Protein complex Arp2/3 has a conserved role in the nucleation of branched actin filaments. It is constituted of seven subunits, including actin-like subunits ARP2 and ARP3 plus five other subunits called Arp2/3 Complex Component 1 to 5, which are not related to actin. Knock-out plant mutants lacking individual plant ARP2/3 subunits have a typical phenotype of distorted trichomes, altered pavement cells shape and defects in cell adhesion. While knock-out mutant Arabidopsis plants for most ARP2/3 subunits have been characterized before, Arabidopsis plant mutants missing ARPC1 and ARPC3 subunits have not yet been described. Using CRISPR/Cas9, we generated knock-out mutants lacking ARPC1 and ARPC3 subunits. We confirmed that the loss of ARPC1 subunits results in the typical ARP2/3 mutant phenotype. However, the mutants lacking ARPC3 subunits resulted in plants with surprisingly different phenotypes. Our results suggest that plant ARP2/3 complex function in trichome shaping does not require ARPC3 subunit, while the fully assembled complex is necessary for the establishment of correct cell adhesion in the epidermis.

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

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Sci Rep. 2023 Apr 12;13(1):6018. doi: 10.1038/s41598-023-32386-x. PubMed

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Machesky LM, Atkinson SJ, Ampe C, Vandekerckhove J, Pollard TD. Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J. Cell Biol. 1994;127:107–115. doi: 10.1083/jcb.127.1.107. PubMed DOI PMC

Veltman DM, Insall RH. WASP family proteins: Their evolution and its physiological implications. Mol. Biol. Cell. 2010;21:2880–2893. doi: 10.1091/mbc.e10-04-0372. PubMed DOI PMC

Mullins RD, Heuser JA, Pollard TD. The interaction of Arp2/3 complex with actin: Nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl. Acad. Sci. U. S. A. 1998;95:6181–6186. doi: 10.1073/pnas.95.11.6181. PubMed DOI PMC

Fäßler F, Dimchev G, Hodirnau V-V, Wan W, Schur FKM. Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction. Nat. Commun. 2020;11:6437. doi: 10.1038/s41467-020-20286-x. PubMed DOI PMC

Goley ED, Welch MD. The ARP2/3 complex: An actin nucleator comes of age. Nat. Rev. Mol. Cell Biol. 2006;7:713–726. doi: 10.1038/nrm2026. PubMed DOI

Galloni, C. et al. MICAL2 enhances branched actin network disassembly by oxidizing Arp3B-containing Arp2/3 complexes. J. Cell Biol.220, (2021). PubMed PMC

Welch MD, DePace AH, Verma S, Iwamatsu A, Mitchison TJ. The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly. J. Cell Biol. 1997;138:375–384. doi: 10.1083/jcb.138.2.375. PubMed DOI PMC

Winter DC, Choe EY, Li R. Genetic dissection of the budding yeast Arp2/3 complex: A comparison of the in vivo and structural roles of individual subunits. Proc. Natl. Acad. Sci. U.S.A. 1999;96:7288–7293. doi: 10.1073/pnas.96.13.7288. PubMed DOI PMC

Brawley SH, et al. Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta) Proc. Natl. Acad. Sci. U.S.A. 2017;114:E6361–E6370. doi: 10.1073/pnas.1703088114. PubMed DOI PMC

Gupta CM, Ambaru B, Bajaj R. Emerging functions of Actins and actin binding proteins in Trypanosomatids. Front. Cell Dev. Biol. 2020;8:587685. doi: 10.3389/fcell.2020.587685. PubMed DOI PMC

Chorev DS, Moscovitz O, Geiger B, Sharon M. Regulation of focal adhesion formation by a vinculin-Arp2/3 hybrid complex. Nat. Commun. 2014;5:3758. doi: 10.1038/ncomms4758. PubMed DOI

Yae K, et al. Sleeping Beauty transposon-based phenotypic analysis of mice: Lack of Arpc3 results in defective trophoblast outgrowth. Mol. Cell. Biol. 2006;26:6185–6196. doi: 10.1128/MCB.00018-06. PubMed DOI PMC

Sun S-C, et al. Actin nucleator Arp2/3 complex is essential for mouse preimplantation embryo development. Reprod. Fertil. Dev. 2013;25:617–623. doi: 10.1071/RD12011. PubMed DOI

Mishra M, Huang J, Balasubramanian MK. The yeast actin cytoskeleton. FEMS Microbiol. Rev. 2014;38:213–227. doi: 10.1111/1574-6976.12064. PubMed DOI

Mathur J, Mathur N, Kernebeck B, Hülskamp M. Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis. Plant Cell. 2003;15:1632–1645. doi: 10.1105/tpc.011676. PubMed DOI PMC

Mathur J, et al. Arabidopsis CROOKED encodes for the smallest subunit of the ARP2/3 complex and controls cell shape by region specific fine F-actin formation. Development. 2003;130:3137–3146. doi: 10.1242/dev.00549. PubMed DOI

El-Din El-Assal S, Le J, Basu D, Mallery EL, Szymanski DB. DISTORTED2 encodes an ARPC2 subunit of the putative Arabidopsis ARP2/3 complex. Plant J. 2004;38(3):526–538. doi: 10.1111/j.1365-313X.2004.02065.x. PubMed DOI

Saedler R, et al. Actin control over microtubules suggested by DISTORTED2 encoding the Arabidopsis ARPC2 subunit homolog. Plant Cell Physiol. 2004;45:813–822. doi: 10.1093/pcp/pch103. PubMed DOI

Hossain MS, et al. Lotus japonicus ARPC1 is required for rhizobial infection. Plant Physiol. 2012;160:917–928. doi: 10.1104/pp.112.202572. PubMed DOI PMC

Kotchoni SO, et al. The association of the Arabidopsis actin-related protein2/3 complex with cell membranes is linked to its assembly status but not its activation. Plant Physiol. 2009;151:2095–2109. doi: 10.1104/pp.109.143859. PubMed DOI PMC

Hülskamp M, Misŕa S, Jürgens G. Genetic dissection of trichome cell development in Arabidopsis. Cell. 1994;76:555–566. doi: 10.1016/0092-8674(94)90118-X. PubMed DOI

El-Assal SE-D, Le J, Basu D, Mallery EL, Szymanski DB. Arabidopsis GNARLED encodes a NAP125 homolog that positively regulates ARP2/3. Curr. Biol. 2004;14:1405–1409. doi: 10.1016/j.cub.2004.06.062. PubMed DOI

Saedler R, Zimmermann I, Mutondo M, Hülskamp M. The Arabidopsis KLUNKER gene controls cell shape changes and encodes the AtSRA1 homolog. Plant Mol. Biol. 2004;56:775–782. doi: 10.1007/s11103-004-4951-z. PubMed DOI

Li S, Blanchoin L, Yang Z, Lord EM. The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis. Plant Physiol. 2003;132:2034–2044. doi: 10.1104/pp.103.028563. PubMed DOI PMC

Sahi VP, et al. Arabidopsis thaliana plants lacking the ARP2/3 complex show defects in cell wall assembly and auxin distribution. Ann. Bot. 2018;122:777–789. PubMed PMC

Yanagisawa M, et al. Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis. Nat Plants. 2015;1:15014. doi: 10.1038/nplants.2015.14. PubMed DOI

Yanagisawa M, Alonso JM, Szymanski DB. Microtubule-dependent confinement of a cell signaling and actin polymerization control module regulates polarized cell growth. Curr. Biol. 2018;28:2459–2466.e4. doi: 10.1016/j.cub.2018.05.076. PubMed DOI

Zhang C, Mallery EL, Szymanski DB. ARP2/3 localization in Arabidopsis leaf pavement cells: A diversity of intracellular pools and cytoskeletal interactions. Front. Plant Sci. 2013;4:238. doi: 10.3389/fpls.2013.00238. PubMed DOI PMC

Cifrová P, et al. Division of labor between two actin Nucleators—the Formin FH1 and the ARP2/3 Complex—in arabidopsis epidermal cell morphogenesis. Front. Plant Sci. 2020;11:148. doi: 10.3389/fpls.2020.00148. PubMed DOI PMC

Wang P, Richardson C, Hawes C, Hussey PJ. Arabidopsis NAP1 regulates the formation of Autophagosomes. Curr. Biol. 2016;26:2060–2069. doi: 10.1016/j.cub.2016.06.008. PubMed DOI

García-González J, et al. Arp2/3 complex is required for auxin-driven cell expansion through regulation of auxin transporter homeostasis. Front. Plant Sci. 2020;11:486. doi: 10.3389/fpls.2020.00486. PubMed DOI PMC

Martinek, J. et al. ARP2/3 complex associates with peroxisomes to participate in pexophagy in plants. bioRxiv 2022.04.07.487451 (2022). PubMed

Menand B, Calder G, Dolan L. Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens. J. Exp. Bot. 2007;58:1843–1849. doi: 10.1093/jxb/erm047. PubMed DOI

Harries PA, Pan A, Quatrano RS. Actin-related protein2/3 complex component ARPC1 is required for proper cell morphogenesis and polarized cell growth in Physcomitrella patens. Plant Cell. 2005;17:2327–2339. doi: 10.1105/tpc.105.033266. PubMed DOI PMC

Finka A, et al. The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens. Cell Motil. Cytoskelet. 2008;65:769–784. doi: 10.1002/cm.20298. PubMed DOI

Perroud P-F, Quatrano RS. The role of ARPC4 in tip growth and alignment of the polar axis in filaments of Physcomitrella patens. Cell Motil. Cytoskelet. 2006;63:162–171. doi: 10.1002/cm.20114. PubMed DOI

Perroud P-F, Quatrano RS. BRICK1 is required for apical cell growth in filaments of the moss Physcomitrella patens but not for gametophore morphology. Plant Cell. 2008;20:411–422. doi: 10.1105/tpc.107.053256. PubMed DOI PMC

Xu Y, Huang S. Control of the Actin cytoskeleton within apical and subapical regions of pollen tubes. Front. Cell Dev. Biol. 2020;8:614821. doi: 10.3389/fcell.2020.614821. PubMed DOI PMC

Chin S, et al. Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development. Plant Cell. 2021;33:2131–2148. doi: 10.1093/plcell/koab115. PubMed DOI PMC

Van Gestel K, et al. Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: Subcellular localization to actin-enriched pit fields and emerging root hairs. Protoplasma. 2003;222:45–52. doi: 10.1007/s00709-003-0004-8. PubMed DOI

Denninger P, et al. Distinct RopGEFs successively drive polarization and outgrowth of root hairs. Curr. Biol. 2019;29:1854–1865.e5. doi: 10.1016/j.cub.2019.04.059. PubMed DOI

Yokota K, et al. Rearrangement of actin cytoskeleton mediates invasion of Lotus japonicus roots by Mesorhizobium loti. Plant Cell. 2009;21:267–284. doi: 10.1105/tpc.108.063693. PubMed DOI PMC

Qiu L, et al. SCARN a novel class of SCAR protein that is required for root-hair infection during legume nodulation. PLoS Genet. 2015;11:e1005623. doi: 10.1371/journal.pgen.1005623. PubMed DOI PMC

Miyahara A, et al. Conservation in function of a SCAR/WAVE component during infection thread and root hair growth in Medicago truncatula. Mol. Plant Microbe Interact. 2010;23:1553–1562. doi: 10.1094/MPMI-06-10-0144. PubMed DOI

Gavrin A, Jansen V, Ivanov S, Bisseling T, Fedorova E. ARP2/3-mediated actin nucleation associated with Symbiosome membrane is essential for the development of Symbiosomes in infected cells of Medicago Truncatula root nodules. Mol. Plant Microbe Interact. 2015;28:605–614. doi: 10.1094/MPMI-12-14-0402-R. PubMed DOI

Isner J-C, et al. Actin filament reorganisation controlled by the SCAR/WAVE complex mediates stomatal response to darkness. New Phytol. 2017;215:1059–1067. doi: 10.1111/nph.14655. PubMed DOI PMC

Jiang K, et al. The ARP2/3 complex mediates guard cell actin reorganization and stomatal movement in Arabidopsis. Plant Cell. 2012;24:2031–2040. doi: 10.1105/tpc.112.096263. PubMed DOI PMC

Sun G, et al. The tomato Arp2/3 complex is required for resistance to the powdery mildew fungus Oidium neolycopersici. Plant Cell Environ. 2019;42:2664–2680. doi: 10.1111/pce.13569. PubMed DOI PMC

Qi T, et al. TaARPC3, contributes to wheat resistance against the stripe rust fungus. Front. Plant Sci. 2017;8:1245. doi: 10.3389/fpls.2017.01245. PubMed DOI PMC

Badet T, et al. Expression polymorphism at the ARPC4 locus links the actin cytoskeleton with quantitative disease resistance to Sclerotinia sclerotiorum in Arabidopsis thaliana. New Phytol. 2019;222:480–496. doi: 10.1111/nph.15580. PubMed DOI

Xing H-L, et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014;14:327. doi: 10.1186/s12870-014-0327-y. PubMed DOI PMC

Lei Y, et al. CRISPR-P: A web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol. Plant. 2014;7:1494–1496. doi: 10.1093/mp/ssu044. PubMed DOI

Zhang X, Henriques R, Lin S-S, Niu Q-W, Chua N-H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 2006;1:641–646. doi: 10.1038/nprot.2006.97. PubMed DOI

Vernet T, Dignard D, Thomas DY. A family of yeast expression vectors containing the phage f1 intergenic region1. Gene. 1987;52:225–233. doi: 10.1016/0378-1119(87)90049-7. PubMed DOI

Clontech Laboratories, Inc. Yeast Protocols Handbook. https://www.takara.co.kr/file/manual/pdf/PT3024-1.pdf (2009).

Ganger MT, Dietz GD, Ewing SJ. A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments. BMC Bioinf. 2017;18:534. doi: 10.1186/s12859-017-1949-5. PubMed DOI PMC

Schindelin J, et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Datla RS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W. Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene. 1992;122:383–384. doi: 10.1016/0378-1119(92)90232-E. PubMed DOI

Gournier H, Goley ED, Niederstrasser H, Trinh T, Welch MD. Reconstitution of human Arp2/3 complex reveals critical roles of individual subunits in complex structure and activity. Mol. Cell. 2001;8:1041–1052. doi: 10.1016/S1097-2765(01)00393-8. PubMed DOI

Suraneni P, et al. The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. J. Cell Biol. 2012;197:239–251. doi: 10.1083/jcb.201112113. PubMed DOI PMC

Cabrera R, Suo J, Young E, Chang EC. Schizosaccharomyces pombe Arc3 is a conserved subunit of the Arp2/3 complex required for polarity, actin organization, and endocytosis. Yeast. 2011;28:495–503. doi: 10.1002/yea.1853. PubMed DOI PMC

Li Y, Sorefan K, Hemmann G, Bevan MW. Arabidopsis NAP and PIR regulate actin-based cell morphogenesis and multiple developmental processes. Plant Physiol. 2004;136:3616–3627. doi: 10.1104/pp.104.053173. PubMed DOI PMC

Szymanski DB. Breaking the WAVE complex: The point of Arabidopsis trichomes. Curr. Opin. Plant Biol. 2005;8:103–112. doi: 10.1016/j.pbi.2004.11.004. PubMed DOI

Balcer HI, Daugherty-Clarke K, Goode BL. The p40/ARPC1 subunit of Arp2/3 complex performs multiple essential roles in WASp-regulated actin nucleation. J. Biol. Chem. 2010;285:8481–8491. doi: 10.1074/jbc.M109.054957. PubMed DOI PMC

Pan F, Egile C, Lipkin T, Li R. ARPC1/Arc40 mediates the interaction of the actin-related protein 2 and 3 complex with Wiskott-Aldrich syndrome protein family activators. J. Biol. Chem. 2004;279:54629–54636. doi: 10.1074/jbc.M402357200. PubMed DOI

Basu D, et al. DISTORTED3/SCAR2 is a putative arabidopsis WAVE complex subunit that activates the Arp2/3 complex and is required for epidermal morphogenesis. Plant Cell. 2005;17:502–524. doi: 10.1105/tpc.104.027987. PubMed DOI PMC

Uhrig JF, et al. The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis. Development. 2007;134:967–977. doi: 10.1242/dev.02792. PubMed DOI

Yanagisawa M, Zhang C, Szymanski DB. ARP2/3-dependent growth in the plant kingdom: SCARs for life. Front. Plant Sci. 2013;4:166. doi: 10.3389/fpls.2013.00166. PubMed DOI PMC

Schwarzerova, K. Divergent: The unique role of the ARPC3 subunit in the plant ARP2/3 complex. (2022). 10.17605/OSF.IO/XNSQF.