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Biochemical and functional characterization of Xylella fastidiosa XadA3 adhesin

Grant number: 11/15217-0
Support type:Scholarships in Brazil - Master
Effective date (Start): March 01, 2012
Effective date (End): July 31, 2013
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Aline Maria da Silva
Grantee:Ana Paula Silva de Souza
Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The plant pathogen Xylella fastidiosa is the causal agent for severe diseases in citrus, grapevines and almond trees, among other plants and presents on its cell surface, several fimbrial and afimbrial adhesins potentially responsible for efficient colonization of its plants and insect hosts. The functionality for some of these adhesins has been demonstrated, as mutants deficient in these proteins show reduced ability to adhere. Among the adhesins encoded in the genome of X. fastidiosa (strain 9a5c, isolated from citrus or Temecula, isolated from grapevines) are three trimeric autotransport adhesins (XadA1, Hsf/XadA2 and Hsf/XadA3). It has been demonstrated that XadA1 and XadA2 transcripts levels are increased during biofilm development. Moreover XadA1 protein is expressed throughout all stages of biofilm formation in strain 9a5c while XadA2 is expressed in later stages. XadA1 appears to be important in the early stages of biofilm formation. On the other hand, until now no biochemical or functional characterization for XadA3 has been done, although this protein was detected by proteomic analysis as being strongly associated with X. fastidiosa membrane and present as >300,000 Da complex. In this project we aim to characterize the biochemistry and function of XadA3 in X. fastidiosa. For this we propose: (1) to obtain anti-XadA3 polyclonal antibodies using recombinant XadA3 expressed in heterologous system; (2) to knockout XadA3 gene in X. fastidiosa and analyse the ability of such mutant to adhere to the substrate and to form biofilm; (3) analyze the sequence of XadA3 and the level of expression of transcript in different strains of X. fastidiosa and (4) evaluate the ability of a anti-XadA3 polyclonal antibody to interfere in the adhesion of X. fastidiosa cells on the surface microfluidic chambers.