Natural history of Arabidopsis thaliana and oomycete symbioses

Molecular ecology of plant–microbe interactions has immediate significance for filling a gap in knowledge between the laboratory discipline of molecular biology and the largely theoretical discipline of evolutionary ecology. Somewhere in between lies conservation biology, aimed at protection of habitats and the diversity of species housed within them. A seemingly insignificant wildflower called Arabidopsis thaliana has an important contribution to make in this endeavour. It has already transformed botanical research with deepening understanding of molecular processes within the species and across the Plant Kingdom; and has begun to revolutionize plant breeding by providing an invaluable catalogue of gene sequences that can be used to design the most precise molecular markers attainable for marker-assisted selection of valued traits. This review describes how A. thaliana and two of its natural biotrophic parasites could be seminal as a model for exploring the biogeography and molecular ecology of plant–microbe interactions, and specifically, for testing hypotheses proposed from the geographic mosaic theory of co-evolution

Lactic Acid Bacteria as Markers for the Authentication of Swiss Cheeses

The manufacture of traditional Swiss-type cheeses adheres to strict rules, so as to guarantee quality and purity of the end product. This raises production costs and means consumers pay more. It also opens the door to cut-rate forgeries claiming to be made to the stringent standards and causing considerable economic losses to the entire dairy sector. In order to combat product counterfeiting, Agroscope has developed proof-of-origin cultures that allow the identification of copycats. Carefully selected lactic acid bacteria, having uniquely located insertion sequence elements, are proliferated by fermentation and subsequently dried by lyophilization. The proof-of-origin culture is added during the cheese production process and sustains maturation. These so-called 'biological markers' can be traced using polymerase chain reaction (PCR) methods, which allow authentication even if the cheese is cut into pieces or grated. They do not lead to any alteration of the cheese's taste or texture, and are compatible with the strict 'protected designation of origin' (PDO) specifications. The proof-of-origin cultures are used for the protection of several traditional Swiss-cheese varieties, such as Emmental PDO, Tête de Moine PDO, and Appenzeller®. A market survey of Emmental PDO showed that the system is effective in revealing fraud and has the power to enforce corrective measures

Ecology and Pathogenicity for Honey Bee Brood of Recently Described Paenibacillus melissococcoides and Comparison With Paenibacillus dendritiformis, Paenibacillus thiaminolyticus.

Honey bee colonies contain thousands of individuals living in close proximity in a thermally homeostatic nest, creating ideal conditions for the thriving of numerous pathogens. Among the bacterial pathogens, Paenibacillus larvae infects larvae via the nutritive jelly that adult workers feed them, causing the highly contagious American foulbrood disease. Further Paenibacillus species were anecdotally found in association with honey bees, including when affected by another disease, European foulbrood (EFB). However, their pathogenicity remains largely unknown. Our results indicate that Paenibacillus dendritiformis, Paenibacillus thiaminolyticus and newly described Paenibacillus melissococcoides are pathogenic towards honey bee brood and that their virulence correlates with their sporulation ability, which confers them resistance to the bactericidal properties of the nutritive jelly. Our survey occasionally but increasingly detected P. melissococcoides in confirmed and idiopathic cases of EFB but never in healthy colonies, suggesting that this bacterium is an emerging pathogen of honey bee brood. Overall, our results suggest that virulence traits allowing a pathogenic or opportunistically pathogenic habit towards honey bee brood are frequent in Paenibacillus spp., but that their degree of adaptation to this host varies. Our study clarifies the ecology of this ubiquitous genus, especially when infecting honey bees

The sterol-binding activity of PATHOGENESIS-RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein

Pathogenesis-related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS-RELATED 1 (PR-1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR-1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR-1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol-auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR-1, whereas sterol-prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR-1 expression are particularly well protected against oomycete pathogens

The Lectin Receptor Kinase LecRK-I.9 Is a Novel Phytophthora Resistance Component and a Potential Host Target for a RXLR Effector

In plants, an active defense against biotrophic pathogens is dependent on a functional continuum between the cell wall (CW) and the plasma membrane (PM). It is thus anticipated that proteins maintaining this continuum also function in defense. The legume-like lectin receptor kinase LecRK-I.9 is a putative mediator of CW-PM adhesions in Arabidopsis and is known to bind in vitro to the Phytophthora infestans RXLR-dEER effector IPI-O via a RGD cell attachment motif present in IPI-O. Here we show that LecRK-I.9 is associated with the plasma membrane, and that two T-DNA insertions lines deficient in LecRK-I.9 (lecrk-I.9) have a ‘gain-of-susceptibility’ phenotype specifically towards the oomycete Phytophthora brassicae. Accordingly, overexpression of LecRK-I.9 leads to enhanced resistance to P. brassicae. A similar ‘gain-of-susceptibility’ phenotype was observed in transgenic Arabidopsis lines expressing ipiO (35S-ipiO1). This phenocopy behavior was also observed with respect to other defense-related functions; lecrk-I.9 and 35S-ipiO1 were both disturbed in pathogen- and MAMP-triggered callose deposition. By site-directed mutagenesis, we demonstrated that the RGD cell attachment motif in IPI-O is not only essential for disrupting the CW-PM adhesions, but also for disease suppression. These results suggest that destabilizing the CW-PM continuum is one of the tactics used by Phytophthora to promote infection. As countermeasure the host may want to strengthen CW-PM adhesions and the novel Phytophthora resistance component LecRK-I.9 seems to function in this process

Extracellular Fibrils of Pathogenic Yeast Cryptococcus gattii Are Important for Ecological Niche, Murine Virulence and Human Neutrophil Interactions

Cryptococcus gattii, an emerging fungal pathogen of humans and animals, is found on a variety of trees in tropical and temperate regions. The ecological niche and virulence of this yeast remain poorly defined. We used Arabidopsis thaliana plants and plant-derived substrates to model C. gattii in its natural habitat. Yeast cells readily colonized scratch-wounded plant leaves and formed distinctive extracellular fibrils (40–100 nm diameter ×500–3000 nm length). Extracellular fibrils were observed on live plants and plant-derived substrates by scanning electron microscopy (SEM) and by high voltage- EM (HVEM). Only encapsulated yeast cells formed extracellular fibrils as a capsule-deficient C. gattii mutant completely lacked fibrils. Cells deficient in environmental sensing only formed disorganized extracellular fibrils as apparent from experiments with a C. gattii STE12α mutant. C. gattii cells with extracellular fibrils were more virulent in murine model of pulmonary and systemic cryptococcosis than cells lacking fibrils. C. gattii cells with extracellular fibrils were also significantly more resistant to killing by human polymorphonuclear neutrophils (PMN) in vitro even though these PMN produced elaborate neutrophil extracellular traps (NETs). These observations suggest that extracellular fibril formation could be a structural adaptation of C. gattii for cell-to-cell, cell-to-substrate and/or cell-to- phagocyte communications. Such ecological adaptation of C. gattii could play roles in enhanced virulence in mammalian hosts at least initially via inhibition of host PMN– mediated killing

A conserved RxLR effector interacts with host RABA-type GTPases to inhibit vesicle-mediated secretion of antimicrobial proteins

Plant pathogens of the oomycete genus Phytophthora produce virulence factors, known as RxLR effector proteins that are transferred into host cells to suppress disease resistance. Here, we analyse the function of the highly conserved RxLR24 effector of Phytophthora brassicae. RxLR24 was expressed early in the interaction with Arabidopsis plants and ectopic expression in the host enhanced leaf colonization and zoosporangia formation. Co‐immunoprecipitation (Co‐IP) experiments followed by mass spectrometry identified different members of the RABA GTPase family as putative RxLR24 targets. Physical interaction of RxLR24 or its homologue from the potato pathogen Phytophthora infestans with different RABA GTPases of Arabidopsis or potato, respectively, was confirmed by reciprocal Co‐IP. In line with the function of RABA GTPases in vesicular secretion, RxLR24 co‐localized with RABA1a to vesicles and the plasma membrane. The effect of RxLR24 on the secretory process was analysed with fusion constructs of secreted antimicrobial proteins with a pH‐sensitive GFP tag. PATHOGENESIS RELATED PROTEIN 1 (PR‐1) and DEFENSIN (PDF1.2) were efficiently exported in control tissue, whereas in the presence of RxLR24 they both accumulated in the endoplasmic reticulum. Together our results imply a virulence function of RxLR24 effectors as inhibitors of RABA GTPase‐mediated vesicular secretion of antimicrobial PR‐1, PDF1.2 and possibly other defence‐related compounds

Arabidopsis-Phytophthora, un pathosystème modèle pour la caractérisation d'une interaction entre une plante et un pathogène oomycète

Les Oomycètes sont de redoutables pathogènes pour les végétaux, particulièrement pour les plantes de culture et les pertes annuelles occasionnées par ces organismes sont considérables. Leur biologie est très différente de celle des champignons, même s’ils partagent avec ces derniers un mode de croissance mycélaire. Ainsi, il n’est pas aisé de les combattre et il n’existe que peu de fongicides capables de stopper une épidémie de manière durable. Leur biologie particulière, encore peu connue, rend ces organismes très intéressants à étudier de même que les mécanismes régissant les interactions avec leurs hôtes respectifs. Un nouveau pathosystème a été élaboré en utilisant Phytophthora porri, un Oomycète infectant, entre autres, les choux (Brassica sp.) comme pathogène et Arabidopsis thaliana, une petite Brassicacée, comme hôte. Ce choix a été motivé par le caractère de plante modèle possédé par A. thaliana ainsi que par la qualité de biotrophe facultatif de P. porri, avantage que l’on ne retrouve pas chez tous les Oomycètes. Ainsi, pour la première fois lors d’une interaction entre un Oomycète et une plante, il est possible d’étudier non seulement l’interaction mais aussi les deux partenaires aux niveaux moléculaires et génétiques. Cette thèse a fait suite à mon travail de diplôme, dans lequel il s’est agi, à partir d’une quinzaine d’écotypes d’A. thaliana et de sept isolats de P. porri, d’une part de déterminer si cette plante pouvait servir d’hôte pour ce pathogène et d’autre part, d’établir une méthode d’inoculation fiable et reproductible. Ainsi, lorsque A. thaliana est infectée par P. porri, deux types d’interactions ont pu être observées lors de la caractérisation cytologique. On trouve une interaction incompatible, dans laquelle P. porri est très rapidement stoppé dans sa progression par les différentes barrières mises en place par A. thaliana, dont par exemple la réaction hypersensible qui représente la mort cellulaire d’une ou de plusieurs cellules végétales à l’endroit où le pathogène a tenté de s’introduire. On observe aussi une interaction compatible, dans laquelle la colonisation du tissu par le pathogène a lieu sans qu’il y ait de réaction visible de la part de la plante. P. porri est ainsi capable d’effectuer son cycle de vie en élaborant ses structures de reproduction végétative et sexuée dans le tissu végétal de l’hôte compatible, ce qui confirme que cet Oomycète est un véritable pathogène pour A. thaliana. Dans cette thèse, l’implication de différentes voies de biosynthèse présentes chez A. thaliana et conduisant à un état résistant dans d’autres pathosystèmes a été étudié dans le cadre de l’interaction entre cette Brassicacée et P. porri. Il a pu être établi par une analyse biochimique et moléculaire, que les principales voies de défense, à savoir celles de l’acide salicylique, de l’acide jasmonique, de l’éthylène ainsi que celle conduisant à la synthèse des phytoalexines sont mises à contribution lors du processus d’infection mais qu’elles ne sont toutefois pas les principales responsables de l’état résistant. Par ailleurs, de l’étude de mutants de ces quatre différentes voies de biosynthèse, un mutant, originellement isolé lors d’un criblage pour la déficience dans l’accumulation de phytoalexines après inoculation avec une bactérie virulente et nommé de ce fait pad 2-1, a été mis en évidence car ce dernier a présenté un phénotype d’extrême susceptibilité envers P. porri alors que les autres mutants (npr 1-1, jar 1-1, etr 1-1, ein 2.1, pad 3-1) et le transformant nahG, de même que l’écotype sauvage sont eux restés résistants suite à leur inoculation avec cet Oomycète. Des expériences d’induction d’une réaction systémique acquise, effectuées à l’aide d’un inducteur biotique et abiotique, ont donné une indication supplémentaire que la voie de l’acide salicylique, qu’elle soit enclenchée ou non, n’a pas le pouvoir d’inverser un phénotype susceptible vers un phénotype résistant. Ceci a aussi permis de confirmer les résultats des analyses biochimiques et moléculaires, desquelles découlent que pad2 est aussi empêché dans la voie de l’acide salicylique. Ainsi, la résistance envers P. porri semble être régie par un mécanisme différent de ce que l’on connaissait jusqu’à présent pour d’autres interactions impliquant une plante et un Oomycètre et nécessite un gène PAD2 fonctionnel. Par ailleurs, une analyse génétique a permis d’établir que la résistance envers P. porri est régie par au moins un gène de résistance et ceci s’est vu confirmé par le phénotype susceptible de deux mutants (ndr 1-1 et eds 1.1) empêchés dans les voies de biosynthèse situées en aval de la reconnaissance gène pour gène. Un autre aspect étudié a été le rôle d’une petite protéine lors du processus d’infection. Cette dernière est abondamment sécrétée par P. porri lorsqu’il est placé en culture liquide. L’infiltration d’un filtrat de culture dans des feuilles de Nicotiana benthamiana, une Solanacée très sensible envers divers éliciteurs, a causé des nécroses sur la surface infiltrée alors que chez A. thaliana une réaction différentielle a pu être mise en évidence, ce qui indique un processus de reconnaissance selon l’écotype testé. La protéine responsable de ces réactions a été identifiée comme étant une élicitine et a été nommée Porrine I. Le gène codant pour cette dernière a été cloné est s’est révélé faire partie d’une famille multigénique à l’instar des autres élicitines isolées chez la plupart des Phytophthora. L'ARN de Porrine I a pu être mis en évidence lors de l’interaction compatible toutefois il n’a pas été possible, dans le cadre de cette thèse, de clarifier la fonction biologique de cette élicitine.Oomycetes are pathogens responsible for many plant diseases over the world and the economical impact of their damage is quite important. Although these organisms show a mycelar growth, their biology is quite different from that of fungi. This makes them not easy to fight against and up to now no fungicide is able to stop an epidemy due to Oomycetes in a durable way. The particular biology of these organisms, which is still not well known, makes them very interesting to study together with the study of the mechanisms of the interactions with their hosts. A novel pathosystem using Phytophthora porri as pathogen, an Oomycete infecting, among other hosts, cabbage (Brassica sp.) and Arabidopsis thaliana, a little weed from the family of the Brassicacea, was established. The choice of both protagonists was justified by the feature of plant model owned by A. thaliana and because P. porri is a facultative biotroph, an advantage not shared by every Oomycete. With this pathosystem, it will be possible, for the first time, to study not only the plant-Oomycete interaction but also both organisms separately because they are amenable to molecular and genetical studies. This thesis is the next step after my diploma work, which aim was to set up the novel pathosystem. For this, 15 accessions of A. thaliana and 7 isolates of P. porri were screened in order to find accessions that could be hosts for P. porri and to establish a reproducible and reliable inoculation method. The cytological characterisation of the interaction enabled to distinguish between two distinct interactions. First, an incompatible interaction in which the growth of P. porri is rapidly stopped by the various hindrances built up by A. thaliana, among them the hypersensitive reaction which represents the death of one or several cells around the location where the pathogen attempted to penetrate. Secondly, a compatible interaction, in which the colonisation of the tissue by the pathogen takes places without any visible reaction of the plant. P. porri is able to complete its whole life cycle by producing its vegetative and sexual structures in the plant tissue of the compatible host, which confirms that this Oomycete is a true pathogen of A. thaliana. In this thesis, the implication of different biosynthetic pathways leading to resistance in A. thaliana, as observed in other plant-pathogen interactions, was investigated during the interaction between this plant and P. porri. Biochemical and molecular analysis established that the main defense pathways of A. thaliana, namely the salicylic acid-, the jasmonic acid- and the ethylene pathways as well as the one leading to the biosynthesis of the phytoalexins are induced during the interaction but are not the principal components of the resistance phenotype. The study of mutants impaired in these four different biosynthetic pathways resulted in the discovery that a mutant originally found in a screen for deficiency in phytoalexin accumulation after inoculation with a virulent bacteria and named pad 2-1, showed a hypersusceptible phenotype towards P. porri when all the other mutants (npr 1-1, jar 1-1, etr 1-1, ein 2.1 and pad 3-1) and the transformant nahG showed, like the wild type, resistance. The induction of systemic acquired resistance, with a biotic and an abiotic inducer, helped to show that the salicylic acid pathway is not involved because there is no switch from the susceptible phenotype towards a resistant phenotype when this pathway is turned on. The biochemical and molecular analysis also showed that pad2 is impaired in the salicylic acid pathway. So, the resistance towards P. porri seems to be under the control of a different mechanism than the one known so far for plant-Oomycete interactions and requires a functional PAD2 gene. A genetical analysis showed that the resistance is under the control of at least one resistance gene and this was confirmed by the susceptible phenotype of two mutants (ndr 1-1 and eds 1.2) which are impaired in the signalling pathways lying downstream of the gene-for-gene recognition events. Another aspect investigated was the role of a small protein during the infection process. This protein is abundantly secreted by P. porri in liquid culture and the infiltration of a culture filtrate in leaves of Nicotiana benthamiana, a Soleanaceous species very sensitive towards different elicitors, caused necrosis in the infiltrated area. When A. thaliana leaves were infiltrated with the same culture filtrate, a differential reaction was observed which indicates a recognition mechanism depending on the accession tested. The protein responsible for these reactions was identified as an elicitin and was named Porrine I. The gene coding for this protein was cloned and shown to belong to a multigene family as other elicitins produced by various Phytophthora species. The RNA of Porrine I could be detected during the compatible interaction but it was not possible, during this thesis, to clarify the biological function of this elicitin