Master 6 month internship in plant science

Development of DESI-HRMS imaging to localize triterpene accumulation in apple seedlings Keywords: DESI-imaging, high resolution mass spectrometry, specialized metabolite localization, histology, plant-pathogen interactions, apple, Venturia inaequalis 1 Information on research institute The Research Institute of Horticulture and Seeds (IRHS) is a large laboratory of 250 staff developing projects focusing on quality and health of horticultural crops and seeds under the auspices of INRAE, Institut Agro and University of Angers. Its main projects deal with ornamental and fruit quality, genetic diversity, epigenetics, sustainable resistance to pathogens, diseases emergence and microbial communities, evolutive ecology of pathogens, seed quality, bioinformatics and plant phenotyping. The plant models are roses, pome-fruits and vegetables, seeds, pathogenic bacteria and fungi. Its projects are designed to develop integrated approaches by coordinating the efforts and expertise of geneticists, breeders, plant pathologists, physiologists, biochemists, eco-physiologists, modelers and statisticians. 2.
Description of the training project 2.1 Scientific context Apple scab is a major disease of apple trees caused by the fungus Venturia inaequalis (Vi). It is characterized by black, oily spots on the surface of leaves and fruit, affecting both gross and marketable yields. Controlling this disease accounts for nearly two-thirds of the treatments applied in orchards during a production season. In recent decades, breeding programs have led to the market release of numerous varieties relying on a single major resistance gene, Rvi6. However, the widespread use of this single resistance gene is leading to the emergence of new scab strains capable of overcoming this resistance. Consequently, there is a search for alternative strategies to control this pathogen—strategies that are more sustainable and require fewer inputs. Recently, our team identified a correlation between the level of resistance to apple scab and the leaf accumulation of triterpene metabolites. Triterpenes generally accumulate constitutively in the cuticle of leaves and fruits. In apple trees, the primary forms are ursolic and oleanolic acids. Based on metabolomic analyses of an apple progeny derived from crossing two genotypes carrying alleles for quantitative scab resistance, we demonstrated that the genomic region corresponding to the resistance QTL qT1 is also involved in the accumulation of approximately ten triterpenes in leaves in response to Vi infection. Several of these metabolites were positively identified, revealing that they all derive from ursolic acid. Accumulation of these same metabolites is also induced in apple leaves treated with a plant resistance inducer (PRI) that confers resistance to scab. In vitro assessments by our team indicate that some of these metabolites can inhibit Vi hyphal elongation, which reinforce the potential role of triterpenes in scab resistance. Our objective is now to more formally demonstrate the role of these molecules in apple tree resistance to scab in planta. To this end, following the tissue-level accumulation of these metabolites during scab infection would bring new comprehensive elements. Technically, it could be achieved through mass spectrometry imaging. The DESI-HRMS (Desorption ElectroSpray Ionization – High Resolution Mass Spectrometry) imaging technique is based on a soft ionization similar to ESI (electrospray ionization) in LC-MS. It enables metabolites to be located on sample surface by recording mass spectra with minimal fragmentations in either positive or negative mode. DESI can be applied to study highly complex sample surfaces, ranging from polymeric materials to biological tissues and even to fluids. This allows for the detection and semi-quantification of a wide variety of small polar and non-polar molecules. Although DESI-MS analysis is one of the simplest ionization-based imaging techniques, its efficiency depends on various parameters, particularly the properties of the analyte, the surface on which it is deposited, the solvent system, and the physical parameters such as the geometry of the ion source (e.g. capillary nebulisation angle and its distance from the surface and at the MS entrance). Similarly, depending on the sample, sample preparation may differ. DESI analysis is generally limited to flat and preferably hard surfaces. Smooth and regular surfaces can generally be analysed directly without sample preparation. In the case of plants, sample preparation can be carried out in different ways. Flowers and leaves can be problematic because their surfaces are smooth, irregular and highly absorbent, which can result in a weak or unstable signal during imagery analysis. Sometimes, indirect analysis is required. Depending on the spatial information required, different sample preparation strategies can therefore be considered, including direct analysis of the leaf surface, indirect analysis following transfer onto a porous PTFE support, and analysis of transverse tissue sections. For transverse tissue imaging, the preparation of plant tissues is a critical step to preserve tissue organization and obtain reproducible mass spectrometry images. The choice of sectioning technique, embedding medium, section thickness and cutting temperature may influence tissue integrity and the spatial preservation of metabolites. Optimizing these parameters will be an important part of the internship. Optical imaging will be performed in parallel with DESI-HRMS imaging to characterize tissue organization and assess the quality and integrity of the prepared samples. Bright-field images will provide complementary anatomical information that will help interpret the molecular distribution maps obtained by DESI-HRMS. 4.2 Objectives The M2 training aims to develop mass spectrometry imaging workflow for investigating the spatial distribution of triterpenes in apple leaves and their potential spatial association with Venturia inaequalis during the early stages of infection. Demonstrating such co-localization would provide strong additional evidence supporting the functional role of these metabolites in the mechanisms of apple tree resistance to scab. 4.3 Implementation The training period will consist in a series of iterative steps aimed at developing the DESI-HRMS method for analysing triterpenes. It will include the following activities:
- preparation of the biological material: apple seedlings will be treated with a PRI in order to increase the level of triterpenes in the leaves.
- development of a DESI-HRMS imaging method applied to the studied pathosystem: o optimisation of sample preparation (sample cuts by freehand, cryotome, PTFE printing) and of sectioning conditions optimization of DESI conditions o localization and identification of metabolites of interest. o comparison with optical imaging (acquisition of complementary bright-field images using the Zeiss Axio Zoom V16 macroscope) o Image correlation and data interpretation Once the parameters have been established on the induced apple seedlings, observation will be realized on apple seedling inoculated with Vi. 5 Desired techniques and skills The candidate should have an interest in chemistry and plant/pathogen interactions, and for multidisciplinary approaches (histology and imaging). Accuracy, motivation, autonomy and communication skills are expected. 6 Information on proposed training period 6.1 Duration : 26 weeks/Six months 6.2 Anticipated starting date : January 2027 6.3 Stipend The hourly internship allowance will be at least €4.50, corresponding to 15% of the French Social Security hourly ceiling (€30 × 0.15), which approximately correspond to 670€/month 7 Application procedure Please send your CV and your motivation letter to the three supervisor of the training (romain.larbat@inrae.fr; severine.boisard@univ-angers.fr ; aurelia.rolland@univ-angers.fr) Deadline 09/11/2026