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

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.