Lipopolysaccharides from Gram-negative bacteria are amphipathic, tripartite molecules consisting of a hydrophobic lipid A portion, a core hetero-oligosaccharide and a repetitive hydrophilic O-antigen polysaccharide region. Through cell : cell interactions, plants can come into contact with LPS originating from root-associated rhizobacteria, bacterial endophytes as well as bacterial pathogens. Biologically active LPS molecules have been shown to act as determinants of bacterial virulence but also as determinants of induced systemic resistance (ISR) and activators of the phenotypically related systemic acquired resistance (SAR), characterised by accelerated and enhanced defence responses. LPS as a ¡¥pathogen associated molecular pattern, PAMP¡¦ molecule, has the ability to activate the innate mammalian immunity system and to act as an immunomodulator of immune ¡V and inflammatory systems via the conserved lipid A region. It is thus believed that LPS is able to promote plant disease resistance through activation of ISR and/or SAR; however here, the O-antigen region is also implicated to play a pivotal role in the signal perception and transduction in response to elicitation by this bio-active lipoglycan. LPS was isolated from the cell walls of the endophyte, Burkholderia cepacia, characterised by denaturing electrophoresis and compared to the equivalent from the pathogen Ralstonia solanacearum. When dissolved in the presence of Ca2+ and Mg2+, the LPS attained its biologically active micellar state through complex formation. The former LPS strongly induced the activation of two MAPKs following treatment of Nicotiana tabacum cv Samsun leaves, while comparative inductions with the R. solanacearum counterpart were extremely weak and might be ascribed to it lacking an extensive O-antigen region. No previous reports on LPS-responsive MAP kinases in plant tissues exist in the literature. The time- and dose dependent activation of the two kinases were therefore investigated and their physico-chemical properties compared. A novel 32 kDa MAP kinase was transiently activated in response to exposure to LPS with optimal activation at 7 min post-elicitation with 100 ƒÝg.ml-1 LPS. Its identity as an ERK (extracellular signal-related) MAPK was confirmed by immunodetection with a pTEpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylated association of activation and inhibition of activation by U0126, an inhibitor of upstream MAPKKs. The kinase did not utilise casein, histone or myelin basic protein as substrates and no endogenous substrate could be identified. The activated MAP kinase exhibited a pI of 6.3, but two charge isomers of 32 kDa respectively were found upon two-dimensional electrophoresis. Although loss of the dual-phosphorylated epitope during purification attempts prevented extensive purification, 30% ammonium sulphate fractionation significantly (33 fold) enriched the MAPK. A second, distinct, 30 kDa MAP kinase was transiently activated in response to 125 ƒÝg.ml-1 LPS at 40 min post-elicitation, and its identity as a p38 MAPK, to date not yet found in plants, was confirmed by immunodetection with a pTGpY-specific (anti-active) MAPK antibody, tyrosine-phosphorylation associated with activation and inhibition of activation by SB203580, a direct inhibitor of p38 MAPKs. The kinase did not utilise casein, histone or MBP as substrates and no endogenous substrate could be identified. The kinase displayed a pI of 6.0, but two charge isomers of 30 kDa respectively were found following two-dimensional electrophoresis. Loss of the dual-phosphorylated epitope again prevented significant purification, but the protein was found to be significantly (83 fold) enriched by 30% ammonium sulphate fractionation. Although LPS has been reported to be capable of altering Ca2+ permeability and perturbation of Ca2+ homeostasis across plasma membranes, Ca2+ did not appear to potentiate or reduce the activation of either the 30 or the 32 kDa kinases. To date other MAP kinases have been shown to act either independently or upstream from reactive oxygen intermediates (ROI) produced during the oxidative burst. It was found that peroxide and concomitant ROI is either not generated in leaf tissue in response to LPS elicitation, or if generated, do not trigger the activation of the two kinases. The identification and partial characterisation of these two novel tobacco MAPKs in the signal perception and transduction response to LPS, significantly contributes to understanding the biochemical basis of the mechanism of action of LPS as a ¡¥resistance elicitor¡¦ involved in the triggering of effective plant defence responses and contributes towards relating the activation of mammalian innate immunity to similar responses in plants. / Prof. I.A. Dubery

Plants respond to pathogen attack by inducing a coordinated resistance strategy, which results in the expression of defense gene products. When a plant-pathogen interaction results in disease establishment, parasite colonization is caused by a delayed plant defense response, not due the absence of any response. Thus, the speed and intensity of the plant response and intracellular signalling determines the outcome of a plant-pathogen interaction. The acceleration of plant responses by the application of resistance inducers could provide a commercially, biologically and environmentally feasible alternative to existing pathogen control methods. Lipopolysaccharides are amphipathic lipoglycans that are attached to the outer bacterial membrane by a lipidic entity inserted into the bacterial phospholipid monolayer, with the saccharidic part oriented towards the exterior. The general structure of this compound is comprised of an anchor named lipid A associated with a core polysaccharide, which bears an O-antigen domain. LPS has been described as one of the pathogen-associated molecular patterns (PAMPs) capable of eliciting the activation of the plant innate immune system. LPS present in the outer membranes of plant growth-promoting rhizobacteria (PBPR) are major determinants of induced systemic resistance (ISR). In addition, LPS may function as an activator of systemic acquired resistance (SAR), providing non-specific immunization against later infection. Evidence suggests that LPS may advance plant disease resistance using the mechanism of ISR or SAR through its application to plants as a sensitizing agent, priming them to respond more effectively to subsequent pathogen attack. Phosphorylation plays a major role during the plant defense response, exemplified by its phosphorylation of transcription factors, required for the expression of defense-related genes. One of the most extensively documented phosphorylation responses is that of MAP kinase activation by phosphorylation in response to elicitation by race-specific and non-racespecific elicitors in various plant species.Proteins that undergo differential phosphorylation as a result of elicitation could be components of signal transduction pathways which connect pathogen perception with defense responses. Thus the identification of protein kinases, protein phosphatases and their substrates is essential in the elucidation of plant defense responses. The hypothesis behind this dissertation is that LPS elicitation results in alterations in the phosphorylation profile of Arabidopsis thaliana proteins. In this study, LPS was extracted from the cell walls of Burkholderia cepacia, a bacterial endophyte, and characterized by SDS-PAGE. The exposure of Arabidopsis callus culture cells to LPS resulted in distinctive changes in the phosphoprotein profile of the cells. Radioactive phosphorous labelling of proteins provided evidence that phosphorylation occurs in Arabidopsis following LPS perception, as part of a defense response related to LPS elicitation. Further investigation of differential protein phosphorylation via immunoblotting with antiphosphotyrosine antibodies revealed that tyrosine phosphorylation of Arabidopsis proteins occurs in response to LPS. One of the tyrosine-phosphorylated proteins was found to be a 42 kDa kinase, activated in response to LPS elicitation. The identity of the kinase as a mitogen-activated protein (MAP) kinase was confirmed by immunoblotting with anti-active MAP kinase antibodies. In addition, an assay of MAP kinase activity demonstrated the ability of the LPS-responsive MAP kinase to phosphorylate the ERK-MAP kinase substrate Elk1. In terms of the global phosphoproteome of Arabidopsis in response to LPS, phosphopeptides were purified from a crude protein digest by immobilized metal affinity chromatography and analyzed by liquid chromatography-tandem mass spectrometry (LCMS/ MS). While LC indicated both quantitative and qualitative differences resulting from LPS elicitation, no peptides could be positively identified as phosphopeptides by MS analysis. This work can however be repeated with further precautions to prevent the loss of phosphate groups prior to analysis. The results obtained in this study indicate that LPS causes specific alterations in Arabidopsis protein phosphorylation as a post-translational modification in response to the perception of LPS during a plant-pathogen interaction, proving the original hypothesis. / Prof. I.A. Dubery

M.Sc. / Plant cells have evolved the ability to detect pathogen ingress and subsequently activate defense-related functions as part of the plant pathogen response. One or more poorly defined signal transduction pathways, initiated upon recognition of the pathogen by the plant host, regulate expression of plant defense genes. Acquired resistance (AR) is an inducible defense mechanism exhibited by many plants that provides protection against a broad range of pathogens.Many chemical and environmental cues can elicit the same defenses or subsets therein. Cytochrome P450 enzymes are heme-dependent, mixed function oxidase systems that utilize dioxygen to produce a functionalized organic substrate and a molecule of water. They play important biosynthetic and detoxicative roles. They have been identified as being involved in the activation (e.g. allene oxide synthase) and execution of plant defense responses. To investigate the involvement of cytochromes P450 in the plant defense response - mainly through the activation of allene oxide synthases in the jamonate signaling pathway — Phaseolus vulgaris L.cv. Contender leaves were treated with chemical elicitors to mimic the plant-pathogen interaction and thereby activate plant defense responses. Through the use of differential display reverse transcription polymerase chain reaction and denaturing polyacrylamide gel electrophoresis, differentially expressed cDNA bands were isolated, cloned and sequenced. One of the cloned cDNA fragment proved to be a previously unreported cytochrome P450 cDNA, and was named CYP98A5. Dot blot analysis of bean leaves treated with various chemicals showed an enhanced expression of CYP98A5 in leaves treated with 3,5- dichlorosalicylic acid. Northern blot analysis of a time dependent induction study of CYP98A5 in bean leaves treated with this chemical compound indicated that 3,5-dichlorosalicylic acid induces CYP98A5 transcripts earlier than it is induced in control leaves. This might be indicative of a possible conditioning and sensitizing effect of 3,5- dichlorosalicylic acid on bean leaves to a more rapid and effective response with defense reactions once attacked by pathogens. CYP98A5, however, did not appear to be an allene oxide synthase when sequence comparison with other allene oxide synthases was performed; isolation and comparison of the complete CYP98A5 sequence could prove this wrong. It is not possible to assign any function to CYP98A5 at this stage; elucidation of the function of this enzyme in plants would provide more insight into this study.

Ralstonia solanacearum est une bactérie phytopathogène à la gamme d'hôte exceptionnellement large et à la répartition mondiale. Cet organisme présente une biologie à facettes multiples et s'est adapté à quasiment tous les types de sols, à la vie planctonique, et à de nombreux hôtes et plantes réservoirs. Cette capacité d'adaptation est attestée par une très forte hétérogénéité des souches qui unifient ce complexe d'espèces, aussi bien au plan de la diversité génétique, phénotypique, que de la gamme d'hôte. Des approches phylogénétiques ont montré une structuration de la population mondiale en quatre phylotypes qui correspondent globalement à l'origine géographique des souches. Les travaux de thèse portent sur des souches du phylotype II qui ont valeur de modèle expérimental car épidémiologiquement inféodées à un hôte particulier : souches Moko pathogènes du bananier, souches ‘Brown rot’ adaptées à la pomme de terre et souches émergentes NPB, un variant du pouvoir pathogène. La question de recherche centrale porte sur la compréhension des mécanismes d'adaptation à l'hôte. Pour cela, une dizaine de génomes ont été séquencés dans une perspective (i) de revisiter la taxonomie de ce complexe d'espèce, (ii) d'en faire une analyse génomique comparative et (iii) d'analyser les paysages transcriptomiques produits lors de l'infection (in planta). L'ensemble des ces approches complémentaires permettent ainsi d'intégrer la complexité génétique et phénotypique de l'organisme de manière plus systémique. / Ralstonia solanacearum is a plant pathogenic bacterium globally distributed with a particularly broad host range. This organism is biologically diverse and is adapted to all types of soil, to planktonic lifestyle and to many plant hosts and natural reservoirs. This bacterium is a species complex and its genetic, phenotypic and host range diversity is a direct consequence of adaptation mechanisms. Phylogenetic analyses have divided this species complex into four distinct phylotypes correlating mostly with strains’ geographical origin. This thesis focuses on using phylotype II strains as an experimental model due to their adaptation to specific hosts: Moko strains pathogenic to banana, ‘Brown rot’ strains adapted to potatoes and emergent pathological variant NPB strains. Our main research topic is the understanding of host adaptation processes. In order to tackle this problematic we sequenced about ten genomes as a starting point of (i) a taxonomic revision of the species complex (ii) a comparative genomic analysis and (iii) an in planta transcriptomic analysis. Together, these complementary approaches allow a more systemic view of this organism’s genetic and phenotypic complexity.

Fusarium is one of the most important genera of fungi on earth. Many species of Fusarium are well-suited for atmospheric dispersal, yet little is known about their aerobiology. Previous research has shown that large-scale features known as atmospheric transport barriers (Lagrangian coherent structures) guide the transport and mixing of atmospheric populations of Fusarium. The overall goal of this work is to expand our knowledge on the movement and structure of atmospheric populations of Fusarium. The first objective was to monitor changes in colony forming units (CFUs) in atmospheric populations of Fusarium over small time intervals (10 min to several hours). We hypothesized that consecutive collections of Fusarium with unmanned aerial vehicles (UAVs) demonstrate small variations in colony counts. To test this hypothesis, sampling devices on UAVs were separated into two groups, four inner sampling devices opened during the first 10 minutes and four outer sampling devices opened during the second 10 minutes. Results indicated that (1) consecutive collections of Fusarium at 100 m demonstrated small variations in counts and (2) the similarity between collections decreased as the time between sampling intervals increased. The second objective was to determine the structure of atmospheric populations of Fusarium species and relate this to potential source regions. We hypothesized that diverse atmospheric populations of Fusarium are associated with multiple source regions. To test this hypothesis, Fusarium samples were collected with UAVs and identified to the level of species by sequencing a portion of the translation elongation factor 1-alpha gene (TEF-1•). Potential source regions were identified using the atmospheric transport model HYSPLIT. Results indicated that (1) diverse atmospheric populations of Fusarium appeared to be associated with multiple source regions, and (2) the number of Fusarium species collected with UAVs increased with back-trajectory distance of the sampled air. The third objective was to examine the associations between concentrations of populations of Fusarium at ground level (1 m) and in the lower atmosphere (100 m). We hypothesized that concentrations of Fusarium in the atmosphere vary between 1m and 100m. To test this hypothesis, Fusarium was collected with a Burkard volumetric sampler (BVS) and UAVs. Colony counts were converted to spore concentrations (spores per cubic meter of air). Sampling efficiency was used to correct spore concentrations. Results indicated that (1) the distribution of spore concentrations was similar for both samplers over different times of the day, (2) spore concentrations were generally higher in the fall, spring, and summer, and lower in the winter, and (3) spore concentrations were generally higher with BVS samplers than those with UAVs for both hourly and seasonal data. The fourth objective was to assess the ability of strains of Fusarium collected in the lower atmosphere to cause plant disease. We hypothesized that certain isolates of Fusarium collected with UAVs cause plant diseases. To test this hypothesis, we randomly selected isolates of three different species (F. circinatum, F. avenaceum, and F. sporotrichioides) of Fusarium collected with UAVs to inoculate three different hosts (wheat, corn, and pine). Known Fusarium strains were obtained from J. Leslie at Kansas State University as controls. Results indicated showed that the three different isolates tested were able to cause plant diseases in three different hosts (wheat, corn, and pine), confirming that these were potential agents of disease. This work sets the stage for future work examining potential source regions, transport distances, and seasonal patterns of Fusarium. An increased understanding of the dynamics and population structure of plant pathogenic Fusarium in the lower atmosphere is essential for predicting the spread of plant disease and optimizing disease management strategies in the future. / Ph. D.

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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22128号 / 農博第2374号 / 新制||農||1073(附属図書館) / 学位論文||R1||N5236(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 田中 千尋, 教授 寺内 良平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p><em>Eutrema salsugineum</em> is an extremophile plant native to the Yukon Territory and coastal China. As an extremophile, Yukon <em>Eutrema</em> is tolerant to highly saline, drought conditions and cold temperatures while Shandong <em>Eutrema</em> can survive in highly saline conditions (Griffith et al., 2007; Guevara et al., 2012; Inan et al., 2004). The disease resistance responses of the Yukon and Shandong accessions of <em>Eutrema</em> were investigated to understand how an abiotic stress-tolerant plant responds to biotic stress. A pathosystem was developed using <em>Pseudomonas</em> <em>syringae</em> pv. <em>tomato</em> DC3000 (<em>Pst</em>) to examine <em>Eutrema</em> defense responses. Compared to <em>Arabidopsis </em>(Col-0), both <em>Eutrema</em> accessions exhibited resistance to <em>Pst,</em> with Shandong <em>Eutrema</em> displaying greater resistance than Yukon <em>Eutrema</em>. Resistance to <em>P. syringae</em> pv. <em>maculicola</em> (<em>Psm</em>) was also observed in both accessions. Furthermore, both <em>Eutrema</em> accessions displayed a differential capacity for effector-triggered immunity (ETI). RNA-Seq data of uninoculated Shandong vs. Yukon <em>Eutrema</em> revealed an overrepresentation of defense genes including <em>PR1</em> (<em>pathogenesis-related1</em>; Champigny et al., 2013). Expression of the <em>Eutrema</em> <em>PR1</em> ortholog in uninoculated Shandong leaves combined with enhanced resistance to <em>Pst</em> compared to Yukon <em>Eutrema</em> or Col-0 <em>Arabidopsis</em> suggests that Shandong plants exist in a defense-primed state. Resistance to other pathogens such as <em>Pectobacterium</em> <em>carotovorum</em> ssp. <em>wasabiae</em> (<em>Pcw</em>) further supported the hypothesis that Shandong <em>Eutrema</em> is primed for pathogen tolerance. The <em>Eutrema</em>-<em>P. syringae</em> pathosystem will facilitate future studies to understand how <em>Eutrema</em> deals with multiple or concurrent stresses and this knowledge will contribute to efforts to improve tolerance to both abiotic and biotic stress in crop plants.</p> / Master of Science (MSc)