Thèse Contribution de l'Autophagie Végétale à la Nutrition Azotée Lors de la Symbiose Maïs-Mycorhize Arbusculaire H/F - 75

Établissement : Université Paris-Saclay GS Biosphera - Biologie, Société, Ecologie & Environnement, Ressources, Agriculture & Alimentation
École doctorale : Sciences du Végétal : du gène à l'écosystème
Laboratoire de recherche : IJPB - Institut Jean-Pierre Bourgin-Sciences du Végétal
Direction de la thèse : Alia DELLAGI ORCID 0000000296299187
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-05-06T23:59:59La symbiose mycorhizienne à arbuscules (SMA) est une association mutualiste répandue impliquant des champignons mycorhiziens arbusculaires (CMA) et 80 % des plantes terrestres. La SMA est reconnue pour améliorer la nutrition phosphorée (P) et azotée (N) des plantes. Cependant, le remodelage et le fonctionnement du métabolisme azoté des plantes au cours de la SMA restent mal connus. Le maïs, culture la plus productive au monde, nécessite d'importants apports d'azote pour atteindre les rendements escomptés. Des analyses transcriptomiques et métabolomiques, combinées à une modélisation métabolique mathématique, ont révélé l'activation de gènes liés à l'autophagie ainsi que des modulations significatives du métabolisme des nucléotides et des uréides au cours de la SMA chez le maïs. Notre principale hypothèse suggère que les CMA orchestrent un remodelage du métabolisme azoté des plantes en manipulant l'autophagie, ce qui pourrait amplifier la croissance des plantes via le recyclage de composés riches en azote tels que les acides aminés et les nucléotides. Les objectifs du projet de doctorat seront d'explorer la contribution de l'autophagie à l'absorption et au métabolisme de l'azote pendant la fermentation malolactique en utilisant du maïs sauvage et des mutants d'autophagie du maïs.

Maize is currently the most productive cereal crop worldwide (www.faostat.org) and its production relies on the use of large amount of N fertilizers. As most terrestrial plant species, maize associates with AMF to form a mutualistic interaction that improve maize N nutrition and that represents thus an interesting potential to reduce N input in agriculture. AMS involves the establishment of nutrient exchange interfaces between partners called arbuscules and these symbiotic structures are surrounded by a plant-derived membrane called peri-arbuscular membrane. At this interface, nutritional exchanges occur: carbon-containing molecules derived from the photosynthesis, such as hexoses and lipids, feed AMF, and in return the AMF provide minerals, especially N and P coming from the extraradical mycelium (MacLean et al., 2017; Choi et al., 2018). Molecular mechanisms underlying the plant nutritional benefit mediated by the AMS in terms of N uptake remains poorly understood (Dellagi et al., 2020; Gojon et al., 2022; Xie et al., 2022). In the team, we performed a molecular and physiological characterization of maize plants inoculated or not with the AMF strain Rhizophagus irregularis DAOM 197198 (R. irregularis) and we found that the AMS relieves the N deficiency stress and profoundly remodels maize N metabolism and transport (Decouard et al., 2023, BioRxiv). Furthermore, mathematical metabolic modeling suggested that nucleotide and ureide metabolism play major roles in plant N nutrition during AMS. We also found that AMS activates autophagy-related genes (autophagic genes ATG, cysteine protease SAG12, ripening proteins, subtilisins, etc...) and strongly modulates the accumulation of N-containing molecules such as pyrimidines and amino acids (Decouard et al, 2023, BioRxiv). This suggests that processes typical of the catabolic and nutrients recycling events described in the context of leaf senescence are induced by AMS in arbuscule containing cells. Macro-autophagy is a ubiquitous process in eukaryotic cells that aims to preserve cell longevity by discarding cellular wastes. It targets unwanted cytoplasmic material towards the vacuole to be degraded by proteases, RNAses and lipases. At the same time, autophagy cleans up the cells from damaged organelles, aggregated proteins and unwanted RNA (Masclaux et al., 2017; Floyd et al., 2015). As recently reported, nucleic acid degradation is one of the major senescence events happening during oilseed rape root senescence (James et al., 2024) and nucleic acid metabolism genes colocalized with maize yield QTLs (Xing et al 2024) pointing to the major role played by nucleic acid metabolism in plant growth. Autophagy has been described as a key player in plant-pathogen interactions as well as in legume rhizobia interactions (Hofius et al., 2017; Thanthrige et al., 2025). However, little is known about the roles of autophagy during AMS and how it contributes to plant N uptake and N metabolism remodeling during this key mutualistic interaction (Estrada-Navarrete et al. 2016).

Objective of the thesis (a few lines)

The objectives of the present PhD project are about to answer the following questions:

-Does plant autophagy impact the success of AM symbiosis?

-Does plant autophagy control arbuscule lifetime?

-Does autophagy play a role in the release and transfer of N metabolites from AMF to plant cells?

-What is the contribution of autophagy and nucleic acid degradation to N mobilization from the AMF to the plant?

Thesis detailled overview (1 ½ page max.)
Outline the different tasks, strategies, tools and technics that will be used to carry out the project

Our work hypothesis is that, along AMS, plant would feed on degenerating arbuscules by recycling part of its N content through autophagy and that this would improve plant N metabolism and nutrition.
In this thesis project, making use of the model organisms maize and R. irregularis, the PhD candidate will investigate how N metabolism is remodeled during AMS, determine the contribution of autophagy to N metabolism and nutrition in maize under AMS and decipher what kind of N-containing molecule are remobilized to feed maize and promote its growth.

Within plant roots, symbiotic arbuscules are subjected to a rapid turnover of about 1-3 days (Gutjahr and Parniske, 2017). The molecular processes involved in the recycling of arbuscules and the fate of recycled N-containing molecules following arbuscular decay is poorly understood. The metabolism of arbuscules is highly active and enriched in N-containing molecules such as DNA, RNA, t-RNA, arginine, ureides, etc... (Xie et al., 2022, Decouard et al., 2024, BioRxiv). Pyrimidines and purines are known be degraded into ureides such as allantoin known to be an important N storage metabolite (Tegeder et al., 2014). These compounds might be released once arbuscules degenerate representing a substantial amount of reusable N for plant. In line with these data, we can note that the autophagy-deficient mutant Zmautophagy12 (Zmatg12, Zm00001D018259, KO in W22 background) is disturbed for nucleotide and ureide contents (McLoughlin et al., 2020).

WP1: What is the contribution of autophagy to maize N uptake during AMS?
The PhD student will study the role of autophagy during AMS. To do so, he/she will take advantage of the available maize autophagy-deficient mutant Zmatg12 and of the maize over-expressor line ZmAUTOPHAGY8a-YFP (ZmOE-ATG8a-YFP, Zm00001d006474 in W22 background). Both lines were obtained from Vierstra's lab and are available in the welcome laboratory (Li et al., 2015).
The kinetics of autophagy will be monitored using ZmATG8a and ZmNBR1 markers by western blot and RTqPCR following plant inoculation and in parallel to the monitoring of arbuscules colonization and development. The kinetics will be based on key developmental stages of maize (from vegetative stages to reproductive stage). This will allow to determine the onset and level of the autophagy activity in the WT and ZmOE-ATG8a-YFP genotypes during AMS.
According to the results obtained, a detailed histological analyses of AMF colonization levels and arbuscules morphology will be performed at several chosen time points on the roots of WT, Zmatg12 and ZmOE-ATG8a-YFP mutants in order to determine whether autophagy level influences AMS establishment and success. Morphological characteristics of arbuscules will be monitored in order to evaluate the senescence status of arbuscular structures depending on host genotype. This will be performed by confocal microscopy using the wheat germ agglutinin conjugated to the fluorescent dye Alexa Fluor 488. To localize autophagosomes in relation to arbuscules, ATG8a-YFP fusion will also be tracked by confocal microscopy. These results will be confirmed by a complementary ATG8a-YFP proteins immunolocalization.
For all these experiments plants will be grown under greenhouse-controlled conditions, under limited N and non-limited N conditions (1 mN and 5 mM NO3-, respectively) and inoculated or not with R. irregularis. The experiment will be performed three times.

WP2. Large-scale characterization of key metabolic pathways modified during AMS and identification of N-containing molecules remobilized.

WP2.1: To better understand processes by which the plant reassorts its N metabolism in the presence of the AMF, the PhD student will monitor enzymatic activities related to C and N maize metabolism, TCA cycle, N assimilation etc., in the roots and leaves of W22, Zmatg12 and ZmOE-ATG8a-YFP mutants, as previously described in Cañas et al., 2017. This will be performed in collaboration with Yves Gibon from the platform Bordeaux Metabolome).

WP2.2: As we hypothesized that nucleotides and ureides represent novel forms of N transferred from AMF to plants, the student will quantify nucleotides and ureides in leaves, roots and xylem saps of inoculated or non-inoculated plants grown from WP1 using metabolomics approaches in collaboration with Caroline Mauve from the plateform Plantes Métabolisme Métabolomes of IPS2 at Saclay. This will be performed on W22, Zmatg12 and ZmOE-ATG8a-YFP mutants.

WP2.3: To determine what N-containing metabolites are transferred from the AMF to the plant, we will make use of a dual compartment system in which AMF will be separated from the plant and fed with 15NO3- or 15N-labelled nucleotides or ureides. Isotopically labelled amino acids, nucleotides and ureide quantification in maize plants colonized by R. irregularis will allow help determining whether AMF is able to transfer these metabolites or if it actually boosts nucleotide biosynthesis. This will be performed using W22, Zmatg12 and ZmOE-ATG8a-YFP genotypes.

WP3: To know whether N transfers occur from decaying arbuscules towards the plant, we will manipulate arbuscules lifetime and monitor the N transfer using 15N-labelled sources such as 15NO3-. For this purpose, we will trigger the death of arbuscules by resupplying plant with P and N. Also, as a complementary approach, we will purchase Zmstr2 and Zmspx3 maize mutants impaired in lipid transfer and P sensing, respectively, in which the development of arbuscules is aborted (Paries and Gutjahr, 2023). By this means we will be able to determine whether degenerating arbuscules can be used by the plant as an N source.

By combining phenotyping, imaging, enzymatic and metabolomic approaches, and using maize autophagy mutant lines, we will be able to better understand the role of autophagy during AMS and the fate of N during the life cycle of AMF. This thesis will help deciphering whether autophagy contributes to maize N uptake from the symbiotic fungal structures.