|KO, NA YEON - Chungnam National University|
|KIM, HYUN-SEUNG - Chungnam National University|
|KIM, JUNG-KYU - Chungnam National University|
|CHO, SEUNGHEE - Chungnam National University|
|SEO, EUN-YOUNG - Chungnam National University|
|KWON, HYE-RI - Chungnam National University|
|YU, YONG MAN - Chungnam National University|
|GOTOH, TAKAFUMI - Kyushu University|
|YOUN, YOUNG NAM - Chungnam National University|
|LIM, HYOUN-SUB - Chungnam National University|
Submitted to: Journal of Faculty of Agriculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/14/2014
Publication Date: 1/22/2015
Citation: Ko, N., Kim, H., Kim, J., Cho, S., Seo, E., Kwon, H., Yu, Y., Gotoh, T., Hammond, J., Youn, Y., Lim, H. 2015. Developing an Alternanthera mosaic virus vector for efficient clonging of Whitefly cDNA RNAi to screen gene function. Journal of Faculty of Agriculture. 60:139-149.
Interpretive Summary: Determination of the function of previously uncharacterized plant genes can be challenging, requiring over-expression or gene-knockout to determine altered phenotypes in order to understand gene function; reverse genetics approaches using Virus-induced gene silencing (VIGS) offer a more rapid alternative to generation of transgenic plants for gene knockdown studies, but suitable VIGS vectors are not available for all crops. Alternanthera mosaic virus (AltMV) has a broad host range, and is also potentially valuable for enhanced knockdown of chloroplast-associated genes, as AltMV replicates in association with the chloroplast. An AltMV VIGS vector was modified with a Gateway cloning system to allow high-throughput insertion of short cDNA fragments suitable for VIGS to examine gene function. A plant chloroplast-associated gene was targeted by VIGS using this vector, in comparison to a Tobacco rattle virus (TRV) vector that does not associate with the chloroplast. The AltMV VIGS vector was shown to be more efficient than the TRV vector for suppression of expression of the selected chloroplast-associated gene. In addition, it was demonstrated that multiple short gene fragments derived from either plants or the whitefly Bemisia tabaci could be simultaneously independently cloned into the AltMV using the Gateway cloning system. This strategy could also be applied to other VIGS vectors, increasing the efficiency of gene discovery, to the benefit of plant geneticists and plant breeders.
Technical Abstract: Alternanthera mosaic virus (AltMV; genus Potexvirus) is distinguished from the type member of the genus, Potato virus X by 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 beta-ATPase gene reduced beta-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 Bemisia 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.