Effects of fluorescent tags and activity status on the membrane localization of ROP GTPases

Plant-specific Rho-type GTPases (ROPs) are master regulators of cell polarity and development. Over the past 30 years, their localization and dynamics have been largely examined with fluorescent proteins fused at the amino terminus without investigating their impact on protein function. The moss Physcomitrium patens genome encodes four rop genes. In this study, we introduce a fluorescent tag at the endogenous amino terminus of ROP4 in wild-type and rop1,2,3 triple mutant via homologous recombination and demonstrate that the fluorescent tag severely impairs ROP4 function and inhibits its localization on the plasma membrane. This phenotype is exacerbated in mutants lacking ROP-related GTPase-activating proteins. By comparing the localization of nonfunctional and functional ROP4 fusion reporters, we provide insight into the mechanism that governs the membrane association of ROPs.