cDNA-AFLP analysis of differential gene expression in the prokaryotic plant pathogen Erwinia carotovora.
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.
Dellagi
Alia
A
Department of Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
Birch
Paul R J
PRJ
Department of Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
Heilbronn
Jacqueline
J
Department of Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
Lyon
Gary D
GD
Department of Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
Toth
Ian K
IK
Department of Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1.
D015152:Blotting, Northern; D003470:Culture Media; D017931:DNA Primers; D018076:DNA, Complementary; D020869:Gene Expression Profiling; D008969:Molecular Sequence Data; D016973:Pectobacterium carotovorum / Q000235:genetics* / Q000254:growth & development; D018515:Plant Leaves / Q000382:microbiology; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011198:Solanum tuberosum / Q000382:microbiology*
D003470:Culture Media; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger