General discussion and
conclusion
In the present study, we show for
the first time the potential of the exogenous application of
acyl-hydroperoxides 13-HPOD and 13-HPOT to protect plants against
phytopathogens. Both forms of HPOs applied on A. thaliana roots
strongly reduce the size of the lesions further to the inoculation ofB. cinerea on leaves. The protection effect without direct
contact with the phytopathogen suggests their capacity to stimulate the
plant immune system. The fact that a significant protection was also
obtained on potatoes, infected by the causal agent of late blight,
settles their effectiveness and indicates an aspecific mode of action.
Other phyto-oxylipins, such as the jasmonic acid precursor
12-oxo-phytodienoic acid and an ⍺-ketol of octadecadienoic acid were
recently identified as mobile signals responsible of ISR, originated in
the plant roots and travelling into the plant vasculature (Wang et
al. , 2020). Our results show that exogenous application of 13-HPOD and
13-HPOT on A. thaliana cell suspension induces an importantin vitro oxidative burst, known as one of the hallmarks of
elicitor recognition by the plant cells
(Yu et al. , 2017).
Beyond their role as signals, we
thus clearly demonstrate that HPO are recognized by the plant cells
triggering a signalling cascade leading to SR and plant protection
against pathogens.
However,
it remains essential to determine whether other immune responses (such
as expression of genes from JA/SA or PTI pathways) are generated in
leaves when roots are treated with HPOs. Moreover, we cannot exclude
that oxylipins might be transported in the xylem of plants, as traces of
oxylipins have been found in aphids gut after ingestion of the phloem
sap (Harmel et al. , 2007). Nothing is known about the xylemic
systemicity of oxylipins but, as there are not soluble in water, this
seems unlikely. The possibility that HPOs application in roots just
primer the plant immunity in shoots, which initiate stronger immune
response when true elicitors from pathogens are perceived, can also not
be ruled out. Their effectiveness in the case of a local immune
induction is also not known.
The extracellular ROS production is initiated earlier and lasts longer
that the one observed with well-known proteic elicitors like flagellin
for which recognition phenomenon involves direct interaction with
membrane proteic receptors (Gómez-Gómez and Boller, 2002). But the
kinetic profile and the active concentration range are similar to the
one observed for other amphiphilic lipid elicitors like surfactin and
RLs (Aranda et al. , 2007; Jourdan et al. , 2009; Henryet al. , 2011; Ma et al. , 2017; Luzuriaga-Loaiza et
al. , 2018) for which a mechanism linked to the perception by the lipid
of the plasma membrane was suggested. From our previous work (Deleuet al. , 2019), we know that HPOs can interact with the lipid
fraction of PPM. In the present study, NR analyses show that both HPOs
are more preferably inserted in the outer leaflet of the bilayer. This
interaction modifies the global morphology of the bilayer as shown by
AFM where bilayer erosion is observed for both HPOs. 13-HPOT has a
higher impact on the PPM structure, but does not affect the integrity of
the membrane according to the calcein release assays. Its insertion
further reduces the thickness of the bilayer according to the NR and AFM
data and fluidifies it more according to the Laurdan GP data than
13-HPOD. This difference between 13-HPOT and 13-HPOD could be explained
by the presence of an additional double bond in 13-HPOT which gives it a
greater structure rigidity. We postulate that this might force the
lipids of the membrane outer leaflet to further reorganize compared to a
more flexible molecule like 13-HPOD, and consequently could have a
stronger impact on the dynamics of the membrane. The higher binding
affinity of 13-HPOT compared to 13-HPOD for PPM bilayer (Deleu et
al. , 2019) could also enhance this reorganization effect.
The relationship between the higher ROS production and the higher impact
on PPM lipid bilayer structure for 13-HPOT compared to 13-HPOD is in
favour of our hypothesis that the PPM lipid fraction plays a key role in
the recognition of HPOs giving rise to plant defence mechanisms.
In
the study of Sandor et al. (2016), it is demonstrated on A.
thaliana and tobacco cells, that the induction of ROS by various
elicitors including cryptogein, flagellin and an oligosaccharide, is
concomitant to the increase in the relative proportion of membrane
ordered domains (Sandor et al. , 2016). According to them, the
recognition of the elicitor at the plasma membrane level triggers the
production of ROS which in turn reorganizes the membrane leading to an
increase of ordered domains. But in the case of cryptogein, they have
also suggested an inverse event sequence. Although cryptogein is known
to trigger immune response, including ROS production, through the
PPM-resident ELR-BAK1 receptor complex (Du et al. , 2015), its
capacity to interact with membrane sterols and to mechanically trap them
was also demonstrated (Gerbeau-Pissot et al. , 2014). The latter
phenomenon was shown to induce a higher membrane fluidity stimulating
ROS production (Sandor et al. , 2016). In agreement with this
study, our results suggest that elicitors that directly act on membrane
lipid dynamics and more particularly on the membrane fluidity are able
to trigger early defence events like ROS production. But the complete
molecular mechanistic view between the change of the membrane structure
and the occurrence of the oxidative burst is not yet identified. The
formation of specific membrane lipid domains recruiting key signalling
proteins (Gronnier et al. , 2018) could be implicated. From our
previous studies (Deleu et al. , 2019; Deboever et al. ,
2020c ), we also know that HPOs modify the organisation of lipid
domains and that plant membrane sphingolipids are privileged partners
for HPO interaction. Therefore, the presence of
glycosyl-inositol-phosphoryl-ceramides (GIPCs), the plant sphingolipids
exclusively located in the outer leaflet of PPM and involved in the
inter-leaflet coupling (Gronnier et al. , 2016), could also play a
role in the signal transduction.
In addition to their eliciting activity evidenced in the present study,
HPOs also retain some antimicrobial activity against various
phytopathogens (Deboever et al. , 2020c ). The dual effect
of HPOs as well as the possibility to produce them at low cost
(Fauconnier and Marlier, 1996) make them attractive compounds to be used
as alternative to conventional pesticides for plant protection.
Author Contributions: Experiments design, E.D., M.D., V.R.,
A.K., M.M.M., M.O. and G.V.A..; experiments and data analysis, E.D.,
M.D., M.M.M., V.R. and G.V.A.; HPOs synthesis, and purification, E.D.;
writing—original draft preparation, E.D.; writing—review and
editing, all authors.
Data availability statement : The data supporting the findings
of this study are available from the corresponding author, Magali Deleu,
upon request.
Funding: E.D. is supported by a « Fonds pour la formation à la
Recherche dans l’Industrie et dans l’Agriculture » (FRIA) grant
(5100617F) from the FRS-FNRS (Fonds National de la Recherche
Scientifique, Belgium). M.D., M. O. and L.L. thank the FRS-FNRS for
their position as Senior Research Associates and for grant CDR
(J.0014.08 and J.0086.18 projects). Work at the Université Catholique de
Louvain was supported by the National Fund for Scientific Research
(FNRS) and the Research Department of the Communauté Française de
Belgique (Concerted Research Action). Y.F.D. is a Research Director at
the FNRS. This research was funded also by the ‘Medical Biotechnologies
and Translational Medicine Department’ of the ‘Università degli Studi di
Milano’, grant number ‘PSR2018’ to V.R. This article was published with
the support of the “Fondation Universitaire de Belgique”.
Acknowledgments: The authors thank the financial support via
the project from University of Liège (ARC-FIELD project 13/17-10).
Authors also thank beamline MARIA Jülich Centre for Neutron Science
(JCNS) at Heinz Maier-Leibnitz Zentrum (MLZ, Garching, Germany) for
allocation of beamtime. Acknowledgements are also due to FytoFend’s
research team for their logistic support in bioassays. Final thanks to
Jelena Prisc for technical support and nice discussion on ISR and ROS
experiment data measured in planta .
Conflicts of Interest: The authors declare no conflict of
interest. The funders had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the
manuscript, or in the decision to publish the results.