The subject of my thesis was to genetically modify a potato for increased resistance against its pathogens and pests. In developing a resistant plant, it is quite common to use the same type of molecules that plants use themselves in their defense reactions. In this work I used the gene SPI-2 originating from a honeycomb moth (Galleria mellonella). The protein SPI-2 is a member of serine protease inhibitors. Since the previous attempts of the team to detect the protein in transformed plants haven't been successful, the basic form of the gene was modified by adding a Kozak sequence near the start codon, which should have increased the translation initiation and hence increase the level of the protein. Two constructs were prepared for the transformation: SPI-2-T a SPI-2-Y. They differ by one amino acid, which slightly changes their inhibitory activity. First, the construct SPI-2-T was used for a transient transformation of tobacco Nicotiana benthamiana by agroinfiltration of its leaves. Then both constructs were used for a stable transformation of Solanum tuberosum cv. Desireé. The detection of the protein has not been successful, although the inserted gene was transcribed and his sequence was verified by sequencing. It is therefore most likely that the protein has a low stability in the cytoplasm....

No description available.

In der vorliegenden Arbeit wurde der lichtinduzierte Reaktionszyklus des bakteriellen Phytochroms Agp2 aus Agrobacterium tumefaciens mit FTIR‑ und UV‑Vis‑Spektroskopie untersucht. Der Photorezeptor besteht aus einem photosensorischen Modul und einer signalgebenden Histidin-Kinase-Domäne. Das photosensorische Modul bindet das Tetrapyrrol Biliverdin als Chromophor. Der Grundzustand von Agp2 (Pfr, 750 nm) ist gegenüber dem lichtaktivierten Zustand (Pr, 700 nm) rotverschoben, weshalb Agp2 den Bathyphytochromen zugeordnet wird. Die Untersuchungen erfolgten unter Verwendung von Isotopenmarkierung, H/D-Austauschexperimenten und ortsspezifischer Mutagenese. Daraus ließen sich folgende molekulare Änderungen charakterisieren, welche im Reaktionszyklus von Agp2 erfolgen: Die lichtinduzierte Isomerisierung des Chromophors führt zu einem Übergang vom Pfr- in den Pr-Zustand, wobei zwei Intermediate, Lumi‑F und Meta‑F, durchlaufen werden. Neben der Konformationsänderung des Chromophor‑D‑Rings ist auch die C‑Ring-Propionsäureseitenkette an der Photoreaktion beteiligt. Die C-Ring-Propionsäureseitenkette ist im Pfr-Zustand protoniert und wird im Übergang von Meta-F zu Pr deprotoniert. Der Pr-Zustand weist eine pH-Abhängigkeit auf, welche auf die pH-abhängige Ladung des Histidins 278 der Chromophortasche zurückzuführen ist. Je nach Ladung des Histidins 278 wird die Keto‑ bzw. Enolform der C(19)=O‑Gruppe des D‑Rings stabilisiert. Die Keto/Enol-Tautomerie ist auf eine innerhalb des Chromophors erfolgende Protontranslokation zurückzuführen und moduliert die Relaxation in den Pfr-Zustand. Änderungen der Amid-I-Absorption im Pfr-Pr-Übergang werden der Umstrukturierung der Tongue-Region des photosensorischen Moduls von einer Alpha-helikalen zu einer Beta‑Faltblatt-Struktur zugeordnet. Diese Strukturänderung wird als möglicher Weg der proteininternen Signaltransduktion zwischen photosensorischem und signalgebendem Modul vorgeschlagen. / In this thesis the light-induced reaction cycle of the bacterial phytochrome Agp2 from Agrobacterium tumefaciens was investigated using FTIR and UV‑vis spectroscopy. The photoreceptor comprises a photosensitive module and a signalling histidine kinase domain. The photosensitive module binds the biliverdin tetrapyrrol as chromophore. The Agp2 ground state (Pfr, 750 nm) is red-shifted in comparison with its light-activated state (Pr, 700 nm). Therefore, Agp2 is assigned to the group of bathy phytochromes. The investigations were conducted using isotopically labelled protein, labelled chromophore as well as hydrogen‑deuterium (H‑D) exchange and site-directed mutagenesis. Based on these the following molecular changes could be characterized that occur in the reaction cycle of Agp2: The light-induced isomerization of the chromophore leads to a transition from the Pfr to the Pr state, involving two intermediates, Lumi-F and Meta-F. Besides conformational changes of the chromophore D-ring, the C-ring propionic side chain is involved in the photoreaction as well. The C-ring propionic side chain is protonated in the Pfr state and gets deprotonated in the Meta-F to Pr transition. The Pr state exhibits pH‑dependent alterations which can be explained by pH dependent polarity changes of histidine 278 in the chromophore pocket. Depending on the charge of histidine, the D‑ring C(19)=O group is stabilized either in keto or enol form. The keto/enol tautomerism involves a proton translocation within the chromophore and modulates the relaxation to the Pfr state. The changes in the amide I region in the Pfr-Pr transition are associated with an alpha‑helix to beta‑sheet secondary structure change of the PHY domain tongue‑region. This structural change is proposed as the potential path of signal transduction between the photosensitive and the signalling module.

Indiana University-Purdue University Indianapolis (IUPUI) / The phototropin photoreceptors are important regulators of plant growth and development and can therefore affect the photosynthetic activity of plants. Phototropin1 and Phototropin2 are versatile protein kinases that become activated when exposed to blue light. Their photobiological actions are best understood in the model plant Arabidopsis thaliana, where they are known to trigger several responses to blue light, one of which is phototropism, the bending of plant organs towards light. Additionally, phot1 and phot2 drive stomatal opening, chloroplast arrangement in leaf cells, leaf expansion, and leaf orientation. The phot1-specific response is rapid inhibition of hypocotyl growth, leaf positioning and mRNA stability whereas phot2 mediates the chloroplast avoidance response to high light. These responses impact a plant’s ability to capture light for photosynthesis, therefore the phototropins play important roles in optimizing a plant’s photosynthetic activity. Soybean (Glycine max) is a very important crop plant in Indiana known for its nutritional versatility and is also utilized for biodiesel production.In spite of soybean being a key crop, there is currently no information about the functionality of soybean phototropins. Also, being a legume, soybean has many structural and functional features that are not present in Arabidopsis. Interestingly, PsPHOT1A (a photoreceptor from garden pea) was found to be a functional phototropin as it was able to complement the phot1 mutation in Arabidopsis. The roles of these proteins in soybean will be elucidated based on the hypothesis that soybean phototropins play essential roles in regulating photosynthetic activity as do the Arabidopsis phototropins. To date, five soybean phototropins, 3 PHOT1s and 2 PHOT2s, are believed to exist. These GmPHOT protein coding regions were amplified by RT-PCR and cloned into pCR8/TOPO or pENTR-D/TOPO vectors via TOPO cloning to utilize Gateway cloning technology to create plant transformation constructs subsequently. The cloned GmPHOT cDNAs from each of the 5 GmPHOTs were sequenced and compared to the GmPHOT sequences from the Phytozome database to assess the accuracy of the gene models. The gene models of all the GmPHOTs were found to be accurate except that of GmPHOT1B-2. The high level of sequence identity between the GmPHOTs and AtPHOTs and the conservation of LOV domains and catalytic domains indicate structural resemblance between them. This suggests that soybean phototropins should encode active photoreceptors. The cloned protein coding regions from soybean were then recombined into a plant expression vector via Gateway technology,which were then used for transformation of Agrobacterium tumefaciens. These plant expression constructs will be utilized in the future to determine the functionality of soybean phototropins in Arabidopsis.

The salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae family and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Despite their important role in plant defense, their biosynthetic pathway is not known, although recent work has suggested that benzyl benzoate acts as a possible biosynthetic intermediate. We identified three candidate genes encoding BAHD-type acyltransferases that are predicted to produce benzylated secondary metabolites, named PtACT47, PtACT49, and PtACT54. Expression of PtACT47 and PtACT49 generally correlated with PG content in a variety of tissues and organs of wild type hybrid poplar plants. This correlation was also found in transgenic hybrid poplar where PG content varied with the level of expression of the condensed tannin regulator MYB134 transcript. In these plants, a suppression of PtACT47 and PtACT49 expression was correlated with lower PG content. In contrast, PtACT54 exhibited very low expression in all tissues tested, and this level of expression was not affected in MYB134 plants. In order to better understand their possible biochemical functions, cDNA cloning, heterologous expression, and in vitro functional characterization was performed on these three BAHD acyltransferases. Recombinant PtACT47 exhibited a low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM-1 sec-1) and lowest Km values (45.1 µM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA:salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, specifically benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM-1 sec-1) and lowest Km (55.3 µM) was observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA:benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters at trace levels, for example hexenyl benzoate. Recombinant PtACT54 shares low sequence identity with PtSABT (52.3%) and PtBEBT (52.5%) and exhibited only moderate BEBT-like properties. PtSABT and PtBEBT appear to be paralogs based on their high sequence identity (90.6%) and closely related yet distinct biochemical functions. They likely arose from gene duplication and subsequent functional diversification possibly by neofunctionalization. Wounding experiments showed that abiotic damage stimulated the synthesis of specific PGs, notably salicin and salicortin within 24-48hrs. This was accompanied by a proportional increase in the expression of PtSABT and PtBEBT. Furthermore, experiments using transgenic RNAi lines with knock-down suppression of PtBEBT, and PtSABT, and both genes simultaneously, provided the first direct evidence that BAHD acyltransferases are important in PG production. PtSABT suppression, both individually and in the double knock-down suppression, significantly lowered salicortin content, particularly in mature leaves. However, a reduced level of PtBEBT expression did not have a significant effect on the PGs measured. This could indicate that BEBT-like activity may be a shared function among closely related BAHDs. The suppression of multiple BEBT-like genes may be necessary to further delineate their functions. / Graduate / rjchedgy@uvic.ca

A cana-de-açúcar é uma das principais culturas agrícolas plantadas no Brasil e apresenta significativa importância sócio-econômica e agroindustrial ao país. O cenário mundial encontrase bastante favorável no que concerne à comercialização de seus dois principais produtos derivados, o açúcar e o álcool, impulsionando o desenvolvimento do setor sucroalcooleiro nacional. Neste sentido, o melhoramento genético da cana-de-açúcar aparece como base fundamental para o desenvolvimento de novas variedades para a manutenção e incremento dos agronegócios da agroindústria sucroalcooleira. Técnicas de engenharia genética, como a transformação genética nuclear, estão trazendo excelentes resultados no melhoramento genético da cultura, permitindo diminuir o custo e o tempo de obtenção de novas variedades. Baseando-se na importância em se obter variedades tolerantes a diferentes estresses bióticos e abióticos que induzem perturbações metabólicas e ativam o processo de morte celular programada, o presente trabalho teve por objetivo transformar geneticamente a variedade de cana-de-açúcar RB835089 com o cDNA do gene AtBI-1 isolado de Arabidopsis thaliana, visando suprimir a indução do mecanismo de morte celular sob condição de estresse. Para isto, calos embriogênicos foram utilizados como explante alvo, empregando-se dois métodos de transformação, a cotransformação por biolística, e o mediado por Agrobacterium tumefaciens no qual foram testadas duas técnicas: (a) inoculação direta dos calos em suspensão bacteriana; e (b) agrobiolística que é o bombardeamento dos calos com partículas de tungstênio seguido da inoculação em suspensão bacteriana. A proteína AtBI-1 (Bax inhibitor-1) apresenta homólogos em outros organismos e está localizada na membrana do retículo endoplasmático. Ela apresenta funções citoprotetoras modulando o mecanismo de morte celular programada induzida por estresses bióticos e abióticos. Como resultados deste trabalho, diferentes taxas de eficiência da transformação genética foram obtidas pelo método mediado por A. tumefaciens nas duas técnicas testadas, sendo que suas taxas foram superiores às alcançadas pelo método de co-transformação por biolística. A expressão heteróloga do cDNA do gene AtBI-1 em cana-de-açúcar atenuou a indução das vias de morte celular em presença do antibiótico tunicamicina, indutor do estresse no retículo endoplasmático, sendo comprovado pela maior tolerância ao estresse das plantas transgênicas quando comparadas com as plantas não transformadas que foram afetas no crescimento do sistema radicular, conteúdo de clorofila total, apresentando sintomas típicos de morte celular programada como clorose foliar e morfologia irregular das raízes, com consequente morte do sistema radicular. / Sugarcane is one of the main crops planted in Brazil and presents significant socioeconomic and agribusiness importance to the country. The world scene is quite favorable as regards the marketing of its two main products, sugar and alcohol, driving the development of the national sugar-alcohol sector. Therefore, the sugarcane genetic breeding appears as the fundamental base for developing new varieties for the maintenance and increase of agribusiness in the sugarcane agroindustry. Genetic engineering techniques, such as the nuclear genetic transformation, are providing excellent results in genetic breeding of this crop allowing reducing the cost and time to obtain new varieties. Based on the importance of obtaining varieties tolerant to different biotic and abiotic stresses that induce metabolic disturbances and activate the process of programmed cell death, this work aimed to transform sugarcane variety RB835089 with the cDNA of AtBI-1 gene, isolated from Arabidopsis thaliana, to suppress the induction of the cell death mechanism under stress condition. For this, embryogenic calli were used as target explant, by using two methods of transformation, the cotransformation by biolistic, and mediated by Agrobacterium tumefaciens in which two techniques were tested: (a) direct inoculation of calli in bacterial suspension; (b) agrobiolistic which is the bombardment of calli with tungstein particles followed by inoculation in bacterial suspension. The AtBI-1 protein (Bax inhibitor-1) presents homologs in other organisms and is located in the endoplasmic reticulum membranes. It has cytoprotective functions by modulating the mechanism of programmed cell death induced by biotic and abiotic stresses. As results of this work, different efficiency rates in genetic transformation were obtained in the method mediated by A. tumefaciens in the two techniques tested, and that their rates were higher than those achieved using the cotransformation by biolistic. The heterologous expression of cDNA of AtBI-1 gene in sugarcane attenuated the induction of cell death pathways in the presence of tunicamycin antibiotic, an inducer of stress in the endoplasmic reticulum, being proven by the increased stress tolerance of transgenic plants compared with sugarcane wild type that were affected in the root growth, total chlorophyll content, showing typical symptoms of programmed cell death such as leaf chlorosis and irregular morphology of the roots, with subsequent death of the root system.

Genotypic differences in growth and yield of two common bean (Phaseolus vulgaris L) cultivars to Rhizobium inoculation and management were investigated. In 2003-04, the two bean cultivars (Scylla and T-49) were combined with three inoculant treatments (strains CC 511 and RCR 3644, and a control of no inoculation), two fertiliser levels (0 and 150 kg N ha⁻¹) and two irrigation treatments (irrigated and rainfed). There was no nodulation on either cultivar. To further investigate the symbiotic relationship, 16 rhizobial isolates, including the two used in the first field experiment, were combined with the cultivar Scylla and evaluated in a greenhouse. Subsequently, five Rhizobium isolates were chosen for further field evaluation, based on signs of early nodulation in the greenhouse trial. The second field experiment in 2004-05 combined the five inoculant strains (RCR 3644, UK 2, H 20, PRF 81, PhP 17 and a control) with two bean cultivars (Scylla and T-49). In the greenhouse, nodule number varied from 7 (UK 2) to 347 (H 441) nodules plant⁻¹ at 51 DAS and from 13 (UK 1) to 335 (CIAT 899) nodules plant⁻¹ at 85 DAS. In 2004-05, in the field, nodulation was also variable, ranging between 1 and approximately 70 nodules plant⁻¹, with higher nodules numbers plant⁻¹ being found on cultivar T-49. Of the isolates used in the field, strains H 20, PRF 81 and PhP 17 produced 70, 25 and 12 nodules plant⁻¹ at 70, 40 and 54 DAS respectively. Nodules formed were of various sizes and more than 80 % were pink to dark red in colour denoting the presence of leghaemoglobin and active N fixation. The remaining nodules were either green or white. The importance of selecting an appropriate cultivar for the growing conditions was highlighted in these experiments. Leaf area index, leaf area duration intercepted radiation and final utilisation efficiency were significantly affected by cultivar. In both seasons cv. T-49 reached maturity (dry seed) before Scylla, while unirrigated plants reached green pod maturity seven days before irrigated plants. Plants of cv. Scylla gave a final TDM of 730 g m⁻²; compared to the 530 g m⁻² produced by T-49. The average growth rate was 7.0 and 5.2 g m⁻² day⁻¹ for Scylla and T-49 respectively (2003-04). Plants receiving 150 kg N ha⁻¹ produced 665 g m⁻² TDM which was 12 % more than was produced by unfertilised plants. The application of 150 kg N ha⁻¹ gave an average growth rate of 6.4 g m⁻² day⁻¹ compared to 5.7 g m⁻² day⁻¹ from plants with no N. Inoculation in the field had no significant effect on TDM in both seasons. Temperature affected growth and DM accumulation. Accumulated DM was highly dependent on cumulative intercepted PAR. Air temperatures below the base temperature (10 °C) affected growth in 2004-05, resulting in plants accumulating just 0.24 g DM MJ⁻¹ PAR during early growth. This increased to 2.26 g DM MJ⁻¹ PAR when the temperature was increased above the base temperature. There was a strong relationship between LAI and intercepted PAR. A LAI of 4.0-4.5 was required to intercept 90-95 % of incident solar radiation. Cultivar significantly (p < 0.001) affected radiation use efficiency (RUE). Scylla had a RUE of 1.02 g DM MJ⁻¹ PAR compared to T-49 at 1.18 g DM MJ⁻¹ PAR. Seed yield was significantly (p < 0.001) affected by cultivar and fertiliser application. Cultivar Scylla produced 467 g m⁻² which was 76 % more than T-49, while a 12 % increase in seed yield was observed in N fertilised plants over unfertilised plants. Only cultivar significantly affected HI, while the yield components that had the greatest effect on seed yield were hundred seed weight and pods plant⁻¹. Inoculation significantly (p< 0.05) affected 100 seed weight (2004-05). Plants inoculated with strain H 20 had the highest 100 seed weight at 25.2 g with cv. Scylla producing larger seeds than T-49. The belief that local environmental conditions play a major role on field survival of bacteria, led to the use of PCR methods to identify field nodulating organisms. Amplification of genomic DNA from parent isolates using primers fC and rD generated a single band for each isolate. Isolates were identified to the species level as either Rhizobium or Agrobacterium, using the highly conserved internally transcribed spacer (ITS) region and are known to nodulate common bean. The DNA extracted from the isolates recovered from nodules of field grown beans gave multiple bands with primers fC and rD. Five distinct banding patterns were observed. All of these were different from those of parent isolates. Sequencing of the 16S rRNA demonstrated that nodules of field grown beans in Canterbury were inhabited by Pseudomonads either alone or in association with other root nodulating organisms. The inability to identify the inoculant strains in nodules of field grown beans does not rule out their infection and nodulating function in the cultivars used. The results suggest the possibility of both Rhizobium and Pseudomonads cohabiting in the nodules of field grown beans. The aggressive nature of Pseudomonads on artificial media, possibly out competing the inoculant rhizobia is proposed, leading to the inability to identify the inoculant strain from the nodules of the field grown beans by PCR methods. The need to identify the nodule forming or nodule inhabiting bacteria in the nodules is necessary to classify the importance of these organisms and their economic benefit to agricultural production. This study also underlines the importance of using PCR methods to gain valuable insights into the ecological behaviour of Rhizobium inoculants and nodule inhabiting organisms.

Wheat is one of the most essential food sources in the world. However, there has been serious yield loss of wheat production due to stripe rust disease caused by the fungal pathogen Puccinia striiformis f. sp. tritici. The cost-effective and long-lasting defense to the disease can be achieved by generating genetically resistant crops against the disease forming pathogens. To accomplish this, first step is to acquire knowledge in the plant pathogen interactions of the crop and the pathogen of interests at the cellular and the molecular level. In this thesis research, PstHa2a5 candidate effector gene from Puccinia striiformis f. sp. tritici is investigated to identify its role and interaction between host factors in yellow rust infected Triticum aestivum L. The gene construct was engineered with FLAG-tag fusion at its N-terminus, and synthesized. This construct was cloned into pJL48-TRBO vector for an expression in Nicotiana benthamiana via agrobacterium-mediated gene transformation. The expressed protein structure with FLAG-tag was purified, and immunoprecipitated with one putative N. benthamiana interactor by immunoprecipitation experiments. This candidate interactor protein will be identified with Mass Spectroscopy. In addition to this, subcellular localization of the effector candidate was examined in N. benthamiana plant. This was achieved by cloning PstHa2a5 gene construct in pK7WGF2 gateway destination vector and localization is determined by GFP expression in N. benthamiana after agrobacterium-mediated gene transformation.

Douglas-fir (Pseudotsuga menziesii) is an economically important softwood that is clonally propagated for reforestation purposes by somatic embryogenesis. The molecular basis of embryogenesis in conifers is largely unknown and this prevents progress in somatic embryogenesis protocols. In angiosperms, the LEAFY COTYLEDON1 (LEC1) gene, encoding the HAP3 subunit of the eukaryotic CCAAT box-binding factor, is important in embryo formation, and necessary for somatic embryogenesis. A candidate gene strategy was employed to isolate the Douglas-fir LEC1 homologue, PmLEC1, via the polymerase chain reaction (PCR) with degenerate primers based on the Arabidopsis conserved domain, and the full-length cDNA sequence was obtained by rapid amplification of cDNA ends-PCR (RACE-PCR). The putative protein sequence shared high sequence identity with Arabidopsis LEC1. Northern analysis and quantitative real-time PCR indicate that this is an embryo-specific gene, expressed with the highest abundance during early embryogenesis. Antibodies were raised against a synthetic 18-amino acid PmLEC1 peptide, and in contrast to mRNA expression, Western blotting shows that PmLEC1 protein expression persists until the seedling stage. To gain insight into modulation of PmLEC1 expression and its inducibility in mature tissues, stress and hormone treatments were performed on mature seed and the promoter sequence was isolated by genome walking. Sorbitol, mannitol and 2,4-epibrassinolide were found to significantly up-regulate PmLEC1 expression. The PmLEC1 promoter contains a 5’ UTR intron with numerous enhancer elements, and factors that bind to these elements mediate responses to auxin, UV light and developmental cues, osmotic stress, biotic stress, and tissue culture. Some of the regulatory elements are binding sites for seed-specific transcription factors that are well known from angiosperms, providing new evidence that AGL15, ABI3 and VP1 proteins have a direct role on LEC1 expression. In investigating the embryogenic capacity of PmLEC1, ectopic expression of PmLEC1 in the embryo lethal Arabidopsis lec1-1 null mutant complemented the mutation and permitted the production of viable, desiccation tolerant seeds. In addition, transgenic seedlings produced embryo-like structures from vegetative organs and expressed seed-specific genes. In wild type plants, ectopic expression of PmLEC1 resulted in a bushy phenotype but expression of seed-specific genes was not observed. Taken together, these results show that PmLEC1 is an embryo-specific gene with an essential role throughout embryogenesis, and PmLEC1 expression may be induced in mature seeds by stress and hormone treatments. Because mature seeds show only trace amounts of PmLEC1 transcripts and Douglas-fir somatic embryogenesis can only be induced from immature embryos, this information provides useful insight into initiation of embryogenesis from vegetative tissues. The identification of binding sites for transcription factors known from angiosperms in the promoter region of PmLEC1 has revealed the identity of several genes which are expected to play pivotal roles in conifer embryogenesis.

No description available.