Want to develop quality science in plant pathology while contributing to something useful for farmers? We are looking for a talented student, to decipher the modes of action of a promising biocontrol antifungal: PhD position in; Toward the understanding of the in vivo mode of action of the 4-phenylbutyric acid, a promising biocontrol molecule with antifungal properties toward Zymoseptoria tritici
Since the 50’s, crop protection against cryptogamic diseases, aiming at securing feed, food, fuel and fibers production, has relied mostly on chemical control. However, in some instances, this has led to the selection of resistant pathogens within populations, resulting in the increase in pesticide use and subsequent breaches.
This situation is a genuine source of concern given the possible toxicity of such chemicals for users, consumers and the environment. Facing this challenge, agroecology promotes the use of alternative solutions, resistant cultivars and biocontrol compounds, some of them stimulating plant immunity. The spatio-temporal combination of the various pathogen control methods used in agricultural systems could, indeed, increase their overall sustainability.
Even though numerous biocontrol solutions have been authorized in France over the past few years, only a few of them aim at controlling fungal diseases, and they generally exhibit variable or weak field efficiency on their own. Thus far, only eight active substances have obtained a market approval as bio-fungicides of the category “Natural substances” and specific options for fungal diseases that affect arable crops are especially lacking. This lack of biocontrol products may be tentatively explained by a poor understanding of their mode-of-action (MoA) at the molecular level, which could prevent their informed improvement.
Indeed, elucidating these underlying molecular mechanisms in both the fungi and plants should help not only improving the use of biocontrol molecules (e.g. optimizing doses, application timing, product formulation), but also assessing risks associated with their use in agroecological systems (e.g. some pests can develop resistance to some natural toxins).
This project aims at studying the biocontrol molecule, 4-phenylbutyric acid (4-PBA), which is produced by telluric and oceanic bacteria, such as Bacillus megaterium and Bacillus pumilus, respectively, for which we patented the use to cope with plant bacterial and cryptogamic diseases.
Our preliminary experiments showed that 4-PBA is able to protect tomato, grapevine and Arabidopsis thaliana plants against Botrytis cinerea. Direct in vitro toxicity was also demonstrated for fourteen additional pathogenic fungi and oomycetes, including Zymoseptoria tritici known as the most severe fungal pathogen on wheat worldwide. In addition, 4-PBA shows a bacteriostatic activity associated with a Plant Defense Stimulation (PDS) activity, proven efficient in planta.
Although 4-PBA has been extensively studied in mammalian systems, its MoA still remains unclear in plant and fungi. In our hands, 4-PBA behaves as a promising powerful bio-fungicide with a broad spectrum of action, with a direct and an indirect MoA, which might guarantee its sustainability for use in agricultural systems. In parallel with this research project, the 4-PBA is currently undergoing operational development by a private collaborator. Interactions between the two projects are expected.
Objectives: In a context where sustainable and efficient biocontrol solutions are expected, the aims of the PhD student will be to:
(1) Elucidate the molecular mechanisms responsible for the fungitoxicity of 4-PBA using Zymoseptoria tritici as model (direct mode of action)
(2) Confirm, decipher and optimize the PDS activity of 4-PBA in wheat plants infected with Zymoseptoria tritici (indirect mode of action)
(3) Find out more about the operational potential of 4-PBA for practical use.
This thesis project encompasses three inter-dependent tasks, mirroring the two objectives previously mentioned. The Z. tritici / wheat model was preferred, among several options, because of its economic relevance for the development of biocontrol fungicides and because it was the only one for which all technical resources were already available.
Task 1– Deciphering molecular mechanisms responsible for the biofungicide activity of 4-PBA on a model species. (University of Exeter/ INRAE BIOGER) Three parallel approaches will be developed, to identify, or at least refine 4-PBA target.
– A cellular biology approach. The PhD candidate will assist our partner at University of Exeter (UK) in understanding the cellular pathways and proteins inhibited by 4-PBA using a live cell imaging approach. After determining sub-lethal concentrations of 4-PBA by live/dead staining for the IPO-323 reference strain, he/she will examine and quantify by live cell imaging the effects of 4-PBA on various structures and organelles (endoplasmic reticulum, plasma membrane, cytoskeleton, secretory pathway, etc…). If appropriate, impacts of 4-PBA on Z. tritici will be confirmed using electron microscopy and biochemical assays. This first set of data will provide information as to whether 4-PBA likely acts on multiple subcellular sites or could target only one site.
– A transcriptomics approach. A comparative transcriptomic approach (RNA sequencing) will be undertaken to gather more insights into 4-PBA MoA. Sensitive and 4-PBA resistance isolates will be grown in vitro in the presence of a sub-lethal concentration of the molecule. Comparing the dynamics of the transcriptomic response to the untreated control will unravel the cellular response to its fungitoxic action. If relevant, changes in expression of interesting candidates will be confirmed by RT-qPCR at different time points or concentrations.
– A genome sequencing approach. The PhD candidate will Illumina-sequence the genome of 4-PBA resistant strains previously generated by the team after directed selection. He/she will compare them to that of the parental strain IPO-323, whose genome is fully sequenced. He/she will screen variations in genomes from resistant isolates, which ultimately will allow to identify mutation(s) responsible for resistance, possibly in the gene encoding or regulating the target.
Task 2– Confirming the plant defense stimulating activity of 4-PBA on the wheat plant model:
The purpose of this task is to characterize and elucidate the indirect MoA of 4-PBA at the molecular level by investigating its PDS activity using the model interaction Z. tritici / wheat.
The phytotoxicity of 4-PBA will be preliminarily evaluated via the measurement of ecophysiological parameters (biomass, plant height, leaf number, cell death assays, etc…) in wheat during the course of its development in greenhouses and according to the 4-PBA concentration and its application procedures.
Based on these data, the optimal conditions will be chosen for the following experiments.
The PDS activity of 4-PBA will be probed by measuring its effects at the local and systemic scales on plant defense mechanisms, and especially by changes in hormonal levels (JA, SA, ABA, ethylene, auxins and cytokinins quantified by HPLC-MS/MS) and in marker gene expression (PR1, PDF1-2, LOX2 to name a few, measured by RT-qPCR), after preventive or curative treatments with 4-PBA, in plants infected or not by Z. tritici.
These results will be analyzed with regards to symptom severity of septoria leaf blotch, quantified by means of the in planta fungal genomic DNA concentration (qPCR) and by means of the symptoms measurement. To help assessing systemic effects, the concentration of 4-PBA and its derivatives present in plant tissues will also be determined using a HPLC-based method.
Task 3- Towards practical use of 4-PBA
In order to anticipate the practical use of 4-PBA, the PhD candidate will compare its efficacy in vivo and in planta with that of biocontrol fungicides (currently available), on a range of Z. tritici strains susceptible and resistant to synthetic fungicides (notably generalist resistances). He/she will also assess the toxicity of 4-PBA, at operational doses, to a number of microorganisms commonly used in biocontrol, as well as to daphnia (a common marker of ecotoxicity; subcontracting to be organized).
Master’s 2 or engineering degree in microbiology, mycology, plant pathology or agronomic sciences.
Scientific and technical skills in microbiology, molecular biology, bioinformatics. Initial experience in fungal genomics would be a plus.
The application should include the following information:
- Detailed CV
- Letter of motivation
- Master 1 and 2 transcripts
- Contact details of two referees and, if possible, a letter of recommendation
Applications should be sent by email to Anne-Sophie Walker (firstname.lastname@example.org)
The doctorate will ideally begin on September 1st 2023, but this date is flexible.
The candidate will be enrolled in the “Plant Sciences: From genes to ecosystems” doctoral school of the University of Paris-Saclay (https://www.universite-paris-saclay.fr/en/doctoral-schools/plant-sciences-genes-ecosystems).
The student will be based in the “Biologie et Gestion du Risques” (BIOGER) laboratory and especially in the AMAR team, on the INRAE Palaiseau (91120) campus (https://www6.versailles-grignon.inrae.fr/bioger). Some experiments will be carried out at the “Institut Jean-Pierre Bourgin” (IJPB), on the INRAE Versailles (78000) campus (https://ijpb.versailles.inrae.fr).
The thesis scholarship is already acquired and is funded by the Ecophyto Plan and the Plant Health Division of INRAE.
Vasselon D, Walker AS, Poinssot B, Lamotte O, Cacas JL (2020) Utilisation de l’acide 4-phénylbutyrique et/ou de l’acide 3-phénylbutyrique et/ou de l’acide 2-phénylbutyrique pour la prévention et le traitement des maladies cryptogamiques. DI-RV-1900-48RS, numéro de dépôt : FR2005221.
Cacas JL, Champion A (2012) Utilisation de l’acide 4-phenylbutyrique pour améliorer la tolérance des plantes aux bioagresseurs. Référence : BFF120267/BA, numéro de demande : 1256660 ; Référence : WO2014/009402A1, numéro de demande : PCT/EP2013/0645/47.
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