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

In 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.Fluorescence 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.In recent years the transient expression of proteins in plants has become a favoured procedure over the generation of stably transformed transgenic plants to achieve high levels of protein expression. In contrast to the time-consuming procedure involved in engineering transgenic plants, transient expression methods are more convenient and allow high level protein production in as little as a few days [1,2]. Transient production of proteins is mediated by the introduction of an expression construct into plants which leads to a strong increase in protein synthesis [3]. For high level protein expression, these constructs are preferably under control of a constitutive promoter, e