The β-glucosidase (Abg) from ATCC 21400, an Agrobacterium species, was purified to homogeneity. The protein was cleaved with cyanogen bromide and the peptides were purified by reversed phase high pressure liquid chromatography. The partial amino-acid sequences for three CNBr peptides, CNBr1, CNBr2 and CNBr3, were determined by automated Edman degradation. A sequence from CNBr2 was used to synthesize a mixture of oligonucleotides which was used as a hybridization probe to identify a recombinant DNA clone carrying the gene for β-glucosidase. A single clone was isolated which expressed an enzymatic activity that hydrolyzed several β-glucosides. The enzymatic activity produced by this clone could be adsorbed by rabbit antiserum raised against the Agrobacterium enzyme. The direction of transcription of the β-glucosidase gene was determined by verifying the DNA sequence 3' to the oligonucleotide probe binding site. After subcloning the gene a high level of expression was obtained in the plasmid vector pUC18 using the lacZ gene promoter. The nucleotide sequence of the 1599 bp insert in pABG5 was determined using the chain terminator method. The start of the protein coding region was determined by aligning the amino terminal sequence of the protein with the predicted amino acid sequence of the cloned gene. The open reading frame was 1387 nucleotides and contained 458 codons. The molecular weight calculated from the deduced amino acid sequence agreed with that observed from both the native and recombinant enzymes. The predicted amino acid composition from the open reading frame matched with that determined for the native β-glucosidase. The stop codon of this coding region was followed by a potential stem loop structure which may be the transcriptional terminator. There was a region of the deduced Abg sequence which had homology to a region from two other β-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A degradação de quitina é importante para o remodelamento da parede celular em fungos filamentosos e crucial para o rompimento da cutícula de hospedeiros artrópodes durante a infecção de fungos entomopatogênicos. Além disso a quitina é uma importante fonte nutricional. Para que a quitina possa ser eficientemente utilizada, a atividade de b-Nacetilglicosaminidases (NAGases) deve estar presente. Após a ação de quitinases sobre a quitina, gerando dímeros de N-acetilglicosamina (GlcNAc)2, NAGases hidrolisam suas ligações β-1-4 produzindo GlcNAc livre. Fungos filamentosos possuem, em média, 15 a 25 quitinases, mas somente duas NAGases, o que leva a questões sobre a real importância destas enzimas. Em escala genômica, foram identificadas no fungo entomopatogênico Metarhizium anisopliae duas NAGases da família GH20 (MaNAG1 e MaNAG2) e duas NAGases da família GH3 (MaNAG3 e MaNAG4) das glicosil hidrolases. Análises filogenéticas sugerem subsequentes duplicações ocorrendo principalmente no clado de MaNAG2, resultando na presença de ortólogos em um amplo espectro de ascomicetos com diferentes estilos de vida. MaNAG1 agrupou majoritariamente com espécies entomopatogênicas. MaNAG3 e MaNAG4 apresentaram alta similaridade de sequências e conservação de domínios com NAGases GH3 de bactérias O perfil transcricional dos genes das NAGases GH20 e GH3 foi avaliado por qPCR, em oito diferentes condições de cultivo, representando diferentes estágios de desenvolvimento ou diferentes estados nutricionais. As NAGases apresentaram perfis de transcrição diferenciais em resposta às diferentes condições, indicando a ausência de um padrão de regulação gênica em comum. Os perfis de expressão variáveis também sugerem que elas não devem possuir funções totalmente redundantes. Ensaios de transcrição relativa mostraram a indução da expressão de MaNAG1, MaNAG2 e MaNAG4 por quitina 1%, enquanto MaNAG3 foi induzida em meio suplementado com GlcNAc 0,25%. As relações evolutivas de MaNAG3 e MaNAG4 e a regulação de suas expressões por substratos quitinosos são a primeira evidência do envolvimento de NAGases GH3 em processos celulares fisiológicos em ascomicetos, apontando para sua potencial relevância na diferenciação celular durante o ciclo de vida de M. anisopliae. Com o objetivo de avançar no estudo funcional das NAGases de M. anisopliae, foram gerados vetores para a construção de mutantes nulos para os quatro genes de NAGases e linhagens transformantes foram obtidas utilizando-se a metodologia de transformação de fungos mediada por Agrobacterium tumefaciens. / Chitin degradation is important for filamentous fungi cell wall remodeling and, in entomopathogenic fungi, this process is pivotal for breaching the arthropod host cuticles during infection. Chitin is an important nutrient and to be efficiently used, β-Nacetylglucosaminidases (NAGases) activity must be present. After chitinase action on chitin generating N-acetylglucosamine dimers (GlcNAc)2, NAGases hydrolyze theirs β-1-4 linkages producing free GlcNAc. Filamentous fungi have between 15 to 25 chitinases, but only two NAGases; then, questions arise about the actual importance of these enzymes. On a genomic scale, were identified in the entomopathogenic fungus Metarhizium anisopliae two GH20 NAGases (MaNAG1 and MaNAG2) and two GH3 NAGases (MaNAG3 and MaNAG4) from glycoside hydrolases. Phylogenetic analysis suggested subsequent duplications occurring mainly in MaNAG2 clade, resulting in ortholog clusters in several ascomycetes with a broad range life style. MaNAG1 clusters mostly with entomopathogenic species clades. MaNAG3 and MaNAG4 showed high sequence similarity and domain conservation with bacterial GH3 NAGases Transcriptional profiles of GH20 and GH3 NAGase genes were evaluated by qPCR from eight culture conditions, representing different stages of development and different nutritional states. NAGases showed differential transcript profiles in response to different conditions, indicating an absence of a common gene regulation pattern. The variable expression profiles also suggest they may not have totally redundant roles. Relative transcription assays showed MaNAG1, MaNAG2 and MaNAG4 expression induction by chitin 1%, while MaNAG3 was induced in medium supplemented with GlcNAc 0.25%. Evolutionary relationships of MaNAG3 and MaNAG4 and their expression regulated by chitinous substrates are the first evidence of GH3 NAGases involvement in physiological cell process in entomopathogenic fungi, therefore, pointing to potential relevance on cell differentiation during M. anisopliae life cycle. In order to proceed on functional studies of M. anisopliae NAGases, vectors were constructed to produce knockout mutants for four NAGases genes and transformant strains were obtained by using fungi transformation mediated by Agrobacterium tumefaciens.

An Agrobacterium-mediated transformation system of purple raspberry ‘Amethyst’ was established after a series of experiments that determined the effect of genotype, inoculum density, and co-cultivation time on transformation. In this study, a plant regeneration protocol was established for ‘Joan J’ and ‘Polana’ (the regeneration protocol of ‘Amethyst’ was previously developed). Agrobacterium-mediated transformation was conducted for all three cultivars. The minimum killing level of hygromycin B and kanamycin was determined. Inoculum density and co-cultivation time were optimized. Polymerase chain reaction (PCR) verified a successful transformation of ‘Amethyst’ with the frequency of 3.3 ~ 4.4 % when leaves were infected with Agrobacterium EHA105 at the cell density of OD600 0.3 and co-cultivated for 3 days in the medium with 25.0 mg∙l-1 kanamycin. Transgenic lines with the PtFIT gene were hydroponically grown under iron sufficiency or deficiency. The real-time quantitative PCR verified the gene expression in response to iron sufficiency and deficiency conditions.

Development of genetic improvement procedures for the bioenergy crop, Miscanthus × giganteus (M×g; triploid), are desired for trait improvement. Agrobacterium tumefaciens has been used in other Miscanthus spp. for genetic improvement, but not in M×g, nor have more than a few genes been introduced at a given time. Transformation procedures for M×g were developed; studies included use of A.tumefaciens (strain LBA4404) and A. rhizogenes (strain A4) for introduction of seven genes isolated (by other researchers) from Sorghum bicolor involved in sorgoleone biosynthesis (an allelopathic compound), and selectable marker gene neomycin phosphotransferase II. Procedural development included generation of embryogenic calli from immature inflorescences, attempts to minimize generation of phenolics by these tissues, transformation procedures for both Agrobacterium spp., selection of transgenic tissues, and tissue screenings via polymerase chain reaction to identify putative transgenic tissues/plants. Although four M×g putative transgenic plants were generated, none were proven to be transgenic.

No description available.

The respiratory rates of Rhizobium trifolii and Agrobacterium tumefaciens were measured by use of a Warburg Respirometer. A non-proliferating or "resting cell" technique was utilized in demonstrating the effect of Krilium on the oxygen consumption of the organisms when Krilium was aided to a given substrate, glucose, succinate or mannitol. Two types of Krilium were used in this investigation, Krilium (Blend #6) and Krilium (Blend #9). Observations were made on the effect of various concentrations (.05%, .10% and 1.0%) of the two blends of Krilium on the respiratory activities of the test organisms. It was observed that Krillium (Blend #6) caused a noticeable increase in the respiratory rate of Rhizobium trifolii, whereas in the case of Agrobacterium tumefaciens, no effect or a slight decrease was observed when Krilium (Blend #9) was added to the substrate, A very noticeable decreases in respiratory rates was observed when both test organisms were permitted to respire on substrates containing Krilium (Blend #9). When Krilium (Blend #6) and Krilium (Blend #9) were used as the only substrates, an increase in respiration rates of the organisms was observed. It is indicative from this work that Krilium (Blend #6) either causes a slight increase In the respiratory activities or has no deleterious effect on the respiration of the test organisms., The results obtained, when using Krilium (Blend #9), seem to indicate that this blend of Krillum has an undesirable effect on the respiration of the two organisms used in this investigation. / Master of Science

Hairy roots are a syndrome of the plant pathogen Agrobacterium rhizogenes, which induces the aggressive growth of roots in the plants it infects. Hairy roots are shown to have increased production of secondary metabolites when compared to roots that are not transformed, especially when they are exposed to plant signaling hormones called elicitors. Two popular elicitors are Methyl Jasmonate (MeJA) and Salicylic Acid (SA), which are also potent plant signaling compounds involved in plant defense and immunity. Many studies have reported on the secondary metabolites of hairy roots and their production of metabolites after exposure to elicitors. However, there is a gap in current knowledge of how hairy roots and non-transformed roots of Helianthus annuus (the common sunflower) compare in their secondary metabolite profiles, which include the volatile terpenoids they produce. This experiment used solid-phase microextraction gas chromatography-mass spectrometry (SPME GC-MS) to compare the volatile terpenoid profiles of hairy roots and non-transformed roots of H. annuus after their exposure to 0.2 mM MeJA and MeSA, the methyl ester of SA. The experiment identified several differences in the production of volatile compounds across elicitor treatments and time points yet hairy roots largely maintained the properties of their native counterparts. This project provides information on the secondary metabolism and volatile terpenoid profiles of hairy roots and explores the biotechnological applications of such insights.

Uma das espécies que atualmente vem despertando interesse econômico por seu elevado valor de mercado é o maracuzajeiro doce (Passiflora alata Curtis). Entretanto, a cultura é afetada por diferentes doenças que prejudicam a produtividade e a qualidade dos frutos, sendo a doença causada pelo Cowpea aphid-borne mosaic virus (CABMV) a que mais afeta a cultura do maracujazeiro no Brasil. O presente trabalho teve como objetivo a obtenção de plantas transgênicas de P. alata visando resistência ao CABMV. O processo de transformação genética utilizado foi via Agrobacterium tumefaciens, estirpe EHA105, contendo o cassete de expressão com um fragmento do gene da proteína capsidial do CABMV, numa construção tipo hairpin e o gene de seleção nptII que confere resistência ao antibiótico canamicina. Para os experimentos de transformação genética foram utilizados como explantes segmentos de hipocótilo e segmentos internodais. Após 2 a 3 dias de co-cultivo em meio de cultura MS (MURASHIGE; SKOOG, 1962) contendo acetosseringona (100 mM), os explantes foram transferidos para meio de cultura de seleção e regeneração constituído de sais minerais e vitaminas de MS, suplementado com benzilaminopurina (BAP - 1mg/L) + thidiazuron (TDZ - 0,5 mg/L) + canamicina (100 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L), pH 5,8. Após 4 a 6 semanas de incubação, determinou-se o número de explantes responsivos e as gemas adventícias desenvolvidas foram transferidas para meio de cultura de alongamento MSM + GA3 (1,0 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L). As plantas desenvolvidas foram aclimatizadas e analisadas por PCR, utilizando primers específicos para a detecção do fragmento do gene da proteína capsidial do CABMV e do gene de seleção (nptII). Foram identificadas 47 plantas transgênicas PCR positivas para do gene nptII. Até o momento, a integração do gene nptII foi confirmada por Southern blot em 9 plantas / One species that is currently attracting interest due to its high economic value is the sweet passionflower (Passiflora alata Curtis). However, the culture is affected by different diseases that harm the productivity and fruit quality. The disease caused by Cowpea aphid-borne mosaic virus (CABMV) is the one that more affect the culture of passionflower in Brazil. This work aimed to obtain transgenic plants of P. alata resistant to the CABMV. The genetic transformation process was via Agrobacterium tumefaciens, strain EHA105, containing the expression cassette with a fragment of the coat protein gene of CABMV, in a hairpin construct and the selection gene nptII, which confers resistance to the antibiotic kanamycin. In the experiments of genetic transformation hypocotyl segments and internodal segments were used as explants. After 2-3 days of co-cultivation in MS medium (MURASHIGE; SKOOG, 1962) containing acetosyringone (100 mM), the explants were transferred to the selection and regeneration culture medium consisting of mineral salts and vitamins of MS medium supplemented with benzylaminopurine (BAP - 1 mg/L) + thidiazuron (TDZ - 0.5 mg/L) + kanamycin (100 mg/L) + cefotaxime (500 mg/L) + silver nitrate (4.0 mg /L), pH 5.8. After 4-6 weeks of incubation, it was determined the number of responsive explants. Shoots developed were transferred to elongating culture medium MSM + GA3 (1.0 mg/L) + cefotaxime (500 mg/L) + nitrate silver (4.0 mg/L). The developed plants were acclimatized and analyzed by PCR using specific primers to detect the fragment of CABMV and the selection gene (nptII). It was identified 47 transgenic plants PCR positive for the gene nptII. Until this moment, the integration of the nptII gene was confirmed by Southern blot in 9 plants

A cultura do maracujazeiro é afetada pela virose causada pelo Cowpea aphid-borne mosaic virus (CABMV), provocando a redução da qualidade e produtividade dos frutos e, em alguns casos, pode inviabilizar o cultivo comercial desta espécie. Uma alternativa para o controle de doenças viróticas é o desenvolvimento de plantas resistentes pela transformação genética. O objetivo deste trabalho foi a obtenção de plantas transgênicas de maracujazeiro (Passiflora edulis f. flavicarpa), utilizando 2 construções gênicas contendo a região codificadora do gene da proteína capsidial do CABMV. A construção pCABMV-asCP, que contém um fragmento na orientação antisenso e a construção pCABMV-dsCP, que contém fragmentos senso e antisenso do gene da proteína capsidial, separados por um íntron, uma construção hairpin. Para os experimentos de transformação genética, via Agrobacterium tumefaciens, foram utilizados os explantes de segmentos de hipocótilo e discos de folhas jovens das variedades FB-100, IAC-275 e IAC-277. Após 2 - 3 dias de co-cultivo em meio de cultura MS (MURASHIGE; SKOOG, 1962) contendo acetosseringona (100 mM), os explantes foram transferidos para meio de cultura de seleção e regeneração constituído de sais minerais e vitaminas de MS, suplementado com canamicina (100 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L), pH 5,8 e os reguladores BAP, TDZ e combinação de BAP e TDZ. Após 4 - 6 semanas, as gemas adventícias desenvolvidas foram transferidas para meio de cultura de alongamento MSM + GA3 (1,0 mg/L) + cefotaxima (500 mg/L) + nitrato de prata (4,0 mg/L). As plantas desenvolvidas foram aclimatizadas e analisadas por PCR, utilizando-se primers específicos para a detecção dos transgenes. Foram identificadas 30 plantas transgênicas PCR positivas para do gene nptII, sendo 11 positivas para o fragmento antisenso da proteína capsidial do CABMV e 2 positivas para o fragmento da construção gênica hairpin. Até o momento, a integração dos transgenes foi confirmada por Southern blot em 4 plantas. Paralelo aos experimentos de transformação genética, foram avaliadas plantas de maracujazeiro das variedades IAC-275 e IAC-277, obtidas em experimentos anteriores, com a construção gênica pCABMV-CP, que contém o gene da proteína capsidial do CABMV. A análise de Southern blot de 14 plantas destes experimentos, confirmou a integração do transgene em 13 plantas, as quais foram propagadas e inoculadas mecanicamente com 3 isolados do CABMV (SP, RJ, CE). A linhagem T16 foi resistente as 3 inoculações, com os 3 isolados testados. Clones desta linhagem foram analisados por RT-PCR, comprovando a transcrição do transgene / The yellow passionfruit is affected by Cowpea aphid-borne mosaic virus (CABMV), which causes a decrease in fruit quality and productivity and in some cases making it unpractical for commercial cultivation of this species. Ann alternative for the control of virus diseases is the development of resistant plants through genetic transformation techniques. The objective of this work was to obtain passion fruit (Passiflora edulis f. flavicarpa) transgenic plants with two different gene constructs derived from the CABMV coat protein coding region. pCABMV-asCP contains the gene fragment in an antisense direction and pCABMV-dsCP contains a sense and antisense coat protein gene fragments, separated by intron, a hairpin construct. The genetic transformation experiments with Agrobacterium tumefaciens, were done in hypocotyl segments and young leaf disks explants from varieties FB-100, IAC-275 and IAC-277. After two to three days in co-culture in MS culture medium (MURASHIGE; SKOOG, 1962) supplemented with acetoseringone (100 mM) the explants were subcultured to medium for selection and regeneration, composed of MS minerals and vitamins, supplemented with kanamycin (100 mg/L), cefotaxime (500 mg/L) and silver nitrate (4.0 mg/L), at pH 5.8, and the growth regulators BAP, TDZ and combination of BAP and TDZ. After four to six weeks, the adventitious buds developed were transferred to an elongating medium composed of MSM salts supplemented with GA3 (1.0 mg/L), cefotaxime (500 mg/L) and silver nitrate (4.0 mg/L). Plants were acclimatized and analyzed by PCR, with specific primers for the transgenes detection. Thirty transgenic plants were identified as PCR positive for the nptII gene, with 11 being positive for the antisense fragment of the CABMV coat protein gene and two positive for the hairpin gene construct fragment. Currently, the transgene integration was confirmed by Southern blot analysis in four plants. Simultaneously to the genetic transformation experiments, passionfruit plants, varieties IAC-275 e IAC-277, obtained from previous experiments with pCABMV-CP, which contains CABMV coat protein gene, were analized. The Southern blot analysis of 14 plants from these experiments confirmed the transgene integration in 13 plants, which were propagated and mecanically inoculated with three CABMV isolates (SP, RJ, CE). Line T16 was resistant to three inoculations with all three isolates tested. Clones from this line were analyzed by RT-PCR which confirmed the transgene transcription

Les opines sont des molécules produites dans les cellules végétales transformées par l’ADN-T d’A. tumefaciens. Ces opines peuvent être utilisées comme nutriments par le phytopathogène et certaines d’entre elles agissent comme signaux moléculaires contrôlant la dissémination du plasmide de virulence Ti via la signalisation quorum-sensing. Le présent travail vise à une compréhension élargie du rôle des opines au cours des interactions A. tumefaciens-plantes hôtes. En se basant d’abord sur des analyses transcriptomiques, le régulon AccR d’A. tumefaciens C58, contrôlé par les opines agrocinopines, a été défini : celui-ci inclut des fonctions associées (i) à l’assimilation des agrocinopines, (ii) à l’assimilation de la nopaline, (iii) à la signalisation quorum-sensing et la conjugaison du plasmide Ti, (iv) à la conjugaison du plasmide At. La corrélation entre la co-régulation des conjugaisons des plasmides Ti et At et le co-transfert des deux réplicons a en outre été mise en évidence. Dans un second temps, associant des approches de génétique fonctionnelle à des travaux collaboratifs en biologie structurale, l’avantage sélectif conféré par les opines nopaline et octopine à A. tumefaciens au sein des tumeurs végétales a été quantifié. Les bases moléculaires sous-jacentes à cet avantage sélectif, notamment celles associées à la perception et l’importation des deux opines dans le cytoplasme bactérien, ont été décrites. Enfin, en combinant des approches de métabolomique et de génétique inverse avec des tests de conjugaison in planta, les effets opposés de la signalisation GABA d’une part et des signalisations opine et quorum-sensing d’autre part sur la dissémination du plasmide Ti ont été démontrés. En conclusion, nos résultats révèlent l’intrication des signalisations opine, quorum-sensing et GABA au cours de l’interaction A. tumefaciens-plantes hôtes. Ils soulignent en particulier les impacts de cette intrication sur la colonisation de l’hôte ainsi que sur la dissémination des gènes de virulence et d’adaptation à l’environnement tumeur portés par les plasmides Ti et At. / Opines are produced in plant cells transformed with the A. tumefaciens T-DNA. These opines can be used as nutrients by the phytopathogen and some of them act as signaling molecules controlling Ti plasmid dissemination through quorum-sensing. The present study aims at an enlarged understanding of the roles of opines during A. tumefaciens/plant host interactions. First, based on transcriptomic investigations, the agrocinopine-controled AccR regulon from A. tumefaciens C58 was thoroughly characterized. This one includes functions associated with (i) agrocinopines assimilation, (ii) nopaline assimilation, (iii) quorum-sensing and Ti plasmid conjugation, (iv) At plasmid conjugation. Moreover our analysis showed that co-regulation of Ti and At plasmid conjugations correlated with the co-transfer of the two replicons. In a second step using functional genetic and structural biology we quantified the selective advantage conferred to colonizing A. tumefaciens populations by the assimilation of the opines nopaline and octopine. The molecular basis which underlies this selective advantage, especially regarding the sensing and import of the two opines within the bacterial cytoplasm, were also described. Finally combining metabolomics and reverse genetic with in planta conjugation assays we demonstrated the opposite effects of GABA and opine/quorum-sensing signaling on the dissemination of Ti plasmids. In conclusion our results reveal the interlink between opine, quorum-sensing and GABA signaling during A. tumefaciens/plant host interactions. They noticeably highlight the impact of this interlink on host colonization and the dissemination of Ti and At plasmid genes which are critical for the virulence and the adaption of the bacteria to the tumor lifestyle.