Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

Abstract

BackgroundTransient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP)-based imaging assay to quantitatively assess suppressor protein activity.ResultsIn a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP) P0 or Plum pox virus (PPV) HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi) when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi) and P0 giving higher long term GFP fluorescence (at 21 dpi). Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas.ConclusionsFluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants.

Disruption of the methionine cycle and reduced cellular gluthathione levels underlie potex–potyvirus synergism in Nicotiana benthamiana

“…Owing to their stability and ease of quantification, fluorescent protein‐based detection methods have garnered wide acceptance in plant studies. They can be imaged as well as quantified accurately from intact leaf tissues without any need for cell lysis or sample preparation (Dhillon et al ., ; Richards et al ., ; Stephan et al ., ). The expression of fluorescent proteins from viral genomes within the host cell can accurately reflect the viral gene expression level.…”

Exploring the Multifunctional Roles of Odontoglossum Ringspot Virus P126 in Facilitating Cymbidium Mosaic Virus Cell-to-Cell Movement during Mixed Infection

“…The agroinfiltrated leaves were illuminated under a long-wavelength UV lamp (Black Ray model B 100 AP) and photographed at 4 days post-agroinfiltration (DPA). The GFP signal intensity was detected and measured by using an IVIS Lumina III LT in vivo Imaging System (XENOGEN Co., Alameda, CA, USA) [ 46 ]. All experiments were repeated three times.…”

Mutational analysis of Arabidopsis thaliana ABCE2 identifies important motifs for its RNA silencing suppressor function

“…Because of high variability in GFP fluorescence intensity between leaves, the levels of GFP expression were always compared separately between infiltrated patches in each leaf. At 5 dpi, GFP fluorescence was quantified and analysed using IVIS Lumina II (Caliper Life Sciences, Waltham, MA, USA) and Living Image (version 4.1) software as described in Stephan et al (2011) including the modifications reported in Kärblane et al (2015). For the local silencing suppression assay, we measured radiant efficiency of GFP in agroinfiltrated leaves and analysed the difference between two patches in the same leaf using a paired two‐tail t ‐test.…”