7
Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein marker (Fermentas SM0669); lane 3, protein extract of FPPS3 in pASK-IBA37plus; lane 4, protein extract of FPPS2 in pASK-IBA37plus; lane 5, empty vector pASK-IBA37plus; lane S2, unstained protein marker (Thermo Scientific 26614). 1 2 S1 3 4 5 S2 50kDA 20kDA A maize (Zea mays) herbivore-induced farnesyl diphosphate synthase supports the sesquiterpene synthesis in leaves. Annett Richter 1 , Irmgard Seidl-Adams 2 , Tobias Köllner 3 , Joerg Degenhardt 1 1 Institute of Pharmacy, Martin Luther University, Hoher Weg 8, 06120 Halle, Germany, 2 Chemical Ecology, The Pennsylvania State University, State College PA 16802, 3 Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, 07745 Jena, Germany e-mail: [email protected]

Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein

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Page 1: Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein

Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein marker (Fermentas SM0669); lane 3, protein extract of FPPS3 in pASK-IBA37plus; lane 4, protein extract of FPPS2 in pASK-IBA37plus; lane 5, empty vector pASK-IBA37plus; lane S2, unstained protein marker (Thermo Scientific 26614).

1 2 S1 3 4 5 S2

50kDA

20kDA

A maize (Zea mays) herbivore-induced farnesyl diphosphate synthase supports the sesquiterpene synthesis in leaves.Annett Richter1, Irmgard Seidl-Adams2, Tobias Köllner3, Joerg Degenhardt1

1Institute of Pharmacy, Martin Luther University, Hoher Weg 8, 06120 Halle, Germany, 2Chemical Ecology, The Pennsylvania State University, State College PA 16802, 3Max Planck Institute for Chemical Ecology, Hans-Knöll-Strasse 8, 07745 Jena, Germanye-mail: [email protected]

Page 2: Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein

FPPS2+ IDP+ DMADP

1

2

3Farnesol

a

FPPS3+ IDP+ DMADP

1

2

3 Farnesolb

Fig. S2 Activity assay of FPPS after heterologous expression in E. coli. FPPS2 (a) and FPPS3 (b) without His-tag and the empty vector control (d) were incubated with the substrates IDP and DMADP for 45 min. The assay in panel c was incubated with both recombinant FPPS3 and TPS23, a maize (E)-ß-caryophyllene synthase that converts the FDP of FPPS3 to (E)-ß-caryophyllene. To the assays in panel a, b and d, hydrochloric acid was added to convert the prenyl phosphate product FDP into the corresponding alcohol farnesol. The volatiles were detected by GC-MS.

Retention time (min)

5 10 15 20

empty vector+ IDP+ DMADP

1

2

3

Farnesol

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lativ

e a

bu

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ce(T

ICx

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)

FPPS3+ IDP+ DMADP+ TPS23

1

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3 (E)-ß-caryophyllenec

d

Page 3: Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein

control Spodoptera treated

control Spodoptera treated

control Spodoptera treated

FPPS1 FPPS2 FPPS3

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0.1

0.2

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b) B73

con

tro

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FPPS1 FPPS2 FPPS3

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3 a) Delprim

Tra

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PT

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Spod. litt.treated

Spod. litt.treated

Spod. litt.treated

Spod. litt.treated

Spod. litt.treated

Fig. S3 Transcript abundance of fpps1, fpps2, and fpps3 genes in leaves after attack by Spodoptera littoralis. Transcript levels were determined in leaves of 14 day-old plants of the variety Delprim (a) and B73 (b) that were undamaged, wounded mechanically, or treated with third instar larvae of S. littoralis. for 24h. The expression was calculated relative to the housekeeping gene APT1. Significance was calculated by one-way-ANOVA of three technical repeats of four biological samples. Significant differences among treatments for each gene for P≤ 0.001 (***).

*** ***

***

Page 4: Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein

FPPS1 FPPS2 FPPS3

0

2

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Control Wounded Elicitor

0

0.1

0.2

0

0.04

0.08

******

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nscr

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PT

1

***

Fig. S4 Transcript abundance of fpps1, fpps2, and fpps3 genes in leaves. Transcript levels were determined in leaves of 14 day-old plants of the inbred line B73 that were undamaged, wounded mechanically, and wounded and treated with elicitor (indanone-derivate) for 24h. The expression was calculated relative to the housekeeping gene APT1. Significance was calculated by one-way-ANOVA of three technical repeats of four biological samples. Significant differences among treatments for each gene were indicated for P≤ 0.001 (***).

FPPS2FPPS1

Page 5: Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein

Control Wound ElicitorTPS23

0

0.5

1

1.5

2

2.5

Tra

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ipt

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o A

PT

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tps23

Fig. S5 Transcript abundance of tps23 in leaves. Transcript levels were determined in leaves of 14 day-old plants of the variety Delprim that were undamaged, wounded mechanically, and wounded and treated with elicitor (indanone-derivate) for 24h. The expression was calculated relative to the housekeeping gene APT1. Significance was calculated by one-way-ANOVA of three technical repeats of four biological samples. Significant differences were indicated for P≤ 0.001 (***).

***

Page 6: Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein

Gene Name Sequence Application

GRMZM2G131907 APT1_F AGGCGTTCCGTGACACCATC APT1 QRT fwd

APT1_R CTGGCAACTTCTTCGGCTTCC APT1 QRT rev

GRMZM2G147721 FPPS3F CCTGGCTAGTTGTGCAAGCT FPPS2 QRT fwd

FPPS8R GAAAACAcTCTGGACTGCCT FPPS2 QRT rev

FPPS2_R GTCGACGGAGCAGCTTCTTC FPPS2 cloning fwd

FPPS2_pASK-IBA33_F ATGGTAGGTCTCAAATGGCGACGGTGGAGGTGGTGGT

FPPS2 cloning (pASK-IBA33+) fwd

FPPS2_pASK-IBA33_R ATGGTAGGTCTCAGCGCTCTTGTCCCTCTTGTAGATCTTGTG

FPPS2 cloning (pASK-IBA33+) rev

FPPS2_pASK-IBA37_F ATGGTAGGTCTCAGCGCATGGCGACGGTGGAGGTGGTG

FPPS2 cloning (pASK-IBA37+) fwd

FPPS2_pASK-IBA37_R ATGGTAGGTCTCATATCACTTGTCCCTCTTGTAGATCTTGTG

FPPS2 cloning (pASK-IBA37+) rev

FPPS2_F CGAGGCCGCAATGGCGACGGT FPPS2 cloning rev

GRMZM2G168681 FPPS8.1F CATGGCTAGTTGTGCAAGCC FPPS1 QRT fwd

FPPS8.1R CAGCACTTTCTGAACCGCAA FPPS1 QRT rev

GRMZM2G098569 FPPS3F CCTGGCTAGTTGTGCAAGCT FPPS3 QRT fwd

FPPS3R GAAAACAGTTTGGACTGCCT FPPS3 QRT rev

FPPS3_F CGAGGCCGCAATGGCGACAGC FPPS3 cloning fwd

FPPS3_R GTCGATGGAGCAGCTTGTCG FPPS3 cloning rev

FPPS3_pASK-IBA33_F ATGGTAACCTGCATTAAATGGCGACAGCGGAGGTGGTGGT FPPS3 cloning (pASK-

IBA33+) fwd

FPPS3_pASK-IBA33_R ATGGTAACCTGCATTAGCGCTCTTGTCCCTCTTGTAGATCTTGTGFPPS3 cloning (pASK-

IBA33+) rev

FPPS3_pASK-IBA37_F ATGGTAACCTGCATTAGCGCATGGCGACAGCGGAGGTGGTGFPPS3 cloning (pASK-

IBA37+) fwd

FPPS3_pASK-IBA37_R ATGGTAACCTGCATTATATCACTTGTCCCTCTTGTAGATCTTGTGFPPS3 cloning (pASK-

IBA37+) rev

Table S1 Primer for QRT-PCR, isolation of open reading frames and cloning into expression vectors of fpps1, fpps2 and fpps3

Page 7: Fig. S1 SDS-page of maize FDPS protein. Lane 1, purified FPPS3 in pASK-IBA37plus; lane 2, purified FPPS2 in pASK-IBA37plus; lane S1, unstained protein

Plant species Prenyldiphosphate synthase Accession number

Zea mays ZmFPPS3 BT085025

ZmFPPS1 BT043171

ZmFPPS2 LOC100192004

ZmGGPPS1 EF417573

zmGGPPS2 EF415754

ZmGGPPS3 EF417575

Oryza sativa OsFPPS1 D85317

OsFPPS2 LOC_Os05g46580

OsFPPS3 LOC_Os04g56230

A.thaliana AtFPS1 NM_124151

AtFPS2L NM_117823

AtGGPPS2 U44876

AtGGPPS4 AC006135

Table S2 Accession numbers of the rooted phylogenetic tree of FPPSs and GGPPs of different plant species