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cDNA-AFLP analysis of differential gene expression in the prokaryotic plant pathogen Erwinia carotovora

Department of Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK¹

Author for correspondence: Ian K. Toth. Tel: +44 1382 562731. Fax: +44 1382 562426. e-mail: itoth{at}scri.sari.ac.uk

For studies of differential gene expression in prokaryotes, methods for synthesizing representative cDNA populations are required. Here, a technique is described for the synthesis of cDNA from the potato pathogens Erwinia carotovora subsp. atroseptica (Eca) and Erwinia carotovora subsp. carotovora (Ecc) using a combination of short oligonucleotide (11-mer) primers that were known to anneal to conserved sequences in the 3' regions of enterobacterial genes. Specific PCR amplifications with primers designed to anneal to 14 known genes from either Eca or Ecc revealed the presence of the corresponding transcripts in cDNA, suggesting that the cDNA represented a broad genomic coverage. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify differentially expressed genes in Eca, including one that shows significant similarity, at the protein level, to an avirulence gene from Xanthomonas campestris pv. raphani. Northern analysis was used to confirm that differentially amplified cDNA fragments were derived from differentially expressed genes. This is the first report of the use of cDNA-AFLP to study differential gene expression in prokaryotes.

Keywords: prokaryotic differential gene expression, Erwinia, plant pathogen, cDNA-AFLP

Abbreviations: Eca, Erwinia carotovora subsp. atroseptica; Ecc, Erwinia carotovora subsp. carotovora; AFLP, amplified fragment length polymorphism

The GenBank accession numbers for the EL1, EL2, EL3, EP5, EP22, EP26, EP11 and EP21 sequences determined in this work are AJ274641–AJ274648, respectively.

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