Resumo: Formigas cortadeiras do gênero Atta, popularmente conhecidas como saúvas, são as mais derivadas dentro da tribo Attini. Apresentam grande importância ecológica, porém, pelo hábito de cortarem folhas para manutenção do fungo simbionte e pelo enorme tamanho das colônias, causam muitos prejuízos às lavouras, pastagens e plantações, sendo consideradas pragas agrícolas. Atta laevigata Smith, 1858 apresenta vasta distribuição pelo Brasil e é responsável pela herbivoria de inúmeras plantas dicotiledôneas, gramíneas e espécies nativas de diferentes biomas. O presente trabalho teve como objetivos a caracterização parcial do transcriptoma e do genoma mitocondrial de A. laevigata. Foram caracterizadas 2006 sequências únicas do transcriptoma, a partir de uma biblioteca de cDNA preparada com indivíduos inteiros da formiga. Entre essas sequências, 16 provavelmente representam genes com grande número de transcritos. Esses 16 genes estão relacionados a três funções celulares: (i) conservação de energia através de reações redox na mitocôndria; (ii) estrutural, pelo citoesqueleto e músculos; (iii) regulação da expressão gênica e metabolismo. Considerando o estilo de vida e processos biológicos chaves para essas formigas, 146 sequências foram identificadas com base na sua utilização para o controle de cortadeiras pragas. A partir de dados da biblioteca de cDNA e procedimentos envolvendo primer walking, o genoma mitocondrial de A. laevigata foi parcialmente caracterizado, apresentandose com 17920 pb, maior, portanto, do que outros já descritos em Hymenoptera, mesmo considerando-se a impossibilidade de determinação da sequência de uma pequena porção do mtDNA, envolvendo a região controle, uma parte do 12S e os tRNAs S1, V e M. Como já descrito para outros mitogenomas, o de A. laevigata apresentou alto conteúdo AT, os mesmos 13 genes codificadores... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leafcutter ants from Atta genus, popularly known as "saúvas", are the most derived of the tribe Attini. They have major ecological importance, but, because of their habit of cutting leaves for the maintenance of the symbiotic fungus and the huge colony size, they impose severe economic damages to plantations, pastures, and agriculture, being considered as agriculture pests. Atta laevigata shows wide distribution in Brazil and it is responsible for the herbivory of many dicots, grass, and native species from different biomes. The present work aimed to characterize the transcriptome and the mitochondrial genome of A. laevigata. 2,006 unique sequences of the transcriptome were characterized from a cDNA library constructed with whole individuals. Among those sequences, 16 are likely from genes with high number of transcripts. Those 16 genes are related with three cellular functions: (i) energy conservation through redox reactions in mitochondria; (ii) cytoskeleton and muscle structuring; (iii) regulation of gene expression and metabolism. Based on lifestyle and key biological processes of these ants, 146 sequences were identified with potential use for controlling pest leafcutters. Using data from cDNA library and primer walking proceedings, the mitochondrial genome of A. laevigata was partially characterized with 17,920 bp, being larger than the others already described for Hymenoptera. A small part of the mtDNA was not sequenced, including the control region, a portion of 12S and tRNAs S1, V, and M. As described before for other mitogenomes, A. laevigata mtDNA displayed high AT contain, the same 13 proteincoding genes and the two ribosomal subunits with length and location according to the hypothetic ancestral mitogenome. Rearrangements were found for the tRNAs, but the most remarkable difference were the high number and longer length of intergenic regions presented in the mtDNA... (Complete abstract click electronic access below) / Orientador: Maurício Bacci Júnior / Coorientador: Henrique Ferreira / Banca: Flavio Henrique da Silva / Banca: Marco Antonio del Lama / Banca: Mariana Lúcio Lyra / Banca: Klaus Hartmann Hartfelder / Doutor

A produção brasileira de laranjas destina-se à indústria de suco concentrado, principal produto citrícola, sendo o estado de São Paulo o maior produtor e exportador do país. Devido à diminuição da variabilidade genética causada pela multiplicação de plantas cítricas por enxertia, esta cultura se tornou alvo constante de inúmeras pragas e doenças. Dentre estas doenças que vem causando crescentes prejuízos para a citricultura brasileira destaca-se a pinta preta causada pelo fungo Guignardia citricarpa Kiely. O combate desta doença é feito por inúmeras pulverizações de fungicidas que aumenta significativamente o custo para os produtores. Uma possível alternativa para o combate a este patógeno seria a produção de plantas cítricas resistentes a doença, através de transformação gênica. Mas para isto, é necessário conhecer os mecanismos de respostas de defesa da planta e identificar quais genes podem estar relacionados com a defesa. Na tentativa de elucidar as respostas de defesa deste hospedeiro foram feitas análises anatômicas de frutos de laranja doce (Citrus sinensis Osbeck cv. Pêra) inoculados com o patógeno e análise transcricional de duas bibliotecas de cDNA, uma de albedo de frutos sadios(BAFS) e outra de albedo de frutos inoculados com o patógeno (BAFC). As análises anatômicas mostraram que os danos causados pelo patógeno na planta iniciam-se 24 horas após a inoculação com as lesões das células do epicarpo, e continuam gradativamente até 72 horas apresentando a diminuição na quantidade de amido e acúmulo de compostos fenólicos. Foi obtido 184 ESTs válidas da BAFS e 370 da BAF. A anotação e categorização destas sequencias apresentaram que o perfil transcricional encontrado nas duas bibliotecas foi semelhante, porém, na BAFC, foram encontrados transcritos pertencentes à defesa da planta como a catalase, a glutationa e monooxygenases. Está analise mostrou que a resposta de defesa da planta inicia-se com lesões nas células, que a interação é custo-intensiva (redução da reserva de amido) para o hospedeiro, e que a defesa da planta está relacionada a muitos genes de defesa. / The Brazilian production of oranges is mainly for the industry of concentrated juice, it is the most important citrus product, and the state of São Paulo is the largest producer and exporter of the country. Due to the low genetic variability caused by the multiplication of citrus plants by grafting, this culture became constant target of many pests and diseases. Among all the diseases that lead to elevated damage to the Brazilian citrus plantation, one is very relevant, the Black Spot disease caused by the fungus Guignardia citricarpa Kiely. The control of this disease is done by many sprays of fungicides that significantly increase the production cost to the producers. A possible alternative to control this pathogen is the development of resistant citrus plants to disease through genetic transformation. To that, it is necessary to understand the mechanisms of plant defense response and identify the genes related to the defense. In an attempt to elucidate the responses of this host both,anatomical analysis of sweet orange fruits (Citrus sinensis Osbeck cv. Pêra) inoculated with the pathogen and transcriptional analysis of two cDNA libraries of albedo section were done. One was from health fruits (BAFS) and another from albedo of fruits inoculated with the pathogen (BAFC). The analysis showed that the anatomical damage caused by the pathogen in the plant shall begin 24 hours after inoculation showing lesions on epicarp cells, and continue gradually until 72 hours also showing a decrease in the amount of starch and accumulation of phenolic compounds. It was obtained 184 valid ESTs of the BAFS and 370 from BAFC. The annotation and categorization of these sequences showed that the transcriptional profile in the two libraries were similar, however, in BAFC, were found transcripts belonging to plant defense such as catalase, the glutathione and monooxygenases. These analyses showed that the plant defense response begins with lesions in the cells, being it cost-intensive interaction for the host, (with reduction of starch reserves) and the plant defense to resistance is related to many genes.

No description available.

This study represents the first comprehensive analysis of gene regulation underlying the distinct categories of resistance afforded to wheat (Triticum aestivum, L.) by different Dn genes. Russian wheat aphid (Diuraphis noxia, Mordv.) feeding on susceptible wheat cultivars causes leaf rolling, chlorosis and the eventual death of the plant. Plants expressing Dn genes are resistant to D. noxia infestation, but different Dn genes afford phenotypically distinct modes of resistance: the Dn1 gene confers an antibiotic effect to lower aphid fecundity; Dn2 confers tolerance to high aphid pressure; and Dn5 confers antixenosis, and aphids do not prefer such plants as hosts. Little is known about the components involved in establishing a successful defence response against D. noxia attack and how these differ between the distinct resistance categories. It is assumed that the Dn genes function as classic R genes in plant defence, being receptors for elicitors in aphid saliva. Upon recognition, defence response signalling is initiated, but the exact mechanics of subsequent cellular events in aphid resistance have only recently come under investigation. Evidence from cDNA microarray and subtractive hybridization experiments indicated the involvement of kinase signalling cascades and photosynthetic proteins in the response against D. noxia. However, expression analysis describing how these processes differ between plants carrying different Dn genes and how these differences account for antibiosis, antixenosis or tolerance had not been conducted. We consequently investigated the downstream components involved in or affected by the generation of these resistance mechanisms by comparing the responses in transcript regulation of Tugela near-isogenic lines with different Dn genes to D. noxia infestation. cDNA-AFLP analysis was selected as an appropriate functional genomics tool, since it is semi-quantitative, does not require prior sequence information and allows for the discovery of novel genes. cDNA-AFLP analysis yielded 121 differentially regulated transcript-derived fragments (TDFs) grouped into eight expression clusters. We cloned and sequenced 49 representative TDFs, which were further classified into five broad functional categories based on inferred similarity to database sequences. Transcripts involved in such diverse processes as stress, signal transduction, photosynthesis, metabolism and gene regulation were found to be differentially regulated during D. noxia feeding. Many TDFs demonstrated homology to proteins with unknown function and several novel transcripts with no similarity to previously published sequences were also discovered. Detailed expression analysis using quantitative RT-PCR and RNA hybridization provided evidence that the time and intensity of induction of specific pathways is critical for the development of a particular mode of resistance. This includes: the generation of kinase signalling cascades and the induction of several ancillary processes such as ubiquitination, leading to a sustained oxidative burst and the hypersensitive response during antibiosis; tolerance as a passive resistance mechanism countering aphid-induced symptoms through the repair or de novo synthesis of photosystem proteins; and the possible involvement of ethylene-mediated wounding pathways in generating volatile organic compounds during antixenosis. This is the first report on the involvement of KCO1, a vacuolar K+ channel, in assisting cytosolic Ca2+-influx and preventing leaf rolling, as well as on the role of iron homeostasis as a gene regulatory mechanism for sustaining the oxidative burst during the antibiotic defence response. This study opens up several areas of investigation heretofore unexplored in cereal-aphid interaction research. Of particular interest is the induction of genes involved in photosynthetic compensation during Dn2 tolerance responses, since these constitute a novel, passive resistance mechanism exclusive to aphid defence as opposed to the active resistance triggered in the presence of the Dn1 gene in the form of a general hypersensitive response. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

Muchos productos hortofrutícolas desarrollan 'daños de frío' durante la conservación a temperaturas inferiores a 12-15 ºC. El tratamiento de 'curado' (3 días 37 ºC) previene la aparición de esta alteración en los frutos cítricos cuando se conservan a 2 ºC. En la mandarina 'Fortune', muy susceptible al frío, los daños se manifiestan como un picado y áreas de color pardo en la parte más externa de la corteza (flavedo). Otra de las alteraciones frecuentes en la postcosecha de los frutos cítricos es el 'colapso de la corteza', que se produce a temperaturas superiores a las que causan 'daños de frío' y cuyos síntomas se caracterizan por la aparición de depresiones en la piel. El acondicionamiento de frutos de 'Navelate' durante 4 días con 10 ?L L-1 de etileno a 22 ºC y 90-95% de humedad relativa (HR) redujo notablemente la incidencia de esta alteración, mientras que la aplicación de 1 ?L L-1 de 1-metilciclopropeno (1-MCP), un inhibidor de la percepción de etileno, la potenció. Para entender los mecanismos asociados al efecto beneficioso del curado reduciendo el 'daño de frío' y del etileno frente al 'colapso de la corteza', se han evaluado cambios globales en la expresión génica en el flavedo de frutos de mandarina 'Fortune' almacenados a 2 ºC, directamente o después del curado, y en el flavedo y albedo de frutos de naranja 'Navelate' almacenados a 22 ºC y 90-95% de HR después de ser tratados con etileno o 1-MCP. Para ello se han empleado dos micromatrices de cDNA generadas en el Consorcio de Genómica Funcional de Cítricos (CFGP). La eficacia del curado reduciendo la incidencia de 'daños de frío' parece estar más relacionada con su efecto evitando la inducción de genes implicados en la degradación de lípidos durante el almacenamiento en frío, que con cambios en la expresión de genes del metabolismo de ácidos grasos que afectan al grado de insaturación de los mismos o a la síntesis de ceras. Además, la expresión de genes del metabolismo de fenilpropanoides, y de otros que / Establés Ortiz, BA. (2008). Estudio transcriptómico de los mecanismos implicados en la tolerancia inducida por el curado al daño de frío y por el etileno al colapso de la corteza en los frutos cítricos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/3782

THAP5 is a protein which was recently isolated in the Zervos Lab as an interactor of a pro-apoptotic protein, Omi/HtrA2. THAP5 is unique because it shares no homology with mouse or rat and can only be found in humans. The only homology it shares with any other protein is its THAP domain. THAP proteins are zinc-dependent sequence specific DNA-binding factors belonging to the zinc-finger family of proteins (2). There are 12 identified members of TIIAP proteins in humans, THAP0-THAP 11. The roles of these THAP proteins include proliferation, apoptosis, cell cycle, chromosome segregation, chromosome modification, and transcriptional regulation (2). The function of THAP5 is still unclear and thus, a Yeast Two-Hybrid experiment will be done to further determine its function. The Yeast Two-Hybrid System is a common molecular biology technique used to identify interactors of a certain protein of interest. By identifying the protein interactors of THAP5 and their functions, it is possible to further determine the function of THAP5.

La famille des Flexiviridae a été créée en 2004 et regroupe plusieurs genres viraux affectant particulièrement des espèces ligneuses dont des arbres fruitiers. Grâce à diverses approches plusieurs nouveaux Flexiviridae ont été partiellement caractérisés au cours de ces dernières années. En revanche la position taxonomique précise de certains d’entre eux et leur contribution à des pathologies particulières restent encore incertaines du fait de difficultés inhérentes à l’étude de ces agents. Dans le présent travail, nous avons obtenu les séquences génomiques complètes pour quatre agents proches de l’Apricot latent virus. Ceci a permis de préciser l’organisation génomique de ces virus et d’en déterminer la position taxonomique. Cette étude a également permis de montrer que la partie C-terminale de la capside et la protéine TGBp1 sont soumises à une pression sélective particulièrement forte. Dans un second volet de ce travail, plusieurs approches permettant l’obtention simple et rapide d’ADNc infectieux, sous forme clonée ou non ont été développées. Travaillant sur plusieurs Flexiviridae, dont le virus des taches foliaires chlorotiques du pommier (Apple chlorotic leaf spot virus, ACLSV), nous avons mis au point l’amplification d’ADNc génomiques complets en une seule étape à partir d’extraits d’acides nucléiques totaux obtenus à partir de plantes infectées. Des amplifiats comportant l’ADNc viral sous le contrôle du promoteur 35S du CaMV ou du promoteur de la RNA polymérase du phage T7 ont été obtenus et utilisés pour infecter des plantes directement par biolistique (promoteur 35S) ou pour obtenir des ARN infectieux par transcription in vitro (promoteur T7). Ces données ont mis en évidence des différences importantes dans le comportement de deux hôtes de l’ACLSV, Chenopodium quinoa et Nicotiana occidentalis 37B. Nous avons également utilisé le système de recombinaison homologue de la levure Saccharomyces cerevisiae simplifier le clonage d’ADNc complets amplifiés par PCR ou pour réaliser en une seule étape la construction d’un vecteur navette ternaire levure-E. coli-A. tumefaciens et l’obtention d’un clone ADNc de l’ACLSV inoculable par agroinfiltration. Ces différentes stratégies devraient trouver une large application, en particulier pour tester plus rapidement des hypothèses d’étiologie pour les virus de plantes réputés "difficiles", tels que ceux infectant des hôtes ligneux. / The Flexiviridae family was created in 2004 and contains several viral genera affecting in particular woody hosts, including fruit trees. Using various strategies several new Flexiviridae have been partially characterized in the past few years. However, due to difficulties inherent in studying these agents, the precise taxonomic position of some of them and their contribution to particular diseases are still uncertain. In the present work, the complete genomic sequences of four Prunus-infecting Apricot latent virus (ApLV) like isolates have been determined. This has allowed to determine the genomic organization and the taxonomic position of these viruses. The results obtained also indicate that the C-terminal half of the coat protein and the TGBp1 are the genomic regions under the strongest purifying selection pressure. In the second part of this work, a set of approaches to simplify and streamline the construction of cloned or uncloned infectious full-length viral cDNAs were developed. working with several Flexiviridae and, in particular, with the Apple chlorotic leaf spot virus (ACLSV), we have developed protocols allowing the one-step amplification from total nucleic acids extracts of full-length cDNAs. under the control of the CaMV 35S or phage T7 RNA polymerase promoters. Successful inoculation of plants with these uncloned amplification products was obtained by biolistic bombardment (35S promoter) or using in vitro synthesized RNA transcripts (T7 promoter). Results obtained showed significant differences in the behavior of the two ACLSV hosts, Chenopodium quinoa and Nicotiana occidentalis 37B. We also used the yeast homologous recombination system for the efficient cloning of full-length cDNAs and for the simultaneous one-step construction of a ternary yeast-E. coli-Agrobacterium tumefaciens shuttle vector and generation of an agroinfiltrable infectious ACLSV construct. These various strategies should find broad applications, in particular for the validation of etiological hypotheses in the case of “difficult” plant viruses, such as those infecting woody hosts.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications. The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line. Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency

La conductivité hydraulique foliaire (KF) est une composante majeure du transport d'eau dans toute la plante. Dans les feuilles de noyer, la KF est stimulée à la lumière et est étroitement liée à l'accroissement du taux des transcrits d'aquaporines JrPIP2s. Par ailleurs, la corrélation entre la stimulation de la KF et des transcrits d'aquaporines à la lumière, n'est pas générale et dépend de l'espèce. Ici, nous étudions cette corrélation chez cinq espèces forestières (Juglans regia, Fagus sylvitica, Quercus robur, Salix alba et Populus tremula) différant par leur réponse à la lumière. Nous démontrons seulement chez le noyer (Juglans regia), la contribution des deux familles d'aquaporines PIP1s et PIP2s. Afin de mieux comprendre le rôle des JrPIP1s et JrPIP2 dans la réponse à la lumière, nous avons isolé 8 nouvelles isoformes dans les feuilles de noyer et nous avons étudié leurs profils d'expression sur une cinétique lumière. Toutes les isoformes étudiées sont accumulées à la lumière et réprimées à l'obscurité. De plus, la KF est dépendante de la qualité de lumière. Elle est réduite de 65% en absence de lumière bleue. Cette diminution serait liée à l'inhibition des transcrits d'aquaporines. Afin de caractériser les mécanismes moléculaires précoces impliqués dans la modulation de KF par la lumière, l'approche globale cDNA-AFLP a été menée sur des feuilles de noyer sous différentes conditions d'éclairement. Nous obtenons 12000 transcrits différentiels dérivés (TDFs) générés par les 128 couples d'amorces. Parmi les 187 séquences obtenues, 93 d'entre elles ont une fonction putative. Leur classification fonctionnelle montre que les gènes relatifs à la régulation cellulaire représentent environ 58% des TDFs identifiés. Les feuilles exposées à la lumière, montrent des changements dans les voies de : signalisation calcique, protéolyse, trafic vésiculaire et l'expression de divers facteurs de transcription et protéines de régulation. Pour mieux comprendre le rôle potentiel de la signalisation calcique dans la modulation de la KF par la lumière, nous avons étudié l'effet d'un inhibiteur des canaux calciques [LaCl3] et d'un antagoniste de calmoduline [W7] sur la KF et les transcrits des 10 JrPIPs. Comparées aux feuilles témoins, les inhibiteurs calciques provoquent une réduction de la KF et de la majorité des JrPIPs étudiées à la lumière. Nos résultats confirment l'implication du complexe Ca2+ /calmoduline dans la transduction du signal lumineux responsable de la stimulation de la KF et des transcrits d'aquaporines chez le noyer.