The Omp85-type outer membrane protein p36 of Arabidopsis
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Supplemental Materials to The Omp85-type outer membrane protein p36 of Arabidopsis thaliana evolved by recent gene duplication Kerstin Nicolaisena,b, Sandra Missbacha,1, Yi-Ching Hsueha, Franziska Ertela,2, Hrvoje Fulgosie, Maik S. Sommera, and Enrico Schleiffa-d, * a Department b Center of Molecular Cell Biology of Plants, Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany of Membrane Proteomics, Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany c Cluster of Excellence Frankfurt, Goethe University, D-60438 Frankfurt, Germany d Buchman e Institute Institute of Molecular Life Sciences, Goethe University, Max-von-Laue Str. 15, D-60438 Frankfurt, Germany Ruđer Bošković, Zavod za molekularnu biology, 10 000 Zagreb, Croatia * Corresponding author: Phone: +49 (0)69 798 29287; Fax: +49 (0)69 798 29286 Email addresses: K. Nicolaisen (kerstin.nicolaisen@gmx.de), S. Missbach (sandramissbach@gmail.com), Y.-C. Hsueh (hsueh@bio.uni-frankfurt.de), F. Ertel (franzi.ertel@gmx.de), H. Fulgosi (hrvoje.fulgosi@irb.hr), M. S. Sommer (sommer@bio.uni-frankfurt.de), E. Schleiff (schleiff@bio.uni-frankfurt.de) 1 Current addresses: Novartis Vaccines and Diagnostics GmbH, Emil-von-Behring-Str. 76, D-35041 Marburg, Germany 2 Current addresses: Department of Biochemistry, McGill University Montreal, Montreal H3G 1Y6 Canada 1 Supplemental Table Table S1 Oligonucleotides used in this study T-DNA analyses qRT-PCR Primer name 069122-FW1 069122-RV1 069122-FW3 069122-RV3 LBa1 LBa2 Sequence 5´to 3´ GAGAAGCTTGATGTTTCATGGG GCCCATACCCGAATCCATAC CTTTAGTGATTGGCAGGCAAG ACCAATGGCAAATGCTTGATAAGGTG TGGTTCACGTAGTGGGCCATCG CCAGGCGGTGAAGGGCAATCAGC Toc75-III_Fw Toc75-III_Rv Toc75-V_Fw Toc75-V_Rv P36_Fw P36_Rv P39_Fw P39_Rv CTGTGGTGCGCAGGGAATTG GACTTCAATGTTAGAGAACAG CGTATAAACTACACTGATCC CTACTATTGTCTGGTTGATTG CAAGATGACAGAAGGGACC GCCCATACCCGAATCCATAC GATGTTAATGTGGACTTCACTC GATGTTTGATACAGAGGCTGC Toc75-III F Toc75-III R Promoter-GUS p36 F assay p36 R p39 F p39 R ACGCGTCGACTCTTTCCATGATTTTCTGCT ACGCGGATCCGGCGGAGAAGATAAGGTTTC ACGCGTCGACGTGGGCTCGATATGTAGAAG CTAGTCTAGATCAGGTCTAGGTCTCCTAAAAG ACGCGTCGACGCATAGAGCAAACCAAACTC ACGCGGATCCGGCGATGGAATGAGAGAAG 2 Supplemental Figures a AT3G48620 AT3G44160 -----------------------------------------------------------atgggagctcagaagagtatccacgctggtagagccaagattgatgttaatgtggacttc AT3G48620 AT3G44160 -----------------------------------------------------------actcacaagctttgtacttctttaatgtttcctgctttcagggacactagcagtcctctt AT3G48620 AT3G44160 -----------------------------------------------------------tctctagtgattggcagcctctgtatcaaacatccaaatttgtttggaggaagcgagaag AT3G48620 AT3G44160 ----atgtttcatgggataaagggttgtatgattccaatgtactggtggcttttaggaga cttgatgtatcatgggataaaggattgtatgattccaatgtacttgtggcttttaggaga AT3G48620 AT3G44160 cctagacctgaatggcgtccgcaacagtgcttcttcatac-------------------cctagacctgaatggcgtccacaacagtgtttcttcatacagcattctctctcacccgag AT3G48620 AT3G44160 ---------------------------------------------agtggaggtgtaaat ataggggtccacggcaccccagtcgacaacttttctcggtcaggaagtggaggtgtaaat AT3G48620 AT3G44160 ctttctaaattggctgttggtttagacttgagtgagccagcgagttcaaaatggagcagc ctgtctaaattggctcttggtttagacttgagtgagccagcgagttcaaaatggagcagc AT3G48620 AT3G44160 acaaccagcgtaaagtttgaggtacccggacattaccttcttcagacattgtacatgtgc acaaccagcataaagtttgag--------------------------------catgtgc AT3G48620 AT3G44160 gtccgattaacgatgacggacgggcgat-atcagagatctggacggatttcctataacat gtccgattaacgatgatggacgcgcgataaccagagatctggacggatttcctataac-- AT3G48620 AT3G44160 gcagaattctctgctatgcagtggaaatacccatgacagtatggtagttttaaagcaaga ---------------atgcagtggaaatacccatgacagtatggtagttttaaagcaaga AT3G48620 AT3G44160 atcccggtttgcaaaggctactgaccaaggtctttctcattttagtatgcaaatagaaca atcccggtttgcaaaggctaccgaccaaggtctttctcattttagcatgcaaatagaaca AT3G48620 AT3G44160 aggtattccggttgtgtccaactggcttatcttcaaccgtttcaaatttgttgcatcaaa aggtattccggttgtgtccaagtggcttatcttcaaccgtttcaaatttgttgcatcaaa AT3G48620 AT3G44160 aggtgtcaggtttggaccggcttttcccttagcaagcttgacaggtggttcaattgtagg aggtgtcaggtttggaccggcttttctcttagcaagcttgacaggtggttcaattgtagg AT3G48620 AT3G44160 agacatgacaccttatcaagcatttgccattggtgggctaggcagtgttcggggatatgg agacatggcaccttatcaagcatttgccattggtgggctaggcagtgttcgcggatatgg AT3G48620 AT3G44160 tgaggttgctgttggatctgggcggtcatgccttgttgctaatacggaattggc-----tgagggtgctgttggatccggtcggtcatgccttgttgccaatacagaattggcgttacc AT3G48620 AT3G44160 ---gaacaagatgacagaagggaccatcttcttggattgtggaacagacctaggctcaag tttgaacaagatgacagaagggaccatcttcttggattgtggaacagacctaggctcaag AT3G48620 AT3G44160 ccgccttgtcccggtaagttcactttaccttctccgaacaagaaccataaagaagttgtt ccgccttgtcc------------------------------------------------- AT3G48620 AT3G44160 gagacacgaaaatgataaagctgttgcggaattggtttcaggaaacccttcgctgagaca --------------------------------------ccggaaacccttcaatgagaca AT3G48620 AT3G44160 agggaagccggggtttgggtatggattcgggtatgggctacggttcaagtctccattggg agggaagccagggtttgggtatggattcggatatgggctacggttcaagtctccattggg AT3G48620 AT3G44160 tcaccttcaggttgactatgccatgaatgctttcaaccaaaagactctttacttcggtat tcaccttcaggttgactatgccataaatgctttcaaccagaagactctttacttcggtgt AT3G48620 AT3G44160 caccaatcttgcttcatcaacatag caccaatcttgcttcatcaacatag 3 b AT3G44160 A_484837 atgggagctcagaagagtatccacgctggtagagccaagattgatgttaatgtggacttc atgggagctcagaagagtatccacgctggtagagccaagattgatgttaatgtggatttc AT3G44160 A_484837 actcacaagctttgtacttctttaatgtttcctgctttcagggacactagcagtcctctt actcacaagctttgtacttctttaatgtttcctgctttcagggacactagcagtcctctt AT3G44160 A_484837 tctctagtgattggcag---------cctctgtatcaaacatccaaatttgtttggagga tctctagtgattggcaggcaagttttcctctgtatcaaacatccaaatttgtttggagga AT3G44160 A_484837 agcgagaagcttgatgtatcatgggataaaggattgtatgattccaatgtacttgtggct agcgagaagcttgatgtatcatgggataaagggttgtatgattccaatgtactggtggct AT3G44160 A_484837 tttaggagacctagacctgaatggcgtccacaacagtgtttcttcatacagcattctctc tttaggagacctagacctgaatggcgtccgcaacagtgcttcttcatacagcattctctt AT3G44160 A_484837 tcacccgagataggggtccacggcaccccagtcgacaacttttctcggtcaggaagtgga tcgcccgagataggggtccatggcaccccagttgacaacttttctcggtcagggagtgga AT3G44160 A_484837 ggtgtaaatctgtctaaattggctcttggtttagacttgagtgagccagcgagttcaaaa ggtgtaaatctttctaaattggctgttggtttagacttgagtgagccagcgagttcaaaa AT3G44160 A_484837 tggagcagcacaaccagcataaagtttgagcatgtgcgtccgattaacgatgatggacgc tggagcagcacaaccagcgtaaagtttgagcatgtgcgtccgattaacgatgacggacgc AT3G44160 A_484837 gcgataaccagagatctggacggatttcctataacatgcagtggaaatacccatgacagt gcgataaccagagatctggacggatttcctataacatgcagtggaaatacccatgacagt AT3G44160 A_484837 atggtagttttaaagcaagaatcccggtttgcaaaggctaccgaccaaggtctttctcat atggtagttttaaagcaagaatcccggtttgcaaaggctactgaccaaggtctttctcat AT3G44160 A_484837 tttagcatgcaaatagaacaaggtattccggttgtgtccaagtggcttatcttcaaccgt tttagcatgcaaatagaacaaggtattccggttgtgtccaagtggcttatcttcaaccgt AT3G44160 A_484837 ttcaaatttgttgcatcaaaaggtgtcaggtttggaccggcttttctcttagcaagcttg ttcaaatttgttgcatcaaaaggtgtcaggtttggaccggcttttctcttagcaagcttg AT3G44160 A_484837 acaggtggttcaattgtaggagacatggcaccttatcaagcatttgccattggtgggcta acaggtggttcaattgtaggagacatggcaccttatcaagcatttgccattggtgggcta AT3G44160 A_484837 ggcagtgttcgcggatatggtgagggtgctgttggatccggtcggtcatgccttgttgcc ggcagtgttcggggatatggtgagggtgctgttggatccgggcgatcatgccttgttgcc AT3G44160 A_484837 aatacagaattggcgttacctttgaacaagatgacagaagggaccatcttcttggattgt aatacagaattggcggtacctttgaacaagatgacagaagggaccatcttcttggattgt AT3G44160 A_484837 ggaacagacctaggctcaagccgccttgtccccggaaacccttcaatgagacaagggaag ggaacagacctaggctcaagccgccttgtccctggaaacccttcgatgagacaagggaag AT3G44160 A_484837 ccagggtttgggtatggattcggatatgggctacggttcaagtctccattgggtcacctt ccagggtatgggtatggattcgggtatgggcttcggttcaagtctccattgggtcacctt AT3G44160 A_484837 caggttgactatgccataaatgctttcaaccagaagactctttacttcggtgtcaccaat caggttgactatgccatgaatgctttcaaccagaagactctgtacttcggtgtcaccaat AT3G44160 A_484837 cttgcttcatcaacatag cttgcttcatcaacatag Fig. S1 Alignment for synonymous substitution rate determination and protein structure prediction. a The alignment between the two coding sequences (CDS) of P39 and P36 generated with MAFFT v6.815b is shown. Mutations (yellow) and deletions/insertions (grey) are highlighted. The alignment shows 30 mutations and 6 deletions/insertions in 1041 positions. b The alignment between the two P39-like CDS from A. thaliana and A. lyrata is shown. Mutations (yellow) and deletions/insertions (grey) are highlighted. The alignment shows 26 mutations and 1 deletion / insertion in 1098 positions 4 Fig. S2 The T-DNA insertion line of P36. a Genomic DNA isolated from A. thaliana wild-type and p36-1 plants was processed as described (Thole et al. 2009), subjected to DNA agarose electrophoresis and sequenced (arrowheads). b Shown are the sequences of the border between T-DNA (in grey) and genomic sequence (in black). c Genomic DNA of A. thaliana wild-type (wt, lane 1-3) and p36-1 (lane 4-6) plants was isolated and PCR performed with P36 (lane 1-6) specific oligonucleotides in combination with the left border primer (LB(a2), lane 1-2 and lane 4-5). d Shown is the qRT-PCR result on RNA of 3-day-old p36-1 or wild-type plants. 5 Fig. S3 2 Analysis of promoter activity as shown in Fig. 2. 60 day-old A. thaliana plants transformed with constructs P36 promoter GUS construct (wt::p(P36)-GUS) were analyzed. Low frequent occurring staining in flowers (left) or roots (right) is shown. We observed in few cases GUS-staining of flowers, while the control line did not show any staining. In roots, staining occurred often at positions of side root development. The frequency of staining found for plants transformed with empty vector (co) or wt::p(P36)-GUS (p(P36)) is indicated for n>30. 6 Fig. S4 Comparison of the transcriptome of wild-type and p36-1. The transcript abundance in leaves of 43 day old plants was analysed by Affymetrix Chip technology and compared between wild type and p36-1 -/- (black spots). Grey lines indicate the border for 2 fold-changes and grey spots represent significant signals determined as described in Materials and Methods. 7
