TY - JOUR
AU - Cvrčková,, Fatima
AB - Abstract Localized delivery of plasma membrane and cell wall components is an essential process in all plant cells. The vesicle-tethering complex, the exocyst, an ancient eukaryotic hetero-octameric protein cellular module, assists in targeted delivery of exocytosis vesicles to specific plasma membrane domains. Analyses of Arabidopsis and later other land plant genomes led to the surprising prediction of multiple putative EXO70 exocyst subunit paralogues. All land plant EXO70 exocyst subunits (including those of Bryophytes) form three distinct subfamilies—EXO70.1, EXO70.2, and EXO70.3. Interestingly, while the basal well-conserved EXO70.1 subfamily consists of multiexon genes, the remaining two subfamilies contain mostly single exon genes. Published analyses as well as public transcriptomic and proteomic data clearly indicate that most cell types in plants express and also use several different EXO70 isoforms. Here we sum up recent advances in the characterization of the members of the family of plant EXO70 exocyst subunits and present evidence that members of the EXO70.2 subfamily are often recruited to non-canonical functions in plant membrane trafficking pathways. Engagement of the most evolutionarily dynamic EXO70.2 subfamily of EXO70s in biotic interactions and defence correlates well with massive proliferation and conservation of new protein variants in this subfamily. Autophagy, defence, evolution, EXO70, exocyst, exocytosis, land plants, unconventional secretion Introduction The exocyst complex and diversity of its EXO70 subunits in plants Specific tethering protein complexes assist in vectorial delivery of trafficking cargos to distinct stations within the eukaryotic endomembrane system, acting mostly as effectors and regulators of small RAB and RHO GTPases (Koumandou et al., 2007; Vukašinović and Žárský, 2016). One of them is the exocyst, an ancient eukaryotic hetero-octameric protein complex originally discovered in the context of polarized secretory vesicles tetheried at the plasma membrane (PM) in budding yeast (TerBush et al., 1996; Munson and Novick, 2006; Novick, 2014). Driven by knowledge of the yeast exocyst, this complex was found also in animals and plants, and proven to be an ancestral cellular regulator traceable back to the last eukaryotic common ancestor (Koumandou et al., 2007; Heider and Munson, 2012; Vaškovičová et al., 2013; Wu and Guo, 2015). The exocyst consists of eight subunits (SEC3, SEC5, SEC6, SEC8, SEC10, SEC15, EXO84, and EXO70). By interacting with activated GTP-bound RHO GTPases, it localizes exocytotic membrane containers to specific PM cortical domains (Robinson et al., 1999; Pommereit and Wouters, 2007). The SEC3 and EXO70 subunits target the complex by direct binding to phosphatidylinositol bisphosphate (PIP2) (He et al., 2007; Liu et al., 2007). The exocyst also catalyses SNARE complex-mediated membrane fusion. This process is mechanistically best understood in the budding yeast, where the t-SNAREs Sso2p and Sec9p directly interact with Sec3p (Yue et al., 2017) and Sec6p (Sivaram et al., 2005), respectively. The cis-SNARE complex formation is further boosted by the direct interaction of Sec6p with the regulatory Sec1 protein (Morgera et al., 2012). Metazoan EXO70 functions within the exocyst complex in processes such as insulin secretion, neurite growth, cell migration, as well as midbody scission (Martin-Urdiroz et al., 2016) and phagosome maturation (Rauch et al., 2014). EXO70 is also involved in the autophagy as a part of an exocyst subcomplex (Bodemann et al., 2011) and has several exocyst-independent roles, including membrane deformation resulting in the formation of actin-free cell protrusions by EXO70 oligomerization-induced negative membrane curvature (Zhao et al., 2013). Independently from the rest of the exocyst, EXO70 stimulates Arp2/3-induced actin polymerization and branching (Liu et al., 2012). Two different isoforms, E-EXO70 and M-EXO70, result from mammalian EXO70 alternative splicing, with only the M-EXO70 splice variant being able to activate the Arp2/3 complex. During the epithelial–mesenchymal transition, cells switch expression from E-EXO70 to M-EXO70, facilitating invadopodia formation and cell migration (Lu et al., 2013). Several additional mammalian EXO70 splice variants with differential tissue expression were documented, but the functional significance of these variants is unclear. Surprisingly, EXO70 even seems to regulate pre-mRNA splicing (Dellago et al., 2011). Bioinformatic analyses predicted surprisingly many paralogues of the EXO70 exocyst subunit encoded by land plant genomes (Eliáš et al., 2003; Synek et al., 2006). The extraordinary evolutionary dynamics of plant EXO70 paralogues is begging for a functional explanation. In the Arabidopsis genome 23 and in rice 47 EXO70-encoding genes were identified (Cvrčková et al., 2012). Land plant EXO70 paralogues can be divided into three well-defined monophyletic subfamilies—EXO70.1, EXO70.2, and EXO70.3. The EXO70.1 subfamily is the least evolutionarily dynamic, with its members closely related to single type EXO70A subunits encoded in Chlorophyta, fungal, and animal genomes (Fig. 1). Interestingly, while the EXO70.1 subfamily consists of multiexon genes, the remaining two subfamilies contain mostly single-exon (or, occasionally, single-intron) genes in Arabidopsis (Synek et al., 2006), suggesting a possible derived character of EXO70.2 and EXO70.3 subfamilies, whose evolutionary history probably involved reverse transcription at some point. The same pattern of intron distribution was also confirmed in a recent study in wheat (Zhao et al., 2018), although the phylogeny presented there, generated by a method known to be prone to produce artefacts for divergent sequences, would require a critical re-assessment. Fig. 1. Open in new tabDownload slide Relationships between EXO70 subfamilies and lower order clades. The maximum likelihood phylogenetic tree was constructed using the method and a subset of data from our previous reports (Cvrčková et al., 2012; Rawat et al., 2017), with inclusion of the SmEXO70.3 sequence (Synek et al., 2006). The FX clade was not included in the present analysis because of loss of resolution affecting mainly the non-angiosperm sequences; its position, based on a previous study (Cvrčková et al., 2012), is indicated by a dashed line. The asterisk denotes the presumed position of the root, as inferred previously (Rawat et al., 2017). For legibility, names of Arabidopsis and rice sequences are abbreviated: A. thaliana EXO70A1 as AtA1, rice EXO70B1 as OsB1, etc. For the remaining sequences, terminology follows our previous reports (Cvrčková et al., 2012, Rawat et al., 2017). Bootstrap support for deep (A to H clade level and below) branches is indicated by symbols (absence of a symbol means support <60%). Fig. 1. Open in new tabDownload slide Relationships between EXO70 subfamilies and lower order clades. The maximum likelihood phylogenetic tree was constructed using the method and a subset of data from our previous reports (Cvrčková et al., 2012; Rawat et al., 2017), with inclusion of the SmEXO70.3 sequence (Synek et al., 2006). The FX clade was not included in the present analysis because of loss of resolution affecting mainly the non-angiosperm sequences; its position, based on a previous study (Cvrčková et al., 2012), is indicated by a dashed line. The asterisk denotes the presumed position of the root, as inferred previously (Rawat et al., 2017). For legibility, names of Arabidopsis and rice sequences are abbreviated: A. thaliana EXO70A1 as AtA1, rice EXO70B1 as OsB1, etc. For the remaining sequences, terminology follows our previous reports (Cvrčková et al., 2012, Rawat et al., 2017). Bootstrap support for deep (A to H clade level and below) branches is indicated by symbols (absence of a symbol means support <60%). Published analyses as well as public transcriptomic and proteomic data clearly indicate that most cell types in plants express and also use several different EXO70 isoforms (Žárský et al., 2009, 2013; Pečenková et al., 2011; Sekereš et al., 2017; Kulich et al., 2018). Ten years ago we proposed a hypothesis on the biological role of EXO70 multiplicity, assuming an exclusively exocytotic function of the exocyst and suggesting, in addition to tissue-specific roles, a function of particular EXO70s in distinct cortical secretory domains of individual plant cells (Žárský et al., 2009). The landscape of exocyst research both in plants and in animals was, however, transformed substantially by independent discoveries that versions of the exocyst complex are involved in the autophagy process and lysosome/vacuolar delivery pathway in mammals and plants (Bodemann et al., 2011; Kulich et al., 2013). Based on these discoveries and the follow-up studies of plant EXO70.2 subfamily exocyst paralogues summarized below, we formulate a new hypothesis implicating the EXO70.2 subfamily in often non-canonical secretory processes derived from, or related to, autophagy. The autophagy-related pathways include processes contributing to secondary cell wall (SCW) biogenesis, biotic interactions, and defence. Engagement of the most evolutionarily dynamic EXO70.2 subfamily of EXO70s in biotic interactions and defence correlates well with massive proliferation and conservation of new protein variants, resulting in six well-defined clades (B, C, D, E, F, and H) within this subfamily in angiosperms, with different clades undergoing massive amplification in monocots (F) and dicots (H). Also in conifers, mosses, and lycophytes, the most identified EXO70 paralogues belong to the EXO70.2 clade (Rawat et al., 2017). The basal and least divergent EXO70.1 subfamily, comprising only one clade—the EXO70A isoforms—is clearly linked to the housekeeping exocytotic function of the exocyst, functionally related especially to cell growth and primary and secondary cell wall biogenesis. The third subfamily, EXO70.3, comprising EXO70G paralogues and in most plant families also the EXO70I clade (lost in Brassicaceae/Arabidopsis) remains currently almost fully uncharacterized. In this review we sum up recent advances in the characterization of the numerous members of the family of plant EXO70 exocyst subunits, with focus on Arabidopsis thaliana (Table 1), and present evidence that members of the EXO70.2 subfamily are often recruited to non-canonical functions in plant secretory pathways. Table 1. Summary of known functions and mutant phenotypes of Arabidopsis thaliana EXO70 paralogues Gene . AGI locus . Function or phenotype . References . EXO70.1 subfamily EXO70A1 At5g03540 Cytokinesis (cell plate initiation) Fendrych et al. (2010); Rybak et al. (2014) Hypocotyl elongation Hála et al. (2008) Root cell growth Cole et al. (2014) Root hair growth Synek et al. (2006); Wu et al. (2013) Smaller and non-receptive stigmatic papillae in mutants Synek et al. (2006); Safavian et al. (2015) Seed coat deposition Kulich et al. (2010) Aberrant hypocotyl development in dark-grown mutants Drdová et al. (2019) Secondary cell wall biogenesis in endodermal Casparian bands Kalmbach et al. (2017) Secondary cell wall deposition in developing xylem Li et al. (2013); Oda et al. (2015); Vukašinović et al. (2017) PIN transporter recycling Drdová et al. (2013); Tan et al. (2016) Binding to phosphatidylinositol 4,5-bisphosphate-rich PM domain in trichomes Kubátová et al. (2019) EXO70A2 At5g52340 High expression in pollen, role unknown Loraine et al. (2013); Synek et al. (2017) EXO70A3 At5g52350 Modulation of auxin-controlled root architecture development Ogura et al. (2019) EXO70.2 subfamily EXO70B1 At5g58430 Autophagy triggered by starvation and autophagy-related anthocyanin trafficking Kulich et al. (2013) Immune response to pathogens, cell death Stegmann et al. (2013; Zhao et al. (2015); Sabol et al. (2017) Light-induced stomatal opening Hong et al. (2016); Seo et al. (2016) EXO70B2 At1g07000 Formation of cell wall appositions in plant defence Pečenková et al. (2011) Regulation of PAMP-induced signalling Stegmann et al. (2012) Mannitol-induced stomatal closure Seo et al. (2016) EXO70C1 At5g13150 High expresion in root trichoblasts and pollen, role unknown Grobei et al. (2009; Li et al. (2010); Synek et al. (2017) EXO70C2 At5g13990 Negative regulation of tip growth Synek et al. (2017) EXO70D1 At1g72470 Unknown EXO70D2 At1g54090 Unknown EXO70D3 At3g14090 Unknown EXO70E1 At3g29400 Unknown EXO70E2 At5g61010 Unconventional secretory pathway or autophagy Wang et al. (2010); Ding et al. (2014); Lin et al. (2015); P. Sabol et al. (unpublished results) EXO70F1 At5g50380 Unknown EXO70H1 At3g55150 Defence against pathogens, mechanism unknown, nuclear localization Pečenková et al. (2011) EXO70H2 At2g39380 Iron transport and homeostasis, mechanism unknown Xing et al. (2015) EXO70H3 At3g09530 High expression in pollen, role unknown Synek et al. (2017) EXO70H4 At3g09520 Delivery of callose synthases to plasma membrane Kulich et al. (2018) Secondary cell wall callose and silica deposition in trichomes Kulich et al. (2015) Callose deposition during reaction to pathogen attack in epidermis Kulich et al. (2018) Binding to phosphatidic acid and phosphatidylserine-rich PM domain in trichomes Kubátová et al. (2019) EXO70H5 At2g28640 High expression in pollen, role unknown Synek et al. (2017) EXO70H6 At1g07725 High expression in pollen, role unknown Synek et al. (2017) EXO70H7 At5g59730 Unknown EXO70H8 At2g28650 Unknown EXO70.3 subfamily EXO70G1 At4g31540 Unknown EXO70G2 At1g51640 Unknown Gene . AGI locus . Function or phenotype . References . EXO70.1 subfamily EXO70A1 At5g03540 Cytokinesis (cell plate initiation) Fendrych et al. (2010); Rybak et al. (2014) Hypocotyl elongation Hála et al. (2008) Root cell growth Cole et al. (2014) Root hair growth Synek et al. (2006); Wu et al. (2013) Smaller and non-receptive stigmatic papillae in mutants Synek et al. (2006); Safavian et al. (2015) Seed coat deposition Kulich et al. (2010) Aberrant hypocotyl development in dark-grown mutants Drdová et al. (2019) Secondary cell wall biogenesis in endodermal Casparian bands Kalmbach et al. (2017) Secondary cell wall deposition in developing xylem Li et al. (2013); Oda et al. (2015); Vukašinović et al. (2017) PIN transporter recycling Drdová et al. (2013); Tan et al. (2016) Binding to phosphatidylinositol 4,5-bisphosphate-rich PM domain in trichomes Kubátová et al. (2019) EXO70A2 At5g52340 High expression in pollen, role unknown Loraine et al. (2013); Synek et al. (2017) EXO70A3 At5g52350 Modulation of auxin-controlled root architecture development Ogura et al. (2019) EXO70.2 subfamily EXO70B1 At5g58430 Autophagy triggered by starvation and autophagy-related anthocyanin trafficking Kulich et al. (2013) Immune response to pathogens, cell death Stegmann et al. (2013; Zhao et al. (2015); Sabol et al. (2017) Light-induced stomatal opening Hong et al. (2016); Seo et al. (2016) EXO70B2 At1g07000 Formation of cell wall appositions in plant defence Pečenková et al. (2011) Regulation of PAMP-induced signalling Stegmann et al. (2012) Mannitol-induced stomatal closure Seo et al. (2016) EXO70C1 At5g13150 High expresion in root trichoblasts and pollen, role unknown Grobei et al. (2009; Li et al. (2010); Synek et al. (2017) EXO70C2 At5g13990 Negative regulation of tip growth Synek et al. (2017) EXO70D1 At1g72470 Unknown EXO70D2 At1g54090 Unknown EXO70D3 At3g14090 Unknown EXO70E1 At3g29400 Unknown EXO70E2 At5g61010 Unconventional secretory pathway or autophagy Wang et al. (2010); Ding et al. (2014); Lin et al. (2015); P. Sabol et al. (unpublished results) EXO70F1 At5g50380 Unknown EXO70H1 At3g55150 Defence against pathogens, mechanism unknown, nuclear localization Pečenková et al. (2011) EXO70H2 At2g39380 Iron transport and homeostasis, mechanism unknown Xing et al. (2015) EXO70H3 At3g09530 High expression in pollen, role unknown Synek et al. (2017) EXO70H4 At3g09520 Delivery of callose synthases to plasma membrane Kulich et al. (2018) Secondary cell wall callose and silica deposition in trichomes Kulich et al. (2015) Callose deposition during reaction to pathogen attack in epidermis Kulich et al. (2018) Binding to phosphatidic acid and phosphatidylserine-rich PM domain in trichomes Kubátová et al. (2019) EXO70H5 At2g28640 High expression in pollen, role unknown Synek et al. (2017) EXO70H6 At1g07725 High expression in pollen, role unknown Synek et al. (2017) EXO70H7 At5g59730 Unknown EXO70H8 At2g28650 Unknown EXO70.3 subfamily EXO70G1 At4g31540 Unknown EXO70G2 At1g51640 Unknown Some of the genes with no experimental data available are not discussed in the text. Open in new tab Table 1. Summary of known functions and mutant phenotypes of Arabidopsis thaliana EXO70 paralogues Gene . AGI locus . Function or phenotype . References . EXO70.1 subfamily EXO70A1 At5g03540 Cytokinesis (cell plate initiation) Fendrych et al. (2010); Rybak et al. (2014) Hypocotyl elongation Hála et al. (2008) Root cell growth Cole et al. (2014) Root hair growth Synek et al. (2006); Wu et al. (2013) Smaller and non-receptive stigmatic papillae in mutants Synek et al. (2006); Safavian et al. (2015) Seed coat deposition Kulich et al. (2010) Aberrant hypocotyl development in dark-grown mutants Drdová et al. (2019) Secondary cell wall biogenesis in endodermal Casparian bands Kalmbach et al. (2017) Secondary cell wall deposition in developing xylem Li et al. (2013); Oda et al. (2015); Vukašinović et al. (2017) PIN transporter recycling Drdová et al. (2013); Tan et al. (2016) Binding to phosphatidylinositol 4,5-bisphosphate-rich PM domain in trichomes Kubátová et al. (2019) EXO70A2 At5g52340 High expression in pollen, role unknown Loraine et al. (2013); Synek et al. (2017) EXO70A3 At5g52350 Modulation of auxin-controlled root architecture development Ogura et al. (2019) EXO70.2 subfamily EXO70B1 At5g58430 Autophagy triggered by starvation and autophagy-related anthocyanin trafficking Kulich et al. (2013) Immune response to pathogens, cell death Stegmann et al. (2013; Zhao et al. (2015); Sabol et al. (2017) Light-induced stomatal opening Hong et al. (2016); Seo et al. (2016) EXO70B2 At1g07000 Formation of cell wall appositions in plant defence Pečenková et al. (2011) Regulation of PAMP-induced signalling Stegmann et al. (2012) Mannitol-induced stomatal closure Seo et al. (2016) EXO70C1 At5g13150 High expresion in root trichoblasts and pollen, role unknown Grobei et al. (2009; Li et al. (2010); Synek et al. (2017) EXO70C2 At5g13990 Negative regulation of tip growth Synek et al. (2017) EXO70D1 At1g72470 Unknown EXO70D2 At1g54090 Unknown EXO70D3 At3g14090 Unknown EXO70E1 At3g29400 Unknown EXO70E2 At5g61010 Unconventional secretory pathway or autophagy Wang et al. (2010); Ding et al. (2014); Lin et al. (2015); P. Sabol et al. (unpublished results) EXO70F1 At5g50380 Unknown EXO70H1 At3g55150 Defence against pathogens, mechanism unknown, nuclear localization Pečenková et al. (2011) EXO70H2 At2g39380 Iron transport and homeostasis, mechanism unknown Xing et al. (2015) EXO70H3 At3g09530 High expression in pollen, role unknown Synek et al. (2017) EXO70H4 At3g09520 Delivery of callose synthases to plasma membrane Kulich et al. (2018) Secondary cell wall callose and silica deposition in trichomes Kulich et al. (2015) Callose deposition during reaction to pathogen attack in epidermis Kulich et al. (2018) Binding to phosphatidic acid and phosphatidylserine-rich PM domain in trichomes Kubátová et al. (2019) EXO70H5 At2g28640 High expression in pollen, role unknown Synek et al. (2017) EXO70H6 At1g07725 High expression in pollen, role unknown Synek et al. (2017) EXO70H7 At5g59730 Unknown EXO70H8 At2g28650 Unknown EXO70.3 subfamily EXO70G1 At4g31540 Unknown EXO70G2 At1g51640 Unknown Gene . AGI locus . Function or phenotype . References . EXO70.1 subfamily EXO70A1 At5g03540 Cytokinesis (cell plate initiation) Fendrych et al. (2010); Rybak et al. (2014) Hypocotyl elongation Hála et al. (2008) Root cell growth Cole et al. (2014) Root hair growth Synek et al. (2006); Wu et al. (2013) Smaller and non-receptive stigmatic papillae in mutants Synek et al. (2006); Safavian et al. (2015) Seed coat deposition Kulich et al. (2010) Aberrant hypocotyl development in dark-grown mutants Drdová et al. (2019) Secondary cell wall biogenesis in endodermal Casparian bands Kalmbach et al. (2017) Secondary cell wall deposition in developing xylem Li et al. (2013); Oda et al. (2015); Vukašinović et al. (2017) PIN transporter recycling Drdová et al. (2013); Tan et al. (2016) Binding to phosphatidylinositol 4,5-bisphosphate-rich PM domain in trichomes Kubátová et al. (2019) EXO70A2 At5g52340 High expression in pollen, role unknown Loraine et al. (2013); Synek et al. (2017) EXO70A3 At5g52350 Modulation of auxin-controlled root architecture development Ogura et al. (2019) EXO70.2 subfamily EXO70B1 At5g58430 Autophagy triggered by starvation and autophagy-related anthocyanin trafficking Kulich et al. (2013) Immune response to pathogens, cell death Stegmann et al. (2013; Zhao et al. (2015); Sabol et al. (2017) Light-induced stomatal opening Hong et al. (2016); Seo et al. (2016) EXO70B2 At1g07000 Formation of cell wall appositions in plant defence Pečenková et al. (2011) Regulation of PAMP-induced signalling Stegmann et al. (2012) Mannitol-induced stomatal closure Seo et al. (2016) EXO70C1 At5g13150 High expresion in root trichoblasts and pollen, role unknown Grobei et al. (2009; Li et al. (2010); Synek et al. (2017) EXO70C2 At5g13990 Negative regulation of tip growth Synek et al. (2017) EXO70D1 At1g72470 Unknown EXO70D2 At1g54090 Unknown EXO70D3 At3g14090 Unknown EXO70E1 At3g29400 Unknown EXO70E2 At5g61010 Unconventional secretory pathway or autophagy Wang et al. (2010); Ding et al. (2014); Lin et al. (2015); P. Sabol et al. (unpublished results) EXO70F1 At5g50380 Unknown EXO70H1 At3g55150 Defence against pathogens, mechanism unknown, nuclear localization Pečenková et al. (2011) EXO70H2 At2g39380 Iron transport and homeostasis, mechanism unknown Xing et al. (2015) EXO70H3 At3g09530 High expression in pollen, role unknown Synek et al. (2017) EXO70H4 At3g09520 Delivery of callose synthases to plasma membrane Kulich et al. (2018) Secondary cell wall callose and silica deposition in trichomes Kulich et al. (2015) Callose deposition during reaction to pathogen attack in epidermis Kulich et al. (2018) Binding to phosphatidic acid and phosphatidylserine-rich PM domain in trichomes Kubátová et al. (2019) EXO70H5 At2g28640 High expression in pollen, role unknown Synek et al. (2017) EXO70H6 At1g07725 High expression in pollen, role unknown Synek et al. (2017) EXO70H7 At5g59730 Unknown EXO70H8 At2g28650 Unknown EXO70.3 subfamily EXO70G1 At4g31540 Unknown EXO70G2 At1g51640 Unknown Some of the genes with no experimental data available are not discussed in the text. Open in new tab The basal EXO70.1 subfamily comprising exclusively EXO70A isoforms Arabidopsis EXO70A1 is the first described and so far best characterized land plant EXO70 isoform, closely related to opisthokont EXO70s (Synek et al., 2006). Published data clearly indicate that the exocyst complex containing EXO70A regulates housekeeping exocytotic functions; this correlates well with rather stable and strong expression across Arabidopsis sporophytic tissues (Synek et al., 2006; Hála et al., 2008; Fendrych et al., 2010). A knockout mutant exo70A1 also phenocopies defects of many core exocyst subunit mutants (Synek et al., 2006; Hála et al., 2008; Fendrych et al., 2010). In the sporophyte, these shared defects include: slower hypocotyl elongation (Hála et al., 2008), thinner and collapsed xylem SCW (Li et al., 2013; Vukašinović et al., 2017), impaired seed coat deposition mainly manifested as pectin deficiency (Kulich et al., 2010), smaller and non-receptive stigmatic papillae (Synek et al., 2006; Safavian et al., 2015), reduced size of the root apical meristem and slower root cell elongation (Cole et al., 2014), defective root hair growth (Wen et al., 2005; Synek et al., 2006), as well as impaired auxin transport due to slower PIN transporter recycling (Drdová et al., 2013; Tan et al., 2016) contributing to aberrant hypocotyl development in the dark (Drdová et al., 2019). The elongation of exo70A1 mutant root hairs is less stimulated in response to Pseudomonas syringae, and the mutant seedling roots are more susceptible to colonization by the bacteria than wild-type (WT) plants (Pečenková et al., 2017). EXO70A1 shares subcellular localization with core exocyst subunits in many tissues, including root epidermis with polarization towards the outer lateral membrane domain (Fendrych et al., 2013). Exocyst function is essential for plant cytokinesis (Fendrych et al., 2010; Rybak et al., 2014). Loss-of-function (LOF) mutants exo84b (Fendrych et al., 2010) and sec6 (complemented in pollen by a lat52:SEC6 construct; Wu et al., 2013) exhibit deformed seedlings and cell wall stubs resulting from defective cytokinesis. Although EXO70A1 co-localizes with the core exocyst subunits during both early cytokinesis and cell plate maturation, only the first phase of initial vesicle fusion is delayed in exo70A1 mutants, but the defect is fully compensated during the cell plate progression. Thus, while loss of core exocyst subunits has dramatic consequences, only a transient defect in cell plate initiation was observed in Arabidopsis exo70A1 LOF mutants, possibly due to functional redundancy with other EXO70 isoforms (Fendrych et al., 2010). Arabidopsis EXO70A2, the sister isoform of EXO70A1, is highly expressed in pollen (Loraine et al., 2013; Synek et al., 2017). It thus probably plays an analogous role in recruiting the exocyst to the area of active secretion around the pollen tube tip. In tobacco, NtEXO70A1a, and to some extent also NtEXO70A2, are both expressed in pollen and localize to the same small exocytotically active subapical region of the pollen tube; however, tobacco NtEXO70A1b, which is not expressed in the male gametophyte, does not bind pollen tube PM (Sekereš et al., 2017). EXO70A isoforms are thus involved in exocytosis in both sporophytes and gametophytes, with pollen-specific isoforms possibly adapted to the extraordinary demand for vesicle fusion during rapid tip growth of pollen tubes. Recently, a GWAS (genome-wide association study) identified Arabidopsis EXO70A3 as a modulator of auxin contribution to overall root architecture development, showing, for the first time, that this EXO70A paralogue is an important factor of natural genetic variability in deep- versus shallow-rooting Arabidopsis accessions (Ogura et al., 2019). A new pharmacological experimental intervention tool into the EXO70 function, endosidin2 (ES2), was described recently (Zhang et al., 2016). This substance, discovered as an inhibitor of Arabidopsis exocytosis, was shown to specifically inhibit EXO70 homologues of eukaryotes, providing a new valuable possibility to address exocyst function in different cell types. Since the EXO70.1 (EXO70A) isoforms are closest to opisthokont EXO70s and target exocysts to the PM in various tissues (Fendrych et al., 2013; Kalmbach et al., 2017), interaction with PM phospholipids can be expected to contribute to EXO70A PM targeting. Indeed, tobacco NtEXO70A1a localizes to a polar secretory domain where PIP2 and phosphatidic acid (PA) overlap in the growing pollen tube (Sekereš et al., 2017). EXO70A1 also co-localizes with PIP2 in a prospective Casparian strip deposition site in differentiating endodermis (Kalmbach et al., 2017). Importantly, besides Arabidopsis, a mutant in the EXO70A1 gene was also characterized in rice (Tu et al., 2015). The mutant plants are dwarfed, with impaired cell elongation and differentiation (and also collapsed vascular xylem cell walls), and show a partial loss of apical dominance, clearly indicating conserved EXO70A function in all basal exocytotic processes across angiosperms. Cellular context-dependent specific functions of Arabidopsis EXO70A1 in endodermis and xylem development Possibly in every plant cell type there are several expressed EXO70 paralogues, resulting in the presence of several specific exocyst complexes. We proposed that this might support formation of distinct cortical exocytotic PM and cell wall domains (Žárský et al., 2009, 2013). Surprisingly, recent studies indicate that even the same EXO70 paralogue, namely Arabidopsis EXO70A1, might function in several specific modes depending on the cellular context. An allele of EXO70A1 with a point mutation altering a putative C-terminal phospholipid-binding domain of the protein was found in a forward screen for genes engaged in Arabidopsis endodermis differentiation. Unlike the exo70A1 LOF mutant, these point mutant plants do not exhibit gross developmental defects but show disturbed local SCW biogenesis in Casparian bands (Kalmbach et al., 2017). Defective xylem development was observed in the Arabidopsis exo70A1 mutant, and was mistakenly interpreted as the sole primary cause of the whole-mutant developmental deviation syndrome (Li et al., 2013). Using an inducible xylogenesis model system in Arabidopsis, Oda et al. (2015) surprisingly found that EXO70A1 protein localization in developing xylem depends on the microtubular (MT) cytoskeleton. This finding contrasts with our previous observations from root epidermal cells, where exocyst dynamics are largely independent of both actin and MT cytoskeletons and not affected even after a chronic pharmacological disturbance of the MTs (Fendrych et al., 2013). In developing xylem, MT-dependent EXO70A1 localization is due to an indirect interaction between EXO70A1 and MTs mediated by xylem precursor cell-specific VESICLE TETHERING 1 and 2 (VETH1/2) proteins and the CONSERVED OLIGOMERIC GOLGI (COG) tethering complex subunit COG2 (Oda et al., 2015). Our subsequent experiments revealed that several core exocyst subunits interact indirectly with the xylem-specific VETH1/2 adaptors via COG2 binding and that MT relocalization to the domains of future SCW deposition precedes exocyst localization to the same domain (Vukašinović et al., 2017). Here we also observed for the first time that the delivery of cellulose synthase (CesA) complexes depends on the exocyst function (Vukašinović et al., 2017). These observations are an important reminder that specific cellular contexts substantially affect protein function—in this case the same EXO70A1 paralogue functions in the biogenesis of different and cell type-specific cell wall domains. EXO70.2 subfamily: a champion in evolutionary multiplication comprising six clades often involved in defence and non-canonical exocyst functions Already the first systematic phylogenetic analysis of the immense land plant EXO70 diversity led us to propose that evolution of this gene family may be driven by competition with parasites, and that some EXO70s may participate in defence (Hála et al., 2008). Indeed, pathogen/elicitor-induced EXO70B2 and EXO70H1 subunits are involved in the resistance against P. syringae and Blumeria graminis infections (Pečenková et al., 2011). Phylogenetic analyses further confirm that the EXO70.2 subfamily, which comprises six well-defined clades, namely EXO70B, EXO70C, EXO70D, EXO70E, EXO70F, and EXO70H, is responsible for most of EXO70 diversification in land plants (see Fig. 1), often linked to defence. Evidence for a tendency of these EXO70s to form homo- or heterodimers slowly accumulates, while paralogues from other subfamilies seem incapable in this respect (Pečenková et al., 2011; Fujisaki et al., 2015; Z. Kubátová, unpublished data). Grasses have a specific, extraordinarily rapidly evolving subgroup FX within the EXO70F clade (Synek et al., 2006; Cvrčková et al., 2012). EXO70B The best characterized members of the EXO70.2 subfamily are both members of the Arabidopsis EXO70B clade, EXO70B1 and EXO70B2. Other angiosperm genomes often encode either only one EXO70B paralogue or paralogues that arose by independent duplications, not related to the evolution of Arabidopsis EXO70B1 and EXO70B2 (Cvrčková et al., 2012). Therefore, some of the functional properties of EXO70B proteins discussed here are probably relevant mostly for Brassicaceae; it is also possible that the EXO70B duplication in Brassicaceae led to separation of functions present in the unique EXO70B paralogue of other plant families. Arabidopsis EXOB1: role in autophagy, stomatal regulation, and defence EXO70B1 is highly expressed across many sporophytic and gametophytic tissues and, surprisingly, has a role in autophagy triggered by starvation (for either photosynthetic/sugar or nitrogen) and in autophagy-related direct import of anthocyanins to the vacuole, bypassing the Golgi apparatus (Kulich et al., 2013). Vesicular autophagy-related anthocyanin trafficking involves EXO70B1 co-localization with the anthocyanin-containing compartments possibly initiated at the endoplasmic reticulum and also with the autophagy marker AUTOPHAGY-RELATED (ATG) protein ATG8f internalized into the vacuole (Kulich et al., 2013). Mutants lacking EXO70B1 are nitrogen starvation sensitive, accumulate fewer autophagic bodies in the vacuole, and generate paramural bodies in the apoplast, possibly due to defective transport of autophagosomes or multivesicular bodies (MVBs) to the vacuole. Importantly, the exo70A1exo70B1 double mutant displays phenotypic deviations of both single mutants, indicating no functional overlap between EXO70A1 and EXO70B1. exo70B1 mutant plants also develop spontaneous leaf hypersensitive response (HR)-like lesions, with cells undergoing programmed cell death linked to salicylic acid (SA) hyperaccumulation (Kulich et al., 2013), and have a reduced threshold for programmed cell death initiation after pathogen infection (Stegmann et al., 2013; Zhao et al., 2015). The onset of the HR-like phenotype varies depending on the cultivation conditions and appears with high incidence in plants older than 6 weeks (Kulich et al., 2013). As a consequence of SA hyperaccumulation, the mutant is more resistant towards strictly biotrophic pathogens but displays reduced overall fitness (Stegmann et al., 2013; Zhao et al., 2015). Since SA hyperaccumulation is a common feature of autophagy mutants, and the SA analogue BTH [benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester] activates autophagy, the hypersensitive phenotype of exo70B1 might be caused by impaired SA clearance due to its defective autophagy-related transport to the vacuole (Yoshimoto et al., 2009; Kulich et al., 2013; Kulich and Žárský, 2014). A direct EXO70B1 interactor, TN2 (a TIR-NBS truncated NLR disease resistance protein), is required for SA and H2O2 accumulation, as well as for a spontaneous HR in exo70B1 mutants (Zhao et al., 2015). The TN2-driven HR involves direct phosphorylation of EXO70B1 by the TN2-interacting calcium-dependent protein kinase 5 (CDPK5), probably with an inhibitory effect (Liu et al., 2017). It was hypothesized that EXO70B1 is targeted by an as yet unknown pathogen effector and that TN2 monitors EXO70B1 integrity, triggering a HR if EXO70B1 becomes compromised (Zhao et al., 2015). Alternatively, TN2 could be normally degraded by an EXO70B1-dependent autophagy pathway to prevent excessive TN2-induced SA accumulation, which would occur in the absence of EXO70B1, since autophagy has been proposed as a possible mechanism for negative regulation of R proteins (Pečenková et al., 2016). These mechanisms postulating either a direct or an indirect role for EXO70B1 in SA level control are not mutually exclusive. EXO70B1 could monitor TN2 activity under standard conditions, as well as facilitate degradation of excessive SA. An autophagy-related pathway involving EXO70B1 has been proposed as a general trafficking route of various secondary metabolites to the vacuole (Kulich and Žárský, 2014). The role of EXO70B1 in plant immunity is not limited to SA-mediated indirect effects. Recently, we have shown (Sabol et al., 2017) that EXO70B1 is translocated to the PM by RIN4 (RPM1 INTERACTING PROTEIN 4), a regulator of plant defence interacting with many plant R proteins. EXO70B1, unlike several other EXO70 isoforms tested, directly binds RIN4. Molecular details of EXO70B1 recruitment to the PM by RIN4 were investigated in a heterologous Nicotiana benthamiana system, showing that full-length RIN4 is required for this process and that upon RIN4 cleavage by the P. syringae effector protease, AvrRpt2, both RIN4 and EXO70B1 are released from the PM (Sabol et al., 2017). Furthermore, EXO70B1 also regulates stomatal opening and closure (Hong et al., 2016). The exo70B1 mutants exhibit slower light-induced stomatal opening than WT plants. EXO70B1 directly interacts with the Rho of plants (ROP) regulator RIC7 (ROP-interactive Cdc42- and Rac-interactive binding motif-containing protein 7). Both proteins are translocated to the PM by active ROP2 and are held inside the cytoplasm by dominant-negative ROP2 in biolistically transformed stomata of Vicia faba (Hong et al., 2016). Since Arabidopsis ric7 mutants display faster light-induced stomatal opening than WT plants, RIC7 probably also acts as a negative regulator of EXO70B1 activity during stomatal opening (Hong et al., 2016). While mutation in the EXO70B1 gene has no effect on abscisic acid (ABA)-induced stomatal closure, upon treatment with 1 µM ABA (Hong et al., 2016) the stomata of exo70B1 close more slowly than those of WT plants upon the application of 10 µM ABA or mannitol; on the other hand, plants overexpressing EXO70B1 close their stomata faster under these conditions (Seo et al., 2016). The tobacco homologue NtEXO70B1 is distinctly expressed in pollen (Conze et al., 2017; Sekereš et al., 2017), and localizes to a specific subapical membrane domain in growing tobacco pollen tube, overlapping with the zone of active endocytosis marked by localization of the endocytic machinery; thus, the protein could play a direct or indirect role in the endocytosis. Alternatively, it could regulate an unknown minor secretory pathway in tobacco pollen, distinct from the bulk secretion regulated by EXO70A clade members (Sekereš et al., 2017). Very low expression of B-clade EXO70 was reported in Arabidopsis pollen (summarized in Synek et al., 2017). It is thus possible that the pollen function of EXO70B1 detected in Solanaceae might not be a general feature of pollen tubes in all Angiosperms. Tobacco NtEXO70B1 could be targeted to the PM via interaction with PA in a growing pollen tube, since its localization largely overlaps with the PA maximum, although direct binding has not been demonstrated so far (Sekereš et al., 2017). In general, PM targeting of Arabidopsis EXO70B1 could be driven by various protein interactors such as RIN4 (Sabol et al., 2017) and RIC7 in stomatal guard cells (Hong et al., 2016). Direct experiments and data from protein–protein interaction databases implicate that different EXO70 isoforms interact with different NO3-induced (NOI)-domain containing proteins to a varying degree (Afzal et al., 2013; Sabol et al., 2017)—certainly an important issue for future studies. Arabidopsis EXOB2: specialized for defence EXO70B2 is the closest paralogue of EXO70B1 in Arabidopsis; on the other hand, the EXO70B1/EXO70B2 pair has the lowest sequence similarity of Arabidopsis EXO70 isoform sister pairs and, for instance, EXO70B1 is less related to EXO70B2 than to the tobacco NtEXO70B1 orthologue (Sekereš et al., 2017). Divergence of EXO70B1 and EXO70B2 may be an evolutionary novelty of the Brassicaceae family, possibly not relevant for other angiosperm groups (for a detailed phylogenetic analysis, see Cvrčková et al., 2012; Sekereš et al., 2017). EXO70B2 mRNA expression is induced by various pathogens and elicitors (Pečenková et al., 2011). The exo70B2 mutant plants are more susceptible to infection by P. syringae (Pečenková et al., 2011; Stegmann et al., 2012) and the oomycete Hyaloperenospora arabidopsidis (Stegmann et al., 2012) than WT plants. The mutants also exhibit deviations in defensive papilla formation upon infection by the fungus B. graminis (Pečenková et al., 2011). Unlike WT plants, the exo70B2 mutants do not up-regulate expression of several defence markers after PAMP (pathogen-associated molecular pattern) treatment, do not build up protection against Pseudomonas infection upon flagellin (flg22) pre-treatment, and inhibition of their primary root growth by flg22 is less pronounced than in WT plants (Stegmann et al., 2012). EXO70B2 is capable of homodimerization and heterodimerization with another pathogen-related isoform, EXO70H1 (Pečenková et al., 2011). In addition, the EXO70B2 role in plant defence may be related to its transient association with SNARE proteins, mainly SNAP33 and SYP121 (Pečenková et al., 2011; Zhao et al., 2015; J. Ortmannová et al., unublished data). Molecular determinants of EXO70B2 recruitment to the membrane are unclear, but, unlike EXO70B1, EXO70B2 is not translocated to the PM when co-expressed with RIN4 in N. benthamiana leaves. It weakly interacts, however, with RIN4-like protein NOI6; EXO70B2 might also be recruited to the PM by one or more of the many other NOI-domain-containing proteins (Afzal et al., 2013; Sabol et al., 2017). EXO70B2 does not seem to participate in ABA-induced stomatal closure, but it is implicated in the mannitol-induced stomatal closure (Seo et al., 2016). To conclude, unlike EXO70A, involved in housekeeping exocytosis, clade B EXO70 isoforms are involved in autophagic trafficking of mostly defence-related cargos through the pre-vacuolar compartment and MVBs to the vacuole but also to the extracellular space (cell wall and apoplast). Current evidence indicates that the EXO70B-assisted trafficking pathway may bypass the Golgi apparatus. For apoplast delivery, EXO70Bs might require a PM-associated recruitment protein. The function of EXO70Bs also varies depending on the cell type (e.g. stomata), age of the plant, and also strongly on environmental conditions. EXO70C—a non-canonical EXO70 protein moderating growth rate in tip-growing cells Arabidopsis EXO70C1 and EXO70C2 are specifically transcribed in root hair trichoblasts and pollen at a very high level, exceeding the transcript levels of core exocyst subunits (Synek et al., 2017). At the protein level, C subfamily EXO70s are the most abundant EXO70 isoforms in both Arabidopsis and tobacco pollen (Grobei et al., 2009; Sekereš et al., 2017), suggesting a possible specific role in tip growth. Surprisingly, unlike EXO70A isoforms or core exocyst subunits, EXO70C proteins do not localize to the PM and seem to be purely cytoplasmic in Arabidopsis root hairs and Arabidopsis and tobacco pollen tubes, even when overexpressed (Sekereš et al., 2017; Synek et al., 2017). However, the effects of their overexpression in tobacco pollen tube indicate misregulated growing tip polarity (Sekereš et al., 2017), and both exo70C1 (Li et al., 2010) and exo70C2 (Synek et al., 2017) mutants display a partial male transmission defect. Mutant exo70C2 pollen tubes growing in the style or in vitro are shorter than WT pollen tubes. Surprisingly, this is not due to a slower growth rate or loss of pollen tube polarity. On the contrary, mutant pollen tubes can elongate up to twice as fast as WT tubes, but cannot sustain such a rapid growth, which often results in bursting of their fast growing tips, evident as local release of cytoplasmic content. Burst pollen tubes cease to grow, regenerate, and often start a new period of fast growth and burst again. This stop and go mode of growth in cycles results in overall shorter pollen tube length compared with WT pollen tubes. This phenomenon of tip bursting, probably resulting from overstretching and weakening of the cell wall at the apex due to disrupted balance between the rate of delivery of cell wall biogenesis components and an excessive speed of elongation, suggests that the C clade of the EXO70.2 subfamily is a negative regulator or moderator of secretion (Synek et al., 2017). EXO70D, EXO70E, and EXO70F (and FX in grasses) These three clades of the EXO70.2 subfamily are so far poorly characterized, with published data only for EXO70E. However, transcriptome data suggest a cell type-specific engagement in the endodermis or stomata for EXO70D (Winter et al., 2007; Hruz et al., 2008). Arabidopsis EXO70E2 localizes to cytoplasmic punctae when overexpressed in cell culture, and this localization is unaffected by inhibitors commonly used to dissect plant cell membrane trafficking. The structures resemble inclusion bodies but turned out to be double-membrane compartments reminiscent of autophagosomes that can fuse with the PM, releasing a large exosome to the apoplast (Wang et al., 2010). These EXO70E2-containing particles sequester exocyst subunits and other proteins upon overexpression in Arabidopsis cell culture (Wang et al., 2010; Ding et al., 2014) and do not co-localize with known membrane compartment markers including the autophagy marker Atg8e under normal conditions (Wang et al., 2010). However, upon autophagy induction, the EXO70E2-containing structures co-localize with Atg8e and Atg8f and move with them into the vacuolar lumen (Lin et al., 2015). The closest tobacco homologue of Arabidopsis EXO70E2, NtEXO70E2, is not naturally expressed in tobacco pollen; however, when ectopically expressed, yellow fluorescent protein (YFP):NtEXO70E2 in pollen tubes localizes to mobile puncta with negligible cytoplasmic signal, even at a low level of expression (Sekereš et al., 2017). Strangely, Arabidopsis EXO70E2 induces the above-described double-membrane compartments even when expressed in mammalian cells—an extremely heterologous system for the plant-specific protein (Ding et al., 2014). It is thus evident that the EXO70E2 protein either hijacks the autophagy machinery in various species, or directly strongly deforms membranes in a manner analogous to the action of BAR domains, as proposed for mammalian EXO70 (Zhao et al., 2013), or forms aggregates that are very potent autophagy cargo in both animal and plant cells, thus resulting in autophagy induction. It is currently difficult to figure out the biological function of EXO70E2, because most subcellular localization data are based on 35S promoter-driven overexpression in Arabidopsis or in cell cultures. Unless the protein is studied in its native environment under a physiological level of expression, overexpression artefacts are difficult to avoid and interpretation of the results is problematic. EXO70E2 could play a role in a specific subtype of autophagic processes connected to an unconventional secretory pathway involving exosome production, especially in the root cap (Lin et al., 2015; P. Sabol et al., unpublished observations). Using strong expression of eight different Arabidopsis EXO70 isoforms in protoplasts under the 35S promoter, Wang et al. (2010) found that three of them (EXO70A1, EXO70B1, and EXO70E2) form punctate autophagosome-like double-membrane structures and co-localize in these structures. However focusing in detail on EXO70E2, they observed that these structures are not targeted to the vacuole upon starvation conditions, but rather are secreted to the apoplast (Wang et al., 2010). Based on these observations, the authors proposed the existence of a new plant-specific endomembrane exocyst-positive compartment in plants—EXPO—involved in unconventional secretion (Wang et al., 2010; Ding and Wang, 2017). We have shown, however, that the EXO70B1 paralogue is involved in autophagy, and EXPO-like structures are also formed when the EXO84b exocyst subunit is expressed under the 35S promoter, while expression controlled by the native promoter does not lead to formation of such structures (supplementary fig. 5 of Kulich et al., 2013). In Arabidopsis exo70b1 mutants, more exosomal structures possibly related to autophagosomes (similar to proposed EXPOs but also to paramural bodies) are secreted to the apoplast (supplementary fig. 3 of Kulich et al., 2013). As indicated above, currently only the EXO70E2 isoform is considered to be localized in some cells (especially in the root cap) to EXPO-like structures; new observations also indicate vacuolar re-targeting of these structures upon autophagy induction (Lin et al., 2015). As autophagosomes are known to be able to fuse with the PM and participate in the unconventional protein secretion, we prefer to interpret the EXO70E2 compartment as also related to the so-called secretory autophagy pathway (for a review, see, for example, Ponpuak et al., 2015). Unfortunately, no mutant-based functional studies of Arabidopsis EXO70E2 or its closest homologue EXO70E1 have been reported so far, and the latter remains entirely uncharacterized. The closest homologue of Arabidopsis EXO70E1, NtEXO70E1b, is expressed in tobacco pollen and localizes to the inverted cone of secretory and recycling vesicles at the tip of growing tobacco pollen tubes, although the functional significance of this subcellular localization is unclear (Sekereš et al., 2017). A very interesting new protein involved in the effective defence against herbivorous planthoppers BPH6 was uncovered recently in rice—it functions by stimulation of exocytosis via interaction with the exocyst mediated by direct binding with EXO7OE1 (Guo et al., 2018). Also the EXO70F clade is still awaiting characterization in Arabidopsis. While in the dicots this clade underwent only moderate diversification, in the grasses its FX subclade underwent a massive evolutionary expansion, suggesting a possible role in defence (Cvrčková et al., 2012). Consistent with this hypothesis, silencing of one of the barley F subfamily EXO70s increased susceptibility towards grass powdery mildew (Ostertag et al., 2013), rice OsEXO70-F2 and OsEXO70-F3 form complexes with the avirulence factor AVR-Pii, and OsEXO70-F3 contributes to Pii-mediated resistance against incompatible Magnaporthe oryzae strains expressing AVR-Pii (Fujisaki et al., 2015). EXO70H While encoded by only a few paralogues in monocot genomes, EXO70H is the most diversified clade in dicots. The H clade EXO70 paralogues display a striking example of independent EXO70 duplications specific for many angiosperm taxa (Cvrčková et al., 2012). It is also noteworthy that the rate of substitutions detected between EXO70 genes from tobacco (Nicotiana tabacum) and its parental species (N. sylvestris and N. tomentosiformis) was very low for the EXO70A clade but highest among all clades for EXO70H (Sekereš et al., 2017). The EXO70H clade thus seems to be subject to rapid evolution in dicots compared with other EXO70 clades. Also, non-orthologous EXO70H isoforms are expressed in Arabidopsis and tobacco pollen (Sekereš et al., 2017; Synek et al., 2017), further pointing to the rapid evolution of this clade. Eight EXO70H isoforms are found in the Arabidopsis genome; thus, it is the most numerous clade of EXO70s in Arabidopsis. Nonetheless, very limited information is available regarding the biological functions of EXO70H isoforms, with only a few of them characterized at least basically. The functionally best described member of the H clade is Arabidopsis EXO70H4. It is one of the most up-regulated genes in developing trichomes (Jakoby et al., 2008), where it regulates polarized callose-rich cell wall deposition during secondary thickening, including a noteworthy structure on the stalk above the trichome base denominated as the Ortmannian ring (Kulich et al., 2015). This SCW ring-like build-up is enriched mainly in callose but also in other cell wall components, and probably forms a physical barrier separating the plant body from the trichome SCW. EXO70H4 localizes to sites of prospective cell wall deposition in trichomes together with other exocyst subunits and recruits callose synthases to these sites, namely GLUCAN SYNTHASE LIKE (GSL) 5, also known as POWDERY MILDEW RESISTANT 4 (PMR4), and GSL10 (Kulich et al., 2018). The EXO70H4-dependent callose deposition is also a pre-requisite for silica deposition in the cell wall. Importantly, this function of EXO70H4 in trichomes seems to be highly specific and cannot be replaced by any other Arabidopsis EXO70H isoforms, including closely related EXO70H3. While limited to trichomes under normal conditions, EXO70H4 expression is induced in pavement epidermal cells upon flg22 or chitin treatments, suggesting a role in callose deposition also during reaction to pathogen attack (Kulich et al., 2018). EXO70H4 expression is negatively regulated by methyl jasmonates (Hruz et al., 2008), conceivably linking EXO70H4 with herbivore resistance. On the other hand, positive regulation by UV-B light through the CONSTITUTIVE PHOTOMORPHOGENETIC 1 (COP1) E3 ubiquitin ligase pathway was reported (Oravecz et al., 2006) and EXO70H4-dependent UV light stimulation of Arabidopsis trichome SCW thickening was observed (Kulich et al., 2015). Recently we described very specific PM lipid domains in mature Arabidopsis trichomes separated by the callose Ortmannian ring. The upper PA- and phosphatidylserine-rich domain above the ring binds the EXO70H4 isoform preferentially, while the basal domain enriched in PIP2 binds preferentially EXO70A1 (Kubátová et al., 2019). This observation supports the hypothesis that some EXO70 isoforms in the same cell might target the exocyst (or exocyst subcomplexes) to specific cortical PM domains (Žárský et al., 2009). In the same report, capturing of EXO70H4-positive membranes within the trichome secondary cell wall was uncovered using electron microscopy (Kubátová et al., 2019). Based on both experimental and bioinformatic data, a hypothesis on miRNA interference with EXO70 mRNAs was proposed for the EXO70H clade (Kulich et al., 2018). An interesting question of stoichiometry arises from transcriptomic and proteomic data (Jakoby et al., 2008; for pollen summarized in Synek et al., 2017), where other exocyst subunits are not as abundant as EXO70H4 itself. In yeast and mammals, each exocyst subunit seems to be present in 1:1 stoichiometry already at the transcript level, but this is not always the case in plants. It was demonstrated that EXO70H4 is trapped within the SCW so that it does not recycle, in contrast to other exocyst subunits (Kubátová et al., 2019)—this may well explain non-stoichiometry of EXO70H4 in trichomes (Kulich et al., 2015, 2018). The Cucumis sativus EXO70H4 isoform was also found to be highly up-regulated during development of its multicellular fruit trichomes (Chen et al., 2014). This suggests a conserved role for EXO70H4 across a variety of eudicot clades and between multicellular and unicellular trichomes (Kulich et al., 2015). A role in defence against pathogens has also been described for the EXO70H1 paralogue, which is expressed in response to treatment with the elf18 peptide. LOF mutation in the EXO70H1 locus results in enhanced susceptibility towards P. syringae. However, the detailed mechanism of EXO70H1 action in defence is unknown. Surprisingly, Arabidopsis EXO70H1 localizes to the nucleus when expressed in N. benthamiana epidermis (Pečenková et al., 2011). Also tobacco NtEXO70H1/2 and NtEXO70H5-8b localize to the nucleus in growing pollen tubes (Sekereš et al., 2017). The high ratio of nuclear to cytoplasmic localization of several tobacco EXO70H isoforms is consistent with their possible function within the nucleus. This is further supported by richness of nuclear proteins within the putative Arabidopsis EXO70H1 interactome (Žárský et al., 2013). EXO70H1pro::GUS (β-glucuronidase) and EXO70H2pro::GUS were found to be expressed specifically in the elongation and root hair formation root region (Li et al., 2010). The paralogue EXO70H2 has been followed in the context of iron transport and homeostasis regulation where it was identified as a potential downstream regulatory target of the histone acetyltransferase GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5; Xing et al., 2015). In Medicago truncatula, an EXO70H clade member (Medtr4g062330) is induced upon Rhizobium infection, co-localizes to the infection thread tip with vapyin (VPN) and its interactor, the E3 ligase LIN, and is required, together with these two proteins, for normal infection thread development (Liu et al., 2019). For unclear reasons, Liu et al. consider Medtr4g062330 an EXO70H4 orthologue, although a previous phylogenetic analysis locates this protein clearly inside a clade that contains Arabidopsis EXO70H5, H6, H7, and H8, but not EXO70H4 (Zhang et al., 2015). The EXO70.3 subfamily The ancestral EXO70.3 subfamily comprises two clades, EXO70G and EXO70I, with clade I apparently lost in the Brassicaceae including Arabidopsis (Eliáš et al., 2003; Cvrčková et al., 2012). However, the reliability of resolution between the G and I clades varies somewhat depending on the methods of phylogenetic analysis used, becoming less distinct with inclusion of divergent sequences that necessitate exclusion of unreliably aligned sequence portions from the analysis (e.g. Cvrčková et al., 2012). A phylogenetic analysis focusing exclusively on angiosperm EXO70.3 subfamily members that can be reliably aligned along their whole length clearly confirms the existence of these two clades, as well as the presence of an EXO70I clade member in grapevine, a basal rosid (Fig. 2). Fig. 2. Open in new tabDownload slide Relationships between clades within the angiosperm EXO70.3 subfamily. The maximum likelihood phylogenetic tree was constructed using the method and data described previously (Cvrčková et al., 2012). One incomplete Sorghum bicolor sequence has been omitted to preserve alignment length. The asterisk denotes the presumed position of the root as inferred from a preliminary tree including also A. thaliana EXO70A1 (not shown). For terminology, see legend to Fig. 1. Bootstrap support is indicated by symbols (branches without a symbol had bootstrap support between 50% and 60%). Fig. 2. Open in new tabDownload slide Relationships between clades within the angiosperm EXO70.3 subfamily. The maximum likelihood phylogenetic tree was constructed using the method and data described previously (Cvrčková et al., 2012). One incomplete Sorghum bicolor sequence has been omitted to preserve alignment length. The asterisk denotes the presumed position of the root as inferred from a preliminary tree including also A. thaliana EXO70A1 (not shown). For terminology, see legend to Fig. 1. Bootstrap support is indicated by symbols (branches without a symbol had bootstrap support between 50% and 60%). In our previously published analysis of EXO70 evolution, based on the final published Selaginella genome assembly, we concluded that lycopods possibly lost the EXO70.3 subfamily (Cvrčková et al., 2012). However, in an earlier study, including data from a pre-release of the Selaginella genome, we identified a gene, SmEXO70.3, which clearly clusters within the EXO70.3 subfamily (Synek et al., 2006). This gene is, however, located on a genomic fragment that has been excluded from the final genome release and its sequence has never been deposited in GenBank [it remains, however, available in the supplementary data of our report (Synek et al., 2006)]. After critical re-evaluation of related Selaginella sequence data, we go back to our original conclusion (also indicated in Rawat et al., 2017), that also lycopods, including Selaginella, harbour all three land plant EXO70 subfamilies (see Fig. 1). Members of the Arabidopsis EXO70G clade are awaiting functional characterization. The sole published report focusing on functional characterization of a member of this particular clade describes the mutant phenotype and protein localization of EXO70.3d in the moss Physcomitrella patens. The exo70.3d LOF mutant shows pleiotropic developmental deviations in protonemata, gametophores, sexual organs, and sporophytes. It is obvious that while the gene is not essential for cell survival, its function is important for multicellular development and possibly for communication between cells, as the egg cell in archegonia is initiated but not differentiated properly, and the mutant is thus unable to form the sporophyte (Rawat et al., 2017). Only a few functional studies exist for members of the EXO70I clade, which is absent in Arabidopsis. Expression of M. truncatula EXO70I is induced by arbuscular mycorrhizal symbiosis; in exo70i mutant plants, the branching of developing arbuscules is aberrant and incorporation of membrane cargo into the peri-arbuscular membrane is impaired. EXO70I:YFP specifically accumulates near the hyphal tips of developing arbuscules. The scaffold protein VPN co-localizes with M. truncatula EXO70I and specifically interacts with this isoform, but not with EXO70A1 or EXO70B2, and is likely to act in EXO70I recruitment to its target membrane domain (Zhang et al., 2015). EXO70I-mediated trafficking to the developing peri-arbuscular membrane is likely to involve the whole EXO70I-containing exocyst complex, because the core exocyst subunit EXO84b also localizes to sites of symbiotic fungal penetration in Medicago and Daucus carota. Unlike mature arbuscules, the developing arbuscules recruit green fluorescent protein (GFP):EXO84b to tips and branches in carrot (Genre et al., 2012). Although the GFP:EXO70I was also reported to accumulate near tips of the infection threads during rhizobial nodulation of Medicago cells (Gavrin et al., 2017), mutation in Medicago EXO70I does not affect symbiotic interactions with nodule bacteria (Zhang et al., 2015). Non-canonical EXO70 domain-containing proteins A legume-specific group of EXO70 domain-containing proteins was discovered in soybean. Their EXO70 domain is close to EXO70B but the 12 proteins form a distinct phylogenetic subgroup, and were named the EXO70J subfamily. Many of them contain a transmembrane domain homologous to one from a WRKY-related protein. The transmembrane domain-containing GmEXO70J1, 6, 7, 10, and 12 localize to the Golgi apparatus when expressed in N. benthamiana, unlike GmEXO70J3, which does not contain the transmembrane domain. While their cellular function is unknown, EXO70J subfamily members were hypothesized to participate in Golgi apparatus restructuralization and fragmentation (Chi et al., 2015). This would be in agreement with the capability of the animal EXO70 to directly deform the PM (Zhao et al., 2013). Expression of EXO70J paralogues increases in leaves during senescence (Chi et al., 2015), but they are also expressed in many other tissues (Wang et al., 2016). Viral-induced silencing of either transmembrane domain GmEXO70J7 or soluble GmEXO70J8 leads to premature leaf senescence. Furthermore, amiRNA silencing of GmEXO70J7 or (also transmembrane) GmEXO70J9 leads to a reduced number of nodules (Wang et al., 2016). Strangely, expression of GmEXO70J7 and GmEXO70J9 decreases upon soybean inoculation with Rhizobium (Wang et al., 2016). While hypotheses about the physiological role of J subfamily EXO70s include nitrogen transport, senescence regulation, nutrient elongation, and cell elongation (Chi et al., 2015; Wang et al., 2016), conclusive functional studies still remain to be done. An EXO70-like domain is also present, together with NB and LRR domains, in a large protein, RGA2a, encoded by the Sr33 locus that confers resistance of Aegilops tauschii to Puccinia graminis (Periyannan et al., 2013); a homologue was also reported from Triticum urartu (Ling et al., 2013). While there is genetic evidence that these proteins have a defence-related role, mechanistic data are still lacking. Regulation of the cellular EXO70 repertoire by targeted degradation Bioinformatic analyses uncovered an over-representation of Atg8-interacting motifs (AIMs) in exocyst subunits, including many EXO70 isoforms (Tzfadfia and Galili, 2013; Cvrčková and Žárský, 2013). Therefore, distinct EXO70 isoforms could be involved in autophagy and autophagy-derived unconventional trafficking pathways that bypass the Golgi apparatus, as shown for EXO70B1 (Kulich et al., 2013). Alternatively, EXO70 isoforms could act as adaptors targeting their specific protein interactors for selective autophagy. Such specific targeting for degradation has been recently demonstrated in the case of the brassinosteroid pathway regulator BES1 (BRASSINOSTEROID INSENSITIVE 1-EMS SUPRESSOR 1), whose degradation is mediated by the DSK2 (DOMINANT SUPRESSOR OF KAR 2) adaptor, which directly interacts with ATG8e via the AIMs (Nolan et al., 2017). The AIMs of EXO70 proteins might also directly target EXO70 isoforms for degradation, as recently suggested for Arabidopsis EXO70B2. Besides induction of its proteasomal degradation, flg22 treatment also induces EXO70B2 relocalization to the microsomal fraction, co-localization with autophagosomes dependent on the C-terminal AIMs, and subsequent vacuolar degradation (Teh et al., 2019). This is in agreement with the hypothesis that EXO70 isoforms within the exocyst complex may be replaced according to the needs of cell differentiation or by biotic or abiotic stimuli-induced expression of required isoforms and regulated proteolysis of those not required under the particular circumstances (Žárský et al., 2013). The degradation of EXO70B2 is regulated by its direct interactors, the plant U-box-type ubiquitin ligase 22 and 23 (PUB22 and PUB23; Stegmann et al., 2012). PUB22 is itself subject to proteasomal degradation but it is stabilized by flg22 treatment; thus flg22 increases ubiquitin ligase activity of PUB22, resulting in a negative feedback loop that clears EXO70B2 once it is no longer needed (Stegmann et al., 2012). Other polyubiquitin ligases, PUB18 and its related isoform PUB19, induce proteolytic degradation of EXO70B1 in an ABA-regulated manner, controlling EXO70B1 function in ABA-induced stomatal closure. Like PUB22, PUB18 is itself targeted to degradation due to self-ubiquitination, resulting thus in a tight regulatory feedback loop between a stress signalling mediator, EXO70, and a specific E3 ubiquitin ligase. Interestingly, the specificity of EXO70B1–PUB18 and EXO70B2–PUB22 interactions depends on the U-box N-terminal (UND) domain of PUB18. Upon swapping of the domain to PUB22, the specificity of interaction with the EXO70 isoforms is reversed (Seo et al., 2016). Since the division of labour between EXO70B1 and EXO70B2 is probably specific to Brassicaceae and thus relatively recent, specific ‘wiring’ of the machinery regulating EXO70 degradation can evolve rapidly and might be taxon specific. Indeed, while the E3 ubiquitin ligase ARMADILLO REPEAT CONTAINING 1 (ARC1) directly binds and primes EXO70A1 for degradation in Brassica (Samuel et al., 2009; Liu et al., 2016), Arabidopsis ARC1 is a non-functional pseudogene (Kitashiba et al., 2011). Thus, EXO70A1 is subject to different regulatory mechanisms even in closely related genera. This indicates that specific degradation of EXO70 isoforms is a well-regulated process possibly allowing exchange of EXO70 isoforms associated with the core exocyst subunits, facilitating formation of alternative exocyst complexes in plants (Žárský et al., 2013; Pečenková et al., 2017). Conclusions Over the last 10 years, new insights into the plant exocyst function in general, and that of EXO70 subunits in particular, indicate the necessity to revise our original model based on the assumption of exocysts being exclusively involved in exocytosis and membrane recycling (Žárský et al., 2009). It turned out that the most evolutionarily dynamic EXO70 subfamily, EXO70.2, often participates in non-canonical (i.e. non-simple exocytosis) functions. EXO70B clade members are involved in autophagy (Kulich et al., 2013), while EXO70Cs even perform a moderating function limiting the speed of cell expansion in tip-growing cell types (Synek et al., 2017). Ten years ago we hypothesized that specialized EXO70s may be responsible for delivering cargos to specific cortical domains of the cell, thus generating compositional and structural diversity of the plasmalemma, cell wall, and the apoplast (Žárský et al., 2009). However, it is nowadays clear that delivery (including, importantly, that of CesA complexes) to a variety of cortical domains can be mediated by exocyst complexes containing the same basal major exocytotic EXO70A subunit, representing the only clade of the EXO70.1 subfamily with specificity determined by cell type-specific protein and lipid interactors (Oda et al., 2015; Kalmbach et al., 2017; Vukašinović et al., 2017). The examples of EXO70H4, but also the EXO70B isoforms, representatives of the class EXO70.2, indicate that these EXO70 paralogues might be important for secondary wall modifications (callose and silica deposition in the case of EXO70H4, and local modifications of the cell wall during biotic interactions in the case of EXO70B2), and possibly also for specific cases of autophagy, as observed for EXO70B1-dependent anthocyanin import to the vacuole. Over-representation of AIMs in class EXO70.2 EXO70 subunits (Cvrčková and Žárský, 2013), in some cases supported by experimental data, suggests a specific mechanism of down-regulation by autophagy for some of these paralogues (and possbly also their interactors). The EXO70.3 subfamily remains mostly uncharacterized. Its EXO70I clade paralogues seem to be important for symbiotic interactions (consistent with this clade being absent in Brassicaceae including Arabidopsis). However, the phenotype of a LOF mutant in the moss P. patens EXO70.3 representative indicates the importance of this subfamily for development of a multicellular body. Since different EXO70 paralogues compete for the same exocyst core subunits, we proposed a hypothesis about the contribution of the exocyst complex to the coordination of the endomembrane dynamics (Žárský et al., 2013; Pečenková et al., 2017). Most of the experimental work on plant exocysts still needs to be done, and we can expect that such an endeavour will be an interesting journey. Abbreviations: Abbreviations: ABA abscisic acid AIM Atg8-interacting motif CesA cellulose synthase flg22 flagellin GFP green fluorescent protein HR hypersensitive response LOF loss of function MT microtubule MVB multivesicular body PA phosphatidic acid PIP2 phosphatidylinositol bisphosphate PM plasma membrane SA salicylic acid SCW secondary cell wall YFP yellow fluorescent protein WT wild type Acknowledgements We thank current and past members of our team, especially Ivan Kulich, Martin Potocký, Peter Sabol, and Jitka Ortmannová, for contributing ideas as well as unpublished observations. 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Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Three subfamilies of exocyst EXO70 family subunits in land plants: early divergence and ongoing functional specialization
JF - Journal of Experimental Botany
DO - 10.1093/jxb/erz423
DA - 2020-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/three-subfamilies-of-exocyst-exo70-family-subunits-in-land-plants-EEvZd4s2ED
SP - 49
VL - 71
IS - 1
DP - DeepDyve
ER -