Supporting information
Supporting figures
Figure S1. Plasmid vector overview.
Plasmid design of the LhGR (a, pSOL9LHGRC, GeneBank JX185747), pOp6irpds
(b, pPOP6IRPDS, GeneBank JX185750) and pOp6ipt (c, pPOP6IPT, GeneBank
JX185749) constructs.
LB/RB, left/right border of T-DNA; PNOS/TNOS, promoter/terminator of the nopaline
synthase gene from the Ti plasmid of A. tumefaciens; P35S/T35S, 35S promoter/
terminator from cauliflower mosaic virus; hptII, hygromycin phosphotransferase
gene from pCAMBIA-1301 (AF234297); PpOp6, six lac operators upstream of a
minimal CaMV 35S promotor (-50 to +8); lacI-His17, high-affinity DNA binding lac
repressor mutant (residues 1-330); sbd-ger, steroid-binding-domain (residues 508795) of a glucocorticoid receptor from rat; Gal4, Gal4 transcription-activatordomain-II from yeast (residues 768-881); i, intron 3 of Flaveria trinervia pdk gene
for pyruvate, orthophosphate dikinase; ’pds’, N. attenuata phytoene desaturase
(positions 847-1195 in GeneBank JX185751); A. t. ipt, isopentenyl transferase of
A. tumifaciens; nptII, aminoglycoside phosphotransferase class II; ori, origin of
replication.
Figure S2. Screening overview.
Workflow for screening of the transgenic lines.
Figure S3. Hygromycin resistance silencing.
Ratio of lines with silenced hptII gene observed within the screening process. HptII
silencing was detected by hygromycin sensitivity. T1 and T2 plants are considered
as silenced if less than 60% of the seedlings were hygromycin resistant.
Homozygous T3 were considered as silenced if hygromycin sensitive seedlings
were observed. N.D., not determined.
Figure S4. Seedling phenotype of i-irpds and i-ovipt.
Wildtype (WT), LhGR, i-irpds and i-ovipt seedlings two weeks after germination on
0 (-) or 20µM (+) DEX-containing GB5 plates.
Figure S5. LhGR-efficiency screening.
(a) LhGR, i-irpds and i-ovipt seedlings two weeks after germination on 0 or 20µM
DEX-containing GB5 plates. Different LhGR lines (76, 88, 92 and 103) were
compared before and after crossing with pOp6irpds (-> i-irpds) and pOp6ipt (-> iovipt).
(b) Overview of the observed phenotypes. Expected phenotype: – absent, + weak,
++ medium, +++ strong.
Figure S6. DEX stability under different temperature and light conditions.
Remaining DEX in a 100% methanol solution after indicated time
(a) in a dark 37°C temperature-chamber,
(b) under glasshouse conditions covered with or without a ~1mm lanolin layer or
(c) at 60°C.
Asterisks indicate significant differences between the same incubation times at
different conditions. (independent samples t-test; *=p< 0.05). Error bars show
standard errors (N=3). For additional statistical analysis, see text.
Figure S7. DEX application method.
Application of DEX-containing lanolin paste with a syringe to the petiole of a leaf.
Arrows indicate the lanolin paste after application.
Figure S8. pOp6/LhGR system in Nicotiana attenuata under glasshouse
conditions.
(a) Inverted repeat PDS (irPDS) and PDS transcript level in i-irpds plants one day
after application of 0 or 100µM DEX-containing lanolin paste to the midvein.
(b) i-irpds plants three days after application of 0 or 100µM DEX-containing lanolin
paste. Red circle indicates the first signs of bleaching.
(c) IPT transcript level and trans-zeatin level (ng*gFM-1) in i-ovipt plants one day
after application of 0 or 100µM DEX-containing lanolin paste to the midvein.
(d) i-ovipt plants three days after application of 0 or 100µM DEX-containing lanolin
paste.
Asterisks indicate significant differences between DEX treated samples and the
corresponding control (0µM) (independent samples t test: *** P<0.001). irPDS, IPT
transcript and trans-zeatin data, 24h after DEX treatment were log10 transformed
before t test analysis. Error bars show standard errors (N≥4). FM, Fresh mass.
Figure S9. Spatial characteristics of the pOp6/LhGR system after induction with
DEX-containing lanolin paste.
Comparison between a single DEX treated leaf with the next oldest untreated
leaves. 20µM DEX-containing lanolin paste was applied to a single leaf for six
days. Arrows indicate the treated leaf. Experiments were performed under
glasshouse conditions.
Figure S10. pOp6/LhGR system in Nicotiana attenuata - Hydroponic culture.
i-irpds (a) and i-ovipt (b, c) plants treated for six days (a, b) respectively 12 days
(c) with 0 (-) or 1µM (+) DEX-containing hydroponic solution. Experiment was
performed under glasshouse conditions.
Figure S11. Inducible gene-silencing in the field.
Field plot in the Great Basin Desert, Utah. Red circle indicates i-irpds plants two
weeks after a single treatment with different concentrations of DEX-containing
lanolin paste (0, 5, 20 and 100µM).
Figure S12. DEX treatments do not influence plant growth.
Representative decapitated plants at the stage of treatment begin (a) and 12d later
(b). Side branch length of LhGR plants 12 days after application of 0, 1, 5 or 20µM
DEX-containing lanolin paste to different side branches. Lanolin paste was applied
every three days. Error bars show standard errors (N≥15).
Figure S13. Chlorophyll content of leaves with different bleaching grades.
Average amount ± SE (N=20) and minimal values (min.) are shown in relative
units. Measurements were done with the SPAD502 Chlorophyll Meter from Konica
Minolta Sensing, Inc. Bleaching was achieved by DEX-induced pds silencing (-> iirpds plants). Experiment was performed under field conditions in the Great Basin
Desert, Utah. n.d., not detected.
Figure S14. Subtle cytokinin induction in the field does not change plant growth.
Side branch lengths of i-ovipt plants after application of 0, 0.05 or 0.1µM DEXcontaining lanolin paste to different side branches. Lanolin paste was refreshed
every three days. Experiment was performed under field conditions in the Great
Basin Desert, Utah. Error bars show standard errors (N≥15).
Figure S15. DEX treatment does not affect Tupiocoris notatus performance.
T. notatus leaf damage on different side branches of LhGR plants treated either
with 0, 1, 5 or 20µM DEX-containing lanolin paste for 12d. Lanolin paste was
refreshed every three days.
Experiments were performed under field conditions in the Great Basin Desert,
Utah. Error bars show standard errors (N≥15). n.s. = no significant difference
(paired samples t test: P>0.05).
Figure S16. Quantification method for Tupiocoris notatus damage.
Leaves of i-ovipt plants were treated with 0 or 5µM DEX-containing lanolin paste
and exposed to T. notatus for 14 days. The red frame indicates the quantified
damaged leaf area. Experiment was performed under glasshouse conditions.
Figure S17. DEX treatment does not affect Manduca sexta performance.
M. sexta caterpillar performance on LhGR plants treated with 0 or 100µM DEXcontaining lanolin paste. Lanolin paste was applied every three days.
Experiments were performed under glasshouse conditions. Error bars show
standard errors (N≥8).
Supporting tables
Table S1. Temperature conditions at the Utah field site at one day within the field
season (4pm, 02.06.2012).
Table S2. Sequences of primers used for qPCR.
Table S3. Sequences of primers used for PCR.
Table S4. Multi-reaction-monitoring settings for DEX quantification in negative
ionization mode.
Table S5. Multi-reaction-monitoring settings for cytokinin quantification in positive
ionization mode.