Expressão diferencial de Xanthomonas axonopodis pv. citri na interação com Citrus sinensis utilizando eletroforese bidimensional de proteinas e cDNA RDA ("Representational Difference Analysis")

AUTOR(ES)
DATA DE PUBLICAÇÃO

2002

RESUMO

The present study reports the differential expression of X. axonopodis pv. citri in response to different growth conditions by two-dimensional gel electrophoresis (2-DE) of proteins and cDNA RDA (Representational Difference Analysis). For the protein analysis, the bacterium was cultured in the basal medium MM1 and in the presence of leaf extracts from a susceptible host plant (sweet orange) as well as a resistant (ponkan) and a non-host plant (passion fruit). The protein profiles revealed 12 differential spots in the citrus extract treatment (susceptible host) when compared to that of MM1. The 2-DE protein profile of the bacterium grown in the complex medium NYG was also obtained and the comparison with that of MM 1 revealed 36 differential spots. Five proteins from the different treatments were successfully N-terminally sequenced and included 2 constitutively expressed proteins, 2 proteins up-regulated in the presence of citrus extracts and 1 up-regulated in the complex medium. A method for the recovery of bacterial cells from the host plant leaves was also developed and validated by the analysis of protein and RNA extracted from the recovered cells. The protein pattem obtained by 2-DE was similar to that obtained for the bacterium grown in the presence of leaf extract of Citrus sinensis. Total RNA was analysed by agarose gel electrophoresis and RT -PCR reactions using specific primers for X. axonopodis pv. citri genes confirmed the quality af the RNA obtained. This method will be useful for in planta expression studies of several phytopathogenic species. In the cDNA RDA technique, cDNAs expressed in the presence of leaf extract of the host plant, as well as in vivo were identified after subtractive hybridizations against cDNAs of the bacterium cultured in the basal medium MM1. Also, a subtractive hybridization of cDNAs of the 3 bacterium grown in complex media against cDNAs expressed in the minimal medium was performed. A total of 37 distinct genes were identified by homology searches in the genome of X. axonopodis pv. citri, including genes that encode hypothetical proteins (12), genes involved in metabolism (13), cellular processes (6) and pathogenicity (4), and mobile genetic elements (2).

ASSUNTO(S)

eletroforese xanthomonas

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