), wherein a ~30- kDa protein band was revealed. Induction by Isopropyl-Thiogalactoside (IPTG) at final concentrations of 0.5 to 2 mM appeared to produce similar results as to the amount of the expressed protein, which was judged by intensity of the band on SDS-PAGE. Results: The identity of the expressed protein was confirmed by immunoassays such as western blot, Dot-Immunobinding Assay (DIBA) and Enzyme-Linked Immunosorbent Assay (ELISA) by the use of anti-CMV antibody. Conclusions: This is the first report of expression of CMV CP gene in Iran, which is important for the preparation of anti-CMV antibody and paving the way for the use of the virus coat protein as a nanomaterial. Keywords: Methods; Cloning; Expression; Gene; Escherichia col

434.qxp

Absract A pair of degenerate primers, GMPF1 and GMPR1, was designed on the basis of alignment of previously reported Grapevine fanleaf virus (GFLV) movement protein (MP) nucleotide sequences from Iran and other parts of the world. cDNA was synthesized by the use of Oligo d(T)18 from total RNA extraction from each diseased grapevine leaf sample and subjected to polymerase chain reaction (PCR) with the degenerate primers under a range of annealing temperatures from 48 to 62°C. It was revealed that 55°C gave the best result in terms of producing exactly the expected fragment (1044 bp) from as many samples as possible although accompanied by few fade non specific fragments. However, by application of "hot-start" PCR and annealing at 60°C the specific fragment was amplified from 41 out of 86 samples. This was the first amplification of the precise MP cDNA from GFLVs in Iran which is very important as to preparation of recombinant anti-GFLV MP antibody to use in studying the GFLVgrapevine interaction, and also for generating pathogen-derived resistant vines

A Dual coat protein construct establishes resistance to Passionfruit woodiness and Cucumber mosaic viruses

There is a high degree (>95%) of intraspecies similarity in the coat protein (CP) amino acid sequences within Passionfruit woodiness virus (PWV) and Cucumber mosaic virus (CMV), both infecting passionfruit vine in New South Wales. On this basis, a dual transgene containing the translatable cDNAs coding for the CPs of PWV and CMV was constructed in the binary vector pBI121 and used for transformation of Nicotiana benthamiana, a susceptible host to both viruses. The transformation was achieved by cocultivation of the agrobacteria with the leaf disks prepared from the surface- sterilized leaves. Five transgenic lines including 1-1, 1-5, 1-7, 1-12 and 1-24 were regenerated. Insertion and transcription of the dual construct were confirmed, however, only the CMV CP was feasibly detectable by DAS-ELISA in the lines. Low level accumulation of CMV and/or PWV was evident in the lines. In the initail challenge trial where 1:10 dilution of plant sap was used, a 5-day delay in symptom was generally shown. Inoculations with 1:100 plant sap also gave similar results as with 1:10 dilution. Lines 1-5 and 1-12, which were inoculated with 1:1000 dilution of sap, remained uninfected by CMV till 27 dpi, whereas with PWV, 1-12 became infected by 11 dpi. Four cuttings of line 1-12 reacted diffferently to the challenge inoculations i.e. three of them resisted PWV, whereas two of them were susceptible to CMV. Since PWV CP was not detectable in the transgenic lines but evidence of resistance to PWV was found in them, this was suggestive of an RNA silencing mechnaism involved in the resistance. Because the CMV CP was detectable in the transgenic lines, this suggested requirement for the CP expression in the resistance. The resistance, or apparent immunity, was manifested by an apparent delay in symptom expression and accumulation of relatively low levels of the viruses.16 page(s

Genomic characterization of Ambrosia asymptomatic virus 1 and evidence of other Tymovirales members in the Oklahoma tallgrass prairie revealed by sequence analysis

The Plant Virus Biodiversity and Ecology project was undertaken to better understand the nature of plant-viral interactions and the occurrence of non-pathogenic viruses. Plants from the Tallgrass Prairie Preserve (TPP), Osage County, Oklahoma, were surveyed from 2005 to 2008 for the presence of viruses, resulting in the detection, using a virus-like particle enrichment method, of the genome a novel virus, Ambrosia asymptomatic virus 1 (AAV1), from Ambrosia psilostachya DC (western ragweed). Here, we present the genomic organization and genetic variability of AAV1. The virus has a single-stranded RNA genome of about 7408 nt, which has six open reading frames (ORFs). Phylogenetic analysis of the replicase and coat protein ORFs of the virus indicates strongly that the virus should be placed in the genus Mandarivirus. No evidence of recombination was detected. We also report the detection in the TPP of two known viruses and seven other putative viruses, members of the order Tymovirales

Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

The genomic region of Grapevine fanleaf virus (GFLV) encoding the movement protein (MP) was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3) to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG) concentrations (1, 1.5, and 2 mM) each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA) before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs) and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection