Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation

2012 ◽  
Vol 92 (3) ◽  
pp. 495-500
Author(s):  
Wei Chuanbao ◽  
Wu Qiyao ◽  
Yang Yu ◽  
Liu Chunyun
Keyword(s):  
Mosaic Virus ◽  
Prokaryotic Expression ◽  
Pcr Primers ◽  
Amino Acid Identity ◽  
Virus Particles ◽  
Specific Pcr ◽  
Cp Gene ◽  
Sds Page ◽  
Virus Coat ◽  
Antiserum Preparation

Wei, C. B., Wu, Q. Y., Yang, Y. and Liu, C. Y. 2012. Prokaryotic expression of a Thunberg fritillary mosaic virus CP gene and antiserum preparation. Can. J. Plant Sci. 92: 495–500. Fritillaria anhuiensis is a valued traditional Chinese medicinal plant. Thunberg fritillary mosaic virus (TFMV) was found to cause mosaic symptoms. Virus particles of the Lu'an isolate were purified and its RNA genome was extracted. cDNA was synthesized from the RNA and specific PCR primers were used to amplify the virus coat protein (CP) gene. It shared 96.8% nucleotide identity and 97.6% amino acid identity with the sequences of CPs from the other TFMV isolates. The CP gene was inserted into pSBET and expressed in Escherichia coli BL21 (DE3) plys S strain. The expressed protein was first purified by 12% (wt/vol) SDS-PAGE and subsequently by 5–20% (wt/vol) gradient SDS-PAGE. Antiserum against the CP was raised in mouse and its specificity was confirmed by Western blot analysis suggesting that it may be useful for detecting the virus.

scholarly journals First Report of Zantedeschia mosaic virus Infecting a Zantedeschia sp. in New Zealand

Plant Disease ◽  
2008 ◽  
Vol 92 (8) ◽  
pp. 1253-1253 ◽  
Author(s):  
T. Wei ◽  
M. N. Pearson ◽  
D. Cohen ◽  
J. Z. Tang ◽  
G. R. G. Clover
Keyword(s):  
New Zealand ◽  
Amino Acid ◽  
Mosaic Virus ◽  
Amino Acid Identity ◽  
Cut Flowers ◽  
Rt Pcr ◽  
Acid Identity ◽  
Virus Particles ◽  
Blast Search ◽  
Cp Gene

In February 2004, leaf yellowing, mottling, and mosaics were observed on a few plants of a Zantedeschia sp. (calla lily) growing in Rangiora, Canterbury, New Zealand. Zantedeschia spp. are known to be susceptible to at least 13 virus species (1). No symptoms were observed on Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis, or N. tabacum when inoculated with sap from symptomatic plants. However, electron microscopy of crude sap preparations from a symptomatic Zantedeschia sp. and inoculated N. clevelandii plants revealed the presence of flexuous, filamentous virus particles approximately 700 nm long and 12 nm wide. No virus particles were seen in the other inoculated indicator species. Nucleic acid was extracted from leaves of the infected Zantedeschia sp. and N. clevelandii plants and tested in reverse transcription (RT)-PCR using published potyvirus-specific primers (4). PCR amplicons of the expected size (327 bp) were obtained from both plant species and sequenced directly. The products were identical, and a BLAST search in GenBank showed 99% nucleotide identity with a Taiwanese isolate of the species Zantedeschia mosaic virus (ZaMV) (GenBank Accession No. AY026463). A product of 1,531 bp (GenBank Accession No. EU544542) was amplified from symptomatic Zantedeschia by RT-PCR using novel forward (5′-GCACGGCAGATAAACACGAC-3′) and reverse (5′-GTGGGCAACCTTCAACTGTG-3′) primers designed to amplify the 3′ untranslated region (3′UTR), coat protein (CP), and partial nuclear inclusion b protein (NIb) genes. The product was sequenced and had 94% nucleotide identity with a South Korean ZaMV isolate (GenBank Accession No. AB081519), with 95% nucleotide (97% amino acid) identity in the CP gene. A second crop of Zantedeschia spp. in Tauranga, New Zealand (approximately 700 km north of Rangiora) was observed to have similar disease symptoms. Symptomatic plants tested positive in ELISA using a potyvirus-specific monoclonal antibody (Agdia Inc., Elkhart, IN). Nucleic acid was extracted from leaves of symptomatic plants and tested in RT-PCR using potyvirus-specific primer pairs, PV2I/T7 and D335 and U335 and PV1/SP6, which amplify overlapping regions within the 3′UTR, CP, and NIb genes (2,3). The products were sequenced and a consensus sequence of 1,793 bp was generated (GenBank Accession No. EU532065). A BLAST search showed that the sequence had 78% nucleotide (88% amino acid) identity with Zantedeschia mild mosaic virus (ZaMMV) (GenBank Accession No. AY626825). However, the sequences had only 73% nucleotide (79% amino acid) identity in the CP gene, and therefore, this second virus may be a distinct species. To our knowledge, this is the first report of ZaMV in New Zealand. Cut flowers are an increasingly important commodity in New Zealand and Zantedeschia is one of the most important crops; in 2005, exports of rhizomes and cut flowers of the genus were worth NZ$10.9 million. These viral diseases may require management to ensure that the quality of production is maintained. References: (1) C. H. Huang et al. Plant Pathol. 56:183, 2007. (2) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (3) A. M. Mackenzie et al. Arch. Virol. 143:903, 1998. (4) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000.

Identification of Two Tobacco rattle virus Sequence Variants Associated with Virus-like Mottle Symptom on Hosta in Ohio

2013 ◽  
Vol 14 (1) ◽  
pp. 24 ◽  
Author(s):  
John R. Fisher
Keyword(s):  
Mosaic Virus ◽  
Tobacco Rattle Virus ◽  
Alfalfa Mosaic Virus ◽  
Pcr Primers ◽  
Ringspot Virus ◽  
Impatiens Necrotic Spot Virus ◽  
Sequence Variants ◽  
Arabis Mosaic Virus ◽  
Specific Pcr ◽  
Wilt Virus

Two Hosta sp. ‘So Sweet’ plants and one Hosta sieboldii (labeled as ‘Albo-marginata’) plant showing a suspected virus-like leaf mottle symptom tested negative for the Potyvirus group, Hosta virus X, Alfalfa mosaic virus, Arabis mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato ringspot virus, and Tomato spotted wilt virus by ELISA. DsRNA analysis produced a banding profile suggestive of a viral infection, and dsRNA was used as template to synthesize cDNAs for use with tobravirus group and Tobacco rattle virus (TRV) specific PCR primers. Amplicons were cloned and sequenced, and results showed two distinct populations of sequences: the two So Sweet isolates were ∼99% identical to each other but only ∼92% identical to the Albo-marginata isolate. These results represent the first confirmed report of TRV in Hosta in Ohio, and further demonstrate that there are at least two nucleotide sequence variants of the virus infecting Ohio Hosta. Accepted for publication 21 December 2012. Published 30 March 2013.

scholarly journals First Report of Pepper veinal mottle virus in Tomato and Pepper in Taiwan

Plant Disease ◽  
2009 ◽  
Vol 93 (1) ◽  
pp. 107-107 ◽  
Author(s):  
Y. H. Cheng ◽  
R. Y. Wang ◽  
C. C. Chen ◽  
C. A. Chang ◽  
F.-J. Jan
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Polyclonal Antibodies ◽  
Amino Acid Identity ◽  
Mottle Virus ◽  
Rt Pcr ◽  
Acid Identity ◽  
First Report ◽  
Cp Gene ◽  
Pepper Veinal Mottle Virus

In May of 2006, samples from tomato plants (Solanum lycopersicum cv. Known-you 301) exhibiting necrotic symptoms on stems, petioles, and leaves were collected from Chiayi County, Taiwan. Double-antibody sandwich-ELISAs were performed using Cucumber mosaic virus, Tomato mosaic virus, Potato virus Y, Watermelon silver mottle virus, and Chilli veinal mottle virus (ChiVMV) polyclonal antibodies. Three of eight samples reacted with antibodies against ChiVMV but not with the others. Using the potyvirus degenerate primers (Hrp 5/Pot 1) (2), an expected 1.5-kb DNA fragment including the 3′-end of the NIb gene, the complete coat protein (CP) gene, and the 3′-nontranslatable region of the virus was amplified from total RNA isolated from these three samples by reverse transcription (RT)-PCR. A homology search in GenBank indicated that the new tomato-infecting virus in Taiwan belongs to Pepper veinal mottle virus (PVMV) since they shared >90% amino acid identity in the CP gene. A virus culture (Tom1) isolated from one of the diseased tomatoes was then established in Chenopodium quinoa and Nicotiana benthamiana and the CP gene was amplified and sequenced (GenBank Accession No. EU719647). Comparisons of the 807-nt CP gene with those of five PVMV isolates available in GenBank showed 81.5 to 93.1% nucleotide and 90.0 to 97.8% amino acid identity. Tom1 induced irregular necrotic lesions on stems, petioles, and leaves of tomato while inducing only mild mottle symptoms on pepper. Serological cross reaction between ChiVMV and PVMV has been observed previously (1,3) and also found in this study. To differentiate these two potyviruses by RT-PCR, primer pair CPVMVup/dw (5′-TATTC(T/C)TCAGTGTGG(A/T/C)T(T/C)CCACCAT and 5′-(T/C)C(A/T)C(A/T)(A/T/G)(A/T)AA(A/G)CCATAA(A/C)(A/C)ATA(A/G)T(T/C)T) was designed on the basis of the comparison of the CP gene and the 3′-nontranslatable region of the PVMV and ChiVMV. DNA fragments of 171 and 259 bp are expected to be amplified from ChiVMV and PVMV, respectively, by RT-PCR with primers CPVMVup/dw. In a field survey done in 2006, samples from diseased peppers (Capsicum annuum) that reacted with the polyclonal antibodies against ChiVMV were further identified by RT-PCR with primers CPVMVup/dw, indicating that both ChiVMV and PVMV infected pepper crops (Capsicum spp.) in Taiwan. A pepper isolate (Pep1) of PVMV was obtained from Nantou County through three times of single lesion passages on C. quinoa and then propagated on N. benthamiana. The CP gene of Pep1 was amplified and sequenced (GenBank Accession No. EU719646) and found to share 99.1% nucleotide and 100% amino acid identity with that of Tom1. Pep1 caused mild mottle symptoms on leaves of both tomato and pepper. To our knowledge, this is the first report of the presence of PVMV in Taiwan as well as in East Asia. References: (1) B. Moury et al. Phytopathology 95:227, 2005. (2) S. S. Pappu et al. Plant Dis. 82:1121, 1998. (3) W. S. Tsai et al. Plant Pathol. 58:408, 2008.

scholarly journals Identification of Tomato mosaic virus Infection in Lisianthus in Taiwan

Plant Disease ◽  
2003 ◽  
Vol 87 (12) ◽  
pp. 1537-1537 ◽  
Author(s):  
F.-J. Jan ◽  
C.-C. Chen ◽  
H. T. Hsu
Keyword(s):  
Amino Acid ◽  
Mosaic Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Systemic Infection ◽  
Amino Acid Identity ◽  
Tomato Mosaic Virus ◽  
Nucleotide Identity ◽  
Acid Identity ◽  
Cp Gene

In recent years, Lisianthus (Eustoma russellianum (Don.) Griseb) has become popular as potted plants and cut flowers in Taiwan. They are grown in the central and southern regions of the island. Since 1998, diseased plants with mosaic symptoms, followed by necrosis of leaf tissues, were observed in commercial greenhouses and field-grown lisianthus. Newly emergent leaves were curled and smaller compared with those on healthy plants. These symptoms greatly decreased the commercial value of the crop. Rigid rods similar to tobamoviruses that measured 300 × 18 nm were found consistently associated with symptomatic plants. In July 2002, a virus culture was isolated from diseased lisianthus from Chiayi County, Taiwan and established and maintained in systemic hosts Nicotiana tabacum L. and N. benthamiana Domin. Chlorotic and necrotic spots developed on lisianthus leaves 1 to 2 weeks after inoculation with the virus; symptoms eventually became systemic. Virions were purified from inoculated N. tabacum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the virus contained one 18-kDa (Mr) polypeptide. The virus reacted positively in agar gel double diffusion tests and enzyme-linked immunosorbent assays with antisera prepared to Tobacco mosaic virus (TMV) or Tomato mosaic virus (ToMV) (gifts of S. D. Yeh, National Chung Hsing University, Taichung, Taiwan). A viral coat protein (CP) gene approximately 0.5 kb was amplified by reverse transcription-polymerase chain reaction from total RNA prepared from infected N. benthamiana using 5′-GCGAGCCATGGATTCTTACTCAATTACT as a forward primer and 5′-ACTCTCGGATCCTTAAGATGCAGGTGCAGA as a reverse primer. Comparison of the 480-nt CP gene region with that of ToMV-OM (GenBank Accession No. X02144) (3) revealed 99.2% nucleotide identity and 99.4% amino acid identity. It shares, however, 74.4% nucleotide identity and 83.9% amino acid identity with CP genes of TMV-U1 (GenBank Accession No. AX040174) and TMV-vulgare (GenBank Accession No. J02415) (1). The virus induced local lesion responses similar to ToMV on inoculated N. tabacum cv. White Burley, N. sylvestris Speg. & Comes, and Datura stramonium L. Inoculation of TMV, however, resulted in a systemic infection in these plants. Results from sequence analysis and diagnosis based on host reaction to the virus inoculation indicated that the tobamovirus infecting lisianthus in Taiwan is an isolate of ToMV. The virus is economically important to lisianthus and tomato in Taiwan. To our knowledge, this is the first report of ToMV causing disease on lisianthus in Taiwan. The disease was previously observed on lisianthus in Italy (2). References: (1) P. Goelet et al. Proc. Natl. Acad. Sci. USA 79:5818, 1982. (2) V. Lisa and A. Gera. Lisianthus. Pages 443–448 in: Virus and Virus-like Diseases of Bulb and Flower Crops. G. Loebenstein et al. eds. John Wiley and Sons, West Sussex, U.K., 1995. (3) T. Ohno et al. J. Biochem. 96:1915, 1984.

scholarly journals Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

2012 ◽  
Vol 47 (1) ◽  
pp. 66-75
Author(s):  
Sebahattin Çürük ◽  
Ebru Meşe
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Zucchini Yellow Mosaic Virus ◽  
Yellow Mosaic Virus ◽  
Flesh Color ◽  
Flesh Firmness ◽  
Cp Gene ◽  
Virus Coat Protein ◽  
Virus Coat ◽  
Parental Cultivar

The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP) and neomycin phosphotransferase II (NPT II) genes to the watermelon 'Crimson Sweet'(CS) genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.

scholarly journals Development of a Co-Dominant Cleaved Amplified Polymorphic Sequences Assay for the Rapid Detection and Differentiation of Two Pathogenic Clarireedia spp. Associated with Dollar Spot in Turfgrass

Agronomy ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1489
Author(s):  
Tammy Stackhouse ◽  
Sumyya Waliullah ◽  
Alfredo D. Martinez-Espinoza ◽  
Bochra Bahri ◽  
Emran Ali
Keyword(s):  
Pcr Primers ◽  
The United States ◽  
Fungal Species ◽  
Dollar Spot ◽  
Direct Pcr ◽  
Specific Pcr ◽  
Specificity And Sensitivity ◽  
Pure Cultures ◽  
Speed Up ◽  
Cleaved Amplified Polymorphic Sequences

Dollar spot is one of the most destructive diseases in turfgrass. The causal agents belong to the genus Clarireedia, which are known for causing necrotic, sunken spots in turfgrass that coalesce into large damaged areas. In low tolerance settings like turfgrass, it is of vital importance to rapidly detect and identify the pathogens. There are a few methods available to identify the genus Clarireedia, but none of those are rapid enough and characterize down to the species level. This study produced a co-dominant cleaved amplified polymorphic sequences (CAPS) test that differentiates between C. jacksonii and C. monteithiana, the two species that cause dollar spot disease within the United States. The calmodulin gene (CaM) was targeted to generate Clarireedia spp. specific PCR primers. The CAPS assay was optimized and tested for specificity and sensitivity using DNA extracted from pure cultures of two Clarireedia spp. and other closely related fungal species. The results showed that the newly developed primer set could amplify both species and was highly sensitive as it detected DNA concentrations as low as 0.005 ng/µL. The assay was further validated using direct PCR to speed up the diagnosis process. This drastically reduces the time needed to identify the dollar spot pathogens. The resulting assay could be used throughout turfgrass settings for a rapid and precise identification method in the US.

Protein-Embedded Metalloporphyrin Arrays Templated by Circularly Permuted Tobacco Mosaic Virus Coat Proteins

ACS Nano ◽  
2020 ◽  
Author(s):  
Jing Dai ◽  
Gavin J. Knott ◽  
Wen Fu ◽  
Tiffany W. Lin ◽  
Ariel L. Furst ◽  
...  
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Coat Proteins ◽  
Virus Coat
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