Construction and characterization of an infectious cDNA clone of potato virus S developed from selected populations that survived genetic bottlenecks

Li, Xin; Hataya, Tatsuji

doi:10.1186/s12985-019-1124-x

Cited by 4 publications

(2 citation statements)

References 36 publications

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“…Co-transcriptional capping of the 5′-terminus was performed as described in previous studies [ 32 ]. First, 5 μg of linearized plasmid DNAs was incubated for 25 min at 37 °C in a 20 µL reaction mixture containing 40 mM Tris-HCl (pH 8.0); 25 mM NaCl; 8 mM MgCl 2 ; 2 mM spermidine; 10 mM dithiothreitol; 2 mM each of ATP, UTP, and CTP; 0.2 mM GTP; 0.5 mM cap analog m 7 G(5′)ppp(5′)G (Invitrogen); 40 U of RNase inhibitor; and 50 U T7 RNA polymerase (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Hataya

Nakada

2021

Cells

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Infectious dimeric RNA transcripts are a powerful tool for reverse genetic analyses in viroid studies. However, the construction of dimeric cDNA clones is laborious and time consuming, especially in mutational analyses by in vitro mutagenesis. In this study, we developed a system to synthesize a precisely monomeric linear RNA that could be transcribed in vitro directly from the cDNA clones of four viroid species. The cDNA clones were constructed such that RNA transcription was initiated at the guanine nucleotide of a predicted processing and ligation site in the viroid replication process. Although the transcribed RNAs were considered to possess 5′-triphosphate and 3′-hydroxyl termini, the RNA transcripts were infectious even without in vitro modifications. Additionally, infectivity was detected in the monomeric RNA transcripts, in which transcription was initiated at guanine nucleotides distinct from the predicted processing/ligation site. Moreover, monomeric viroid RNAs bearing 5′-monophosphate, 5′-hydroxyl, or 5′-capped termini were found to be infectious. Northern blot analysis of the pooled total RNA of the plants inoculated with the 5′-terminal modified RNA of potato spindle tuber viroid (PSTVd) indicated that maximum PSTVd accumulation occurred in plants with 5′-monophosphate RNA inoculation, followed by the plants with 5′-triphosphate RNA inoculation. Our system for synthesizing an infectious monomeric linear viroid RNA from a cDNA clone will facilitate mutational analyses by in vitro mutagenesis in viroid research.

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Section: Methodsmentioning

confidence: 99%

Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Hataya

Nakada

2021

Cells

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“…Despite infectious clones for plant viruses closely related to BVF, included in the Flexiviridae family have been reported [ 42 , 43 , 44 , 45 , 46 ], reverse genetic systems based on BVF genome have not been developed yet. Since BVF is a small (+)ssRNA virus and has no effects on the fungal growth rate or virulence of B. cinerea , the development of an infectious clone of BVF would be highly feasible and useful in identifying determinants responsible for pathogenesis and those involved in fungus-plant interaction.…”

Section: Introductionmentioning

confidence: 99%

Construction and Characterization of a Botrytis Virus F Infectious Clone

Córdoba

Ruiz-Padilla

Rodríguez‐Romero

et al. 2022

JoF

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Botrytis virus F (BVF) is a positive-sense, single-stranded RNA (+ssRNA) virus within the Gammaflexiviridae family of the plant-pathogenic fungus Botrytis cinerea. In this study, the complete sequence of a BVF strain isolated from B. cinerea collected from grapevine fields in Spain was analyzed. This virus, in this work BVF-V448, has a genome of 6827 nt in length, excluding the poly(A) tail, with two open reading frames encoding an RNA dependent RNA polymerase (RdRP) and a coat protein (CP). The 5′- and 3′-terminal regions of the genome were determined by rapid amplification of cDNA ends (RACE). Furthermore, a yet undetected subgenomic RNA species in BVF-V448 was identified, indicating that the CP is expressed via 3′ coterminal subgenomic RNAs (sgRNAs). We also report the successful construction of the first BVF full-length cDNA clone and synthesized in vitro RNA transcripts using the T7 polymerase, which could efficiently transfect two different strains of B. cinerea, B05.10 and Pi258.9. The levels of growth in culture and virulence on plants of BVF-V448 transfected strains were comparable to BVF-free strains. The infectious clones generated in this work provide a useful tool for the future development of an efficient BVF foreign gene expression vector and a virus-induced gene silencing (VIGS) vector as a biological agent for the control of B. cinerea.

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Construction of an infectious full-length cDNA clone of potato aucuba mosaic virus

et al. 2021

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Construction and characterization of an infectious cDNA clone of potato virus S developed from selected populations that survived genetic bottlenecks

Cited by 4 publications

References 36 publications

Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Precisely Monomeric Linear RNAs of Viroids Belonging to Pospiviroid and Hostuviroid Genera Are Infectious Regardless of Transcription Initiation Site and 5′-Terminal Structure

Construction and Characterization of a Botrytis Virus F Infectious Clone

Construction of an infectious full-length cDNA clone of potato aucuba mosaic virus

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