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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]
FPPS2+ IDP+ DMADP
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3Farnesol
a
FPPS3+ IDP+ DMADP
1
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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
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FPPS3+ IDP+ DMADP+ TPS23
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3 (E)-ß-caryophyllenec
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control Spodoptera treated
control Spodoptera treated
control Spodoptera treated
FPPS1 FPPS2 FPPS3
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b) B73
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FPPS1 FPPS2 FPPS3
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3 a) Delprim
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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 (***).
*** ***
***
FPPS1 FPPS2 FPPS3
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Control Wounded Elicitor
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******
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***
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
Control Wound ElicitorTPS23
0
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1.5
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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 (***).
***
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
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
