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, *

aDepartment of Molecular Cell Biology of Plants, Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany

bCenter of Membrane Proteomics, Goethe University, Max-von-Laue Str. 9, D-60438 Frankfurt, Germany

cCluster of Excellence Frankfurt, Goethe University, D-60438 Frankfurt, Germany

dBuchman Institute of Molecular Life Sciences, Goethe University, Max-von-Laue Str. 15, D-60438 Frankfurt, Germany

eInstitute 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 (), S. Missbach (), Y.-C. Hsueh (), F. Ertel (), H. Fulgosi (), M. S. Sommer (), E. Schleiff ()

1Current addresses: Novartis Vaccines and Diagnostics GmbH, Emil-von-Behring-Str. 76, D-35041 Marburg, Germany

2Current addresses: Department of Biochemistry, McGill University Montreal, Montreal H3G 1Y6 Canada

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Supplemental Table

Table S1 Oligonucleotides used in this study
Primer name / Sequence 5´to 3´
T-DNA analyses / 069122-FW1 / GAGAAGCTTGATGTTTCATGGG
069122-RV1 / GCCCATACCCGAATCCATAC
069122-FW3 / CTTTAGTGATTGGCAGGCAAG
069122-RV3 / ACCAATGGCAAATGCTTGATAAGGTG
LBa1 / TGGTTCACGTAGTGGGCCATCG
LBa2 / CCAGGCGGTGAAGGGCAATCAGC
qRT-PCR / Toc75-III_Fw / CTGTGGTGCGCAGGGAATTG
Toc75-III_Rv / GACTTCAATGTTAGAGAACAG
Toc75-V_Fw / CGTATAAACTACACTGATCC
Toc75-V_Rv / CTACTATTGTCTGGTTGATTG
P36_Fw / CAAGATGACAGAAGGGACC
P36_Rv / GCCCATACCCGAATCCATAC
P39_Fw / GATGTTAATGTGGACTTCACTC
P39_Rv / GATGTTTGATACAGAGGCTGC
Promoter-GUS
assay / Toc75-III F / ACGCGTCGACTCTTTCCATGATTTTCTGCT
Toc75-III R / ACGCGGATCCggcggagaagataaggtttc
p36 F / ACGCGTCGACGTGGGCTCGATATGTAGAAG
p36 R / CTAGTCTAGATCAGGTCTAGGTCTCCTAAAAG
p39 F / ACGCGTCGACGCATAGAGCAAACCAAACTC
p39 R / ACGCGGATCCGGCGATGGAATGAGAGAAG

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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

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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

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Fig. S2The T-DNA insertion line of P36.a Genomic DNA isolated from A. thalianawild-type and p36-1plants was processed as described (Thole et al. 2009), subjected to DNA agaroseelectrophoresis 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.

Fig. S32 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.

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.

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