Biocontrol proteomics:Implication of the pentoses phosphates pathway in the antagonist effect of Pichia anomala against Botrytis cinerea on apple.

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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}

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cDNA-AFLP

: 11 transcripts of P. anomala overexpressed in presence of B. cinerea cell walls

Objective

Material and methods

Results

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

: Implication of PAEXG1 et PAEXG2 in the mechanism of action of P. anomala

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

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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 methods

Results

Background

Mechanisms of action:

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Objective

Results

Background

Interaction Model

Material and methods

Wash and

Overnight dry _{Exponential phase}7h of incubation

24h of incubation Stationary phase Membrane 47mm / 0.45µm P. anomala 400µL / 107_ufc/mL B. cinerea 400µL / 106_sp/mL OR mock inoculation After 1h YEAST INOCULATION YEAST INOCULATION Membrane in

isotonic water _{Store at}

-20°C

vortex 20s _{ultra pure water}Wash with

YEAST RECOVERY

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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

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

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K1= P. anomala alone KB1= P.anomala + B.cinerea Exponential phase Mitochondria Cytoplasm Glucose G6P NADPH,H+

Pentose Phosphate Pathway Pentose Phosphate Pathway

Glycolysis Glycolysis

K1 vs. KB1

Objective

Results

Background

Material and methods

EXPONENTIAL PHASE 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 Citric acid cycle cycle Oxidative phosphorylation Oxidative phosphorylation S-Co-S PDH ATPase Cyt Bc1 Cyt c

Use of Glycolysis pathway

Nucleic acids

ATP

Energetic metabolism orientated to

Oxidative phosphorylation

Nucleic acids

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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

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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