Chemokine-like MDL proteins modulate flowering time and innate immunity in plants

Human macrophage migration inhibitory factor (MIF) is an atypical chemokine implicated in intercellular signaling and innate immunity. MIF orthologs (MIF/D-DT-like proteins, MDLs) are present throughout the plant kingdom, but remain experimentally unexplored in these organisms. Here, we provide an in planta characterization and functional analysis of the three-member gene/protein MDL family in Arabidopsis thaliana. Subcellular localization experiments indicated a nucleo-cytoplasmic distribution of MDL1 and MDL2, while MDL3 is localized to peroxisomes. Protein–protein interaction assays revealed the in vivo formation of MDL1, MDL2, and MDL3 homo-oligomers, as well as the formation of MDL1-MDL2 hetero-oligomers. Functionally, Arabidopsis mdl mutants exhibited a delayed transition from vegetative to reproductive growth (flowering) under long-day conditions, but not in a short-day environment. In addition, mdl mutants were more resistant to colonization by the bacterial pathogen Pseudomonas syringae pv. maculicola. The latter phenotype was compromised by the additional mutation of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), a gene implicated in the defense-induced biosynthesis of the key signaling molecule salicylic acid. However, the enhanced antibacterial immunity was not associated with any constitutive or pathogen-induced alterations in the levels of characteristic phytohormones or defense-associated metabolites. Interestingly, bacterial infection triggered relocalization and accumulation of MDL1 and MDL2 at the peripheral lobes of leaf epidermal cells. Collectively, our data indicate redundant functionality and a complex interplay between the three chemokine-like Arabidopsis MDL proteins in the regulation of both developmental and immune-related processes. These insights expand the comparative cross-kingdom analysis of MIF/MDL signaling in human and plant systems. Human macrophage migration inhibitory factor (MIF) is an atypical chemokine implicated in intercellular signaling and innate immunity. MIF orthologs (MIF/D-DT-like proteins, MDLs) are present throughout the plant kingdom, but remain experimentally unexplored in these organisms. Here, we provide an in planta characterization and functional analysis of the three-member gene/protein MDL family in Arabidopsis thaliana. Subcellular localization experiments indicated a nucleo-cytoplasmic distribution of MDL1 and MDL2, while MDL3 is localized to peroxisomes. Protein–protein interaction assays revealed the in vivo formation of MDL1, MDL2, and MDL3 homo-oligomers, as well as the formation of MDL1-MDL2 hetero-oligomers. Functionally, Arabidopsis mdl mutants exhibited a delayed transition from vegetative to reproductive growth (flowering) under long-day conditions, but not in a short-day environment. In addition, mdl mutants were more resistant to colonization by the bacterial pathogen Pseudomonas syringae pv. maculicola. The latter phenotype was compromised by the additional mutation of SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2), a gene implicated in the defense-induced biosynthesis of the key signaling molecule salicylic acid. However, the enhanced antibacterial immunity was not associated with any constitutive or pathogen-induced alterations in the levels of characteristic phytohormones or defense-associated metabolites. Interestingly, bacterial infection triggered relocalization and accumulation of MDL1 and MDL2 at the peripheral lobes of leaf epidermal cells. Collectively, our data indicate redundant functionality and a complex interplay between the three chemokine-like Arabidopsis MDL proteins in the regulation of both developmental and immune-related processes. These insights expand the comparative cross-kingdom analysis of MIF/MDL signaling in human and plant systems. Human macrophage migration inhibitory factor (MIF) is a small (114 amino acids, 12.345 kDa) multifunctional protein that is best known for its role as an atypical cytokine/chemokine to regulate innate immunity. In fact, MIF was the first human cytokine to be discovered over five decades ago (1Calandra T. Roger T. Macrophage migration inhibitory factor: A regulator of innate immunity.Nat. Rev. Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1309) Google Scholar, 2David J.R. Delayed hypersensitivity in vitro: Its mediation by cell-free substances formed by lymphoid cell-antigen interaction.Proc. Natl. Acad. Sci. U. S. A. 1966; 56: 72-77Crossref PubMed Scopus (1089) Google Scholar, 3Kapurniotu A. Gokce O. Bernhagen J. The multitasking potential of alarmins and atypical chemokines.Front. Med. 2019; 6: 3Crossref PubMed Scopus (44) Google Scholar). Dysregulation of MIF has been associated with acute and chronic inflammatory diseases such as septic shock, rheumatoid arthritis, and atherosclerosis, as well as autoimmune conditions and cancer (4Kang I. Bucala R. The immunobiology of MIF: Function, genetics and prospects for precision medicine.Nat. Rev. Rheumatol. 2019; 15: 427-437Crossref PubMed Scopus (83) Google Scholar, 5Morand E.F. Leech M. Bernhagen J. Mif: A new cytokine link between rheumatoid arthritis and atherosclerosis.Nat. Rev. Drug Discov. 2006; 5: 399-410Crossref PubMed Scopus (294) Google Scholar, 6Sinitski D. Kontos C. Krammer C. Asare Y. Kapurniotu A. Bernhagen J. Macrophage Migration Inhibitory Factor (MIF)-based therapeutic concepts in atherosclerosis and inflammation.Thromb. Haemost. 2019; 119: 553-566Crossref PubMed Scopus (42) Google Scholar, 7Tilstam P.V. Qi D. Leng L. Young L. Bucala R. MIF family cytokines in cardiovascular diseases and prospects for precision-based therapeutics.Expert Opin. Ther. Targets. 2017; 21: 671-683Crossref PubMed Scopus (48) Google Scholar). The expression of MIF in the human organism is not limited to immune cells but is broadly detectable in various tissues and cell types. Unlike other cytokines/chemokines, MIF lacks a canonical N-terminal signal peptide and is released from preformed intracellular stores into the extracellular environment via stimulus-regulated unconventional secretion (1Calandra T. Roger T. Macrophage migration inhibitory factor: A regulator of innate immunity.Nat. Rev. Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1309) Google Scholar, 8Flieger O. Engling A. Bucala R. Lue H. Nickel W. Bernhagen J. Regulated secretion of macrophage migration inhibitory factor is mediated by a non-classical pathway involving an ABC transporter.FEBS Lett. 2003; 551: 78-86Crossref PubMed Scopus (182) Google Scholar). MIF’s cytokine/chemokine activities are mediated through interaction with its cognate receptor CD74 and/or by noncognate engagement of one of its three CXC chemokine receptors in a cell-, tissue-, or disease-specific manner (1Calandra T. Roger T. Macrophage migration inhibitory factor: A regulator of innate immunity.Nat. Rev. Immunol. 2003; 3: 791-800Crossref PubMed Scopus (1309) Google Scholar, 3Kapurniotu A. Gokce O. Bernhagen J. The multitasking potential of alarmins and atypical chemokines.Front. Med. 2019; 6: 3Crossref PubMed Scopus (44) Google Scholar, 9Leng L. Metz C.N. Fang Y. Xu J. Donnelly S. Baugh J. Delohery T. Chen Y. Mitchell R.A. Bucala R. MIF signal transduction initiated by binding to CD74.J. Exp. Med. 2003; 197: 1467-1476Crossref PubMed Scopus (810) Google Scholar, 10Bernhagen J. Krohn R. Lue H. Gregory J.L. Zernecke A. Koenen R.R. Dewor M. Georgiev I. Schober A. Leng L. Kooistra T. Fingerle-Rowson G. Ghezzi P. Kleemann R. McColl S.R. et al.MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment.Nat. Med. 2007; 13: 587-596Crossref PubMed Scopus (919) Google Scholar). CD74 is a single-pass transmembrane protein, also known as the human HLA class II histocompatibility antigen γ chain (9Leng L. Metz C.N. Fang Y. Xu J. Donnelly S. Baugh J. Delohery T. Chen Y. Mitchell R.A. Bucala R. MIF signal transduction initiated by binding to CD74.J. Exp. Med. 2003; 197: 1467-1476Crossref PubMed Scopus (810) Google Scholar, 11Borghese F. Clanchy F.I.L. CD74: An emerging opportunity as a therapeutic target in cancer and autoimmune disease.Expert Opin. Ther. Targets. 2011; 15: 237-251Crossref PubMed Scopus (103) Google Scholar). Upon MIF binding, signaling from cell-surface-expressed CD74 prominently regulates cell proliferation, apoptosis, and inflammatory gene expression. The chemokine receptors of MIF, CXCR2, CXCR4, and CXCR7, belonging to the class of heptahelical membrane proteins, function as G-protein-coupled receptors (GPCRs) and are the bona fide receptors for classical chemokines such as CXCL8, CXCL12, and CXCL11, respectively. MIF engages these receptors via a structural mimicry mechanism to promote leukocyte recruitment responses that can play an important pathogenic role in cardiovascular and inflammatory diseases (6Sinitski D. Kontos C. Krammer C. Asare Y. Kapurniotu A. Bernhagen J. Macrophage Migration Inhibitory Factor (MIF)-based therapeutic concepts in atherosclerosis and inflammation.Thromb. Haemost. 2019; 119: 553-566Crossref PubMed Scopus (42) Google Scholar, 7Tilstam P.V. Qi D. Leng L. Young L. Bucala R. MIF family cytokines in cardiovascular diseases and prospects for precision-based therapeutics.Expert Opin. Ther. Targets. 2017; 21: 671-683Crossref PubMed Scopus (48) Google Scholar, 10Bernhagen J. Krohn R. Lue H. Gregory J.L. Zernecke A. Koenen R.R. Dewor M. Georgiev I. Schober A. Leng L. Kooistra T. Fingerle-Rowson G. Ghezzi P. Kleemann R. McColl S.R. et al.MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment.Nat. Med. 2007; 13: 587-596Crossref PubMed Scopus (919) Google Scholar). In addition to its cytokine/chemokine functions in the extracellular space (“MIF the cytokine”), MIF has a number of suggested intracellular functions mediated by protein–protein interactions or enzymatic activity. This is in line with its high degree of evolutionary conservation, and it has been speculated that intracellular, enzymatic activities of MIF are evolutionarily ancient (“MIF the enzyme”) (3Kapurniotu A. Gokce O. Bernhagen J. The multitasking potential of alarmins and atypical chemokines.Front. Med. 2019; 6: 3Crossref PubMed Scopus (44) Google Scholar). Reported enzymatic activities comprise a tautomerase activity linked to an N-terminal proline-containing catalytic pocket (3Kapurniotu A. Gokce O. Bernhagen J. The multitasking potential of alarmins and atypical chemokines.Front. Med. 2019; 6: 3Crossref PubMed Scopus (44) Google Scholar, 12Rosengren E. Bucala R. Åman P. Jacobsson L. Odh G. Metz C.N. Rorsman H. The immunoregulatory mediator macrophage Migration Inhibitory Factor (MIF) catalyzes a tautomerization reaction.Mol. Med. 1996; 2: 143-149Crossref PubMed Google Scholar), an oxidoreductase activity that is dependent on a central CXXC motif (3Kapurniotu A. Gokce O. Bernhagen J. The multitasking potential of alarmins and atypical chemokines.Front. Med. 2019; 6: 3Crossref PubMed Scopus (44) Google Scholar, 13Kleemann R. Kapurniotu A. Frank R.W. Gessner A. Mischke R. Flieger O. Jüttner S. Brunner H. Bernhagen J. Disulfide analysis reveals a role for macrophage migration inhibitory factor (MIF) as thiol-protein oxidoreductase.J. Mol. 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Marsala M. et al.Macrophage migration inhibitory factor as a chaperone inhibiting accumulation of misfolded SOD1.Neuron. 2015; 86: 218-232Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). However, the precise molecular mechanisms of these activities and their potential interplay are incompletely understood and their physiological significance remains elusive. Many vertebrates including humans possess a paralog of MIF, termed D-dopachrome tautomerase (D-DT) or MIF-2. In humans, D-DT/MIF-2 shares approximately 35% amino acid identity and a high degree of architectural similarity with MIF, and the protein recapitulates some of MIF’s pathogenic activities. However, the physiological and pathogenic functions of D-DT are less well characterized than those of MIF (16Merk M. Mitchell R.A. Endres S. Bucala R. D-dopachrome tautomerase (D-DT or MIF-2): Doubling the MIF cytokine family.Cytokine. 2012; 59: 10-17Crossref PubMed Scopus (110) Google Scholar, 17Merk M. Zierow S. Leng L. Das R. Du X. Schulte W. Fan J. Lue H. Chen Y. Xiong H. Chagnon F. Bernhagen J. Lolis E. Mor G. Lesur O. et al.The D-dopachrome tautomerase (DDT) gene product is a cytokine and functional homolog of macrophage migration inhibitory factor (MIF).Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 85Crossref PubMed Scopus (151) Google Scholar). MIF-like proteins are conserved in most eukaryotes, including plants, with signs of neofunctionalization in some taxa (18Michelet C. Danchin E.G.J. Jaouannet M. Bernhagen J. Panstruga R. Kogel K.-H. Keller H. Coustau C. Cross-kingdom analysis of diversity, evolutionary history, and site selection within the eukaryotic macrophage migration inhibitory factor superfamily.Genes. 2019; 10: E740Crossref PubMed Scopus (13) Google Scholar, 19Sparkes A. Baetselier P. de Roelants K. Trez C. de Magez S. van Ginderachter J.A. Raes G. Bucala R. Stijlemans B. The non-mammalian MIF superfamily.Immunobiology. 2017; 222: 473-482Crossref PubMed Scopus (33) Google Scholar). Previously, we performed a comprehensive in silico analysis of plant MIF/D-DT-like (MDL) proteins, focusing on the dicotyledonous reference plant species Arabidopsis thaliana (20Panstruga R. Baumgarten K. Bernhagen J. Phylogeny and evolution of plant macrophage migration inhibitory factor/D-dopachrome tautomerase-like proteins.BMC Evol. Biol. 2015; 15: 64Crossref PubMed Scopus (25) Google Scholar). We found that seed plants typically express two (gymnosperms) or three (angiosperms) different paralogs, which in Arabidopsis have been named MDL1 (AT5G57170), MDL2 (AT5G01650), and MDL3 (AT3G51660). Publicly accessible microarray data indicate that MDL1 and MDL2 are essentially expressed constitutively in aerial plant organs with little responsiveness to abiotic or biotic stress cues, while expression of MDL3 appears to be stress-inducible. The latter gene exhibits strongly enhanced transcript accumulation in leaves upon various abiotic (cold treatment, osmotic and oxidative stress, wounding, UV-B exposure) and biotic (microbial elicitors, various pathogens) stress factors. MDL3 shows in addition coexpression with a number of prominent genes involved in plant immunity (20Panstruga R. Baumgarten K. Bernhagen J. Phylogeny and evolution of plant macrophage migration inhibitory factor/D-dopachrome tautomerase-like proteins.BMC Evol. Biol. 2015; 15: 64Crossref PubMed Scopus (25) Google Scholar). Structure prediction and preliminary experimental data suggest that all three Arabidopsis MDL proteins resemble the secondary and tertiary structure of human MIF (20Panstruga R. Baumgarten K. Bernhagen J. Phylogeny and evolution of plant macrophage migration inhibitory factor/D-dopachrome tautomerase-like proteins.BMC Evol. Biol. 2015; 15: 64Crossref PubMed Scopus (25) Google Scholar, 21Sinitski D. Gruner K. Brandhofer M. Kontos C. Winkler P. Reinstädler A. Bourilhon P. Xiao Z. Cool R. Kapurniotu A. Dekker F.J. Panstruga R. Bernhagen J. Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs.J. Biol. Chem. 2020; 295: 850-867Abstract Full Text Full Text PDF PubMed Google Scholar). In contrast to human and murine MIF, very little is known about the function of plant MDLs. Analysis of recombinant epitope-tagged Arabidopsis MDLs revealed an unexpected lack of tautomerase activity, which is possibly conditioned by an amino acid polymorphism in their catalytic clefts. Surprisingly, the three MDLs can bind to the human MIF receptors CD74 and CXCR4 (which are absent from plants), activate signaling activities downstream of these in human immune cells, and substitute for human MIF in leukocyte recruitment. These findings disclose cross-kingdom mimicry of human MIF by these plant orthologs and reflect their (partial) functional conservation (21Sinitski D. Gruner K. Brandhofer M. Kontos C. Winkler P. Reinstädler A. Bourilhon P. Xiao Z. Cool R. Kapurniotu A. Dekker F.J. Panstruga R. Bernhagen J. Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs.J. Biol. Chem. 2020; 295: 850-867Abstract Full Text Full Text PDF PubMed Google Scholar, 22Foley J.F. Plant chemokine mimics.Sci. Signal. 2020; 13: eabb0387Crossref Scopus (2) Google Scholar). Reminiscent of the situation of human immune cells and some parasitic pathogens (19Sparkes A. Baetselier P. de Roelants K. Trez C. de Magez S. van Ginderachter J.A. Raes G. Bucala R. Stijlemans B. The non-mammalian MIF superfamily.Immunobiology. 2017; 222: 473-482Crossref PubMed Scopus (33) Google Scholar, 23Kamir D. Zierow S. Leng L. Cho Y. Diaz Y. Griffith J. McDonald C. Merk M. Mitchell R.A. Trent J. Chen Y. Kwong Y.-K.A. Xiong H. Vermeire J. Cappello M. et al.A Leishmania ortholog of macrophage migration inhibitory factor modulates host macrophage responses.J. Immunol. 2008; 180: 8250-8261Crossref PubMed Scopus (82) Google Scholar), plant-feeding aphids secrete an MIF ortholog into host cells to suppress plant immune responses, which is necessary for their survival and nourishment (24Naessens E. Dubreuil G. Giordanengo P. Baron O.L. Minet-Kebdani N. Keller H. Coustau C. A secreted MIF cytokine enables aphid feeding and represses plant immune responses.Curr. Biol. 2015; 25: 1898-1903Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Here, we functionally characterized the three Arabidopsis MDL proteins in planta by a comprehensive set of biochemical, cell biological, and genetic experiments. We validated the respective gene models, determined subcellular protein localizations, and studied their interactions, revealing the formation of MDL homo- and, in part, hetero-oligomers. Using a set of mdl mutants, we further uncovered roles for MDLs in the control of flowering time and bacterial pathogenesis. The TAIR (The Arabidopsis Information Resource; https://www.arabidopsis.org/) database catalogs two different gene models for MDL1 (designated MDL1.1 and MDL1.2), four different gene models for MDL2 (MDL2.1, MDL2.2, MDL2.3, and MDL2.4, leading to three different predicted protein variants), and a single gene model for MDL3 (Fig. S1A, (20Panstruga R. Baumgarten K. Bernhagen J. Phylogeny and evolution of plant macrophage migration inhibitory factor/D-dopachrome tautomerase-like proteins.BMC Evol. Biol. 2015; 15: 64Crossref PubMed Scopus (25) Google Scholar)). Protein structure prediction suggests that these transcript variants give rise to MDL forms with different C-terminal tail regions (Fig. S1B). To confirm experimentally the existence of the respective MDL transcript versions and to explore a putative tissue specificity of their expression, we first developed sets of splice variant-specific oligonucleotide primer pairs for reverse transcriptase–polymerase chain reaction (RT-PCR) (Fig. S1C). We validated the specificity of these primer pairs by using plasmid DNA harboring cloned versions of the various predicted MDL transcripts (except MDL2.4) as a template (Fig. S1D). Semiquantitative RT-PCR analysis of RNA samples from various Arabidopsis organs indicates that (i) all five tested MDL1 and MDL2 transcript variants exist (the used primer pairs did not discriminate between MDL2.2 and MDL2.3; Fig. S1C), (ii) MDL1.1 and MDL2.1 appear to be the predominant transcript versions, and (iii) that there is no pronounced organ specificity in the accumulation of any of the various splice variants (Fig. S1E). We previously performed in silico analysis of the three MDL proteins regarding the presence of subcellular targeting signals. This revealed a putative nuclear localization signal for MDL1 and a predicted C-terminal peroxisomal targeting sequence (PTS1) for MDL3 (20Panstruga R. Baumgarten K. Bernhagen J. Phylogeny and evolution of plant macrophage migration inhibitory factor/D-dopachrome tautomerase-like proteins.BMC Evol. Biol. 2015; 15: 64Crossref PubMed Scopus (25) Google Scholar). Proteomic studies further reported MDL1 and MDL2 as being stromal chloroplast proteins (25Zybailov B. Rutschow H. Friso G. Rudella A. Emanuelsson O. Sun Q. van Wijk Klaas J. Sorting signals, N-terminal modifications and abundance of the chloroplast proteome.PLoS One. 2008; 3: e1994Crossref PubMed Scopus (517) Google Scholar, 26Olinares Paul Dominic B. Ponnala L. van Wijk Klaas J. Megadalton complexes in the chloroplast stroma of Arabidopsis thaliana characterized by size exclusion chromatography, mass spectrometry, and hierarchical clustering.Mol. Cell. Proteomics. 2010; 9: 1594-1615Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) and MDL3 as being a peroxisomal protein (27Reumann S. Babujee L. Ma C. Wienkoop S. Siemsen T. Antonicelli G.E. Rasche N. Lüder F. Weckwerth W. Jahn O. Proteome analysis of Arabidopsis leaf peroxisomes reveals novel targeting peptides, metabolic pathways, and defense mechanisms.Plant Cell. 2007; 19: 3170-3193Crossref PubMed Scopus (268) Google Scholar, 28Pan R. Reumann S. Lisik P. Tietz S. Olsen L.J. Hu J. Proteome analysis of peroxisomes from dark-treated senescent Arabidopsis leaves.J. Integr. Plant Biol. 2018; 60: 1028-1050Crossref PubMed Scopus (24) Google Scholar). Accordingly, the Arabidopsis Cell electronic Fluorescent Pictograph (eFP) Browser at BAR ePlant (https://bar.utoronto.ca/eplant/) presents MDL1 and MDL2 preferably localizing to chloroplasts and MDL3 to peroxisomes (Fig. S2). To explore the subcellular localization of the MDLs experimentally, we generated N- and C-terminally mCherry-tagged variants and expressed these transiently in Arabidopsis leaf mesophyll protoplasts under the control of the constitutive cauliflower mosaic virus 35S promoter. We recorded mCherry fluorescence and chlorophyll autofluorescence in transformed protoplasts by confocal laser scanning microscopy (CLSM). This revealed mainly cytoplasmic and frequently nuclear localization of MDL1 (both MDL1.1 and MDL1.2) and MDL2 (both MDL2.1 and MDL2.2), irrespective of the site of tagging (N- or C-terminal). We further noted a punctate mCherry fluorescence pattern for the N-terminally tagged MDL3 fusion protein and nucleocytoplasmic localization for the C-terminally tagged MDL3 variant. No mCherry-derived fluorescence was observed in chloroplasts, which could be unequivocally identified by their characteristic chlorophyll-mediated autofluorescence, for any of the MDL fusion proteins (Fig. 1A). Colocalization of mCherry-MDL3 with a canonical peroxisomal marker (GFP-PTS1; (29Jedd G. Chua N.H. Visualization of peroxisomes in living plant cells reveals acto-myosin-dependent cytoplasmic streaming and peroxisome budding.Plant Cell Physiol. 2002; 43: 384-392Crossref PubMed Scopus (122) Google Scholar)) upon transient expression in leaf mesophyll protoplasts derived from a transgenic GFP-PTS1 marker line supports residence of the MDL3 fusion protein in peroxisomes (Fig. 1B). We next generated transgenic lines stably expressing N-terminally mCherry-tagged MDL variants (MDL1.1, MDL2.2, and MDL3) under the control of the viral 35S promoter in the background of Arabidopsis accession Col-0. CSLM analysis of rosette leaves of these lines revealed a similar pattern of subcellular MDL localization as in the leaf mesophyll protoplasts, with the mCherry-MDL1.1 and mCherry-MDL2.2 fusions localizing to the cytoplasm and partly the nucleus and mCherry-MDL3 locating to mobile punctate structures, likely representing peroxisomes. Again, we did not detect any of the fluorophore-tagged MDLs in chloroplasts (as identified by their autofluorescence; Fig. 1C). CLSM-based imaging of root tips of the same transgenic lines in combination with labeling of nuclei via the fluorescent DAPI (4′,6-diamidino-2-phenylindole) dye further corroborated nucleo-/cytoplasmic localization of mCherry-tagged MDL1.1 and MDL2.2 and likely peroxisomal localization of MDL3 (Fig. 1D). To identify in vivo protein interaction partners of MDL proteins, we first performed a yeast two-hybrid (Y2H) screen using the three MDLs as bait proteins. For this and all following protein interaction experiments, we limited our analysis to the prevalent MDL1.1 and MDL2.1 splice variants (Fig. S1E), referred to as MDL1 and MDL2 in the following. The screen was performed in an automated manner on a one-by-one basis against a prey library (full-length ORFeome) of Arabidopsis comprising 12,000 full-length cDNAs (30Weßling R. Epple P. Altmann S. He Y. Yang L. Henz S.R. McDonald N. Wiley K. Bader K.C. Gläßer C. Mukhtar M.S. Haigis S. Ghamsari L. Stephens A.E. Ecker J.R. et al.Convergent targeting of a common host protein-network by pathogen effectors from three kingdoms of life.Cell Host Microbe. 2014; 16: 364-375Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). This yeast-based approach yielded MDL1 and MDL2 (preys) as interaction partners of MDL1 (bait), MDL1 (prey) as an interaction partner of MDL2 (bait), and PEX5 (AT5G56290), a protein of unknown function (AT5G64160), as well as a cysteine/histidine-rich C1 domain family protein (AT2G42060) (preys) as interaction partners of MDL3 (bait). We subsequently validated these candidate interactions identified in the high-throughput screen by targeted one-on-one Y2H analysis (Fig. 2A). In summary, these data suggest that MDL1 has the capacity to form homo-oligomers, whereas MDL1 and MDL2 might also form hetero-oligomers. Consistent with its peroxisomal localization and the presence of a C-terminal PTS1 targeting signal, MDL3 interacted with the PTS1 receptor PEX5. Its second interacting protein, AT5G64160, lacks any recognizable protein domains but was already found previously as an interactor of MDL3 (31Arabidopsis Interactome Mapping ConsortiumEvidence for network evolution in an Arabidopsis interactome map.Science. 2011; 333: 601-607Crossref PubMed Google Scholar). In the following, we focused on the putative homo- and hetero-oligomerization of the MDLs by exploring all possible pairwise interactions between the three proteins using three different in planta experimental approaches. As a first approach, we used the split luciferase assay, which is based on the complementation of N- and C-terminal luciferase fragments translationally fused to the proteins of interest. Successful establishment of luciferase activity by fragment complementation can be assessed quantitatively by luminometric measurements. We transiently coexpressed the three MDLs N-terminally tagged with cLuc (C-terminal luciferase fragment) and nLuc (N-terminal luciferase fragment) in Nicotiana benthamiana and scored the resulting luminescence. This revealed noticeable luciferase activities upon coexpression of cLuc-MDL1 in combination with nLuc-MDL1 and nLuc-MDL2, cLuc-MDL2 in combination with nLuc-MDL1 and nLuc-MDL2, as well as cLuc-MDL3 in combination with nLuc-MDL2 and nLuc-MDL3. Accordingly, the respective pooled luminescence intensity values of multiple experimental replicates differed in a statistically significant manner from empty vector controls, which was not the case for the other tested pairwise combinations (Fig. 2, B and C). As a second approach, we transiently coexpressed epitope-tagged (FLAG and mCherry) versions of various combinations of the three MDLs in N. benthamiana and performed co-immunoprecipitation (co-IP) assays using epitope-directed antibodies. This revealed pull-down of mCherry-MDL1 together with FLAG-MDL1 and FLAG-MDL2, of mCherry-MDL2 together with FLAG-MDL1, as well as of mCherry-MDL3 together with FLAG-MDL3 (Fig. 2D). Finally, as a third approach, we took advantage of the transgenic Arabidopsis Col-0 lines constitutively expressing one of the three mCherry-tagged MDLs (Fig. 1C) and a monoclonal antibody directed against MDL2 (designated ATM 20C8; Fig. S3A) to perform co-IP experiments under seminative conditions in Arabidopsis leaf extract. This procedure yielded pull-down of mCherry-MDL1 together with MDL2 as well as mCherry-MDL2 (here MDL2.2) together with MDL2 (Fig. 2E). In summary, data of four independent protein interaction assays (Y2H, split luciferase, co-IP in N. benthamiana, and co-IP in Arabidopsis) in essence consistently indicate MDL1-MDL1, MDL1-MDL2, and MDL2-MDL2 interactions (Table 1). Moreover, results from split luciferase and co-IP experiments in N. benthamiana further suggest MDL3-MDL3 interactions. These data are thus indicative of the in planta formation of MDL1, MDL2, and MDL3 homo-oligomers as well as the formation of MDL1-MDL2 hetero-oligomers.Table 1Summary of MDL protein–protein interaction dataaY2H, yeast two-hybrid assay; co-IP (Nb), co-immunoprecipitation in N. benthamiana; split luc, split luciferase assay; co-IP (At), co-immunoprecipitation in A. thaliana. Protein 1 refers to the MDL bait protein in Y2H analysis, the mCherry-tagged MDL