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.
Highlights
Transient 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
We report here the differences in efficiency of two plant viral suppressors, Beet mild yellowing virus P0 (BMYV-P0) and Plum pox virus HC-Pro (PPV-HC-Pro), using a novel whole leaf fluorescent imaging methodology, and demonstrate that over time both viral suppressor proteins can significantly effect green fluorescent protein (GFP) expression for up to 21 days post-agroinfiltration
Quantitative analysis of transgene and transient GFP expression from intact leaves In order to verify whether our imaging system was suitable to detect GFP expression from plant tissue, leaves of GFP-transgenic and non-transgenic N. benthamiana plants of the same age were harvested and observed simultaneously side-by-side
Summary
Transient 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. 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. 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]. Interference by autofluorescence of plant tissues can often be reduced or eliminated by specific optical filters [15]
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