In vivo Rac/Rop localization as well as interaction with Rho GAP and Rho GDI in tobacco pollen tubes: analysis by low-level expression of fluorescent fusion proteins and bimolecular fluorescence complementation.

Polarized Rac/Rop GTPase signaling plays a key role in polar cell growth, which is essential for plant morphogenesis. The molecular and cellular mechanisms responsible for the polarization of Rac/Rop signaling during polar cell growth are only partially understood. Mutant variants of Rac/Rop GTPases lacking specific functions are important tools to investigate these mechanisms, and have been employed to develop a model suggesting that Rho GAP ( GTPase activating protein) and Rho GDI (Guanine Nucleotide Dissociation Inhibitor) mediated recycling of Rac/Rop GTPases maintains apical polarization of Rac/Rop activity in pollen tubes, which elongate by 'tip growth' (an extreme form of polar cell growth). Despite the importance of these mutant variants for Rac/Rop functional characterization, their distinct intracellular distributions have not been thoroughly comparatively and quantitatively analyzed. Furthermore, support for the proposed Rho GAP and Rho GDI functions in apical polarization of Rac/Rop activity based on the analysis of in vivo interactions between these proteins and Rac/Rop GTPases has been missing. Here, extensive fluorescent protein tagging and bimolecular fluorescence complementation (Bi FC) analyses are described of the intracellular distributions of wild type and mutant variants of the tobacco pollen tube Rac/Rop GTPase Nt-Rac5, as well as of interactions of these Nt-Rac5 variants with Rho GAP and Rho GDI proteins, in normally growing transiently transformed pollen tubes. Presented results substantially enhance our understanding of apical dynamics of pollen tube Rac/Rop signaling proteins, confirm previously proposed Rho GAP and Rho GDI functions in Rac/Rop polarization and provide important technical insights facilitating future in vivo protein localization and Bi FC experiments in pollen tubes. [ABSTRACT FROM AUTHOR]