Implication of the pentoses phosphates pathway in the antagonist effect of P.anomala. Anthony Kwasiborski 1, Jenny Renaut 2, Pierre Delaplace 3, Philippe Lepoivre 1 & Haïssam M. Jijakli 1 1 Plant Pathology Unit, GxABT, Ulg, Gembloux 2 Proteomic plateform, CRPGL, Luxembourg 3 Plant Biology Unit, GxABT, Ulg, Gembloux

Mechanism of action cDNA-AFLP cDNA-AFLP : 11 transcripts of P. anomala overexpressed in presence of B. cinerea cell walls Objective Material and methodsResults Background Apples production 5-20% loss B. cinerea P. expansum Gloeosporium spp. Chemical fungicide - Resistant Fungal Biological control - P. anomala Kh6 B. cinerea 90% of protection GENE DISRUPTION GENE DISRUPTION : Implication of PAEXG1 et PAEXG2 in the mechanism of action of P. anomala MUTANTS paexg1 and/or paexg2 MUTANTS paexg1 and/or paexg2 : Decrease of protective level to 8% Study of mode of action, without a priori, at the ultime expression level of the genome Complexicity of the mechanism of action Restoration of the protective level: ! - Yeast inoculum concentration - Maturation of apples

Proteomic tool: Global view without a priori of the metabolic actors: PROTEINS Conditions closed to the natural infection: Tripartite interaction: HOST / ANTAGONIST / PATHOGEN Objective Material and methodsResults Background Mechanisms of action: P. anomala vs. B. cinerea

Objective Results Background Interaction Model Material and methods Wash and Overnight dry 7h of incubation Exponential phase 24h of incubation Stationary phase Membrane 47mm / 0.45µm P. anomala 400µL / 10 7 ufc/mL B. cinerea 400µL / 10 6 sp/mL OR mock inoculation After 1h YEAST INOCULATION Membrane in isotonic water Store at -20°C vortex 20s Wash with ultra pure water YEAST RECOVERY

Proteins study Objective Results Background Interaction Model Material and methods Protein extraction Hot SDS buffer Mechanical Lysis (Homogenization of 2.5min) Thermal Lysis (70°C for 3min + 15min on ice) Acetone precipitation 2D-electrophoresis 1 st dimension: 100µg of proteins 24cm / pH 4-7 IPG strips 2 nd dimension: SDS-PAGE 12.5% Proteins identification Gel analysis: Decyder v 7.0 Identification: MALDI-ToF Proteins influenced by the presence of B. cinerea

K1= P. anomala alone KB1= P.anomala + B.cinerea Exponential phase Mitochondria Cytoplasm Glucose G6P NADPH,H + Pentose Phosphate Pathway Glycolysis K1 vs. KB1 Objective Results Background Material and methods EXPONENTIAL PHASE 6-PGD Ru5P R5P Xu5P DHAP GA3P F6P TK F-1,6-BP TPI New orientation of the genomic expression Pyruvate Citric acid cycle Oxidative phosphorylation S-Co-S PDH ATPase Cyt Bc1 Cyt c Use of Glycolysis pathway Nucleic acids ATP Energetic metabolism orientated to Oxidative phosphorylation Kh6 + Bc Kh6 Nucleic acids ATP

K2 vs. KB2 K1 vs. KB1 Objective Results Background Material and methods STATIONARY PHASE Energetic metabolism orientated to Alcoholic fermentation Metabolic delay of P. anomala in presence of B. cinerea K2= P. anomala alone KB2= P.anomala + B.cinerea Stationary phase

Conclusion Exponential phase Set up the pentose phosphate pathway to answer to its needs EFFECTIVE COLONIZATION OF THE SUBSTRACT Stationary phase Orientation of energetic metabolism from glycolysis to oxidative phosphorylation of P. anomala In presence of B. cinerea Orientation of energetic metabolism to alcoholic fermentation of P. anomala In absence and presence of B. cinerea Metabolic delay due to the presence of B. cinerea In presence of B. cinerea