Functional characterization of ROP GTPase interactive proteins in Arabidopsis thaliana

ROP GTPase have emerged to be key molecular switches regulating multiple processes including polar growth, diffuse growth, hormone responses, defense responses and organogenesis. The diversity of ROP GTPases signaling is presumably achieved partly through its diverse targets or effector proteins. A genome-wide identification revealed 11 ROP targets, termed RICs (R&barbelow;OP-i&barbelow;nteractive C&barbelow;RIB motif-containing proteins), which contain a CRIB (CDC42/RAC interactive binding) motif required for their specific interaction with GTP-bound ROP1.{09}RICs are divergent and classified into five groups that share little sequence homology outside of the conserved CRIB domain. Overexpression in tobacco pollen tubes of the 9 RIC genes that are expressed in Arabidopsis pollen causes distinct phenotypes, implying distinct functions for various RICs. I propose that RICs function as ROP GTPase targets that control various ROP-dependent signaling pathways in plants. Based on the observation that overexpression of RIC3 and RIC4 phenocopy ROP1OX in certain degree, I further tested whether RIC and RIC4 fall into ROP1-dependent pathway and how does ROP regulate pollen tube growth through RIC3 and RIC4-mediated pathway. I demonstrate that ROP-dependent tip growth and actin dynamics requires a balance action of two opposing ROP1 downstream pathways. ROP1 activation of RIC4 promotes F-actin assembly in the tip of pollen tubes, whereas ROP1 activation of RIC3 controls calcium signaling that promotes actin depolymerization. Therefore this is the first demonstration of a Rho GTPase-mediated signaling pathway with two counteracting branches, acting coordinately to control actin dynamics and coordinating spatial and temporal regulation of polar growth.; To identify RICs' function in other ROP-dependent pathway, I generated various ric gain-of-function and loss-of-function mutants. My main effort was to address whether any of RICs are involved in ROP2 controlled leaf epidermal cell morphogenesis. This study reveals two putative ROP2 targets (RIC1 and RIC4) in the controlling of actin- and MT-mediated pathways. RIC1, associated with cortical MTs, negatively regulate the neck expansion possibly through the formation of transverse MTs. RIC4, colocalized with cortical F-actin, positively regulate the lobe formation as well as cell expansion possibly through the promotion of F-actin formation in the cell cortex of pavement cells.; In my initial attempt to unravel the mystery how ROP signaling is activated upon extracellular stimuli, I isolated several putative ROP activators through yeast two-hybrid screen. The functional analysis of these putative ROP activators will reveal new insights on ROP signaling.