Localized delivery of plasma-membrane and cell-wall components is a crucial process for plant cell growth. One of the regulators of secretory-vesicle targeting is the exocyst tethering complex. The exocyst mediates first interaction between transport vesicles and the target membrane before their fusion is performed by SNARE proteins. In land plants, genes encoding the EXO70 exocyst subunit underwent an extreme proliferation with 23 paralogs present in the Arabidopsis (Arabidopsis thaliana) genome. These paralogs often acquired specialized functions during evolution. Here, we analyzed functional divergence of selected EXO70 paralogs in Arabidopsis. Performing a systematic cross-complementation analysis of exo70a1 and exo70b1 mutants, we found that EXO70A1 was functionally substituted only by its closest paralog, EXO70A2. In contrast, none of the EXO70 isoforms tested were able to substitute EXO70B1, including its closest relative, EXO70B2, pointing to a unique function of this isoform. The presented results document a high degree of functional specialization within the EXO70 gene family in land plants.

Department of Experimental Plant Biology Faculty of Science Charles University Viničná 5 12844 Prague Czech Republic

Institute of Experimental Botany Czech Academy of Sciences Rozvojová 263 16502 Prague Czech Republic

See more in PubMed

Novick P., Field C., Schekman R. Identification of 23 Complementation Groups Required for Post-Translational Events in the Yeast Secretory Pathway. Cell. 1980;21:205–215. doi: 10.1016/0092-8674(80)90128-2. PubMed DOI

Guo W., Grant A., Novick P. Exo84p Is an Exocyst Protein Essential for Secretion. J. Biol. Chem. 1999;274:23558–23564. doi: 10.1074/jbc.274.33.23558. PubMed DOI

TerBush D.R., Maurice T., Roth D., Novick P. The Exocyst Is a Multiprotein Complex Required for Exocytosis in Saccharomyces Cerevisiae. EMBO J. 1996;15:6483–6494. doi: 10.1002/j.1460-2075.1996.tb01039.x. PubMed DOI PMC

Elias M. The Exocyst Complex in Plants. Cell Biol. Int. 2003;27:199–201. doi: 10.1016/S1065-6995(02)00349-9. PubMed DOI

Cvrčková F., Grunt M., Bezvoda R., Hála M., Kulich I., Rawat A., Zárský V. Evolution of the Land Plant Exocyst Complexes. Front. Plant Sci. 2012;3:159. doi: 10.3389/fpls.2012.00159. PubMed DOI PMC

Žárský V., Sekereš J., Kubátová Z., Pečenková T., Cvrčková F. Three Subfamilies of Exocyst EXO70 Family Subunits in Land Plants: Early Divergence and Ongoing Functional Specialization. J. Exp. Bot. 2020;71:49–62. doi: 10.1093/jxb/erz423. PubMed DOI

Fendrych M., Synek L., Pecenková T., Drdová E.J., Sekeres J., de Rycke R., Nowack M.K., Zársky V. Visualization of the Exocyst Complex Dynamics at the Plasma Membrane of Arabidopsis Thaliana. Mol. Biol. Cell. 2013;24:510–520. doi: 10.1091/mbc.e12-06-0492. PubMed DOI PMC

Fendrych M., Synek L., Pecenková T., Toupalová H., Cole R., Drdová E., Nebesárová J., Sedinová M., Hála M., Fowler J.E., et al. The Arabidopsis Exocyst Complex Is Involved in Cytokinesis and Cell Plate Maturation. Plant Cell. 2010;22:3053–3065. doi: 10.1105/tpc.110.074351. PubMed DOI PMC

Synek L., Schlager N., Eliás M., Quentin M., Hauser M.-T., Zárský V. AtEXO70A1, a Member of a Family of Putative Exocyst Subunits Specifically Expanded in Land Plants, Is Important for Polar Growth and Plant Development. Plant J. 2006;48:54–72. doi: 10.1111/j.1365-313X.2006.02854.x. PubMed DOI PMC

Drdová E.J., Synek L., Pečenková T., Hála M., Kulich I., Fowler J.E., Murphy A.S., Zárský V. The Exocyst Complex Contributes to PIN Auxin Efflux Carrier Recycling and Polar Auxin Transport in Arabidopsis. Plant J. 2013;73:709–719. doi: 10.1111/tpj.12074. PubMed DOI

Tan X., Feng Y., Liu Y., Bao Y. Mutations in Exocyst Complex Subunit SEC6 Gene Impaired Polar Auxin Transport and PIN Protein Recycling in Arabidopsis Primary Root. Plant Sci. 2016;250:97–104. doi: 10.1016/j.plantsci.2016.06.001. PubMed DOI

Cole R.A., McInally S.A., Fowler J.E. Developmentally Distinct Activities of the Exocyst Enable Rapid Cell Elongation and Determine Meristem Size during Primary Root Growth in Arabidopsis. BMC Plant Biol. 2014;14:386. doi: 10.1186/s12870-014-0386-0. PubMed DOI PMC

Wen T.-J., Hochholdinger F., Sauer M., Bruce W., Schnable P.S. The roothairless1 Gene of Maize Encodes a Homolog of sec3, Which Is Involved in Polar Exocytosis. Plant Physiol. 2005;138:1637–1643. doi: 10.1104/pp.105.062174. PubMed DOI PMC

Kalmbach L., Hématy K., De Bellis D., Barberon M., Fujita S., Ursache R., Daraspe J., Geldner N. Transient Cell-Specific EXO70A1 Activity in the CASP Domain and Casparian Strip Localization. Nat. Plants. 2017;3:17058. doi: 10.1038/nplants.2017.58. PubMed DOI

Li S., Chen M., Yu D., Ren S., Sun S., Liu L., Ketelaar T., Emons A.-M.C., Liu C.-M. EXO70A1-Mediated Vesicle Trafficking Is Critical for Tracheary Element Development in Arabidopsis. Plant Cell. 2013;25:1774–1786. doi: 10.1105/tpc.113.112144. PubMed DOI PMC

Vukašinović N., Oda Y., Pejchar P., Synek L., Pečenková T., Rawat A., Sekereš J., Potocký M., Žárský V. Microtubule-Dependent Targeting of the Exocyst Complex Is Necessary for Xylem Development in Arabidopsis. New Phytol. 2017;213:1052–1067. doi: 10.1111/nph.14267. PubMed DOI

Marković V., Cvrčková F., Potocký M., Kulich I., Pejchar P., Kollárová E., Synek L., Žárský V. EXO70A2 Is Critical for Exocyst Complex Function in Pollen Development. Plant Physiol. 2020;184:1823–1839. doi: 10.1104/pp.19.01340. PubMed DOI PMC

Beuder S., Dorchak A., Bhide A., Moeller S.R., Petersen B.L., MacAlister C.A. Exocyst Mutants Suppress Pollen Tube Growth and Cell Wall Structural Defects of Hydroxyproline O-Arabinosyltransferase Mutants. Plant J. 2020;103:1399–1419. doi: 10.1111/tpj.14808. PubMed DOI PMC

Ogura T., Goeschl C., Filiault D., Mirea M., Slovak R., Wolhrab B., Satbhai S.B., Busch W. Root System Depth in Arabidopsis Is Shaped by EXOCYST70A3 via the Dynamic Modulation of Auxin Transport. Cell. 2019;178:400–412.e16. doi: 10.1016/j.cell.2019.06.021. PubMed DOI

Kulich I., Pečenková T., Sekereš J., Smetana O., Fendrych M., Foissner I., Höftberger M., Zárský V. Arabidopsis Exocyst Subcomplex Containing Subunit EXO70B1 Is Involved in Autophagy-Related Transport to the Vacuole. Traffic. 2013;14:1155–1165. doi: 10.1111/tra.12101. PubMed DOI

Hong D., Jeon B.W., Kim S.Y., Hwang J.-U., Lee Y. The ROP2-RIC7 Pathway Negatively Regulates Light-Induced Stomatal Opening by Inhibiting Exocyst Subunit Exo70B1 in Arabidopsis. New Phytol. 2016;209:624–635. doi: 10.1111/nph.13625. PubMed DOI

Zhao T., Rui L., Li J., Nishimura M.T., Vogel J.P., Liu N., Liu S., Zhao Y., Dangl J.L., Tang D. A Truncated NLR Protein, TIR-NBS2, Is Required for Activated Defense Responses in the exo70B1 Mutant. PLoS Genet. 2015;11:e1004945. doi: 10.1371/journal.pgen.1004945. PubMed DOI PMC

Pecenková T., Hála M., Kulich I., Kocourková D., Drdová E., Fendrych M., Toupalová H., Zársky V. The Role for the Exocyst Complex Subunits Exo70B2 and Exo70H1 in the Plant-Pathogen Interaction. J. Exp. Bot. 2011;62:2107–2116. doi: 10.1093/jxb/erq402. PubMed DOI PMC

Stegmann M., Anderson R.G., Ichimura K., Pecenkova T., Reuter P., Žársky V., McDowell J.M., Shirasu K., Trujillo M. The Ubiquitin Ligase PUB22 Targets a Subunit of the Exocyst Complex Required for PAMP-Triggered Responses in Arabidopsis. Plant Cell. 2012;24:4703–4716. doi: 10.1105/tpc.112.104463. PubMed DOI PMC

Synek L., Vukašinović N., Kulich I., Hála M., Aldorfová K., Fendrych M., Žárský V. EXO70C2 Is a Key Regulatory Factor for Optimal Tip Growth of Pollen. Plant Physiol. 2017;174:223–240. doi: 10.1104/pp.16.01282. PubMed DOI PMC

Acheampong A.K., Shanks C., Cheng C.-Y., Schaller G.E., Dagdas Y., Kieber J.J. EXO70D Isoforms Mediate Selective Autophagic Degradation of Type-A ARR Proteins to Regulate Cytokinin Sensitivity. Proc. Natl. Acad. Sci. USA. 2020;117:27034–27043. doi: 10.1073/pnas.2013161117. PubMed DOI PMC

Wang J., Ding Y., Wang J., Hillmer S., Miao Y., Lo S.W., Wang X., Robinson D.G., Jiang L. EXPO, an Exocyst-Positive Organelle Distinct from Multivesicular Endosomes and Autophagosomes, Mediates Cytosol to Cell Wall Exocytosis in Arabidopsis and Tobacco Cells. Plant Cell. 2010;22:4009–4030. doi: 10.1105/tpc.110.080697. PubMed DOI PMC

Ding Y., Wang J., Chun Lai J.H., Ling Chan V.H., Wang X., Cai Y., Tan X., Bao Y., Xia J., Robinson D.G., et al. Exo70E2 Is Essential for Exocyst Subunit Recruitment and EXPO Formation in Both Plants and Animals. Mol. Biol. Cell. 2014;25:412–426. doi: 10.1091/mbc.e13-10-0586. PubMed DOI PMC

Ostertag M., Stammler J., Douchkov D., Eichmann R., Hückelhoven R. The Conserved Oligomeric Golgi Complex Is Involved in Penetration Resistance of Barley to the Barley Powdery Mildew Fungus. Mol. Plant Pathol. 2013;14:230–240. doi: 10.1111/j.1364-3703.2012.00846.x. PubMed DOI PMC

Fujisaki K., Abe Y., Ito A., Saitoh H., Yoshida K., Kanzaki H., Kanzaki E., Utsushi H., Yamashita T., Kamoun S., et al. Rice Exo70 Interacts with a Fungal Effector, AVR-Pii, and Is Required for AVR-Pii-Triggered Immunity. Plant J. 2015;83:875–887. doi: 10.1111/tpj.12934. PubMed DOI

Kulich I., Vojtíková Z., Glanc M., Ortmannová J., Rasmann S., Žárský V. Cell Wall Maturation of Arabidopsis Trichomes Is Dependent on Exocyst Subunit EXO70H4 and Involves Callose Deposition. Plant Physiol. 2015;168:120–131. doi: 10.1104/pp.15.00112. PubMed DOI PMC

Kulich I., Vojtíková Z., Sabol P., Ortmannová J., Neděla V., Tihlaříková E., Žárský V. Exocyst Subunit EXO70H4 Has a Specific Role in Callose Synthase Secretion and Silica Accumulation. Plant Physiol. 2018;176:2040–2051. doi: 10.1104/pp.17.01693. PubMed DOI PMC

Zhang X., Pumplin N., Ivanov S., Harrison M.J. EXO70I Is Required for Development of a Sub-Domain of the Periarbuscular Membrane during Arbuscular Mycorrhizal Symbiosis. Curr. Biol. 2015;25:2189–2195. doi: 10.1016/j.cub.2015.06.075. PubMed DOI

Synek L., Pleskot R., Sekereš J., Serrano N., Vukašinović N., Ortmannová J., Klejchová M., Pejchar P., Batystová K., Gutkowska M., et al. Plasma membrane electrostatic signature targets the plant exocyst complex via the EXO70 subunit. Proc. Natl. Acad. Sci. USA. under review. PubMed PMC

Wu C., Tan L., van Hooren M., Tan X., Liu F., Li Y., Zhao Y., Li B., Rui Q., Munnik T., et al. Arabidopsis EXO70A1 Recruits Patellin3 to the Cell Membrane Independent of Its Role as an Exocyst Subunit. J. Integr. Plant Biol. 2017;59:851–865. doi: 10.1111/jipb.12578. PubMed DOI

Wang W., Liu N., Gao C., Cai H., Romeis T., Tang D. The Arabidopsis Exocyst Subunits EXO70B1 and EXO70B2 Regulate FLS2 Homeostasis at the Plasma Membrane. New Phytol. 2020;227:529–544. doi: 10.1111/nph.16515. PubMed DOI

Pourcel L., Irani N.G., Lu Y., Riedl K., Schwartz S., Grotewold E. The formation of anthocyanic vacuolar inclusions in Arabidopsis thaliana and implications for the sequestration of anthocyanin pigments. Mol. Plant. 2010;3:78–90. doi: 10.1093/mp/ssp071. PubMed DOI PMC

Pečenková T., Potocká A., Potocký M., Ortmannová J., Drs M., Drdová E.J., Pejchar P., Synek L., Soukupová H., Žárský V., et al. Redundant and Diversified Roles Among Selected Arabidopsis Thaliana EXO70 Paralogs During Biotic Stress Responses. Front. Plant Sci. 2020;11:960. doi: 10.3389/fpls.2020.00960. PubMed DOI PMC

Sabol P., Kulich I., Žárský V. RIN4 Recruits the Exocyst Subunit EXO70B1 to the Plasma Membrane. J. Exp. Bot. 2017;68:3253–3265. doi: 10.1093/jxb/erx007. PubMed DOI PMC

Vukašinović N., Cvrčková F., Eliáš M., Cole R., Fowler J.E., Žárský V., Synek L. Dissecting a Hidden Gene Duplication: The Arabidopsis Thaliana SEC10 Locus. PLoS ONE. 2014;9:e94077. PubMed PMC

Hála M., Cole R., Synek L., Drdová E., Pecenková T., Nordheim A., Lamkemeyer T., Madlung J., Hochholdinger F., Fowler J.E., et al. An Exocyst Complex Functions in Plant Cell Growth in Arabidopsis and Tobacco. Plant Cell. 2008;20:1330–1345. doi: 10.1105/tpc.108.059105. PubMed DOI PMC

Karimi M., Bleys A., Vanderhaeghen R., Hilson P. Building Blocks for Plant Gene Assembly. Plant Physiol. 2007;145:1183–1191. doi: 10.1104/pp.107.110411. PubMed DOI PMC

Clough S.J., Bent A.F. Floral Dip: A Simplified Method forAgrobacterium-Mediated Transformation ofArabidopsis Thaliana. Plant J. 1998;16:735–743. doi: 10.1046/j.1365-313x.1998.00343.x. PubMed DOI