Transcript PowerPoint-Präsentation - Springer Static Content Server

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, Germany
e-mail: [email protected]
1
2
S1
3
4
5
S2
50kDA
20kDA
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).
3
2
a
Farnesol
FPPS2+ IDP+ DMADP
Relative abundance(TICx 107)
1
3
2
b
Farnesol
FPPS3+ IDP+ DMADP
1
3
2
c
(E)-ß-caryophyllene
FPPS3+ IDP
+ DMADP+ TPS23
1
3
2
d
empty vector+ IDP+ DMADP
Farnesol
1
5
10
15
Retention time (min)
20
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)-ßcaryophyllene. 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.
3
***
a) Delprim
2.5
2
1.5
1
0.5
0
control
Spodoptera
Spod. litt.
treated
FPPS1
control
Spodoptera
treated
FPPS2
control
Spodoptera
Spod. litt.
treated
treated
FPPS3
Transcript level relative to APT1
Transcript level relative to APT1
3.5
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
b) B73
***
***
control
Spodoptera
Spod. litt.
treated
treated
FPPS1
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 (***).
control
Spodoptera
Spod. litt.
treated
treated
FPPS2
control
Spodoptera
Spod. litt.
treated
treated
FPPS3
Transcript level relative to APT1
Control
10
Wounded
Elicitor
0.08
0.2
***
***
8
0.04
0.1
6
4
0
0
FPPS1
FPPS2
2
***
0
FPPS1
FPPS2
FPPS3
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 (***).
Transcript level relative to APT1
3.5
***
3
2.5
2
1.5
1
0.5
0
Control
Wound
Elicitor
TPS23
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 (***).
Table S1 Primer for QRT-PCR, isolation of open reading frames and cloning into expression vectors
of fpps1, fpps2 and fpps3
Gene
Name
Sequence
Application
GRMZM2G131907
APT1_F
AGGCGTTCCGTGACACCATC
APT1 QRT fwd
APT1_R
CTGGCAACTTCTTCGGCTTCC
APT1 QRT rev
FPPS3F
CCTGGCTAGTTGTGCAAGCT
FPPS2 QRT fwd
GRMZM2G147721
GRMZM2G168681
GRMZM2G098569
FPPS8R
GAAAACAcTCTGGACTGCCT
FPPS2 QRT rev
FPPS2_R
GTCGACGGAGCAGCTTCTTC
FPPS2_pASK-IBA33_F
ATGGTAGGTCTCAAATGGCGACGGTGGAGGTGGTGGT
FPPS2_pASK-IBA33_R
ATGGTAGGTCTCAGCGCTCTTGTCCCTCTTGTAGATCTTGTG
FPPS2_pASK-IBA37_F
ATGGTAGGTCTCAGCGCATGGCGACGGTGGAGGTGGTG
FPPS2_pASK-IBA37_R
FPPS2_F
ATGGTAGGTCTCATATCACTTGTCCCTCTTGTAGATCTTGTG
CGAGGCCGCAATGGCGACGGT
FPPS2 cloning fwd
FPPS2 cloning (pASKIBA33+) fwd
FPPS2 cloning (pASKIBA33+) rev
FPPS2 cloning (pASKIBA37+) fwd
FPPS2 cloning (pASKIBA37+) rev
FPPS2 cloning rev
FPPS8.1F
CATGGCTAGTTGTGCAAGCC
FPPS1 QRT fwd
FPPS8.1R
CAGCACTTTCTGAACCGCAA
FPPS1 QRT rev
FPPS3F
CCTGGCTAGTTGTGCAAGCT
FPPS3 QRT fwd
FPPS3R
GAAAACAGTTTGGACTGCCT
FPPS3 QRT rev
FPPS3_F
CGAGGCCGCAATGGCGACAGC
FPPS3 cloning fwd
FPPS3_R
GTCGATGGAGCAGCTTGTCG
FPPS3_pASK-IBA33_F
ATGGTAACCTGCATTAAATGGCGACAGCGGAGGTGGTGGT
FPPS3_pASK-IBA33_R
ATGGTAACCTGCATTAGCGCTCTTGTCCCTCTTGTAGATCTTGTG
FPPS3_pASK-IBA37_F
ATGGTAACCTGCATTAGCGCATGGCGACAGCGGAGGTGGTG
FPPS3_pASK-IBA37_R
ATGGTAACCTGCATTATATCACTTGTCCCTCTTGTAGATCTTGTG
FPPS3 cloning rev
FPPS3 cloning (pASKIBA33+) fwd
FPPS3 cloning (pASKIBA33+) rev
FPPS3 cloning (pASKIBA37+) fwd
FPPS3 cloning (pASKIBA37+) rev
Table S2 Accession numbers of the rooted phylogenetic tree of FPPSs and GGPPs of different plant
species
Plant species
Prenyldiphosphate
synthase
Accession number
Zea mays
ZmFPPS3
BT085025
ZmFPPS1
BT043171
ZmFPPS2
LOC100192004
ZmGGPPS1
EF417573
zmGGPPS2
EF415754
ZmGGPPS3
EF417575
OsFPPS1
D85317
OsFPPS2
LOC_Os05g46580
OsFPPS3
LOC_Os04g56230
AtFPS1
NM_124151
AtFPS2L
NM_117823
AtGGPPS2
U44876
AtGGPPS4
AC006135
Oryza sativa
A.thaliana