SILVA, Maria Zelandia Rocha. Isolamento em cromatografia de imunoafinidade e atividade antifúngica de osmotinas de fluidos laticíferos. 2015. 85 f. Dissertação (Mestrado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2015. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-09-01T19:36:22Z No. of bitstreams: 1 2015_dis_mzrsilva.pdf: 2515574 bytes, checksum: 4dcf1607c0ce726fde9bddf9f92b2d80 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-09-02T20:25:43Z (GMT) No. of bitstreams: 1 2015_dis_mzrsilva.pdf: 2515574 bytes, checksum: 4dcf1607c0ce726fde9bddf9f92b2d80 (MD5) / Made available in DSpace on 2016-09-02T20:25:44Z (GMT). No. of bitstreams: 1 2015_dis_mzrsilva.pdf: 2515574 bytes, checksum: 4dcf1607c0ce726fde9bddf9f92b2d80 (MD5) Previous issue date: 2015 / Plants are constantly exposed to a variety of stresses conditions. However, they react to biotic stresses by triggering a set of defense mechanisms including the synthesis of defensive substances as the pathogenesis-related (PR) proteins. The PR-protein named osmotin can be induced under osmotic stress and water shortage conditions. Osmotin-like proteins have been purified from latex and some of them are related to antifungal activity. The aim of this study was to investigate the osmotin in the following species laticifers: C. grandiflora, P. rubra, T. peruviana, H. drasticus and C. papaya to isolate and evaluate its antifungal activities. Immunoaffinity column chromatography with anti-CpOsm antibodies were performed in order to purify these osmotin-like. They were detected in latex of C. grandiflora and P. rubra by immunoassays the ELISA, Dot Blot and Western Blot using anti-CpOsm antibody (the osmotin of C. procera latex). Osmotin of C. procera, C. grandiflora, P. rubra and H. drasticus were identified by mass spectrometry. However, the osmotin from C. procera was co-purified with cysteine proteases. The co-purified cysteine protease from C. procera was identified as Procerain B. The alignment and the 3-D structure analysis of Procerain B and CpOsm revealed the presence of a similar sequence in both proteins. This sequence might be an epitope which allows the anti-antibody recognition. The osmotin from C. grandiflora, and P. rubra did not show antifungal activity against Fusarium solani and Colletotrichum gloesporioides. Since no correlation between the antifungal activity and the presence of these osmotins were found, the proteolytic activities of these latex protein fractions were evaluated in order to correlate with the antifungal activity C. procera and C. grandiflora showed a strong proteolytic activity. In latex, the cysteine proteases are more often related to antifungal activity than osmotin, which might explain, at least in part, the antifungal activity performed by C. grandifora and not for its osmotin. Further studies on the role of osmotin in physiology laticifers plants are needed. / As plantas estão constantemente sujeitas a diversos tipos de estresse, tanto bióticos como abióticos, resultando em respostas de defesa. Decorrente disto, os vegetais sintetizam certas proteínas denominadas de proteínas relacionadas à patogênese (PR proteína). As Pr-proteínas chamadas de osmotinas podem ser induzidas sob condições de estresse osmótico, frio e escassez de água. Osmotinas tem sido purificadas de fluidos laticíferos e algumas delas estão relacionadas com a atividade antifúngica. O objetivo do presente trabalho foi prospectar osmotinas, bem como isolá- las e avaliar suas atividades antifúngicas, nos fluidos laticíferos das seguintes espécies: C. grandiflora, P. rubra, T. peruviana, H. drasticus e C. papaya. Nos látex de C. grandiflora e P. rubra foram detectadas osmotinas através de imunoensaios em placa de ELISA, Dot Blot e Westen Blot, utilizando os anticorpos anti-CpOsm (osmotina do látex de C. procera). Cromatografia de imunoafinidade em coluna com anticorpos anti-CpOsm foram realizadas com o intuito de purificar estas osmotinas. Análises por meio de espectrometria de massas, revelaram a presença de osmotina em C. procera, C. grandiflora, P. rubra e H. drasticus. No entanto, a osmotina de C. procera foram co-purificadas com proteases cisteínicas. A protease cisteínica co- purificada no látex de C. procera foi identificada como Proceraina B. O alinhamento e a análise da estrutura tridimensional da Proceraina B e CpOsm revelaram a presença de uma sequência semelhante em ambas as proteínas, que pode ser um epítopo disponível ao reconhecimento do anticorpo anti-CpOsm. As osmotinas isoladas de C. grandiflora e P. rubra não apresentaram atividade antifúngica contra F. solani e C. gloesporioides. Desde que não houve correlação entre a atividade antifúngica e à presença destas osmotinas, as atividades proteolíticas das frações proteicas foram avaliadas a fim de correlaciona-las à atividade antifúngica. Nos fluidos laticíferos, as proteases cisteínicas estão mais frequentemente relacionadas à atividade antifúngica do que as osmotinas. Estudos mais aprofundados sobre a função das osmotinas na fisiologia de plantas laticíferas são necessários.

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CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Plants are constantly exposed to a variety of stresses conditions. However, they react to biotic stresses by triggering a set of defense mechanisms including the synthesis of defensive substances as the pathogenesis-related (PR) proteins. The PR-protein named osmotin can be induced under osmotic stress and water shortage conditions. Osmotin-like proteins have been purified from latex and some of them are related to antifungal activity. The aim of this study was to investigate the osmotin in the following species laticifers: C. grandiflora, P. rubra, T. peruviana, H. drasticus and C. papaya to isolate and evaluate its antifungal activities. Immunoaffinity column chromatography with anti-CpOsm antibodies were performed in order to purify these osmotin-like. They were detected in latex of C. grandiflora and P. rubra by immunoassays the ELISA, Dot Blot and Western Blot using anti-CpOsm antibody (the osmotin of C. procera latex). Osmotin of C. procera, C. grandiflora, P. rubra and H. drasticus were identified by mass spectrometry. However, the osmotin from C. procera was co-purified with cysteine proteases. The co-purified cysteine protease from C. procera was identified as Procerain B. The alignment and the 3-D structure analysis of Procerain B and CpOsm revealed the presence of a similar sequence in both proteins. This sequence might be an epitope which allows the anti-antibody recognition. The osmotin from C. grandiflora, and P. rubra did not show antifungal activity against Fusarium solani and Colletotrichum gloesporioides. Since no correlation between the antifungal activity and the presence of these osmotins were found, the proteolytic activities of these latex protein fractions were evaluated in order to correlate with the antifungal activity C. procera and C. grandiflora showed a strong proteolytic activity. In latex, the cysteine proteases are more often related to antifungal activity than osmotin, which might explain, at least in part, the antifungal activity performed by C. grandifora and not for its osmotin. Further studies on the role of osmotin in physiology laticifers plants are needed. / As plantas estÃo constantemente sujeitas a diversos tipos de estresse, tanto biÃticos como abiÃticos, resultando em respostas de defesa. Decorrente disto, os vegetais sintetizam certas proteÃnas denominadas de proteÃnas relacionadas à patogÃnese (PR proteÃna). As Pr-proteÃnas chamadas de osmotinas podem ser induzidas sob condiÃÃes de estresse osmÃtico, frio e escassez de Ãgua. Osmotinas tem sido purificadas de fluidos laticÃferos e algumas delas estÃo relacionadas com a atividade antifÃngica. O objetivo do presente trabalho foi prospectar osmotinas, bem como isolÃ- las e avaliar suas atividades antifÃngicas, nos fluidos laticÃferos das seguintes espÃcies: C. grandiflora, P. rubra, T. peruviana, H. drasticus e C. papaya. Nos lÃtex de C. grandiflora e P. rubra foram detectadas osmotinas atravÃs de imunoensaios em placa de ELISA, Dot Blot e Westen Blot, utilizando os anticorpos anti-CpOsm (osmotina do lÃtex de C. procera). Cromatografia de imunoafinidade em coluna com anticorpos anti-CpOsm foram realizadas com o intuito de purificar estas osmotinas. AnÃlises por meio de espectrometria de massas, revelaram a presenÃa de osmotina em C. procera, C. grandiflora, P. rubra e H. drasticus. No entanto, a osmotina de C. procera foram co-purificadas com proteases cisteÃnicas. A protease cisteÃnica co- purificada no lÃtex de C. procera foi identificada como Proceraina B. O alinhamento e a anÃlise da estrutura tridimensional da Proceraina B e CpOsm revelaram a presenÃa de uma sequÃncia semelhante em ambas as proteÃnas, que pode ser um epÃtopo disponÃvel ao reconhecimento do anticorpo anti-CpOsm. As osmotinas isoladas de C. grandiflora e P. rubra nÃo apresentaram atividade antifÃngica contra F. solani e C. gloesporioides. Desde que nÃo houve correlaÃÃo entre a atividade antifÃngica e à presenÃa destas osmotinas, as atividades proteolÃticas das fraÃÃes proteicas foram avaliadas a fim de correlaciona-las à atividade antifÃngica. Nos fluidos laticÃferos, as proteases cisteÃnicas estÃo mais frequentemente relacionadas à atividade antifÃngica do que as osmotinas. Estudos mais aprofundados sobre a funÃÃo das osmotinas na fisiologia de plantas laticÃferas sÃo necessÃrios.

Melampsora larici-Populina est un champignon biotrophe qui infecte le peuplier et cause la maladie de la rouille foliaire, entraînant d'importants dégâts dans les peupleraies. Un des objectifs de l'UMR Interactions Arbres/Microorganismes est de caractériser les déterminants moléculaires de ce pathosystème. Au cours de cette thèse, des approches post-Génomiques ont permis de mener à bien quatre projets de recherche. Premièrement, l'analyse du transcriptome des temps précoces de l'interaction peuplier/M. larici-Populina a révélé un transporteur de sulfate de peuplier fortement induit par l'infection (chapitre II). Deuxièmement, l'analyse phylogénomique de la famille des thaumatin-Like proteins (TLP) a entre autres mis en évidence certains clades spécifiquement associés aux réponses aux stress chez le peuplier (chapitre III). Troisièmement, le gène codant la petite protéine sécrétée Risp de fonction inconnue est fortement induit lors des réponses de défense du peuplier et n'a pas d'homologue chez les autres plantes. La protéine recombinante est intrinsèquement désordonnée et présente une double activité de protéine antifongique envers M. larici-Populina et d'éliciteur endogène des réponses de défense chez le peuplier (chapitre IV et V). La combinaison de ces deux propriétés n'a jamais été rapportée chez une protéine de plante. Enfin, les gènes MlpP4.1 et MlpH1.1 de M. larici-Populina codent des petites protéines sécrétées riches en cystéines et de fonction inconnue, considérées comme des effecteurs candidats (chapitre VI). L'expression de MlpP4.1 et MlpH1.1 est très fortement induite lors de l'infection des feuilles de peupliers et des activités de virulence ont été observées chez Arabidopsis thaliana. Les analyses biochimique et structurale des protéines recombinantes sont en cours et ont déjà permis de démontrer la forte stabilité de MlpP4.1, probablement liée à la présence de plusieurs ponts disulfures. A l'aide des protéines recombinantes, plusieurs partenaires protéiques ont été identifiés chez les plantes permettant d'établir des hypothèses quant à leur rôle / Melampsora larici-Populina is a biotrophic fungus that infects poplar and causes the foliar rust disease, leading to severe damages in plantations. A major aim of the Tree- Microbe Interactions department is to characterize molecular determinants of the pathosystem. During this thesis, four research projects were achieved through post-Genomic approaches. First, transcriptome analysis of the early interaction between poplar and M. larici-Populina revealed a fungal-Induced host sulfate transporter (chapter II). Secondly, the phylogenomic analysis of the thaumatin-Like protein (TLP) family uncovered some clades specifically associated with stress responses in poplar (chapterIII). Thirdly, the gene encoding the small secreted protein of unknown function Risp is strongly induced during poplar defense reponses and has no homolog in other plants. The recombinant protein is intrinsically disordered and presents a dual activity as an antifungal protein against M. larici-Populina and as an endogenous elicitor of defense responses in poplar (chapter IV and V). The combination of both properties in a single protein has never been reported in plants. Finally, M. larici-Populina MlpP4.1 and MlpH1.1 genes encode cysteine-Rich small-Secreted proteins of unknown fonction, considered as candidate effectors (chapter VI). MlpP4.1 and MlpH1.1 expression is strongly induced during poplar leaf colonization, and virulence activities were observed in Arabidopsis thaliana. Biochemical and structural analyses of recombinant proteins are ongoing and already revealed the strong stability of MlpP4.1, likely due to the presence of several disulfide bridges. Several plant partners of the recombinant proteins were identified and have allowed for setting hypotheses about their role

When tomato Craigella is infected with Verticillium dahliae Dvd-E6 (Dvd-E6), a tolerant state is induced with substantial pathogen load, but few symptoms. Unexpectedly, these plants are more robust and taller with Dvd-E6 behaving as an endophyte. Some endophytes can protect plants from virulent pathogens. This research was undertaken to improve understanding of the cellular and molecular nature of Verticillium tolerance in tomato, especially whether infection by Dvd-E6 can protect Craigella from virulent V. dahliae, race 1 (Vd1). To permit mixed infection experiments a restriction fragment length polymorphism (RFLP)-based assay was developed and used for differentiating Dvd-E6 from Vd1, when present in mixed infections. The results suggested that protection involves molecular interplay between Dvd-E6 and Vd1 in susceptible Craigella (CS) tomatoes, resulting in restricted Vd1 colonization. Further studies showed a dramatic reduction of Vd1 spores and mycelia. To examine genetic changes that account for these biological changes, a customized DNA chip (TVR) was used to analyze defense gene mRNA levels. The defense gene response was categorized into four groups. Group 1 was characterized by strong induction of defense genes followed by suppression. However, Vd1-induced gene suppression was blocked by Dvd-E6 in mixed infections. These genes included some transcription factors and PR proteins such as class IV chitinases and beta glucanases which are known to target fungal spores and mycelia. Experiments also were repeated with a Craigella resistant (CR) isoline containing a fully active Ve locus (Ve1+ and Ve2+). The biological results showed that the presence of the Ve1+ allele resulted in restricted Vd1 colonization and, in a mixed infection with Dvd-E6, Vd1 was completely eliminated from the plant stem. Surprisingly, there was no significant increase in defense gene mRNAs. Rather, elevated basal levels of defense gene products appeared sufficient to combat pathogen attack. To investigate functional effects of the genetic changes observed, an inducible RNAi knockdown vector for a defense gene (TUS15G8) with unknown function (pMW4-TUS15G8) as well as the Ve2 resistance gene (pMW-Ve2) was prepared as a initial step for future transformation analyses. Taken together the results reveal intriguing but complex biological and molecular changes in mixed infections, which remain a basis for future experiments and potential agricultural benefits. / Canadian Commonwealth Scholarship and Fellowship Plan