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Implication of the pentoses phosphates pathway in the
antagonist effect of P.anomala.
Anthony Kwasiborski1, Jenny Renaut2, Pierre Delaplace3, Philippe Lepoivre1 & Haïssam M. Jijakli1
1
Plant Pathology Unit, GxABT, Ulg, Gembloux
Proteomic plateform, CRPGL, Luxembourg
3 Plant Biology Unit, GxABT, Ulg, Gembloux
2
Objective
Background
5-20%
loss
Material and methods
-
B. cinerea
P. expansum
Gloeosporium spp.
Results
Chemical fungicide
Resistant
Fungal
Biological control
-
P. anomala Kh6
90% of
protection
B. cinerea
cDNA-AFLP: 11 transcripts of P. anomala overexpressed in presence of B. cinerea cell walls
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%
Restoration of the protective level: -
!
-
Yeast inoculum concentration
Maturation of apples
Complexicity of the mechanism of action
Study of mode of action, without a priori, at the ultime expression level of the genome
Mechanism of action
Apples
production
Background
Objective
Material and methods
Results
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
Background
Objective
Material and methods
Results
Interaction Model
YEAST INOCULATION
Wash and
Overnight dry
P. anomala
400µL / 107 ufc/mL
B. cinerea
400µL / 106 sp/mL
OR
mock inoculation
7h of incubation
Exponential phase
After 1h
Membrane
47mm / 0.45µm
24h of incubation
Stationary phase
YEAST RECOVERY
Membrane in
isotonic water
vortex 20s
Wash with
ultra pure water
Store at
-20°C
Objective
Background
Material and methods
Interaction Model
Proteins study
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
Results
Background
Proteins
synthesis
Energetic
metabolism
EXPONENTIAL PHASE
Objective
Material and methods
Results
K1 vs. KB1
S-Co-S
PDH
Protein function
ATPase
Glycolysis
Cyt Bc1
Citric acid cycle
Cyt c
Oxidative phosphorylation
Pentose phosphate pathway
Nucleotide synthesis
Transcription
Amino acid synthesis
Ribosome synthesis
Translation
Cell division
6-PGD
TK
K1
KB1
3
2
1
0
1
0
4
2
7
5
2
2
5
3
4
0
5
1
7
3
Exponential phase K1= P. anomala alone
KB1= P.anomala + B.cinerea
F-1,6-BP
TPI
Use of Glycolysis pathway
Energetic metabolism orientated to
Oxidative phosphorylation
New orientation of the genomic expression
Cytoplasm
Pentose Phosphate Pathway
Glucose
NADPH,H+
Nucleic acids
G6P
Ru5P
Nucleic acids
F6P
Xu5P
DHAP
R5P
ATP
Oxidative phosphorylation
GA3P
ATP
Citric acid
cycle
Glycolysis
Pyruvate
Mitochondria
Background
Objective
Material and methods
Results
K1 vs. KB1
Stationary phase
K2 vs. KB2
K2= P. anomala alone
KB2= P.anomala + B.cinerea
STATIONARY PHASE
Protein
Energetic
synthesis metabolism
Protein function
K2
KB2
Glycolysis
3
5
Alcoholic fermentation
3
2
Nucleotide synthesis
Amino acid synthesis
Ribosome synthesis
Traduction
0
0
0
0
1
3
1
1
Energetic metabolism orientated to
Alcoholic fermentation
Metabolic delay of P. anomala in presence of B. cinerea
Conclusion
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