The Omp85-Type Outer Membrane Protein P36 of Arabidopsis Thaliana Evolved by Recent Gene

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 of Molecular Cell Biology of Plants, Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany b Center 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 Institute of Molecular Life Sciences, Goethe University, Max-von-Laue Str. 15, D-60438 Frankfurt, Germany e Institute 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 ([email protected]), S. Missbach ([email protected]), Y.-C. Hsueh ([email protected]), F. Ertel ([email protected]), H. Fulgosi ([email protected]), M. S. Sommer ([email protected]), E. Schleiff ([email protected]) 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 Primer name Sequence 5´to 3´ 069122-FW1 GAGAAGCTTGATGTTTCATGGG 069122-RV1 GCCCATACCCGAATCCATAC T-DNA 069122-FW3 CTTTAGTGATTGGCAGGCAAG analyses 069122-RV3 ACCAATGGCAAATGCTTGATAAGGTG LBa1 TGGTTCACGTAGTGGGCCATCG LBa2 CCAGGCGGTGAAGGGCAATCAGC

Toc75-III_Fw CTGTGGTGCGCAGGGAATTG Toc75-III_Rv GACTTCAATGTTAGAGAACAG Toc75-V_Fw CGTATAAACTACACTGATCC Toc75-V_Rv CTACTATTGTCTGGTTGATTG qRT-PCR P36_Fw CAAGATGACAGAAGGGACC P36_Rv GCCCATACCCGAATCCATAC P39_Fw GATGTTAATGTGGACTTCACTC P39_Rv GATGTTTGATACAGAGGCTGC

Toc75-III F ACGCGTCGACTCTTTCCATGATTTTCTGCT Toc75-III R ACGCGGATCCGGCGGAGAAGATAAGGTTTC Promoter-GUS p36 F ACGCGTCGACGTGGGCTCGATATGTAGAAG assay p36 R CTAGTCTAGATCAGGTCTAGGTCTCCTAAAAG p39 F ACGCGTCGACGCATAGAGCAAACCAAACTC p39 R ACGCGGATCCGGCGATGGAATGAGAGAAG

2 Supplemental Figures

a AT3G48620 ------AT3G44160 atgggagctcagaagagtatccacgctggtagagccaagattgatgttaatgtggacttc AT3G48620 ------AT3G44160 actcacaagctttgtacttctttaatgtttcctgctttcagggacactagcagtcctctt AT3G48620 ------AT3G44160 tctctagtgattggcagcctctgtatcaaacatccaaatttgtttggaggaagcgagaag AT3G48620 ----atgtttcatgggataaagggttgtatgattccaatgtactggtggcttttaggaga AT3G44160 cttgatgtatcatgggataaaggattgtatgattccaatgtacttgtggcttttaggaga AT3G48620 cctagacctgaatggcgtccgcaacagtgcttcttcatac------AT3G44160 cctagacctgaatggcgtccacaacagtgtttcttcatacagcattctctctcacccgag AT3G48620 ------agtggaggtgtaaat AT3G44160 ataggggtccacggcaccccagtcgacaacttttctcggtcaggaagtggaggtgtaaat AT3G48620 ctttctaaattggctgttggtttagacttgagtgagccagcgagttcaaaatggagcagc AT3G44160 ctgtctaaattggctcttggtttagacttgagtgagccagcgagttcaaaatggagcagc AT3G48620 acaaccagcgtaaagtttgaggtacccggacattaccttcttcagacattgtacatgtgc AT3G44160 acaaccagcataaagtttgag------catgtgc AT3G48620 gtccgattaacgatgacggacgggcgat-atcagagatctggacggatttcctataacat AT3G44160 gtccgattaacgatgatggacgcgcgataaccagagatctggacggatttcctataac-- AT3G48620 gcagaattctctgctatgcagtggaaatacccatgacagtatggtagttttaaagcaaga AT3G44160 ------atgcagtggaaatacccatgacagtatggtagttttaaagcaaga AT3G48620 atcccggtttgcaaaggctactgaccaaggtctttctcattttagtatgcaaatagaaca AT3G44160 atcccggtttgcaaaggctaccgaccaaggtctttctcattttagcatgcaaatagaaca AT3G48620 aggtattccggttgtgtccaactggcttatcttcaaccgtttcaaatttgttgcatcaaa AT3G44160 aggtattccggttgtgtccaagtggcttatcttcaaccgtttcaaatttgttgcatcaaa AT3G48620 aggtgtcaggtttggaccggcttttcccttagcaagcttgacaggtggttcaattgtagg AT3G44160 aggtgtcaggtttggaccggcttttctcttagcaagcttgacaggtggttcaattgtagg AT3G48620 agacatgacaccttatcaagcatttgccattggtgggctaggcagtgttcggggatatgg AT3G44160 agacatggcaccttatcaagcatttgccattggtgggctaggcagtgttcgcggatatgg AT3G48620 tgaggttgctgttggatctgggcggtcatgccttgttgctaatacggaattggc------AT3G44160 tgagggtgctgttggatccggtcggtcatgccttgttgccaatacagaattggcgttacc AT3G48620 ---gaacaagatgacagaagggaccatcttcttggattgtggaacagacctaggctcaag AT3G44160 tttgaacaagatgacagaagggaccatcttcttggattgtggaacagacctaggctcaag AT3G48620 ccgccttgtcccggtaagttcactttaccttctccgaacaagaaccataaagaagttgtt AT3G44160 ccgccttgtcc------AT3G48620 gagacacgaaaatgataaagctgttgcggaattggtttcaggaaacccttcgctgagaca AT3G44160 ------ccggaaacccttcaatgagaca AT3G48620 agggaagccggggtttgggtatggattcgggtatgggctacggttcaagtctccattggg AT3G44160 agggaagccagggtttgggtatggattcggatatgggctacggttcaagtctccattggg AT3G48620 tcaccttcaggttgactatgccatgaatgctttcaaccaaaagactctttacttcggtat AT3G44160 tcaccttcaggttgactatgccataaatgctttcaaccagaagactctttacttcggtgt AT3G48620 caccaatcttgcttcatcaacatag AT3G44160 caccaatcttgcttcatcaacatag

3 b AT3G44160 atgggagctcagaagagtatccacgctggtagagccaagattgatgttaatgtggacttc A_484837 atgggagctcagaagagtatccacgctggtagagccaagattgatgttaatgtggatttc AT3G44160 actcacaagctttgtacttctttaatgtttcctgctttcagggacactagcagtcctctt A_484837 actcacaagctttgtacttctttaatgtttcctgctttcagggacactagcagtcctctt AT3G44160 tctctagtgattggcag------cctctgtatcaaacatccaaatttgtttggagga A_484837 tctctagtgattggcaggcaagttttcctctgtatcaaacatccaaatttgtttggagga AT3G44160 agcgagaagcttgatgtatcatgggataaaggattgtatgattccaatgtacttgtggct A_484837 agcgagaagcttgatgtatcatgggataaagggttgtatgattccaatgtactggtggct AT3G44160 tttaggagacctagacctgaatggcgtccacaacagtgtttcttcatacagcattctctc A_484837 tttaggagacctagacctgaatggcgtccgcaacagtgcttcttcatacagcattctctt AT3G44160 tcacccgagataggggtccacggcaccccagtcgacaacttttctcggtcaggaagtgga A_484837 tcgcccgagataggggtccatggcaccccagttgacaacttttctcggtcagggagtgga AT3G44160 ggtgtaaatctgtctaaattggctcttggtttagacttgagtgagccagcgagttcaaaa A_484837 ggtgtaaatctttctaaattggctgttggtttagacttgagtgagccagcgagttcaaaa AT3G44160 tggagcagcacaaccagcataaagtttgagcatgtgcgtccgattaacgatgatggacgc A_484837 tggagcagcacaaccagcgtaaagtttgagcatgtgcgtccgattaacgatgacggacgc AT3G44160 gcgataaccagagatctggacggatttcctataacatgcagtggaaatacccatgacagt A_484837 gcgataaccagagatctggacggatttcctataacatgcagtggaaatacccatgacagt AT3G44160 atggtagttttaaagcaagaatcccggtttgcaaaggctaccgaccaaggtctttctcat A_484837 atggtagttttaaagcaagaatcccggtttgcaaaggctactgaccaaggtctttctcat AT3G44160 tttagcatgcaaatagaacaaggtattccggttgtgtccaagtggcttatcttcaaccgt A_484837 tttagcatgcaaatagaacaaggtattccggttgtgtccaagtggcttatcttcaaccgt AT3G44160 ttcaaatttgttgcatcaaaaggtgtcaggtttggaccggcttttctcttagcaagcttg A_484837 ttcaaatttgttgcatcaaaaggtgtcaggtttggaccggcttttctcttagcaagcttg AT3G44160 acaggtggttcaattgtaggagacatggcaccttatcaagcatttgccattggtgggcta A_484837 acaggtggttcaattgtaggagacatggcaccttatcaagcatttgccattggtgggcta AT3G44160 ggcagtgttcgcggatatggtgagggtgctgttggatccggtcggtcatgccttgttgcc A_484837 ggcagtgttcggggatatggtgagggtgctgttggatccgggcgatcatgccttgttgcc AT3G44160 aatacagaattggcgttacctttgaacaagatgacagaagggaccatcttcttggattgt A_484837 aatacagaattggcggtacctttgaacaagatgacagaagggaccatcttcttggattgt AT3G44160 ggaacagacctaggctcaagccgccttgtccccggaaacccttcaatgagacaagggaag A_484837 ggaacagacctaggctcaagccgccttgtccctggaaacccttcgatgagacaagggaag AT3G44160 ccagggtttgggtatggattcggatatgggctacggttcaagtctccattgggtcacctt A_484837 ccagggtatgggtatggattcgggtatgggcttcggttcaagtctccattgggtcacctt AT3G44160 caggttgactatgccataaatgctttcaaccagaagactctttacttcggtgtcaccaat A_484837 caggttgactatgccatgaatgctttcaaccagaagactctgtacttcggtgtcaccaat AT3G44160 cttgcttcatcaacatag A_484837 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.