Results
In planta protective effect of HPOs
against
phytopathogens
The
capacity of HPOs to induce SR in A. thaliana against B.
cinerea was tested under controlled conditions by treating plant roots
with HPO solutions and inoculating plant leaves with the pathogen. The
disease severity with HPO treatment was measured after 4 days, compared
to controls (treatment with water containing 1% DMSO) and we attributed
a disease severity scores from 1 to 4 representing no symptoms (score 1)
to full development of the disease (score
4).
Treatment with both HPOs provided similar obvious disease reduction as
shown by the decreased size of the lesions (Figure 1 ). About
40% of the plants had no symptoms which leads to disease severity
scores mainly between 1 and 2. Another host-pathogen couple
(Solanum tuberosum var. Bintje infected byPhytophthora infestans) was also considered in order to check if
the protective action of HPOs is pathosystem-dependent. It was found
that 13-HPOT offers more than 50% protection against the pathogen (seeFigure S1 ).
Given
the experimental design (treatment and infection on two different plant
organs or upstream of an infection), this cannot be a direct biocidal
effect but rather a systemic signalling in the plant. One can therefore
wonder about the signalling mechanism initiated by HPOs and more
particularly their initial perception by the plant cells and the
responses they induce. HPOs were efficient in both pathosystems studied,
suggesting that the mechanism of perception is not dependent on specific
recognition receptors.
Perception of HPOs and early defenses responses
activation
Very often, ROS production is a biphasic process with a first transient
phase within minutes after the infection and a second more intense and
sustained phase that can last for many hours (Wang et al. , 2019).
This first wave, which is linked to the activation of early defence
responses, has been investigated to determine whether the two HPOs are
perceived by plants and can induce an immune response.
Cell suspensions cultures are a valuable model system for studying
elicitor-induced defence reactions in plants and they easily allow
studying early signalling events like oxidative bursts (Khonon et
al. , 2011; Jogaiah et al. , 2019). Here, photoautotrophicA. thaliana cell suspensions were used to detect
H2O2 production after treatment with
HPOs. Extracellular ROS accumulation was detected using a luminol-based
assay (van Aubel et al. , 2016; Monnier et al. , 2018).
The elicitor
FytoSave® was used as positive control as its active
substance, COS-OGA made of pectin-derived oligogalacturonides (OGA) and
chitooligosaccharides (COS) (Cabrera et al. , 2010), is known to
induce a significant production of extracellular ROS at a concentration
of 25 ppm (Ledoux et al. , 2014; van Aubel et al. , 2016,
2018). In our experiment, a range of six different concentrations
(0.5 µM to 100 µM) was tested and ROS production was monitored for 90
min.
The
ROS production after 13-HPOD or 13-HPOT treatment was
concentration-dependent (Figure 2A). It was higher than the
negative control but also higher than the positive control, for
concentrations 50 µM and 100µM. In those cases, the response to 13-HPOT
treatment is higher than the 13-HPOD one. Also, the oxidative burst peak
occurred quicker (30 min instead of 40 min) for 13-HPOT than for 13-HPOD
(Figure 2B). The same conclusion was drawn from experiments
performed on foliar disks of A. thaliana , a plant model closer to
the reality (see supplementary data – Figure S2 ).
Comparatively to COS-OGA, the kinetics of ROS production induced by HPOs
in plant cells was slower (within 5 min only for COS-OGA vs 30-40
min for HPOs) but the oxidative burst lasted longer with a total
duration of 60-70 min before returning to the basal level. Such long
lasting response profile was also observed with synthetic RL bolaforms,
for which it was suggested that their perception occurred via the lipid
fraction of the plasma membrane (Bahar et al. , 2016;
Luzuriaga-Loaiza et al. , 2018; Come et al. , 2021). On the
contrary, the elicitor flagellin, known to be perceived by membrane
pattern recognition receptors (Meindl et al. , 2000; Smith and
Heese, 2014), induces a quicker oxidative burst initiated within 4-6 min
with a peak at ~10min (Yu et al. , 2017),
similarly to the OGA in the COS-OGA composition (van Aubel et
al. , 2013; Smith and Heese, 2014).
Moreover,
the induction of ROS production occurs at concentrations much higher
than concentrations usually active when a proteic receptor is involved
as for flagellin which still binds to its receptor at femtomolar
concentrations (Meindl et al. , 2000; Zhao et al. , 2010).
The observation of different kinetic profiles and different
concentration ranges for HPOs comparatively to classical elicitors would
suggest that a proteic receptor is not directly involved in their
recognition. Due to the ability of HPOs to interact with PPM lipids
(Deleu et al. , 2019), we hypothesized that the HPOs would rather
be recognized via the lipid phase of the membrane.
Changes of PPM biophysical properties induced by
HPOs
The interaction of HPOs with PPM
characteristic lipids has already been found to modify the lateral
organization of membrane bilayer in terms of lipid domain size and
distribution (Deleu et al. , 2019). It is also known that the
plant sphingolipid GluCer is a privileged partner for the interaction
and that 13-HPOT has a higher interaction affinity than 13-HPOD.
In the present study, further analysis of the effects of HPOs
interaction with lipids on PPM structure was carried out. Simplified
biomimetic models with two different lipid compositions were studied,
the first mimicking the PPM, namely PLPC:sito:GluCer (60/20/20), and the
second made of d62DPPC, a classic deuterated model.
Effect of HPOs on membrane
permeability
First, we have investigated the ability of HPOs to permeabilize model
membranes by measuring the release of calcein. If the membrane is
permeabilized by a bioactive molecule, the self-quenched calcein
initially encapsulated within the LUV is released into the external
medium and gives rise to an increase of fluorescence emission.
Very little permeabilization
effect was observed (values less than 10%) for both HPOs on the
PLPC:sito:GluCer membrane model (Figure S3 ) suggesting that
HPOs would not derive their mode of action from a mechanism of
solubilization of the membrane or pore formation, but rather a more
subtle modification of the membrane organization that could lead to the
activation of a signalling cascade.
Change in bilayer fluidity induced by
HPOs
The effect of HPOs on the bilayer
fluidity was investigated by monitoring the lipid phase-dependent
emission spectrum shift of Laurdan, a fluorescent probe that readily
locates at the hydrophilic/hydrophobic interface of bilayers (Harriset al. , 2002; Sanchez et al. , 2007). Its fluorescence
depends on the physical state of the environment. When present in a gel
phase bilayer, its maximum fluorescence intensity is close to 440 nm
emission wavelength. When the bilayer is in a fluid state, the Laurdan
maximum fluorescence is observed at higher wavelengths (around 490 nm).
This ”red-shift” phenomenon is due to a higher quantity and mobility of
the water molecules located around the probe. This is directly related
to the lower order within the bilayer and is measured by the Generalized
Polarization (GP): a decreasing GP value corresponds to a higher
fluidity of the bilayer (Parasassi et al. , 1991). This method has
been previously applied for investigating the effect of drugs, natural
herbicides or other elicitors on lipid membrane organization (Deleuet al. , 2013; Sautrey et al. , 2014; Lebecque et
al. , 2019; Furlan et al. , 2020).
The effect of HPOs on PLPC:sito:GluCer MLV membrane fluidity was
investigated for a range of temperatures from 20°C to 50°C
(Figure 3 ). In presence of 13-HPOT, the Laurdan GP values
decreased significantly compared to those observed on pure MLVs with no
significant effect of temperature. This indicates a fluidifying effect
of 13-HPOT on the bilayer. On the contrary, 13-HPOD did not induce
significant change in lipid order at any temperature as its curve almost
superimposes to that of pure PLPC:sito:GluCer vesicles.
Effect of HPOs on the bilayer transversal
structure
The effect of HPOs on the
transversal structure of PPM was analysed by NR, a technique of choice
to study the transverse structure of layered samples within a few Å
resolution (Mattauch et al. , 2018) and to evidence the structural
effects of the interaction of incoming molecules on biological membranes
(Rondelli et al. , 2016, 2018). Neutrons interaction with matter
depends on the isotopic species. Therefore, neutron-based experiments
can profit by the use of deuterated molecules to enhance the visibility
of molecules within a mixed complex system. As the lipids representative
of the PPM are not commercially available in their deuterated form,
d62DPPC was used to form SLB and to highlight the
presence and location of the H-bringing HPOs within the membrane.Figure 4A-D show the reflectivity curves together with their
fittings in two contrasts and the corresponding fit parameters are
summarized in Figure 4E . NR spectra were not drastically
changed by the addition of HPOs. However, the data analysis revealed
that HPOs always insert into the outer hydrophilic leaflet of the
d62DPPC SLB without flipping into the inner layer
attached to the silicon block (this is evident from the variation of the
external polar leaflet SLD and roughness, as reported in Figure 4E). A
slight modification of the SLD profiles was observed while adding
13-HPOT but not with 13-HPOD. This gave rise to a small but significant
decrease of the membrane thickness (approximately 2 Å) and roughness
without modification of the solvent penetration and of lipid chains SLD,i.e ., no alteration of the bilayer nor external molecules deep
penetration. To confront these results, obtained on a
d62DPPC bilayer, to a more realistic PPM model, another
experiment was performed with the deuterated version of 13-HPOD and the
ternary mixture of non-deuterated lipids representative of PPM. It
confirms that 13-HPOD interacts with PPM SLB and localizes on top of the
outer leaflet without no major change of the membrane organization as
observed from SLD profile and NR spectra (Figure S4 andTable S2 ).
Lateral erosion of plant lipid bilayers by
HPOs
To further analyse the effect of
HPOs on the lipid bilayer organization, atomic force microscopy (AFM)
was used to investigate their impact on the lateral nanoscale morphology
of SLB. As shown in Figure 5A , the ternary mixture of plant
lipids reconstituted in SLB did not reveal any phase separation within
the thickness resolution limit of AFM (0.1 nm), rather homogeneous
smooth patches (bright areas) distributed on the mica (dark areas).
Though large patches were mainly found to cover the entire scanned area,
defects in the SLB were used as a “visualization control” to confirm
the presence of the lipid bilayer. Its thickness of ~4-5
nm, determined by measuring section profiles on the AFM height images,
is in agreement with previous studies (Figure 5B ) (Dufrêne and
Lee, 2000; Mingeot-Leclercq et al. , 2008). The presence of the
lipid bilayer was further confirmed by recording AFM force curves on
areas of high vs low heights. A typical breakthrough of the lipid
bilayer in the bright areas was observed while no such force signature
was found in dark areas without lipid bilayers and associated with mica
(Figure 5C ).
After confirming the presence of the SLB, the sample was incubated with
either 13-HPOT or 13-HPOD and AFM images were recorded every 10-15 min
on a defined area. Incubation 13-HPOT or 13-HPOD resulted in a
time-dependent alteration of the lipid patches (Figure 5D ).
Results showed that very small SLB patches (green arrows) disappeared
after the addition of 13-HPOT, but that it had not a drastic impact on
the large ones. On the contrary, 13-HPOD completely removed part of a
large angular domain after 75 min (see green arrows). Nonetheless, after
75 min treatment with 13-HPOT, most of the lipid domains were thinner by
approximately 2 nm as compared to the initial ones, suggesting that
13-HPOT flattened lipids or part of the upper leaflet in a time- and
zone-dependent way, which was not observed for 13-HPOD (Figure
5E-F ).
In brief, AFM studies revealed three major effects of HPOs on plant
mimetic lipid bilayers (i) ”erosion” of angular protrusions of large
lipid domains, (ii) total erosion of small domains, and (iii) reduction
in the thickness of the bilayer between 0.5 and 2 nm. 13-HPOT has also a
greater effect on membrane organization and bilayer thickness than
13-HPOD.