Genome-based proteomics: analysis of protein-protein interactions of Xanthomonas axonopodis pv. citri
Winck, F.V.*†; Amaral, A.M.*; Baptista, J.C.*; Cabrera, O.G.#; Missawa,S.K#; Marango, S. †; Novello,J.C. †; Machado, M.A.*
* Laboratório de Biotecnologia – Centro de citricultura “Silvio Moreira” –IAC- Cordeirópolis- S.P. ;† Laboratório de Química de Proteínas – Depto. de Bioquímica – IB –UNICAMP – S.P.;# Laboratório de Genômica e expressão – Depto. de Genética –IB –UNICAMP –S.P.
Xanthomonas axonopodis is the causative agent of citrus canker, a serious bacterial disease that affects citrus plant species and leads to significant economical losses to the citrus industry. In the post-genomic era, new perspectives have been open for proteomics based on information from genome databases, such as studies towards protein-protein interactions. In this way, we proposed to evaluate the interactions between two proteins supposedly involved with pathogenicity and other cytoplasmic proteins from of X. axonopodis pv. citri, through an affinity purification method and the co-purification of proteins. To analyze such interactions, two genes were chosen [hrpF (XAC0394) and avrXACE2 (XAC3224)] and cloned into the pET15b expression vector, which yields the fusion of a polihistidine-tag in the N-terminal position of the protein. In addition, site-directed mutants were produced for the two endogenous genes to allow only the interaction of expressed proteins containing His-tag with their cytoplasmic proteins. By using the co-purification of these cytoplasmic proteins that associate to the proteins containing the polihistidine residue, we aim to identify, by mass spectrometry, proteins that are involved in the occurrence of citrus canker.
|