Expanded View Figures

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1 Expanded View Figures m/z , 2+ y6 y5 y4 y3 y2 y1 H I T G I E R b1 b2 b3 b5 b6 Figure EV1. MS/MS spectra of peptides mapped to MKKK7., LTQ-Orbitrap MS/MS spectra of MKKK7 peptides identified in FLS2-GFP-co-immunoprecipitated samples. Peptide sequence and fragmentation pattern are shown above the spectra together with the observed m/z and charge state of the precursor ion. m/z , 2+ y9 y8 y7 y6 y5 y4 y3 y2 y1 V D L L L E F R b2 b3 b4 b5 b6 b7 b8 b9 ª 2016 The uthors EMO reports EV1

2 EMO reports MKKK7 negatively regulates FLS2 signaling Sharon C Mithoe et al Kinase Domain S337 S452 S503 RM/HET domain S775 S854 Figure EV2. MKKK7 domain structure and phosphorylated residues. Protein structure of MKKK7 with the protein kinase domain shown in yellow and an RM/HET repeat domain shown in blue. The position of the phosphorylated serine residues (S) is shown with triangles. The green triangles indicate non-differentially phosphorylated sites. The red triangles indicate phosphorylated serine (ps) sites that were targeted for mutagenesis. Protein sequence of MKKK7, highlighted in yellow are all (phospho-) peptides measured by mass spectrometry. Highlighted in green are modified residues, and the red box around the S residues indicates phosphorylated serine residues that were targeted for mutagenesis. EV2 EMO reports ª 2016 The uthors

3 tmkkk6 NP_ SEKDE-GSQEVVESVSEKVEVTKTNS-----KSKLPVIGGSFRSEKDQSSPSDLGEE 354 nm3ke1 XP_ SEKDE-GSEEVTESLSEEKGMSKSDS-----KSKL---GVSFRSEKDPSSSSDLGEE 351 CsM3KE XP_ SEKDDE-GNQDVESLSEKVGMSETDS-----KSKLPLVGVSFRSEKDQPTPSDLGED 354 tmkkk7 NP_ SEKDDE-GSQDESLSGENVGISKTDS-----KSKLPLVGVSSFRSEKDQSTPSDLGEE 354 lmkkk7 XP_ SEKDDE-GSQDESLSENVGMSKSDS-----KSKLPLLGVSSFRSEKDQSTPSDLGEE 350 SlM3KE NP_ IRESNEEDKGGSSSSDKKESSTTL SPEVLETSKSEEVDGSSIRIEG 352 NbM3KE DK36643 DTDSNEDDKGGSSSSDKKESCSVL SPEVSEISKSEEFDGSTSNHLEG 352 MdM3KE XP_ GEISNGDNQGSESPSEKVEVSTIKDSGKELLSTEVPDMGRSDDNPSDVKSVEE 357 PtM3KE XP_ EEILTGDNQRTVQINSVDRTKSVDFKGSRKESLP-DSEDVSKSDKNTSSDGDVVEE 358..:. * :.. : : :*:. : * tmkkk6 NP_ LETESEGRRNTLTKLVGKE-YSIQSS---HSFSQKGE-DGLRKVKTPSSFGGNELTR 466 nm3ke1 XP_ SETESSKNGKNT-LEKQVGKE-SSIHVDQPSHSVGQKGEDRRLRKVRTPSSVGGNELTR 462 CsM3KE XP_ LETESERKN----KQVGKE-CSIQVDQTSHSSGLKGEDRGIRKVKTPSSLGGNELR 464 tmkkk7 NP_ LVTETSERKNTSIKHVGKE-LSIPVDQTSHSFGRKGEERGIRKVKTPSSVSGNELR 461 lmkkk7 XP_ LETETSERKNTSKKQVGKE-LSIPVDQTSHSFGQKGEERGIRKVKTPSSVSGNELR 457 SlM3KE NP_ GELESSESRGRNTVGRKVEDKGHGVNYSSSSSGQKNTDYSPRKVKTSVVPQGNELSR 467 NbM3KE DK36643 GELESSESKGGNNVGKKEEEKRGINYSSSSSGQKNPDHSPRKMKISVVPRGNELSR 467 MdM3KE XP_ GEVRSPELTTKNVSGKQGGK-----GVGYRFGFGTRNQDGSFQKKMPVLLGGNELSK 471 PtM3KE XP_ DDLESPDRGKNIERRNGGKTSS-RVENGSFGFTRNQDNGLRKVKTSMTSGGNELSK 476.:. :... : :** : ****:: tmkkk6 NP_ FSDPPGDSLHDLFHPLDKVPEGKTNESTSTPTNVNQGDSPVDGGKNDLTKLRRI 526 nm3ke1 XP_ FSDPPGDSLHDLFQPLDKVPEGKPNESTSPTSNVIQGDSPVDGGKNDLTKLRTI 522 CsM3KE XP_ FSDPPGDSLHDLFHPLDKVPEGKPNESTSMPTSNINQGDSPVDGGKNDLTKLRTI 524 tmkkk7 NP_ FSDPPGDSLHDLFHPLDKVSEGKPNESTSMPTSNVNQGDSPVDGGKNDLTKLRTI 521 lmkkk7 XP_ FSDPPGDSLHDLFHPLDKVSEGKPNESTSMPTSNVNQGDSPVDGGKNDLTKLRTI 517 SlM3KE NP_ FSDPPGDSLDDLFHPLEKNLENREVSLSSSSQIQNNI-ETGKNDLTKLRTI 526 NbM3KE DK36643 FSDPPGDSLDDLFHPLEKNLENREVSLSSSSSQIQSNV-SETGKNDLTKLRTI 526 MdM3KE XP_ FSDTPGDSLDDLFHPLDKHPEDRTESTSSMSQSNQGNTPGNDGKSDLTKLRTI 531 PtM3KE XP_ FSDTPRDSLDDLFHPLDKNPEDRESTSTSSHMNQGNIMDGKNDLILRTI 536 *** * ****.***:**:* * : *.* * :. * :: : **.***: *** * tmkkk6 NP_ NTLYSLSETRLSISG-DLILDGQTPRRSGQLDPNNPIFSQRET-SPSVIDHPDGLK 823 nm3ke1 XP_ NTLYSLNETRLSISG-GPLSVDGLPRLRSGQLDPNNPIFSHHES-SLGVIDHPDLK 819 CsM3KE XP_ NTLYSLNETRLSISG-GLIVDGQPRVRSGQLDPNNPIFTQHET-SLSMIDQPDVLK 821 tmkkk7 NP_ NTLYSLNETRLSISG-G---LDGQPRVRSGQLDPNNPIFGQNETSSLSMIDQPDVLK 816 lmkkk7 XP_ NTLYSLNETRLSISG-G-IVDGQPRRSGQLDPNNPIFGQNET-SLSMIDQPDVLK 813 SlM3KE NP_ NTLYSLNERLSSGGGGFPPDGLPRPRSGPLDHGNSSFMQTEV-PPYGTDQPDMLK 821 NbM3KE DK36643 NTLYSLNERLSSGGSGFPPDGLPRPRSGPLDPGNSSFMQTEM-PPYGTDQPDMLK 821 MdM3KE XP_ NTLYSLNETRLSISVGGGFPLEGSQRPRSGSLDSGHPIFQSDV-LLSTTDQHDLSK 828 PtM3KE XP_ NTLYSLNETRLSISVGTGFPLDGLSQRPRSGPLDSNHPIFIQSET-LSSDQPDVFK 832 ******.**:**** * :* : * *** **. * : : *: * * tmkkk6 NP_ TRNG GGEEPSHLTSNSQSSDVHQPD--LHPDGDRPRLSSVVD nm3ke1 XP_ TKHV GGEEPSHSTSNSQRSDIHQP DGDRPRLSSDGS CsM3KE XP_ TRHG GGEEPSHSTSNSQRSDVHQPD--LHPDGDRPRLSSVTPDSTS tmkkk7 NP_ TRHG GGEEPSHSTSNSQRSDVHQPD--LHPDGDKPRVSSVPDSTS lmkkk7 XP_ TRHG VGEEPSHSTSNSQRSDVHQPD--LHPDGDRPRVSSVPDSTS SlM3KE NP_ IKNGD--RVLPSGMQEPSRNSSHS PDSPFFRQDGERPRSSNTMESGLSRL 872 NbM3KE DK36643 IKNGE--RVLPGMQELSRTSSHS PDSPYFRQDFERPRSSNTVEVSGPSKL 872 MdM3KE XP_ VRHGLIDFHLSTGTEPRSTSNSQRSDNQSDPRYLHLDTDRQSSNVVVEIVPSKL 888 PtM3KE XP_ VRHGMIDHSLPFGTLEPSRSTSHSQRLDIQPDRFFGTDTDGSQSNETIEISKL 892 :. * :: :*.* * : : *.. : Figure EV3. Multiple sequence alignment of MKKK7 and related MP3K. mino acid sequences for rabidopsis thaliana MKKK6 (tmkkk6) and MKKK7 (tmkkk7), rabidopsis lyrata MKKK7 (lmkkk7), rassica napus MP3K epsilon protein kinase 1 (nm3ke1), Camelina sativa MP3K epsilon protein kinase (CsM3KE), Solanum lycopersicum MP3K epsilon protein kinase (SlM3KE), Nicotiana benthamiana MP3K epsilon protein kinase (NbM3KE), Malus domestica MP3K epsilon protein kinase (MdM3KE), and Populus trichocarpa MP3K epsilon protein kinase (PtM3KE) were aligned with Clustal Omega ( Only alignments of amino acid sequences surrounding the phosphorylated residues in MKKK7 are shown. Residues phosphorylated in tmkkk7 are indicated with a red arrow and highlighted in red, and conserved residues in other MP3Ks are highlighted in green. Protein names and Genank accession numbers are indicated on the left side of the alignments, while to the right the number of the last amino acid in the alignment is indicated. ª 2016 The uthors EMO reports EV3

4 EMO reports MKKK7 negatively regulates FLS2 signaling Sharon C Mithoe et al Figure EV4. EV4 EMO reports ª 2016 The uthors

5 Figure EV4. MP kinase activation loop phosphorylation. Selected reaction monitoring (SRM) of MPK6 activation loop phosphorylation in response to flg22 stimulation in cultured cells. SRM of MPK3 activation loop phosphorylation in response to flg22 stimulation in cultured cells. Monophosphorylated and doubly phosphorylated versions of the tryptic MPK activation loop peptides were monitored at 0, 5, 10, 20, and 30 min after stimulation with 1 lm flg22. Data information: Sequences are shown on the left, with lower case p indicating phosphorylation of the residue to the right. Left panels show integrated peak area data for three biological replicates (,, and C). Middle panels show examples of transitions measured for endogenous 15 N-labeled peptides. Right panels show examples of total integrated peak area for endogenous 15 N-labeled peptides and 14 N-labeled synthetic peptides, which were spiked into the samples at a constant amount. Fold induction FRK1 Figure EV5. Phosphorylation of MKKK7 on specific serine residues is required for negative regulation of flg22-induced FRK1 gene expression. Transient co-expression of MKKK7 in rabidopsis mesophyll protoplasts shows the suppression of FRK1 gene expression in protoplasts after flg22 treatment. Protoplasts were isolated from 4-week-old plants and transfected with pfrk1: fluc, 35S:rLUC, and overexpression constructs of MKKK7 (OE-MKKK7, OE-MKKK7, or OE-MKKK7 DD ) as indicated on the horizontal axis. Sixteen hours later, protoplasts were treated with 10 lm flg22 for 4 h. ll measurements were normalized to the rluc activity, and expression levels were calculated relative to the mock-treated control sample as shown as fold induction represented on the vertical axis flg22 Col-0 OE-MKKK7 OE-MKKK7 OE-MKKK7 DD Col-0 mkkk7/35s:mkkk7-gfp mkkk7 mkkk7/35s:mkkk7-gfp Figure EV6. MKKK7 negatively regulates basal resistance to virulent bacterial infection. Example of symptom development at 2 dpi in the p35s:mkkk7-gfp in mkkk7 background as compared to Col-0 (left panel). Example of symptom development at 2 dpi in mkkk7 and p35s:mkkk7-gfp in mkkk7 background (right panel). Overexpression of MKKK7 DD reduces resistance to Pst infection. Quantification of bacterial growth in Col-0 and two independent imkkk7 DD lines. Four- to five-week-old plants were pressure-infiltrated with virulent Pst DC3000, and at 2 dpi, leaf disks were harvested and bacteria re-isolated. The number of colony-forming units (cfu/cm 2 ) was determined at t = 2 days post-inoculation (dpi). Data represent mean values SEM (n = 6; *P < 0.05; paired t-test). Pst reisolation at 2 dpi 1.00E+04 CFU/cm E E+02 Col-0 imkkk7dd L1 imkkk7dd L3 1.00E E+00 ª 2016 The uthors EMO reports EV5