M2-Identification and use of promoter sequences for resistance to Venturia inaequalis in apple

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INRAE presentation

The French National Research Institute for Agriculture, Food, and Environment (INRAE) is a major player in research and innovation. It is a community of 12,000 people with 272 research, experimental research, and support units located in 18 regional centres throughout France. Internationally, INRAE is among the top research organisations in the agricultural and food sciences, plant and animal sciences, as well as in ecology and environmental science. It is the world’s leading research organisation specialising in agriculture, food and the environment. INRAE’s goal is to be a key player in the transitions necessary to address major global challenges. Faced with a growing world population, climate change, resource scarcity, and declining biodiversity, the Institute has a major role to play in building solutions and supporting the necessary acceleration of agricultural, food and environmental transitions.

Work environment, missions and activities

The main objective of ResPom team is to improve apple and pear resistance against pests with a focus on the three major pests of apple, Venturia inaequalis (apple scab), Erwinia amylovora (fire blight) and Dysaphis plantaginea (rosy apple aphid), either genetically or by the application of Plant Resistance Inducers (PRIs). Promotion of apple immunity is thought as a conceptual agroecological model, integrating not only intrinsic (genetic) and induced (PRIs) resistances, but also other sources of variability such as crop management constraints, climatic factors and additional biotic factors. The main goal is to achieve a level of immunity of cropping systems as well as a growth/immunity trade-off that guarantees the highest level of production in quality and quantity.

Scientific context

Apple is one of the most heavily treated crops, due to the fact that most commercial varieties are susceptible to pests, and apple scab, caused by the fungus Venturia inaequalis (Vi), is the main disease affecting this species in France, in terms of IFT (Indice de Fréquence de Traitement - Treatment Frequency Index: 15-20 on average per year [1, 2]). In the current context of regular withdrawal by the European Union of authorisations for the use of chemical compounds previously used to combat diseases, particularly in fruit trees (e.g. Mancozeb in 2021) [3], it seems crucial for European and national economies to find alternatives, among which the deployment of genetically resistant varieties remains a major option.

Conventional varietal improvement is faster than 50 years ago [4], but needs to be accelerated further to meet the urgent need for alternatives to pesticides. NBTs (New Breeding Technics), including cis/intragenesis and CRISPR/Cas, have recently emerged as effective methods [4]. Ideally, NBT-generated apple varieties resistant to Vi would modify the expression of the genes involved only when necessary, i.e. at the time of pathogen attack, and not constitutively, in order to preserve the plant's physiological balance and development [5]. This can be achieved by developing an artificial transcriptional induction system. However, in perennial crops such as apple, knowledge is still lacking about the cis-regulatory DNA regions that could be used to design such innovative strategies, and thus speed up the creation of genotypes that are better adapted to changing environments.

Objectives

The project therefore aims to generate this missing knowledge in the context of Vi resistance. To this end, we will previously 1) characterise infection-responsive genes using RNAseq data, and 2) determine the over-represented cis-regulatory elements (pTFBS for putative Transcription Factor Binding Site) in the promoter regions of these genes, with the PLMdetect method [6].

The envisioned internship will  then 3) functionally validate the 10 best candidate TFBSs identified as a source of inducibility of gene expression.

Implementation

Vi induction of these 10 best candidate TFBSs will be validated in a generic screening system including them in a minimal promoter (pNOPALINE SYNTHASE [pNOS]) and attaching them to the GFP-GUS reporter cassette [7]. The 288 bp pNOS sequence will be copied from the binary vector pKGWFS7 [7] and the sequences of the selected TFBSs will be added before synthesis. Subsequent cloning of the chimeric pNOS + TFBS promoters will use the Gateway method. The agro-infiltration method will then be used to transiently test the reactivity of the 10 pNOS+TFBS::GFP-GUS constructs to inoculation with Vi. This method for transient testing of constructs in the context of Vi infection, is mastered by the team on greenhouse-grown plants and will also be adapted on vitroplants as part of the internship, in order to speed up the testing of future constructs (see [8] for a similar adaptation in a context of infection by the pathogenic bacterium Erwinia amylovora). Five days after agro-infiltration of leaf apices, plants (grown in the greenhouse) carrying the different pNOS+TFBS::GFP-GUS constructs, and pNOS::GFP-GUS as a control, will be sprayed with a suspension of strain 104 at 2.5 × 10⁵ conidia.ml-1 or water as a control. Samples will be taken just before treatment (nt: untreated) and then 24, 72 and 96 hpt. The samples will then be analysed by QPCR (nt, 24 and 72 hpt samples) to check the induction of the GUS gene, and by fluorimetry (nt, 72 and 96 hpt samples) to check the activity of the GUS protein [8]. This experiment will be carried out identically on vitroplants in order to test its feasibility on this smaller-scale material, which is quicker to produce and easier to handle. At the end of the internship, from the 10 TFBSs tested, we will select the two most effective in the sense that they should allow a rapid and strong signal without leading to a constitutive expression profile. The validation of these two TFBSs will be continued by the team.

The student will acquire different skills by hands-on experience in:

• Molecular biology: cloning, miniprep extraction, DNA and RNA extraction, PCR, QPCR
• Biochemistry: protein extraction, fluorimetry
• Microbiology: E. coli and A. tumefaciens cultures and transformations
• Greenhouse experiment management: set up of the experiments, plant follow-up and phenotyping
• Phytopathology: preparation and inoculation of V. inaequalis strains, symptom scoring

Keywords: Malus domestica, apple scab, cis-regulatory elements, agro-infiltration.

 

Cited Literature:

1. Soufflet-Freslon et al. Inheritance studies of apple scab resistance and identification of Rvi14, a new major gene that acts together with other broad-spectrum QTL. Genome. 2008 51:657.

2. Bus et al. Revision of the Nomenclature of theDifferential Host-Pathogen Interactions of Venturia inaequalis and Malus. Annual Review of Phytopathology. 2011 49:391.

3. ANSES, Saisine n° 2018-SA-0163.

4. Aglawe et al. New breeding technique "genome editing" for crop improvement: applications, potentials and challenges. 3 Biotech. 2018 8:336.

5. Tian et al. Fitness costs of R-gene-mediated resistance in Arabidopsis thaliana. Nature. 2003 423:74.

6. Bernard et al. Improved detection of motifs with preferential location in promoters. Genome. 2010 53:739.

7. Karimi et al. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 2002 7:193.

8. Gaucher et al. An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance. Plant Cell Rep. 2022 4:1499.

Training and skills

We are looking for a student in its last year of engineer degree or Master program with a major in plant biology, showing:

• strong appeal for functional genetics,
• skills in molecular biology,
• basic knowledge in statistics,
• oral and written communication aptitudes. 

INRAE's life quality

By joining our teams, you benefit from (depending on the type of contract and its duration):

- 2 days of per month;
- parenting support: CESU childcare, leisure services;
- social support: advice and listening, social assistance and loans;
- holiday and leisure services: holiday vouchers, accommodation at preferential rates;
- sports and cultural activities;
- collective catering.

How to apply

I send my CV and my motivation letter