Identification of Metabolic Pathways Expressed by Pichia anomala KH6 in the Presence of the Pathogen Botrytis cinerea on Apple: New Possible Targets for Biocontrol Improvement

peer reviewe

Evaluation of two protein extraction protocols for Pichia anomala proteome analysis.

Pichia anomala (strain Kh6) was isolated from the surface of apple fruits and selected for its high and reliable biocontrol activity against Botrytis cinerea and Penicillium expansum. Its main modes of action have until now been studied using essentially microbiological and molecular approaches. The study continues now using the proteomic approach and considering the in situ P. anomala/B. cinerea/apple interaction. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of the most powerful tools used for proteomic analysis. It combines two sequential separation steps, the first dimension via isoelectric focusing (IEF) and the second one by SDS PAGE. Although recent advances in 2-D PAGE, the extraction of the whole proteome and the removal of interfering contaminants still limit its application. Sample preparation constitutes indeed a critical influential step for IEF which in turn affects 2-D gel quality. The objective of the present work was thus to develop an effective protein extraction protocol designed for 2-D PAGE analysis of the proteome of P. anomala strain Kh6. As a starting point, two contrasting protein extraction protocols were chosen to be evaluated in terms of protein yield and one-dimensional (1-D) SDS PAGE and 2-D PAGE gel patterns. The first protocol uses a urea/thiourea-based lysis buffer whereas the second protocol utilizes a hot SDS-based lysis buffer with an additional precipitation step. The comparison model used consisted of apples treated with strain Kh6 alone (K) and apples first treated with Kh6 and then inoculated with B. cinerea conidia (KB). Growth kinetics of strain Kh6 on wounded apples was determined and found to be not affected by the presence of B. cinerea conidia. Proteins were extracted from yeast pellets collected at both the exponential and stationary phases of strain Kh6 growth on apples. The evaluation of both extraction protocols indicates that more proteins were extracted with the SDS protocol and, according to 1-D assays, higher molecular weight proteins were obtained with the ‘urea/thiourea’ protocol and, regardless of the protocol used, more bands were obtained during the exponential phase. 2-D assays are currently underway and the corresponding results will be presented

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

The growing interest of the consumers for the wholesome food and the protection of the environment as well as the development of resistant pathogens to pesticides, stimulate the interest of growers to apply biological control methods. Pichia anomala strain K was previously identified as an efficient biocontrol agent of the main apple pathogens, Botrytis cinerea and Penicillum expansum. Further study demonstrated the complexicity of the mode of action of P. anomala against B. cinerea. A cDNA-AFLP and gene disruption study revealed implication of exo-β-1,3-glucanases in the mode of action of P. anomala strain Kh6 (a haploid form of P. anomala strain K displaying the same biocontrol properties). However, these studies suggested also implication of other factors. The present study aims to increase our knowledge of the mode of action of P. anomala strain Kh6 against B. cinerea using an in situ approach allowing the triple interaction, host/pathogen/antagonist and the proteomic tool allowing to study the ultime expression of the genome without a priori. One 50mm wound per apple were covered by a membrane and inoculated by a P. anomala suspension then by B. cinerea or not. Samples were collected during the exponential and stationary phase to identify the early and later responses to the presence of B. cinerea. After extraction, proteins were separated on 2-D gels. Spots influenced by the presence of B. cinerea in exponential and stationary phases were identified by MALDI-ToF. One hundred five and sixty spots of proteins were influenced by the presence of B. cinerea in exponential and stationary phases respectively. In exponential phase, P. anomala Kh6 in absence of B. cinerea uses mainly the glycolysis pathway, whereas in presence of pathogen, it orientates its energetic metabolism to the oxidative phosphorylation and sets up the pentose phosphate pathway. Thanks to this new orientation, P. anomala Kh6 probably obtains energy and nucleic acids allowing to colonize the wound as fast as in absence of B. cinerea and prevents the use of nutrients by the pathogen. In stationary phase, no differences in the P. anomala Kh6 energetic metabolism, in absence and in presence of B. cinerea were observed. During that phase, P. anomala Kh6 seems to use the alcoholic fermentation in order to face the nutrients impoverishment of the substrate

Responses to hydric stress in the seed-borne necrotrophic fungus Alternaria brassicicola

Alternaria brassicicola is a necrotrophic fungus causing black spot disease and is an economically important seed-borne pathogen of cultivated brassicas. Seed transmission is a crucial component of its parasitic cycle as it promotes long-term survival and dispersal. Recent studies, conducted with the Arabidopsis thaliana/A. brassicicola pathosystem, showed that the level of susceptibility of the fungus to water stress strongly influenced its seed transmission ability. In this study, we gained further insights into the mechanisms involved in the seed infection process by analyzing the transcriptomic and metabolomic responses of germinated spores of A. brassicicola exposed to water stress. Then, the repertoire of putative hydrophilins, a group of proteins that are assumed to be involved in cellular dehydration tolerance, was established in A. brassicicola based on the expression data and additional structural and biochemical criteria. Phenotyping of single deletion mutants deficient for fungal hydrophilin-like proteins showed that they were affected in their transmission to A. thaliana seeds, although their aggressiveness on host vegetative tissues remained intact

PROTEOME ET TRANSCRIPTOME DU MUSCLE LONGISSIMUS LUMBORUM DE PORC : INFLUENCE DU MODE D'ELEVAGE, DE L'ORIGINE GENETIQUE ET DU SEXE. RELATIONS AVEC LES QUALITES DES VIANDES ANTHONY KWASIBORSKI

Pork is the most consumed meat in Europe with a mean consumption of 19 600 thousand ton of carcass equivalent in 2005. However, it shows that meat quality variability is in part due to variations in post-mortem muscular energy metabolism defined as whole biochemical pathways and their interactions occurring in muscle from the animal death to the consumption. Many factors depending of animals (genetic, rearing method, feeding, stress reactivity...) or technology (stunning...) can influence post-mortem metabolism and consequently meat quality. A 2x2x2 factorial design compared proteome and some interesting gene expression in female or castrated males pigs, from 2 genetic background (Large White or Duroc sires, Large White x Landrace dams), and 2 rearing conditions (conventional indoors or alternative outdoors). Results underlined important effect of treatment factors on protein quantities and on biochemical pathways involved in meat quality. They allowed identification of specific proteins of genetic and rearing background. Treatment factors did not influence studied gene expression. Meat quality may not be a consequence of gene expression modifications, but a regulation of translational process.Avec une consommation de 19600 milliers de tonnes équivalent carcasses en 2005, la viande de porc est la plus consommée en Europe. Elle montre, toutefois, une forte variabilité, en partie due à des variations dans le métabolisme énergétique musculaire post-mortem. Celui-ci est défini comme l'ensemble des réactions biochimiques et leurs interactions survenant dans le muscle au cours de la période allant de la mort de l'animal jusqu'à la préparation de la viande avant consommation. De nombreux facteurs, dépendant de l'animal (génétique, mode d'élevage, alimentation, réactivité au stress d'abattage...) ou de la technologie (mode d'étourdissement...), peuvent influencer le métabolisme post-mortem et par conséquent, les qualités de viande. Un plan expérimental 2x2x2 comparait le protéome et l'expression de certains gènes d'intérêt chez des porcs femelles ou mâles castrés, de 2 origines génétiques différentes (pères Large White ou Duroc, mères Large White x Landrace), élevés dans 2 conditions différentes (conventionnelle en intérieur ou alternative en extérieur). Les résultats obtenus mettent en évidence l'important effet des facteurs de variation sur les quantités de protéines ainsi que sur les voies biochimiques impliquées dans le déterminisme des qualités des viandes. Ils ont également permis l'identification des marqueurs protéiques caractéristiques de l'origine génétique ou environnementale de l'animal. Les facteurs de variations n'influence pas l'expression des gènes étudiés. Le déterminisme des qualités des viandes n'est probablement pas une conséquence des modifications dans le niveau d'expression des gènes, mais plutôt dans la régulation des processus traductionnels

Gene expression in Large White or Duroc-sired female and castrated male pigs and relationships with pork quality

Chantier qualité GAThis study assessed expression of 12 genes in 24 pig longissimus samples earlier subjected to a proteomic study by our group. Genes were selected on the basis of the earlier proteomic results. Pigs differed in rearing environment (indoors or outdoors), sire breed (Duroc or Large White) and gender (female or castrated male). At slaughter they experienced different stress conditions. The proportion of gene expression changes influenced by treatment factors was consistent with the proportion of protein changes in an earlier proteomic analysis of the same pigs. Expression levels of genes were often correlated. Gene expression was generally not correlated with the levels of the corresponding protein. Finally, most meat quality traits were correlated with the expression of at least one of the studied genes. The most meaningful of these was the association of a slower pH decline with lower levels of HSP72 expression and higher levels of HSP72 protein. ANXA2 and cMDH expression were also associated with various meat quality traits. These relationships may be related to pre-slaughter stress levels and fibre type composition

In situ proteome study of Pichia anomala strain K, an antagonist of the apple pathogen Botrytis cinerea

Postharvest fungal pathogens, mainly Botrytis cinerea, Penicillum expansum and Gloeosporium spp., annually cause 5-20% losses of fruit. Control measures against these diseases include chemical fungicide applications, but the development of resistant fungal explains the growing interest for biological control. Pichia anomala strain K was previously identified as an efficient antagonist of pathogens on apples. Indeed, the percentage of protection of P.anomala against B.cinerea reached from 90 to 100% on apple wounds according to the experimental conditions. Microbiological, biochemical and molecular approaches demonstrated the implication of exo-β-1,3-glucanases in the mechanism of action of P.anomala. However, study of these mechanisms could be improved by observations under natural infection conditions in order to take into account the tripartite interactions host/antagonist/pathogen. The proteomic tool allowed an overview of process implicated in the antagonism against B.cinerea in such conditions. One 50mm wound per apple were covered by a membrane and inoculated by a P.anomala suspension then by B.cinerea or not. Samples were collected during the exponential and stationary phase to identify the early and later responses to the presence of B.cinerea. After extraction, proteins were separated on 2DE gels. Spots influenced by the presence of B.cinerea in exponential and stationary phases were identified by MALDI-ToF. One hundred five and sixty spots of proteins were influenced by the presence of B.cinerea in exponential and stationary phase respectively. In exponential phase, influenced proteins were implicated in the different steps of the proteins biosynthesis: amino acid synthesis, translation or mRNA maturation and in energy synthesis. On the other hand, in stationary phase, influenced proteins were mainly implicated in energy metabolic pathway: glycolysis, alcoholic fermentation or gluconeogenesis. The presence of B.cinerea seemed to induce a slowdown in the metabolism of P.anomala without influenced its growth. Further studies have to be realized to understand the complexity of these modes of action

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

Pichia anomala strain K was previously identified as an efficient biocontrol agent of the apple pathogen, Botrytis cinerea. Further study demonstrated the complexicity of the mode of action of P. anomala against B. cinerea. A molecular study revealed implication of exo-β-1,3-glucanases in the mode of action of P. anomala but suggested implication of other factors. The present study aims to increase our knowledge of the mode of action of P. anomala strain Kh6 against B. cinerea using an in situ approach allowing interactions between organisms, and the proteomic tool allowing to study the ultime expression of the genome. One wound per apple were covered by a membrane and inoculated by P. anomala then by B. cinerea or not. Samples were collected at different incubation times to identify the responses of P. anomala to the presence of B. cinerea. After extraction, proteins were separated on 2-D gels. Spots influenced were identified by MALDI-ToF. In exponential phase, P. anomala Kh6 orientates its energetic metabolism from the glycolysis to the oxidative phosphorylation and sets up the pentose phosphate pathway. Thanks to this new pathway, P. anomala probably obtains energy and nucleic acids allowing to colonize the wound as fast as in absence of B. cinerea. In stationary phase, no differences in the P. anomala energetic metabolism, in absence and in presence of B. cinerea were observed. During that phase, P. anomala seems to use the alcoholic fermentation in order to face the nutrients impoverishment of the substrate

Pig Longissimus lumborum proteome: Part I. Effects of genetic background, rearing environment and gender

International audienceA 2 x 2 x 2 factorial experiment on Longissimus lumborum of 24 pigs found that rearing environment (indoors or outdoors), breed of sire (Duroc or Large White), and gender (female or castrated male) influenced 22, 10, and 88 proteins of the soluble fraction, respectively, containing 220 matched spots in total. Some proteins were influenced by more than one main effect. Outdoor rearing resulted in lower levels of enzymes of the glycolytic pathway suggesting a more oxidative metabolism. Breed of sire slightly altered the balance of enzymes of the glycolytic pathway. Gender had profound effects. In particular, different enzyme levels suggest a more lipid oriented energy metabolism, and a higher extractability of myofibrillar proteins suggest altered control of the contractile apparatus, in castrated males. Differences in extractability did not explain the profound gender effects. Glycogen content, ultimate pH, drip and thawing losses showed main or interactive effects of the three treatment factors