Regulation of Cytokinesis by Exocyst Subunit SEC6 and KEULE in Arabidopsis thaliana

Abstract

Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formation, but the exact mechanisms still remain to be explored. In this study, using yeast two-hybrid (Y-2-H) assay, we found that SEC6 interacted with KEULE, and that a small portion of C-terminal region of KEULE was required for the interaction. The direct SEC6-KEULE interaction was supported by further studies using in vitro pull-down assay, immunoprecipitation, and in vivo bimolecular fluorescence complementation (BIFC) microscopy. sec6 mutants were male gametophytic lethal as reported; however, pollen-rescued sec6 mutants (PRsec6) displayed cytokinesis defects in the embryonic cells and later in the leaf pavement cells and the guard cells. SEC6 and KEULE proteins were co-localized to the cell plate during cytokinesis in transgenic Arabidopsis. Furthermore, only SEC6 but not other exocyst subunits located in the cell plate interacted with KEULE in vitro. These results demonstrated that, like KEULE, SEC6 plays a physiological role in cytokinesis, and the SEC6-KEULE interaction may serve as a novel molecular linkage between arriving vesicles and membrane fusion machinery or directly regulate membrane fusion during cell plate formation in plants.

RNA silencing of exocyst genes in the stigma impairs the acceptance of compatible pollen in Arabidopsis

“…Localization studies of specific Arabidopsis exocyst subunits also supported conserved roles in polarized exocytosis and cytokinesis in plants. Localization studies have shown EXO70, SEC6, and SEC8 at the growing tip of pollen tubes (Hála et al, 2008), EXO70A1 at the stigmatic papillar plasma membrane (Samuel et al, 2009), SEC3a, SEC6, SEC8, SEC15b, EXO70A1, and EXO84b at the root epidermal cell plasma membrane and developing cell plate (Fendrych et al, 2010Wu et al, 2013;Zhang et al, 2013;Rybak et al, 2014), and SEC3a at the plasma membrane in the embryo and root hair (Zhang et al, 2013). Similar to the yeast exocyst mutants, vesicle accumulation has also been observed in the exo70A1 and exo84b mutants (Fendrych et al, 2010;Safavian and Goring, 2013).…”

“…A number of Arabidopsis exocyst subunit loss-offunction mutants display severe growth defects; thus, flowering is compromised in these mutants (Fendrych et al, 2010;Wu et al, 2013;Zhang et al, 2013). As we were interested in investigating if the exocyst subunits are required in the stigmatic papilla to accept compatible pollen, a focused strategy was adopted to knockdown the expression of individual exocyst subunit genes.…”

“…At the cellular level, these phenotypes have been associated with decreased cell elongation and polar growth (Cole et al, 2005(Cole et al, , 2014Wen et al, 2005;Synek et al, 2006), defects in cytokinesis and cell plate formation (Fendrych et al, 2010;Wu et al, 2013;Rybak et al, 2014), and disrupted Pin-Formed (PIN) auxin efflux carrier recycling and polar auxin transport . Several Arabidopsis subunit mutants display strong growth defects such as the sec3a mutant with an embryo-lethal phenotype (Zhang et al, 2013), sec6, sec8, and exo84b mutants with severely dwarfed phenotypes and defects in root growth (Fendrych et al, 2010;Wu et al, 2013;Cole et al, 2014), and exo70A1 with a milder dwarf phenotype (Synek et al, 2006). The Arabidopsis exo70A1 mutant has also been reported to have defects in root hair elongation, hypocotyl elongation, compatible pollen acceptance, seed coat deposition, and tracheary element differentiation (Synek et al, 2006;Samuel et al, 2009;Kulich et al, 2010;Li et al, 2013).…”

“…The Arabidopsis exo70A1 mutant has also been reported to have defects in root hair elongation, hypocotyl elongation, compatible pollen acceptance, seed coat deposition, and tracheary element differentiation (Synek et al, 2006;Samuel et al, 2009;Kulich et al, 2010;Li et al, 2013). Essential roles for other exocyst subunits include Arabidopsis SEC5a/SEC5b, SEC6, SEC8, and SEC15a/SEC15b in male gametophyte development and pollen tube growth (Cole et al, 2005;Hála et al, 2008;Wu et al, 2013), SEC8 in seed coat deposition (Kulich et al, 2010), SEC5a, SEC8, EXO70A1, and EXO84b in root meristem size and root cell elongation (Cole et al, 2014), and a maize (Zea mays) SEC3 homolog in root hair elongation (Wen et al, 2005). Finally, the Arabidopsis EXO70B1, EXO70B2, and EXO70H1 subunits have been implicated in plant defense responses (Pecenková et al, 2011;Stegmann et al, 2012;Kulich et al, 2013;Stegmann et al, 2013).…”

Exocyst SEC3 and phosphoinositides define sites of exocytosis in pollen tube initiation and growth

“…Studies in Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) have implicated the exocyst in the regulation of pollen tube and root hair growth, seed coat deposition, response to pathogens, cytokinesis, and meristem and stigma function (Cole et al, 2005;Synek et al, 2006;Hála et al, 2008;Fendrych et al, 2010;Kulich et al, 2010;Pecenková et al, 2011;Safavian and Goring, 2013;Wu et al, 2013;Safavian et al, 2015;Zhang et al, 2016). The growth arrest of pollen tubes in sec8, sec6, sec15a, and sec5a/sec5b single and double mutants (Cole et al, 2005;Hála et al, 2008) or following treatment with the EXO70 inhibitor ENDOSIDIN2 (Zhang et al, 2016), and of root hairs in maize root hairless1 (rth1) SEC3 mutant (Wen et al, 2005), the inhibition of seed coat deposition in the sec8 and exo70A1 mutants (Kulich et al, 2010), and stigmatic papillae function in exo70A1 mutant plants (Safavian and Goring, 2013;Safavian et al, 2015) have implicated the exocyst in polarized exocytosis in plants.…”