The heterooctameric vesicle-tethering complex exocyst is important for plant development, growth, and immunity. Multiple paralogs exist for most subunits of this complex; especially the membrane-interacting subunit EXO70 underwent extensive amplification in land plants, suggesting functional specialization. Despite this specialization, most Arabidopsis exo70 mutants are viable and free of developmental defects, probably as a consequence of redundancy among isoforms. Our in silico data-mining and modeling analysis, corroborated by transcriptomic experiments, pinpointed several EXO70 paralogs to be involved in plant biotic interactions. We therefore tested corresponding single and selected double mutant combinations (for paralogs EXO70A1, B1, B2, H1, E1, and F1) in their two biologically distinct responses to Pseudomonas syringae, root hair growth stimulation and general plant susceptibility. A shift in defense responses toward either increased or decreased sensitivity was found in several double mutants compared to wild type plants or corresponding single mutants, strongly indicating both additive and compensatory effects of exo70 mutations. In addition, our experiments confirm the lipid-binding capacity of selected EXO70s, however, without the clear relatedness to predicted C-terminal lipid-binding motifs. Our analysis uncovers that there is less of functional redundancy among isoforms than we could suppose from whole sequence phylogeny and that even paralogs with overlapping expression pattern and similar membrane-binding capacity appear to have exclusive roles in plant development and biotic interactions.

Department of Experimental Plant Biology Faculty of Science Charles University Prague Czechia

Institute of Experimental Botany CAS Prague Czechia

See more in PubMed

Baker N. A., Sept D., Joseph S., Holst M. J., McCammon J. A. (2001). Electrostatics of nanosystems: Application to microtubules and the ribosome. Proc. Natl. Acad. Sci. U. S. A. 98, 1019–1137.  10.1073/pnas.181342398 PubMed DOI PMC

Balla T. (2013). Phosphoinositides: Tiny lipids with giant impact on cell regulation. Physiol. Rev. 93, 1019–1137.  10.1152/physrev.00028.2012 PubMed DOI PMC

Boyd C., Hughes T., Pypaert M., Novick P. (2004). Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p. J. Cell Biol. 167, 889–901.  10.1083/jcb.200408124 PubMed DOI PMC

Chong Y. T., Gidda S. K., Sanford C., Parkinson J., Mullen R. T., Goring D. R. (2010). Characterization of the Arabidopsis thaliana exocyst complex gene families by phylogenetic, expression profiling, and subcellular localization studies. New Phytol. 185, 401–419.  10.1111/j.1469-8137.2009.03070.x PubMed DOI

Cole R. A., Synek L., Zarsky V., Fowler J. E. (2005). SEC8, a subunit of the putative arabidopsis exocyst complex, facilitates pollen germination and competitive pollen tube growth. Plant Physiol. 138, 2005–2018.  10.1104/pp.105.062273 PubMed DOI PMC

Cvrčková F., Grunt M., Bezvoda R., Hála M., Kulich I., Rawat A., et al. (2012). Evolution of the land plant exocyst complexes. Front. Plant Sci. 3, 159.  10.3389/fpls.2012.00159 PubMed DOI PMC

Drdová E. J., Synek L., Pečenková T., Hála M., Kulich I., Fowler J. E., et al. (2013). The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis. Plant J. 73, 709–719  10.1111/tpj.12074 PubMed DOI

Du Y., Mpina M. H., Birch P. R. J., Bouwmeester K., Govers F. (2015). Phytophthora infestans RXLR effector AVR1 interacts with exocyst component Sec5 to manipulate plant immunity. Plant Physiol. 169, 1975–1990.  10.1104/pp.15.01169 PubMed DOI PMC

Du Y., Overdijk E. J. R., Berg J. A., Govers F., Bouwmeester K. (2018). Solanaceous exocyst subunits are involved in immunity to diverse plant pathogens. J. Exp. Bot. 69, 655–666.  10.1093/jxb/erx442 PubMed DOI PMC

Dubuke M. L., Maniatis S., Shaffer S. A., Munson M. (2015). The exocyst subunit Sec6 interacts with assembled exocytic SNARE complexes. J. Biol. Chem. 290, 28245–28256.  10.1074/jbc.M115.673806 PubMed DOI PMC

Dvořáková L., Cvrčková F., Fischer L. (2007). Analysis of the hybrid proline-rich protein families from seven plant species suggests rapid diversification of their sequences and expression patterns. BMC Genomics. 8, 412.  10.1186/1471-2164-8-412 PubMed DOI PMC

Elias M., Drdova E., Ziak D., Bavlnka B., Hala M., Cvrckova F., et al. (2003). The exocyst complex in plants. Cell Biol. Int. 27, 199–201.  10.1016/S1065-6995(02)00349-9 PubMed DOI

Fendrych M., Synek L., Pecenková T., Toupalová H., Cole R., Drdová E., et al. (2010). The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation. Plant Cell 22, 3053–3065.  10.1105/tpc.110.074351 PubMed DOI PMC

Fendrych M., Synek L., Pečenková T., Drdová E. J., Sekereš J., de Rycke R., et al. (2013). Visualization of the exocyst complex dynamics at the plasma membrane of Arabidopsis thaliana . Mol. Biol. Cell. 24, 510-520. 10.1091/mbc.E12-06-0492 PubMed DOI PMC

Guo W., Roth D., Walch-Solimena C., Novick P. (1999). The exocyst is an effector for Sec4P, targeting secretory vesicles to sites of exocytosis. EMBO J.f. 18, 1071–1080.  10.1093/emboj/18.4.1071 PubMed DOI PMC

Hála M., Cole R., Synek L., Drdová E., Pečenková T., Nordheim A., et al. (2008). An exocyst complex functions in plant cell growth in Arabidopsis and tobacco. Plant Cell. 20, 1330–1345.  10.1105/tpc.108.059105 PubMed DOI PMC

Hála M., Soukupová H., Synek L., Žárský V. (2010). Arabidopsis RAB geranylgeranyl transferase β-subunit mutant is constitutively photomorphogenic, and has shoot growth and gravitropic defects. Plant J. 62, 615–627.  10.1111/j.1365-313X.2010.04172.x PubMed DOI

Hall T. A. (1999). BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symp. Ser. 41, 95–98.

He B., Xi F., Zhang X., Zhang J., Guo W. (2007). Exo70 interacts with phospholipids and mediates the targeting of the exocyst to the plasma membrane. EMBO J. 26, 4053–4065.  10.1038/sj.emboj.7601834 PubMed DOI PMC

Hekkelman M. L., te Beek T. A. H., Pettifer S. R., Thorne D., Attwood T. K., Vriend G. (2010). WIWS: A protein structure bioinformatics web service collection. Nucleic Acids Res. 38, W719–W723.  10.1093/nar/gkq453 PubMed DOI PMC

Hruz T., Laule O., Szabo G., Wessendorp F., Bleuler S., Oertle L., et al. (2008). Genevestigator V3: A reference expression database for the meta-analysis of transcriptomes. Adv. Bioinf. 2008, 420747.  10.1155/2008/420747 PubMed DOI PMC

Hsu S. C., TerBush D., Abraham M., Guo W. (2004). The exocyst complex in polarized exocytosis. Int. Rev. Cytol. 21, 537–542.  10.1016/S0074-7696(04)33006-8 PubMed DOI

Ishiga Y., Ishiga T., Uppalapati S. R., Mysore K. S. (2011). Arabidopsis seedling flood-inoculation technique: A rapid and reliable assay for studying plant-bacterial interactions. Plant Methods. 7, 32.  10.1186/1746-4811-7-32 PubMed DOI PMC

Janková Drdová E., Klejchová M., Janko K., Hála M., Soukupová H., Cvrćková F., et al. (2019). Developmental plasticity of Arabidopsis hypocotyl is dependent on exocyst complex function. J. Exp. Bot. 70, 1255–1265.  10.1093/jxb/erz005 PubMed DOI PMC

Jones J. D. G., Dangl J. L. (2006). The plant immune system. Nature. 444, 323–329.  10.1038/nature05286 PubMed DOI

Kalachova T., Janda M., Šašek V., Ortmannová J., Nováková P., Dobrev I. P., et al. (2019). Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant. Ann. Bot. 125, 775–784.  10.1093/aob/mcz112 PubMed DOI PMC

Koumandou V. L., Dacks J. B., Coulson R. M. R., Field M. C. (2007). Control systems for membrane fusion in the ancestral eukaryote; Evolution of tethering complexes and SM proteins. BMC Evol. Biol. 7, 29.  10.1186/1471-2148-7-29 PubMed DOI PMC

Kulich I., Pečenková T., Sekereš J., Smetana O., Fendrych M., Foissner I., et al. (2013). Arabidopsis exocyst subcomplexcontaining subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic. 14, 1155–1165.  10.1111/tra.12101 PubMed DOI

Kulich I., Vojtíková Z., Glanc M., Ortmannová J., Rasmann S., Zárský V. (2015). Cell wall maturation of arabidopsis trichomes is dependent on exocyst subunit EXO70H4 and involves callose deposition. Plant Physiol. 168, 120–131.  10.1104/pp.15.00112 PubMed DOI PMC

Kulich I., Vojtíková Z., Sabol P., Ortmannová J., Neděla V., Tihlaříková E., et al. (2018). Exocyst subunit EXO70H4 has a specific role in callose synthase secretion and silica accumulation. Plant Physiol. 176, 2040–2051.  10.1104/pp.17.01693 PubMed DOI PMC

Laxalt A. M., Munnik T. (2002). Phospholipid signalling in plant defence. Curr. Opin. Plant Biol. 5, 332–338.  10.1016/s1369-5266(02)00268-6 PubMed DOI

Liu J., Zuo X., Yue P., Guo W. (2007). Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells. Mol. Biol. Cell. 18, 4483–4492.  10.1091/mbc.E07-05-0461 PubMed DOI PMC

Ma J., Chen J., Wang M., Ren Y., Wang S., Lei C., et al. (2018). Disruption of OsSEC3A increases the content of salicylic acid and induces plant defense responses in rice. J. Exp. Bot. 69, 1051–1064.  10.1093/jxb/erx458 PubMed DOI PMC

Markovic V., Cvrckova F., Potocky M., Pejchar P., Kolarova E., Kulich I., et al. (2020). EXO70A2 is critical for the exocyst complex function in Arabidopsis pollen. bioRxiv.  10.1101/831875 PubMed DOI PMC

Martin-Urdiroz M., Deeks M. J., Horton C. G., Dawe H. R., Jourdain I. (2016). The exocyst complex in health and disease. Front. Cell Dev. Biol. 4, 24.  10.3389/fcell.2016.00024 PubMed DOI PMC

Moskalenko S., Henry D. O., Rosse C., Mirey G., Camonis J. H., White M. A. (2002). The exocyst is a Ral effector complex. Nat. Cell Biol. 4, 66–72.  10.1038/ncb728 PubMed DOI

Pečenková T., Janda M., Ortmannová J., Hajná V., Stehlíková Z., Žárský V. (2017. a). Early Arabidopsis root hair growth stimulation by pathogenic strains of Pseudomonas syringae. Ann. Bot. 120, 437–446.  10.1093/aob/mcx073 PubMed DOI PMC

Pečenková T., Marković V., Sabol P., Kulich I., Zárský V. (2017. b). Exocyst and autophagy-related membrane trafficking in plants. J. Exp. Bot. 69, 47–57.  10.1093/jxb/erx363 PubMed DOI

Pecenková T., Hála M., Kulich I., Kocourková D., Drdová E., Fendrych M., et al. (2011). The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction. J. Exp. Bot. 62, 2107–2116.  10.1093/jxb/erq402 PubMed DOI PMC

Picco A., Irastorza-Azcarate I., Specht T., Böke D., Pazos I., Rivier-Cordey A. S., et al. (2017). The in vivo architecture of the exocyst provides structural basis for exocytosis. Cell. 168, 400–412.e18.  10.1016/j.cell.2017.01.004 PubMed DOI

Platre M. P., Noack L. C., Doumane M., Bayle V., Simon M. L. A., Maneta-Peyret L., et al. (2018). A combinatorial lipid code shapes the electrostatic landscape of plant endomembranes. Dev. Cell. 45, 465–480.  10.1016/j.devcel.2018.04.011 PubMed DOI

Pleskot R., Cwiklik L., Jungwirth P., Žárský V., Potocký M. (2015). Membrane targeting of the yeast exocyst complex. Biochim. Biophys. Acta Biomembr. 1848, 1481–1489.  10.1016/j.bbamem.2015.03.026 PubMed DOI

Rawat A., Brejšková L., Hála M., Cvrčková F., Žárský V. (2017). The Physcomitrella patens exocyst subunit EXO70.3d has distinct roles in growth and development, and is essential for completion of the moss life cycle. New Phytol. 216, 438–454.  10.1111/nph.14548 PubMed DOI

Redditt T. J., Chung E. H., Zand Karimi H., Rodibaugh N., Zhang Y., Trinidad J. C., et al. (2019). AvrRpm1 functions as an ADP-ribosyl transferase to modify NOI-domain containing proteins, including Arabidopsis and soybean RPM1-interacting protein 4. Plant Cell. (Epub ahead of print).  10.1105/tpc.19.00020 PubMed DOI

Rossi G., Lepore D., Kenner L., Czuchra A. B., Plooster M., Frost A., et al. (2020). Exocyst structural changes associated with activation of tethering downstream of Rho/Cdc42 GTPases. J. Cell Biol. 219, e201904161.  10.1083/jcb.201904161 PubMed DOI PMC

Roth D., Guo W., Novick P. (1998). Dominant negative alleles of SEC10 reveal distinct domains involved in secretion and morphogenesis in yeast. Mol. Biol. Cell. 9, 1725–1739.  10.1091/mbc.9.7.1725 PubMed DOI PMC

Sabol P., Kulich I., Žárský V. (2017). RIN4 recruits the exocyst subunit EXO70B1 to the plasma membrane. J. Exp. Bot. 68, 3253–3265.  10.1093/jxb/erx007 PubMed DOI PMC

Schindelin J., Rueden C. T., Hiner M. C., Eliceiri K. W. (2015). The ImageJ ecosystem: An open platform for biomedical image analysis. Mol. Reprod. Dev. 82, 518–529.  10.1002/mrd.22489 PubMed DOI PMC

Sekereš J., Pejchar P., Šantrůček J., Vukasinovic N., Žárský V., Potocký M. (2017). Analysis of exocyst subunit EXO70 family reveals distinct membrane polar domains in Tobacco pollen tubes. Plant Physiol. 173, 1659–1675.  10.1104/pp.16.01709 PubMed DOI PMC

Stegmann M., Anderson R. G., Ichimura K., Pecenkova T., Reuter P., Žárský V., et al. (2012). The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in arabidopsis c w. Plant Cell. 24, 4703–4716.  10.1105/tpc.112.104463 PubMed DOI PMC

Stegmann M., Anderson R. G., Westphal L., Rosahl S., McDowell J. M., Trujillo M. (2014). The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death. Plant Signal. Behav. 8, e27421.  10.4161/psb.27421 PubMed DOI PMC

Synek L., Schlager N., Eliáš M., Quentin M., Hauser M. T., Žárský V. (2006). 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. 48, 54–72.  10.1111/j.1365-313X.2006.02854.x PubMed DOI PMC

Synek L., Vukašinović N., Kulich I., Hála M., Aldorfová K., Fendrych M., et al. (2017). EXO70C2 is a key regulatory factor for optimal tip growth of pollen. Plant Physiol. 174, 223–240.  10.1104/pp.16.01282 PubMed DOI PMC

TerBush D. R., Maurice T., Roth D., Novick P. (1996). The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J. 15, 6483–6494.  10.1002/j.1460-2075.1996.tb01039.x PubMed DOI PMC

van Wersch R., Li X., Zhang Y. (2016). Mighty dwarfs: Arabidopsis autoimmune mutants and their usages in genetic dissection of plant immunity. Front. Plant Sci. 7, 1717.  10.3389/fpls.2016.01717 PubMed DOI PMC

Vukašinović N., Oda Y., Pejchar P., Synek L., Pečenková T., Rawat A., et al. (2017). Microtubule-dependent targeting of the exocyst complex is necessary for xylem development in Arabidopsis. New Phytol. 213, 1052–1067.  10.1111/nph.14267 PubMed DOI

Wang W., Liu N., Gao C., Cai H., Romeis T., Tang D. (2020). The Arabidopsis exocyst subunits EXO70B1 and EXO70B2 regulate FLS2 homeostasis at the plasma membrane. New Phytol. 227, 529–544.  10.1111/nph.16515 PubMed DOI

Wang X. (2004). Lipid signaling. Curr. Opin. Plant Biol. 7, 329–336.  10.1016/j.pbi.2004.03.012 PubMed DOI

Webb B., Sali A. (2016). Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinforma. 1654, 39–54.  10.1002/cpbi.3 PubMed DOI PMC

Wiederstein M., Sippl M. J. (2007). ProSA-web: Interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res. 35, W407–W410.  10.1093/nar/gkm290 PubMed DOI PMC

Wu H., Turner C., Gardner J., Temple B., Brennwald P. (2010). The Exo70 subunit of the exocyst is an effector for both Cdc42 and Rho3 function in polarized exocytosis. Mol. Biol. Cell. 21, 430–442.  10.1091/mbc.E09-06-0501 PubMed DOI PMC

Yuan J., He S. Y. (1996). The Pseudomonas syringae Hrp regulation and secretion system controls the production and secretion of multiple extracellular proteins. J. Bacteriol. 178, 6399–6402.  10.1128/jb.178.21.6399-6402.1996 PubMed DOI PMC

Yuan S., Chan H. C. S., Filipek S., Vogel H. (2016). PyMOL and inkscape bridge the data and the data visualization. Structure. 24, 2041–2042.  10.1016/j.str.2016.11.012 PubMed DOI

Žárský V., Cvrčková F., Potocký M., Hála M. (2009). Exocytosis and cell polarity in plants - Exocyst and recycling domains: Tansley review. New Phytol. 183, 255–272.  10.1111/j.1469-8137.2009.02880.x PubMed DOI

Žárský V., Kulich I., Fendrych M., Pečenková T. (2013). Exocyst complexes multiple functions in plant cells secretory pathways. Curr. Opin. Plant Biol. 16, 726–733.  10.1016/j.pbi.2013.10.013 PubMed DOI

Žárský V., Sekereš J., Kubátová Z., Pečenková T., Cvrčková F. (2020). Three subfamilies of exocyst EXO70 family subunits in land plants: early divergence and ongoing functional specialization. J. Exp. Bot. 71, 49–62.  10.1093/jxb/erz423 PubMed DOI

Zhang Z., Feechan A., Pedersen C., Newman M. A., Qiu J. L., Olesen K. L., et al. (2007). A SNARE-protein has opposing functions in penetration resistance and defense signalling pathways. Plant J. 49, 302–312.  10.1111/j.1365-313X.2006.02961.x PubMed DOI

Zhao Y., Liu J., Yang C., Capraro B. R., Baumgart T., Bradley R. P., et al. (2013). Exo70 generates membrane curvature for morphogenesis and cell migration. Dev. Cell. 26, 266–278.  10.1016/j.devcel.2013.07.007 PubMed DOI PMC

Zhao T., Rui L., Li J., Nishimura M. T., Vogel J. P., Liu N., et al. (2015). A Truncated NLR protein, TIR-NBS2, is required for activated defense responses in the exo70B1 mutant. PloS Genet. 11, e1004945.  10.1371/journal.pgen.1004945 PubMed DOI PMC

Chitosan stimulates root hair callose deposition, endomembrane dynamics, and inhibits root hair growth

Small secreted proteins and exocytosis regulators: do they go along?

Functional Specialization within the EXO70 Gene Family in Arabidopsis