Abstract：Rop(Rho-related GTPase of plant)is the plant-specific signaling small GTPase. It can start and terminate multiple cell behavious by converting its active guanosine triphosphate(GTP)-bound form and inactive guanosine diphosphate(GDP)-bound form. The interconversion of the two forms of Rop GTPases in plant is strictly regulated by three types of regulators: GTPase activating proteins (RopGAPs), guanine nucleotide exchange factors (RopGEFs) and guanine nucleotide dissociation inhibitors (RopGDIs). In this study, with the bait of CA-CaRop1 which is the GTP-bound form mutant of a Capsicum annuum L. Rop, CaRop1, we screened a regulator of Rho GTPase activating protein, designated as CaRopGAP3, from the prey library of the pepper seedling by ProQuestTM two-hybrid system. Blastp analysis showed that the deduced amino acid sequence of CaRopGAP3 not only had conserved domains of GAP but also had the Cdc42/Rac-interactive binding (CRIB) domain, which indicated that the CaRopGAP3 was a CRIB-containing RopGAP subfamily that was unique to the regulation of the Rop GTPase of plant. Yeast two-hybrid analysis showed that CaRopGAP3 could interact with constitutively active(CA)-CaRop1 but not with dominant negative(DN)-CaRop1, and the interaction was not obviously regulated by its CRIB-containing N-terminus. Subcellular localization analysis showed that the full-length CaRopGAP3 focused mainly on the cytomembrane while the truncated mutant that the CRIB-containing N-terminus was deleted dispersively distributed in the whole cytoplasm, which indicated that the CRIB-containing N-terminus may play important role in the membrane localization of CaRopGAP3. Real-time PCR analysis showed that the expression level of CaRopGAP3 in different tissues and organs were obvious different, the level in the young leaf was highest and was about 17-fold higher than that in the young root, 8-fold higher than that in the mature root and flower. The structure characterizations and the expression pattern of the CaRopGAP3 may be closely associated with the signaling pathway it involved, and may be important for clarifing the signaling regulation mechanism of the CaRop1 in pepper further.
