1
Appendix S1. Method details
2
Analysis of leaf secondary metabolites by HPLC. We quantified the levels of nicotine, CP,
3
CA, rutin and DTGs by high performance liquid chromatography and photo diode array
4
detection (HPLC-DAD; Shimadzu SPD-M20A, Tokyo, Japan) as previously described
5
(Keinänen et al., 2001) with minor modifications. Harvested leaf samples (100 mg fresh mass)
6
were flash-frozen in liquid nitrogen and powdered using mortar and pestle in liquid nitrogen.
7
After vortexing samples in 1 mL extraction buffer (40% methanol, 0.5% acidic acid) for 1 h
8
and centrifuging them twice (16,000 rcf for 10 min), we injected 20 µL of a sample which was
9
separated on C18-column (Inertsil ODS 3.3 µm: 4.6 x 150 mm and pre-column: 4.6 x 15 mm)
10
in a gradient mode (eluents: A - 0.25% H3PO4 in H2O, B - acetonitrile; gradient: 0-6 min, 0-
11
12% B; 6-10 min, 12-25% B; 10-30 min, 25-80% B; flow rate: 0.5 mL min-1). We assigned the
12
compounds according to retention times and UV spectra. Based on the peak areas at
13
wavelengths 254 nm (nicotine), 320 nm (CP, CA) and 360 nm (rutin), we quantified the
14
compounds according to external standard curves of nicotine, CA and rutin, while quantities of
15
DTGs detected at 210 nm were expressed as summed peak areas per fresh leaf mass.
16
Analysis of TPI activity. We determined the activity of trypsin proteases inhibitors (TPI) from
17
100 mg leaf material (fresh mass) which had been flash-frozen and powdered. We measured
18
TPI activity either by a radial diffusion assay (exp. III) or a microwell plate assay (exp. VII) as
19
previously described (Bode et al., 2013, van Dam et al., 2001). For both assays, we vortexed
20
100 mg of powdered leaf material in extraction buffer and transferred the supernatant after
21
centrifugation (20 min at 12,000 rcf and 4°C) into 1.5 mL tubes and stored them not longer than
22
a few days at -20°C.
23
For the radial diffusion assay, we used 0.3 mL extraction buffer (50 g L-1 polyvinyl
24
polypyrrolidone (PVPP), 18.6 g L-1 Na2EDTA, 2 g L-1 phenylthiourea, 5 g L-1
25
diethyldithiocarbamate in 0.1 M Tris-HCl, pH 7.6). For analysis, we filled protein extracts into
1
26
wells (ø 0.5 mm) in agar plates (12 x 12 cm, 25 mL of 1.8% agar containing 2 mg bovine
27
trypsin). After 16 to 17 h diffusion time at 4°C, we incubated the plates at 37°C for 1 h with
28
25 mL of a staining solution containing 6 mg N-acetyl-DL-phenylalanine-naphthyl ester, 12
29
mg Fast Blue B salt (CAS 14263-94-6) in 20% DMF/0.1 M Tris and determined the Feret
30
diameter of the clear inhibition zones.
31
For the microwell plate assay, we used 0.6 mL extraction buffer (5% (w/v) PVPP in 0.1 M
32
K3PO4, pH 7.3) and added 50 µL of 10% Triton X-100. Into each well of a 96-well plate, we
33
added 20 µL of the extract to 30 µL of 0.1 M Tris-HCl (pH 8.0) containing and 10 ng trypsin.
34
After 5 min of incubation at 37°C, we added 20 µL substrate solution (3.1 g L-1 N-benzoyl-DL-
35
arginine-β-naphthylamide in dimethyl sulfoxide). After 20 min of incubation, absorbance at
36
550 nm was read with a microwell plate reader (MultiskanTM GO, Thermo Scientific, Waltham,
37
Massachusetts, USA). Then we stopped the reaction by adding 100 µL 2% HCl in ethanol and
38
added 100 µL dye reagent (0.06% p-dimethylaminocinnamaldehyde in ethanol) and read the
39
absorbance again at 550 nm after 20 min.
40
We calculated TPI activity according to linear standard curves of a dilution series of soybean
41
trypsin inhibitor (STI) that was run on each plate and normalised it to fresh mass of leaf material
42
and the total protein content of the leaf extracts as determined in a microwell plate reader using
43
Roti®-Quant according to the manufacturer’s instruction (http://www.carlroth.com).
44
Treatment and sampling for phytohormone analysis. We collected the M. sexta OS that
45
contain fatty acid-amino acid conjugates as elicitors from 4th instar larvae that had been feeding
46
on Wt N. attenuata leaf material. OS was collected with a Teflon tube in 5 mL glass vials that
47
were connected to a vacuum pump. We removed solid particles from the OS by centrifugation
48
and stored the OS supernatant (1:1 diluted with water) at -80°C for one day. Using a pattern
49
wheel we inflicted puncture wounds in three rows on each side of the oviposited leaf and of a
50
corresponding leaf of egg-free plants. Instantly, we added 20 µL OS (W+OS) or water (W+W)
2
51
into the wounds. After either 30, 60 and 90 minutes, we harvested the treated leaves of
52
independent plant replicates (nPlant per time point was 5-7 in case of W+OS and 4-6 in case of
53
W+W treatments). In order to obtain non-induced phytohormone levels from oviposited and
54
from egg-free control plants, we also sampled leaf material from untreated plants (nPlant = 3) at
55
the same time points. All leaf material was flash frozen in liquid nitrogen and stored at -80°C.
56
Analysis of JA and JA-Ile. We determined the concentration of JA and its conjugate to
57
isoleucine (JA-Ile) as previously described (Gaquerel et al., 2012). We homogenised 200 mg
58
leaf material in 1 mL ethyl acetate spiked with 200 ng D2-JA and 40 ng JA-13C6-Ile as internal
59
standards for JA and JA-Ile using a FastPrep®-24 instrument (MP Biomedicals, Solon, USA).
60
We transferred the supernatant into 2 mL tubes after centrifugation (10 min at 12,000 rcf and
61
4°C) and re-extracted the pellet with 0.5 mL ethyl acetate. After centrifugation both
62
supernatants were combined and evaporated to dryness in a vacuum concentrator (concentrator
63
5301, Eppendorf, Hamburg, Germany). We suspended the residue in 0.5 mL of 70% methanol
64
(v/v) and transferred the supernatant into glass vials after centrifugation. We used a 1200L
65
Triple-Quadrupole-MS (Varian, Palo Alto, USA) for LC/ESI-MS/MS analysis. Compounds
66
were separated from 10 µL of sample injected on a C8-column (ProntoSIL C18-ace-EPS, 50 x 2
67
mm, 5 μm, 120 Å) in a gradient mode (eluents: A - 0.05% formic acid in H2O, B - methanol;
68
gradient: 0-4.5 min, 15-98% B; 4.5-12 min, 98-15% B; 12-15 min, 15% B; flow rate: 0-1.5 min,
69
0.4 mL min-1; 1.5-10.5 min, 0.2 mL min-1; 10.5-15 min, 0.4 mL min-1). We used negative
70
ionisation mode and tandem mass spectrometry with parent ion/daughter ion selections of
71
209/59 for JA, 213/59 for D2-JA (12 V collision energy), 322/130 for JA-Ile and 328/136 for
72
JA-13C6-Ile (19 V collision energy). For quantification we divided the peak area of the
73
endogenous phytohormone by that of its respective internal standard and multiplied with the
74
amount of internal standard.
3
75
Analysis of antimicrobial activity. We used 2 µL of the haemolymph samples that were
76
diluted 10-fold with phosphate buffered saline (PBS) in the radial diffusion assay in agar plates
77
inoculated with Micrococcus luteus (DSM20030; DMSZ, Brunswick, Germany). A single
78
colony of M. luteus was grown in LB broth (2 g yeast extract, 2 g peptone, pH = 7.0) at 30°C
79
and the overnight cultures were washed twice in PBS (7 mM KH2PO4, 3 mM Na2HPO4, 0.13
80
M NaCl, pH = 7.4). After discarding the supernatant, the cells were diluted to an optimal density
81
OD = 0.6 at 620 nm and 2.5 mL of this culture were mixed with 25 mL medium at 45°C (1 L:
82
6 g Agar, 2 g yeast extract, 2 g peptone, pH = 7.0) before agar plates were poured. After
83
hardening we used a cork borer (ø 0.5 mm) to form wells in the agar plate that were filled up
84
with about 20 µL diluted haemolymph samples. On each plate we run a lysozyme (Sigma-
85
Aldrich, St. Luis, USA) dilution series (500, 250, 125, 62.5, 31.3 and 15.6 mg mL-1) as standard
86
curve. Plates were incubated for 24 h at 30°C. We determined antimicrobial activity from the
87
Feret diameter of the inhibition zones as lysozyme equivalents.
4