scholarly journals Presence of polyadenylated 3’ tail in RNA of Pepper mild mottle virus allows the Oligo(dT)18 in Priming cDNA Synthesis of its genomic RNA template

2020 ◽  
Author(s):  
Nidhi Kumari ◽  
Vivek Sharma ◽  
Sneha Choudhary ◽  
P.N. Sharma
Keyword(s):  
Pcr Amplification ◽  
Bean Common Mosaic Virus ◽  
Mottle Virus ◽  
Infection Cycle ◽  
Genomic Rna ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Pepper Mild Mottle Virus ◽  
Or Gene ◽  
Prime End

AbstractThe PCR amplification of majority of the ssRNA of both genomic and non-genomic mRNA is accomplished by RT-PCR. The mRNA is subjected to cDNA synthesis using reverse transcriptase and either Oligo(dT)18, or random or gene specific reverse primers based priming strategies. The choice of primer largely depends on the nature of 3 prime terminus of mRNA and length of cDNA synthesized. In general, oligo(dT)18 is the preferred choice for mRNAs having poly(A) tail at 3 prime terminus. In general, tobamoviruses lack any poly(A) tail at their 3 prime untranslated region (UTR) which forms a tRNA like structure and upstream pseudoknot domain except tow viruses viz., Hibiscus latent Fort Pierce virus (HLFPV) and Hibiscus latent Singapore virus (HLSV) which accommodate internal poly(A) sequences of 46 and 87 nucleotides long, respectively in their 3 prime UTR. However, determination of full nucleotide sequence of Pepper mild mottle virus (PMMoV) using an oligo(dT)18 primed cDNA as template indicated the libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template which are known to lack poly(A) tail. at the end or internally at its 3 prime end. Moreover, coat protein (CP) gene of PMMoV and bean common mosaic virus (BCMV) (Potyvirus with a poly(A) tract at its 3 prime end) was amplified using cDNA primed with random primer as well as oligo(dT)18 was successfully amplified but with significant variation in the intensity of the amplification band in case of PMMoV but not in BCMV. This clearly indicated the presence of PMMoV mRNA with polyadenylated 3 prime tail in total RNA isolated from PMMoV infected capsicum leaves with abundance of non-polyadenylated PMMoV genomic RNA (gRNA). Hence, we hypothesize that the generation of polyadenylated RNA population during the infection cycle of PMMoV in pepper may be possible reason for libertinism of oligo(dT)18 in priming cDNA synthesis of RNA template isolated from PMMoV infected leaves followed by amplification of entire PMMoV genome through RT-PCR. This is first study indicating the presence of polyadenylated or polyadenylated rich regions in PMMoV gRNA acquired during the infection cycle in pepper.

scholarly journals Nucleotide Sequence of the Genomic RNA of Pepper Mild Mottle Virus, a Resistance-breaking Tobamovirus in Pepper

1991 ◽  
Vol 72 (12) ◽  
pp. 2875-2884 ◽  
Author(s):  
E. Alonso ◽  
I. Garcia-Luque ◽  
A. de la Cruz ◽  
B. Wicke ◽  
M. J. Avila-Rincon ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Mottle Virus ◽  
Genomic Rna ◽  
Pepper Mild Mottle Virus ◽  
Resistance Breaking

scholarly journals Genomic RNA sequence of feline coronavirus strain FCoV C1Je

2007 ◽  
Vol 9 (3) ◽  
pp. 202-213 ◽  
Author(s):  
Charlotte Dye ◽  
Stuart G. Siddell
Keyword(s):  
Consensus Sequence ◽  
Pcr Amplification ◽  
Laboratory Strain ◽  
Open Reading Frames ◽  
Viral Rna ◽  
Genomic Rna ◽  
Rt Pcr ◽  
Rna Sequences ◽  
Rna Sequence ◽  
Feline Coronavirus

This paper reports the first genomic RNA sequence of a field strain feline coronavirus (FCoV). Viral RNA was isolated at post mortem from the jejunum and liver of a cat with feline infectious peritonitis (FIP). A consensus sequence of the jejunum-derived genomic RNA (FCoV C1Je) was determined from overlapping cDNA fragments produced by reverse transcriptase polymerase chain reaction (RT-PCR) amplification. RT-PCR products were sequenced by a reiterative sequencing strategy and the genomic RNA termini were determined using a rapid amplification of cDNA ends PCR strategy. The FCoV C1Je genome was found to be 29,255 nucleotides in length, excluding the poly(A) tail. Comparison of the FCoV C1Je genomic RNA sequence with that of the laboratory strain FCoV FIP virus (FIPV) 79-1146 showed that both viruses have a similar genome organisation and predictions made for the open reading frames and cis-acting elements of the FIPV 79-1146 genome hold true for FCoV C1Je. In addition, the sequence of the 3′-proximal third of the liver derived genomic RNA (FCoV C1Li), which encompasses the structural and accessory protein genes of the virus, was also determined. Comparisons of the enteric (jejunum) and non-enteric (liver) derived viral RNA sequences revealed 100% nucleotide identity, a finding that questions the well accepted ‘internal mutation theory’ of FIPV pathogenicity.

scholarly journals Occurrence of Lily mottle virus on Lilium in Italy

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 771-771 ◽  
Author(s):  
D. Rizzo ◽  
L. Stefani ◽  
M. Paoli ◽  
S. Lazzereschi ◽  
B. Nesi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leaf Tissue ◽  
Flowering Plant ◽  
Mottle Virus ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Vein Clearing ◽  
Cp Gene ◽  
Necrotic Spots

Lily mottle virus (LMoV), a member of the genus Potyvirus, is one of the main viruses infecting lily. Symptoms on lily differ according to the susceptibility and sensitivity of different cultivars and hybrids. They range from leaf mottle or mosaic, vein clearing, chlorotic and yellow streaking, leaf curling, and necrotic spots, to milder forms of leaf symptoms. Plants may even be symptomless at some stages of growth. A varietal collection of Lilium from the early 1990s is held in Pistoia Province (Tuscany, Italy) and is composed of Asian hybrids obtained from intraspecific breeding of commercial cultivars. During a survey conducted from May to June 2010, several plants showing vein clearing, leaf mottle, leaf mosaic, and reddish brownish necrotic spots were observed. Leaf samples from 60 symptomatic or symptomless lily plants, belonging to 20 cultivars, were collected and tested for the presence of LMoV. Samples were assayed by double-antibody sandwich (DAS)-ELISA and eight of them, belonging to four different cultivars, tested positive. Total RNA was extracted from 2 g of leaf tissue of every collected sample according to the protocol described earlier (3) and cDNA synthesis was performed with an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Samples were tested by reverse transcription (RT)-PCR and real-time PCR assays using primers LMoV1 (5′-GCAAATGAGACACTCAATGCTG-3′) and LMoV2 (5′-CGTGCGTGAAGTAACTTCATAG-3′) designed to amplify 651 bp of the coat protein (CP) gene of LMoV (1). Results obtained with RT-PCR and real-time PCR exactly matched those achieved with ELISA assay, and the eight positive samples showed amplicons of the expected size. PCR products from five infected samples were directly sequenced from both directions and submitted in GenBank (Accessions Nos. JQ655106 to JQ655110). Our isolates share more than 99% nucleotide identity among each other. Comparison with other LMoV-CP gene sequences present in GenBank showed nucleotide identities ranging from 93 to 94% with LMoV isolates from South Korea (GenBank Accession Nos. GQ150683 to GQ150686), China (GenBank Accession Nos. EU348826, AJ748256, AJ564636, and AJ564637), Australia (GenBank Accession No. JN127341), and Japan (GenBank Accession No. AB570195). To our knowledge, this is the first report of LMoV on Lilium in Italy where this virus was already reported to infect escarole (2). Considering the economic importance of Lilium production as a flowering plant in Pistoia Province, and in several other areas of Italy, the report of LMoV present on lilies suggests the use of healthy propagation material and the adoption of preventive measures to avoid its diffusion. References: (1) J.-H. Lim et al. Korean J. Microbiol. 45:251, 2009. (2) V. Lisa et al. Plant Dis. 86:329, 2002. (3) D. J. MacKenzie et al. Plant Dis. 81:222, 1997.

scholarly journals Caracterização de um isolado do Pepper mild mottle virus que não quebra a resistência do gene L3 em Capsicum sp.

2004 ◽  
Vol 29 (6) ◽  
pp. 670-675 ◽  
Author(s):  
Marcelo Eiras ◽  
Alexandre L. R. Chaves ◽  
Silvia R. Moreira ◽  
Jansen de Araujo ◽  
Addolorata Colariccio
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Ringspot Virus ◽  
Mottle Virus ◽  
Tomato Mosaic Virus ◽  
Rt Pcr ◽  
Pepper Mild Mottle Virus ◽  
Capsicum Baccatum ◽  
Odontoglossum Ringspot Virus ◽  
Capsicum Spp

Sementes de pimenta (Capsicum baccatum) 'Dedo de Moça' destinadas ao plantio comercial e adquiridas no município de São Paulo, SP, analisadas quanto à presença de vírus, por meio de testes biológicos e sorológicos revelaram-se infetadas por uma estirpe do Pepper mild mottle virus (PMMoV). Para confirmar a identidade do isolado, promoveu-se a RT-PCR com oligonucleotídeos que flanqueiam a ORF da capa protéica de espécies do gênero Tobamovirus do subgrupo 1. Os fragmentos de DNA amplificados, quando seqüenciados e comparados com outros isolados de tobamovírus depositados no GenBank, apresentaram valores de identidade de nucleotídeos entre 94 e 100% com outras seqüências de PMMoV, inferiores a 75% para as demais espécies de tobamovírus do subgrupo I (Tobacco mosaic virus, Tomato mosaic virus e Odontoglossum ringspot virus) e 65% para os tobamovírus dos subgrupos II e III. O PMMoV-BR revelou 100% de identidade com isolados japoneses, sugerindo que este patógeno pode ter sido introduzido daquele país. A seqüência de aminoácidos deduzidos da capa protéica indicou também, que este isolado não é capaz de quebrar a resistência do gene L3 de Capsicum spp. Fato confirmado pelos sintomas causados nas hospedeiras diferenciais de Capsicum spp., verificando-se que este isolado não foi capaz de infetar plantas de C. chinense (L3) e C. chacoense (L4). Estes resultados confirmaram a importância da caracterização dos isolados de tobamovírus, fundamental para adequação de medidas de controle, principalmente, prevenindo a entrada e posterior disseminação do patógeno em novas áreas de cultivo.

scholarly journals First Report of Cherry green ring mottle virus on Sweet Cherry in Poland

Plant Disease ◽  
2005 ◽  
Vol 89 (12) ◽  
pp. 1363-1363 ◽  
Author(s):  
B. Komorowska ◽  
M. Cieślińska
Keyword(s):  
Nucleotide Sequence ◽  
Sweet Cherry ◽  
Pcr Amplification ◽  
Sour Cherry ◽  
Mottle Virus ◽  
Rt Pcr ◽  
Green Ring ◽  
Bacterial Vector ◽  
Primer Sets ◽  
Cherry Trees

Cherry green ring mottle virus (CGRMV), a member of the genus Foveavirus, infects several Prunus species including sweet cherry, sour cherry, ornamental cherry, peach, and apricot throughout North America and Europe. On sour cherry, the virus causes leaf yellowing and dark mottle around secondary veins. Sweet cherry trees are symptomless hosts of CGRMV. During the 2004 growing season, 27 sour and sweet cherry trees were tested for the presence of CGRMV. RNA was isolated from leaves using an RNeasy kit (Qiagen GmbH, Hilden, Germany) and then evaluated by reverse transcription-polymerase chain reaction (RT-PCR) amplification. Two primer sets, GRMV7956/GRMV8316 (1) and NCP5/NCP3 (2), were used for amplification of the CGRMV coat protein gene (807 bp) or its fragment (366 bp), respectively. The cDNA fragments were cloned into bacterial vector pCR 2.1-TOPO, sequenced and analyzed using the Lasergene (DNASTAR, Madison, WI) computer program. Nucleotide sequence of the C328 isolate (GenBank Accession No. AY841279) was compared with corresponding regions of published sequences of CGRMV isolates. The nucleotide sequence of this isolate was 98% identical to the Leb isolate (GenBank Accession No. AF533157) from sour cherry. The lowest similarity (80%) was between the CP sequences of isolate C328 and an isolate from apricot (GenBank Accession No. AY172334.1). Results of biological indexing on Prunus serrulata ‘Shirofugen’ and ‘Kwanzan’ confirmed the infection of ‘Star’ sweet cherry with CGRMV. The indicators showed leaf epinasty and necrosis of fragments of midrib or veins characteristic for CGRMV (2). The CGRMV infection of the indicators was confirmed using RT-PCR. References: (1) M. E. Rott and W. Jelkmann. Eur. J. Plant Pathol. 107:411, 2001. (2) Y. Zhang et al. J. Gen. Virol. 79:2275, 1998.

scholarly journals Complete Genome Sequence of a Pepper mild mottle virus Isolate from Northeast China

2018 ◽  
Vol 6 (9) ◽  
Author(s):  
Man Yu ◽  
Tao Zhou ◽  
Yuanhua Wu ◽  
Mengnan An
Keyword(s):  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Northeast China ◽  
Virus Isolate ◽  
Mottle Virus ◽  
Rt Pcr ◽  
Pepper Mild Mottle Virus ◽  
Reverse Transcription Pcr ◽  
Rapid Amplification

ABSTRACT The complete genome sequence of a Pepper mild mottle virus (PMMoV) isolate obtained from Northeast China was determined by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE). Phylogenetic analysis showed that the virus isolate is closely related to Japanese, Chinese, Spanish, and U.S. isolates.

Variability and evolution of the plant RNA virus pepper mild mottle virus.

1989 ◽  
Vol 63 (5) ◽  
pp. 2198-2203 ◽  
Author(s):  
E Rodríguez-Cerezo ◽  
A Moya ◽  
F García-Arenal
Keyword(s):  
Rna Virus ◽  
Mottle Virus ◽  
Pepper Mild Mottle Virus

scholarly journals Duplex-RT-PCR assay for the simultaneous detection and discrimination of Brome mosaic virus and Cocksfoot mottle virus in cereal plants

2021 ◽  
Author(s):  
Katarzyna Trzmiel
Keyword(s):  
Mosaic Virus ◽  
Simultaneous Detection ◽  
Brome Mosaic Virus ◽  
Mottle Virus ◽  
Grass Species ◽  
Replicase Gene ◽  
Rt Pcr ◽  
Pcr Products ◽  
Cereal Plants ◽  
Cocksfoot Mottle Virus

AbstractBrome mosaic virus (BMV) and cocksfoot mottle virus (CfMV) are pathogens of grass species including all economically important cereals. Both viruses have been identified in Poland therefore they create a potential risk to cereal crops. In this study, a duplex—reverse transcription—polymerase chain reaction (duplex-RT-PCR) was developed and optimized for simultaneous detection and differentiation of BMV and CfMV as well as for confirmation of their co-infection. Selected primers CfMVdiag-F/CfMVdiag-R and BMV2-F/BMV2-R amplified 390 bp and 798 bp RT-PCR products within coat protein (CP) region of CfMV and replicase gene of BMV, respectively. Duplex-RT-PCR was successfully applied for the detection of CfMV-P1 and different Polish BMV isolates. Moreover, one sample was found to be co-infected with BMV-ML1 and CfMV-ML1 isolates. The specificity of generated RT-PCR products was verified by sequencing. Duplex-RT-PCR, like conventional RT-PCR, was able to detect two viruses occurring in plant tissues in very low concentration (as low as 4.5 pg/µL of total RNA). In contrast to existing methods, newly developed technique offers a significant time and cost-saving advantage. In conclusion, duplex-RT-PCR is a useful tool which can be implemented by phytosanitary services to rapid detection and differentiation of BMV and CfMV.

scholarly journals The coat protein of tobamovirus acts as elicitor of both L 2 and L 4 gene-mediated resistance in Capsicum

2004 ◽  
Vol 85 (7) ◽  
pp. 2077-2085 ◽  
Author(s):  
P. Gilardi ◽  
I. García-Luque ◽  
M. T. Serra
Keyword(s):  
Hypersensitive Response ◽  
Host Response ◽  
Expression System ◽  
Potato Virus X ◽  
Mottle Virus ◽  
Coat Proteins ◽  
Pepper Mild Mottle Virus ◽  
Defence Genes ◽  
Glucuronidase Reporter ◽  
Capsicum Chacoense

In Capsicum, the resistance conferred by the L 2 gene is effective against all of the pepper-infecting tobamoviruses except Pepper mild mottle virus (PMMoV), whereas that conferred by the L 4 gene is effective against them all. These resistances are expressed by a hypersensitive response, manifested through the formation of necrotic local lesions (NLLs) at the primary site of infection. The Capsicum L 2 gene confers resistance to Paprika mild mottle virus (PaMMV), while the L 4 gene is effective against both PaMMV and PMMoV. The PaMMV and PMMoV coat proteins (CPs) were expressed in Capsicum frutescens (L 2 L 2) and Capsicum chacoense (L 4 L 4) plants using the heterologous Potato virus X (PVX)-based expression system. In C. frutescens (L 2 L 2) plants, the chimeric PVX virus containing the PaMMV CP was localized in the inoculated leaves and produced NLLs, whereas the chimeric PVX containing the PMMoV CP infected the plants systemically. Thus, the data indicated that the PaMMV CP is the only tobamovirus factor required for the induction of the host response mediated by the Capsicum L 2 resistance gene. In C. chacoense (L 4 L 4) plants, both chimeric viruses were localized to the inoculated leaves and produced NLLs, indicating that either PaMMV or PMMoV CPs are required to elicit the L 4 gene-mediated host response. In addition, transient expression of PaMMV CP into C. frutescens (L 2 L 2) leaves and PMMoV CP into C. chacoense (L 4 L 4) leaves by biolistic co-bombardment with a β-glucuronidase reporter gene led to the induction of cell death and the expression of host defence genes in both hosts. Thus, the tobamovirus CP is the elicitor of the Capsicum L 2 and L 4 gene-mediated hypersensitive response.

Sign in / Sign up

Export Citation Format

Share Document