M.Sc. (Biochemistry) / A 14-member, closely-spaced cluster of genes coding for leucine-rich repeat receptor-like kinases (LRR-RLKs) is located on chromosome 1 of Arabidopsis thaliana. Following on from previous microarray studies that found some of the members of this cluster to be upregulated in response to biotic stressors, including the bacterial elicitor flg22, the present study sought to confirm, using a luciferase-based protoplast assay, that flg22 does in fact induce the expression of the genes, and then to investigate the promoters of the genes. The promoters of At1g51790, At1g51850 and At1g51890 responded positively in this particular assay, and bioinformatic analyses determined that W-boxes are over-represented in the cloned regions. Mutational inactivation of individual W-boxes in the promoter of At1g51790 drastically reduced the flg22 response, except for the W-box closest to the start site, which seemed to increase both basal and flg22-inducible expression. In the promoter of At1g51850, mutational inactivation of either or both of its W-box dyads resulted in virtually no flg22 inducibility. The deletion of 6 W-boxes in the promoter of At1g51890, done via truncation, drastically reduced both its basal expression and its inducible response to flg22. These results provide evidence that W-box cis-elements are responsible for the upregulation of these LRR-RLKs in response to flg22. WRKYs -7, -11, -22,and -26 were found bioinformatically to have similar expression patterns to some of the genes in the cluster, and are thus good candidates to investigate as transcriptional regulators of the cluster in future studies.

Because plants lack a circulating adaptive immune system, they have evolved multicomponent defense mechanisms to protect themselves against pathogen attack. These defense mechanisms/responses are either constitutively active in the plant, or they are inducible by pathogens. Understanding of the plant response to pathogen attack has advanced rapidly in recent years. Bacterial and fungal pathogenicity factors have been isolated, and mechanisms that are utilized by the plant to recognize the pathogen and initiate a plethora of defense mechanisms have been identified. In contrast to the well-documented effects of LPS on mammalian cells, the effects of LPS on plant cells have been far less studied. The present study focused on the involvement of lipopolysaccharides (LPS) isolated from the outer cell wall of the Gram-negative bacteria, Burkholderia cepacia (strain ASP B 2D), and yeast elicitor (YE, a cell wall preparation from Saccharomyces cerevisiae) on the molecular mechanisms and components involved in signal transduction and defense-related responses in suspension cultured cells derived from tobacco plants (Nicotiana tabacum cv. Samsun). LPS was extracted, analyzed by denaturing electrophoresis and characterized with regard to 2-keto-3-deoxyoctonate (KDO) content, carbohydrate content, and protein content. The purified LPS and YE were found to trigger defense- and resistance-related responses in the tobacco cells. These responses included a rapid influx of Ca2+ into the cytoplasm of transgenic aequorin-transformed tobacco cells, the production of reactive oxygen species (ROS) during the oxidative burst, alkalinization of the extracellular culture medium of the cells, and changes in the protein phosphorylation patterns of the cells. Time- and concentration-dependent studies for the induction of perception and signal transduction-related responses by YE and LPS indicated that 100 µg.ml-1 of either elicitor was sufficient to induce significant responses in the cells. YE and LPS both induced a rapid transient increase in cytosolic Ca2+ levels, returning to basal levels after seconds, followed by a second, larger and long-term increase in cytosolic Ca2+. The YE-induced cytosolic Ca2+ influx was 7.5 fold higher than that of LPS. Luminol-dependent chemiluminescence measurements of hydrogen peroxide (H2O2) produced during the YE- and LPS-induced oxidative burst reactions indicated 3.5 fold higher levels of H2O2 induced by YE than that induced by LPS. Total inhibition of H2O2 production by YE- and LPS-induced cells was observed upon treatment of the cells with the H2O2-degrading enzyme, catalase. ROS production was also analyzed by the H2DCF-DA-derived fluorescence assay. The degree of ROS production by YE-treated cells was larger than that of cells treated with LPS, suggesting that YE is a more potent inducer of plant defense responses than LPS. Categorization of the origins of the oxidative bursts, induced by YE and LPS, by the addition of a ROS scavenger (NAC), inhibitors of ROS production (DPI and DDC) and a nitric oxide scavenger (PTIO) indicated that YE and LPS induced different quantities of the same ROS species. The induced ROS included O2-·, H2O2 and perhaps other ROS species as well. In addition, both YE and LPS induced a remarkable burst of nitric oxide (NO), as determined by the 97% and 95% respective inhibitions of the H2DCF-DA-derived fluorescence by the nitric oxide scavenger PTIO. Alkalinization of the extracellular culture medium of the tobacco cells was observed after treatment of the cells with YE and LPS. Both of these elicitors induced a significant increase in extracellular pH from resting pH values of 5.7 to pH 6.3 by YE, and 6.0 by LPS. Notably, the YE-induced response returned to near basal pH levels after 50 min, while the LPS-induced response showed no signs of declining and fluctuated around pH 5.9 for the duration of the experiment. YE and LPS both induced the hyperphosphorylation of two distinct proteins with approximate molecular masses of 28 kDa and 2 kDa. Changes in the pattern of the [32P]-radiolabeled proteins pp28 became visible after 20 min of YE-elicitation and 30 min of LPS-elicitation and changes in pp2 phosphorylation became visible after 20 min treatment of the cells with both elicitors. Addition of the protein kinase inhibitor, staurosporine, to the cells followed by subsequent elicitation by YE or LPS, resulted in inhibition or abolishment of the elicitor-induced responses during the oxidative burst, extracellular alkalinization, and protein phosphorylation. In contrast, the addition of the protein phosphatase inhibitor, calyculin A, was found to mimic elicitor action in several aspects, including extracellular alkalinization, the oxidative burst and protein phosphorylation, even in the absence of elicitors or any other stimulus. Thus, a fine balance between the actions of certain protein kinases and protein phosphatases is an essential component of signal transduction during YE and LPS elicitation of tobacco cells but the identification and characterization of the staurosporine-sensitive protein kinases and their substrates are necessary to gain a better understanding of the chemosensory perception and signal transduction of the YE and LPS elicitor signals in plant cells. Moreover, the question of whether these perception and transduction mechanisms are connected with a reduced activity of a protein phosphatase, or with the increased activity of a protein kinase, or even a combination of both, remains to be elucidated. / Prof. I.A. Dubery

M.Sc. (Biochemistry) / Our comprehension of pathogen perception and defense response mechanisms that play key roles in the resistance of plants against pathogen attack have progressed substantially within the recent years. Recognizing the molecular mechanisms involved in pathogen perception is the basis of understanding the signalling networks that are involved, including the transcriptional regulation of plant defense genes. This has proven to be a great challenge in plant pathology and, as such, has attracted much attention. The receptor-like kinases (RLKs) constitute one of the largest classes of plant defense genes in Arabidopsis thaliana, and contains, inter alia, the well-known leucine-rich repeats-RLKs (LRR-RLK), as well as the S-domain receptor-like kinases (SD-RLKs) that have been shown to be involved in pathogen perception and not only self-incompatibility (SI) as originally discovered. Some members of these RLKs are able to detect pattern-associated molecular patterns (PAMPs), which are conserved pathogen-derived molecules, and trigger a battery of basal defense responses. The transcriptional activation and expression levels of RLKs are dependent on the variation in promoter architecture as a result of the number, location, order and class of cis-elements found in a promoter sequence. It is hypothesized that candidate RLK genes involved in PAMP surveillance are triggered and transcriptionally regulated in response to perception of PAMPs, and that the intensity of response is relative to the promoter architecture. The primary objective was to identify SD-RLKs and LRR-RLKs which demonstrated up-regulation in response to PAMPs. The SD-RLKs (At1g11330, At1g61430 and At1g61610) and LRR-RLKs (At1g51850, At2g19190 and At5g45840) were selected on the basis of microarray data (Nürnberger - TAIR accession set 100808727) and the Genevestigator database, and characterized utilizing bioinformatics tools. Here, molecular techniques were used to show that the selected RLK genes were responsive to PAMP inductions. Furthermore, this study explored which cis-elements and their corresponding transcription factors (TFs) are found in the promoter of plant defense genes and that may be involved in transcriptional regulation thereof...

Antibiotic resistance is a major threat to public health. Understanding how it evolves, and the genes that underlie resistance, is the main goal of my Ph.D. research. After a resistance mutation arises, it’s fate within a pathogen population will be etermined in part by its fitness: mutations that suffer little or no fitness cost are more likely to persist in the absence of antibiotic treatment. My research centers on understanding this process better by gaining knowledge about the spectrum of fitness effects associated with antibiotic resistance mutations. Using a meta-analysis framework I find that, across a range of antibiotics and pathogens, on average single resistance mutations exhibit fitness costs in the absence of drug, however, there are instances of cost-free mutations. To evaluate the conditions leading to the persistence of resistance in the absence of antibiotic, I use experimental evolution of the opportunistic pathogen Pseudomonas aeruginosa and the antibiotic ciprofloxacin to investigate the phenotypic and genetic differences associated with constant and fluctuating drug treatment. I find that fluctuating drug treatment leads to the evolution of cost-free resistance. At the genetic level, cost-free resistance is the result of second-site mutations that compensate for the fitness cost associated with ciprofloxacin-resistance mutations. Further examination of the resistance mutations shows a lack of epistatic interactions between co-occurring mutations that confer resistance within a single isolate. To investigate the repeatability of the genetic causes of resistance, I execute a second evolution experiment using multiple clinical strains of P. aeruginosa adapting to a constant ciprofloxacin selective pressure. I find a remarkable lack of parallel evolution at the genomic level both within and between different P. aeruginosa strains. I have shown that antibiotic resistance is costly, and that these costs can be ameliorated by second-site mutations that readily arise over short time scales. Additionally, different strains of the same bacteria can gain resistance through a diverse set of genetic mutations. On an applied level these results are not positive; combating resistance evolution will be difficult because pathogens can easily compensate fitness costs of resistance, and resistance itself can be gained via a large number of genetic targets.

Investigations into plant-pathogen interactions have provided us with several models underlying the genetic basis of host resistance in plants. In the past decade, tens of resistance genes have been isolated from numerous crop and model plant species and these form a few distinct classes when classified by domain structure, the majority being nucleotide-bindingsite- leucine-rich-repeat (NBS-LRR) genes. The NBS-LRR family consists of two sub-families based on the N-terminal domain: the coiled-coil (CC) NBS-LRRs and the Toll Interleukin Receptor homology domain (TIR) NBS-LRRs. The potential of these genes for future and current agricultural breeding programs has driven a large number of studies exploring the members of these gene families in the genomes of a variety of crop species. In the present study I focused on the NBS-LRR family in the allohexaploid wheat genome and obtained a comprehensive set of Triticeae NBS-LRR homologues using a combination of data-mining approaches. As starting point I detected conserved motifs in the dataset, finding all six previously characterized in the core-NBS domain of other plant NBS-LRRs. Phylogenetic analysis was performed to study relationships between the Triticeae NBS-LRR family and the 25 CC-NBS-LRR (CNL) R genes identified to date. I found the Triticeae CNL family to be highly divergent, containing ancient clade lineages, as seen in all angiosperm 120 taxa previously studied, and found a number of “ancient” dicotyl R genes grouped with Triticeae clades. The evolution of recent NBS-LRR gene duplications in the Triticeae was studied at the hand of two modes of duplication - firstly individual gene duplications yielding paralogous loci and secondly gene duplication by allopolyploidy. Current models of NBS-LRR family evolution predict that functional divergence occurs after gene duplication. An alternative is that divergence takes place at allele level, followed by a locus duplication that fixes heterozygosity in a single haplotype by unequal recombination. I investigated this hypothesis by studying the evolution of gene duplicates in two different contexts – paralogous duplications in the diploid barley genome and homeologous duplications in the allohexaploid genome of wheat. Nonsynonymous to synonymous substitution rate ratios were estimated for paralogous gene duplications in three recently diverged NBS-LRR clades. All pairwise comparisons yielded Ka:Ks ratios strongly indicative of purifying selection. Given that R gene mediated resistance is inherited qualitatively rather than quantitatively, I interpret this as evidence that even closely related paralogous copies (90-95% identity) should have independent recognition specificities maintained by purifying selection. Homeologous duplications were studied in allohexaploid wheat (AABBDD) using a section of the go35 NBS-LRR gene (2L) of the B and D diploid donor species of wheat. Numerous synonymous substitutions distinguished the B and D genome copies, with an absence of nonsynonymous substitutions. In contrast, single unique nonsynonymous substitutions were found in four out of five polyploid wheat go35 alleles, indicating that selection pressure was indeed relaxed across the homeolocus. Recent studies on polyploid genomes have shown that duplicated resistance genes are far more likely to be eliminated than highly transcribed genes such as tRNAs and rRNAs. These results are in agreement with the view that functional divergence takes place before duplication for NBS-LRR genes, as the loci duplicated by polyploidy appear not to evolve under purifying selection, as I found for the paralogous loci investigated. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

Escherichia coli produtoras de toxina de Shiga (STEC) são patógenos veiculados por alimentos capazes de causar desde diarréia branda até severa e sanguinolenta e evoluir para complicações graves como colite hemorrágica, síndrome hemolítica urêmica e púrpura trombótica trombocitopênica. Esses microrganismos têm sido associados a numerosos surtos e vários casos esporádicos de infecções em todo o mundo devido ao consumo de alimentos contaminados. O sorogrupo O157 desse grupo de microrganismos é considerado o principal devido ao seu envolvimento em surtos de doença por STEC, entretanto, muitos casos vêm ocorrendo em todo o mundo devido a cepas patogênicas de STEC não-O157, como O26, O103, O111 e O145. Os objetivos do presente trabalho foram avaliar a ocorrência de STEC em três pontos da linha de abate de bovinos destinados à exportação e em cortes refrigerados de aves e de bovinos comercializados na região da Grande São Paulo; pesquisar a presença dos fatores de virulência dos isolados através dos genes stx1, stx2, eaeA e ehxA; identificar isolados de E. coli O157:H7 pela pesquisa dos genes uidA, rfbO157 e flicH7; verificar a citotoxicidade em células Vero; pesquisar a atividade enterohemolítica dos isolados; avaliar o perfil de suscetibilidade a antimicrobianos; identificar os sorotipos e avaliar a diversidade genética dos isolados de STEC obtidos. Na linha de abate, 201 animais foram amostrados, obtendo-se 603 amostras que compreenderam 201 amostras provenientes do couro, 201 de carcaça e 201 de meia carcaça. No varejo, foram analisadas 100 amostras de cortes de carne bovina e 100 de cortes de frango. A metodologia utilizada para detecção de E. coli sorogrupo O157 foi a preconizada pela ISO 16654, enquanto para os sorogrupos O26, O103, O111 e O145 foi empregada a metodologia descrita pelo \"Surveillance Group for Diseases and Infections of Animals\" (NRM 006). Os isolados obtidos foram confirmados como STEC pela técnica de PCR. Dos 201 animais amostrados, dois (1,0%) foram positivos para STEC, obtendo-se sete isolados (três do animal número 399 e quatro do animal 401) do couro. Não houve o isolamento do microrganismo nas amostras de carcaça e meia carcaça. Os sete isolados apresentaram o perfil stx2, uidA, eaeA, ehxA, rfbO157 e fliCH7 podendo, assim, ser considerados E. coli enterohemorrágica (EHEC) pertencentes ao sorotipo O157:H7. Na avaliação da atividade enterohemolítica, nenhum dos isolados expressou essa proteína e, com relação ao teste de suscetibilidade antimicrobiana, três (42,8%) isolados apresentaram resistência ao ácido nalidíxico e um (14,3%) ao cloranfenicol. O PFGE revelou que as sete cepas de STEC isoladas apresentaram dois perfis genéticos distintos, com similaridade entre eles de 75,3%. STEC não foi detectada nas amostras de carne bovina e de aves comercializadas no varejo. Estes resultados sugerem que, apesar de presente no couro dos animais, o emprego de medidas sanitárias eficientes ao longo da cadeia de produção da carne bovina até sua comercialização na forma de corte, contribui para que o isolamento de STEC nas etapas posteriores seja raro. / Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that can cause since mild or severe and bloody diarrhea to serious complications, such as hemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. These microorganisms have been associated with numerous outbreaks and several sporadic cases worldwide due to consumption of contaminated food, especially meat. E. coli O157 is considered the main serogroup involved in disease outbreaks of STEC, however, many cases have been occurred worldwide due to non-O157, such as O26, O103, O111 and O145 strains. The aims of the present study were to determine the occurrence of STEC at three points of cattle slaughter for exporting and in refrigerated beef and poultry cuts commercialized in the Metropolitan area of Sao Paulo, Brazil; identify the genes that code for the virulence factors stx1, stx2, eaeA and ehxA; detect E. coli O157:H7 strains using uidA, rfbO157 and flicH7 genes; verify the citotoxicity in Vero cells and the enterohemolytic activity; evaluate the antimicrobial susceptibility profile; identify the STEC serotypes and evaluate the genetic diversity of STEC isolates. A total of 603 samples were collected from 201 animals at slaughter. The samples were taken from hide (201), carcass (201) and half-carcass (201) and were always collected from the breast region. At retail, 100 refrigerated beef cuts and 100 chicken cuts were analyzed. The detection of E. coli O157 samples were conducted according to the ISO methodology (16654) and for detection of O26, O103, O111 e O145 serogroups the Surveillance Group for Diseases and Infections of Animals methodology (NRM 006) was used. The isolates were confirmed as STEC by PCR technique. Among 201 animals sampled, two (1,0%) were positive for STEC, obtaining seven isolates from hide (three from animal number 399 and four from animal number 401). The microrganism was not detect in carcass and half carcass samples. The seven isolates carried stx2, uidA, eaeA, ehxA, rfbO157 and fliCH7 genes, so, they can be considered as enterohaemorrhagic E. coli (EHEC) O157:H7 serotype. None of the isolates produced the enterohemolytic activity and three (42,8%) isolates showed resistence to nalidixic acid and one (14,3%) to chloramphenicol. PFGE revealed that the seven STEC strains showed two distinct genetic profiles, with 75.3% of similarity between them. STEC was not detected from beef and poultry cuts commercialized at retail. These results suggest that, although present in animals hides, the STEC isolation at later stages of food chain was rare, probably due to effective sanitary measures to control contamination and transmission of this pathogen along the beef production chain until commercialization.

Cette étude a pour objet la mise en évidence de traces d'ADN bactérien pathogène dans des échantillons animaux et humains anciens, et ainsi améliorer les connaissances sur l'évolution des maladies au cours du temps. En parallèle, nous avons étudié les phénomènes de dégradation de l'ADN dans le sol sur des cadavres de souris enterrées après avoir été contaminées par des bactéries non pathogènes. Cette étude des processus taphonomiques s'est étalée sur trois ans et a permis de montrer une disparition rapide des bactéries simulantes, remplacé par l'ADN des bactéries du sol, qui colonisent rapidement la dépouille et dégradent tant l'ADN endogène (murin) qu'exogène (bactérien). Cette disparition rapide explique la grande difficulté à mettre en évidence des pathogènes dans des échantillons anciens, à de rares exceptions près. Notre étude n'a pas permis de détecter d'agents pathogènes particuliers dans les échantillons que nous avons étudié, mais nous avons mis en évidence l'intérêt d'analyser certains types de restes pour accéder à une information génétique préservée. Le tartre dentaire indique est un bon indicateur de la flore buccale de l'hôte et les kystes calcifiés assurent une bonne préservation de l'ADN endogène, moins soumis à contamination et digestion par les bactéries de l'environnement. Les kystes présentent en règle générale une teneur en ADN endogène supérieure à tous les autres tissus étudiés. / The aim of this study was the identification of pathogenic bacterial DNA traces in ancient animal and human samples, and thus improve knowledge of past diseases that affect humankind over time. In parallel, we studied the DNA degradation phenomena in the soil on the buried corpses of mice after being contaminated by non-pathogenic bacteria. This study of taphonomic processes was spread over three years and has shown a rapid disappearance of simulant bacteria, replaced with the DNA of soil bacteria that colonize the body quickly after burial and degrade both the endogenous DNA (murine) that exogenous (bacteria). This quick degradation can explain the high difficulty to detect and identify bacterial pathogens in old samples, with very few exceptions. Despite the fact in our study we were not able to detect specific pathogens in the samples we have studied, we have shown the interest to analyze certain types of remnants to access preserved and informative genetic data. Dental calculus is a good indicator of the oral flora of the host and calcified cysts ensure good preservation of the endogenous DNA, less subject to contamination and digestion by bacteria from the environment. Cysts generally have an endogenous DNA content higher than all other tissues examined.

Infectious diseases from novel viruses have become a major public health concern. Rapid identification of virus–host interactions can reveal mechanistic insights into infectious diseases and shed light on potential treatments. Current computational prediction methods for novel viruses are based mainly on protein sequences. However, it is not clear to what extent other important features, such as the symptoms caused by the viruses, could contribute to a predictor. Disease phenotypes (i.e., signs and symptoms) are readily accessible from clinical diagnosis and we hypothesize that they may act as a potential proxy and an additional source of information for the underlying molecular interactions between the pathogens and hosts. We developed DeepViral, a deep learning based method that predicts protein– protein interactions (PPI) between humans and viruses. Motivated by the potential utility of infectious disease phenotypes, we first embedded human proteins and viruses in a shared space using their associated phenotypes and functions, supported by formalized background knowledge from biomedical ontologies. By jointly learning from protein sequences and phenotype features, DeepViral significantly improves over existing sequence-based methods for intra- and inter-species PPI prediction. Lastly, we propose a novel experimental setup to realistically evaluate prediction methods for novel viruses.

Abiotic stresses such as salt stress are the major limiting factors for agricultural productivity, and cause global food insecurity. It is well known that plant associated beneficial microorganisms can stimulate plant growth and enhance resistance to abiotic stresses. In this context, bacterial endophytes are a group of bacteria that colonize the host plant and play a fundamental role in plant growth enhancement under stress condition. Recently, our group reported that the beneficial bacteria Enterobacter sp.SA187 induces plant growth in Arabidopsis under salt stress conditions by manipulation of the plant ethylene signaling pathway. We therefore compared inoculation of plants by SA187 with virulent and non-virulent strains Pst DC3000. Although both strains inhibit plant growth at ambient conditions, Pst DC3000 hrcC-, but not Pst DC3000, induced salt stress tolerance, suggesting that Pst DC3000 hrcC- also contains plant growth promoting activity under stress conditions. Our results indicate that Pst DC3000 hrcC- shares features with beneficial bacteria by inducing salt tolerance through reduction of the shoot and root Na+/K+ ratio. To further elucidate the underlying mechanisms of this interaction with Arabidopsis, RNAseq, hormone and biochemical analyses were performed. Genetic studies also show that Pst DC3000 hrcC- induced salt stress tolerance involving several phytohormone pathways, including auxin, ethylene and salicylic acid. Transcriptome and genetic analyses indicate that glucosinolates play an important role in this beneficial interaction. We found that indolic and alkyl glucosinolates act as negative factors on Pst DC3000 hrcC-, alkyl glucosinolates are positive and indolic glucosinolates negative regulators in SA187 interaction with Arabidopsis. These results reveal that besides a repertoire of effectors, Pst DC3000 hrcC- also produces factors that can be beneficial for plant growth under certain stress conditions, as observed with Enterobacter sp. SA187.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21829号 / 農博第2342号 / 新制||農||1068(附属図書館) / 学位論文||H31||N5201(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 田中 千尋, 教授 本田 与一, 准教授 刑部 正博 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM