The Arabidopsisthaliana pathogenesis-related 1 (PR1) is an important defense protein, so far it has only been detected in extracellular space and its subcellular sorting and transport remain unexplained. Using a green fluorescent protein (GFP) tagged full length, as well as a C-terminus truncated version of PR1, we observed that when expressed ectopically in Nicotiana benthamiana leaves, PR1 co-localizes only partially with Golgi markers, and much more prominently with the late endosome (LE)/multivesicular body (MVB) FYVE marker. The C-truncated version PR1ΔC predominantly localized to the endoplasmic reticulum (ER). The same localizations were found for stable Arabidopsis transformants with expression of PR1 and PR1ΔC driven by the native promoter. We conclude that the A. thaliana PR1 (AtPR1) undergoes an unconventional secretion pathway, starting from the C-terminus-dependent sorting from the ER, and utilizing further transportation via phosphatidyl-inositol-3-phosphate (PI(3)P) positive LE/MVB-like vesicles. The homology model of the PR1 structure shows that the cluster of positively charged amino acid residues (arginines 60, 67, 137, and lysine 135) could indeed interact with negatively charged phospholipids of cellular membranes. It remains to be resolved whether Golgi and LE/MVB localization reflects an alternative sorting or trafficking succession, and what the role of lipid interactions in it will be.

Laboratory of Cell Biology Institute of Experimental Botany Academy of Sciences of the Czech Republic Rozvojova 263 165 02 Prague 6 Czech Republic

Laboratory of Cell Morphogenesis Department of Experimental Plant Biology Faculty of Science Charles University Prague Vinicna 5 128 44 Prague 2 Czech Republic

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Stintzi A., Heitz T., Prasad V., Wiedemann-Merdinoglu S., Kauffmann S., Geoffroy P., Legrand M., Fritig B. Plant ‘pathogenesis-related’ proteins and their role in defense against pathogens. Biochimie. 1993;75:687–706. doi: 10.1016/0300-9084(93)90100-7. PubMed DOI

Van Loon L.C., Pierpoint W.S., Boller T., Conejero V. Recommendations for naming plant pathogenesis-related proteins. Plant Mol. Biol. Rep. 1994;12:245–264. doi: 10.1007/BF02668748. DOI

Van Loon L.C., van Kammen A. Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. “Samsun” and “Samsun NN”. II. Changes in protein constitution after infection with tobacco mosaic virus. Virology. 1970;40:190–211. doi: 10.1016/0042-6822(70)90395-8. PubMed DOI

Van Loon L.C., Van Strien E.A. The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol. Mol. Plant Pathol. 1999;55:85–97. doi: 10.1006/pmpp.1999.0213. DOI

Schreiber M.C., Karlo J.C., Kovalick G.E. A novel cDNA from Drosophila encoding a protein with similarity to mammalian cysteine-rich secretory proteins, wasp venom antigen 5, and plant group 1 pathogenesis-related proteins. Gene. 1997;191:135–141. doi: 10.1016/S0378-1119(97)00010-3. PubMed DOI

Van Loon L.C., Rep M., Pieterse C.M. Significance of inducible defense-related proteins in infected plants. Annu. Rev. Phytopathol. 2006;44:135–162. doi: 10.1146/annurev.phyto.44.070505.143425. PubMed DOI

Mitsuhara I., Iwai T., Seo S., Yanagawa Y., Kawahigasi H., Hirose S., Ohkawa Y., Ohashi Y. Characteristic expression of twelve rice PR1 family genes in response to pathogen infection, wounding, and defense-related signal compounds (121/180) Mol. Genet. Genom. 2008;279:415–427. doi: 10.1007/s00438-008-0322-9. PubMed DOI PMC

Gu Y., Innes R.W. The KEEP ON GOING protein of Arabidopsis regulates intracellular protein trafficking and is degraded during fungal infection. Plant Cell. 2012;24:4717–4730. doi: 10.1105/tpc.112.105254. PubMed DOI PMC

Stone S.L., Williams L.A., Farmer L.M., Vierstra R.D., Callis J. KEEP ON GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling. Plant Cell. 2006;18:3415–3428. doi: 10.1105/tpc.106.046532. PubMed DOI PMC

Haseloff J., Siemering K.R., Prasher D.C., Hodge S. Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc. Natl. Acad. Sci. USA. 1997;94:2122–2127. doi: 10.1073/pnas.94.6.2122. PubMed DOI PMC

Hayashi Y., Yamada K., Shimada T., Matsushima R., Nishizawa N.K., Nishimura M., Hara-Nishimura I. A proteinase-storing body that prepares for cell death or stresses in the epidermal cells of Arabidopsis. Plant Cell Physiol. 2001;42:894–899. doi: 10.1093/pcp/pce144. PubMed DOI

Watanabe S., Shimada T.L., Hiruma K., Takano Y. Pathogen infection trial increases the secretion of proteins localized in the endoplasmic reticulum body of Arabidopsis. Plant Physiol. 2013;163:659–664. doi: 10.1104/pp.113.217364. PubMed DOI PMC

Broekaert W.F., Terras F.R.G., Cammue B.P.A. Induced and preformed antimicrobial proteins. In: Slusarenko A.J., Fraser R.S.S., Van Loon L.C., editors. Mechanisms of Resistance to Plant Diseases. Dordrecht; Kluwer, The Netherlands: 2000. pp. 371–477.

Rivière M.P., Marais A., Ponchet M., Willats W., Galiana E. Silencing of acidic pathogenesis-related PR-1 genes increases extracellular β-(1–>3)-glucanase activity at the onset of tobacco defence reactions. J. Exp. Bot. 2008;59:1225–1239. doi: 10.1093/jxb/ern044. PubMed DOI

Chen Y.L., Lee C.Y., Cheng K.T., Chang W.H., Huang R.N., Nam H.G., Chen Y.R. Quantitative peptidomics study reveals that a wound-induced peptide from PR-1 regulates immune signaling in tomato. Plant Cell. 2014;26:4135–4148. doi: 10.1105/tpc.114.131185. PubMed DOI PMC

Chien P.S., Nam H.G., Chen Y.R. A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis. J. Exp. Bot. 2015;66:5301–5313. doi: 10.1093/jxb/erv263. PubMed DOI PMC

Nelson B.K., Cai X., Nebenführ A. A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007;51:1126–1136. doi: 10.1111/j.1365-313X.2007.03212.x. PubMed DOI

Saint-Jore-Dupas C., Nebenführ A., Boulaflous A., Follet-Gueye M.L., Plasson C., Hawes C., Driouich A., Faye L., Gomord V. Plant N-glycan processing enzymes employ different targeting mechanisms for their spatial arrangement along the secretory pathway. Plant Cell. 2006;18:3182–3200. doi: 10.1105/tpc.105.036400. PubMed DOI PMC

Dorokhov Y.L., Skurat E.V., Frolova O.Y., Gasanova T.V., Ivanov P.A., Ravin N.V., Skryabin K.G., Mäkinen K.M., Klimyuk V.I., Gleba Y.Y., et al. Role of the leader sequence in tobacco pectin methylesterase secretion. FEBS Lett. 2006;580:3329–3334. doi: 10.1016/j.febslet.2006.04.090. PubMed DOI

Honig A., Avin-Wittenberg T., Ufaz S., Galili G. A new type of compartment, defined by plant-specific Atg8-interacting proteins, is induced upon exposure of Arabidopsis plants to carbon starvation. Plant Cell. 2012;24:288–303. doi: 10.1105/tpc.111.093112. PubMed DOI PMC

Voigt B., Timmers A.C., Samaj J., Hlavacka A., Ueda T., Preuss M., Nielsen E., Mathur J., Emans N., Stenmark H., et al. Actin-based motility of endosomes is linked to the polar tip growth of root hairs. Eur. J. Cell Biol. 2005;84:609–621. doi: 10.1016/j.ejcb.2004.12.029. PubMed DOI

Sessa G., Yang X.Q., Raz V., Eyal Y., Fluhr R. Dark induction and subcellular localization of the pathogenesis-related PRB-1b protein. Plant Mol. Biol. 1995;28:537–547. doi: 10.1007/BF00020400. PubMed DOI

Berson T., von Wangenheim D., Takáč T., Šamajová O., Rosero A., Ovečka M., Komis G., Stelzer E.H., Šamaj J. Trans-Golgi network localized small GTPase RabA1d is involved in cell plate formation and oscillatory root hair growth. BMC Plant Biol. 2014;14:252. doi: 10.1186/s12870-014-0252-0. PubMed DOI PMC

Kim D.H., Eu Y.J., Yoo C.M., Kim Y.W., Pih K.T., Jin J.B., Kim S.J., Stenmark H., Hwang I. Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells. Plant Cell. 2001;13:287–301. doi: 10.1105/tpc.13.2.287. PubMed DOI PMC

Kolb C., Nagel M.K., Kalinowska K., Hagmann J., Ichikawa M., Anzenberger F., Alkofer A., Sato M.H., Braun P., Isono E. FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis. Plant Physiol. 2015;167:1361–1373. doi: 10.1104/pp.114.253377. PubMed DOI PMC

Van Galen J., van Balkom B.W., Serrano R.L., Kaloyanova D., Eerland R., Stüven E., Helms J.B. Binding of GAPR-1 to negatively charged phospholipid membranes: Unusual binding characteristics to phosphatidylinositol. Mol. Membr. Biol. 2010;27:81–91. doi: 10.3109/09687680903507080. PubMed DOI

Van Galen J., Olrichs N.K., Schouten A., Serrano R.L., Nolte Hoen E.N., Eerland R., Kaloyanova D., Gros P., Helms J.B. Interaction of GAPR-1 with lipid bilayers is regulated by alternative homodimerization. Biochim. Biophys. Acta. 2012;1818:2175–2183. doi: 10.1016/j.bbamem.2012.04.016. PubMed DOI

Montesinos J.C., Sturm S., Langhans M., Hillmer S., Marcote M.J., Robinson D.G., Aniento F. Coupled transport of Arabidopsis p24 proteins at the ER-Golgi interface. J. Exp. Bot. 2012;63:4243–4261. doi: 10.1093/jxb/ers112. PubMed DOI PMC

Axe E.L., Walker S.A., Manifava M., Chandra P., Roderick H.L., Habermann A., Griffiths G., Ktistakis N.T. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 2008;182:685–701. doi: 10.1083/jcb.200803137. PubMed DOI PMC

Bhattacharjee S., Speicher KD., Stahelin R.V., Speicher D.W., Haldar K. PI(3)P-independent and -dependent pathways function together in a vacuolar translocation sequence to target malarial proteins to the host erythrocyte. Mol. Biochem. Parasitol. 2012;185:106–113. doi: 10.1016/j.molbiopara.2012.07.004. PubMed DOI PMC

Nakagawa T., Kurose T., Hino T., Tanaka K., Kawamukai M., Niwa Y., Toyooka K., Matsuoka K., Jinbo T., Kimura T. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 2007;104:34–41. doi: 10.1263/jbb.104.34. PubMed DOI

Clough S.J., Bent A.F. Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998;16:735–743. doi: 10.1046/j.1365-313x.1998.00343.x. PubMed DOI

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

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., 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

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

Biasini M., Bienert S., Waterhouse A., Arnold K., Studer G., Schmidt T., Kiefer F., Cassarino T.G., Bertoni M., Bordoli L., Schwede T. SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 2014;42:W252–W258. doi: 10.1093/nar/gku340. PubMed DOI PMC

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

Baker N.A., Sept D., Joseph S., Holst M.J., McCammon J.A. Electrostatics of nanosystems: Application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA. 2001;98:10037–10041. doi: 10.1073/pnas.181342398. PubMed DOI PMC

Humphrey W., Dalke A., Schulten K. VMD: Visual molecular dynamics. J. Mol. Graph. 1996;14:33–38. doi: 10.1016/0263-7855(96)00018-5. PubMed DOI

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

Immunity functions of Arabidopsis pathogenesis-related 1 are coupled but not confined to its C-terminus processing and trafficking

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