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Doctoral Thesis
DOI
10.11606/T.11.2005.tde-16052005-162932
Document
Author
Full name
Leonardo Sousa Cavalcanti
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2005
Supervisor
Committee
Pascholati, Sergio Florentino (President)
Alves, Eduardo
Duarte, Keila Maria Roncato
Lambais, Marcio Rodrigues
Wulff, Nelson Arno
Title in Portuguese
Papel da celulase XF-0818 na interação Xylella fastidiosa X Citros.
Keywords in Portuguese
bactéria fitopatogênica
clorose variegada dos citros
enzima
escherichia coli
expressão gênica
fruticultura
genoma
imunohistoquímica
xilema
Abstract in Portuguese
A partir do sequenciamento do genoma da bactéria Xylella fastidiosa, agente causal da clorose variegada dos citros (CVC), estudos sobre os mecanismos de patogenicidade expressos durante a doença foram iniciados em projetos de genômica funcional, com o objetivo de confirmar a função das sequências gênicas identificadas no projeto Genoma de X. fastidiosa. Dentre esses mecanismos destacam-se as enzimas envolvidas no processo de ataque da bactéria. O presente trabalho teve como alvo de estudo a seqüência gênica denominada Xf-0818, clonada em Escherichia coli através da inserção em plasmídeo pET 28b(+), que presumivelmente codifica uma celulase de cerca de 60 kDa, possivelmente envolvida na degradação de fibras de celulose presentes nas membranas dos vasos pontuados do xilema de plantas de citros. A degradação desta membrana permitiria a movimentação de células bacterianas entre os vasos do xilema, o que é determinante para o desenvolvimento dos sintomas da CVC. A proteína foi superexpressada em E. coli por meio de indução com lactose e purificada através de ultrafiltração associada à cromatografia de afinidade a metal. A enzima apresentou alta atividade sobre a celulose carboximetilada 1%, avaliada através da quantificação de açúcares redutores. A obtenção de anticorpos foi realizada mediante injeção da enzima purificada em camundongos, os quais foram avaliados através de ELISA. A atividade da enzima sobre celulose carboximetilada foi reduzida após prévia incubação com os anticorpos. Esses anticorpos representam uma poderosa ferramenta em pesquisas visando à identificação das condições que favoreçam a expressão desta enzima "in vivo". A presença de partículas de ouro coloidal em seções de tecido provenientes de plantas infectadas com X. fastidiosa indica a atuação da celulase durante a infecção, porém fazse necessária a confirmação do papel da proteína Xf-0818, através de novas análises com imunolocalização, além de estudos utilizando linhagens mutantes de X. fastidiosa para o gene que codifica esta celulase.
Title in English
Role of the Xf-0818 cellulase in the Xylella fastidiosa X citrus interaction.
Keywords in English
citrus variegated chlorosis
emmunohistochemistry
enzyme
escherichiacoli
fruticulture
genetic expression
genome
phytopathoghic bacteria
xylen
Abstract in English
From the genome sequencing of the bacteria Xylella fastidiosa, the agent responsible for the citrus variegated chlorosis (CVC), studies on the pathogenicity mechanisms expressed during the occurrence of the disease were started in genomic function projects, aiming to confirm the function of the genic sequences identified in the project X. fastidiosa Genome. Among these mechanisms, the enzymes involved in the bacterium attaching process are highlighted. The present study had, as a goal, the orf denominated Xf-0818, cloned in Escherichia coli by means of the insertion into pET 28b(+) plasmid, which codifies a cellulase of a size nearly 60 kDa, possibly involved in the degradation of cellulose fibers present in the membranes of the xylem vessels of citrus plants. The degradation of this membrane would allow for the movement of bacterial cells among the xylem vessels, which would be important for the development of CVC symptoms. The enzyme was over expressed into E. coli by means of their induction using lactose and purified using ultra-filtration associated to metal affinity chromatography. The enzyme presented high activity over 1%-carboximethyl cellulose (CMC), which was evaluated through quantification of released reduced sugars. The antibodies were obtained by injection of the purified enzyme into mice, which were evaluated by using the ELISA. The enzyme activity over CMC was reduced after previous incubation with the antibodies. These antibodies represent a tool for the researches aiming the identification of conditions that favor the expression of this enzyme in vivo. The presence of gold particles on the citrus diseased tissue samples indicate the participation of this cellulase on the disease, but other studies using immunocytochemistry and mutants of X. fastidiosa are necessary to confirm this hypothesis.
 
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Publishing Date
2005-08-08
 
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