I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves

Correct subcellular targeting is crucial for protein function. Protein location can be visualized in vivo by fusion to a fluorescent protein ( FP ). Nevertheless, despite intense engineering efforts, most FP s are dim or completely quenched at low pH (<6). This is particularly problematic for the study of proteins targeted to acidic compartments such as vacuoles ( pH  ~ 3–6) or plant cell walls ( pH  ~ 3.5–8.3). Plant cell walls play important roles (e.g. structural/protective role, control of growth/morphogenesis), are diverse in structure and function, and are highly dynamic (e.g. during cell growth, in response to biotic/abiotic stresses). To study and engineer plant cell walls, it is therefore critical to identify robust tools which can be used to locate proteins expressed in the apoplast. Here we used a transient expression assay in Nicotiana benthamiana leaves to test a range of FP s in vivo , and determined which ones retained strong fluorescence in the acidic environment of the apoplast. We selected 10 fluorescent proteins with a range of in vitro properties; two historical FP s and eight FP s with in vitro properties suggesting lower pH sensitivity or improved brightness, some of which had never been tested in plants prior to our study. We targeted each FP to the cytosol or the apoplast and compared the fluorescence in both compartments, before testing the in vivo pH sensitivity of FP s across a pH 8–4 gradient. Our results suggest that mT urquoise2, mN eonGreen, and mC herry are suited to tracking proteins in the apoplast under dynamic pH conditions. These fluorescent proteins may also be useful in other acidic compartments such as vacuoles.