A two-component RUBY reporter system to assess plant virus infection

The use of plant virus vectors to express reporter genes has significantly enhanced the study of virus biology. In this communication we expand the list of virus vectored reporter systems to include the RUBY visible reporter. RUBY utilizes a three-enzyme system to produce betalain from the amino acid tyrosine, a natural pigment responsible for the bright red coloration in beets. To adapt this system for virus expression one of the three enzymes, L-DOPA 4,5-dioxygenase (DODA), was engineered into tobacco mosaic virus (TMV) and turnip mosaic virus (TuMV) vectors. The two remaining enzymes required for betalain production, P450 oxygenase CYP76AD1 and glucosyltransferase (GT), were transformed for expression in the host plant Nicotiana benthamiana. Inoculation of TMV and TuMV vectors expressing DODA onto transgenic CYP76AD1-GT plants resulted in the production of visible red tissues that corresponded with virus infection. The development of this system provides a non-invasive and robust means to directly visualize virus infection without the need for destructive sampling, special lighting equipment or enzymatic substrates.