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A broadly conserved NERD genetically interacts with the exocyst to affect root growth and cell expansion

A broadly conserved NERD genetically interacts with the exocyst to affect root growth and cell... The exocyst, a conserved, octameric protein complex, helps mediate secretion at the plasma membrane, facilitating specific developmental processes that include control of root meristem size, cell elongation, and tip growth. A gen- etic screen for second-site enhancers in Arabidopsis identified NEW ENHANCER of ROOT DWARFISM1 (NERD1) as an exocyst interactor. Mutations in NERD1 combined with weak exocyst mutations in SEC8 and EXO70A1 result in a synergistic reduction in root growth. Alone, nerd1 alleles modestly reduce primary root growth, both by shortening the root meristem and by reducing cell elongation, but also result in a slight increase in root hair length, bulging, and rup- ture. NERD1 was identified molecularly as At3g51050, which encodes a transmembrane protein of unknown function that is broadly conserved throughout the Archaeplastida. A functional NERD1–GFP fusion localizes to the Golgi, in a pattern distinct from the plasma membrane-localized exocyst, arguing against a direct NERD1–exocyst interaction. Structural modeling suggests the majority of the protein is positioned in the lumen, in a β-propeller-like structure that has some similarity to proteins that bind polysaccharides. We suggest that NERD1 interacts with the exocyst indirectly, possibly affecting polysaccharides destined for the cell wall, and influencing cell wall characteristics in a developmentally distinct manner. Keywords: Arabidopsis, cell elongation, cell wall, exocyst, genetic interaction, root development, root hair, root meristem, secretory pathway, tip growth. Introduction The secretory system in plants is a fundamental determi- sexual reproduction in flowering plants. Additionally, secre- nant of plasma membrane composition and cell wall forma- tion of substances into the apoplast and delivery of receptors tion (Luschnig and Vert, 2014; Ebine and Ueda, 2015; Kim and transporters to the plasma membrane allow for intercel- and Brandizzi, 2016). Consequently, secretory events drive lular communication and coordination. Ultimately, the secre- cell growth and morphogenesis, and influence plant devel- tory system’s intimate influence on the plasma membrane and opment. For example, selective localization of secretion to extracellular activities facilitates responsiveness and survival specific regions of the cell periphery is essential for polar- within variable abiotic and biotic environments. However, it ized growth of pollen tubes, enabling sperm cell delivery and remains unclear how the secretory process in plants is spatially © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3626 | Cole et al. and temporally regulated to direct particular cargos to spe- factors are likely to be involved, including cellular components cific locations of the plasma membrane, cell wall, or apoplast that interact with the exocyst indirectly, e.g. by enhancing the at the appropriate time. Both the conventional secretory sys- activity of an exocyst-trafficked protein. tem, i.e. vesicular transport from endoplasmic reticulum to To advance the investigation of exocyst-mediated secre- Golgi to plasma membrane, and non-conventional pathways tory events in plants, we performed a mutagenesis screen to are involved (Drakakaki and Dandekar, 2013; Robinson et al., identify interactors linked to the exocyst’s role in Arabidopsis 2016; van de Meene et al., 2017), but how these pathways are root growth. In this screen, we identified NEW ENHANCER tailored to the dynamic requirements of different cell types is OF ROOT DWARFISM (NERD1), a protein of unknown only beginning to be revealed. function that, based on genetic interaction data, acts with the The exocyst, an evolutionarily conserved octameric com- exocyst to facilitate root and hypocotyl elongation and to plex, tethers secretory vesicles to specific sites on the plasma influence root hair morphology. NERD1 is expressed through- membrane prior to exocytosis and modulates secretory activ- out the plant, suggesting a potential role beyond the root and ity to achieve an array of specialized functions. In plants, hypocotyl. NERD1 homologs are found throughout the plant components of the exocyst have been implicated in a range kingdom and beyond. Interestingly, the functional interaction of processes including pollen tube germination and growth of the exocyst with NERD1 is likely to be indirect, and var- (Cole et  al., 2005; Hála et  al., 2008; Li et  al., 2010), cytokin- ies dependent on developmental context. We speculate that esis (Fendrych et al., 2010; Rybak et al., 2014), secondary cell NERD1 is involved in the modification of cell wall polysac- wall deposition during tracheary element development (Li charides that are important for cell wall expansion and are a et al., 2013; Oda et al., 2015), hypocotyl elongation in etiolated cargo for exocyst-mediated transport to the apoplast. seedlings (Hála et  al., 2008), determination of meristem size and cell elongation during primary root growth (Cole et  al., Materials and methods 2014), Casparian strip formation (Kalmbach et al., 2017), local- ized disposition of seed coat pectin (Kulich et al., 2010), callose Plant materials and growth conditions deposition in trichomes (Kulich et  al., 2015), and the polar Lines of Landsberg erecta-0 and Columbia-0 ecotype of Arabidopsis with growth of root hairs (Wen et al., 2005; Synek et al., 2006). T-DNA insertions were obtained from the SALK Institute (Alonso et al., The regulation, assembly, and functioning of the exocyst 2003): nerd1-2 (At3g51050, SALK 018060C); nerd1-3 (At3g51050, SALK complex in non-plant eukaryotes has been linked to its inter- 051660); exo70A1-2 (At5g03540, SALK 135462); sec8-3 (At3g10380, SALK 026204); sec8-4 (At3g10380, SALK 118129); sec8-6 (At3g 10380, actions with small GTPases of the Rho, Ral, and Rab fami- SALK 091118); and myo XI-K (At5g20490, SALK 067972). The exo84b-1 lies (Mukherjee et al., 2014), membrane phospholipids (Thapa line was a GABI-Kat line (Rosso et  al., 2003; Fendrych et  al., 2010). et  al., 2012; Pleskot et  al., 2015), plasma membrane scaffold- The EXO84b-GFP and GFP-SEC8 lines were previously described ing proteins (Liu and Novick, 2014), and the actin cytoskel- (Fendrych et  al., 2010). The nerd1-1 mutant was generated in an ethyl eton (Jin et  al., 2011; Liu et  al., 2012). This interactive milieu methanesulfonate (EMS) screen that treated ~5000 sec8-6 seeds with 0.2% EMS for 15 h. M2 generation seed from 4500 M1 plants was collected in helps define exocyst function in yeast and mammals, provid- pools derived from 16 plant lots. The effectiveness of the mutagenesis was ing post-translational regulation of key secretory events (Wu verified by observing greater than 64% of M2 plants with one-quarter and Guo, 2015; Pleskot et  al., 2015). In plants, the molecular aberrant seed, with the gene mutation rate estimated at 1/3000. mechanisms that integrate the exocyst into distinct secretory Arabidopsis seeds were surface-sterilized, stratified at 4 °C for 3–5 d, processes are less well understood. One regulatory mechan- and planted on growth medium (1× MS, 2% (w/v) sucrose, and vitamins in 1% (w/v) Bacto-agar) or soil as previously described (Cole et al., 2005). ism unique to plants is the proliferation and diversification of Plants were grown in a climate chamber at 22 °C under long-day condi- homologs of the exocyst subunit Exo70 (23 in Arabidopsis), tions (16 h of light per day; 7500 lx), with the exception of those used which is hypothesized to allow for specification of particular in hypocotyl elongation experiments. For these, seeds were placed in a exocyst functions (Synek et al., 2006; Li et al., 2010; Cvrčková lighted incubator at 228C for 2–4 h to stimulate germination and then et  al., 2012; Vukašinović and Žárský, 2016). In support of this wrapped in foil, oriented vertically, and placed in a dark box in a 22 °C incubator. After 5 d in the dark, digital images were captured and hypo- hypothesis, different Exo70 paralogs have been associated with cotyl lengths measured. specific cellular processes: Exo70B1 with autophagy (Kulich To evaluate the effect of Endosidin2, three groups were germinated on et  al., 2013), Exo70I with arbuscular mycorrhizal symbiosis MS plates: seedlings that were homozygous for nerd1-2; nerd1-2 siblings (Zhang et  al., 2015), and Exo70E with the EXPO secretory complemented by NERD1–green fluorescent protein (GFP); and Col-0 pathway (Poulsen et  al., 2014). Furthermore, in growing pol- plants. Plants of each genotype were transferred approximately 3 d after germination to plates containing 0, 20, or 40 μM Endosidin2 (ES2), and len tubes, members of the EXO70A, EXO70B and EXO70C grown for an additional 4 d before imaging to determine primary root subgroups show differential localization patterns and apparent growth rates. DMSO-dissolved ES2 (or DMSO alone as a control, at activities (Sekereš et al., 2017; Synek et al., 2017). Other factors 0.5% (v/v)) was added to media during plate preparation. that help regulate the exocyst in specific developmental con- texts are the scaffolding protein Interactor of Constitutive active High-throughput sequencing and analysis ROPs 1 (ICR1) in roots (Lavy et al., 2007); the phosphoinosit- A plant homozygous for nerd1-1 and lacking a sec8 allele in a Col-0 ide PIP2 in pollen tubes (Bloch et  al., 2016); ROP2 GTPase background was backcrossed to Ler-0, and the progeny were self- (with its effector RIC7) in stomata (Hong et al., 2016); and the crossed to generate an F2 population. Pooled genomic DNA from 150 combined activities of VETH1–VETH2–COG2 and cortical F2 plants with the nerd phenotype (i.e. homozygotes) was sequenced microtubules in xylem cells (Oda et al., 2015). However, given via an Illumina HiSeq 2000 to generate 58 million paired-end reads. SHOREmap software (Schneeberger et al., 2009) was used to align the the breadth of functions known for the plant exocyst, other Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3627 reads to the Arabidopsis genome and assess the frequency of Col (the Results mutagenized parent) and Ler single nucleotide polymorphisms (SNPs) across the population. All variant SNPs in the ~200 kb region of chromo- Screening for exocyst interactors some 3 harboring nerd1-1 (Supplementary Fig. S1 at JXB online) were then searched against genes to identify candidate mutations with likely Mutations in genes encoding components of the exocyst com- deleterious effects. plex in Arabidopsis result in root growth defects that vary from a mild decrease in growth rate in some mutants (e.g. sec8-6 and exo70A1) to severe dwarfism in others (e.g. exo84b-1 and Genetic and molecular analyses sec8-3) (Cole et  al., 2014). Additionally, mutations of some DNA extraction from leaves and PCR genotyping for mutants contain- exocyst components reduce the length of root hairs (Synek ing T-DNA insertions was performed as previously described (Cole et al., 2005). Primers used in PCR and RT-PCR are shown in Supplementary et  al., 2006). We reasoned that a protein that interacts with Table S1. PCR-based genotyping to detect the EMS-generated nerd1-1 the exocyst could be revealed if its mutation accentuated the mutation required use of the restriction enzyme AvaII after amplifica- root growth defect of an exocyst mutation that by itself results tion, as a target cleavage site in the At3g51050 genomic sequence was in only a mild phenotype. Therefore, we screened an EMS- eliminated by the G→A transition in the mutant. To evaluate expression treated population of seedlings homozygous for the mild sec8-6 via RT-PCR, roots from approximately 50 10-day-old seedlings of each genotype were harvested from plates and frozen in liquid nitrogen. RNA mutation in a Col-0 background to identify such second-site was extracted from the pooled sample for each genotype using a phenol– enhancers. chloroform procedure, followed by DNase treatment. First strand cDNA Plants from M2 pools exhibiting both short roots and aber- synthesis was performed using Superscript II as per the manufacturer’s rant root hairs—dubbed the new enhancer of root dwarf- specifications (Thermo Fisher Scientific), followed by removal of RNA ism (nerd) phenotype—were outcrossed to a wild-type line, with RNaseH. The cDNA was used as a template for PCR with pri- mer pairs that amplified the sequence to the 5′ of the mutations, to the self-pollinated, and screened again for the phenotype in ~1/16 3′ of the mutations, or spanning the sites of the mutations. Primers for of the progeny, as expected for second site enhancers. After ACTIN2 were included as an internal control. screening 45 pools, we recovered exactly one such mutation, nerd1-1, which, when combined with sec8-6, leads to profound Generation of NERD1–GFP and imaging dwarfism throughout the plant and shorter primary roots A 5597-bp-long genomic fragment encompassing the NERD1 gene (~25% of wild-type length, with some demonstrating termi- along with its putative promoter was PCR amplified from the genomic nated growth). In addition, root hairs in nerd1 sec8-6 double DNA using KOD Hot Start high-fidelity DNA polymerase (Novagen) mutants are occasionally misshapen (Supplementary Fig.  S2). and cloned into a modified pMDC32 plasmid using SbfI and Pac I sites. Enhanced GFP (EGFP; Clontech) cDNA was added downstream from To verify that the genetic interaction was not specific to a par- the NERD1 open reading frame (ORF) to yield pMDC-NERD1-GFP. ticular SEC8 allele, the nerd1-1 mutation (isolated after a ser- The plasmid was mobilized into Agrobacterium tumefaciens strain GV3101. ies of backcrosses to Col-0) was combined with another mild Transient expression or coexpression of NERD1 construct and fluor- allele, sec8-4, yielding a similar result (Fig.  1A). Intriguingly, escent Golgi markers in Nicotiana benthamiana leaf epidermal cells was initial observations indicated that nerd1 plants, in the absence performed by co-infiltrating with Agrobacterium strains carrying NERD1- GFP and either STtmd-YFP or NAG-mTurq (Peremyslov et al., 2012) at of a sec8-6 or sec8-4 mutation, have a similar, but less severe concentrations equal to 0.2 OD . Imaging was conducted 2 d post- phenotype—e.g. root length ~75% of wild-type, and less fre- infiltration. Leaf fragments were immersed in water and observed using quently misshapen root hairs. a Zeiss LSM 780 NLO confocal microscope equipped with a Plan- Apochromat ×63 1.4 NA lens. mTurquoise, GFP, and mCherry were excited with the 405  nm diode laser line, 488-nm argon laser line, or Molecular identification of NERD1 561-nm He–Ne laser line, respectively. For the simultaneous visualization of two fluorophores, dual channel acquisition of signal for either GFP and To identify the nerd1-1 lesion, we used high-throughput mTurquoise or GFP and mCherry was performed sequentially to min- sequencing of a pooled population of mutant plants to iden- imize crosstalk. For Brefeldin A  (BFA) sensitivity, Arabidopsis seedlings tify a ~200  kb region of chromosome 3 tightly linked to expressing fluorophore-tagged proteins were treated with 50  μM BFA nerd1 (Supplementary Fig. S1). This region encompassed ~65 for 90 min, and the BFA-sensitive endomembrane compartments were imaged in root epidermal cells. protein-coding genes, only two of which harbored putative Evaluation of cortical cell files and root growth parameters in nerd1 EMS-generated G→A mutational differences from the Col-0 mutants using confocal microscopy was performed as previously reference sequence linked to nerd1-1. Both mutations were described (Cole et  al., 2014). Briefly, images of roots grown on vertical validated via Sanger sequencing. The best candidate for nerd1-1 plates were captured on day 5 and day 7 after germination to determine appeared to be a change in a conserved splice acceptor site at root growth rates. The 7-day-old seedlings were stained with propidium iodine and then imaged with a Zeiss LSM 780 NLO confocal micro- the ninth exon of At3g51050 (Fig. 1B). To confirm the molec- scope system. Multiple digital images were taken to capture two cortical ular identity of NERD1, two independent T-DNA insertion cell files for each root, one on each side of the longitudinal midline from alleles in At3g51050 (Fig.  1B) were obtained from the Salk the quiescent center to the differentiation zone. Cell widths and lengths mutant collection, both of which were associated with short were measured. The cortical cell length profile combined with the root root and root hair defects. Subsequent complementation tests growth rate allowed estimations of the number of cells in the meristem, the cell production rate, and the length of the cell cycle for each root. between heterozygotes for all three alleles showed the nerd1 Measurements (e.g. root lengths, hypocotyl lengths, root hair dimensions, root growth and root hair phenotype appearing in approxi- and root cortical cell lengths and widths) from confocal digital images mately 25% of the progeny, verifying that the two insertion were achieved using ImagePro analysis software (MediaCybernetics). alleles (designated nerd1-2 and -3) were indeed inactivating Transmitted light images of hypocotyl and root hair specimens were cap- this same locus affected in the original nerd1-1 line, and prov- tured with a Leica DFC 295 digital camera attached to a Zeiss Stemi SV 11 dissecting microscope, utilizing Leica Application Suite v3.8. ing that At3g51050 corresponds to the NERD1 gene. This Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3628 | Cole et al. Fig. 1. NERD1 encodes a transmembrane domain protein important for wild-type root development. (A) The combination of nerd1-1 and sec8-4 mutations results in a more severe root growth defect than either single mutation. Scale bar: 1 cm. (B) Map showing exons of NERD1 (At3g51050), the site of the G→A point mutation at a splice junction in nerd1-1, and the sites of the T-DNA insertions nerd1-2 (triangle 2: SALK_018060) and nerd1-3 (triangle 3: SALK_051660). (C) Schematic representation of NERD1 primary sequence features, showing an N-terminal signaling peptide (green), FG–GAP domains (purple), transmembrane domain (aqua), cytoplasmic domain (yellow), and residues predicted to form a calcium-binding pocket by RaptorX (red). The blue diagonal line between (B) and (C) shows where the splice site mutation in nerd1-1 is predicted to affect the NERD1 polypeptide. (D) NERD1 tertiary structure predicted by RaptorX, showing a β-propeller in yellow and α-helices in magenta. (E) Side view of the model in (D) with locations of residues in the predicted calcium-binding pocket (green). segregation pattern further shows that nerd1 mutants do not level of expression, suggests that NERD1 function is not as have a significant gametophyte-derived transmission defect. central to polarized growth in pollen tubes as it is in root hairs. Notably, pollen is the developmental stage, across 105 stages In addition to the similar phenotypic severity of each of assessed in the Genevestigator database (Grennan, 2006), asso- the three alleles, RT-PCR assays suggested that all three were ciated with lowest expression of At3g51050. The absence of a nulls, each generating aberrant transcripts that likely prod- significant transmission defect in nerd1 mutants, and the low uce non-functional protein. NERD1 transcripts in nerd1-1 Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3629 homozygotes were shorter than wild-type in the region span- Localization of NERD1 ning the point mutation, and thus were likely mis-spliced, Previous large-scale proteomic analyses detect NERD1 at the whereas transcripts in nerd1-2 and -3 were detected upstream, plasma membrane and/or Golgi (Mitra et al., 2009; Zhang and but not downstream from their respective T-DNA insertions Peck, 2011; Parsons et  al., 2012; Heard et  al., 2015). To valid- (Supplementary Fig. S3). ate and refine these findings, a genomic clone harboring the The 698 amino acid-long NERD1 sequence is broadly con- native promoter and complete ORF of NERD1 was tagged served in plants, and homologs are detectable in non-plant spe- with GFP at the 3′ end and expressed transiently in Nicotiana cies (Supplementary Dataset S1). The Gramene EnsemblPlants benthamiana or used to generate transgenic Arabidopsis plants. database (Kersey et al., 2016) identifies NERD1 homologs in 42 Confocal microscopy of leaf epidermis cells of N.  benthami- Viridiplantae species, primarily angiosperms, but also includ- ana revealed that the tagged protein is present in small motile ing more distantly related Archaeplastida species, including bodies of ca 1  μm in diameter that resembled Golgi stacks members of Bryophyta (Physcomitrella patens), Lycopodiophyta (Fig. 2A). To investigate the nature of these bodies, we exam- (Selaginella moellendorffii), and Chlorophyta (Chlamydomonas ined cells of N. benthamiana co-expressing NERD1–GFP and reinhardtii and Ostreococcus lucimarinus). In Arabidopsis and 26 one of two different Golgi markers: STtmd–Cherry or an other Viridiplantae species, NERD1 is identified as a single N-acetylglucosaminyl transferase fused to fluorescent protein copy gene. Certain regions of NERD1 also show notable simi- mTurquoise (NAG–mTurq) (Peremyslov et  al., 2012). In all larity to proteins in both Metazoan and Amoebozoan species cells examined, the GFP signal co-localized with these Golgi (Supplmentary Dataset S1). markers, indicating that NERD1 is primarily present in Golgi Protein modeling software was used to predict potential (Fig. 2C–E; Supplementary Fig. S6). structural characteristics of NERD1 (Fig. 1C; Supplementary The functionality of the NERD1–GFP fusion protein Table  S2; Supplementary Figs  S4 and S5; Supplmentary was validated by genetic complementation. To this end, the Datasets S2 and S3). Arabidopsis NERD1 contains an NERD1–GFP expression cassette was stably transformed N-terminal signaling peptide that is well conserved across all into an Arabidopsis nerd1-2 heterozygote line, which was sub- plant species and is indicative of association with the secre- sequently self-crossed. A  progeny plant homozygous for the tory system (TargetP 1.1, Emanuelsson et al., 2000). A single- nerd1-2 mutation but phenotypically wild-type was identi- pass transmembrane domain is identified near the C-terminus fied and self-crossed. PCR genotyping verified that all the of the protein, leaving a short 18–23 amino acid cytoplas- resultant seedlings were homozygous for the nerd1-2 muta- mic tail. Homology threading programs (3DLigandSite, tion. One-fourth of these plants (13 of 52) exhibited the nerd Wass et  al., 2010; Raptor-X, Källberg et  al., 2012; SWISS- root phenotype (shorter roots: 7.6 ± 1.3 mm versus wild-type Model, Biasini et  al., 2014; Phyre2, Kelley et  al., 2015) that –12 13.0 ± 2.6 mm, t-test P<10 ; and altered root-hair morph- compared NERD1’s primary and secondary structure with ology); all plants exhibiting the nerd phenotype were nega- proteins with tertiary structures solved by x-ray crystallog- tive for the cassette presence and NERD1–GFP expression. raphy (Supplmentary Dataset S2) predict (with 90–98% con- In contrast, the fusion cassette was present and expressed in fidence) that the non-cytoplasmic portion of NERD1 folds all the seedlings that were phenotypically wild-type, indicat- into a globular protein with a β-propeller structure: seven ing that it provides a functional NERD1 protein. Confocal predicted β-sheets arranged radially and pseudosymmetrically microscopy revealed that the NERD1–GFP in a nerd1-2 around a central axis (Fig.  1D–E; Supplementary Fig.  S5). mutant background was localized to mobile punctate struc- β-Propellers are widely used as structural scaffold, providing tures in the cytoplasm (Fig.  2B), similar to the observations a surface for ligand binding and enzymatic activity (Kopec in N.  benthamiana, and consistent with NERD1 localization and Lupas, 2013). In addition, NERD1’s β-propeller con- in the Golgi. To further validate association of NERD1–GFP tains a putative calcium-binding pocket. The predicted ter- with Golgi, we investigated sensitivity of the fluorescent bod- tiary structure resembles templates for some pyrroloquinoline ies to BFA, which disrupts Golgi architecture and induces quinone-dependent enzymes (e.g. alcohol dehydrogenases) formation of an endoplasmic reticulum (ER)–Golgi hybrid and, intriguingly, shares similarity to proteins that interact compartment (Ritzenthaler et  al, 2002). Arabidopsis seedlings with polysaccharides or glycoproteins, including lectins, inte- stably expressing either NERD1–GFP or, as a control, NAG– grins, carbohydrate binding proteins, and perhaps most not- mTurq, were incubated with this drug. As expected, BFA treat- ably, some pectin lyases and xyloglucanases (Supplmentary ment resulted in formation of a typical BFA compartment Dataset S2). Overall, however, the full length of NERD1 is not marked by either NAG–mTurq or NERD1–GFP in each line strictly homologous to members of any known protein family. (Supplementary Fig.  S7), strongly supporting the Golgi resi- Although the exact 3D structure and molecular function of dence of NERD1–GFP. NERD1 remain uncertain, these predictions raise the intrigu- The localization of NERD1–GFP in the Golgi is notably ing possibility that NERD1 is an integral membrane protein distinct from that observed for components of the exocyst at that interacts with polysaccharides, potentially in a calcium- the plasma membrane and in the cytoplasm (Fendrych et  al., dependent manner. Notably, NERD1 (At3g51050) mRNA is 2010, 2013; Li et al., 2013; Oda et al., 2015). This suggests that expressed throughout most of the Arabidopsis plant, with little the interaction between NERD1 and the exocyst is not a dir- change induced by developmental or environmental variables ect interaction at the plasma membrane, as we had initially (Grennan, 2006), suggesting that NERD1 is a component of hypothesized. One possibility is that NERD1 in the Golgi is most plant cells. Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3630 | Cole et al. Fig. 2. NERD1–GFP localizes to the Golgi. (A) NERD1–GFP in Nicotiana benthamiana leaf cells. (B) Root tip of a NERD1-GFP-complemented nerd1-2 mutant. (C–E) Co-localization of NERD1 with the Golgi marker: (C) STtmd::mCherry (sialyltransferase transmembrane domain), (D) NERD1–GFP, and (E) merged image. Scale bars: 5 μm (A, C, D, E) and 20 μm (B). important for correct transit of exocyst components to the exocyst components were examined for their effects on pri- plasma membrane. Thus, we tested whether the nerd1 mutation mary root growth, cell elongation in etiolated hypocotyls, and causes a mislocalization of the exocyst by imaging GFP-labeled the polarized growth of root hairs. Intriguingly, while all three exocyst components. Both EXO84–GFP and SEC8–GFP developmental contexts involve some degree of cell expansion localization patterns at the plasma membrane in nerd1 mutant and are impacted by both NERD1 and the exocyst, as detailed roots were indistinguishable from their localization in wild- below, the specific genetic interactions varied, depending upon type controls (Fig. 3A and B, and 3C and D, respectively). Thus, context. the nerd1 mutant root phenotype is not explained by a mislo- calization of the exocyst. The converse was also considered, i.e. Growth of primary root and etiolated hypocotyl do exocyst mutations result in altered localization of NERD1? Primary root growth was examined in plants harboring the Observation of NERD1–GFP in the roots of exo70A1 and nerd1-1 or nerd1-3 mutation in combination with a mutation exo84b mutants revealed that NERD1 localization (i.e. in in an exocyst component, exo70A1 or sec8-4. A comparison of punctate structures within the cytoplasm) was not altered by sibling plants confirmed that the primary root growth defect was mutation of these exocyst components (Fig. 3E–H). These data more severe in the double mutants than in either of the single further argue that the genetic interaction of NERD1 and exo- mutants (Fig. 4A). Additive and multiplicative models were used cyst mutants is indirect. to predict the severity of the root growth defect that would be observed if the mutations were non-interacting (Hála et al., 2008; Genetic interactions with NERD1 mutants depends on Mani et al., 2008). The observed growth rate defect in the double developmental context mutants was much more severe than predicted by either model, The apparent absence of co-localization of NERD1 and the verifying a synergistic interaction between NERD1 and exocyst exocyst motivated a quantitative assessment of their pheno- in root growth. To further substantiate the functional interaction types and genetic interactions in three distinct developmental between NERD1 and the exocyst, nerd1-2 mutants were treated contexts. Mutations of NERD1 combined with mutations of with the chemical Endosidin2 (ES2), which inhibits exocyst Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3631 Fig. 3. Subcellular localizations of NERD1 and exocyst markers are independent of each other. The localization of exocyst markers to the outer surface of root epidermal cells of nerd1-2 mutants (A, C) is similar to that in wild-type siblings (B, D). Conversely, the predominant localization of NERD1–GFP in the cytoplasm of root epidermal cells of exocyst mutants (E, G) is similar to that of wild-type siblings (F, H). Shown are epidermal cells in the root transition zone (A–D, G, H) and meristem (E, F). Confocal images provide radial longitudinal sections through the center of the root (A, B, E, F) in which the upper portion of cells shown are on the root surface. In tangential sections (C, D, G, H) parallel with the root surface the lateral walls of the epidermal cells are shown. Scale bars: 20 µm. (This figure is available in colour at JXB online.) function by interacting with EXO70A1 (Zhang et  al., 2016). number of cells dividing in shorter meristems. These defects As expected, root growth rates of control nerd1-2 plants harbor- were quantitatively similar for all three nerd1 alleles, and less ing the NERD1–GFP construct were not significantly different severe than in exo84b-1. Notably, mature cortical cell widths in from those of Col-0 seedlings grown on media containing 0, 20, nerd1 mutants are similar to those in Col-0, indicating that the or 40 μM ES2 (Fig. 4B). In contrast, nerd1-2 homozygotes are nerd1 defect is specific to cell elongation. This contrasts with significantly more sensitive to the effect of ES2 on root growth exocyst mutant cells, in which overall mature cortical cell size, rate, at both 20 and 40  μM ES2. Thus, similar to the genetic both length and width, is reduced. Thus, NERD1 appears to be interaction, the effect of pharmacological inhibition of the exo- more specifically involved in longitudinal expansion of lateral cyst on root growth in nerd1-2 mutants is more than would be walls in the root elongation zone. predicted by multiplicative or additive models (Fig. 4B). Elongation of the hypocotyl in etiolated seedlings, in con- The primary root growth defect in exocyst mutants is due trast to the more complex process of root growth, is due solely to both a reduced number of cells dividing in a shorter meri- to cell elongation, thereby providing a second and more spe- stem, and a slower rate of cell expansion in the elongation zone cific system to evaluate the genetic interaction of NERD1 (outside the meristem) (Cole et al., 2014). Given the functional and exocyst mutants in cell elongation. Hypocotyl lengths and interaction between NERD1 and the exocyst in the primary epidermal cell lengths in the hypocotyls of 5-day-old dark root, we were curious to know if nerd1 mutant effects could grown nerd1-3 sec8-4 double mutant seedlings were evaluated be attributed to one or both of these underlying mechanisms. and compared with similar measurements in single mutant Consequently, cortical cell files in the root tips of nerd1 mutants and wild-type siblings (Supplementary Fig.  S8). As in roots, were examined by confocal microscopy and compared with nerd1-3 and sec8-4 interact synergistically to reduce hypo- those in the root tips of Col-0 and exo84b (an exocyst mutant cotyl lengthening (Supplementary Fig. S8B). Furthermore, the with a severe root growth defect) grown on the same vertical effect on the hypocotyl was associated with a synergistic defect plates (Table  1). Similar to exocyst mutants with severe root in cell elongation (Supplementary Fig. S8C), again similar to growth defects (e.g. exo84b-1) (Cole et al., 2014), the reduced the results in the primary root. These data are consistent with primary root growth in nerd1 mutants arises from both less expression data pointing to a role for NERD1 and the exocyst cell elongation, leading to shorter mature cells, and a reduced in cell growth throughout the plant. Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3632 | Cole et al. Fig. 4. NERD1 acts synergistically with exocyst components to affect primary root growth. (A) Mutation of exocyst components sec8-4 or exo70A1-2 results in a mild reduction in primary root growth rate compared with wild-type (blue bars), while mutation of nerd1 (nerd1-1 or nerd1-3, red bars) results in an approximately 50% reduction in growth rate. The combination of a mutation in an exocyst component with a nerd1 mutation (green bars) results in a severe root growth defect that is more severe than predicted by additive (P<0.001, z-test) or multiplicative models (P<0.0001, z-test), indicating a synergistic interaction. (Error bars: SE; n=19–67 roots for each genotype.) (B) Compared with DMSO-treated controls, root growth was significantly more reduced in nerd1-2 mutants when 20 or 40 μM Endosidin2, an exocyst inhibitor, was added to the growth medium, compared with either Col-0 or nerd1- 2; NERD-GFP complemented seedlings. At both concentrations, the reduced growth rate in the nerd1-2 mutant treated with Endosidin2 was more than would be predicted by additive or multiplicative models (P<0.001, z-test). (Error bars: SE; n=13–17 roots for each genotype/Endosidin2 concentration.) Table 1. Primary root growth parameters of nerd1 mutants compared with Col-0 and exo84b-1 Genotype Roots Root growth Meristem Mature cell Mature cell Cell Estimated −1 evaluated (μm h ) size (no. of length (μm) width (μm) production length of −1 cells) (cells h ) cell cycle (h) a a Mean SD Mean SD n Mean SD n Mean SD Mean SD Mean SD Col-0 9 469.3 43.8 49.6 4.4 264 200.9 43.0 84 28.0 2.57 2.33 0.24 14.7 1.5 exo84b-1 10 51.2 6.6 10.0 1.9 278 86.6 21.8 84 13.2 1.53 0.59 0.08 11.4 1.1 nerd1-1 6 248.1 30.9 33.8 4.0 82 144.0 30.1 89 27.0 3.98 1.77 0.23 13.3 1.5 nerd1-2 6 231.2 38.8 32.4 3.0 112 153.3 38.5 87 28.4 3.34 1.50 0.17 14.5 0.8 nerd1-2 6 262.7 21.6 35.3 0.9 89 130.7 36.5 75 29.9 3.81 2.08 0.41 12.0 2.3 Values highlighted in bold are significantly different from Col-0 and exo84b-1 (P<0.001, t-test). Values highlighted in italic are significantly different from exo84b-1, but not Col-0 (P<0.001, t-test). n=number of cells measured. Polarized growth of root hairs (Synek et  al., 2006), whereas root hairs that are wild-type in length are observed in other exocyst mutants (e.g. sec8-4). As One phenotype leading to selection of the initial nerd1 allele in the primary root, abrogating exocyst function does not alter was altered root hair morphology. Short root hairs are charac- NERD1 localization patterns in the root hair (Fig.  5A, B). teristic of several exocyst mutants (e.g. exo70A1 and exo84b) Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3633 Fig. 5. NERD1 is epistatic to the exocyst in the root hair. Root hair lengths were examined for plants growing on vertical plates so that their roots were on the surface of the growth medium, i.e. at the medium–air interface. NERD1–GFP is localized to mobile punctate structures in both wild-type (A) and exo70A1 (B) root hairs (scale bars for A and B: 20 μm). Mature root hairs of nerd1-3 and nerd1-1 mutants are longer than for wild-type siblings (P<0.0001, t-test). (C) Extremely short root hair lengths are observed in exo70A1 mutants compared with wild-type (P<0.0001), and this phenotype is not altered by the addition of a nerd1-3 mutation in the double mutant (P=0.3). (n=190 for WT, nerd1-3, and exo70A1, n =51 for double mutant.) (D) The myo xi-k mutant has short root hairs compared with wild-type (P<0.0001). In nerd1-1; myo xi-k double mutants, root hairs are longer than those of myo xi-k (P<<0.0001), but still far shorter than wild-type (P<0.001). (n>164 for WT, nerd1-1 and myo xi-k; n=79 for double mutant; error bars: SD.) (This figure is available in colour at JXB online.) Nevertheless, comparison of root hairs in exo70A1 nerd1-3 and the exocyst was specific, we tested for interactions with double mutants and sibling single mutants revealed a genetic another mutation that affects the secretory pathway in root interaction (Fig. 5C). Surprisingly, the average root hair length hairs, myosin xi-k. Mutation of myosin xi-k results in shorter in the single nerd1 mutants was significantly longer than that root hairs, likely due to inhibition of cytoplasmic streaming of their wild-type siblings, indicating that NERD1 limits cell that drives secretory vesicle transport (Peremyslov et al., 2008; growth in this context. As a second surprise, in contrast to the Peremyslov et al., 2012; Park and Nebenführ, 2013; Peremyslov synergistic interaction in primary root growth, exo70A1 nerd1- et al., 2015). The double nerd1-1 myo xi-k mutant demonstrates 3 double mutants have short root hairs of similar size to those an additive phenotype: root hairs are longer than with the myo of exo70A1 single mutants. That is, the effect of the nerd1-3 xi-k mutation alone, but not as long as wild-type (Fig.  5D). mutation in increasing average root hair length is masked (epis- Thus, the epistatic interaction of nerd1 and exo70A1 mutants tasis), suggesting that, in root hairs, the exocyst is required for is specific, further arguing for a close functional relationship manifestation of NERD1’s root hair length limiting activity. between NERD1 and the exocyst. Moreover, the differing To determine whether the epistatic interaction of NERD1 outcomes of the interaction in root hairs versus primary roots Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3634 | Cole et al. bases, or bulbous shapes, morphologies that are rare in wild- type siblings (Supplementary Fig. S2). These deviant morphol- ogies are more consistently observed in roots that are growing within agar medium, rather than on the agar surface (where root hairs predominantly extend into the air). Consequently, root hair morphology within the medium was evaluated in 18 roots for each of five genotypes: Col-0 (wild-type), exo84b-1, nerd1-1, nerd1-2, and nerd1-3 (Fig. 6; Supplementary Table S3). Root hairs in nerd1 mutants, as in wild-type, grow out of the apical end of the trichoblasts at a single location, thus indicat- ing that root hair initiation per se is unaffected in nerd1 mutants. However, nerd1 root hairs are often more bulbous, with wider bases and shanks on average, compared with Col-0 (Fig. 6E). Because nerd1 roots exhibit both wild-type and bulbous root hair morphologies, high standard deviations are associated with nerd1 root hair measurements. Rupture of root hairs, evidenced by the extrusion of cytoplasmic contents into the medium from the root hair tip, is also notable in nerd1 mutants (Fig. 6C and D), occurring in 26–34% of root hairs evaluated, compared with a rare incidence (0–1.8%) in Col-0 or exo84b-1 (Fig. 6F; Supplementary Table  S3). Average root hair length for nerd1 root hairs growing within the medium is similar to that in wild-type (nerd1-1: 227  μm; nerd1-3: 247  μm; WT: 230  μm), even though rupture presumably stopped growth in some of the mutant root hairs. Overall, nerd1 root hair morphology is consistent with a role for NERD1 in establishing the structural stability, and perhaps limiting compliance, of the cell wall in growing root hairs. Increased compliance of the cell wall upon loss of NERD1 function might allow for more rapid expan- sion, leading to increased root hair lengths. But such an effect might also make root hairs vulnerable to bulbous expansion and bursting, as is observed. Discussion Secretory events upon which plant growth and development depend are manifested and regulated by a complex network of cellular components interacting both directly and indirectly. Facilitating secretion in many circumstances is an octameric protein complex, the exocyst (Cole and Fowler, 2006; Žárský et al., 2013; Kulich et al., 2015; Vukašinović and Žárský, 2016). To search for unknown components of the exocyst-mediated secretory network, we used a second-site enhancer screen, and identified nerd1 mutants via their genetic interaction with Fig. 6. NERD1 affects morphology of root hairs growing within agar exocyst mutants, influencing primary root growth, root hair medium. (A) Col-0 (wild-type), (B) exo84b-1, (C) nerd1-2, (D) nerd1-3. expansion, and hypocotyl elongation in Arabidopsis. Notably, Arrowheads: ruptured root hair tips; scale bar: 200 µm. (E, F) Root hairs of the interaction between NERD1 and the exocyst appears to nerd1 mutants (n=~200 root hairs for each allele) were significantly wider be indirect, and thus would not have been detected by other at the base and at the widest part of their shaft than either Col-0 (n=203) or exo84b-1 (n=79) (E), and showed a significantly higher incidence of root methods, e.g. yeast two-hybrid screening. Mutation of NERD1 hair rupture (F). (Root hairs measured from 18 roots for each genotype, leads to a shortened root meristem and reduced cell elong- P<0.001, t-test; error bars: SD.) ation in both the primary root and etiolated hypocotyls. These defects are also seen in plants with mutations affecting exocyst (epistatic versus synergistic, respectively) argue that this rela- components, but are synergistically accentuated when both tionship depends on cellular and developmental context. nerd1 and exocyst mutations are combined. Additionally, muta- Additional insight into the role of NERD1 in root hair tions of both NERD1 and components of the exocyst affect growth was gained by a closer examination of the morphology root hair morphology. The nearly ubiquitous expression of of root hairs in nerd1 mutants, which exhibit branches, inflated NERD1 throughout the plant (similar to that of most exocyst Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3635 components) and its conservation throughout the plant king- polysaccharides. Homology modeling suggests that this lume- dom underline its potential importance in a broader context. nal portion of NERD1 resembles proteins that interact with Notably, we were unable to generate a doubly homozygous polysaccharides, i.e. lectins, integrins, and carbohydrate bind- nerd1/exocyst mutant plant from the self-cross of a double het- ing proteins. Perhaps most notably, threading programs iden- erozygote combining nerd1-3 with severe exocyst mutants (i.e. tify certain NERD1 regions as similar to bacterial RGI pectin exo84b-1 and sec8-3; 0 out of 58 and 0 out of 66 individuals lyases, and to a lesser extent xyloglucanases (Supplmentary genotyped from nerd/exocyst segregating populations, respect- Dataset S2). Interestingly, RGI pectin lyases are activated by ively; P<0.05 by chi-square test for each). This suggests that calcium, consistent with the calcium-binding pocket predicted mutations of NERD1 combined with severe exocyst muta- for NERD1. Examination of the cell wall in nerd1 roots by, tions lead to lethality due to very early developmental defects. for example, histochemical staining for specific components We initially hypothesized that NERD1 directly interacts should help test the hypothesis that NERD1’s impact arises with the exocyst at the plasma membrane, where exocyst from a role influencing cell wall structure. components are known to localize (Fendrych et  al., 2010). It is noteworthy that the phenotypes observed in nerd1 However, the majority of proteomic studies identify NERD1 mutants are consistent with cell wall pectins as a target of in the ER or Golgi (Parsons et al., 2012; Nikolovski et al., 2014; NERD1 activity. Altering the synthesis of pectic polysaccha- Heard et  al., 2015), and not in the plasma membrane (Mitra rides is known to cause a dwarfed phenotype with develop- et  al., 2009). Direct examination of the functional NERD1– mental defects that include shorter primary roots and reduced GFP fusion validated its prominent localization in the Golgi elongation of etiolated hypocotyls (Reboul et al., 2011), rem- (Fig. 2; Supplementary Fig. S6), in contrast to the preferential iniscent of defects observed in nerd1 mutants. RGI pectins association of exocyst components with the plasma membrane impact the same aspects of root hair morphology (e.g. swelling (Fig. 3), suggesting that NERD1–exocyst interaction is indir- and branching) as those altered in nerd1 mutants (Diet et  al., ect. Although it remains possible that NERD1 is present at 2006; Reboul et  al., 2011). The accelerated cell elongation the plasma membrane transiently or at a low level, the most phase observed in etiolated hypocotyls is the result of cell wall likely interpretation of our results is that the synergistic genetic modification, and in particular has been associated with altered interaction between NERD1 and exocyst components does pectins (Derbyshire et al., 2007; Pelletier et al., 2010), possibly not involve direct physical contact. independent of cellulose synthesis. Thus, a role of NERD1 One alternative hypothesis that does not rely on direct in the synthesis or modification of cell wall pectins might contact to explain the observed NERD1–exocyst genetic explain the range of phenotypes observed in nerd1 mutants interaction is that NERD1 is required for correct exocyst and deserves further investigation. localization; or vice versa, NERD1 is a cargo for exocyst- Intriguingly, the effect of NERD1 on cell expansion is not mediated trafficking. A few specific cargos requiring the plant uniform throughout development: nerd1 mutations result in exocyst for correct delivery have been identified (pectinacious reduced cell elongation in the root tip and etiolated hypocotyl, mucilage in Arabidopsis seed coats (Kulich et al., 2010); callose but increased elongation of root hairs, along with increased in leaf trichomes (Kulich et al., 2015); and the integral plasma likelihood of rupture at the growing root hair tip. It is also of membrane proteins PEN3/ABCG36 and NIP5;1 (Mao et al., note that a mutation altering Arabidopsis EXO70C2, another 2016)). However, no mislocalization of fluorescently tagged potential indirect exocyst interactor, leads to a similar pheno- exocyst components in nerd1 mutants, or of NERD1–GFP type in the tip-growing pollen tube: more rapid growth and in exocyst mutants, was observed (Fig.  3), arguing against increased tube rupture (Synek et  al., 2017). The contrasting this possibility. On the other hand, these experiments do not effects of nerd1 mutants on root hair tip growth versus pri- exclude genetic interaction via a currently unknown cargo that mary root cell elongation could manifest because the cell wall requires both NERD1 and exocyst-mediated vesicle transport matrices in the two cell types are structurally distinct from each for its proper function. other, generated by fundamentally different processes: delivery The localization of NERD1 to the Golgi, the site of syn- of non-cellulosic cell wall components to a narrowly focused thesis of non-cellulose polysaccharides incorporated into the region versus more broadly distributed modification of a pre- cell wall matrix (e.g. pectin and hemicellulose; Driouich et al., existing cell wall matrix, respectively. Additional indirect evi- 2012; Kim and Brandizzi, 2016), is tantalizing. NERD1 could dence that cell walls are differentially altered is the increased be involved in the formation or function of trans-Golgi-local- incidence of bulging in nerd1 root hair shafts, although neither ized protein complexes, such as the ECHIDNA/YIP4 complex bulging nor rupture is characteristic of cells in mutant root that plays a role in post-Golgi secretion of pectin and hemi- meristematic and elongation zones. In the root, the compos- cellulose to the cell wall, and which also influences cell elong- ition and structure of the cell wall changes as cells progress ation in roots and hypocotyls (Gendre et al., 2013). However, through division, elongation, and differentiation zones. For we currently favor a working hypothesis, based on predicted example, the rhamnogalacturonan I pectin in cell walls is mod- structure, in which NERD1 directly affects a cell wall matrix ified during the transition from cell proliferation to cell elong- polysaccharide, glycoprotein or proteoglycan that is ultimately ation in roots (Willats et al., 1999), and pectins in root hairs are secreted via exocyst-mediated trafficking to influence cell wall structurally distinct from those in the lateral cell walls elsewhere growth and expansion. Most of NERD1 is predicted to be in the primary root (Muszyński et  al., 2015). Thus, the com- located in the Golgi lumen, folded into a β-propeller-like ter- position of cell wall components available for interaction with tiary structure that could serve as a scaffold for interactions with NERD1 within the Golgi likely varies as root cells progress Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3636 | Cole et al. Cole RA, Synek L, Zarsky V, Fowler JE. 2005. SEC8, a subunit of the from elongation to differentiation. Such differences could alter putative Arabidopsis exocyst complex, facilitates pollen germination and NERD1’s impact on cell wall extensibility, elongation, and fra- competitive pollen tube growth. Plant Physiology 138, 2005–2018. gility in a developmentally dependent manner. Revealing the Cvrčková F, Grunt M, Bezvoda R, Hála M, Kulich I, Rawat A, Žárský molecular function of NERD1 should help define how it is V. 2012. Evolution of the land plant exocyst complexes. 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A broadly conserved NERD genetically interacts with the exocyst to affect root growth and cell expansion

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Oxford University Press
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10.1093/jxb/ery162
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Abstract

The exocyst, a conserved, octameric protein complex, helps mediate secretion at the plasma membrane, facilitating specific developmental processes that include control of root meristem size, cell elongation, and tip growth. A gen- etic screen for second-site enhancers in Arabidopsis identified NEW ENHANCER of ROOT DWARFISM1 (NERD1) as an exocyst interactor. Mutations in NERD1 combined with weak exocyst mutations in SEC8 and EXO70A1 result in a synergistic reduction in root growth. Alone, nerd1 alleles modestly reduce primary root growth, both by shortening the root meristem and by reducing cell elongation, but also result in a slight increase in root hair length, bulging, and rup- ture. NERD1 was identified molecularly as At3g51050, which encodes a transmembrane protein of unknown function that is broadly conserved throughout the Archaeplastida. A functional NERD1–GFP fusion localizes to the Golgi, in a pattern distinct from the plasma membrane-localized exocyst, arguing against a direct NERD1–exocyst interaction. Structural modeling suggests the majority of the protein is positioned in the lumen, in a β-propeller-like structure that has some similarity to proteins that bind polysaccharides. We suggest that NERD1 interacts with the exocyst indirectly, possibly affecting polysaccharides destined for the cell wall, and influencing cell wall characteristics in a developmentally distinct manner. Keywords: Arabidopsis, cell elongation, cell wall, exocyst, genetic interaction, root development, root hair, root meristem, secretory pathway, tip growth. Introduction The secretory system in plants is a fundamental determi- sexual reproduction in flowering plants. Additionally, secre- nant of plasma membrane composition and cell wall forma- tion of substances into the apoplast and delivery of receptors tion (Luschnig and Vert, 2014; Ebine and Ueda, 2015; Kim and transporters to the plasma membrane allow for intercel- and Brandizzi, 2016). Consequently, secretory events drive lular communication and coordination. Ultimately, the secre- cell growth and morphogenesis, and influence plant devel- tory system’s intimate influence on the plasma membrane and opment. For example, selective localization of secretion to extracellular activities facilitates responsiveness and survival specific regions of the cell periphery is essential for polar- within variable abiotic and biotic environments. However, it ized growth of pollen tubes, enabling sperm cell delivery and remains unclear how the secretory process in plants is spatially © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3626 | Cole et al. and temporally regulated to direct particular cargos to spe- factors are likely to be involved, including cellular components cific locations of the plasma membrane, cell wall, or apoplast that interact with the exocyst indirectly, e.g. by enhancing the at the appropriate time. Both the conventional secretory sys- activity of an exocyst-trafficked protein. tem, i.e. vesicular transport from endoplasmic reticulum to To advance the investigation of exocyst-mediated secre- Golgi to plasma membrane, and non-conventional pathways tory events in plants, we performed a mutagenesis screen to are involved (Drakakaki and Dandekar, 2013; Robinson et al., identify interactors linked to the exocyst’s role in Arabidopsis 2016; van de Meene et al., 2017), but how these pathways are root growth. In this screen, we identified NEW ENHANCER tailored to the dynamic requirements of different cell types is OF ROOT DWARFISM (NERD1), a protein of unknown only beginning to be revealed. function that, based on genetic interaction data, acts with the The exocyst, an evolutionarily conserved octameric com- exocyst to facilitate root and hypocotyl elongation and to plex, tethers secretory vesicles to specific sites on the plasma influence root hair morphology. NERD1 is expressed through- membrane prior to exocytosis and modulates secretory activ- out the plant, suggesting a potential role beyond the root and ity to achieve an array of specialized functions. In plants, hypocotyl. NERD1 homologs are found throughout the plant components of the exocyst have been implicated in a range kingdom and beyond. Interestingly, the functional interaction of processes including pollen tube germination and growth of the exocyst with NERD1 is likely to be indirect, and var- (Cole et  al., 2005; Hála et  al., 2008; Li et  al., 2010), cytokin- ies dependent on developmental context. We speculate that esis (Fendrych et al., 2010; Rybak et al., 2014), secondary cell NERD1 is involved in the modification of cell wall polysac- wall deposition during tracheary element development (Li charides that are important for cell wall expansion and are a et al., 2013; Oda et al., 2015), hypocotyl elongation in etiolated cargo for exocyst-mediated transport to the apoplast. seedlings (Hála et  al., 2008), determination of meristem size and cell elongation during primary root growth (Cole et  al., Materials and methods 2014), Casparian strip formation (Kalmbach et al., 2017), local- ized disposition of seed coat pectin (Kulich et al., 2010), callose Plant materials and growth conditions deposition in trichomes (Kulich et  al., 2015), and the polar Lines of Landsberg erecta-0 and Columbia-0 ecotype of Arabidopsis with growth of root hairs (Wen et al., 2005; Synek et al., 2006). T-DNA insertions were obtained from the SALK Institute (Alonso et al., The regulation, assembly, and functioning of the exocyst 2003): nerd1-2 (At3g51050, SALK 018060C); nerd1-3 (At3g51050, SALK complex in non-plant eukaryotes has been linked to its inter- 051660); exo70A1-2 (At5g03540, SALK 135462); sec8-3 (At3g10380, SALK 026204); sec8-4 (At3g10380, SALK 118129); sec8-6 (At3g 10380, actions with small GTPases of the Rho, Ral, and Rab fami- SALK 091118); and myo XI-K (At5g20490, SALK 067972). The exo84b-1 lies (Mukherjee et al., 2014), membrane phospholipids (Thapa line was a GABI-Kat line (Rosso et  al., 2003; Fendrych et  al., 2010). et  al., 2012; Pleskot et  al., 2015), plasma membrane scaffold- The EXO84b-GFP and GFP-SEC8 lines were previously described ing proteins (Liu and Novick, 2014), and the actin cytoskel- (Fendrych et  al., 2010). The nerd1-1 mutant was generated in an ethyl eton (Jin et  al., 2011; Liu et  al., 2012). This interactive milieu methanesulfonate (EMS) screen that treated ~5000 sec8-6 seeds with 0.2% EMS for 15 h. M2 generation seed from 4500 M1 plants was collected in helps define exocyst function in yeast and mammals, provid- pools derived from 16 plant lots. The effectiveness of the mutagenesis was ing post-translational regulation of key secretory events (Wu verified by observing greater than 64% of M2 plants with one-quarter and Guo, 2015; Pleskot et  al., 2015). In plants, the molecular aberrant seed, with the gene mutation rate estimated at 1/3000. mechanisms that integrate the exocyst into distinct secretory Arabidopsis seeds were surface-sterilized, stratified at 4 °C for 3–5 d, processes are less well understood. One regulatory mechan- and planted on growth medium (1× MS, 2% (w/v) sucrose, and vitamins in 1% (w/v) Bacto-agar) or soil as previously described (Cole et al., 2005). ism unique to plants is the proliferation and diversification of Plants were grown in a climate chamber at 22 °C under long-day condi- homologs of the exocyst subunit Exo70 (23 in Arabidopsis), tions (16 h of light per day; 7500 lx), with the exception of those used which is hypothesized to allow for specification of particular in hypocotyl elongation experiments. For these, seeds were placed in a exocyst functions (Synek et al., 2006; Li et al., 2010; Cvrčková lighted incubator at 228C for 2–4 h to stimulate germination and then et  al., 2012; Vukašinović and Žárský, 2016). In support of this wrapped in foil, oriented vertically, and placed in a dark box in a 22 °C incubator. After 5 d in the dark, digital images were captured and hypo- hypothesis, different Exo70 paralogs have been associated with cotyl lengths measured. specific cellular processes: Exo70B1 with autophagy (Kulich To evaluate the effect of Endosidin2, three groups were germinated on et  al., 2013), Exo70I with arbuscular mycorrhizal symbiosis MS plates: seedlings that were homozygous for nerd1-2; nerd1-2 siblings (Zhang et  al., 2015), and Exo70E with the EXPO secretory complemented by NERD1–green fluorescent protein (GFP); and Col-0 pathway (Poulsen et  al., 2014). Furthermore, in growing pol- plants. Plants of each genotype were transferred approximately 3 d after germination to plates containing 0, 20, or 40 μM Endosidin2 (ES2), and len tubes, members of the EXO70A, EXO70B and EXO70C grown for an additional 4 d before imaging to determine primary root subgroups show differential localization patterns and apparent growth rates. DMSO-dissolved ES2 (or DMSO alone as a control, at activities (Sekereš et al., 2017; Synek et al., 2017). Other factors 0.5% (v/v)) was added to media during plate preparation. that help regulate the exocyst in specific developmental con- texts are the scaffolding protein Interactor of Constitutive active High-throughput sequencing and analysis ROPs 1 (ICR1) in roots (Lavy et al., 2007); the phosphoinosit- A plant homozygous for nerd1-1 and lacking a sec8 allele in a Col-0 ide PIP2 in pollen tubes (Bloch et  al., 2016); ROP2 GTPase background was backcrossed to Ler-0, and the progeny were self- (with its effector RIC7) in stomata (Hong et al., 2016); and the crossed to generate an F2 population. Pooled genomic DNA from 150 combined activities of VETH1–VETH2–COG2 and cortical F2 plants with the nerd phenotype (i.e. homozygotes) was sequenced microtubules in xylem cells (Oda et al., 2015). However, given via an Illumina HiSeq 2000 to generate 58 million paired-end reads. SHOREmap software (Schneeberger et al., 2009) was used to align the the breadth of functions known for the plant exocyst, other Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3627 reads to the Arabidopsis genome and assess the frequency of Col (the Results mutagenized parent) and Ler single nucleotide polymorphisms (SNPs) across the population. All variant SNPs in the ~200 kb region of chromo- Screening for exocyst interactors some 3 harboring nerd1-1 (Supplementary Fig. S1 at JXB online) were then searched against genes to identify candidate mutations with likely Mutations in genes encoding components of the exocyst com- deleterious effects. plex in Arabidopsis result in root growth defects that vary from a mild decrease in growth rate in some mutants (e.g. sec8-6 and exo70A1) to severe dwarfism in others (e.g. exo84b-1 and Genetic and molecular analyses sec8-3) (Cole et  al., 2014). Additionally, mutations of some DNA extraction from leaves and PCR genotyping for mutants contain- exocyst components reduce the length of root hairs (Synek ing T-DNA insertions was performed as previously described (Cole et al., 2005). Primers used in PCR and RT-PCR are shown in Supplementary et  al., 2006). We reasoned that a protein that interacts with Table S1. PCR-based genotyping to detect the EMS-generated nerd1-1 the exocyst could be revealed if its mutation accentuated the mutation required use of the restriction enzyme AvaII after amplifica- root growth defect of an exocyst mutation that by itself results tion, as a target cleavage site in the At3g51050 genomic sequence was in only a mild phenotype. Therefore, we screened an EMS- eliminated by the G→A transition in the mutant. To evaluate expression treated population of seedlings homozygous for the mild sec8-6 via RT-PCR, roots from approximately 50 10-day-old seedlings of each genotype were harvested from plates and frozen in liquid nitrogen. RNA mutation in a Col-0 background to identify such second-site was extracted from the pooled sample for each genotype using a phenol– enhancers. chloroform procedure, followed by DNase treatment. First strand cDNA Plants from M2 pools exhibiting both short roots and aber- synthesis was performed using Superscript II as per the manufacturer’s rant root hairs—dubbed the new enhancer of root dwarf- specifications (Thermo Fisher Scientific), followed by removal of RNA ism (nerd) phenotype—were outcrossed to a wild-type line, with RNaseH. The cDNA was used as a template for PCR with pri- mer pairs that amplified the sequence to the 5′ of the mutations, to the self-pollinated, and screened again for the phenotype in ~1/16 3′ of the mutations, or spanning the sites of the mutations. Primers for of the progeny, as expected for second site enhancers. After ACTIN2 were included as an internal control. screening 45 pools, we recovered exactly one such mutation, nerd1-1, which, when combined with sec8-6, leads to profound Generation of NERD1–GFP and imaging dwarfism throughout the plant and shorter primary roots A 5597-bp-long genomic fragment encompassing the NERD1 gene (~25% of wild-type length, with some demonstrating termi- along with its putative promoter was PCR amplified from the genomic nated growth). In addition, root hairs in nerd1 sec8-6 double DNA using KOD Hot Start high-fidelity DNA polymerase (Novagen) mutants are occasionally misshapen (Supplementary Fig.  S2). and cloned into a modified pMDC32 plasmid using SbfI and Pac I sites. Enhanced GFP (EGFP; Clontech) cDNA was added downstream from To verify that the genetic interaction was not specific to a par- the NERD1 open reading frame (ORF) to yield pMDC-NERD1-GFP. ticular SEC8 allele, the nerd1-1 mutation (isolated after a ser- The plasmid was mobilized into Agrobacterium tumefaciens strain GV3101. ies of backcrosses to Col-0) was combined with another mild Transient expression or coexpression of NERD1 construct and fluor- allele, sec8-4, yielding a similar result (Fig.  1A). Intriguingly, escent Golgi markers in Nicotiana benthamiana leaf epidermal cells was initial observations indicated that nerd1 plants, in the absence performed by co-infiltrating with Agrobacterium strains carrying NERD1- GFP and either STtmd-YFP or NAG-mTurq (Peremyslov et al., 2012) at of a sec8-6 or sec8-4 mutation, have a similar, but less severe concentrations equal to 0.2 OD . Imaging was conducted 2 d post- phenotype—e.g. root length ~75% of wild-type, and less fre- infiltration. Leaf fragments were immersed in water and observed using quently misshapen root hairs. a Zeiss LSM 780 NLO confocal microscope equipped with a Plan- Apochromat ×63 1.4 NA lens. mTurquoise, GFP, and mCherry were excited with the 405  nm diode laser line, 488-nm argon laser line, or Molecular identification of NERD1 561-nm He–Ne laser line, respectively. For the simultaneous visualization of two fluorophores, dual channel acquisition of signal for either GFP and To identify the nerd1-1 lesion, we used high-throughput mTurquoise or GFP and mCherry was performed sequentially to min- sequencing of a pooled population of mutant plants to iden- imize crosstalk. For Brefeldin A  (BFA) sensitivity, Arabidopsis seedlings tify a ~200  kb region of chromosome 3 tightly linked to expressing fluorophore-tagged proteins were treated with 50  μM BFA nerd1 (Supplementary Fig. S1). This region encompassed ~65 for 90 min, and the BFA-sensitive endomembrane compartments were imaged in root epidermal cells. protein-coding genes, only two of which harbored putative Evaluation of cortical cell files and root growth parameters in nerd1 EMS-generated G→A mutational differences from the Col-0 mutants using confocal microscopy was performed as previously reference sequence linked to nerd1-1. Both mutations were described (Cole et  al., 2014). Briefly, images of roots grown on vertical validated via Sanger sequencing. The best candidate for nerd1-1 plates were captured on day 5 and day 7 after germination to determine appeared to be a change in a conserved splice acceptor site at root growth rates. The 7-day-old seedlings were stained with propidium iodine and then imaged with a Zeiss LSM 780 NLO confocal micro- the ninth exon of At3g51050 (Fig. 1B). To confirm the molec- scope system. Multiple digital images were taken to capture two cortical ular identity of NERD1, two independent T-DNA insertion cell files for each root, one on each side of the longitudinal midline from alleles in At3g51050 (Fig.  1B) were obtained from the Salk the quiescent center to the differentiation zone. Cell widths and lengths mutant collection, both of which were associated with short were measured. The cortical cell length profile combined with the root root and root hair defects. Subsequent complementation tests growth rate allowed estimations of the number of cells in the meristem, the cell production rate, and the length of the cell cycle for each root. between heterozygotes for all three alleles showed the nerd1 Measurements (e.g. root lengths, hypocotyl lengths, root hair dimensions, root growth and root hair phenotype appearing in approxi- and root cortical cell lengths and widths) from confocal digital images mately 25% of the progeny, verifying that the two insertion were achieved using ImagePro analysis software (MediaCybernetics). alleles (designated nerd1-2 and -3) were indeed inactivating Transmitted light images of hypocotyl and root hair specimens were cap- this same locus affected in the original nerd1-1 line, and prov- tured with a Leica DFC 295 digital camera attached to a Zeiss Stemi SV 11 dissecting microscope, utilizing Leica Application Suite v3.8. ing that At3g51050 corresponds to the NERD1 gene. This Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3628 | Cole et al. Fig. 1. NERD1 encodes a transmembrane domain protein important for wild-type root development. (A) The combination of nerd1-1 and sec8-4 mutations results in a more severe root growth defect than either single mutation. Scale bar: 1 cm. (B) Map showing exons of NERD1 (At3g51050), the site of the G→A point mutation at a splice junction in nerd1-1, and the sites of the T-DNA insertions nerd1-2 (triangle 2: SALK_018060) and nerd1-3 (triangle 3: SALK_051660). (C) Schematic representation of NERD1 primary sequence features, showing an N-terminal signaling peptide (green), FG–GAP domains (purple), transmembrane domain (aqua), cytoplasmic domain (yellow), and residues predicted to form a calcium-binding pocket by RaptorX (red). The blue diagonal line between (B) and (C) shows where the splice site mutation in nerd1-1 is predicted to affect the NERD1 polypeptide. (D) NERD1 tertiary structure predicted by RaptorX, showing a β-propeller in yellow and α-helices in magenta. (E) Side view of the model in (D) with locations of residues in the predicted calcium-binding pocket (green). segregation pattern further shows that nerd1 mutants do not level of expression, suggests that NERD1 function is not as have a significant gametophyte-derived transmission defect. central to polarized growth in pollen tubes as it is in root hairs. Notably, pollen is the developmental stage, across 105 stages In addition to the similar phenotypic severity of each of assessed in the Genevestigator database (Grennan, 2006), asso- the three alleles, RT-PCR assays suggested that all three were ciated with lowest expression of At3g51050. The absence of a nulls, each generating aberrant transcripts that likely prod- significant transmission defect in nerd1 mutants, and the low uce non-functional protein. NERD1 transcripts in nerd1-1 Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3629 homozygotes were shorter than wild-type in the region span- Localization of NERD1 ning the point mutation, and thus were likely mis-spliced, Previous large-scale proteomic analyses detect NERD1 at the whereas transcripts in nerd1-2 and -3 were detected upstream, plasma membrane and/or Golgi (Mitra et al., 2009; Zhang and but not downstream from their respective T-DNA insertions Peck, 2011; Parsons et  al., 2012; Heard et  al., 2015). To valid- (Supplementary Fig. S3). ate and refine these findings, a genomic clone harboring the The 698 amino acid-long NERD1 sequence is broadly con- native promoter and complete ORF of NERD1 was tagged served in plants, and homologs are detectable in non-plant spe- with GFP at the 3′ end and expressed transiently in Nicotiana cies (Supplementary Dataset S1). The Gramene EnsemblPlants benthamiana or used to generate transgenic Arabidopsis plants. database (Kersey et al., 2016) identifies NERD1 homologs in 42 Confocal microscopy of leaf epidermis cells of N.  benthami- Viridiplantae species, primarily angiosperms, but also includ- ana revealed that the tagged protein is present in small motile ing more distantly related Archaeplastida species, including bodies of ca 1  μm in diameter that resembled Golgi stacks members of Bryophyta (Physcomitrella patens), Lycopodiophyta (Fig. 2A). To investigate the nature of these bodies, we exam- (Selaginella moellendorffii), and Chlorophyta (Chlamydomonas ined cells of N. benthamiana co-expressing NERD1–GFP and reinhardtii and Ostreococcus lucimarinus). In Arabidopsis and 26 one of two different Golgi markers: STtmd–Cherry or an other Viridiplantae species, NERD1 is identified as a single N-acetylglucosaminyl transferase fused to fluorescent protein copy gene. Certain regions of NERD1 also show notable simi- mTurquoise (NAG–mTurq) (Peremyslov et  al., 2012). In all larity to proteins in both Metazoan and Amoebozoan species cells examined, the GFP signal co-localized with these Golgi (Supplmentary Dataset S1). markers, indicating that NERD1 is primarily present in Golgi Protein modeling software was used to predict potential (Fig. 2C–E; Supplementary Fig. S6). structural characteristics of NERD1 (Fig. 1C; Supplementary The functionality of the NERD1–GFP fusion protein Table  S2; Supplementary Figs  S4 and S5; Supplmentary was validated by genetic complementation. To this end, the Datasets S2 and S3). Arabidopsis NERD1 contains an NERD1–GFP expression cassette was stably transformed N-terminal signaling peptide that is well conserved across all into an Arabidopsis nerd1-2 heterozygote line, which was sub- plant species and is indicative of association with the secre- sequently self-crossed. A  progeny plant homozygous for the tory system (TargetP 1.1, Emanuelsson et al., 2000). A single- nerd1-2 mutation but phenotypically wild-type was identi- pass transmembrane domain is identified near the C-terminus fied and self-crossed. PCR genotyping verified that all the of the protein, leaving a short 18–23 amino acid cytoplas- resultant seedlings were homozygous for the nerd1-2 muta- mic tail. Homology threading programs (3DLigandSite, tion. One-fourth of these plants (13 of 52) exhibited the nerd Wass et  al., 2010; Raptor-X, Källberg et  al., 2012; SWISS- root phenotype (shorter roots: 7.6 ± 1.3 mm versus wild-type Model, Biasini et  al., 2014; Phyre2, Kelley et  al., 2015) that –12 13.0 ± 2.6 mm, t-test P<10 ; and altered root-hair morph- compared NERD1’s primary and secondary structure with ology); all plants exhibiting the nerd phenotype were nega- proteins with tertiary structures solved by x-ray crystallog- tive for the cassette presence and NERD1–GFP expression. raphy (Supplmentary Dataset S2) predict (with 90–98% con- In contrast, the fusion cassette was present and expressed in fidence) that the non-cytoplasmic portion of NERD1 folds all the seedlings that were phenotypically wild-type, indicat- into a globular protein with a β-propeller structure: seven ing that it provides a functional NERD1 protein. Confocal predicted β-sheets arranged radially and pseudosymmetrically microscopy revealed that the NERD1–GFP in a nerd1-2 around a central axis (Fig.  1D–E; Supplementary Fig.  S5). mutant background was localized to mobile punctate struc- β-Propellers are widely used as structural scaffold, providing tures in the cytoplasm (Fig.  2B), similar to the observations a surface for ligand binding and enzymatic activity (Kopec in N.  benthamiana, and consistent with NERD1 localization and Lupas, 2013). In addition, NERD1’s β-propeller con- in the Golgi. To further validate association of NERD1–GFP tains a putative calcium-binding pocket. The predicted ter- with Golgi, we investigated sensitivity of the fluorescent bod- tiary structure resembles templates for some pyrroloquinoline ies to BFA, which disrupts Golgi architecture and induces quinone-dependent enzymes (e.g. alcohol dehydrogenases) formation of an endoplasmic reticulum (ER)–Golgi hybrid and, intriguingly, shares similarity to proteins that interact compartment (Ritzenthaler et  al, 2002). Arabidopsis seedlings with polysaccharides or glycoproteins, including lectins, inte- stably expressing either NERD1–GFP or, as a control, NAG– grins, carbohydrate binding proteins, and perhaps most not- mTurq, were incubated with this drug. As expected, BFA treat- ably, some pectin lyases and xyloglucanases (Supplmentary ment resulted in formation of a typical BFA compartment Dataset S2). Overall, however, the full length of NERD1 is not marked by either NAG–mTurq or NERD1–GFP in each line strictly homologous to members of any known protein family. (Supplementary Fig.  S7), strongly supporting the Golgi resi- Although the exact 3D structure and molecular function of dence of NERD1–GFP. NERD1 remain uncertain, these predictions raise the intrigu- The localization of NERD1–GFP in the Golgi is notably ing possibility that NERD1 is an integral membrane protein distinct from that observed for components of the exocyst at that interacts with polysaccharides, potentially in a calcium- the plasma membrane and in the cytoplasm (Fendrych et  al., dependent manner. Notably, NERD1 (At3g51050) mRNA is 2010, 2013; Li et al., 2013; Oda et al., 2015). This suggests that expressed throughout most of the Arabidopsis plant, with little the interaction between NERD1 and the exocyst is not a dir- change induced by developmental or environmental variables ect interaction at the plasma membrane, as we had initially (Grennan, 2006), suggesting that NERD1 is a component of hypothesized. One possibility is that NERD1 in the Golgi is most plant cells. Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3630 | Cole et al. Fig. 2. NERD1–GFP localizes to the Golgi. (A) NERD1–GFP in Nicotiana benthamiana leaf cells. (B) Root tip of a NERD1-GFP-complemented nerd1-2 mutant. (C–E) Co-localization of NERD1 with the Golgi marker: (C) STtmd::mCherry (sialyltransferase transmembrane domain), (D) NERD1–GFP, and (E) merged image. Scale bars: 5 μm (A, C, D, E) and 20 μm (B). important for correct transit of exocyst components to the exocyst components were examined for their effects on pri- plasma membrane. Thus, we tested whether the nerd1 mutation mary root growth, cell elongation in etiolated hypocotyls, and causes a mislocalization of the exocyst by imaging GFP-labeled the polarized growth of root hairs. Intriguingly, while all three exocyst components. Both EXO84–GFP and SEC8–GFP developmental contexts involve some degree of cell expansion localization patterns at the plasma membrane in nerd1 mutant and are impacted by both NERD1 and the exocyst, as detailed roots were indistinguishable from their localization in wild- below, the specific genetic interactions varied, depending upon type controls (Fig. 3A and B, and 3C and D, respectively). Thus, context. the nerd1 mutant root phenotype is not explained by a mislo- calization of the exocyst. The converse was also considered, i.e. Growth of primary root and etiolated hypocotyl do exocyst mutations result in altered localization of NERD1? Primary root growth was examined in plants harboring the Observation of NERD1–GFP in the roots of exo70A1 and nerd1-1 or nerd1-3 mutation in combination with a mutation exo84b mutants revealed that NERD1 localization (i.e. in in an exocyst component, exo70A1 or sec8-4. A comparison of punctate structures within the cytoplasm) was not altered by sibling plants confirmed that the primary root growth defect was mutation of these exocyst components (Fig. 3E–H). These data more severe in the double mutants than in either of the single further argue that the genetic interaction of NERD1 and exo- mutants (Fig. 4A). Additive and multiplicative models were used cyst mutants is indirect. to predict the severity of the root growth defect that would be observed if the mutations were non-interacting (Hála et al., 2008; Genetic interactions with NERD1 mutants depends on Mani et al., 2008). The observed growth rate defect in the double developmental context mutants was much more severe than predicted by either model, The apparent absence of co-localization of NERD1 and the verifying a synergistic interaction between NERD1 and exocyst exocyst motivated a quantitative assessment of their pheno- in root growth. To further substantiate the functional interaction types and genetic interactions in three distinct developmental between NERD1 and the exocyst, nerd1-2 mutants were treated contexts. Mutations of NERD1 combined with mutations of with the chemical Endosidin2 (ES2), which inhibits exocyst Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3631 Fig. 3. Subcellular localizations of NERD1 and exocyst markers are independent of each other. The localization of exocyst markers to the outer surface of root epidermal cells of nerd1-2 mutants (A, C) is similar to that in wild-type siblings (B, D). Conversely, the predominant localization of NERD1–GFP in the cytoplasm of root epidermal cells of exocyst mutants (E, G) is similar to that of wild-type siblings (F, H). Shown are epidermal cells in the root transition zone (A–D, G, H) and meristem (E, F). Confocal images provide radial longitudinal sections through the center of the root (A, B, E, F) in which the upper portion of cells shown are on the root surface. In tangential sections (C, D, G, H) parallel with the root surface the lateral walls of the epidermal cells are shown. Scale bars: 20 µm. (This figure is available in colour at JXB online.) function by interacting with EXO70A1 (Zhang et  al., 2016). number of cells dividing in shorter meristems. These defects As expected, root growth rates of control nerd1-2 plants harbor- were quantitatively similar for all three nerd1 alleles, and less ing the NERD1–GFP construct were not significantly different severe than in exo84b-1. Notably, mature cortical cell widths in from those of Col-0 seedlings grown on media containing 0, 20, nerd1 mutants are similar to those in Col-0, indicating that the or 40 μM ES2 (Fig. 4B). In contrast, nerd1-2 homozygotes are nerd1 defect is specific to cell elongation. This contrasts with significantly more sensitive to the effect of ES2 on root growth exocyst mutant cells, in which overall mature cortical cell size, rate, at both 20 and 40  μM ES2. Thus, similar to the genetic both length and width, is reduced. Thus, NERD1 appears to be interaction, the effect of pharmacological inhibition of the exo- more specifically involved in longitudinal expansion of lateral cyst on root growth in nerd1-2 mutants is more than would be walls in the root elongation zone. predicted by multiplicative or additive models (Fig. 4B). Elongation of the hypocotyl in etiolated seedlings, in con- The primary root growth defect in exocyst mutants is due trast to the more complex process of root growth, is due solely to both a reduced number of cells dividing in a shorter meri- to cell elongation, thereby providing a second and more spe- stem, and a slower rate of cell expansion in the elongation zone cific system to evaluate the genetic interaction of NERD1 (outside the meristem) (Cole et al., 2014). Given the functional and exocyst mutants in cell elongation. Hypocotyl lengths and interaction between NERD1 and the exocyst in the primary epidermal cell lengths in the hypocotyls of 5-day-old dark root, we were curious to know if nerd1 mutant effects could grown nerd1-3 sec8-4 double mutant seedlings were evaluated be attributed to one or both of these underlying mechanisms. and compared with similar measurements in single mutant Consequently, cortical cell files in the root tips of nerd1 mutants and wild-type siblings (Supplementary Fig.  S8). As in roots, were examined by confocal microscopy and compared with nerd1-3 and sec8-4 interact synergistically to reduce hypo- those in the root tips of Col-0 and exo84b (an exocyst mutant cotyl lengthening (Supplementary Fig. S8B). Furthermore, the with a severe root growth defect) grown on the same vertical effect on the hypocotyl was associated with a synergistic defect plates (Table  1). Similar to exocyst mutants with severe root in cell elongation (Supplementary Fig. S8C), again similar to growth defects (e.g. exo84b-1) (Cole et al., 2014), the reduced the results in the primary root. These data are consistent with primary root growth in nerd1 mutants arises from both less expression data pointing to a role for NERD1 and the exocyst cell elongation, leading to shorter mature cells, and a reduced in cell growth throughout the plant. Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3632 | Cole et al. Fig. 4. NERD1 acts synergistically with exocyst components to affect primary root growth. (A) Mutation of exocyst components sec8-4 or exo70A1-2 results in a mild reduction in primary root growth rate compared with wild-type (blue bars), while mutation of nerd1 (nerd1-1 or nerd1-3, red bars) results in an approximately 50% reduction in growth rate. The combination of a mutation in an exocyst component with a nerd1 mutation (green bars) results in a severe root growth defect that is more severe than predicted by additive (P<0.001, z-test) or multiplicative models (P<0.0001, z-test), indicating a synergistic interaction. (Error bars: SE; n=19–67 roots for each genotype.) (B) Compared with DMSO-treated controls, root growth was significantly more reduced in nerd1-2 mutants when 20 or 40 μM Endosidin2, an exocyst inhibitor, was added to the growth medium, compared with either Col-0 or nerd1- 2; NERD-GFP complemented seedlings. At both concentrations, the reduced growth rate in the nerd1-2 mutant treated with Endosidin2 was more than would be predicted by additive or multiplicative models (P<0.001, z-test). (Error bars: SE; n=13–17 roots for each genotype/Endosidin2 concentration.) Table 1. Primary root growth parameters of nerd1 mutants compared with Col-0 and exo84b-1 Genotype Roots Root growth Meristem Mature cell Mature cell Cell Estimated −1 evaluated (μm h ) size (no. of length (μm) width (μm) production length of −1 cells) (cells h ) cell cycle (h) a a Mean SD Mean SD n Mean SD n Mean SD Mean SD Mean SD Col-0 9 469.3 43.8 49.6 4.4 264 200.9 43.0 84 28.0 2.57 2.33 0.24 14.7 1.5 exo84b-1 10 51.2 6.6 10.0 1.9 278 86.6 21.8 84 13.2 1.53 0.59 0.08 11.4 1.1 nerd1-1 6 248.1 30.9 33.8 4.0 82 144.0 30.1 89 27.0 3.98 1.77 0.23 13.3 1.5 nerd1-2 6 231.2 38.8 32.4 3.0 112 153.3 38.5 87 28.4 3.34 1.50 0.17 14.5 0.8 nerd1-2 6 262.7 21.6 35.3 0.9 89 130.7 36.5 75 29.9 3.81 2.08 0.41 12.0 2.3 Values highlighted in bold are significantly different from Col-0 and exo84b-1 (P<0.001, t-test). Values highlighted in italic are significantly different from exo84b-1, but not Col-0 (P<0.001, t-test). n=number of cells measured. Polarized growth of root hairs (Synek et  al., 2006), whereas root hairs that are wild-type in length are observed in other exocyst mutants (e.g. sec8-4). As One phenotype leading to selection of the initial nerd1 allele in the primary root, abrogating exocyst function does not alter was altered root hair morphology. Short root hairs are charac- NERD1 localization patterns in the root hair (Fig.  5A, B). teristic of several exocyst mutants (e.g. exo70A1 and exo84b) Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3633 Fig. 5. NERD1 is epistatic to the exocyst in the root hair. Root hair lengths were examined for plants growing on vertical plates so that their roots were on the surface of the growth medium, i.e. at the medium–air interface. NERD1–GFP is localized to mobile punctate structures in both wild-type (A) and exo70A1 (B) root hairs (scale bars for A and B: 20 μm). Mature root hairs of nerd1-3 and nerd1-1 mutants are longer than for wild-type siblings (P<0.0001, t-test). (C) Extremely short root hair lengths are observed in exo70A1 mutants compared with wild-type (P<0.0001), and this phenotype is not altered by the addition of a nerd1-3 mutation in the double mutant (P=0.3). (n=190 for WT, nerd1-3, and exo70A1, n =51 for double mutant.) (D) The myo xi-k mutant has short root hairs compared with wild-type (P<0.0001). In nerd1-1; myo xi-k double mutants, root hairs are longer than those of myo xi-k (P<<0.0001), but still far shorter than wild-type (P<0.001). (n>164 for WT, nerd1-1 and myo xi-k; n=79 for double mutant; error bars: SD.) (This figure is available in colour at JXB online.) Nevertheless, comparison of root hairs in exo70A1 nerd1-3 and the exocyst was specific, we tested for interactions with double mutants and sibling single mutants revealed a genetic another mutation that affects the secretory pathway in root interaction (Fig. 5C). Surprisingly, the average root hair length hairs, myosin xi-k. Mutation of myosin xi-k results in shorter in the single nerd1 mutants was significantly longer than that root hairs, likely due to inhibition of cytoplasmic streaming of their wild-type siblings, indicating that NERD1 limits cell that drives secretory vesicle transport (Peremyslov et al., 2008; growth in this context. As a second surprise, in contrast to the Peremyslov et al., 2012; Park and Nebenführ, 2013; Peremyslov synergistic interaction in primary root growth, exo70A1 nerd1- et al., 2015). The double nerd1-1 myo xi-k mutant demonstrates 3 double mutants have short root hairs of similar size to those an additive phenotype: root hairs are longer than with the myo of exo70A1 single mutants. That is, the effect of the nerd1-3 xi-k mutation alone, but not as long as wild-type (Fig.  5D). mutation in increasing average root hair length is masked (epis- Thus, the epistatic interaction of nerd1 and exo70A1 mutants tasis), suggesting that, in root hairs, the exocyst is required for is specific, further arguing for a close functional relationship manifestation of NERD1’s root hair length limiting activity. between NERD1 and the exocyst. Moreover, the differing To determine whether the epistatic interaction of NERD1 outcomes of the interaction in root hairs versus primary roots Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3634 | Cole et al. bases, or bulbous shapes, morphologies that are rare in wild- type siblings (Supplementary Fig. S2). These deviant morphol- ogies are more consistently observed in roots that are growing within agar medium, rather than on the agar surface (where root hairs predominantly extend into the air). Consequently, root hair morphology within the medium was evaluated in 18 roots for each of five genotypes: Col-0 (wild-type), exo84b-1, nerd1-1, nerd1-2, and nerd1-3 (Fig. 6; Supplementary Table S3). Root hairs in nerd1 mutants, as in wild-type, grow out of the apical end of the trichoblasts at a single location, thus indicat- ing that root hair initiation per se is unaffected in nerd1 mutants. However, nerd1 root hairs are often more bulbous, with wider bases and shanks on average, compared with Col-0 (Fig. 6E). Because nerd1 roots exhibit both wild-type and bulbous root hair morphologies, high standard deviations are associated with nerd1 root hair measurements. Rupture of root hairs, evidenced by the extrusion of cytoplasmic contents into the medium from the root hair tip, is also notable in nerd1 mutants (Fig. 6C and D), occurring in 26–34% of root hairs evaluated, compared with a rare incidence (0–1.8%) in Col-0 or exo84b-1 (Fig. 6F; Supplementary Table  S3). Average root hair length for nerd1 root hairs growing within the medium is similar to that in wild-type (nerd1-1: 227  μm; nerd1-3: 247  μm; WT: 230  μm), even though rupture presumably stopped growth in some of the mutant root hairs. Overall, nerd1 root hair morphology is consistent with a role for NERD1 in establishing the structural stability, and perhaps limiting compliance, of the cell wall in growing root hairs. Increased compliance of the cell wall upon loss of NERD1 function might allow for more rapid expan- sion, leading to increased root hair lengths. But such an effect might also make root hairs vulnerable to bulbous expansion and bursting, as is observed. Discussion Secretory events upon which plant growth and development depend are manifested and regulated by a complex network of cellular components interacting both directly and indirectly. Facilitating secretion in many circumstances is an octameric protein complex, the exocyst (Cole and Fowler, 2006; Žárský et al., 2013; Kulich et al., 2015; Vukašinović and Žárský, 2016). To search for unknown components of the exocyst-mediated secretory network, we used a second-site enhancer screen, and identified nerd1 mutants via their genetic interaction with Fig. 6. NERD1 affects morphology of root hairs growing within agar exocyst mutants, influencing primary root growth, root hair medium. (A) Col-0 (wild-type), (B) exo84b-1, (C) nerd1-2, (D) nerd1-3. expansion, and hypocotyl elongation in Arabidopsis. Notably, Arrowheads: ruptured root hair tips; scale bar: 200 µm. (E, F) Root hairs of the interaction between NERD1 and the exocyst appears to nerd1 mutants (n=~200 root hairs for each allele) were significantly wider be indirect, and thus would not have been detected by other at the base and at the widest part of their shaft than either Col-0 (n=203) or exo84b-1 (n=79) (E), and showed a significantly higher incidence of root methods, e.g. yeast two-hybrid screening. Mutation of NERD1 hair rupture (F). (Root hairs measured from 18 roots for each genotype, leads to a shortened root meristem and reduced cell elong- P<0.001, t-test; error bars: SD.) ation in both the primary root and etiolated hypocotyls. These defects are also seen in plants with mutations affecting exocyst (epistatic versus synergistic, respectively) argue that this rela- components, but are synergistically accentuated when both tionship depends on cellular and developmental context. nerd1 and exocyst mutations are combined. Additionally, muta- Additional insight into the role of NERD1 in root hair tions of both NERD1 and components of the exocyst affect growth was gained by a closer examination of the morphology root hair morphology. The nearly ubiquitous expression of of root hairs in nerd1 mutants, which exhibit branches, inflated NERD1 throughout the plant (similar to that of most exocyst Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 NERD1 interacts with the exocyst in root development | 3635 components) and its conservation throughout the plant king- polysaccharides. Homology modeling suggests that this lume- dom underline its potential importance in a broader context. nal portion of NERD1 resembles proteins that interact with Notably, we were unable to generate a doubly homozygous polysaccharides, i.e. lectins, integrins, and carbohydrate bind- nerd1/exocyst mutant plant from the self-cross of a double het- ing proteins. Perhaps most notably, threading programs iden- erozygote combining nerd1-3 with severe exocyst mutants (i.e. tify certain NERD1 regions as similar to bacterial RGI pectin exo84b-1 and sec8-3; 0 out of 58 and 0 out of 66 individuals lyases, and to a lesser extent xyloglucanases (Supplmentary genotyped from nerd/exocyst segregating populations, respect- Dataset S2). Interestingly, RGI pectin lyases are activated by ively; P<0.05 by chi-square test for each). This suggests that calcium, consistent with the calcium-binding pocket predicted mutations of NERD1 combined with severe exocyst muta- for NERD1. Examination of the cell wall in nerd1 roots by, tions lead to lethality due to very early developmental defects. for example, histochemical staining for specific components We initially hypothesized that NERD1 directly interacts should help test the hypothesis that NERD1’s impact arises with the exocyst at the plasma membrane, where exocyst from a role influencing cell wall structure. components are known to localize (Fendrych et  al., 2010). It is noteworthy that the phenotypes observed in nerd1 However, the majority of proteomic studies identify NERD1 mutants are consistent with cell wall pectins as a target of in the ER or Golgi (Parsons et al., 2012; Nikolovski et al., 2014; NERD1 activity. Altering the synthesis of pectic polysaccha- Heard et  al., 2015), and not in the plasma membrane (Mitra rides is known to cause a dwarfed phenotype with develop- et  al., 2009). Direct examination of the functional NERD1– mental defects that include shorter primary roots and reduced GFP fusion validated its prominent localization in the Golgi elongation of etiolated hypocotyls (Reboul et al., 2011), rem- (Fig. 2; Supplementary Fig. S6), in contrast to the preferential iniscent of defects observed in nerd1 mutants. RGI pectins association of exocyst components with the plasma membrane impact the same aspects of root hair morphology (e.g. swelling (Fig. 3), suggesting that NERD1–exocyst interaction is indir- and branching) as those altered in nerd1 mutants (Diet et  al., ect. Although it remains possible that NERD1 is present at 2006; Reboul et  al., 2011). The accelerated cell elongation the plasma membrane transiently or at a low level, the most phase observed in etiolated hypocotyls is the result of cell wall likely interpretation of our results is that the synergistic genetic modification, and in particular has been associated with altered interaction between NERD1 and exocyst components does pectins (Derbyshire et al., 2007; Pelletier et al., 2010), possibly not involve direct physical contact. independent of cellulose synthesis. Thus, a role of NERD1 One alternative hypothesis that does not rely on direct in the synthesis or modification of cell wall pectins might contact to explain the observed NERD1–exocyst genetic explain the range of phenotypes observed in nerd1 mutants interaction is that NERD1 is required for correct exocyst and deserves further investigation. localization; or vice versa, NERD1 is a cargo for exocyst- Intriguingly, the effect of NERD1 on cell expansion is not mediated trafficking. A few specific cargos requiring the plant uniform throughout development: nerd1 mutations result in exocyst for correct delivery have been identified (pectinacious reduced cell elongation in the root tip and etiolated hypocotyl, mucilage in Arabidopsis seed coats (Kulich et al., 2010); callose but increased elongation of root hairs, along with increased in leaf trichomes (Kulich et al., 2015); and the integral plasma likelihood of rupture at the growing root hair tip. It is also of membrane proteins PEN3/ABCG36 and NIP5;1 (Mao et al., note that a mutation altering Arabidopsis EXO70C2, another 2016)). However, no mislocalization of fluorescently tagged potential indirect exocyst interactor, leads to a similar pheno- exocyst components in nerd1 mutants, or of NERD1–GFP type in the tip-growing pollen tube: more rapid growth and in exocyst mutants, was observed (Fig.  3), arguing against increased tube rupture (Synek et  al., 2017). The contrasting this possibility. On the other hand, these experiments do not effects of nerd1 mutants on root hair tip growth versus pri- exclude genetic interaction via a currently unknown cargo that mary root cell elongation could manifest because the cell wall requires both NERD1 and exocyst-mediated vesicle transport matrices in the two cell types are structurally distinct from each for its proper function. other, generated by fundamentally different processes: delivery The localization of NERD1 to the Golgi, the site of syn- of non-cellulosic cell wall components to a narrowly focused thesis of non-cellulose polysaccharides incorporated into the region versus more broadly distributed modification of a pre- cell wall matrix (e.g. pectin and hemicellulose; Driouich et al., existing cell wall matrix, respectively. Additional indirect evi- 2012; Kim and Brandizzi, 2016), is tantalizing. NERD1 could dence that cell walls are differentially altered is the increased be involved in the formation or function of trans-Golgi-local- incidence of bulging in nerd1 root hair shafts, although neither ized protein complexes, such as the ECHIDNA/YIP4 complex bulging nor rupture is characteristic of cells in mutant root that plays a role in post-Golgi secretion of pectin and hemi- meristematic and elongation zones. In the root, the compos- cellulose to the cell wall, and which also influences cell elong- ition and structure of the cell wall changes as cells progress ation in roots and hypocotyls (Gendre et al., 2013). However, through division, elongation, and differentiation zones. For we currently favor a working hypothesis, based on predicted example, the rhamnogalacturonan I pectin in cell walls is mod- structure, in which NERD1 directly affects a cell wall matrix ified during the transition from cell proliferation to cell elong- polysaccharide, glycoprotein or proteoglycan that is ultimately ation in roots (Willats et al., 1999), and pectins in root hairs are secreted via exocyst-mediated trafficking to influence cell wall structurally distinct from those in the lateral cell walls elsewhere growth and expansion. Most of NERD1 is predicted to be in the primary root (Muszyński et  al., 2015). Thus, the com- located in the Golgi lumen, folded into a β-propeller-like ter- position of cell wall components available for interaction with tiary structure that could serve as a scaffold for interactions with NERD1 within the Golgi likely varies as root cells progress Downloaded from https://academic.oup.com/jxb/article/69/15/3625/4990813 by DeepDyve user on 18 July 2022 3636 | Cole et al. Cole RA, Synek L, Zarsky V, Fowler JE. 2005. SEC8, a subunit of the from elongation to differentiation. Such differences could alter putative Arabidopsis exocyst complex, facilitates pollen germination and NERD1’s impact on cell wall extensibility, elongation, and fra- competitive pollen tube growth. Plant Physiology 138, 2005–2018. gility in a developmentally dependent manner. Revealing the Cvrčková F, Grunt M, Bezvoda R, Hála M, Kulich I, Rawat A, Žárský molecular function of NERD1 should help define how it is V. 2012. Evolution of the land plant exocyst complexes. 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Journal of Experimental Botany – Oxford University Press

Published: Jun 27, 2018

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