New insights into the dimerization of small GTPase Rac/ROP guanine nucleotide exchange factors in rice

Abstract

Molecular links between receptor-kinases and Rac/ROP family small GTPases mediated by activator guanine nucleotide exchange factors (GEFs) govern diverse biological processes. However, it is unclear how the Rac/ROP GTPases orchestrate such a wide variety of activities. Here, we show that rice OsRacGEF1 forms homodimers, and heterodimers with OsRacGEF2, at the plasma membrane (PM) and the endoplasmic reticulum (ER). OsRacGEF2 does not bind directly to the receptor-like kinase (RLK) OsCERK1, but forms a complex with OsCERK1 through OsRacGEF1 at the ER. This complex is transported from ER to the PM and there associates with OsRac1, resulting in the formation of a stable immune complex. Such RLK-GEF heterodimer complexes may explain the diversity of Rac/ROP family GTPase signalings.

The Rac/ROP small GTPases constitute a plant-specific Rho subfamily and participate in diverse signal transduction processes, including disease resistance, pollen tube growth, root hair development, ROS production, cell wall patterning and hormone responses.^1-5 Small GTPases undergo exchange reactions between an inactive GDP-bound form and an active GTP-bound form to actuate functional switches that play key roles in cell dynamics. In these reactions, guanine nucleotide exchange factors (GEFs) facilitate the exchange of GDP for GTP, allowing the small GTPases to interact with downstream target proteins. Accumulating evidence suggests that signaling involving Rac/ROPs is positively regulated by various receptor-like kinases (RLKs) through Rac/ROPGEFs. The RLK-Rac/ROPGEF-Rac/ROP module appears to be widely used as the core machinery of Rac/ROP signaling in diverse plant activities.^6-8

Pattern recognition receptors (PRRs) at the cell surface are the first line of defense against pathogen infections.⁹ Microbe-derived molecules recognized by PRRs are called microbe-associated molecular patterns (MAMPs).¹⁰ Host perception of MAMPs activates a variety of immune responses, termed MAMP-triggered immunity (MTI). Most PRRs are RLKs or receptor-like proteins (RLPs). Chitin derived from pathogenic and non-pathogenic fungi is one of the best-characterized MAMPs,¹⁰ and 2 lysine motif (LysM)-containing PRRs, OsCEBiP and OsCERK1, play a key role in chitin signaling.^11,12 OsCEBiP is an RLP that lacks an intracellular kinase domain and directly binds chitin, while OsCERK1 is an RLK and does not directly bind chitin. These two receptor proteins form a receptor complex to transduce chitin signals to downstream target proteins.¹²

The rice small GTPase OsRac1 participates in defense signaling induced by fungal pathogen-derived chitin.^13,14 OsRac1 regulates ROS production through its interaction with the N-terminal region of NADPH oxidase.^2,15,16 Furthermore, OsRac1 is also involved in defense responses induced by nucleotide binding domain and leucine-rich repeat (NLR) family resistance (R) proteins such as Pit and Pia.^1,13,17,18 A family of Rac/ROP GEFs whose members contain the plant-specific Rac/ROP nucleotide exchange factor (PRONE) domain for GEF activity has been identified in plants.^6,19 We recently characterized a PRONE GEF for OsRac1, termed OsRacGEF1, which forms a complex with OsRac1 as well as the rice chitin receptor OsCERK1 or the flagellin receptor OsFLS2 at the plasma membrane (PM) of rice cells.^12,20,21 OsRacGEF1 is rapidly phosphorylated by the cytoplasmic domain of OsCERK1 upon chitin treatment at the PM, resulting in enhanced activation of OsRac1 by OsRacGEF1 to implement resistance to rice blast fungus.²¹ This OsCEBiP/OsCERK1-OsRacGEF1-OsRac1 module contributes to the immune response induced by chitin in rice.²¹ Previous structural studies showed that the PRONE domain of AtROPGEF8 is a constitutive homodimer, and that its dimerization is essential for ROP activation.²² However, the physiological roles of the dimerization of GEFs in plant cells remain elusive.

To investigate the molecular mechanism of OsRac1 activation by OsRacGEF1, we searched for interacting partner(s) of OsRacGEF1 by yeast 2-hybrid screens using a rice cDNA library constructed from chitin-treated rice suspension cells.²³ Two OsRacGEFs, Os09g0544800 (OsRacGEF2) and Os01g0849100 (OsRacGEF1), were found to interact with OsRacGEF1. OsRacGEF2 comprises 546 amino acids, and possesses a highly conserved PRONE domain (aa 72–443) and variable N (aa 1–71) and C termini (aa 444–546) (Fig. 1A). We performed yeast 2-hybrid assays using WT, the PRONE domain, and the N- and C-terminal regions of both OsRacGEF1 (Fig. 1B, left) and OsRacGEF2 (Fig. 1B, right) as prey, and WT OsRacGEF1 and OsRacGEF2 as bait. The yeast 2-hybrid results showed that OsRacGEF1 interacted not only with OsRacGEF1 but also with OsRacGEF2 via the PRONE domain (Fig. 1B, left); likewise, OsRacGEF2 also associated with both OsRacGEF1 and OsRacGEF2 through its PRONE domain (Fig. 1B, right). The PRONE domain of GEFs is known to bind directly with, and to display GEF activity toward, small GTPases.¹⁹ Consistent with this, OsRac1 interacted with the PRONE domain of OsRacGEF2 (Fig. 1C) as well as of OsRacGEF1.²¹ A previous structural study has demonstrated that the PRONE domain of AtROPGEF8 forms a homodimer, and that this dimerization is essential for GEF activity toward the small GTPase Arabidopsis ROP4 (AtROP4) in vitro.²² Four residues, F18 and L20-L22, of AtROPGEF8 are important for the formation of this homodimer; indeed, compared to WT, a mutant of AtROPGEF8 (L23D) displays reduced dimer formation and significantly decreased GEF activity toward ROP4.²² These amino acid residues are perfectly conserved in both OsRacGEF1 and OsRacGEF2 (Fig. 1D, dotted-line boxes). Therefore, these results suggest that both OsRacGEF1 and OsRacGEF2 form homodimers and heterodimers with each other through their PRONE domains, in a similar fashion to AtROPGEF8.

Figure 1.

Interaction analysis of OsRacGEFs. (A) Schematic representation of OsRacGEF1 and OsRacGEF2. (B), (C) Yeast cells containing prey and bait constructs were tested for their interaction, and growth on selective plates without histidine and with 3 mM aminotriazole (His(−) 3 mM 3-AT) indicates a positive interaction. The images show growth of each line after 3 d of incubation at 30°C. Empty indicates the vector controls. (B) Representative yeast 2-hybrid assay plates showing interaction between WT, N terminus (aa 1–84), PRONE (aa 85–456) or C terminus (aa 457–561) of OsRacGEF1 and either OsRacGEF1 (WT) or OsRacGEF2 (WT) (left); and between WT, N terminus (aa 1–71), PRONE (aa 72–443) or C terminus (aa 444–546) of OsRacGEF2 and either OsRacGEF1 (WT) or OsRacGEF2 (WT) (right). (C) Representative yeast 2-hybrid assay plates showing interaction between OsRac1 (WT) and WT, N terminus, PRONE or C terminus of OsRacGEF2. (D) Portion of a multiple sequence alignment of OsRacGEF1, OsRacGEF2 and AtROPGEF8. Highlighted residues are important for the interaction between ROP and PRONE.²² Boxes bordered with dotted, blue, red and green lines indicate the GEF-GEF interaction domain, subdomain I, subdomain II and β-turn, respectively.

The aforementioned structural study also demonstrated that the PRONE dimer forms a tetrameric complex with 2 ROP GTPases.²² The PRONE domain of AtROPGEF8 is composed almost entirely of α helical structure (α1–13), except for a β turn that protrudes from the main body of the PRONE domain. The overall structure of the AtROPGEF8 PRONE domain can thus be divided into 3 parts: subdomain I (α1–5 and α13), subdomain II (α6–12) and the β turn.²² AtROPGEF8 has 2 interfaces that make contact with 2 ROP molecules: subdomains I and II make the interface to contact one AtROP4, and the β turn binds to another AtROP4 molecule.²² We therefore next compared PRONE domain amino acid sequences among OsRacGEF1, OsRacGEF2 and AtROPGEF8. The blue, red and green boxes in Figure 1D highlight the important residues in AtROPGEF8 for binding to AtROP4 in subdomain I, subdomain II and the β turn, respectively. The important residues for the interaction between AtROPGEF8 and AtROP4 are highly conserved. Together, these results suggest that OsRac1 is also capable of forming a tetrameric complex, which consists of 2 OsRacGEFs and 2 OsRac1 molecules.

We have previously shown that OsRacGEF1 localizes to the cytoplasm and the endoplasmic reticulum (ER) and is transported to the PM to form a receptor complex with OsCERK1 and OsRac1. This OsCERK1-OsRacGEF1-OsRac1 module triggers rapid activation of OsRac1 upon chitin treatment to induce rice immunity.²¹ We next examined the intracellular localization of OsRacGEF2 using fluorescent probes in rice protoplasts. Protoplasts were transformed with OsRacGEF2-Venus and organelle markers, and analyzed under a microscope (Fig. 2). The fluorescent signals of OsRacGEF2 overlapped highly with punctate structures visualized by the ER marker CFP-HDEL, but not in the nucleus (Fig. 2A and B), indicating that OsRacGEF2 localizes mainly to the ER (Fig. 2B). However, some signals were also detected at the cis-Golgi network (CGN) (Fig. 2C), the trans-Golgi network (TGN) (Fig. 2D) and the late endosome (LE) (Fig. 2E), but not at the mitochondria (Fig. 2F), the peroxisome (Fig. 2G) or the plastid (Fig. 2H). However, only a small part of GEF2 is overlapped with CGN, TGN, and LE markers and there are many independent signals. Further studies are necessary to clarify the localization of those signals.

Figure 2.

Localization of OsRacGEF2 in rice cells. Rice protoplasts, isolated from rice cultured cells, were cotransformed with the fluorescent constructs OsRacGEF2-Venus and mCherry (A) or CFP-HDEL as an ER marker (B), and OsRacGEF2-mCherry and GFP-Erd2 as a CGN marker (C), GFP-SYP61 as a TGN marker (D), GFP-ARA6 as an LE marker (E), GFP-ATPase delta-prime subunit as a mitochondria maker (F), GFP-PTS1 as a peroxisome marker (G) or GFP-L12 as a plastid marker (H). Ten to 16 h after transformation, the cells were imaged using a Leica SP5 inverted microscope. White arrows indicate merged signals. Bars in (A-H), 5 μm. N, ER, CGN, TGN and LE indicate nucleus, endoplasmic reticulum, cis-Golgi network, trans-Golgi network and late endosome, respectively.

To further confirm the interaction between OsRacGEF2 and OsRac1 in rice cells, we conducted bimolecular fluorescence complementation (BiFC) assays.^14,24 When OsRac1 tagged with the N-terminal domain (aa 1–154) of Venus (Vn-OsRac1) and OsRacGEF2 tagged with the C-terminal domain (aa 155–238) of Venus (OsRacGEF2-Vc) were co-expressed in rice protoplasts, the fluorescent signal of the reconstituted Venus was detected at the PM, indicating that OsRacGEF2 associates there with OsRac1 (Fig. 3A). Further BiFC results showed that OsRacGEF2 homodimerizes mainly at the ER (Fig. 3B a), and that it heterodimerizes with OsRacGEF1 at the PM, the cytoplasm and the ER (Fig. 3B k).²¹ Likewise, OsRacGEF1 homodimerizes at the ER, the cytoplasm and the PM (Fig. 3B f). We next examined whether the expression of CFP-OsRac1 alters the localization of the OsRacGEF2 and OsRacGEF1 homodimers, or of the OsRacGEF1-OsRacGEF2 heterodimer. Interestingly, in the presence of CFP-OsRac1, all 3 dimers dramatically moved to the PM (Fig. 3B b, g and l), where OsRac1 localizes (Fig. 3B c, h and m), indicating that OsRac1 tethers OsRacGEF1 and OsRacGEF2 at the PM. Overall, these results suggest strongly that 2 OsRac1 molecules form a complex with the OsRacGEF2 or OsRacGEF1 homodimer, or with the OsRacGEF1-OsRacGEF2 heterodimer, at the PM as reported for AtROPGEF8 and AtRop4.²²

Figure 3.

Interaction analysis using BiFC assays. Rice protoplasts were isolated from rice cultured cells and co-transformed with BiFC constructs, (A) OsRacGEF2-Vc and Vn-OsRac1. mCherry was used as an internal control. (B) Upper images, OsRacGEF2-Vc and OsRacGEF2-Vn without (a) or with CFP-OsRac1 WT (b-e). Middle images, OsRacGEF1-Vc and OsRacGEF1-Vn without (f) or with CFP-OsRac1 WT (g-j). Lower images, OsRacGEF2-Vc and OsRacGEF1-Vn without (k) or with CFP-OsRac1 WT (l-o). (C) OsRacGEF2-Vn and OsCERK1-Vc. CFP-HDEL was used as an ER marker. (D) Upper images, OsCERK1-Vc, OsRacGEF2-Vn and CFP-OsRacGEF1. Lower images, OsFLS2-Vc, OsRacGEF2-Vn and CFP-OsRacGEF1. (A-D) Expression of these genes was driven by the CaMV 35S promoter. GUS served as a negative control (data not shown). Bars in (A-D), 5 μm. (E) Yeast-2 hybrid assay of OsCERK1 and OsRacGEF2. Yeast cells containing prey and bait constructs were tested for their interaction, growth on selective plates without histidine (His(−)) indicates a positive interaction. The results show the growth of each line after 3 d of incubation at 30°C. Representative plates of yeast 2-hybrid assay showing interaction between the cytoplasmic domain (CD) of OsCERK1 and OsRacGEF2 (WT). Empty indicates vector controls. (F) Localization patterns of OsRacGEF2 in the presence of WT- or CA-AtSar1. Rice protoplasts were co-transformed with OsRacGEF2-mCherry and either WT-AtSar1-Rer1B-GFP or CA-AtSar1-Rer1B-GFP. Rer1B was used as a Golgi marker. Bars, 5 μm.

We next investigated the interaction between OsRacGEF2 and the chitin receptor OsCERK1 (Fig. 3C and D). The BiFC signal of OsRacGEF2 and OsCERK1 was mainly detected at the PM and the ER. More than 70% of OsRacGEF1 expressing cells showed the BiFC signal whereas only 45% of OsRacGEF2 expressing cells demonstrated this effect (Fig. 3C, right). We also examined the interaction between OsRacGEF2 and the flagellin receptor OsFLS2. The BiFC signal of OsRacGEF2 and OsFLS2 was mainly detected at the PM and the ER (Fig. 3D, lower). These results suggest that OsRacGEF2 forms a complex with OsCERK1 and OsFLS2 at the ER and the PM (Fig. 3D). These interactions may have been mediated by endogenous OsRacGEF1, because OsRacGEF2 could not directly bind to the cytoplasmic domain (CD) of OsCERK1 in the yeast 2-hybrid assay (Fig. 3E and Fig. S1), and coexpression of OsRacGEF1-CFP increased the complex formation between OsRacGEF2 and OsCERK1 by approximately 40% when compared with control CFP (Fig. 3D, right, bar 3 and 4). Similarly, in the presence of OsRacGEF1, complex formation between OsRacGEF2 and OsFLS2 was also elevated from 45% to 78% (Fig. 3D, right, bar 7 and 8). We have previously found that OsRacGEF1 is phosphorylated at S549 by the kinase domain of OsCERK1 in response to chitin.²¹ Together, these results show that OsRacGEF2 interacts with the RLKs OsCERK1 and OsFLS2 through OsRacGEF1.²¹

We have demonstrated previously that OsCERK1 and OsRacGEF1 are transported from the ER to the PM through a Sar1-dependent vesicle trafficking pathway.^14,21,25 Since OsRacGEF2 forms a complex with OsCERK1 at the ER in rice protoplasts (Fig. 3D), we examined whether the transport of OsRacGEF2 is also regulated by Sar1-dependent vesicle trafficking. It is known that a constitutively active Arabidopsis Sar1 (CA-AtSar1) mutant inhibits the transport of the Golgi membrane protein AtRer1B from the ER to the Golgi.²⁵ Protoplasts were co-transformed with OsRacGEF1-CFP and GFP-AtRer1B, together with either WT-AtSar1 or CA-AtSar1 (Fig. 3F). In the presence of WT-AtSar1, OsRacGEF2-mCherry predominantly localized to the ER and the cytosol (Fig. 3F, left), while AtRer1B-GFP was found in Golgi-like organelles of protoplasts. In contrast, in cells transfected with CA-AtSar1, OsRacGEF2-mCherry and the AtRer1B-GFP signal were restricted to the ER. Therefore, it seems likely that OsRacGEF2 is also regulated by a Sar1-dependent vesicle trafficking pathway.

Here, we have identified OsRacGEF2 as an interactor of OsRacGEF1 and found that the 2 GEFs heterodimerize via their PRONE domains in vivo and in vitro. OsRacGEF2 did not directly bind to OsCERK1 or OsFLS2, but did form a complex with these receptors through OsRacGEF1. This OsRacGEF1-OsRacGEF2 heterodimer complex interacts not only with OsRac1 but also with OsCERK1 or OsFLS2 at the PM in rice cells. Thus, we demonstrate that the OsCERK1-OsRac1-OsRacGEF1-OsRacGEF2 complex is formed at the PM in rice cells.

Although previous studies have revealed the dimerization of PRONE GEFs by in vitro experiments, how the dimer functions have not hitherto been investigated in plant cells. We have demonstrated here that OsRacGEF1 and OsRacGEF2 form homodimers and also heterodimerize with each other in rice protoplasts (Fig. 3B a, f and k), and these complexes associate stably with OsRac1 at the PM in the absence of external stimulations (Fig. 3B b, g and l). OsRac1 has a truncated, but functional GC-CG box, which is known to be important for lipid modification and membrane association; thus, OsRac1 localizes predominantly at the PM.²⁶ On the other hand, OsRacGEF1 and OsRacGEF2 lack such a plasma membrane retention signal. Therefore, it seems likely that OsRacGEFs are tethered to the PM by OsRac1 (Fig. 3B b, g and l). Interestingly, it has been reported that, in animals, Dbl homology and pleckstrin homology domains (DH-PH) and Dedicator of cytokinesis 2 (DOCK2) work as GEF domains for Rac proteins and also form oligomers to carry out their functions.^27-29 Structural studies have indicated that 2 AtROP4 molecules form a tetrameric complex with 2 ROPGEF8s.²² Dimerization of the PRONE domain is essential for GEF activity, since PRONE L23D, which is mutated at the dimerization interface of RopGEF8, shows no measurable dimerization or GEF activity. It is likely that, in both plants and animals, dimerization is a common phenomenon in GEFs for their biological activities.

Subcellular trafficking of RLKs is important for plant immunity as well as other plant activities.³⁰ We have shown previously that OsRacGEF1 is transported, together with OsCERK1 and (co)chaperones such as Hsp90 and Hop/Sti1, from the ER to the PM through Sar1-dependent vesicle trafficking; they form a complex with OsRac1 at the PM.^14,21,25 Although OsRacGEF2 also forms a complex with OsCERK1 at the ER (Fig. 3C), OsRacGEF2 does not directly interact with the cytoplasmic domain of OsCERK1 in a yeast 2-hybrid assay (Fig. 3E and Fig. S1). Therefore, it seems likely that OsRacGEF1 mediates the interactions between OsCERK1 and OsRacGEF2 at the ER, because the C terminus of OsRacGEF1 can bind to the cytoplasmic domain of OsCERK1.²¹ Indeed, the expression of OsRacGEF1 facilitates the association between OsRacGEF2 and OsCERK1 (Fig. 3D, right). Furthermore, BiFC results showed that OsFLS2 also associates with OsRacGEF2 and that OsRacGEF1 enhances their interaction in a manner similar to OsCERK1.

Evidence has been accumulating that the molecular links between RLKs and Rac/ROP family small GTPases, through their activator Rac/ROPGEFs, are involved in various signaling systems such as those governing pollen tube growth, root hair development, auxin signaling and disease resistance.^1,2,4,5,8 The Rac/Rop family apparently contributes to signaling downstream of CLAVATA1, an RLK that regulates the balance between cell differentiation and cell division in aerial meristems.³¹ An unidentified Rac/Rop was detected in an immunoprecipitate of the active CLAVATA1 fraction. Kaothien et al. reported that a PRONE-type GEF associates with 2 pollen-specific RLKs of tomato, LePRK1 and LePRK2.³² Their group characterized AtPRK2a, an Arabidopsis ortholog of LePRK2, and confirmed the physical interaction between AtPRK2a and the Arabidopsis PRONE-type AtRopGEF12, thus indicating that RopGEF activity is regulated by RLKs. Phosphorylation of AtRopGEF12 by AtPRK2a appears to be important for regulating GEF activity.⁶ A phospho-mimicking mutation at a highly conserved serine residue in the C terminus of AtRopGEF12 results in the loss of C-terminal autoinhibition. Our results suggest that the PRONE GEFs use a common transport system with the various RLKs which play vital roles in signal transduction. RLK-GEF-small GTPase complexes may therefore be common key components of numerous plant activities. The ability of the PRONE GEFs to heterodimerize may provide an explanation for the diversity of signaling that is displayed by the Rac/ROP family GTPases.

To summarize, in rice, OsRacGEF2 forms a heterodimer with OsRacGEF1 and interacts with RLK through OsRacGEF1 at the ER, and the resultant complex is then transported to the PM by a vesicle trafficking system. There, this complex associates with OsRac1, resulting in the formation of a stable immune complex at the PM. The RLK-GEF-small GTPase complex identified in this study and previous structural analyses may therefore be a common and fundamental feature of ROP GTPase-related signaling pathways in plants.

Funding

A.A. was supported by a fellowship from the Japan Society for the Promotion of Science (JSPS). This research was supported by MAFF Genomics for Agricultural Innovation Grant Number PMI0007 to K.S., by JSPS KAKENHI Grant Numbers 19108005 to K.S. and 2377044, 24113515, 26113712 and 26450055 to Y.K., by NAIST Foundation to Y. K., and by the Takeda Science Foundation to Y.K.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Masaru Fujimoto for providing GFP-L12, GFP-ATPase delta-prime subunit and GFP-PTS1, and also Drs. Akihiko Nakano, Takashi Ueda and Masaru Fujimoto for the organelle markers GFP-Erd2, GFP-SYP61 and GFP-ARA6. We appreciate Dr. Minoru Nagano for insightful discussion and Ms. Tomoko Aoi for excellent technical assistance. We also thank the members of the Laboratory of Plant Molecular Genetics for invaluable comments and discussions.

Supplemental Material

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