Bacteriophages cohabit with their bacterial hosts and shape microbial communities. To initiate infection, phages use bacterial components as receptors to recognize and attach to hosts. Flagellotropic phages utilize bacterial flagella as receptors. Studies focused on uncovering mechanistic details of flagellotropic phage infection are lacking. As the number of phage-based applications grows, it is important to understand these details to predict the potential outcomes of phage therapy. To this end, we studied two flagellotropic phages: Agrobacterium phage 7-7-1 and bacteriophage χ. Phage 7-7-1 infects Agrobacterium spp., while bacteriophage χ infects Salmonella and Escherichia coli. Chapter 1 consists of a literature review. Chapter 2 addresses factors underlying phage-bacteria coexistence. We document the emergence of a sector-shaped lysis pattern following co-inoculation of phage χ and one of its Salmonella hosts on swim plates. We propose that this pattern serves as a reporter for balanced phage-bacteria coexistence. Using a combined experimental and mathematical modelling approach, we discovered that variations to intrinsic factors (i.e., bacterial motility, phage adsorption) skews the pattern towards either bacterial or phage predominance. Thus, this computational model may be used to predict phage therapy application outcomes. Chapter 3 details the identification of cell surface receptors essential for phage 7-7-1 infection using a transposon mutagenesis approach. We identified three Agrobacterium sp. H13-3 genes involved in phage 7-7-1 infection. Using mass spectrometry and other analyses, we determined that the LPS profiles of strains lacking these genes varied compared to the wild type. Thus, LPS is a secondary cell surface receptor for phage 7-7-1. Chapter 4 focuses on the discovery of phage encoded receptor binding proteins (RBPs) in Agrobacterium phage 7-7-1. Using an RBP screen, we discovered three candidate RBPs. We learned that our top candidate, Gp4, inhibits the growth of Agrobacterium sp. H13-3 cells in a motility and glycan dependent manner. Because of its bacteriostatic activities, this protein is a promising candidate for therapeutic use. Overall, the described works contribute to a deepened understanding of flagellotropic phage infection and the factors influencing their coexistence with motile bacteria. These works will contribute towards the development of phage therapies using whole phage or their components. / Doctor of Philosophy / Bacteriophages, or phages for short, are the natural killers of bacteria. Like antibiotics, they can also be used as medicines to treat bacterial infections. Their attack on bacteria begins by recognizing specific parts of the bacterial cell and attaching to them. These parts are called receptors. To use phages as medicines it is important to understand how they recognize and kill bacteria. This information is helpful when deciding which phage should be given to treat a bacterial infection and to predict the outcomes of these treatments. In this work, we focused on two phages to answer different questions. Both phages use long helical thread-like structures, called flagella, as receptors. Flagella help the bacteria to move through the environment and reach new areas with more nutrients. One of these flagella-dependent phages, called phage 7-7-1, infects plant pathogens that cause tumor-like growth in plants. We found that this phage uses two very different host cell components during infection and identified one of the phage proteins that interacts with these receptors. This protein prevents the growth of the plant pathogen, which makes it a promising candidate for therapeutic use. We also investigated how another bacterial virus, bacteriophage χ, is spread throughout the environment and co-exists with its motile bacterial host. We built a computational model that can predict how altering different variables affects phage-bacteria coexistence. With additional research, this model will be a useful tool for predicting the outcomes following phage treatment.

No description available.

As urease de plantas catalisam a hidrólise da ureia e apresentam efeitos tóxicos a fungos patogênicos e insetos fitófagos. Em soja [Glycine max L. Merrill] foram descritas duas ureases estruturais: a embrião-específica, codificada pelo gene Eu1, e a ubíqua, codificada pelo gene Eu4. Sabe-se que a urease embrião-específica purificada apresenta efeito inibitório sobre o crescimento in vitro de fungos filamentosos e desenvolvimento de insetos. A urease ubíqua é responsável pela reciclagem de toda a ureia proveniente do metabolismo, mas não há informações sobre seu envolvimento no sistema de defesa das plantas. A transformação genética é uma ferramenta importante em estudos de genômica funcional e, portanto, a disponibilidade de sistemas eficientes é um pré-requisito essencial. O objetivo deste trabalho foi a obtenção de plantas estavelmente transformadas a partir de embriões somáticos de soja submetidos ao sistema integrado bombardeamento/Agrobacterium, bem como a identificação e caracterização funcional dos genes que codificam as ureases estruturais de soja, especialmente a urease ubíqua em relação aos processos de resposta a fungos patogênicos. Inicialmente, testamos a eficiência de transformação de embriões somáticos secundários por um método que combina o bombardeamento de partículas livres de DNA com o sistema Agrobacterium. Plantas transgênicas férteis foram regeneradas de vários experimentos independentes de transformação utilizando diferentes plasmídios. Posteriormente, foi realizada a caracterização dos genes que codificam ureases presentes no genoma da soja. O gene Eu4 apresentou um padrão de expressão diferencial para genótipos suscetível e resistente ao longo do período de infecção por Phakopsora pachyrhizi, o agente etiológico da ferrugem asiática. Plantas transgênicas foram geradas visando a superexpressão de Eu4. Contudo, apenas uma planta apresentou níveis aumentados de expressão desse gene, enquanto que as demais plantas apresentaram o fenômeno de co-supressão dos genes endógeno e transgene. Avaliou-se o crescimento vegetativo dos fungos Rhizoctonia solani, Phomopsis sp., Fusarium solani, Colletotrichum gossypii e Penicillium herguei em meio de cultura contendo extrato protéico bruto de plantas transgênicas expressando maiores e menores níveis de urease e de plantas não-transgênicas. O crescimento dos fungos foi inversamente proporcional a quantidade da urease presente no extrato protéico das plantas. Quando infectadas por uredósporos de P. pachyrhizi, folhas destacadas das plantas co-suprimidas desenvolveram um número significativamente maior de lesões, pústulas e pústulas abetas do que folhas com níveis normais da enzima. Em conjunto estes resultados indicam um 15 importante envolvimento da urease ubíqua da soja na resposta à infecção da planta por fungos patogênicos. Além disso, um terceiro gene que codifica urease foi encontrado no banco de dados com a sequência completa do genoma da soja. O gene foi denominado Eu5 e seu produto SBU-III. A análise filogenética mostra que SBU-III está fortemente relacionada à isoforma embrião-específica. Apesar da grande similaridade na seqüência primária da proteína, SBU-III apresenta uma mutação em um aminoácido altamente conservado entre as ureases, sugerindo ausência da atividade ureolítica. O padrão de expressão do gene Eu5 em diferentes órgãos e estágios de desenvolvimento foi determinado por RT-qPCR. Transcritos foram detectados em sementes um dia após a quebra de dormência, em raízes de plantas jovens e em embriões em desenvolvimento. As evidências sugerem que SBU-III não está envolvida na disponibilização de nitrogênio para as plantas, mas esta pode ter função de defesa. / Plants ureases catalyze urea hydrolysis and display toxic effects against pathogenic fungi and phytophagous insects. For soybean [Glycine max L. Merrill] two structural ureases have been described: the embryo-specific, encoded by Eu1 gene, and the ubiquitous, encoded by Eu4 gene. The toxic property of purified embryo-specific urease against filamentous fungi and insects was demonstrated in vitro. The ubiquitous urease is responsible for recycling all metabolically-derived urea, but there were no information about its putative defense role. Plant genetic transformation offers significant advancement in functional genomics. Therefore an efficient transformation system is required. This study aims to obtain stable transformed plants derived from somatic embryos submitted to the integrated bombardment/ Agrobacterium system, as well as identify and functionally characterize the soybean structural urease-encoding genes, specially the ubiquitous urease gene response to fungi. First, the transformation of soybean proliferating somatic embryos by a procedure that combines DNA-free particle bombardment and Agrobacterium was evaluated. Transgenic fertile plants were recovered from many transformation experiments using different plasmids. After, a study of ureases enconding genes present in the soybean genome was carried out. In the present work, Eu4 gene showed a differential expression pattern in susceptible and resistant genotypes over the course of Phakopsora pachyrhizi infection, the Asian rust causal agent. Transgenic plants aiming Eu4 overexpression were obtained. However, a single transgenic plant exhibited Eu4 overexpression, whereas the other ones showed co-suppression of endogenous and transgenes urease genes. The growth of Rhizoctonia solani, Phomopsis sp., Fusarium solani, Colletotrichum gossypii and Penicillium herguei in media containing crude protein extract from either transgenic or non-transgenic leaves was evaluated. Fugal growth was inversely proportional to ubiquitous urease amount in plant crude extracts. When infected by P. pachyrhizi uredospores, detached leaves of co-suppressed plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves containing normal levels of the enzyme. These results suggested an important role of soybean ubiquitous urease in plant response against fungal infection. Furthermore, by searching the completed soybean genome sequence, a third urease-encoding locus was identified. The gene was designated Eu5 and its product, SBU-III. Phylogenetic analysis shows that SBU-III is closely related to the embryo-specific isoform. Although a high similarity in amino acid sequence was observed, a mutation in a highly conserved residue suggests absence of ureolytic activity. Expression profile of Eu5 gene in different organs and developmental stages was determined by RT-qPCR. Transcripts were detected in seeds one day after dormancy break, roots of young plants and embryos of developing seeds. Evidences suggest that SBU-III may not be involved in nitrogen availability to plants, but a defense role was proposed.

Virulent Agrobacterium tumefaciens strains transfer and integrate a DNA region of the tumor-inducing (Ti) plasmid, the T-DNA, into the plant genome and thereby cause crown gall disease. The most essential genes required for crown gall development are the T-DNA-encoded oncogenes, IaaH (indole-3-acetamide hydrolase), IaaM (tryptophan monooxygenase) for auxin, and Ipt (isopentenyl transferase) for cytokinin biosynthesis. When these oncogenes are expressed in the host cell, the levels of auxin and cytokinin increase and cause cell proliferation. The aim of this study was to unravel the molecular mechanisms, which regulate expression of the agrobacterial oncogenes in plant cells. Transcripts of the three oncogenes were expressed in Arabidopsis thaliana crown galls induced by A. tumefaciens strain C58 and the intergenic regions (IGRs) between their coding sequences (CDS) were proven to have promoter activity in plant cells. These promoters possess eukaryotic sequence structures and contain cis-regulatory elements for the binding of plant transcription factors. The high-throughput protoplast transactivation (PTA) system was used and identified the Arabidopsis thaliana transcription factors WRKY18, WRKY40, WRKY60 and ARF5 to activate the Ipt oncogene promoter. No transcription factor promoted the activity of the IaaH and IaaM promoters, despite the fact that the sequences contained binding elements for type B ARR transcription factors. Likewise, the treatment of Arabidopsis mesophyll protoplasts with cytokinin (trans-zeatin) and auxin (1-NAA) exerted no positive effect on IaaH and IaaM promoter activity. In contrast, the Ipt promoter strongly responded to a treatment with auxin and only modestly to cytokinin. The three Arabidopsis WRKYs play a role in crown gall development as the wrky mutants developed smaller crown galls than wild-type plants. The WRKY40 and WRKY60 genes responded very quickly to pathogen infection, two and four hours post infection, respectively. Transcription of the WRKY18 gene was induced upon buffer infiltration, which implicates a response to wounding. The three WRKY proteins interacted with ARF5 and with each other in the plant nucleus, but only WRKY40 together with ARF5 increased activation of the Ipt promoter. Moreover, ARF5 activated the Ipt promoter in an auxin-dependent manner. The severe developmental phenotype of the arf5 mutant prevented studies on crown gall development, nevertheless, the reduced crown gall growth on the transport inhibitor response 1 (TIR1) tir1 mutant, lacking the auxin sensor, suggested that auxin signaling is required for optimal crown gall development. In conclusion, A. tumefaciens recruits the pathogen defense related WRKY40 pathway to activate Ipt expression in T-DNA-transformed plant cells. IaaH and IaaM gene expression seems not to be controlled by transcriptional activators, but the increasing auxin levels are signaled via ARF5. The auxin-depended activation of ARF5 boosts expression of the Ipt gene in combination with WRKY40 to increase cytokinin levels and induce crown gall development. / Virulente Bakterien des Stamms Agrobakterium tumefaciens, transferieren und integrieren einen Teil ihrer DNA, die T-DNA aus dem Tumor induzierenden Plasmid (Ti), in das Pflanzengenom. Dadurch wird die Tumorbildung induziert und die Krankheit bricht aus. Die wichtigsten Gene, die für die Entwicklung eines Tumors benötigt werden, sind auf der T-DNA lokalisierte Onkogene: IaaH (indole-3-aceetamide hydrolase), IaaM (tryptophan monooxygenase) für die Auxin Biosynthese und Ipt (isopentenyl transferase) für die Cytokinin Biosynthese. Werden diese Onkogene in der Wirtszelle exprimiert, steigt der Gehalt an Auxin und Cytokinin und fördert die Zellteilung. Das Ziel dieser Arbeit war es die molekularen Mechanismen, die die Expression der agrobakteriellen Onkogene in Pflanzenzellen regulieren, aufzuklären. Transkripte der drei Onkogene wurden in Tumoren an Arabidopsis thaliana exprimiert. Die Tumore wurden durch den A. tumefaciens Stamm C58 induziert. Dabei konnte gezeigt werden, dass die Sequenzabschnitte zwischen den Onkogenen (IGRs: intergenic regions) eine Promoteraktivität in der Pflanzenzelle besitzen. Diese Promoter haben eukaryotische Sequenzstrukturen und enthalten cis-Elemente, an die pflanzliche Transkriptionsfaktoren binden. Mit Hilfe der PTA (high-throughput protoplast transactivation) Methode wurden die pflanzlichen Transkriptionsfaktoren WRKY18, WRKY40, WRKY60 und ARF5 von Arabidopsis thaliana identifiziert, welche den Promoter des Ipt Onkogens aktivieren. Für IaaH und IaaM konnte kein Transkriptionsfaktor, der die Promotersequenzen aktiviert, identifiziert werden, obwohl die Promotersequenzen Bindedomänen für den Typ B ARR Transkriptionsfaktor enthalten. Ebenso zeigte die Behandlung von Arabidopsis Protoplasten aus dem Mesophyll mit Cytokinin (trans-zeatin) und Auxin (1-NAA) keinen positiven Effekt auf die Aktivität des IaaH und des IaaM Promoters, wohingegen der Ipt Promoter stark auf eine Behandlung mit Auxin und leicht auf eine Behandlung mit Cytokinin reagierte. Die drei WRKYs aus Arabidopsis spielen eine Rolle in der Tumorentwicklung, da die wrky Mutante kleinere Tumore zeigt, als die Wild Typ Pflanzen. Die Gene WRKY40 und WRKY60 reagieren sehr schnell, innerhalb von zwei, beziehungsweise vier Stunden, auf eine Pathogen Infektion. Die Transkription des WRKY18 Gens wurde durch die Infiltration von Puffer in Blätter induziert, dies lässt auf eine Reaktion im Zusammenhang mit Wunderzeugung schließen. Die drei WRKY Proteine interagieren mit einander und mit ARF5 im Zellekern der Pflanzenzelle, aber nur WRKY40 und ARF5 können gemeinsam den Ipt Promoter aktivieren. Zusätzlich kann ARF5 den Ipt Promoter, in Abhängigkeit von Auxin, aktivieren. Wegen starker Entwicklungsstörungen der arf5 Mutante, konnte das Tumorwachstum an dieser Mutante nicht untersucht werden. Das reduzierte Tumorwachstum an der tri1 (transport inhibitor response, TIR) Mutante, der ein Auxinsensor fehlt, deutet auf die Notwendigkeit des Auxinsignalwegs für optimales Tumorwachstum hin. Zusammengefasst benutzt A. tumefaciens den WRKY40 Signalweg, der mit der Pathogen Abwehr verbunden ist, um die Ipt Expression in der mit T-DNA transformierten Pflanzenzelle zu aktivieren. Die Genexpression von IaaH und IaaM schein nicht von Transkriptionsfaktoren abhängig zu sein, aber erhöhte Auxin Werte werden von ARF5 erkannt. Die Auxin abhängige Aktivierung von ARF5 verstärkt die Expression des Ipt Gens gemeinsam mit WRKY40 um die Cytokin Werte in der Pflanzenzelle zu erhöhen und somit die Tumorentwicklung einzuleiten.

Copy of author's previously published work inserted. Bibliography: leaves 114-136. The study concentrates on: investigation of the biological properties and role of agrocin 434 and other factors in the bicontrol process of crown gall disease; isolation of the complete range of K84 derivatives carrying different combinations of plasmids pAgK84, pAgK434 and pAtK84b in the same host background; development of a rapid plant bioassay to assess the efficacy of biocontrol strains by leaf disc tumorigenesis assay; and characterization of agrocin 434 synthesis and immunity genes.

A method for Agrobacterium-mediated transformation of hybrid poplar (Populus alba x P. grandidentata) suspension cultures and regeneration of transformed plants is described. The best protocol was one in which suspension cultures were inoculated with Agrobacterium tumefaciens to a density of 10⁷ cfu's/ml, cocultivated for 48 hours, plated to cellulose acetate filters at a density of 14 colonies/mm², and cultured on medium containing 1 mg/1 2,4-D. Although cefotaxime inhibited callus growth, it was used in the plating medium to suppress proliferation of Agrobacterium. Selection appeared to be more reliable using hygromycin as compared to kanamycin or geneticin (G418). Transgenic plants were regenerated by culturing the calli on media containing thidiazuron, but no shoots could be regenerated using BA. / Graduation date: 1992

Plant transformation provides a promising methodology of introducing new genes that encode desirable traits to a wide range of crop plants. Success in genetic transformation has been achieved in many of the important crop species, such as soybean, cotton, rice, corn. However, wheat, one of the major crops of the world, has been considered to be difficult to transform via either Agrobacterium or biolistic bombardment (Rakszegi et al., 2001). There have been limited studies on A. tumefaciens-mediated transformation of cereals, including wheat, because of the overall refractory character of host-pathogen interactions between Agrobacterium and the cereal plants (Gould et al., 1991; Hiei et al., 1994; Cheng et al., 1997). While the genetic transformation of rice using Agrobacterium has become routine, only a few successful studies of Agrobacterium- mediated transformation of wheat have been reported, and these involved a model spring wheat, Triticum aestivum cultivar Bobwhite (Cheng et al., 1997). Model genotypes are developed for ease of plant regeneration in tissue culture and both Agrobacterium and biolistic mediated transformation methods require regeneration of plants in tissue culture. More success has been achieved in obtaining fertile transgenic wheat plants by particle bombardment, or biolistics method (Vasil et al., 1992; Weeks et al., 1993; Becker et al., 1994; Zhou et al., 1995; Altpeter et al., 1996). Wheat plants of the model system cultivar Bobwhite were used in most of these studies as well. The primary objective of this study was to use the callus-based transformation procedures mentioned above with a non-model cultivar of hexaploid spring wheat Saratovskaya-29, widely grown in Kazakhstan, to test the genotype dependence of the previously developed transformation protocols with respect to stable transfer of DNA and regeneration of transgenic plants. The spring wheat cultivar Saratovskaya-29 (Albidum-24/ Lutescens-55-11) was chosen for the study as being one of the most widely grown wheat cultivars both in Russia and Kazakhstan. It was bred in early 50??s in the Research Institute of the South-East, Saratov. Because of its drought resistance and good baking quality traits, Saratovskaya-29 reached a peak of nearly 21.2 mln ha in the former USSR in 1996 (Martynov and Dobrotvorskaya, 1996). Economical importance of this cultivar makes it an appropriate candidate for further improvement of economically significant traits. Another objective of the study described was to compare the transformation efficiencies and inheritance in the transgenic plants produced.

Thèse doctorat : Biologie Végétale et Forestière : Nancy 1 : 2006. / Titre provenant de l'écran-titre.

An understanding of plant pathogen-host interactions is essential to design efficient strategies to control disease in crops. Magnaporthe oryzae, an ascomyceteous fungus and causal agent of rice blast disease, is a model organism to study host-microbe interactions. The overall aim of this dissertation project was to identify genes involved in pathogenicity through the construction and characterization of a random insertional mutagenesis library. In order to saturate the genome with DNA inserts, a collection of >54,000 insertion lines of the M. oryzae strain 70-15 was generated via two transformation methods, PEG/CaCl2 (polyethylene glycol)-mediated protoplast transformation and Agrobacterium tumefaciens-mediated transformation. The first part of this dissertation describes the optimization of both transformation approaches, compares their efficiency and provides a description of the high-throughput processing and phenotypic analysis of the insertion lines. An in vitro appressorium assay of 12,000 T-DNA insertion strains allowed the identification of 135 lines that were classified as morphologically or functionally different than wild-type. Rice infection assays demonstrated that 112 of these strains exhibited defects in pathogenicity.The second part of this dissertation project analyzed the T-DNA integration patterns in a subset of pathogenicity mutants. This section aimed to identify the disrupted genes via recovery of M. oryzae sequences adjacent to the sites of T-DNA insertion. Genomic mapping of 61 T-DNA insertions in pathogenicity mutants via rescuing M. oryzae chromosomal T-DNA flanking sequences using inverse PCR resulted in the identification of 22 conserved hypothetical genes with predicted function, 11 predicted open reading frames without a GenBank significant match, two unannotated regions of the genome assembly and seven intergenic regions. The final part of this dissertation describes the characterization of a M. oryzae pathogenicity mutant that contains a T-DNA insertion in the upstream region of two divergently transcribed genes that encode the vacuolar type-ATPase subunit c`` and the general transcription factor TFIIA subunit γ. Genetic complementation demonstrated the insertion of the T-DNA in the promoter region of the general transcription factor TFIIA subunit γ is responsible for observed defects in conidiation, appressorium morphogenesis, and appressorium function. This is the first report relating the function of TFIIA subunit γ to pathogenicity.

Regeneration and transformation protocols for 'Comet' red raspberry were optimized with the purpose of making the Agrobacterium-mediated gene transfer system efficient for this crop. Adventitious shoot regeneration from leaf discs was improved using explants 10 mm in diameter and transferring to fresh medium at the fourth week of incubation. Additions of liquid medium to solid medium during incubation decreased regeneration and attempts to release the suppressive influence of larger shoots on initials (apical dominance) did not succeed. The presence of claforan did not affect shoot regeneration, but inoculations with Agrobacterium and the presence of kanamycin decreased regeneration moderately or considerably, respectively. The threshold for kanamycin concentration for screening for kanamycin resistant transformed raspberry tissue was 30 to 40 mg l$ sp{-1}.$ The best co-incubation interval between wild-type Agrobacterium and 'Comet' leaf discs ranged from 2 days for highly virulent strains to 3 or more days for moderate to low virulent strains. Among several wild-type strains, C58 was chosen as the most appropriate partially because a disarmed form was commercially available for use as a non-oncogenic vector for transformation of red raspberry. / The binary plasmid pBI121 containing the marker genes NPTII and GUS encoding kanamycin resistance and $ beta$-glucuronidase activity, respectively, was successfully introduced into the Agrobacterium strain LBA4404, which is a disarmed C58 derivative. Transformation of 'Comet' red raspberry was apparently achieved by inoculating leaf disc explants with LBA4404 containing pBI121. The probable integration and expression of the foreign genes into the plant cells were confirmed by screening for kanamycin resistance, GUS assays and Southern blot analyses. This transformation system appears to be effective and may be useful in further studies on red raspberry for both introduction of genes for desirable agronomic traits and basic studies of gene expression.