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

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.

A TOM1 homologue is required for multiplication of Tobacco mosaic virus in Nicotiana benthamiana

The AtTOM1 gene of Arabidopsis thaliana had been shown to be essential for the efficient multiplication of Tobacco mosaic virus(TMV) in A.thaliana.In this study,we cloned an AtTOM1-like gene from Nicotiana benthamiana named as NbTOM1.Sequence alignment showed that NbTOM1 is closely related to AtTOM1 homologues of N.tabacum and Lycopersicon esculentum with 97.2% and 92.6% nucleotide sequence identities,respectively.Silencing of NbTOM1 by a modified viral satellite DNA-based vector resulted in complete inhibition of the multiplication of TMV in N.benthamiana.The result suggests that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants.

The bHLH transcription factor GhPAS1 mediates BR signaling to regulate plant development and architecture in cotton

Cotton (Gossypium spp.) is the most important natural textile fiber crop in the world.The ideal plant architecture of cotton is suitable for mechanical harvesting and productivity in modern agricultural production.However,cotton genes regulating plant development and architecture have not been fully identified.We identified a basic helix-loop-helix (b HLH) transcription factor,GhPAS1 (PAGODA1 SUPPRESSOR1) in G.hirsutum (Upland cotton).GhPAS1 was located in the nucleus and showed a strong transcription activation effect.Tissue-specific expression patterns showed that GhPAS1 was highly expressed in floral organs,followed by high expression in early stages of ovule development and rapid fiber elongation.GhPAS1 overexpression in Arabidopsis and BRZ (brassinazole,BR biosynthesis inhibitor) treatment indicated that GhPAS1 positively regulates and responds to the BR (brassinosteroid) signaling pathway and promotes cell elongation.GhPAS1 overexpression in Arabidopsis mediated plant development in addition to increasing plant biomass.Virus-induced gene silencing of GhPAS1 indicated that down-regulation of GhPAS1 inhibited cotton growth and development,as plant height,fruit branch length,and boll size of silenced plants were lower than in control plants.Fiber length and seed yield were also lower in silenced plants.We conclude that GhPAS1,a b HLH transcription factor,regulates plant development and architecture in cotton.These findings may help breeders and researchers develop cotton cultivars with desirable agronomic characteristics.