Development and application of an efficient virus-induced gene silencing system in Nicotiana tabacum using geminivirus alphasatellite

Abstract

Virus-induced gene silencing (VIGS) is a recently developed technique for characterizing the function of plant genes by gene transcript suppression and is increasingly used to generate transient loss-of-function assays. Here we report that the 2mDNA1, a geminivirus satellite vector, can induce efficient gene silencing in Nicotiana tabacum with Tobacco curly shoot virus. We have successfully silenced the β-glucuronidase (GUS) gene in GUS transgenic N. tabacum plants and the sulphur desaturase (Su) gene in five different N. tabacum cultivars. These pronounced and severe silencing phenotypes are persistent and ubiquitous. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants and silencing could be induced in a variety of tissues and organs including leaf, shoot, stem, root, and flower, and achieved at any growth stage. This system works well between 18-32 °C. We also silenced the NtEDS1 gene and demonstrated that NtEDS1 is essential for N gene mediated resistance against Tobacco mosaic virus in N. tabacum. The above results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in N. tabacum.

Suppression of RNA Silencing by a Plant DNA Virus Satellite Requires a Host Calmodulin-Like Protein to Repress RDR6 Expression

“…To further examine the function of Nbrgs-CaM in RNA silencing suppression in the context of a virus infection, we tested whether Nbrgs-CaM could suppress VIGS by using a previously established VIGS vector based on 10A helper virus in association with a geminivirus alphasatellite derivative (2mDNA1) [52] , [53] . The alphasatellite vector (2mDNA1- NbSu ) was designed to express a fragment of the N. benthamiana sulfur ( Su ) reporter gene that encodes a component required for chlorophyll II biosynthesis.…”

“…The alphasatellite vector (2mDNA1- NbSu ) was designed to express a fragment of the N. benthamiana sulfur ( Su ) reporter gene that encodes a component required for chlorophyll II biosynthesis. The recombinant viral vector (10A+2mDNA1- NbSu ) triggered Su silencing in leaves of infected plants as evidenced by yellow-white leaf spots [52] , [53] . In wt N. benthamiana infected by the VIGS vector, the typical Su silencing phenotype first appeared along the main veins of the newly expanded leaves at 10 dpi, and later expand to the lateral veins to produce a white lattice-like phenotype at 30 dpi ( Fig.…”

Plant begomoviruses subvert ubiquitination to suppress plant defenses against insect vectors

“…A fragment (300-500bp) of each targeted gene was amplified from tobacco leaf cDNA using a gene-specific primer pair (S1 Table) and cloned into pBIN2mDNA1 plasmid to generate the gene-silencing vectors [72]. Then we transformed the vectors into A .…”

“…tumefaciens strain EHA105 by electroporation. The method for VIGS was as described previously [72]. All plants were grown at the same conditions as described above.…”

NbPHAN, a MYB transcriptional factor, regulates leaf development and affects drought tolerance in Nicotiana benthamiana

“…Using the geminivirus alphasatellite-based silencing vector and VIGS (Huang et al 2009, Huang et al 2011, we have shown that silencing of NbPHAN resulting in wrinkled and downward curling leaves, and outgrowth from the adaxial side of the midribs in N. benthamiana leaves (Fig. 3A, B).…”