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.