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1 Supplemental material Table 1- Segregation analysis of sgt1a sgt1b double mutant plants Parental genotype No. of lines Normal seeds Phenotype Abnormal seeds Ratio of abnormal seeds (%) WT sgt1a sgt1a / SGT1b sgt1b SGT1a sgt1a / sgt1b sgt1b Table 2- Resistance phenotypes of Arabidopsis lines in response to H. parasitica isolate Noco2 Lines TN SP La-er La-er (sgt1b-3) La-er (Δrpp5) sgt1b-3 (patsgt1b::gatsgt1b) sgt1b-3 (patsgt1a::gatsgt1b) sgt1b-3 (patsgt1b::gatsgt1a) sgt1b-3 (35S::gAtSGT1a) The incidence of plant hypersensitive cell death (), trailing necrosis (TN) or pathogen sporulation in the absence of host cell necrosis (SP) was scored in at least 40 leaves per line in three independent experiments and shown here as +, ++ or +++ to represent their frequency. -, not detected. Supplemental Material and Methods Plasmid construction PCR amplified SGT1 fragments were cloned into pgemt (Promega), then sub-cloned into ppam_mcs for agro-infiltration (Witte et al., 2004) and plexa and/or pb42ad (Clontech) for yeast two hybrid analysis. The AtSGT1a (T91A+T100A), AtSGT1b (A91T+A100T) mutations were introduced using site-directed mutagenesis with QuickChange kit (Stratagene). The different chimeric constructs were generated by PCR. To generate AtSGT1a/AtSGT1b promoter-swaps, the coding regions and the 1.3kb promoter regions of AtSGT1a and AtSGT1b were amplified from Col-0 genomic DNA. The sequences of primers used for these constructs are available upon request. The patsgt1b::gatsgt1b, patsgt1a::gatsgt1b, p35s::gatsgt1a and
2 patsgt1b::gatsgt1a were constructed in pentr/d-topo vector (Invitrogen, Carlsbad, CA) by PCR. Those swap constructs were then transferred into a Gateway based ppam-pat vector (sequence available upon request). In order to generate AtSGT1a and AtSGT1b promoter-gus fusion constructs, 1.3kb upstream promoter regions of AtSGT1a and AtSGT1b were amplified and cloned into pentr/d-topo vector and transfer to pjawohl11-gw-gus vector (unpublished, sequence available upon request). Landsberg erecta (Ler) sgt1b-1 or sgt1b-3 null mutant plants (Austin et al., 2002) were transformed by the Agrobacterium-mediated floral-dipping method (Clough and Bent, 1998). Agrobacterium-mediated transient expression (agro-infiltration) in N. benthamiana. All AtSGT1a and AtSGT1b derivatives in ppam_mcs (Rademacher et al., 2002) were transformed into Agrobacterium GV3101 (pmp90rk). The AtSGT1a gene was also cloned in pbin19 vector and transformed into Agrobacterium C58C1. Agro-infiltration to transiently express genes in N. benthamiana was done as described by Peart et al. (2002). Briefly, 20 days after Agrobacterium-mediated TRV infection, the upper leaves of the plants were co-infiltrated with Agrobacterium carrying test constructs and pathogen elicitor producing plasmids (PVX:GFP, TMV:GFP or PVX-Tk) as follows: the left side of the leaf was infiltrated with an Agrobacterium mixture carrying the empty vector control (ppam_mcs) and Agrobacterium carrying the elicitor producing plasmid; the right side of the same leaf was infiltrated with an Agrobacterium mixture carrying the test constructs and the elicitor producing plasmid. Concentrations of Agrobacterium were OD 600nm =0.25 for the PVX coat protein (Tk), a 1000x dilution from OD 600nm =1 for PVX:GFP and a 40X dilution from an OD 600nm =1 for TMV:GFP. The Agrobacterium solution was infiltrated into leaves using a syringe as described previously. Virus accumulation was monitored 5 days later for PVX:GFP, and 7 days for TMV:GFP under UV or white light. was assessed under white light 5-7 days after infiltration. Each experiment was repeated at least four times with at least three independent plants each time. References Austin, M.J., Muskett, P., Kahn, K., Feys, B.J., Jones, J.D. and Parker, J.E. (2002) Regulatory Role of SGT1 in Early R Gene-Mediated Plant Defenses. Science, 295, Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J, 16, Rademacher, T., Hausler, R.E., Hirsch, H.J., Zhang, L., Lipka, V., Weier, D., Kreuzaler, F. and Peterhansel, C. (2002) An engineered phosphoenolpyruvate carboxylase redirects carbon and nitrogen flow in transgenic potato plants. Plant J, 32, Witte, C.P., Noel, L.D., Gielbert, J., Parker, J.E. and Romeis, T. (2004) Rapid one-step protein purification from plant material using the eight-amino acid StrepII epitope. Plant Mol Biol, 55,
3 Supplemental Figure legend Figure 1. Quantitation of AtSGT1a and AtSGT1b protein levels in Arabidopsis sgt1b-3 stable transgenic lines (A) Top panel: Western blot analysis of AtSGT1a in total leaf extracts of the Arabidopsis lines shown in Figure 4A that either fully (line ), partially (line ) or weakly (line ) complement resistance defects of sgt1b-3 (see Supplemental Table 2). s of extracts (8 µl) were made as indicated, separated by SDS-PAGE, transferred to a membrane and probed with anti-sgsa antibody. Extracts from La-er, sgt1a-1 and sgt1b-3 served as controls and loading was monitored by Ponceau S staining of Rubisco on the blot. SGT1 signals were detected with the ECL system (Pierce) on film. An exposure period is shown in which signal detection is linear, as monitored by ImageJ analysis (Software version 1.34s; Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA; Middle panel: Quantitation of signals on the Western blot by ImageJ. The software calculates the area and pixel value statistics of defined selections on the image. First, an area was selected that covered the strongest signal (TIFF format, 8-bit, Grayscale). Using this area each signal of interest as well as a background signal was selected and measured for its area statistics. The mean background value was subtracted from the mean sample value to give the relative expression level shown. Bottom panel: Linear regression analysis for ImageJ-derived values obtained from different dilutions of extracts of lines, and shows that the relative Western blot signal intensities calculated by ImageJ are linear. (B) Western blot analysis of SGT1b protein leaf extracts of sgt1b-3 transgenic lines and that fully complement and line 2.3 that partially complements the sgt1b-3 resistance defect (Supplemental Table 2). Data shown in the top, middle and bottom panels were generated as described for SGT1a protein in (A). Figure 2. Differential affinity of α-sgsa against AtSGT1a and AtSGT1b. AtSGT1a-SII and AtSGT1b-SII were expressed in N. benthamiana and purified with α-sii antibody. AtSGT1b-SII was loaded on lane 1. Two and four volumes of AtSGT1a-SII were loaded on lane 2 and 3, respectively. Proteins were visualised by silver staining, or probing with α-sii, or α-sgsa antibodies, as indicated. Filled and open arrows indicate AtSGT1a-SII and AtSGT1b-SII, respectively. Figure 3. Sequence comparison of SGT1 proteins from different organisms. Boxed in red are the three threonines in AtSGT1a that are alanines in all other plant SGT1 proteins. The red arrow indicates the region in ScSGT1 that is able to bind to SKP1 in yeast. The red line with an asterisk indicates the swap positions for chimeras AtSGT1b/a/a and AtSGT1a/b/b. Figure 4. The TPR domain is dispensable for disease resistance and SCF TIR1 -mediated auxin response in Arabidopsis. (A) Western blot analysis using α-sgsa antibody of three independent homozygous sgt1-b transgenic lines expressing TPRb or TPRa constructs. (B) RPP5 resistance assay. Lactophenol trypan blue staining of H. parasitica (isolate Noco2) infected Arabidopsis leaves 7 days after inoculation. Transgenic plants
4 expressing TPRb (lines 5936, 5935, 5941) or TPRa (7014, 7017) contain the sgt1b-1 allele. (C) Root growth assay with three independent TPRb (sgt1b-1) segregating lines. Plants in lanes 7, 10, 13, 16, 19, 22 do not contain the transgenes. Seedlings were grown vertically on MS media for 5 days and then transferred onto the MS containing 0.1µM 2,4-D and grown for an additional three days. Arrow indicates the position of the root at the time after transfer.
5 A AtSGT1a abundance B AtSGT1b abundance 8 µl 8 µl sgt1a-1 La-er sgt1b-3 1/4 1/16 1/64 La-er sgt1a-1 sgt1b /2 1/4 α-sgs AtSGT1b AtSGT1a α-sgs AtSGT1b AtSGT1a Rubisco Rubisco Quantitation of AtSGT1a signal by ImageJ sgt1b-3 1/4 1/16 1/64 8 µl Quantitation of AtSGT1b signal by ImageJ /2 1/4 8 µl Linear regression analysis of AtSGT1a signal detection by ImageJ Line Linear regression analysis of AtSGT1b signal detection by ImageJ Line Line Line Line Supplemental Figure 1
6 silver α-sii α-sgsa Supplemental Figure 2
7 AtSGT1a : AtSGT1a : AtSGT1a : AtSGT1a : AtSGT1a : MAKELADKAKEAFVDDDFDVAVDLYSKAIDLDPNCAEFFADRAQAY--IKLESFTEAVADANKAIELDPSL MAKELAEKAKEAFLDDDFDVAVDLYSKAIDLDPNCAAFFADRAQAN--IKIDNFTEAVVDANKAIELEPTL MASDLETRAKEAFIDDHFELAVDLYTQAIAMTPKNAELFADRAQAN--IKLNYFTEAVVDANKAIELDPSM MATAAASDLESKAKAAFVDDDFELAAELYTQAIEASPATAELYADRAQAH--IKLGNYTEAVADANKAIELDPSM MAAAAASDLESKAKEAFVDDDFELAAELYTQAIEAGPATAELYADRAQAH--IKLGSYTEAVADANKAIELDPSM ----MAAAAAGTATSQRFFQSFSDALIDEDPQAALEELTKALEQKPDDAQYYCQRAYCH--ILLGNYCVAVADAKKSLELNPNN MPVEKDLKTAYKALYDEKEPLKALHLYDEILKGSPTNLTALIFKAACLEKLYFGFSDWHSDATMENAKELLDKALMTAEGRGDR TKAYL---RKGTACMKLEEYRTAKTALEKGASITPSESKFKKLIDECNFLITEEEKDLVQPVPSTLPSSVTAPPVSEL AKAYL---RKGTACMKLEEYSTAKAALEKGASVAPNEPKFKKMIDECDLRIAEEEKDLVQPMPPSLPSSSTTPLATEA SKAYL---RKGLACMKLEEYQTAKAALETGASLAPAESRFTKLIKECDERIAEEAGELPNQSVDKTSGNVVAPPASESLDNVAV HKAYL---RKGAACIRLEEYQTAKAALELGYSFASGDSRFTRLMKECDERIAEELSEVP---VKKAEDGAAAPSVASFVE---- HKAYL---RKGSACIKLEEYQTAKAALEVGSSYASGDSRFTRLMKECDDRIAEEASQAP---VKNAAAAVAPATSSGATTVVTE STAML---RKGICEYHEKNYAAALETFTEGQKLDSADANFSVWIKRCQEAQNGSESEVWTH SKIGLVNFRYFVHFFNIKDYELAQSYFKKAKNLGYVDDTLPLWEDRLETKLNKKNKKQKDSTNKHTIKPVESIENRGD * ---DVTPT-----A KYRHEYYQKPEEVVVTVFAKGIPKQNVNIDF-----GEQILSVVIEVP-GEDAYYLQPRLFG ---DAPPV-----PIPAAPAKPMFRHEFYQKPEEAVVTIFAKKVPKENVTVEF-----GEQILSVVIDVA-GEEAYHLQPRLFG APKDAQPTVNLSYQGSAA--RPKYRHEFYQKPEEVVVTIFAKGIPAKNVVVDF-----GEQILSVSIDVP-GDETYSFQPRLFG -EKDDAANMDNTPPMVEV--KPKYRHDFYNSATEVVLTIFAKGVPAENVVVDF-----GEQMLSVSIEVP-GEEPYHFQPRLFS AEDQDGENMENAQPTVEVPSKPKYRHDYYNTPTEVVLTIFAKGVPADSVVVDF-----GEQMLSVSIELP-GEEPYHFQPRLFS QSKIKYDWYQTESQVVITLMIKNVQKNDVNVEF-----SEKELSALVKLP-SGEDYNLKLELLH -NNSSHSPISPLKIETAPQESPKFKIDWYQSSTSVTISLFTVNLPESKEQVNIYISPNDRRTLSISYQVPKSGSEFQYNAKLSH KIIPDKCKYEVLSTKIEIRLAKADIITWASLEH G-KGPAVLPKPNVSSEVSQRPAYPSSKKVK-DWDKLEAE KIIPEKCRFEVLSTKVEIRLAKAEIITWASLEY G-KGQSVLPKPNVSSALSQRPVYPSSKPAK-DWDKLEAE KITPAKCRYEVMSTKIEIRLAKAEPLHWTSLEY T-RASAVVQRPNVSSD-APRPSYPSSKLRHVDWDKLEAE KIIPEKSRYQVLSTKVEIRLAKAEQITWTSLDY DKKPKAVPQKIIPPAESAQRPSYPSSKSKK-DWDKLEAE KIVPDKCKYTVLSTKVEIRLAKAEPVTWTSLDY TGKPKA-PQKINVPAESAQRPSYPSSKSKK-DWDKLEAE PIIPEQSTFKVLSTKIEIKLKKPEAVRWEKLEG QGDVPTPKQFVADV---KNLYPSSSPYTRNWDKLVGE EVDPKAVSLKIFPKKLEITLSKIDSTQWKKLEEDILTESSRLSDEGKNSDSATRLLSAETASKERLSYPSSSKKKIDWSKL--D SGS TPR VR2 VKKQEKDEKLEGDAALNKFFREIYQNADEDMRRAMSKSFVESNGTVLSTNWQEVGTKTIESTPPDGMELKKWEI VKKQEKDEKLDGDAAMNKFFSDIYSSADEDMRRAMNKSFAESNGTVLSTNWKEVGTKKVESTPPDGMELKKWEY VKKEEKDEKLDGDAALNKFFRDIYKDADEDTRRAMMKSFVESNGTVLSTNWKEVGTKKVEGSPPDGMELKKWEI VKKEEKEEKLEGDAALNKFFRDIYSDADEDMRRAMMKSFVESNGTVLSTNWKDVGSKKVEGSPPDGMELKKWEY VKKQEKDEKLDGDAALNKFFREIYSDADEDMRRAMMKSFVESNGTVLSTNWKDVGKKTVEGSPPDGMELKKWEY IKEEEKNEKLEGDAALNRLFQQIYSDGSDEVKRAMNKSFMESGGTVLSTNWSDVGKRKVEINPPDDMEWKKY-- IDEEADEEAGSADS----FFQKLYAGADPDTKRAMMKSFIESNGTALSTDWEDVSKGTVKTSPPEGMEPKHW-- CS VR1 : 350 : 358 : 370 : 367 : 373 : 333 : 395 : 69 : 69 : 69 : 73 : 73 : 78 : 84 : 144 : 144 : 150 : 147 : 151 : 136 : 162 : 206 : 214 : 226 : 222 : 229 : 194 : 245 : 276 : 284 : 296 : 293 : 299 : 261 : 327 Supplemental Figure 3
8 A La-er sgt1b-1 35S:ΔTPRb(sgt1b-1) #5936 #5941 # S:ΔTPRa(sgt1b-1) #7018 #7014 # AtSGT1a ΔTPRb ΔTPRa 32.5 α-sgsa Rubisco Rubisco B Ler sgt1b-1 sgt1b-1 (35S::ΔTPRb) #5936 #5941 #5935 TN TN #7014 sgt1b-1 (35S::ΔTPRa) #7017 C sgt1b-1 35S:ΔTPRb La-er sgt1b-1 #5936 #5941 # S:ΔTPRa #7018 #7014 # µM 2,4-D Supplemental Figure 4
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