SEC3A acts as an effector of RabE1b to regulate auxin transport in arabidopsis

TL;DR

This study shows that SEC3A, a part of the exocyst complex, regulates auxin transport in Arabidopsis by acting as an effector of RabE1b, impacting plant growth and development.

Contribution

SEC3A is identified as a novel effector of RabE1b, revealing a new regulatory mechanism in PIN protein recycling and auxin transport.

Findings

PRsec3a mutants showed reduced root elongation, disrupted gravitropism, and lower auxin accumulation.

Endocytic recycling of PIN1, PIN2, and BRI1 proteins was impaired in PRsec3a mutants.

SEC3A was found to function as an effector of RabE1b, establishing a new regulatory axis in plant development.

Abstract

The establishment and architectural growth of terrestrial plants critically depend on polar auxin transport, a process primarily driven by the asymmetric distribution of PIN proteins within the plasma membrane. In Arabidopsis, the exocyst, an evolutionarily conserved octameric vesicle-tethering complex, orchestrates the recycling of PIN proteins, with subunits such as SEC6, SEC8, and EXO70A1 having established roles in this process. However, how the exocyst is mechanistically integrated into PIN recycling networks and the regulatory hierarchy remain unsolved. Here, SEC3A driven by pollen-specific ProLAT52 promoter was transformed into sec3a/ + background, generating two independent pollen rescued (PRsec3a) mutant lines PRsec3a-1 and PRsec3a-2, and enabling investigation of SEC3A function in sporophytic growth related to PIN protein dynamics. PRsec3a exhibited stunted primary root elongation, disrupted gravitropism, and reduced auxin accumulation. Notably, endocytic recycling of PIN1, PIN2, and BRI1 proteins from the Brefeldin A-induced compartments was compromised in PRsec3a. Furthermore, SEC3A was identified as an effector of the small GTPase RabE1b, defining a novel regulatory axis. Together, these findings advance the understanding of Exocyst-mediated membrane trafficking and its pleiotropic roles in plant development.

Figures

Primary root growth was compromised in PRsec3a.(A) WT and PRsec3a seedlings at 5 DAG. (B) Quantification of primary root length in WT and PRsec3a seedlings at 3, 5 and 7 DAG. (C) Meristem cell numbers at 5 DAG. (D) Quantification of meristem cell numbers at 5 DAG. (E) WT and PRsec3a seedlings grown in darkness and observed at 5 DAG. (F) Statistics of the etiolated hypocotyl length at 5 DAG. Statistical analysis was performed using SPSS software. Student’s t test was used for comparisons between two groups. Data are presented as mean ± SD (n ≥ 90) (*p < 0.05). Bars = 2 mm in (A, E) and 50 μm in (C).

Primary root gravitropism was compromised in PRsec3a.(A) The PRsec3a mutant exhibited impaired root gravitropism. (B) Schematic diagram of the root gravitropism in WT and PRsec3a. Deviation angles from the gravity vector 24 h after gravistimulation were quantified in 15° sectors (24 sectors total). (C) Lugol’s (KI) staining of starch granules in roots at 3, 5 and 7 DAG. Representative images are shown from at least three biological replicates, with 90 seedlings analyzed per genotype. Bars = 1.5 mm in (A), 25 μm in (C).

Auxin activity decreased in primary root of PRsec3a.(A) DR5-GFP signals (B) GUS staining. (C) PIN1-GFP signals. (D) PIN2-GFP signals. (E) PIN3-GFP signals. (F) PIN4-GFP signals. (G) PIN7-GFP signals. (H) BRI1-GFP signals. The horizontal captions in Fig 3 indicate the various markers analyzed, including DR5, PIN1, PIN2, PIN3, PIN4, PIN7, and BRI1. Statistical analysis was performed using SPSS software. Values represent the means ± SD, * indicates P < 0.05 by Student’s t-test. (n ≥ 15). Bars = 25 μm.

SEC3A regulated recycling of the plasm membrane proteins.(A), (B) and (C) The recycling of PIN1-GFP (A), PIN2-GFP (B), and BRI1-GFP (C) from the BFA compartments to the PM was slowed down. BFA bodies were quantified using SPSS software. Seedlings were treated with CHX and BFA for 2 h, followed by washout for 30 or 90 min as indicated. BFA compartments were quantified in 90 cells from fifteen plants per treatment. Data are presented as mean ± SD; *P < 0.05 (Student’s t-test). (n ≥ 15) Bars = 10 μm.

SEC3A interacted with RabE1b in vitro and in vivo.(A) SEC3A interacted with RabE1b and RabE1b[Q74L] in a Y-2-H assay. The transformed yeast cells exhibited growth on QDO medium, indicating a positive protein-protein interaction. The fourth panel represented serial decimal dilutions. (B) SEC3A interacted with RabE1b and RabE1b[Q74L] in a GST pull-down assay. The GST and GST fusion proteins bound to GST beads were incubated with MBP-SEC3A, respectively, and the bound MBP-SEC3A was detected with an anti-MBP antibody. (C) BiFC assay indicated that the SEC3A protein interacted with RabE1b/ RabE1b[Q74L], but not with RabE1b[S29N] in tobacco leaf cells. Different combinations of plasmids were co-transformed with plasm membrane marker into tobacco leaves. Bars = 10 μm.

Full text

Introduction

Auxin serves as a key signaling molecule that regulates tissue polarity and pattern formation in plants [1]. The polar transport and asymmetric distribution of auxin within tissues, manifested as auxin maxima and gradients, are crucial for the normal development of roots and shoots. PIN-FORMED (PIN) proteins, which function as key auxin efflux carriers, exhibit polar localization in the plasma membrane and mediate short-distance polar auxin transport [2,3]. In Arabidopsis roots, different PIN proteins exhibit distinct subcellular localizations and functions [4].

The establishment of PINs polar localization within plasma membrane domains is achieved through multiple factors, such as endocytic recycling, a process dependent on clathrin-mediated endocytosis [5,6]. The ARF GTPase guanine nucleotide exchange factor (ARF-GEF) GNOM could mediate the recycling process, which is sensitive to BFA [7,8]. Furthermore, the recycling of PIN proteins from early endosomes back to the PM for polarized exocytosis is precisely controlled through the synergistic regulation of multiple protein families [9,10], including Rab GTPases, tethering factors [11,12], SNARE proteins [13,14]. Together, these components ensure the precise and dynamic redistribution of PIN proteins, enabling their critical role in auxin transport and plant development.

Exocyst, an evolutionarily conserved octameric complex (comprising SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO70, and EXO84), acts as a tethering factor for exocytosis at the plasma membrane and participates in diverse cellular processes in plants [15–17]. In cytokinesis, for example, pollen rescued sec6 (PRsec6), exo70a1, and exo84b mutants exhibit defective cytokinesis, with SEC6 functions at early-stage, while EXO70A1 and EXO84b at late-stage membrane fusion events [18,19]. In seed coat development, The SEC8-EXO70A1-ROH1 module orchestrates the localized pectin deposition [20], and EXO70A1 regulates patterned secondary cell wall thickening [21]. The complex also contributes autophagy. EXO70B1 is involved in autophagosome membrane trafficking to the vacuole [22], while MPK3-mediated phosphorylation redirects EXO70B2 from the secretory pathway towards the autophagy [23]. Nutrient stress triggers RalB-dependent recruitment of EXO84B to promotes autophagosome-vacuole fusion [24]. During pollen development, exocyst subunits are essential for germination and pollen tube growth. Mutations in SEC5, SEC6, SEC8, SEC15A, and SEC3A lead to male sterility, disrupting a series of processes including early pollen hydration, vesicle fusion to cell wall modification and polarized secretion [25–30]. While SEC3A’s role in pollen tip growth is well-documented, its broad expression in vegetative tissues suggests important, yet unexplored, sporophytic functions that warrant further investigation.

Rab small GTPase functions as a molecular switch in vesicle trafficking by cycling between activate GTP-bound and inactive GDP-bound states. Mutations at conserved residues can lock Rab proteins into constitutively active or dominant-negative forms. Direct Rab-exocyst interactions are observed across systems: in mammals, Sec15 interacts with Rab11 to facilitate vesicle generation at the trans-Golgi network/recycling endosomes and delivery to the plasma membrane [31,32]. In yeast, activated Sec4p interacts with Sec15p and, via targeting hub Sec3p, directs secretory vesicles to specific plasma membrane sites [33,34]. Moreover, Sec15 associates with the myosin motor Myo2p, which links both Sec4 and Ypt32, indicating that Rab proteins help coordinate exocyst-mediated vesicle transport along the cytoskeleton [35,36]. RabEs play important roles in plant development and stress adaptation [37]. Functional studies show that RabE1d (downstream of RabD2) controls secretion and plant morphology, with its active mutant enhancing bacterial pathogen resistance [38,39]. Similarly, RabE1c promotes drought tolerance by degrading the ABA receptor PYL4 to regulates stomatal closure [40]. Moreover, it has been demonstrated that the SCD complex, which functions in post-Golgi trafficking, is required for recruiting RabE1 and the exocyst to secretory vesicles, and defects in this recruitment consequently disrupt both exocytic and recycling pathways [41]. However, the effector molecules acting downstream of RabE in plants remain poorly characterized.

This study revealed that in the PRsec3a mutant, reduced auxin activity impairs primary root growth and gravitropism. Moreover, SEC3A, acting as an effector of RabE1b, primarily reduces auxin activity by regulating the recycling of PIN proteins.

Materials and methods

Plant materials and growth conditions

All experiments were performed using Arabidopsis thaliana ecotype Columbia-0. The T-DNA insertion mutants sec3a/+ (GK_652H12) were obtained from the Arabidopsis Biological Resource Centre (ABRC). Seeds were sterilized in 2% (v/v) NaClO for 10 min, rinsed five times with ddH_2_O, and placed on Murashige and Skoog (MS) medium containing 1% (w/v) agar. After stratification in the dark at 4^o^C for 2 days, plates were transferred to a growth room set at 22^o^C under a 16-h-light/8-h-dark photoperiod with a photosynthetic photon flux density (PPFD) of 100 μmol·m ⁻ ²·s ⁻ ¹. Seven days after germination (DAG), the seedlings were transplanted to soil.

Root gravitropism assays

Seedlings were grown vertically on plates for 4 days, then rotated 90^o^ and maintained in the vertical orientation for an additional 24 h. After gravistimulation, digital images were taken and root bending angles relative to the gravity vector were analyzed using ImageJ software.

Chemical treatments

For the BFA treatment, seedlings were first pretreated with 20 μg/mL CHX (Sigma) in 1/2 MS liquid media for 30 min, then incubated with 20 μg/mL CHX and 50 μΜ BFA (Sigma) for 120 min. After treatment, seedlings were washed with 1/2 MS liquid medium containing CHX for different periods of time (0, 30, 90 min) [11,12], and observed under a laser scanning confocal microscope (CLSM800, Carl Zeiss).

Yeast two-hybrid assays

Yeast two-hybrid assays were performed using the Matchmaker GAL4 Two-Hybrid System (Clontech) following the manufacturer’s instructions. Coding sequences of genes of interest were cloned into the pGBKT7 or pGADT7 vector. Different plasmid combinations were transformed into yeast strain AH109, and positive transformants were selected on SD medium lacking Trp, Leu, His, and Ade (QDO).

BiFC assay

The coding sequence of SEC3A was cloned into VN13 vector, while RabE1b and RabE1b^[Q74L]^ were cloned into VN12 vector to generate SEC3A-VN13, RabE1b-VN12, RabE1b^[Q74L]^-VN12, respectively. Various combinations of plasmids, together with p19 and the plasm membrane marker PIP2-mCherry, were transformed into Agrobacterium tumefaciens EHA105. Bacterial suspensions (OD_600_ = 0.5) were infiltrated into leaves of 5-week-old N. benthamiana plants. After 3 days, fluorescence signals were captured using a laser scanning confocal microscope (CLSM800, Carl Zeiss) with the following settings: YFP, excitation 514 nm/emission 517–524 nm; mCherry, excitation 561 nm/emission 587–610 nm. [19].

GST pull-down

The coding sequences of RabE1b and RabE1b^[Q74L]^ were cloned into the pGEX4T-1 (Novagen) to generate GST-RabE1b and GST-RabE1b^[Q74L]^, while SEC3A CDS was cloned into pMAL-C2X (Novagen) to generate MBP-SEC3A. Recombinant proteins were expressed in E. coli and purified. For each reaction, 5 μg GST, GST-RabE1b, or GST-RabE1b^[Q74L]^ proteins were bound to 100 μL of glutathione sepharose resin (Genscript) in GST binding buffer (1x PBS supplemented with protease inhibitors). The, 2.5 μg the corresponding MBP-fusion proteins were added and incubated in at 4^o^C for 2 h. After washing, bound proteins were separated by SDS-PAGE and analyzed by western blotting using anti-MBP antibodies (1:5000 dilution, Abmart) and anti-GST antibodies (1:5000 dilution, Abmart), followed by HRP conjugated secondary antibodies (1:5000 dilution, Abmart). Signals were detected using a chemiluminescence imager (Tanon) [19].

All primer sequences used in this study are listed in Supplementary S1 Table.

Results

Primary root growth is retarded in PRsec3a

Loss of SEC3A resulted in male gametophytic lethality, as both pollen germination and pollen tube growth were severely compromised. [29,30]. To investigate the function of SEC3A during sporophytic development, a construct containing the SEC3A coding sequence driven by the pollen-specific ProLAT52 promoter was transformed into sec3a/ + plants. This transformation restored male gametophytic development but resulted in homozygous lines with minimal SEC3A expression in vegetative tissues (S1 Fig). These lines, termed Pollen-Rescued sec3a mutants (PRsec3a-1 and PRsec3a-2), were used to examine the impact of SEC3A deficiency on vegetative growth.

The PRsec3a mutants exhibited multiple developmental defects during sporophytic growth, including retarded primary root development (Fig. 1A). Primary roots of PRsec3a mutants were significantly shorter than wild-type at 3, 5 and 7 DAG (Days After Germination), measuring 2.17 ± 0.09 mm vs. 3.03 ± 0.09 mm at 3 DAG, 8.28 ± 0.83 mm vs. 10.02 ± 0.21 mm at 5 DAG, and 14.44 ± 0.35 mm vs. 15.74 ± 0.33 mm at 7 DAG (Fig. 1A-B). Analysis of the root meristem revealed that PRsec3a had fewer cortex cells in the meristem zone compared with the wild type (26 ± 1.78 vs. 30 ± 0.99) at 5 DAG (Fig. 1C-D). In addition, when grown in darkness, PRsec3a mutants exhibited significantly shorter etiolated hypocotyls than wild-type seedlings (1.08 ± 0.19 cm vs. 1.21 ± 0.21 cm) (Fig. 1E-F). Taken together, these results indicate that SEC3A regulates primary root growth by modulating both cell division and cell elongation.

*Primary root growth was compromised in PRsec3a.(A) WT and PRsec3a seedlings at 5 DAG. (B) Quantification of primary root length in WT and PRsec3a seedlings at 3, 5 and 7 DAG. (C) Meristem cell numbers at 5 DAG. (D) Quantification of meristem cell numbers at 5 DAG. (E) WT and PRsec3a seedlings grown in darkness and observed at 5 DAG. (F) Statistics of the etiolated hypocotyl length at 5 DAG. Statistical analysis was performed using SPSS software. Student’s t test was used for comparisons between two groups. Data are presented as mean ± SD (n ≥ 90) (p < 0.05). Bars = 2 mm in (A, E) and 50 μm in (C).

The gravitropism is defective in PRsec3a

To assess whether SEC3A was involved in root gravitropism, seedlings were grown vertically for 4 days and then reoriented by 90^o^. Wild-type roots showed a strong gravitropic curvature, whereas PRsec3a roots exhibited significantly reduced bending (Fig. 2A-B). We next examined starch granule distribution in the root cap using Lugol’s iodine staining (Fig. 2C). At 3 DAG, starch granules were present in two cell layers in both genotypes. At 5 DAG, wild-type root caps exhibited staining in three cell layers, while PRsec3a caps retained only two stained layers. At 7 DAG, Wild-type caps showed three to four distinct stained layers, while PRsec3a caps varied in staining, with some displaying only two layers (Fig. 2C). These results indicate that columella cell development and starch granule accumulation are disrupted in PRsec3a, likely contributing to its defective gravitropic response.

Primary root gravitropism was compromised in PRsec3a.(A) The PRsec3a mutant exhibited impaired root gravitropism. (B) Schematic diagram of the root gravitropism in WT and PRsec3a. Deviation angles from the gravity vector 24 h after gravistimulation were quantified in 15° sectors (24 sectors total). (C) Lugol’s (KI) staining of starch granules in roots at 3, 5 and 7 DAG. Representative images are shown from at least three biological replicates, with 90 seedlings analyzed per genotype. Bars = 1.5 mm in (A), 25 μm in (C).

Auxin activity and polar transport are impaired in PRsec3a

Given the defects in root elongation and gravitropism, we hypothesized that auxin signaling or transport may be disrupted in PRsec3a. To test this, we first examined the auxin activity reporter DR5-GFP and observed a marked reduction in GFP signals in PRsec3a root tips at 5 DAG compared to WT (Fig. 3A). This decrease was further confirmed by DR5-GUS staining (Fig. 3B). Time-course imaging showed consistently lower DR5-GFP fluorescence in PRsec3a (S2 Fig), supporting the conclusion that auxin activity was compromised in the mutant.

Auxin activity decreased in primary root of PRsec3a.(A) DR5-GFP signals (B) GUS staining. (C) PIN1-GFP signals. (D) PIN2-GFP signals. (E) PIN3-GFP signals. (F) PIN4-GFP signals. (G) PIN7-GFP signals. (H) BRI1-GFP signals. The horizontal captions in Fig 3 indicate the various markers analyzed, including DR5, PIN1, PIN2, PIN3, PIN4, PIN7, and BRI1. Statistical analysis was performed using SPSS software. Values represent the means ± SD, * indicates P < 0.05 by Student’s t-test. (n ≥ 15). Bars = 25 μm.

We next analyzed the localization of key PIN auxin transporters. While PIN1-GFP and PIN2-GFP localization in PRsec3a appeared similar to the wild type (Fig. 3C-D), but PIN3-GFP and PIN4-GFP signals were reduced (Fig. 3E-F). PIN7-GFP and BRI1-GFP showed comparable abundance in both genotypes (Fig. 3G-H).

SEC3A is required for efficient recycling of plasma membrane proteins

To assess whether SEC3A influences membrane protein trafficking, we performed Brefeldin A (BFA) washout assays in the presence of cycloheximide (CHX) to block de novo protein synthesis. Seedlings expressing ProPIN1::PIN1-GFP, ProPIN2::PIN2-GFP, or ProBRI1::BRI1-GFP were pretreated with CHX, then exposed to BFA to induce the formation of BFA bodies. Following BFA washout, wild-type seedlings displayed nearly complete disappearance of BFA compartments within 90 min. In contrast, PRsec3a seedlings retained significantly more BFA bodies over the same period (Fig. 4A-C). These results indicate that SEC3A is necessary for the efficient recycling of plasma membrane proteins.

*SEC3A regulated recycling of the plasm membrane proteins.(A), (B) and (C) The recycling of PIN1-GFP (A), PIN2-GFP (B), and BRI1-GFP (C) from the BFA compartments to the PM was slowed down. BFA bodies were quantified using SPSS software. Seedlings were treated with CHX and BFA for 2 h, followed by washout for 30 or 90 min as indicated. BFA compartments were quantified in 90 cells from fifteen plants per treatment. Data are presented as mean ± SD; P < 0.05 (Student’s t-test). (n ≥ 15) Bars = 10 μm.

SEC3A physically interacts with RabE1b

To identify potential interaction partners of SEC3A, a yeast two-hybrid (Y2H) screen was performed using its C-terminal domain (571 aa) as bait. This screen identified RabE1b, a Rab GTPase involved in vesicle trafficking. The interaction was confirmed by retransformation in yeast (Fig. 5A). Further analysis using mutant variants of RabE1b revealed that SEC3A specifically interacted with the constitutively active form (RabE1b^[Q74L]^), but not the inactive forms (RabE1b^[S29N]^, RabE1b^[N121I]^), suggesting that SEC3A may act as an effector of GTP-bound RabE1b. This physical interaction was validated by GST pull-down assays, in which GST-RabE1b and GST-RabE1b^[Q74L]^, but not GST alone, pulled down full-length SEC3A (Fig. 5B). Additionally, bimolecular fluorescence complementation (BiFC) assays demonstrated in vivo interaction between SEC3A and RabE1b or RabE1b^[Q74L]^, with fluorescence detected at the plasma membrane and in the cytosol (Fig. 5C).

SEC3A interacted with RabE1b in vitro and in vivo.(A) SEC3A interacted with RabE1b and RabE1b[Q74L] in a Y-2-H assay. The transformed yeast cells exhibited growth on QDO medium, indicating a positive protein-protein interaction. The fourth panel represented serial decimal dilutions. (B) SEC3A interacted with RabE1b and RabE1b[Q74L] in a GST pull-down assay. The GST and GST fusion proteins bound to GST beads were incubated with MBP-SEC3A, respectively, and the bound MBP-SEC3A was detected with an anti-MBP antibody. (C) BiFC assay indicated that the SEC3A protein interacted with RabE1b/ RabE1b[Q74L], but not with RabE1b[S29N] in tobacco leaf cells. Different combinations of plasmids were co-transformed with plasm membrane marker into tobacco leaves. Bars = 10 μm.

Co-localization studies in tobacco epidermal cells revealed that SEC3A-GFP co-localized with RabE1b-mCherry, regardless of activation state of RabE1b (S3 Fig). This indicates that while co-localization does not require GTP-loading of RabE1b, their physical interaction was activation-dependent. These findings provide mechanistic insight into how SEC3A cooperates with RabE1b to regulate vesicle trafficking and membrane dynamics.

Discussion

Our study demonstrates that the exocyst subunit SEC3A plays a crucial role in sporophytic development by regulating vesicle trafficking, which in turn affects auxin transport and response. Utilizing pollen-rescued PRsec3a lines, we bypassed male gametophytic lethality and revealed that SEC3A deficiency leads to impaired primary root growth and gravitropism. These defects are phenotypically similar to those observed in other exocyst mutants, such as exo70a1 [11,42], reinforcing the conserved function of this complex in plant development.

The root defects in PRsec3a were correlated with a significant reduction in auxin response, as indicated by diminished DR5 reporter activity, while PIN1 and PIN2 localization remained largely unchanged. It has been reported that the steady-state plasma membrane localization of PIN2 is maintained in exocyst subunit mutants because its secretory pathway from the TGN to the PM primarily depends on the RABA2a-SNARE route, which operates independently of the exocyst complex [43] However, the observed defective recycling of PINs in mutants like PRsec3, PRsec6 [12], exo70A1 and sec8 [11], suggests that the exocyst may play a crucial role in the endosome-to-PM recycling step. This indicates that PIN trafficking may be coordinately regulated by two pathways: RABA2a mediates forward secretion, while the exocyst likely facilitates recycling. Supporting the recycling defect hypothesis, BFA washout assays revealed a general delay in the recycling of PIN1, PIN2, and the non-PIN marker BRI1 to the plasma membrane in PRsec3a.

An intriguing contrast emerges from comparisons across species. While we observed reduced auxin response in Arabidopsis PRsec3a roots, rice sec3a mutants accumulate excess IAA in floral organs [44]. This discrepancy likely arises from the integration of tissue-specific and species-specific factors. The root, acting as an auxin sink reliant on polar transport, may be particularly vulnerable to defects in secretory trafficking. In contrast, floral organs with strong local auxin biosynthesis might exhibit compensatory accumulation when efflux is impaired. Furthermore, functional diversification of cargo proteins (e.g., predominant PINs in roots vs. AUX/LAX influx carriers in flowers) and lineage-specific adaptations of the exocyst complex itself could contribute to these divergent phenotypic outcomes.

To elucidate the molecular mechanism, we identified RabE1b, a plant-specific Rab GTPase, as a direct interactor of SEC3A. Biochemical and BiFC assays confirmed that SEC3A preferentially binds the active, RabE1b^[Q74L]^, positioning SEC3A as a potential RabE1b effector. This interaction aligns with the conserved functional partnership between Rabs and exocyst subunits in vesicle tethering. The phenotypic similarities between PRsec3a and mutants of the known ROP effector ICR1—which interacts with SEC3A and affects PIN recycling [45,46]—further support the existence of a regulatory network involving ROP, RabE, and the exocyst to coordinate membrane trafficking for polar growth. Although our data demonstrate a direct SEC3A–RabE1b interaction the in planta functional relevance of this interaction requires genetic validation.

Conclusions

Our work integrates SEC3A into a growing framework of exocyst-mediated vesicle trafficking that is essential for auxin transport and plant development. By identifying RabE1b as an interactor, we provide a mechanistic link between Rab signaling and exocyst function, opening new avenues for understanding how membrane trafficking is precisely regulated to sustain plant growth and environmental adaptation.

Supporting information

S1 FigGenotyping of PRsec3a mutants.(A) Schematic diagram of the SEC3A gene structure and the insertion site of the T-DNA. The black and white boxes represent the exons and non-coding region, respectively. The black lines between exons represent introns, and the inverted triangle indicates the T-DNA insertion site. (B) Schematic diagram of ProLAT52::SEC3A construct and its plant transformation. (C) Genotyping of PRsec3a mutant. P1 + GK-LB was used to verify the T-DNA insertion, and P1 + P2 was used to verify homozygous PRsec3a mutant, P3 + P4 was used to identify ProLAT52, and ACTIN was used to verify DNA quality and amplification efficiency. (D) qRT-PCR analysis of PRsec3a-1 and PRsec3a-2 mutants. Note that SEC3A expression was significantly reduced.(TIF)

S2 FigAuxin distribution of WT and PRsec3a at 3DAG, 5DAG and 7DAG.Bars = 50 μm.(TIF)

S3 FigSEC3A colocalized with RabE1b in tobacco epidermal cells.(A) Subcellular localization of SEC3A, RabE1b, RabE1b^[Q74L]^ and RabE1b^[S29N]^ in tobacco epidermal cells. (B–D) Colocalization of SEC3A with RabE1b (B), RabE1b^[Q74L]^ (C), and RabE1b^[S29N]^ (D) in tobacco epidermal cells. Bars = 2 μm.(TIF)

S1 TablePrimers used in this study.(DOCX)

S1 FileThe original images for the GST pull-down assay.(PDF)