Infectious Barley stripe mosaic virus RNA transcribed in Vitro from full-length genomic cDNA clones

Petty, I.T.D.; Hunter, Brenda G.; Wei, Ning; Jackson, A.O.

doi:10.1016/0042-6822(89)90601-6

Cited by 125 publications

(105 citation statements)

References 19 publications

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“…Uncapped FoMV synthetic transcripts were not infectious. Potexvirus genomes have a 5 -cap structure and the use of capped transcripts has been found essential for infectivity with other viruses as well [6,15,16,28,42]. The percentage of transcripts that are actually capped during in vitro synthesis was not determined, but it is estimated to be about 67% by the Ambion kit used to generate the transcripts.…”

Section: Discussionmentioning

confidence: 99%

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The open reading frame 5A of foxtail mosaic virus is expressed in vivo and is dispensable for systemic infection

Robertson

French

Morris

2000

Archives of Virology

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Summary. Infectious transcripts were successfully derived from full-length cDNA clones of foxtail mosaic potexvirus (FoMV). Full-length clones were constructed by RT-PCR whereby 5 and 3 genomic segments of 2.7 and 3.4 kb, respectively, were ligated into Bluescript II KS. The in vitro RNA transcripts were infectious to moncotyledonous (barley) and dicotyledonous (Chenopodium amaranticolor) plant species. Individual mutation studies on clones of each of the five major ORFs confirmed predicted gene function for the polymerase, TGB (triple gene block), and coat protein (CP) genes. Protoplast studies on expression of a unique open reading frame, ORF 5A, which initiates 143 nts upstream of the CP before it "reads through" the CP, revealed that the 5A protein was produced in vivo. Mutation analysis of the 5A ORF indicated, however, that it was not required for either replication or for productive infection of plants. However, the nucleic acid sequences encoding the extended CP segment were shown to be important for CP expression. Additional mutations in 5A had no effect on FoMV replication in protoplasts but rendered the virus noninfectious to plants. A correlation with diminished CP production from both mutant clones implies that synthesis of subgenomic CP mRNA was compromised, and this limited systemic infection.

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Section: Discussionmentioning

confidence: 99%

“…It can infect at least 56 plant species in the Gramineae as well as a number of species in 11 dicotyledonous families [27,28]. The genome of FoMV consists of a capped, messenger sense ssRNA of 6,151 nucleotides in length and poly(A) tail [5].…”

Section: Introductionmentioning

confidence: 99%

The open reading frame 5A of foxtail mosaic virus is expressed in vivo and is dispensable for systemic infection

Robertson

French

Morris

2000

Archives of Virology

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“…AltMV TGB3 was amplified and inserted in place of eGFP in AltMVeGFPDTGB3 to form AltMVDTGB3(TGB3+); AltMVDTGB3(TGB3-GFP+), expressing TGB3-GFP as an added gene, was created by replacing eGFP with TGB3-GFP, amplified from pHVLG-TGB3 (see below), into BamHI/NheI-digested AltMV-eGFP. AltMV TGB3 was amplified and inserted as a BamHI/MluI fragment into infectious clone PVX-MCS, forming PVX(TGB3 AltMV +); PVX-MCS is a derivative of PVX infectious clone pPC2S, described elsewhere (Lim et al, 2010b Full-length cDNA clones were linearized by using XbaI (AltMV clones) or SpeI (PVX clones) before in vitro transcription of infectious RNA for N. benthamiana inoculation, using T7 RNA polymerase as described by Petty et al (1989). Q-RT-PCR to quantify TGB3 RNA was performed as described by Bae et al (2006) using TGB3-specific primers.…”

Section: Methodsmentioning

confidence: 99%

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement

Lim¹,

Vaira²,

Bae

et al. 2010

Journal of General Virology

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Cell-to-cell movement of potexviruses requires coordinated action of the coat protein and triple gene block (TGB) proteins. The structural properties of Alternanthera mosaic virus (AltMV) TGB3 were examined by methods differentiating between signal peptides and transmembrane domains, and its subcellular localization was studied by Agrobacterium-mediated transient expression and confocal microscopy. Unlike potato virus X (PVX) TGB3, AltMV TGB3 was not associated with the endoplasmic reticulum, and accumulated preferentially in mesophyll cells. Deletion and sitespecific mutagenesis revealed an internal signal VL(17,18) of TGB3 essential for chloroplast localization, and either deletion of the TGB3 start codon or alteration of the chloroplastlocalization signal limited cell-to-cell movement to the epidermis, yielding a virus that was unable to move into the mesophyll layer. Overexpression of AltMV TGB3 from either AltMV or PVX infectious clones resulted in veinal necrosis and vesiculation at the chloroplast membrane, a cytopathology not observed in wild-type infections. The distinctive mesophyll and chloroplast localization of AltMV TGB3 highlights the critical role played by mesophyll targeting in virus longdistance movement within plants.

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“…Run-off transcription yielded a 190 nt probe containing the 167 nt intergenic sequence plus 23 residues of plasmid sequence. For filter binding assays, a full-length BSMVfl genome transcript was prepared by run-off transcription from the ND18 strain cDNA clone f142Spl after linearization with SpeI (Petty et al, 1989).…”

Section: G T C C T G a T G T T T A A A T C T A C T C G C C C G G G A mentioning

confidence: 99%

RNA-binding activities of barley stripe mosaic virus b fusion proteins

Donald

Jackson

1996

Journal of General Virology

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Infectious Barley stripe mosaic virus RNA transcribed in Vitro from full-length genomic cDNA clones

Cited by 125 publications

References 19 publications

The open reading frame 5A of foxtail mosaic virus is expressed in vivo and is dispensable for systemic infection

The open reading frame 5A of foxtail mosaic virus is expressed in vivo and is dispensable for systemic infection

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement

RNA-binding activities of barley stripe mosaic virus b fusion proteins

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