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Implication of the pentoses phosphates pathway in the antagonist effect of P.anomala.

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

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Implication of the pentoses phosphates pathway in the antagonist effect of P.anomala.

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  1. Implication of the pentoses phosphates pathway in the antagonisteffect of P.anomala. Anthony Kwasiborski1, Jenny Renaut2, Pierre Delaplace3, Philippe Lepoivre1 & Haïssam M. Jijakli1 1 Plant Pathology Unit, GxABT, Ulg, Gembloux 2 Proteomic plateform, CRPGL, Luxembourg 3 Plant Biology Unit, GxABT, Ulg, Gembloux

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

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

  4. YEAST INOCULATION B. cinerea 400µL / 106 sp/mL OR mock inoculation Wash and Overnight dry 7h of incubation Exponential phase P. anomala 400µL / 107 ufc/mL After 1h Membrane 47mm / 0.45µm 24h of incubation Stationary phase YEAST RECOVERY Membrane in isotonic water Wash with ultra pure water vortex 20s Store at -20°C Objective Material and methods Results Background Interaction Model

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

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

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

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

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