Tobacco RIC11pt differentially interacts with multiple RAC/ROP-GTPases providing a link to CAR4/GAP1

Author:  Octavian O. H. Stephan

Pre-published:  16 October 2019

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To date our knowledge about plant RAC/ROP-GTPase effectors and their downstream targets is still limited. This work describes the identification of a novel tobacco RAC-effector using yeast two-hybrid methodology. Nt-RIC11pt belongs to the RIC-effector family and shares an N-terminal CRIB-motif (Cdc42/Rac interactive binding) and a conserved C-terminal consensus sequence with its uncharacterized homolog At-RIC11. Y2H and pull down assays show that GST-tagged RIC11pt specifically interacts with constitutively active as well as GTPγS loaded myc:RAC5. Quantitative RT-PCR analysis of different tobacco tissues reveals that Nt-RIC11pt is specifically expressed in mature pollen and growing pollen tubes. Transiently expressed Nt-RIC11pt:YFP is homogenously localized in the cytoplasm of pollen tubes. Remarkably, RIC11 binds with differential affinity to various additional RACGTPases, such as RAC1 and RAC3, implicating a multifaceted role in an interconnective RIC-RAC network. Moreover, Y2H-screening for downstream targets identified Nt-CAR4, which is phylogenetically assigned to a multifaceted family of unusual GTPase activating proteins (GAP) that contain a C2-phospholipid-binding domain. In conclusion, a signal transduction network related to ABA-response is proposed, in which the scaffold RIC11 connects active RAC3 with membrane-bound CAR4, thus relaying GAP-activity, whereby the Ca2+-dependency of CAR4 membrane association implies a role for the pollen tube tip directed calcium gradient.

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