Phosphatidic acid binds to and regulates guanine nucleotide exchange factor 8 (GEF8) activity in Arabidopsis

Phosphatidic acid (PA) forms part of plant lipid metabolism and is a signalling molecule used in response to various external stresses. Guanine nucleotide exchange factors (GEFs) activate small GTPase ROPs, serving as molecular switches in a wide range of signalling pathways. However, the interaction between PA and GEFs in plants has not yet been reported. Here we show that PA bound specifically to GEF8 by using fat-Western blot and isothermal titration calorimetry assays. A C-terminal truncation of GEF8 exhibited strong PA binding, and mutation of lysines 13 and 18 in GEF8 PRONE domain caused a total loss of binding to PA. Two ROPs, ROP7 and ROP10, were identified as preferred substrates of GEF8 by pull-down and bimolecular fluorescence complementation assays. GEF8 activity towards ROP7, but not ROP10, was stimulated by PA both in vitro and in cells. Moreover, the PA- or ABA-induced activation of GEF8 was completely lost in the mutant GEF8, which did not bind to PA. Together, these findings identify a direct interconnection between PA-mediated GEFs activity and small GTPase signalling in plants and provide evidence for a synergistic activation of GEF8 by direct PA-binding to its PRONE domain.