Identification of differentially expressed genes of Xanthomonas axonopodis pv. citri by representational difference analysis of cDNA

Angela Mehta,Yoko B Rosato

doi:10.1590/s1415-47572005000100024

Abstract

Xanthomonas axonopodis pv. citri is a phytopathogenic bacterium responsible for citrus canker, a serious disease which causes severe losses in citriculture around the world. In this study we report the differential expression of X. axonopodis pv. citri in response to specific treatments by using Representational Difference Analysis of cDNA (cDNA RDA). cDNAs from X. axonopodis pv. citri cultured in the presence of leaf extract of the host plant (Citrus sinensis), in vivo, as well as in the complex medium were hybridized against cDNA of the bacterium grown in the minimal medium. Sequencing of the difference products obtained after the second and third hybridizations revealed a total of 37 distinct genes identified by homology searches in the genome of X. axonopodis pv. citri. These genes were distributed in different functional categories, including genes that encode hypothetical proteins, genes involved in metabolism, cellular processes and pathogenicity, and mobile genetic elements. Most of these genes are likely related to growth and/or acquisition of nutrients in specific treatments whereas others might be important for the bacterium pathogenicity.

Highlights

Citrus canker, caused by Xanthomonas axonopodis pv. citri, is a serious disease which causes severe losses in the production of citrus in many areas around the world (Rosetti, 1977; Stall and Seymour, 1983)
The cDNA RDA technique was employed for X. axonopodis pv. citri in an attempt to discriminate genes under regulation of different conditions
The cDNAs of the bacterium recovered directly from infiltrated leaves, grown in medium containing leaf extract or in complex medium were used in 3 rounds of subtractive hybridization against cDNAs of the bacterium cultured in the minimal medium

Summary

Introduction

Citrus canker, caused by Xanthomonas axonopodis pv. citri, is a serious disease which causes severe losses in the production of citrus in many areas around the world (Rosetti, 1977; Stall and Seymour, 1983). Citri, is a serious disease which causes severe losses in the production of citrus in many areas around the world (Rosetti, 1977; Stall and Seymour, 1983). The Representational Difference Analysis of cDNA (cDNA RDA) technique has been considered efficient for differential expression studies. This method is based on the subtractive hybridization of two cDNA populations followed by enrichment of the differential products by PCR amplification (Bowler et al, 1999). CDNA RDA was initially developed for eucaryotic cells and has successfully identified several differentially expressed genes in different tissues (Cooper et al, 2000; Kim et al, 2001). In the present study we have identified differentially expressed genes of X. axonopodis pv. The modified MM1 medium (Schulte and Bonas, 1992), described in the induction of the hrp genes in X. campestris, was used as a basal medium. cDNA from the bacterium grown in the presence of leaf extract from a susceptible host (sweet orange) as well as in the host plant leaves and complex medium was used in successive rounds of subtractive hybridizations against cDNA from the MM1 treatment

Materials and Methods

Results and Discussion

60 KDa chaperonin