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

Biocontrol proteomics:

Role of the pentose phosphate pathway in the antagonist effect of Pichia anomala.

A. Kwasiborski

1

_{, P. Delaplace}

2

_{, J. Renaut}

3

_{, P. Lepoivre}

1

_{and M. H. Jijakli}

1

1_{Plant Pathology Unit, GxABT, ULg, Gembloux, Belgium.} 2_{Plant Biology Unit, GxABT, Ulg, Gembloux, Belgium.} 3_{Proteomic plateform, CRPGL, Belval, Luxembourg.}

Membrane

Protein extraction

Hot SDS buffer Mechanical/thermal lysis Acetone precipitation Exponential phase (=1) Stationary phase (=2)

Method

Wound 50mm P. anomala alone (=K) P. anomala + B. cinerea (=KB) or 2-DE 24cm / pH4-7 strips SDS-PAGE 12.5% Protein identification MALDI-ToF mass spectrometry

Context

Apple production 20% loss B. cinerea P. expansum Gloeosporium spp. Fungicides Biological control

-P. anomala

B. cinerea

Previous found on the modes of action

Modes of action of P.anomala is a complex mechanism implicating different factors

90% protection

Development of resistance

Objective

This work aims to study the modes of action of Pichia anomala strain Kh6 with proteomic tools. P. anomala is an antagonistic yeast on post-harvest apples against Botrytis cinerea.

The proteomic is a method without a priori able to offer a global view of the host/ antagonist/pathogen interactions. The proteins being the metabolic actors, the application of that method will lead to the identification of the implicated metabolic pathways under in situ conditions.

K : Samples In the absence of B. cinerea KB: Samples In the presence of B. cinerea

Using microbiological, biochemical and molecular approches, we identified differents mechanisms implicated in the modes of action of P. anomala against B. cinerea:

•Competition for nutrients between the antagonist and the pathogen

•Implication of β-glucanases secreted by P. anomala

•No antibiosis

Results

Conclusion

In the exponential phase

Protein function K1 KB1 Glycolysis 3 4 m e ta b o lis m Glycolysis 3 4

Citric acid cycle 2 2

Oxidative phosphorylation 1 7

Pentose phosphate pathway 0 5

Nucleotide synthesis 1 2 E n e rg e ti c m e ta b o lis m Nucleotide synthesis 1 2 Transcription 0 2

Amino acid synthesis 5 5

Ribosome synthesis 3 1 Translation 4 7 Cell division 0 3 P ro te in s s y n th e s is

New orientation of

the genome

expression

Set up of the pentose

phosphate pathway

Increase of cell division

Orientation from

glycolysis to oxydative

phosphorylation

In the stationary phase

Protein function K2 KB2

Glycolysis 3 5

Alcoholic fermentation 3 2

Nucleotide synthesis 0 1

Amino acid synthesis 0 3

Ribosome synthesis 0 1 Traduction 0 1 E n e rg e ti c m e ta b o lis m P ro te in s y n th e s is

Metabolic delay due to the

introduction of B. cinerea

Orientation to the

alcoholic fermentation

Oxidative phosphorylation Citric acid cycle Glucose G6P F6P DHAP GA3P Pyruvate NADPH Ru5P Xu5P R5P Nucleic acids G ly co ly si s ATP

Cell growth

and division

Pentose phosphate pathway

Implication of the Pentose Phosphate Pathway

P. anomala

_{needs nucleic acids and energy to stimulate a novel genome expression in presence of B. cinerea. These novel}

orientations lead to use oxidative phosphorylation and set up the pentose phosphate pathway and allow P. anomala to

colonize efficiently the wound and to prevent the growth of B. cinerea .

P. anomala

_{in absence and presence of B. cinerea uses the alcoholic}

fermentation to face the nutrients impoverishment. However, a metabolic

delay due to the introduction of B. cinerea is observed.

+ + +

+

+

+ ₊

This proteomic study strengthens the hypothesis of competition for nutrients as one of the modes of action of

P. anomala against B. cinerea.

The novel orientation of P. anomala metabolism to pentose phosphate pathway during the exponential phase might allowing the yeast to be an efficient nutrient competitor against B. cinerea. When the wound is colonized, P. anomala uses the alcoholic fermentation which might help the yeast to face the nutrient impoverishment and to maintain its competition against

B. cinerea. Number of proteins influenced by the absence

(K1) or presence (KB1) of B. cinerea

Number of proteins influenced by the absence (K2) or presence (KB2) of B. cinerea