Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDSrelated Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers. Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxelaccumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation. A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa- 4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium-mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines.

No description available.

Impatiens walleriana, commonly grown as a herbaceous annual, is susceptible to Impatiens Necrotic Spot Virus (INSV). A lack of resistant cultivars leaves growers with the sole option of destroying infected plants before INSV spreads throughout their entire crop. Therefore, the introduction of INSV resistant cultivars would have the potential to save Impatiens growers a substantial amount of money. Virus resistance has been successfully conveyed in several crops by insertion of pathogen DNA into the host plant. One method of generating transgenic plants involves the use of Agrobacterium-mediated gene transfer. A commonly used technique involves transformation of explant tissue and subsequent regeneration in vitro under aseptic conditions. However, prior to our research there was no regeneration protocol suitable for Agrobacterium-mediated transformation of Impatiens walleriana available. Herein we report the development of a new method for regeneration of Impatiens walleriana using cotyledonary node culture. Using this technique, four regeneration media amended with 1, 3, 5, or 7µM of thidiazuron were evaluated for their ability to induce de novo shoot production in cotyledonary node explants, and evaluated for number of shoots produced per explant. Results showed a significantly greater frequency of regeneration and number of shoots per explant using media amended with 1µM of thidiazuron. This technique has shown to be repeatable and is not susceptible to ploidy instability. Unfortunately, damage to the cotyledonary node explants during Agrobacterium inoculation and transfection prevented regeneration of transformed shoots in several attempts. However, transient GFP expression after transfection of shoot pads derived from cotyledonary nodes with Agrobacterium strain LBA 4404 containing plasmid pHB2829 with nptII and S-GFP was obtained, indicating the possibility for this regeneration protocol to derive stably transformed Impatiens with INSV resistance. / Master of Science

The Rosaceae contains many important commercially grown fruit crops. No comprehensive genomics platform is currently under development for fruit crops, giving functional genomics studies with wild strawberry (Fragaria vesca L.) the potential of identifying genes important in fruit crops. Fragaria vesca has a small genome size compared to the cultivated strawberry, Fragaria à ananassa Duch. (164 vs. 600 Mbp per 1C nucleus). This feature, in addition to a short life cycle (12-16 weeks) and small plant size make F. vesca a good candidate for a model plant for genetic and molecular studies. The specific objective of this work was to develop an efficient high-throughput Agrobacterium-mediated transformation protocol to generate an insertional mutant population to support the justification of F. vesca as a model organism for rosaceous crops. The transformation techniques described by Alsheikh et al. (2002) and Oosumi et al. (2005) were modified and applied to a range of germplasm obtained from the USDA National Germplasm Repository. We found that the modifications made to the Alsheikh protocol were unsuccessful when applied to our germplasm. With the Oosumi et al. (2005) protocol, transformation efficiencies ranging from 11 to 100% were obtained for two accessions when explants were exposed to varying durations on TDZ containing medium during shoot regeneration. The transformation efficiency was given as the mean number of GFP+ plants obtained per primary explant cultured. Multiplex PCR, for amplification of the hptII and GFP genes, was performed on a random sample of GFP+ plants to verify insertion of the T-DNA. The statistical power of our experiment was insufficient to detect treatment effect but based on our findings the transformation efficiencies were high enough to justify PI 551572 for use in the high throughput transformations that are required to generate a population of insertional mutants large enough for gene discovery in F. vesca. / Master of Science

Tomato (Solanum lycopersicum) is a member of the Solanaceae family. In this research project tomato, more specifically the M82 cultivar was chosen as a model plant for Agrobacterium-mediated gene transfer by cotyledon inoculation. Our objective was to transform tomato with a T-DNA construct bearing a transposon from maize that can be used for mutagenesis when it transposes or moves around the genome of the tomato. The vector used is a two-component in-cis Ac-Ds system which needs a single transformation event. It was proved that it worked in Arabidopsis and rice according to Trijatmiko (2005). The construct consists of the BAR gene conferring resistance to herbicide Basta, hygromycin (HYG) gene conferring resistance to the antibiotic hygromycin and the green fluorescent protein (GFP) gene, which are driven by specific plant promoters. The selectable marker genes such as HYG and BAR were used to select the rare transformation events by making the transformed tomato tissue resistant to the toxic chemicals (antibiotic and herbicide) compared to the untransformed tissue in which growth was inhibited. The results described consist of developing a transformation protocol which enabled the production of transgenic tomato lines by the help of the antibiotic augmetin (amoxicillin/clavulanic acid). The transgenic lines were tested through polymerase chain reaction (PCR) and herbicide bioassays. / Master of Science

Brucella abortus and Agrobacterium tumefaciens are pathogenic bacteria that infect animals and plants, respectively. These bacteria are genetically similar and are found within the same Class, Alphaproteobacteria, and Order, Rhizobiales, of the domain Eubacteria; however, they survive and replicate in vastly different environmental niches. In Order to adapt to different environments, bacteria utilize several mechanisms of gene regulation to tightly control gene expression. Two of these mechanisms include transcriptional regulators and small regulatory RNAs (sRNAs), which can activate and repress gene expression through various interactions with DNA, mRNA, and proteins. A well-conserved transcriptional regulator among the Rhizobiales is VtlR, a virulence-associated transcriptional LysR regulator. The objectives of this dissertation were three fold: 1) characterize the known regulon of VtlR in B. abortus with regards to gene regulatory function and virulence, 2) determine the regulon of VtlR in A. tumefaciens and define the mechanism by which this regulation occurs, and 3) define the role of an ABC-type transport system indirectly regulated by VtlR in B. abortus that putatively imports the non-proteinogenic amino acid gamma-aminobutyric acid (GABA). VtlR was characterized in B. abortus as a virulence-associated transcriptional regulator that directly activates four genes: the sRNA AbcR2, and the three small hypothetical proteins BAB1_0914, BAB2_0512, and BAB2_0574; and deletion of vtlR led to a significant defect in the ability of B. abortus to cause infection in vitro and in vivo. Since dysregulation of abcR2 alone could not account for the defect in virulence, it was hypothesized that one or all three hypothetical proteins could be responsible for a virulence phenotype observed in ΔvtlR. This turned out to not be the case, as a deletion of the entire VtlR regulon displayed no difference in virulence compared to the parental strain. Further characterization of the small hypothetical proteins is outlined in Chapter 2 and the data revealed bona fide translation of each small protein, and the deletion strain of the VtlR regulon displayed a growth defect when grown in the presence of the sugar fucose. This phenotype was subsequently observed in ΔvtlR as well. This led to the identification of a putative fucose transport and metabolism locus in B. abortus that has yet to be studied. In A. tumefaciens, VtlR is necessary for proper attachment to plant cells and biofilm formation and regulates over 200 genes, significantly more than the four genes VtlR regulates in B. abortus. The mechanism by which this occurs was unknown, and the relationship between VtlR and AbcR1 or AbcR2 was uncharacterized. The data in Chapter 3 outline the VtlR network by showing that VtlR regulation of myriad genes in A. tumefaciens is primarily indirect via the direct regulation of a few sRNAs. This direct interaction was shown experimentally and a VtlR binding box was identified in the A. tumefaciens genome. This project outlines the divergence of a regulatory element between phylogenetically related organisms that occupy different environmental niches. The AbcR sRNAs are conserved throughout the Rhizobiales and regulate numerous ABC-type transport systems within these bacteria. In A. tumefaciens, one of these transport systems specifically transports the amino acds proline and GABA. B. abortus contains homologs of this system, which led to the hypothesis that the brucellae may also transport GABA but for a yet unknown purpose. The data in Chapter 4 revealed that B. abortus also transports GABA in vitro and this transport is under the regulation of AbcR1 and AbcR2. This transport was increased under extreme nutrient limitations and was uninhibited by the presence of other amino acids. Metabolic studies showed GABA is not utilized by B. abortus under aerobic conditions, and transcriptomic data revealed increased expression of several loci in the presence of GABA. Altogether, this study uncovers a putative signaling role for the amino acid GABA that has been understudied in bacterial pathogens that infect animal hosts. Overall, the work presented in this dissertation is focused on further elucidating the biological role of downstream regulatory targets of both VtlR and the sRNAs AbcR1 and AbcR2 in the related organisms Brucella abortus and Agrobacterium tumefaciens. Findings show that while there are similarities between the two systems, there are also many differences that may be attributed to the vastly different lifestyles of each organism. / Doctor of Philosophy / Brucella abortus and Agrobacterium tumefaciens are two highly related bacterial pathogens that infect mammals and plants, respectively. Although genetically related, both organisms survive and replicate in vastly different environmental niches with one living in the soil (i.e., A. tumefaciens) and the other living within immune cells of the infected host (i.e., B. abortus). In Order to quickly adapt to changing environmental conditions, the bacteria must rapidly control gene expression through multiple regulatory mechanisms. The works presented in this dissertation will focus on further characterizing one of these regulatory systems and comparing the homologous systems shared by B. abortus and A. tumefaciens. This includes uncovering a putative sugar transport and metabolism system, as well as discovering the potential for host-pathogen signaling via the well-studied neurotransmitter GABA.

Résumé Les hémoglobines tronquées (Hbtrs) sont retrouvées chez plusieurs organismes et leurs fonctions sont encore inconnues pour la plupart. Les Hbtrs du pathogène de plante Agrobacterium tumefaciens C58 ont été inactivées pour vérifier leurs implications dans la production de tumeur chez les plantes. Les expériences ont montré que les souches mutantes pour les Hbtrs AtuHb2 et AtuHb3 étaient capables d'induire la production de tumeur chez la plante Kalanchoe daigremontiana. La protéine recombinante AtuHb2 a été caractérisée par spectroscopie d'absorption et de résonance Raman. L'analyse, par spectroscopie d'absorption, montre que la protéine est hexacoordonnée dans la forme ferrique et ferreuse, qu'il est possible pour AtuHb2 de former des complexes stables avec les ligands CO, CN- et NO, mais pas avec l'O2 et que le complexe formé entre la forme ferreuse et le NO est pentacoordonné. L'analyse par spectroscopie de résonance Raman a montré que le ligand CO est peu stabilisé

A bacteriose, ou mancha oleosa, doença causada por Xanthomonas axonopodis pv. passiflorae, é um sério problema em muitas áreas de produção de maracujá no Brasil, especialmente se associada à antracnose. A transformação genética é uma alternativa para obter plantas resistentes. Proteínas bactericidas, como as atacinas encontradas na hemolinfa de insetos, têm sido usadas para conferir resistência a espécies vegetais. Como as atacinas têm um peptídeo sinal que as direciona para o espaço extracelular em insetos, nós iniciamos este estudo investigando o direcionamento da atacina A em plantas. A seqüência do gene da atacina A (attA) com e sem o peptídeo sinal foi fusionada com os genes repórteres uidA e gfp e epidermes de cebola foram transformadas, via biobalística, com essas construções gênicas. A atacina A, de fato, é acumulada no apoplasto onde, justamente, bactérias fitopatogênicas se multiplicam antes de invadir as células vegetais. Visando obter plantas transgênicas resistentes à bacteriose, foram transformados tecidos foliares e hipocotiledonares com as linhagens LBA 4404 e EHA 105 de Agrobacterium tumefaciens contendo o gene attA. De um total de 313 explantes infectados, foram obtidos 31 brotos PCR+, o que representa uma eficiência de transformação da ordem de 10%. A expressão do transgene foi confirmada por RT-PCR e a resistência ao patógeno foi avaliada pela inoculação de X. axonopodis pv. passiflorae em folhas destacadas de plantas mantidas in vitro. Em dez plantas não houve formação de lesão foliar, indicando uma possível resistência ao patógeno. / Bacterial spot disease caused by Xanthomonas axonopodis pv. passiflorae is a serious problem in many passion fruit production areas in Brazil, especially if associated with anthracnose. Genetic transformation provides an alternative for obtaining resistant plants. Bactericide proteins such as attacins, found in the haemolymph of insects, have been used to confer resistance on plant species. As the attacins have a sign peptide that dispatches them to extracellular space in insects, we initiated our studies investigating the attacin A directing in plants. The attacin A gene (attA) sequence, with and without the sign peptide, was fused to uidA and gfp reporter genes, and onion epidermis were transformed using bioballistics with gene constructions. The protein did accumulate in the apoplast, where bacteria multiply before attacking plant cells. With the aim of obtaining transgenic plants of yellow passion fruit resistant to bacterial disease, leaf and hypocotyl-derived tissues were transformed with LBA 4404 and EHA 105 strains of Agrobacterium tumefaciens containing the attA gene. From a total of 313 infected explants, we obtained 31 PCR+ shoots, a transformation efficiency of 10%. Expression of the attA gene was confirmed by RT-PCR, and pathogen resistance evaluated by X. axonopodis pv. passiflorae inoculation in leaves obtained from in vitro plants. Leaf lesions were not observed in 10 shoots, suggesting a possible resistance to pathogen.

No Brasil, doenças causadas pelo Papaya ringspot virus - type Watermelon (PRSV-W) e Zucchini yellow mosaic virus (ZYMV) reduzem a produção e a qualidade dos frutos de abobrinha-de-moita (Cucurbita pepo) e melancia (Citrullus lanatus), assim como em outras cucurbitáceas. O objetivo deste trabalho foi a obtenção de plantas transgênicas de abobrinha-de-moita e melancia resistentes ao PRSV-W e ao ZYMV. Um sistema eficiente de regeneração in vitro é necessário para a obtenção de plantas transgênicas. O sistema de organogênese in vitro de abobrinha-de-moita foi desenvolvido utilizando como explantes a região basal do cotilédone e um segmento do hipocótilo obtidos a partir de sementes germinadas in vitro. Os explantes foram cultivados em meio de cultura MS (MURASHIGE; SKOOG, 1962), suplementado com diferentes concentrações de BAP (benzilaminopurina). A indução de gemas adventícias foi mais eficiente nas concentrações de 1,0 e 1,25 mg/L de BAP. Este protocolo foi usado para regenerar plantas em experimentos de transformação genética de abobrinha-de-moita cv. Caserta e melancia cv. Crimson Sweet, via Agrobacterium tumefaciens. O vetor binário pCAMBIA2201, contendo fragmentos dos genes da proteína capsidial do ZYMV e do PRSV-W, numa construção gênica do tipo hairpin e o gene de seleção nptII, sob controle do promotor 35S, foi usado nos experimentos de transformação genética. Após 2 dias de co-cultivo, em meio de cultura MS, suplementado com BAP (1 mg/L), os explantes foram transferidos para meio de cultura de seleção, suplementado com BAP (1 mg/L), timentin (400 mg/L) e canamicina (100 mg/L) e cultivados por 3 a 4 semanas, sob fotoperíodo de 16 horas de luz. Plantas regeneradas foram analisadas por PCR, usando primers específicos para detecção dos fragmentos dos genes da proteína capsidial do PRSV-W e ZYMV. Foram utilizados 1050 explantes de abobrinha-de-moita e de 973 explantes de melancia, resultando em 36 e 59 plantas PCR positivas, respectivamente. A eficiência de transformação foi de 3,4% para abobrinha-de-moita e 6,1% para melancia. Plantas PCR positivas foram aclimatizadas, gradualmente, em sala de luz e transferidas para casa-de-vegetação. Pela análise de Southern blot foi confirmada a integração dos fragmentos dos genes da proteína capsidial do ZYMV e PRSV-W em 3 plantas de abobrinha-de-moita. Depois de desenvolvidas, flores femininas foram polinizadas manualmente e sementes foram coletadas de frutos maduros. Plantas R1 de abobrinha-de-moita e melancia foram inoculadas com o PRSV-W e o ZYMV por meio de Myzus nicotianae virulíferos. Não foram identificadas, até o momento, plantas resistentes aos patógenos em estudo / Diseases caused by the potyviruses Papaya ringspot virus - type Watermelon (PRSV-W) and Zucchini yellow mosaic virus (ZYMV) significantly reduce the yield and fruit quality of zucchini squash (Cucurbita pepo), watermelon (Citrullus lanatus), as well as other cucurbit crops in Brazil. The purpose of this work was to obtain zucchini squash and watermelon transgenic plants resistant to PRSV-W and ZYMV. An efficient in vitro regeneration system which can be associated with the protocol is necessary to obtain transgenic plants. In vitro organogenesis system was successfully developed using comprised of distal region of hypocotyl and the base of cotyledon of a germinated seed. The explants were cultured in MS medium (MURASHIGE; SKOOG, 1962), supplemented with different concentraction of BAP (benzylaminopurine). The induction of adventitious buds was more efficient at concentrations of 1.0 and 1.25 mg.L-1 BAP. This protocol was used to regenerate plants from genetic transformation experiments with zucchini squash cv. Caserta and watermelon cv. Crimson Sweet via Agrobacterium tumefaciens. For transformation, the binary vector pCAMBIA 2201, containing sequences of the coat protein coding regions of ZYMV and PRSV-W in a hairpin construct and the nptII gene, driven by 35S promoter was used. After 2 days of co-culture in MS medium supplemented with BAP (1.0 mg.L-1), explants were transferred to the MS selection culture medium, supplemented with BAP (1.0 mg.L-1), timentin (400 mg.L-1) and kanamycin (100 mg.L-1), and incubated for 3 to 4 weeks at 27 oC, under 16 h photoperiod. Regenerated plants were analyzed by PCR, using specific pairs of primers for the detection of the coat protein gene segments of PRSV-W and ZYMV. A total of 1,050 zucchini squash and 973 watermelon explants were used in the transformation experiments, resulting in 36 and 59 PCR positive plants, respectively. The genetic transformation efficiency was 3.4% for zucchini squash and 6.1% for watermelon. The PCR positive plants were slowly acclimatized in the culture room and transferred to the greenhouse for further growth. Southern blot analysis confirmed the genome integration of the the ZYMV and PRSV-W coat protein gene fragments in three zucchini squash plants which survived the acclimatization step. Later in development, female flowers were were manually pollinated and seeds were collected from mature fruits. R1 transgenic zucchini squash and watermelon plants were inoculated with PRSV-W and ZYMV by means of viruliferous Myzus nicotianae. Resistant plants were not yet observed among the R1 plants available

L’identification, chez divers organismes, d’enzymes de dégradation des N-acyl homosérineslactones (NAHLs) impliquées dans la signalisation QS pose la question de leurs rôles dans lesinteractions bactéries-eucaryotes. Dans une première partie, une synthèse bibliographique analyse lesconnaissances acquises sur ces enzymes dégradant les NAHLs. Dans une seconde partie, la croissancedes bactéries dégradant les signaux NAHLs a été stimulée par l’application de g-caprolactone (GCL)dans la rhizosphère de plants de pommes de terre à des fins de phytoprotection. L’effet de cetraitement sur la diversité des communautés bactériennes rhizosphériques a été évalué en combinantdifférentes approches d’écologie microbienne moléculaire comme la DGGE, le pyroséquençaged’amplicons rrs, et la métagénomique fonctionnelle. Cette dernière approche appliquée à une banquede 30 000 clones environ a conduit à l’identification d’un gène qsdB codant la dégradation des signauxNAHL. Ce travail révèle ainsi l’existence d’une nouvelle classe d’enzymes de dégradation des NAHLsappartenant à la famille des enzymes possédant une signature amidase (AS) dont des membres sontpar ailleurs impliqués dans la dégradation de composés xénobiotiques. Dans une troisième partie, unsystème expérimental a été développé afin de mesurer le transfert conjugatif du plasmide de virulenceTi (tumor inducing) chez des dérivés du pathogène Agrobacterium tumefaciens, appelés «tricheurs»,incapables de produire des signaux NAHLs mais utilisateurs de ceux produits par les autres bactéries.Ce modèle a permis de montrer l’effet modérateur de lactonases dégradant les NAHLs exprimées chezdes agrobactéries produisant les NAHLs, chez des bactéries réceptrices du plasmide Ti, ou des planteshôtes des agrobactéries sur le transfert conjugatif initié par les tricheurs. L’ensemble de ce travailrévèle à la fois une nouvelle famille d’enzymes impliquées dans la dégradation des NAHLs, ainsiqu’un nouveau rôle de ces enzymes dans la modulation des flux de gènes entre bactériesphytopathogènes en interaction avec une plante hôte. / Identification of bacterial and eukaryotic enzymes that degrade N-acyl homoserine lactones(NAHLs) involved in QS signaling raises the question of their roles in bacteria-eucaryotesinteractions. In a first part of this study, a bibliographic report analyzes the current data on thoseNAHL-degrading enzymes. In a second part, the growth of NAHL-degrading bacteria was stimulatedby g-caprolactone (GCL) amendment in potato rhizosphere to protect this plant against the soft-rotpathogen Pectobacterium. The effect of the GCL treatment on rhizospheric bacterial communities wasevaluated by a combination of different molecular microbial ecology techniques such as DGGE,pyrosequencing and functional metagenomic. This last approach was applied to generate ametagenomic library of ca. 30,000 clones and lead to the identification of the qsdB gene that encodesNAHL degradation, This work revealed the occurrence of a novel class of NAHL-degrading enzymesthat belong to the amidase signature (AS) family, some members of which being involved inxenobiotic compound degradation. In a third part, an experimental system was developed to measurethe conjugative transfer of Ti plasmid in various strains of the pathogen Agrobacterium tumefaciens,including “cheaters”, i.e. bacteria unable to produce NAHL signals but capable to use signals producedby other bacteria. Using this model; variations of the plasmid transfer of cheaters were measured whenNAHL-degrading lactonases were expressed in agrobacteria that produce NAHL signals, in recipientbacteria of Ti plasmid, or in agrobacterial host plant. Taken together, thesis experiments revealed anovel class of enzymes involved in NAHL-degradation and a new role for thoses enzymes in themodulation of gene transfer between pathogenic bacteria interacting with host plants.