The fluorescence-activating and absorption-shifting tag (FAST), a versatile protein marker for live plant cell...

The development of real-time imaging has progressed spectacularly in recent years, largely due to the availability of multiple fluorophores with distinct spectral properties. This progress enables the simultaneous imaging of an increasing number of targets, providing access to complex biological processes occurring during development, signaling, and metabolism. In this study, we successfully tested the transfer of three different fluorescence-activating and absorption-shifting tags (FAST), originally developed in animal systems and yeast, to plants. These small (14 kDa) proteins associate non-covalently and reversibly with specific chemicals known as fluorogens, generating bright fluorescence signals that can be reversibly eliminated by washing. This property offers versatile applications. Green- and Red-FAST proteins were successfully used to label proteins targeted to several plant cellular compartments. In contrast, the latest generation of FAST proteins, identified in Rheinheimera sp. A13L (RspA), which accepts multiple fluorogens and offers broad versatility in excitation and emission spectra, was used with a split FAST version to demonstrate cytosolic protein-protein interactions.