Developing an Alternanthera Mosaic Virus vector for efficient cloning of whitefly cDNA RNAi to screen gene function

Na Yeon Ko, Hyun Seung Kim, Jung Kyu Kim, Seunghee Cho, Eun Young Seo, Hye Ri Kwon, Yong Man Yu, Takafumi Gotoh, John Hammond, Young Nam Youn, Hyoun Sub Lim

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Plant viral vectors have shown significant promse for studies of gene function, through either up-regu-lation or down-regulation of gene expression. However, there have remained issues of efficiency of generating constructs, and of subcellular localization of expression; both issues are addressed here. Alternanthera mosaic virus (AltMV; genus Potexvirus) is distinguished from the type member of the genus, Potato virus Xby features of viral movement and variation within triple gene block protein 1 (TGB1). AltMV TGB1 variants TGB1L88 and TGB1P88 confer strong and weak silencing suppression, respectively, depending on the presence of L or P at residue 88. Because AltMV replication is associated with chloroplasts, we compared the relative efficiency of RNA interference (RNAi) vectors derived from AltMV and Tobacco rattle virus (TRV) to silence a chloroplast-encoded gene. An AltMV RNAi vector expressing a fragment of the chloroplast β ATPase gene reduced β-ATPase expression 1.5 times more than the TRV RNAi vector expressing the same fragment. In addition, we used AltMV (TGB1P88) to create a whitefly (Bemisia tabaci) RNAi vector. For this purpose, we first introduced the Gateway cloning cassette into the AltMV multiple cloning site, into which polymerase chain reaction (PCR) products from a whitefly cDNA library could be easily cloned. Second, a mixture of five different PCR fragments of about 250 bp were used to test cloning efficiency of the newly-created AltMV-P-att vector. Third, random 250 bp fragments of Gateway cDNA libraries from B. tabaci and Nicotiana benthamiana were efficiently cloned into the Gateway-modified AltMV-att vector, demonstrating for the first time a high throughput RNAi system based on AltMV. This strategy could be applied to other RNAi systems.

Original languageEnglish
Pages (from-to)139-149
Number of pages11
JournalJournal of the Faculty of Agriculture, Kyushu University
Volume60
Issue number1
Publication statusPublished - Feb 1 2015

Fingerprint

Alternanthera mosaic virus
Mosaic Viruses
Genetic Vectors
Hemiptera
RNA Interference
Aleyrodidae
RNA interference
molecular cloning
Complementary DNA
Chloroplast Genes
Genes
Tobacco
Organism Cloning
genes
Viruses
Gene Library
Tobacco rattle virus
Chloroplast Proton-Translocating ATPases
Potexvirus
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Agronomy and Crop Science

Cite this

Developing an Alternanthera Mosaic Virus vector for efficient cloning of whitefly cDNA RNAi to screen gene function. / Ko, Na Yeon; Kim, Hyun Seung; Kim, Jung Kyu; Cho, Seunghee; Seo, Eun Young; Kwon, Hye Ri; Yu, Yong Man; Gotoh, Takafumi; Hammond, John; Youn, Young Nam; Lim, Hyoun Sub.

In: Journal of the Faculty of Agriculture, Kyushu University, Vol. 60, No. 1, 01.02.2015, p. 139-149.

Research output: Contribution to journalArticle

Ko, NY, Kim, HS, Kim, JK, Cho, S, Seo, EY, Kwon, HR, Yu, YM, Gotoh, T, Hammond, J, Youn, YN & Lim, HS 2015, 'Developing an Alternanthera Mosaic Virus vector for efficient cloning of whitefly cDNA RNAi to screen gene function', Journal of the Faculty of Agriculture, Kyushu University, vol. 60, no. 1, pp. 139-149.
Ko, Na Yeon ; Kim, Hyun Seung ; Kim, Jung Kyu ; Cho, Seunghee ; Seo, Eun Young ; Kwon, Hye Ri ; Yu, Yong Man ; Gotoh, Takafumi ; Hammond, John ; Youn, Young Nam ; Lim, Hyoun Sub. / Developing an Alternanthera Mosaic Virus vector for efficient cloning of whitefly cDNA RNAi to screen gene function. In: Journal of the Faculty of Agriculture, Kyushu University. 2015 ; Vol. 60, No. 1. pp. 139-149.
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AU - Seo, Eun Young

AU - Kwon, Hye Ri

AU - Yu, Yong Man

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AU - Hammond, John

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