Visual Tracking of Plant Virus Infection and Movement Using a Reporter MYB Transcription Factor That Activates Anthocyanin Biosynthesis  

Abstract

Insertion of reporter genes into plant virus genomes is a common experimental strategy to research many aspects of the viral infection dynamics. Their numerous advantages make fluorescent proteins the markers of choice in most studies. However, the use of fluorescent proteins still has some limitations, such as the need of specialized material and facilities to detect the fluorescence. Here, we demonstrate a visual reporter marker system to track virus infection and movement through the plant. The reporter system is based on expression of Antirrhinum majus MYB-related Rosea1 (Ros1) transcription factor (220 amino acids; 25.7 kD) that activates a series of biosynthetic genes leading to accumulation of colored anthocyanins. Using two different tobacco etch potyvirus recombinant clones tagged with Ros1, we show that infected tobacco (Nicotiana tabacum) tissues turn bright red, demonstrating that in this context, the sole expression of Ros1 is sufficient to induce pigment accumulation to a level readily detectable to the naked eye. This marker system also reports viral load qualitatively and quantitatively by means of a very simple extraction process. The Ros1 marker remained stable within the potyvirus genome through successive infectious passages from plant to plant. The main limitation of this marker system is that color output will depend on each particular plant host-virus combination and must be previously tested. However, our experiments demonstrate accurate tracking of turnip mosaic potyvirus infecting Arabidopsis (Arabidopsis thaliana) and either tobacco mosaic virus or potato X virus infecting Nicotiana benthamiana, stressing the general applicability of the method.

Tobacco etch virus Protein P1 Traffics to the Nucleolus and Associates with the Host 60S Ribosomal Subunits during Infection

“…1A). In this clone, the GFP is released from the viral polyprotein through the proteolytic activities of P1 and NIaPro proteinases (28). In TEVVenusP1-mCherryNIb, in addition to the Venus fusion to P1, the red fluorescent protein mCherry (30) was fused to the amino terminus of NIb (Fig.…”

“…TEV recombinant clones containing mutations in the P1 cistron (see Fig. S3 in the supplemental material) and the transcription factor Rosea1 (Ros1) as a reporter marker (28) were agroinoculated in two different leaves of batches of 10 N. benthamiana plants. Infection symptoms in these plants were recorded over time by visual inspection.…”

“…Viral load was estimated from the anthocyanin accumulation induced by the Ros1 reporter marker activity. Anthocyanins in infected tissues from three different plants for each viral construct were extracted in acidified methanol and quantified spectrophotometrically at 530 nm (28).…”

Relocation of the NIb Gene in the Tobacco Etch Potyvirus Genome

“…The parental and recombinant TEV clones, constructed from the binary plasmid pGTEV-Ros1 (10,11), were agroinoculated (12) into two leaves of 20 3-week-old wild-type plants and 20 transgenic plants constitutively expressing TEV NIb (8). Plants were grown in a glasshouse at 25°C with 16 h light, and Ros1 expression was visually monitored for 4 weeks.…”

“…For each clone, systemic leaves from three wild-type and three transgenic plants were harvested at 15 days postinoculation (d.p.i. ), photographed, and used to estimate viral load by measuring the anthocyanin accumulation induced by the Ros1 marker (10).…”