Virus-induced gene silencing for functional characterization of genes in Petunia

Abstract

Although functional analysis of genes can be readily carried out in Petunia using standard transformation/regeneration techniques, this process is time- and labor-consuming. High throughput analysis of gene knockouts has been made possible by the use of virus-induced gene silencing (VIGS): fragments of target plant genes are included in the genome of a viral vector, the plant silences them as part of its viral defense mechanism, and the consequences of gene inactivation can be readily analyzed. In Petunia, we use a modified tobacco rattle virus (TRV) vector for VIGS. Infection typically results in chimeric plants, and it is therefore desirable to have a reporter that can show where target genes have been silenced. Inserting a fragment of the gene encoding PHYTOENE DESATURASE (PDS) results in silencing-induced photobleaching of leaves; inserting a fragment of the gene encoding CHALCONE SYNTHASE (CHS) allows us to visualize silencing in floral tissues of purple-flowered Petunia cultivars as white patches, sectors or even entire corollas. We have shown that the VIGS system can silence as many as five independent genes at one time. We describe here the methods that we have found to be efficient and effective for VIGS in Petunia, and describe some results obtained by silencing a range of genes, including some transcription factors.