DIP Database of Interacting Proteins 611 Young Drive East; Los Angeles CA 90095; USA http://dip.doe-mbi.ucla.edu/ dip@mbi.ucla.edu Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2009 imex curation Bethke G., Unthan T., Uhrig JF., Poschl Y., Gust AA., Scheel D., Lee J. The authors or references in the text do not supply enough information to unambiguously identify the myelin basic protein used in this study. Arabidopsis mesophyll protoplasts were prepared as described in Asai et al., 2002. jlee@ipb-halle.de, dscheel@ipb-halle.de orchard Only protein-protein interactions arath-mesophyll Arabidopsis thaliana mesophyll mesophyll Mesophyll of plant leaf The photosynthetic parenchyma between the epidermal layers of a foliage leaf. fret fluorescent resonance energy transfer FRET RET FRET analysis FRET RET FRET analysis fret tag fluorescence tag visualisation by fluorescence tag fluorescence Bethke G., Unthan T., Uhrig JF., Poschl Y., Gust AA., Scheel D., Lee J. The authors or references in the text do not supply enough information to unambiguously identify the myelin basic protein used in this study. Arabidopsis mesophyll protoplasts were prepared as described in Asai et al., 2002. jlee@ipb-halle.de, dscheel@ipb-halle.de Accepted 2009-SEP-10 by ORCHARD. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2009 orchard IMEx Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2009 imex curation Bethke G., Unthan T., Uhrig JF., Poschl Y., Gust AA., Scheel D., Lee J. The authors or references in the text do not supply enough information to unambiguously identify the myelin basic protein used in this study. Arabidopsis mesophyll protoplasts were prepared as described in Asai et al., 2002. jlee@ipb-halle.de, dscheel@ipb-halle.de orchard Only protein-protein interactions in vitro In vitro protein kinase assay protein kinase assay autoradiography autoradiography Bethke G., Unthan T., Uhrig JF., Poschl Y., Gust AA., Scheel D., Lee J. The authors or references in the text do not supply enough information to unambiguously identify the myelin basic protein used in this study. Arabidopsis mesophyll protoplasts were prepared as described in Asai et al., 2002. jlee@ipb-halle.de, dscheel@ipb-halle.de Accepted 2009-SEP-10 by ORCHARD. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2009 orchard IMEx Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2009 imex curation Bethke G., Unthan T., Uhrig JF., Poschl Y., Gust AA., Scheel D., Lee J. The authors or references in the text do not supply enough information to unambiguously identify the myelin basic protein used in this study. Arabidopsis mesophyll protoplasts were prepared as described in Asai et al., 2002. jlee@ipb-halle.de, dscheel@ipb-halle.de orchard Only protein-protein interactions arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress ch-ip chromatin immunoprecipitation assay ch-ip primer specific pcr primer specific pcr Bethke G., Unthan T., Uhrig JF., Poschl Y., Gust AA., Scheel D., Lee J. The authors or references in the text do not supply enough information to unambiguously identify the myelin basic protein used in this study. Arabidopsis mesophyll protoplasts were prepared as described in Asai et al., 2002. jlee@ipb-halle.de, dscheel@ipb-halle.de Accepted 2009-SEP-10 by ORCHARD. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2009 orchard IMEx Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Flg22 regulates the release of an ethylene response factor substrate from MAP kinase 6 in Arabidopsis thaliana via ethylene signaling. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2009 imex curation Bethke G., Unthan T., Uhrig JF., Poschl Y., Gust AA., Scheel D., Lee J. The authors or references in the text do not supply enough information to unambiguously identify the myelin basic protein used in this study. Arabidopsis mesophyll protoplasts were prepared as described in Asai et al., 2002. jlee@ipb-halle.de, dscheel@ipb-halle.de orchard Only protein-protein interactions arath-mesophyll Arabidopsis thaliana mesophyll mesophyll Mesophyll of plant leaf The photosynthetic parenchyma between the epidermal layers of a foliage leaf. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Bethke G., Unthan T., Uhrig JF., Poschl Y., Gust AA., Scheel D., Lee J. The authors or references in the text do not supply enough information to unambiguously identify the myelin basic protein used in this study. Arabidopsis mesophyll protoplasts were prepared as described in Asai et al., 2002. jlee@ipb-halle.de, dscheel@ipb-halle.de Accepted 2009-SEP-10 by ORCHARD. Proc. Natl. Acad. Sci. U.S.A. (0027-8424) 2009 orchard IMEx ef104_arath Ethylene-responsive transcription factor ERF104 ERF104 K11J9.13 At5g61600 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MATKQEALAIDFISQHLLTDFVSMETDHPSLFTNQLHNFHSETGPRTITNQSPKPNSTLNQRKPPLPNLSVSRTVSTKTEKEEEERHYRGVRRRPWGKYAAEIRDPNKKGCRIWLGTYDTAVEAGRAYDQAAFQLRGRKAILNFPLDVRVTSETCSGEGVIGLGKRKRDKGSPPEEEKAARVKVEEEESNTSETTEAEVEPVVPLTPSSWMGFWDVGAGDGIFSIPPLSPTSPNFSVISVT 086C0930A63339DC mpk6_arath Mitogen-activated protein kinase 6 MPK6 At2g43790 F18O19.10 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MDGGSGQPAADTEMTEAPGGFPAAAPSPQMPGIENIPATLSHGGRFIQYNIFGNIFEVTAKYKPPIMPIGKGAYGIVCSAMNSETNESVAIKKIANAFDNKIDAKRTLREIKLLRHMDHENIVAIRDIIPPPLRNAFNDVYIAYELMDTDLHQIIRSNQALSEEHCQYFLYQILRGLKYIHSANVLHRDLKPSNLLLNANCDLKICDFGLARVTSESDFMTEYVVTRWYRAPELLLNSSDYTAAIDVWSVGCIFMELMDRKPLFPGRDHVHQLRLLMELIGTPSEEELEFLNENAKRYIRQLPPYPRQSITDKFPTVHPLAIDLIEKMLTFDPRRRITVLDALAHPYLNSLHDISDEPECTIPFNFDFENHALSEEQMKELIYREALAFNPEYQQ 296D2BD753C6DDA4 pdf1_2_arath_gene PDF1.2/ Defensin-like protein 16 Arabidopsis Gene gene gene arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress mpk6-erf104-3 1 5 unspecified role unspecified role neutral component neutral component region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role neutral component neutral component region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction Figure 1 flg22 peptide (QRLSTGSRINSAKDDAAGLQIA) Kinase inactive MPK6 ( Lys92Arg or Ser229Ala , Ser232Ala) interacted with ERF104 independently of flg22 treatment. Treatment with aminoethoxyvinylglycine or silver ions removed flg22 inhibition of the interaction. The authors concluded that inhibition of interaction between MPK6 and ERF104 by flg22 required ethylene bio-synthesis and signalling. In vivo protein-protein interaction based on FRET analysis mpk6-erf104-1 2 5 enzyme target enzyme target substrate substrate neutral component neutral component region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Q9FKG1:p.[Ser229Ala;Ser232Ala] Alters kinetics of interaction mutation mutation certain certain sequence position certain certain certain sequence position certain S A certain certain sequence position certain certain certain sequence position certain S A Fri Jul 22 11:07:06 BST 2016 This feature has been corrected as a result of our quality control procedures. The original label was 'ser232ala,ser229ala' Wed May 02 17:17:40 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser229ala,ser232ala' Wed May 02 18:08:47 BST 2018 This feature has been corrected as a result of our quality control procedures. The original label was 'ser229ala,ser232ala' Tue Jun 30 18:21:58 BST 2020 This feature has been corrected as a result of our quality control procedures. The original label was 'p.[Ser229Ala;Ser232Ala]' region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain ecoli Escherichia coli (strain K12) 6 enzyme enzyme neutral component neutral component arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress phosphorylation phosphorylation reaction phosphorylation MPK6 was immunoprecipitated from flg22 peptide (QRLSTGSRINSAKDDAAGLQIA) treated Arabidopsis plants. Figure 3C Figure 3E Phosphorylation of ERF104 (Q9FKG1) MPK6 phosphorylates ERF104. mpk6-erf104-2 4 5 unspecified role unspecified role bait bait region ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 6 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association SFigure 1D Transfected protoplasts were immunoprecipitated with anti-HA and probed with anti-myc. It should be noted that ERF104 is highly recalcitrant to extraction with standard buffers (without detergents) and that, here, high salt of 500mMNaCl in the extraction buffer (without SDS) is required to extract the nuclear protein, ERF104. Consequently, Co-IP detects only weak signals for the interacting MPK6 but could be reproduced in two independent experiments. More importantly, the ��weak�� MPK6 signal is lost after 15 minutes of flg22 treatment, suggesting complex disruption after elicitation. flg22 erf104-pdf1 3 5 unspecified role unspecified role bait bait region Strep tag strep tag strep tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role prey prey promoter_region binding region binding-associated region binding region undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined association association Figure 5A Chromatin immunoprecipitation (ChIP) shows higher levels of the PDF1.2 promoter in immunoprecipitates of Strep-tagged ERF104 from ERF104OE compared with Col-0 plants.