Abstract
Ratiometric reporter systems enable comparisons of the abundance of a protein of interest, or “target,” relative to a reference protein. Both proteins are encoded on a single transcript but are separated during translation. This arrangement bypasses the potential for discordant expression that can arise when the target and reference proteins are encoded by separate genes. We generated a set of 18 Gateway‐compatible vectors termed pRATIO that combine a variety of promoters, fluorescent, and bioluminescent reporters, and 2A “self‐cleaving” peptides. These constructs are easily modified to produce additional combinations or introduce new reporter proteins. We found that mScarlet‐I provides the best signal‐to‐noise ratio among several fluorescent reporter proteins during transient expression experiments in Nicotiana benthamiana. Firefly and Gaussia luciferase also produce high signal‐to‐noise in N. benthamiana. As proof of concept, we used this system to investigate whether degradation of the receptor KAI2 after karrikin treatment is influenced by its subcellular localization. KAI2 is normally found in the cytoplasm and the nucleus of plant cells. In N. benthamiana, karrikin‐induced degradation of KAI2 was only observed when it was retained in the nucleus. These vectors are tools to easily monitor in vivo the abundance of a protein that is transiently expressed in plants, and will be particularly useful for investigating protein turnover in response to different stimuli.
Highlights
Dynamic monitoring of protein abundance in vivo requires an detectable reporter system
We found that KAI2-mScarlet-I had retained its 2A peptide in pRATIO1212-KAI2 samples, indicating the LP4 linker component of *FMDV 2A (F2A) was not effective (Supplemental Figure 2)
Having developed ratiometric dual-fluorescent and dual-luminescent reporters, we investigated whether KAR-induced degradation of Arabidopsis KAI2 can be observed in tobacco
Summary
Dynamic monitoring of protein abundance in vivo requires an detectable reporter system. Isolation of multiple homozygous transgenic lines is typically required to identify one with consistent and readily detectable expression of the target. The cost and time involved in developing many transgenic reporter lines can discourage rapid progress. This can be resolved with transient transformation and expression of a reporter construct, which in plant biology research is often carried out in protoplasts or Nicotiana benthamiana (hereafter referred to as tobacco) leaves (Yang et al, 2000; Wroblewski et al, 2005). A second reporter protein that can function as a reference is useful to normalize for differences in transformation efficiency or transgene expression across samples
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