Experimental procedures
Materials
As described in our previous works (Fauconnier and Marlier, 1996; Deleuet al. , 2019; Deboever et al. , 2020c ), HPOs were
enzymatically synthetized from the reaction of LOX-1 on linoleic
(13-HPOD) or linolenic acid (13-HPOT). The purity (higher than 98%) was
checked by high-performance liquid chromatography. For deuterated
13-HPOD, we used only deuterated reactants and solvents. Nuclear
magnetic resonance and mass spectrometry were used for a full chemical
characterization of the samples (data not shown).
1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC),
β-sitosterol, C16 glucosyl(ß) ceramide (d18:1/16:0) (GluCer), lipoxidase
from Glycine max (soybean) type I-B (LOX-1), the linoleic and α-
linolenic acids, 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan),
horseradish peroxidases, luminol were purchased from Sigma-Aldrich
(Belgium). 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine
(d62DPPC) was purchased from Avanti Polar Lipids
(Italy). Deuterium oxide (D2O) of 99.8% purity was
purchased from ARMAR (Europa) GmbH. Chloroform and methanol were both
purchased from Scharlau Lab Co. Dimethylsulfoxide (DMSO) and
tri(hydroxymethyl)aminomethane (TRIS) were provided by Sigma Chemical.
The ultrapure water was produced by Millipore systems available in our
laboratory, the resistivity was 18.2 MΏ cm. The active substance COS-OGA
was provided by FytoFend S.A. (Belgium) under the composition FytoSave®
(12.5 g/L COS-OGA). Botrytis cinerea was grown on oat-based
medium (25 g/L oat flour, 12 g/L agar) at room temperature.
Induction of systemic resistance
In Arabidopsis thaliana seedlings
The capacity of HPOs to trigger systemic resistance (SR) was tested onA. thaliana infected by B. cinerea according to the
procedure described in (Ongena et al. , 2000). HPOs were applied
on the root system and the bacteria was inoculated on the leaves in
order to assess the systemicity of the response. Seeds were sterilized
with ethanol (70% v/v) and bleach (15% v/v) before multiple rinsing
with sterile water, sowed in a square Petri Dish filled with agar medium
(6-8 g/L) and transferred to a growth room at 22°C under a 16 h light/8
h dark photoperiod. After one week, seedlings were transferred to a
sterile Araponics system filled with hydroponic solution (5 mL/10 L of
Hydroponic Nutrient Solution 3-part Mix). After approximately 5 weeks in
the growth room, the plants were transferred to 10 mL vials containing
10 mL hydroponic and kept in the dark wrapped in aluminium foil then
transferred to adapt for one day before elicitation. The next day, half
of the plant roots were treated in vials with 10 mL hydroponic solution
supplemented with 20 mM HPOs in 1% DMSO. The other half (control) was
treated with 10 mL hydroponic solution with 1% DMSO. After 24 h, four
leaves of each plant were infected with B. cinerea . A 3-μL
droplet containing 2 500 spores was deposited on the adaxial face of
each leaf. Four days after inoculation, the disease was scored as the
percentage of B. cinerea lesions having extended beyond the
inoculum drop zone to produce spreading lesions (Ongena et al. ,
2000, 2007). Three independent experiments were carried out, with 8
plants per treatment (n=24).
On Solanum tuberosum plants
The capacity of HPOs to trigger SR was tested on Solanum
tuberosum var. Bintje infected by Phytophthora infestansaccording to the procedure described in (Clinckemaillie et al. ,
2017). Plantlets of potatoes were produced in vitro , subcultured
on MS medium, then delicately removed from the media and transplanted
into pots containing sterilized compost. The plants were irrigated daily
with Hoagland’s solution and maintained under greenhouse conditions with
a photoperiod of 16 h at 20/16 °C (day/night temperature). After 4 or 5
weeks’ growth (at the five-leaflet stage), they were separated in
different modalities and transferred in an incubator (Snijders,
LabVision, Germany) to maintain <90% humidity. Plants were
treated with 1% (v/v) of FytoSol® as positive
control, containing 12.5 g L/1 of the active ingredient (COS-OGA), with
13-HPOD or 13-HPOT at a concentration of 50 mM or with water (negative
control plants) by spraying the upper and lower leaf surfaces until
run-off. This was performed at 11, 5 and 1 days before inoculation of
plants with P. infestans . Before and after these treatments, a
high relative humidity was maintained in the incubator to ensure good
adsorption of HPOs/COS-OGA/water by the leaves. Subsequently, the plants
were inoculated with P. infestans by spraying 1 mL of sporangial
suspension (2x104 sporangia mL/1) on each leaf of the
plant using a sterile glass sprayer (Merck). Two independent experiments
were carried out, with 4 plants per treatment (n=8).
Production of
H2O2 by Arabidopsis
thaliana
On plant cell suspensions
Photoautotrophic cell suspensions
from A. thaliana ecotype Landsberg ecotype were cultured on a
rotary shaker at 100 rpm, in Murashige and Skoog (MS) medium (4.4 g/L)
with 0.5 mg/L naphthalene acetic acid, 0.05 mg/L kinetin, pH 5.7 and
maintained at 24°C with approximately 2% CO2 under a 16
h/8 h light/dark photoperiod.
Extracellular
H2O2 production was assessed using
luminol-dependent chemiluminescence on seven-day-old cells directly
after the addition of the elicitors in the growth medium, according to
the method described by Baker and Mock (Baker and Mock, 2004).
Luminescence (relative light units, (RLU)) was measured every three min
for 90 min. Eight technical replicates were carried out for each test
compound and three independent measurements were performed (n=24).
Results were expressed as means ± standard deviations of the area under
the H2O2 production curves. ROS
production values were analysed using Tukey Honest Significant
Differences (THSD) test for multiple comparisons (p values <
0.1).
On foliar discs
A. thaliana plants were grown as described by Smith and Heese
(Smith and Heese, 2014). For all ROS experiments, measurements were
performed on 5-mm disks prepared from leaves from 4-week old plants
using a method adapted from Smith and Heese (Smith and Heese, 2014). The
day before the experiment, disks were placed in water in a 96 wells
plate. The day of measurements, the water was replaced by 150 µL of
treatment solutions (20 µg/mL horseradish POX, 0.2 mM luminol and HPO)
including test compounds. Luminescence (RLU) was monitored every 3 min
for 90 min. Two independent biological repetitions were obtained with
six foliar disks each (6 technical replicates/treatment). Results were
then expressed as means ± standard deviations (n = 12) of the area under
the H2O2 production curves. ROS production values were analyzed using
Tukey Honest Significant Differences (THSD) test for multiple
comparisons (p< 0.05).
Calcein leakage
PLPC/sito/GluCer (60:20:20) small unilamellar vesicles (SUVs) were
prepared as described previously (Deleu et al. , 2013, 2019;
Deboever et al. , 2020c ). PLPC, sitosterol and GluCer in
proportion 60:20:20 were dissolved in a chloroform/methanol mixture
(2/1, v/v). The solvent was evaporated under a gentle stream of nitrogen
to obtain a dried lipid film which was maintained under vacuum
overnight. 10 mM calcein in 10 mM TRIS-HCl buffer pH 7.4 was added to
hydrate the dried lipid film. The lipid dispersion was maintained at
37°C for at least 1 h and vortexed every 10 min. 5 cycles of
freeze-thawing were applied to spontaneously form multilamellar
vesicles. To obtain SUVs, this suspension was sonicated to clarity (5
cycles x 2 min) using a titanium probe with 400W amplitude keeping the
suspension in an ice bath. Finally, generated titanium particles were
removed from SUV solution by centrifuging during 10 min at 6200 rpm. The
unencapsulated calcein was removed from the SUV dispersion by the
Sephadex G65 mini-column separation technique (Fu and Singh, 1999). The
actual phospholipid content of each preparation was determined by
phosphorus assay (Bartlett, 1958) and the concentration of liposomes was
adjusted for each type of experiment to 5 µM in 10 mM TRIS-HCl buffer at
pH 7.4.
Fluorescence was measured as previously described in (Bartlett, 1958)
with a Perkin Elmer (model LS50B) fluorescence spectrometer equipped
with polarizers. Total amount of calcein release was determined by
adding Triton-X100 (0.2%) to a liposome suspension that dissolved the
lipid membrane without interfering with the fluorescence signals. The
emission and excitation wavelengths were set at 517 nm and 467 nm,
respectively. A fluorescence signal of 750 µL of SUV was first recorded
as a baseline, followed by the addition of 13-HPOD/T (at t=30 sec) in 7
different concentrations while continuing the recording for 900 s. The
amount of calcein released after time t was calculated according to
(Shimanouchi et al. , 2009):
\begin{equation}
\text{RF}\ \left(\%\right)=100\ \frac{(I_{t}-I_{o})}{(I_{\max}-I_{o})}\nonumber \\
\end{equation}where RF is the fraction of calcein released, Io,
It and Imax are the fluorescence
intensities measured at the beginning of the experiment, at time t and
after the addition of 0.2% Triton X-100, respectively. All experiments
were carried out at least three times, each time with freshly prepared
SUVs.
Laurdan generalized
polarization
For Laurdan generalized polarization experiments, multilamellar vesicles
(MLVs) were prepared based on (Parasassi and Gratton, 1995; Deboeveret al. , 2020c ). PLPC, sitosterol and GluCer in proportion
60:20:20 were dissolved in a chloroform/methanol mixture (2/1, v/v).
HPOs were added to the lipid mixture to reach a lipid:HPO molar ratio of
5:1. The solvent was evaporated under a gentle stream of nitrogen to
obtain a dried lipid film which was maintained under vacuum overnight.
The resulting film was hydrated with 10 mM Tris-HCl buffer at pH 7.4
prepared from Milli-Q water and 1 µL of Laurdan solution prepared in
DMSO was added to reach a final concentration of 5 nM. The lipid
dispersion was maintained at a temperature well above the transition
phase temperature of the lipid for at least 1 h and vortexed every 10
min.
Fluorescence of Laurdan in MLVs was monitored at various temperatures
(between 20 and 50°C by steps of 5°C) with a Perkin Elmer LS50B
fluorescence spectrometer. Samples were placed in 10 mm pathlength
quartz cuvettes under continuous stirring and the cuvette holder was
thermostated with a circulating bath. Samples were equilibrated at each
temperature for 10-15 min prior to the measurements.
The excitation wavelength was set to 360 nm (slit = 2.5 nm), and at
least 10 measurements of emission intensities at 440 nm and 490 nm were
recorded and averaged for each sample and the blank (DMSO) at each
temperature. An emission spectrum from 400 nm to 600 nm (slit = 4.5 nm)
was also recorded for each sample-temperature combination. Generalized
polarization (GP) of Laurdan was then calculated according to (Parasassiet al. , 1992; Harris et al. , 2002):
\begin{equation}
\text{GP}=\ \frac{I_{440}-I_{490}}{I_{440}+\ I_{490}}\nonumber \\
\end{equation}where I440 and I490 are the
blank-subtracted emission intensities at 440 nm and 490 nm,
respectively. All experiments were carried out at least three times,
each time with freshly prepared MLVs.
Neutron reflectometry
Neutron reflectometry (NR)
measurements were performed at the MARIA neutron reflectometer (Mattauchet al. , 2018) operated by Jülich Centre for Neutron Science at
Heinz Maier-Leibnitz Zentrum in Garching (Germany) while using custom
temperature-regulated (through a connected Julabo F12-ED circulator)
liquid cells (Koutsioubas, 2016). Two different wavelengths were used,
10 Å for the low-q region and 5 Å for the high-q region, and the
reflected intensity has been collected at different angles, up to 0.25
Å−1 q values, with a 10% wavelength spread. Using a
peristaltic pump combined with valves (flow rate ~0.5
mL/min) solvent exchange was possible without moving the measuring cells
from the instrument.
Specular NR measures the thickness and scattering length density (SLD)
profile of layered structures along the surface normal (z). The SLD
distribution along the normal, represented as ρ(z), is specific of the
chemical composition of materials along the normal and depends on the
coherent nuclear scattering lengths (bi) of its
constituent atoms and their number density along the normal
(ni (z)) so that ρ(z) = ∑ibi ni (z). In reflectivity data
measurements, the intensity of reflected neutrons is recorded relatively
to the incident beam as a function of the momentum transfer vector (q =
4πsin θ/λ), where θ is the incidence angle and λ the wavelength of
incident neutrons. The variation of reflectivity as a function of
momentum transfer
R(qz) is related
to the square modulus of the one-dimensional Fourier transform of the
SLD profile along the normal to the interface (ρ(qz))
through the relation:
\begin{equation}
R(q_{z})\ \sim\ (16\pi\ /q_{z}\ )|\rho(q_{z})|\nonumber \\
\end{equation}Following the characterization by neutron reflectivity of
silicon/solution interface, we deposited by vesicle fusion the membrane
of interest (Koutsioubas et al. , 2017). After its full
characterization in 150 mM NaCl solutions in D2O and
H2O, 2 µg of 13-HPOD/T, deuterated or not depending on
the membrane studied, were injected in the measuring cell (6 mL total
volume), to a final concentration lower than their critical micelle
concentration (CMC = 25.4 ± 1.9 μM and 24.0 ± 1.3 μM for 13-HPOT and
13-HPOD, respectively, according to (Deleu et al. , 2019).
Reflectivity was measured after letting the systems equilibrate for 1 h,
again in the two contrasts condition with 10 mM Tris (pH 7.4).
To analyse the specular reflection data, the interface is modelled as a
series of parallel layers where each layer is characterized by an
average SLD and a thickness. Based on these parameters, a model
reflectivity profile is calculated by means of the optical matrix method
(Névot and Croce, 1980). The interfacial roughness between two
consecutive layers is included in the model by the Abeles method, as
described by Nevot and Croce (Névot and Croce, 1980).
Finally, the calculated model
profile is compared to the measured profile and the quality of the fit
is assessed by using the χ2 in minimum-squares method.
Errors on parameters values have been estimated from the maximum
variation in the acceptable fit subject to the constraints of space
filling and stoichiometry. NR is a technique suited to collect
structural information about the different layers of the studied
membrane (Rondelli et al. , 2019). Thus, the silicon support and
the bulk water are seen as bulk infinite layer, the silicon oxide layer,
the water layer between the silicon oxide and the membrane and the
diverse hydrophilic/hydrophobic layers of the lipid membranes are
modelled as defined layers with a proper thickness, roughness with
respect to the previous layer, compactness, composition and consequently
contrast. Supported lipid bilayers (SLB) were formed using both the same
lipid mixture as previously (PLPC/sito/GluCer in molar ratio 60/20/20)
and d62DPPC. Injections were done at 47°C and
measurements at room temperature (RT). The reflectivity profile of the
silicon support and of the samples has been measured in different
contrasts (H2O and D2O) and data
analysis was performed with the fit program MOTOFIT (Nelson, 2006). SLD
used for the specific components are reported in Supplementary DataTable S1 .
Atomic force microscopy
To probe the nanoscale effects of HPOs on lipid membranes, SLB (ternary
mixture of PLPC/sito/GluCer (60/20/20)) were reconstructed on freshly
cleaved mica substrates by allowing the fusion of a 2 mM lipid vesicles
solution (V = 100 µL) at 55°C for 45 min. Samples were then left for
thermalization at room temperature for 30 min without dewetting and
immersed in 3 mL Tris buffer (pH 7.5).
To avoid damaging the samples, atomic force microscopy (AFM) images were
obtained in the quantitative imaging (QI) mode of a JPK Nanowizard III
setup, with a minimal applied force of 200 pN and a speed of 50 µm/s.
Soft sharpened silicon nitride cantilevers (MSCT, Bruker) were used and
calibrated before any experiment using the thermal noise method (k
~ 0.02 N/m). HPOs, prepared in Tris buffer, were
injected to reach a final concentration of 3 µM below their critical
micellar concentration. AFM images were then recorded at different time
points in different areas to follow the HPOs impact on the lipid
bilayer.