Pathogenic plant-microbe interactions. What we know and how we benefit

Plants, like humans and other animals, also get sick, exhibit disease symptoms, and die. Plant diseases are caused by environmental stress, genetic or physiological disorders and infectious agents including viroids, viruses, bacteria and fungi. Plant pathology originated from the convergence of microbiology, botany and agronomy; its ultimate goal is the control of plant disease. Microbiologists have been attracted to this field of research because of the need for identification of the agents causing infectious diseases in economically important crops. In 1878—only two years after Pasteur and Koch had shown for the first time that anthrax in animals was caused by a bacteria—Burril, in the USA, discovered that the fire blight disease of apple and pear was also caused by a bacterium (nowadays known as Erwinia amylovora). In 1898, Beijerinck concluded that tobacco mosaic was caused by a “contagium vivum fluidum” which he called a virus. In 1971, Diener proved that a potato disease named potato spindle tuber was caused by infectious RNA which he called viroid

Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection.

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.

Early life host-microbe interactions : implications for the development of asthma

Plus de 300 millions de personnes dans le monde souffrent de l'asthme. L'asthme est une maladie inflammatoire chronique des voies respiratoires caractérisée par des symptômes variables et récurrents, une obstruction bronchique réversible et des bronchospasmes. Les symptômes communs incluent une respiration sifflante, de la toux, une oppression thoracique et de la dyspnée. Normalement, la maladie commence à se manifester pendant l'enfance. Pourtant, facteurs génétiques héréditaires et événements environnementaux survenant au cours de la petite enfance sont responsables de sa manifestation, indiquant que le développement de la maladie est lié à des événements qui se produisent bien avant son déclenchement. L'infection respiratoire virale aiguë constitue un de ces facteurs environnementaux jouant un rôle prépondérant. Un des virus les plus communs est le virus respiratoire syncytial (VRS), qui infecte presque tous les enfants avant l'âge de 2 ans. Ce virus, s'il infecte des tout-petits, peut en effet provoquer une bronchiolite aiguë, un phénomène qui a été épidémiologiquement lié à l'apparition d'asthme plus tard dans la vie. Dans le premier chapitre de cette thèse, nous avons étudié, chez la souris, comment une infection avec le VRS influe sur l'asthme allergique. Nous avons constaté que seule l'infection des souris à l'état de nouveau-né prédispose à un asthme allergique plus sévère chez l'adulte. En effet, si des souris adultes étaient infectées, elles étaient protégées contre l'apparition des symptômes asthmatiques. Cela nous a mené à investiguer les mécanismes immunitaires spécifiques durant cette courte période du début de la vie. Deux événements se produisent en parallèle au cours de la petite enfance: (1) Le système immunitaire, qui est encore immature immédiatement après la naissance, commence à se développer pour être en mesure de jouer son rôle protecteur contre les agents infectieux. (2) Le corps, y compris les poumons, est colonisé par des bactéries commensales, qui vivent en symbiose avec leur hôte humain. Chez l'adulte, ces bactéries sont connues pour influencer notre système immunitaire, l'éduquant à générer des réponses immunitaires adéquates et efficaces. Dans la deuxième partie de cette thèse, nous avons voulu déterminer si ces bactéries symbiotiques étaient impliquées dans l'éducation du système immunitaire du nouveau-né et quelles conséquences cela pourrait avoir sur les réponses immunitaires engendrées par ce dernier. Pour étudier l'effet de ces bactéries symbiotiques, nous avons utilisé des souris stériles, en d'autres termes des souris qui n'hébergent pas ces bactéries symbiotiques. En comparant ces souris stériles à des souris qui abritent une flore microbienne normale, nous avons constaté que les bactéries symbiotiques sont vitales pour la bonne éducation du système immunitaire du nouveau-né. Nous avons démontré que le contact direct des cellules immunitaires avec la flore microbienne dans les poumons modifie le phénotype de ces cellules immunitaires, ce qui change probablement leur réaction au cours de réponses immunitaires. Nous avons donc vérifié si l'éducation immunitaire induite par cette microflore est importante pour prévenir les maladies pulmonaires telles que l'asthme allergique, affections qui sont causées par une réaction excessive du système immunitaire envers des agents inoffensifs. En effet, nous avons observé que le processus de maturation du système immunitaire néonatal, lequel a été déclenché et façonné par la flore microbienne, est important pour éviter une réaction asthmatique exagérée chez la souris adulte. Ce phénomène est dû aux lymphocytes T régulateurs. Ces cellules, dont la présence est induite dans les poumons, ont des capacités immunosuppressives et atténuent donc les réponses immunitaires pour prévenir une inflammation excessive. En conclusion, nous avons montré dans cette thèse que la colonisation par des bactéries symbiotiques tôt dans la vie est un événement décisif pour la maturation du système immunitaire et pour prévenir le développement de l'asthme. Dans l'avenir, il serait intéressant de découvrir quelles bactéries sont présentes dans les poumons du nouveau-né et lesquelles sont directement impliquées dans ce processus de maturation immunitaire. Une prochaine étape serait alors de favoriser la présence de ces bactéries au début de la vie au moyen d'un traitement avec des agents pré- ou probiotiques, ce qui pourrait éventuellement contribuer à une prévention précoce du développement de l'asthme. -- L'asthme est une maladie chronique inflammatoire des voies respiratoires affectant près de 300 millions d'individus dans le monde. Bien que les traits caractéristiques du phénotype asthmatique s'établissent généralement pendant l'enfance, la prédisposition au développement de la maladie est intimement liée à des événements survenant durant la petite enfance, comme le sont par exemple les infections virales respiratoires aiguës. Les mécanismes par lesquels ces événements provoquent un dysfonctionnement immunitaire et, par conséquent, conduisent au développement de l'asthme n'ont pas encore été entièrement décelés. La dysbiose du microbiote des voies respiratoires a été récemment associes au phénotype asthmatique, touisTcis, la cuûoboiatioî! d un lien cause à effet entre la dysbiose microbienne et l'apparition des symptômes asthmatiques reste à être démontrée. Dans cette thèse, nous avons étudié le rôle que joue la colonisation microbienne des voies respiratoires au cours de la petite enfance dans la maturation du système immunitaire ainsi que dans la protection contre l'inflammation pulmonaire de type allergique. Nous avons de surcroît développé un modèle expérimental pour comprendre comment les infections virales respiratoires interfèrent avec ce processus. Dans la première partie de cette thèse, nous avons évalué l'effet d'infections causées par le virus respiratoire syncytial (VRS) sur le développement de l'asthme. En accord avec des études épidémiologiques, nous avons constaté qu'une infection au VRS lors de la période néonatale exacerbait les réponses pulmonaires allergiques ultérieures. Par contraste, une infection à l'âge adulte avait un effet protecteur. Nous avons ainsi démontré que l'influence d'une infection à VRS sur l'issue et la sévérité de l'asthme respiratoire était strictement dépendante de l'âge. Ces résultats nous ont conduit à émettre l'hypothèse que des différences dans le phénotype homéostatique des cellules immunitaires pourraient être responsables de ces disparités liées à l'âge. Par conséquent, dans la deuxième partie de cette thèse, nous avons suivi et caractérisé le processus de maturation des cellules immunitaires dans les poumons du nouveau-né en condition d'homéostasie. Nous avons découvert que leur phénotype change de façon dynamique pendant le développement néonatal et que la colonisation par des microbes était déterminante pour la maturation des cellules immunitaires dans les poumons. Dans la dernière partie de cette thèse, nous avons démontré comment le microbiote pulmonaire éduque le développement immunitaire durant la période néonatale l'orientant de manière à induire une tolérance face aux aéroallergènes. Nous avons découvert que la colonisation microbienne des voies respiratoires provoque une expression transitoire de PD-L1 sur les cellules dendritiques (CD) pulmonaires du type CD11b+ dans les deux premières semaines de la vie. Cet événement engendre par la suite la génération de lymphocytes T régulateurs (TREG) dans les poumons, lesquels sont responsables de la protection contre une réponse inflammatoire allergique exagérée chez la souris adulte. Par conséquent, nous proposons un rôle pivot de la maturation immunitaire induite par le microbiote pulmonaire dans l'établissement de la tolérance aux aéroallergènes. En conclusion, les résultats présentés dans cette thèse fournissent de nouveaux indices révélant comment des événements se produisant lors de la petite enfance peuvent façonner les réponses du système immunitaire dirigées contre les allergènes et soulignent le rôle central joué par le microbiote pulmonaire dans l'édification d'une réponse immunitaire équilibrée. En résumé, notre travail met en évidence le microbiote pulmonaire comme étant une cible potentielle pour la prévention de certaines maladies respiratoires. -- Asthma is a chronic inflammatory disorder of the respiratory tract and affects approximately 300 million individuals world-wide. Although the asthmatic phenotype commonly establishes during childhood, predisposition towards disease development has been linked to events in early infancy, such as severe respiratory viral infections. However, the mechanisms by which these events cause immune dysfunction and, therefore, lead to the development of asthma have yet to be fully deciphered. Dysbiosis of the airway microbiota has recently been associated with the asthmatic phenotype; however, conclusive evidence for a causal link between microbial dysbiosis in the ail ways and asthma development is still missing. In this thesis we investigated the role of early-life microbial airway colonization in immune maturation and the protection against allergic airway inflammation and established an experimental model to address how respiratory viral infections interfere in this process. In the first part of this thesis we evaluated the effect of Respiratory syncytial virus (RSV) infections on the development of asthma. In concurrence with epidemiological studies, we found that neonatal infection exacerbated subsequent allergic airway inflammation. In contrast, adult infection was protective in the same context. Thus, we could demonstrate that the influence of RSV infection on subsequent allergic airway responses was strictly age-dependent. These findings led us to the hypothesis that differences in the homeostatic phenotype of immune cells could be responsible for the age-related disparities seen within the context of RSV. Therefore, in a second part of this thesis, we followed the process of homeostatic immune cell maturation in the neonatal lung. Immune cell phenotypes changed dynamically during neonatal development. We discovered that the colonization with microbes was central to the maturation of immune cells in the lung. In the last part of this thesis, we demonstrated how microbiota-driven immune development during the neonatal period induces tolerance against aeroallergens. We discovered that microbial colonization led to a transient programmed death-ligand (PD-L) 1 expression on CD11b+ pulmonary dendritic cells (DCs) during the first two weeks of life. This in turn induced regulatory T (TREG) cells in the lung, which were responsible for the protection against exaggerated allergic airway inflammation in adult mice. Thus, we propose a key role for microbiota-driven immune maturation in the establishment of tolerance towards aeroallergens. In conclusion, the results presented in this thesis provide new insights into how early-life events shape pulmonary immune responses towards allergens and suggest the airway microbiota as a key player in establishing a balanced immune response. Overall, our work highlights the airway microbiota as potential target for disease prevention.

Plant-microbe interactions and the new biotechnological methods of plant disease control

Plants constitute an excellent ecosystem for microorganisms. The environmental conditions offered differ considerably between the highly variable aerial plant part and the more stable root system. Microbes interact with plant tissues and cells with different degrees of dependence. The most interesting from the microbial ecology point of view, however, are specific interactions developed by plant-beneficial (either non-symbiotic or symbiotic) and pathogenic microorganisms. Plants, like humans and other animals, also become sick, but they have evolved a sophisticated defense response against microbes, based on a combination of constitutive and inducible responses which can be localized or spread throughout plant organs and tissues. The response is mediated by several messenger molecules that activate pathogen-responsive genes coding for enzymes or antimicrobial compounds, and produces less sophisticated and specific compounds than immunoglobulins in animals. However, the response specifically detects intracellularly a type of protein of the pathogen based on a gene-for-gene interaction recognition system, triggering a biochemical attack and programmed cell death. Several implications for the management of plant diseases are derived from knowledge of the basis of the specificity of plant-bacteria interactions. New biotechnological products are currently being developed based on stimulation of the plant defense response, and on the use of plant-beneficial bacteria for biological control of plant diseases (biopesticides) and for plant growth promotion (biofertilizers)

Tissue culture propagation alters plant-microbe interactions in tobacco rhizosphere

We have compared properties of roots from different lines (genotypes) of tobacco raised either in tissue culture or grown from seed. The different lines included unmodified plants and plants modified to express reduced activity of the enzyme cinnamoyl-CoA reductase, which has a pivotal role in lignin biosynthesis. The size and structure of the rhizosphere microbial community, characterized by adenosine triphosphate and phospholipid fatty acid analyses, were related to root chemistry (specifically the soluble carbohydrate concentration) and decomposition rate of the roots. The root material from unmodified plants decomposed faster following tissue culture compared with seed culture, and the faster decomposing material had significantly higher soluble carbohydrate concentrations. These observations are linked to the larger microbial biomass and greater diversity of the rhizosphere communities of tissue culture propagated plants.

Tissue culture propagation alters plant-microbe interactions in tobacco rhizosphere

We have compared properties of roots from different lines (genotypes) of tobacco raised either in tissue culture or grown from seed. The different lines included unmodified plants and plants modified to express reduced activity of the enzyme cinnamoyl-CoA reductase, which has a pivotal role in lignin biosynthesis. The size and structure of the rhizosphere microbial community, characterized by adenosine triphosphate and phospholipid fatty acid analyses, were related to root chemistry (specifically the soluble carbohydrate concentration) and decomposition rate of the roots. The root material from unmodified plants decomposed faster following tissue culture compared with seed culture, and the faster decomposing material had significantly higher soluble carbohydrate concentrations. These observations are linked to the larger microbial biomass and greater diversity of the rhizosphere communities of tissue culture propagated plants.

Cell Host-Microbe Interactions: Turning Pathogen Mechanisms Into Cell's Advantages

Host-Pathogen Interaction is a very vast field of biological sciences, indeed every year many un- known pathogens are uncovered leading to an exponential growth of this field. The present work lyes between its boundaries, touching different aspects of host-pathogen interaction: We have evaluate the permissiveness of Mesenchimal Stem cell (FM-MSC from now on) to all known human affecting herpesvirus. Our study demonstrate that FM-MSC are full permissive to HSV1, HSV2, HCMV and VZV. On the other hand HHV6, HHV7, EBV and HHV8 are susceptible, but failed to activate a lytic infection program. FM-MSC are pluripotent stem cell and have been studied intensely in last decade. FM-MSC are employed in some clinical applications. For this reason it is important to known the degree of susceptibility to transmittable pathogens. Our atten- tion has then moved to bacterial pathogens: we have performed a proteome-wide in silico analy- sis of Chlamydiaceae family, searching for putative Nuclear localization Signal (NLS). Chlamy- diaceae are a family of obligate intracellular parasites. It’s reasonably to think that its members could delivered to nucleus effector proteins via NLS sequences: if that were the case the identifi- cation of NLS carrying proteins could open the way to therapeutic approaches. Our results strengthen this hypothesis: we have identified 72 protein bearing NLS, and verified their func- tionality with in vivo assays. Finally we have conceived a molecular scissor, creating a fusion protein between HIV-1 IN protein and FokI catalytic domain (a deoxyexonuclease domain). Our aim is to obtain chimeric enzyme (trojIN) which selectively identify IN naturally occurring target (HIV LTR sites) and cleaves subsequently LTR carrying DNA (for example integrated HIV1 DNA). Our preliminary results are promising since we have identified trojIN mutated version capable to selectively recognize LTR carrying DNA in an in vitro experiments.

Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease

Crohn's disease and ulcerative colitis, the two common forms of inflammatory bowel disease (IBD), affect over 2.5 million people of European ancestry, with rising prevalence in other populations. Genome-wide association studies and subsequent meta-analyses of these two diseases as separate phenotypes have implicated previously unsuspected mechanisms, such as autophagy, in their pathogenesis and showed that some IBD loci are shared with other inflammatory diseases. Here we expand on the knowledge of relevant pathways by undertaking a meta-analysis of Crohn's disease and ulcerative colitis genome-wide association scans, followed by extensive validation of significant findings, with a combined total of more than 75,000 cases and controls. We identify 71 new associations, for a total of 163 IBD loci, that meet genome-wide significance thresholds. Most loci contribute to both phenotypes, and both directional (consistently favouring one allele over the course of human history) and balancing (favouring the retention of both alleles within populations) selection effects are evident. Many IBD loci are also implicated in other immune-mediated disorders, most notably with ankylosing spondylitis and psoriasis. We also observe considerable overlap between susceptibility loci for IBD and mycobacterial infection. Gene co-expression network analysis emphasizes this relationship, with pathways shared between host responses to mycobacteria and those predisposing to IBD.

The Roles of Phenotypic Plasticity and Plant-Microbe Interactions in the Evolution of Complex Traits in Boechera stricta

All organisms live in complex habitats that shape the course of their evolution by altering the phenotype expressed by a given genotype (a phenomenon known as phenotypic plasticity) and simultaneously by determining the evolutionary fitness of that phenotype. In some cases, phenotypic evolution may alter the environment experienced by future generations. This dissertation describes how genetic and environmental variation act synergistically to affect the evolution of glucosinolate defensive chemistry and flowering time in Boechera stricta, a wild perennial herb. I focus particularly on plant-associated microbes as a part of the plant’s environment that may alter trait evolution and in turn be affected by the evolution of those traits. In the first chapter I measure glucosinolate production and reproductive fitness of over 1,500 plants grown in common gardens in four diverse natural habitats, to describe how patterns of plasticity and natural selection intersect and may influence glucosinolate evolution. I detected extensive genetic variation for glucosinolate plasticity and determined that plasticity may aid colonization of new habitats by moving phenotypes in the same direction as natural selection. In the second chapter I conduct a greenhouse experiment to test whether naturally-occurring soil microbial communities contributed to the differences in phenotype and selection that I observed in the field experiment. I found that soil microbes cause plasticity of flowering time but not glucosinolate production, and that they may contribute to natural selection on both traits; thus, non-pathogenic plant-associated microbes are an environmental feature that could shape plant evolution. In the third chapter, I combine a multi-year, multi-habitat field experiment with high-throughput amplicon sequencing to determine whether B. stricta-associated microbial communities are shaped by plant genetic variation. I found that plant genotype predicts the diversity and composition of leaf-dwelling bacterial communities, but not root-associated bacterial communities. Furthermore, patterns of host genetic control over associated bacteria were largely site-dependent, indicating an important role for genotype-by-environment interactions in microbiome assembly. Together, my results suggest that soil microbes influence the evolution of plant functional traits and, because they are sensitive to plant genetic variation, this trait evolution may alter the microbial neighborhood of future B. stricta generations. Complex patterns of plasticity, selection, and symbiosis in natural habitats may impact the evolution of glucosinolate profiles in Boechera stricta.

Weights in the balance: jasmonic acid and salicylic acid signaling in root-biotroph interactions.

Work on the interaction of aerial plant parts with pathogens has identified the signaling molecules jasmonic acid (JA) and salicylic acid (SA) as important players in induced defense of the plant against invading organisms. Much less is known about the role of JA and SA signaling in root infection. Recent progress has been made in research on plant interactions with biotrophic mutualists and parasites that exclusively associate with roots, namely arbuscular mycorrhizal and rhizobial symbioses on one hand and nematode and parasitic plant interactions on the other hand. Here, we review these recent advances relating JA and SA signaling to specific stages of root colonization and discuss how both signaling molecules contribute to a balance between compatibility and defense in mutualistic as well as parasitic biotroph-root interactions.

A molecular analysis of the interactions of escherichia coli and macrophages

Escherichia coli (E.coli) is a diverse bacterial species that primarily forms a beneficial symbiotic relationship with the host in the human lower gastrointestinal track (GIT), however it can also be pathogenic in this environment. Furthermore, some strains can diverge from the GIT and occupy niches such as the urinary tract. In all these environments, E. coli interacts with the immune system and macrophages represent the front line of the innate immune system. In this study we characterise the immune response by macrophages to E. coli infection. It was shown that E. coli broadly provoke a similar cytokine response during macrophages infection and furthermore are degraded primarily by the phagocytosis pathway. Recently a new group of E. coli called Adherent Invasive Escherichia coli (AIEC) has been described. AIEC are present in the guts of Crohn’s disease (CD) patients at a higher frequency than in healthy patients. AIEC can replicate in macrophages but the mechanism for this is not fully understood. The processing of AIEC by macrophages was investigated and it was shown that AIEC only replicated in permissive macrophages. Furthermore, even in a permissive macrophages AIEC are trafficked through macrophages in a similar manner to commensal E. coli. This supports the hypothesis that AIEC are highly similar to commensal E. coli and only cause pathogenicity when present in the permissive environment of the gut of CD patients. Replication in macrophages requires functioning metabolic pathways and it was identified that glycolysis is important for AIEC survival in macrophages. AIEC mutants without a fully functioning glycolysis pathway induced less IL-1β cytokine release from macrophages than wild type strain suggesting that metabolism plays a role in inflammasome activation. Furthermore, AIEC mutants that could not produce the glycolytic end product acetate induced significantly reduced IL-1β release during infection. This suggest that the acetate molecule or a phenotypic effect of its production may be a driver of IL-1β release from AIEC infected macrophages. The interaction of uropathogenic E. coli (UPEC) with macrophages was also investigated. UPEC induced very high levels of cytotoxicity in human macrophages which was shown to be dependent on the production of the pore forming toxin α-hemolysin. However, UPEC did not induced high levels of cytotoxicity in murine macrophages suggesting there are species specific sensitivity to α-hemolysin that should be considered when studying UPEC pathogenicity in murine models.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.