Organisms and model

All the experiments were carried out in situ on Malus x domestica cv. Golden Delicious purchased from a local supplier (Van Dyck Freres SA, Namur, Belgium). To study the yeast proteome within the triple apple/antagonist/pathogen interaction, an in situ model was recently developed [27] that allows for nutrient exchanges between apples and microorganisms and for the extraction of proteins compatible with a 2-DE study, both quantitatively and qualitatively. Forty and ten apples were used in the exponential (7 hrs) and stationary (24 hrs) growth phases, respectively. Four different samples corresponding to P. anomala incubated alone for 7 hrs (Kh6-1) or for 24 hrs (Kh6-2) and P. anomala co-inoculated with B. cinerea for 7 hrs (Kh6B-1) or for 24 hrs (Kh6B-2) were produced in five biological replicates.

Protein extraction, two-dimensional electrophoresis, image analysis and protein identification by mass spectrometry

The hot SDS/acetone protein extraction protocol and 24 cm 2-DE procedure based on Delaplace et al. (2006) [28] were adapted and are detailed by Kwasiborski et al. [27]. The protein content was determined using the Bradford method. The obtained 2-DE gels were scanned using the Typhoon Variable Mode Imager 9400 (GE Healtcare) at a resolution of 100 µm and analysed using 2-DE image analysis software Decyder v7 (GE Healthcare). The proteome of P. anomala inoculated alone on apple wounds was compared to the proteome of P. anomala in the presence of B. cinerea in the exponential and stationary growth phases: Kh6-1 vs. Kh6B-1 after 7hrs and Kh6-2 vs. Kh6B-2 after 24hrs.

Statistical procedures for the selection of spots of interest were carried out using XLstat addinsoft version 7.5.3 (Microsoft Corporation, Seattle, USA). The normal distribution of our data was verified using the Shapiro-Wilcoxon test and proteins with abnormal distributions were normalised using the Box-Cox procedure. Protein abundances were considered as statistically different between our conditions using a one way analysis of variance (p<0.05). Then differentially expressed proteins across all biological replicates with an absolute ratio of at least 1.5-fold were selected.

The mass spectrometry identifications were carried out by the proteomic platform based in the Centre de Recherche Public G. Lippmann (Belvaux, Luxembourg). Spots of interest were excised using the Ettan Spot Picker from the Ettan Spot Handling Workstation (GE Healthcare). After washing and desalting in 50 mM ammonium bicarbonate/50% v/v methanol, followed by 75% v/v ACN, spots were then digested with 10ng/µL Trypsin Gold (MSgrade, Promega, Madison, WI, USA) in 20 mM ammonium bicarbonate. The peptides were extracted using a 50% ACN containing 0.1% TFA and analysed using the Applied Biosystems 4800 Proteomics Analyser (Applied Biosystems, Foster City, CA, USA). Calibration was carried out with the peptide mass calibration kit for 4700 (Applied Biosystems). Proteins were identified from their peptide mass fingerprintings and searching the NCBI protein sequence database using the MASCOT software (Matrix Science, http://www.matrixscience.com, London, UK). The search parameters allowed for carboxyamidomethylation of cysteine as fixed modification and oxidation of methionine, oxidation, dioxydation and kynurenin of tryptophan as variable modifications. After manual checking of the identifications, proteins with at least two matching peptides and a total ion score above 60 were considered as significantly identified.

Proteome changes during the exponential phase

Table 2 shows the P. anomala proteins up- or down-regulated by the absence or presence of the pathogen during the exponential phase. The abundance of forty four proteins was decreased, while the abundance of 63 was increased in the presence of B. cinerea. These proteins are implied in a broad range of metabolic pathways: genome expression, carbohydrate, energy and cellular metabolisms and cell process (Table 1).

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Table 2. Identification of proteins exhibiting different levels of expression according to the absence (Kh6-1) or the presence (Kh6B-1) of B. cinerea in the exponential phase.

https://doi.org/10.1371/journal.pone.0091434.t002

Proteome changes during the stationary phase

Table 3 shows proteins influenced by the absence or presence of B. cinerea during the stationary phase. Twenty proteins were down-represented, while 40 were over-represented in the presence of the pathogen.

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Table 3. Identification of proteins exhibiting different expression levels according to the absence (Kh6-2) or the presence (Kh6BB-2) of B. cinerea in the stationary phase.

https://doi.org/10.1371/journal.pone.0091434.t003

Proteins down-represented in the presence of B. cinerea. Seven proteins are implied in the carbohydrate and energy metabolisms. Glyceraldehyde-3-phosphate dehydrogenase (spot 1177) and fructose-1,6-biphosphate aldolase (spots 1350 and 1377) are glycolytic enzymes. Pyruvate decarboxylase (spots 241, 247 and 322) is implied in alcoholic fermentation. Mitochondrial F-ATPase β (spot 1135) is a constituent of the respiratory chain.

Moreover, thioredoxin peroxidase (spot 939) and protein BMH2 (spot 1079) are involved in regulation processes.

The pentose phosphate pathway: a possible early answer to the introduction of B. cinerea

In the early stages after inoculation, i.e. in the exponential growth phase, our results may indicate that P. anomala modified its energetic metabolism in order to respond to its needs.

In the absence of B. cinerea, the over-representation of two isoforms of enolase 1 (spots 441 and 452) and 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (spot 333) suggests that the glycolysis pathway is activated during the growth and wound colonization. Indeed, glyceraldehyde-3-phosphate dehydrogenase and other glycolytic enzymes were the most abundant proteins isolated from Saccharomyces cerevisiae grown on a glucose medium in the exponential phase [29]. Apples are composed of 12.6% carbohydrates, mainly in the form of glucose-derived molecules such as fructose (a glucose isomer), sorbitol and pectin (a heteropolysaccharide of galacturonic acid, arabinose, galactose, rhamnose and xylose [30]) that could enter the glycolytic pathway to provide P. anomala with the energy required for its growth. Likewise; the higher proportion of proteins involved in the protein synthesis process in the absence of B. cinerea should be the consequence of the high rate of protein synthesis that occurs in the exponential phase. Indeed, the highest incorporation rate of 35S-amino acids in a culture of S. cerevisiae on YPD medium was observed during the exponential phase [31].

The proteomic map of P. anomala in the presence of B. cinerea during the exponential phase showed that proteins involved in the carbohydrate and energy metabolisms, genome expression and cellular metabolism were highly represented, suggesting a high metabolic activity. This high activity may be related to the capacity of P. anomala to exhibit similar growth time-courses in the absence or presence of B. cinerea (Figure 1) [27]. In the presence of the pathogen, proteins involved in the PPP are over-represented and may allow P. anomala to efficiently use apple nutrients and support growth. Arabinose, an apple constituent, can be converted into xylose by L-xylulose reductase (spot 724, Table 2 and Fig. 2), an enzyme of the pentose and glucuronate interconversion pathway [32] that is over-represented in the presence of B. cinerea. PPP has been demonstrated as the main route for xylose catabolism in yeast [33]. The newly produced amount of xylose from arabinose or xylose derived from apple could then enter the PPP (Fig. 2) under the xylulose-5P form and be converted into fructose-6P and glyceraldehyde-3P, two glycolysis intermediates, via the action of transketolase (spots 120 and 121) and transaldolase (spot 631, Fig. 2), two enzymes over-represented in the presence of B. cinerea. The over-representation of five glycolysis proteins, particularly triosephosphate isomerase (spot 814) and fructose-1,6-biphosphate aldolase (spot 686) suggests the entry of fructose-6P and glyceraldehyde-3P into glycolysis and the production of high amount of pyruvate. Then pyruvate may be metabolised to supply substrates for oxidative phosphorylation inside mitochondria as suggested by the over-representation of seven proteins involved in that pathway (Table 2 and Fig. 2).

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Figure 2. Diagram showing the implication of the pentose phosphate pathway in the mode of action of the antagonist P. anomala.

https://doi.org/10.1371/journal.pone.0091434.g002

Moreover, fructose, sorbitol, galactose and galacturonic acid, other apple constituents could be converted into glucose-6P. The over-representation of 6-phosphogluconate dehydrogenase (spots 351 and 390), the first enzyme of the PPP which catalyses the irreversible decarboxylation of 6-phosphogluconate into ribulose-5-phosphate, suggests that the newly-formed glucose-6P could enter the PPP to form ribulose-5P together with synthesis of NADPH,H+ (Fig. 2), a powerful reducing-oxidative agent involved in protection against the toxicity of reactive oxygen species and in glutathione regeneration [34], [35]. The newly synthesised NADPH,H+ may probably be used during oxidative phosphorylation in order to protect the yeast against reactive oxygen species (Fig. 2). Indeed, PPP plays an important role in the oxidative stress response in Saccharomyces cerevisiae [36].

These results are linked to the over-representation of 17 proteins involved in nucleotide metabolism, transcription, amino acid metabolism and translation. Via the PPP, newly-synthesised ribulose-5P may be converted into ribose-5P, the precursor of nucleic acids (Fig. 2), in order to respond to its high metabolic activity in the presence of B. cinerea. Moreover, the capacity of P. anomala to exhibit similar growth time-courses in the absence or presence of B. cinerea (Fig. 1) and the over-representation of proteins involved in cell division, cell division control protein Cdc48 (spot 53) implicated in protein processing in the endoplasmic reticulum, ATP-dependent molecular chaperone HSC82 (spot 98), a constitutively expressed protein involved in the cellular cycle [37], and Nucleosome assembly protein (spot 341), a protein involved in mitosis [38], suggest that P. anomala could mainly use ribose-5-P for its multiplication and growth.

Thus, in the presence of B. cinerea, our results suggest that the PPP may supply the yeast with an efficient consumption of apple nutrients and consequently an adaptation of its metabolism to meet its immediate needs. Then, we could hypothesize that P. anomala may be an efficient coloniser of the wound and a nutrient competitor for B. cinerea. Actually, competition for space of specific infection sites is admitted as a mode of action of various biocontrol agents [7]–[12]. Our results may link the PPP with the protective effect of P. anomala against B. cinerea on apple. However, additional experiments have to be done in order to confirm this hypothesis. For example, the gene coding for 6-phosphogluconate dehydrogenase could be disrupted in order to inhibit the PPP and observe its influence on the protection level.

The alcoholic fermentation: the energy metabolism in the later stages

In the later stages after inoculation, i.e. in the stationary growth phase, the proteome of P. anomala in the absence or the presence of B. cinerea seems to present no differences in the carbohydrate and energy metabolism. Proteins implied in glycolysis (spots 363, 534, 765, 814, 1177, 1344, 1350 and 1377) and alcoholic fermentation (pyruvate decarboxylase, spots 241, 247, 261, 264 and 322) are expressed. Alcoholic fermentation was further identified as a metabolic pathway providing energy in the post-diauxic phase that precedes the stationary phase. On the contrary, when yeast was grown on a rich medium for a long time, energy was provided by oxidative respiration in the stationary phase [39]. According to our growth time-course (Figure 1), 24h after inoculation, P. anomala presented stagnation in its population suggesting that yeast entered the stationary phase [27]. Then P. anomala should use the oxidative respiration and not the alcoholic fermentation. However, another study demonstrates that P. anomala, when inoculated on a glucose-rich medium, used alcoholic fermentation as a way to provide energy in response to oxygen limitation [40]. In our model, inoculated apples were enclosed in plastic boxes in order to maintain a high relative humidity. Moreover, wounded apple sites represent media that are rich in glucose-derived molecules. During the first growth steps, the two microorganisms may possibly consume a large part of the available oxygen, or oxygen proportion may have been modified by microorganism carbon dioxide release or apple ethylene release. In these conditions, oxygen limitation may induce fermentative metabolism in P. anomala [40].

These last results could be a new way to explore in biocontrol. Indeed; in enclosed conditions, as in our experiment, yeast could use alcoholic fermentation and may produce volatile compounds like ethanol or ethyl acetate recently described as possible antimicrobial and antifungal compounds [41]. Then we could hypothesize that this volatile compound production could then contribute to the B. cinerea biocontrol by P. anomala. Finally, monitoring oxygen availability in storage rooms could improve the efficiency of biocontrol by P. anomala. However, we cannot rule out the implication of competition for space and nutrients in the inhibitory effect against the pathogen in the later stages. In the stationary phase, yeast was totally established on the wound site, leaving no space or nutrients for B. cinerea growth. Thus, P. anomala could maintain its inhibitory effect on the pathogen without necessarily inducing any specific metabolic pathway involved in the biocontrol modes of action, as in the exponential phase. However, additional experiments have to be done in order to confirm this hypothesis.

Observation of a higher protein synthesis induced by the introduction of the pathogen in the later stages

In the later stages after the co-inoculation, proteins implied in genome expression seem to be over-represented: glutamine synthetase (spot 471), which catalyses the fixation of a second amine group on glutamate to synthetise glutamine (ter Schure et al., 2000), orotate phosphoribosyltransferase (spot 850), which catalyses orotidine monophosphate formation during pyrimidine metabolism [42], NADP-dependent glutamate dehydrogenase (spot 343), involved in the conversion of α-ketoglutarate into glutamate [34], [43], acetohydroxy-acid isomeroreductase (spot 530), involved in valine, leucine and isoleucine synthesis [44], [45], 40S ribosomal protein S0 (spot 662), which is required for the assembly and/or stability of the 40S ribosomal subunit [46] and eukaryotic translation initiation factor 5A (spot 973), which plays a role in the formation of the first peptide bond [47]. This would suggest a higher protein synthesis rate than in the absence of the pathogen. Introducing the pathogen induced metabolic modifications such as the implementation of new metabolic pathways during the exponential phase. Here, the over-representation of proteins implied in genome expression could be the first signs of a new metabolic pathway or alternatively, in the presence of the pathogen, yeast may have maintained a high level of alcoholic fermentation activity implying high corresponding enzyme synthesis levels.

Finally, this study aimed to identify P. anomala metabolic pathways that are differentially expressed after the introduction of B. cinerea on apple. In the early stages of the co-inoculation P. anomala seems to set up the pentose phosphate pathway in order to supply higher amounts of energy and nucleic acid and support its high metabolic activity. This result suggests that PPP supports the efficient apple wound colonization. In the later stages, P. anomala seems to use alcoholic fermentation which is associated with an increased protein synthesis potential.

Nevertheless, this study confirms the complexity of the interaction between B. cinerea and P. anomala. Different mechanisms are influenced by growth stages and experimental conditions. However, these results suggested new targets in the study of the yeast mode of action against the pathogen in apple. Different pathways could be investigated in order to improve our knowledge of the biocontrol agent P. anomala.