AGC Kinases Regulate The Tip Growth Of Pollen Tubes And Root Hairs Through Phosphorylation Of RopGEFs

Tip growth is a specialized type of cell polarization in eukaryotes,whose controlling toolbox inevitably includes the asymmetric distribution of Rho GTPases.Rho of Plants?ROP?represents the only Rho GTPases in plants,participating in a full spectrum of developmental and environmental responses,especially in tip-growing pollen tubes and root hairs,by regulating an array of intracellular activities such as dynamic polymerization of actin microfilaments?MF?.Twenty years of extensive studies on ROP signaling uncovered the downstream effectors?such as ICRs?and regulators?such as guanine nucleotide exchange factors?RopGEFs??of ROPs.However,positive feedback controlling their dynamic asymmetry,a necessity for all signaling systems,is elusive.We report here an elegant regulate mechanism formed by sequential oligomerization and phosphorylation.Briefly,initial accumulation of ROP-GTP at a restricted membrane domain results in the establishment of tip growth.ROP-GTP interacts with its effector ICR2,which in turn interacts with cytoplasmic AGC kinases for their membrane recruitment and activation toward substrates,and which depend on dynamic of microtuble.Then the catalytic PRONE domains of RopGEFs?plant-specific guanine nucleotide exchange factors?interact with and are phosphorylated by AGC kinases,resulting in spatiotemporal accumulation and activation of RopGEFs in vivo.ROP activation by RopGEFs then reinforce the ROP-GTP domain to ensure a single polarization site during tip growth.Such a positive feedback loop is essential to maintain a single and constant polarity cluster in plant tip-growing cells.The main results and conclusions presented in this thesis are as follows:?1?AGC kinases function through phosphorylationAGC1.5 as a functional kinase was firstly tested by an autophosphorylation assay.By contrast,the K214Q mutation abolished its autophosphorylation.Indeed,only wild-type AGC1.5 but not AGC1.5K214Q was able to suppress the meandering growth of agc1.5;agc1.7 pollen tubes.On the other hand,overexpressing wild-type AGCs,but not their kinase-dead mutants,resulted in 3D helical root hairs.These results demonstrated that AGC kinases are active kinases.?2?ROP signaling and AGC kinases are interdependentOn the one hand,ROP signaling depends on AGC activity.The fluorescent probe for ROP-GTP,CRIB_RIC1-mRFP dynamically localize at the apical plasma membrane?PM?of growing pollen tubes in wild type,which is consistent with their specific interaction with GTP-bound ROPs.However,in growing pollen tubes of agc1.5agc1.7,their localization patterns were much expanded at the apical PM.On the other hand,the function of AGC kinases relies on ROP signaling.OX of AGC1.5resulted in 3D helical growth of root hairs,indicating altered growth polarity.Introducing mutants in which ROP signaling was compromised,such as scn1-1,a mutant of guanine nucleotide dissociation inhibitor?RhoGDI?,ROP2DN,or PRONE1L103D,abolished the 3D helical morphology in root hairs.These results demonstrate that AGC activity also relies on ROP signaling.?3?AGC1.5 interacts with activators and effectors of ROP GTPasesAGC1.5 but not the AGC1.5K214Q mutant,interacted with the catalytic PRONE domains of RopGEFs.And,a mutant version that abolished the ability of PRONE1 to dimerize?PRONE1L103D?failed to interact with AGC1.5.ICR2 interacted only with wild-type AGC1.5 but not with AGC1.5K214Q.AGC1.5 interacts with the C-terminal coiled-coil domain?CT or?NT?of ICR2,where active ROP GTPases bind.Mutations that abolished the ROP-ICR2 interaction also abolished the interaction between ICR2 and AGC1.5.?4?AGC kinases phosphorylates RopGEFsAGC1.5 phosphorylates the PRONE domains of RopGEF1 and RopGEF12?PRONE1and PRONE12?in vivo.Mutation at S333 of RopGEF1 substantially reduced AGC1.5-dependent PRONE1 phosphorylation.However,the interaction between ROPs and RopGEFs and the ability of PRONE1 to dimerize were not affected by the mutantion of PRONE1 or PRONE12.Besides,using an in vitro GEF activity assay,we showed PRONE1S333A catalyzed guanine nucleotide exchange of ROP2 comparably to wild-type PRONE1.We tested the effects on the function of PRONE1 in vivo.Both PRONE1 and PRONE1S333D OX resulted in bulged growth and localization restricted to the apical PM of growing pollen tubes.By contrast,the pollen tubes of PRONE1S333A OX grew comparably to non-transformed pollen tubes,but,PRONE1S333A localized randomly into several PM patches instead of being restricted at the apical PM.Besides,the expression of PRONE1 in agc1.5agc1.7 was similar to the expression of PRONE1S333A in wild-type pollen tubes.The results suggested that phosphorylation of PRONE1S333 relies on AGC kinases.Similar results were obtained in root hairs.These results further confirmed that the phosphorylation of PRONE1S333 is critical for its spatiotemporal restriction at a single membrane domain.?5?ICR2 can recruit and activate AGC kinases depending on the dynamic polymerization of microtubleTo determine the biological relevance of the ICR2-AGC interaction,we analyzed AGC localization with or without ICR2.When expressed alone in root hairs,AGC1.5 targeted to the subapical PM,but was excluded from the very apex of initiating or elongating root hairs,in addition to cytoplasmic signals.By contrast,AGC1.5K214Q was cytosolic.Co-expression with ICR2 resulted in the re-distribution of AGC1.5 but not AGC1.5K214Q to the apical PM,correlating with the growth axis.Consistent with the overexpression results,functional loss of ICR2 and MIDD1 rendered AGC1.5 cytosolic.In addition,the 3D helical growth of root hairs overexpressing AGC1.5 was abolished in icr2midd1.Besides,Taxol and oryzalin treatmeat both change the localization of AGC1.5 and suppress the phenotype of AGC1.5 OX in root hairs.These results suggest that ICR2 recruits AGC1.5 to active ROP membrane domains depending on the dynamic polymerization of microtuble.