1.5.13
A NOVEL APPROACH TO DETERMINING PATHOGENESIS-RELATED BACTERIAL GENES: DIFFERENTIAL DISPLAY RT-PCR OF ERWINIA CAROTOVORA SUBSP. ATROSEPTICA AND ERWINIA CAROTOVORA SUBSP. CAROTOVORA

A DELLAGI, IK TOTH, GD LYON and PRJ BIRCH

Fungal and Bacterial Plant Pathology, Scottish Crop Research Institute, Invergowerie, Dundee DD2 5DA, UK

Background and objectives
Erwinia carotovora subsp. atroseptica (Eca) causes blackleg, a seedborne disease of potatoes. Whereas Eca
has a host range limited to potato, the closely related Erwinia carotovora subsp. carotovora (Ecc) has a wide host range, which includes potato. Although a number of genes involved in disease development in Ecc have been defined (for example, pectolytic enzymes), as yet genes involved in the onset of infection or in host specificity in these organisms are unknown. In both these cases, a fundamental knowledge of these mechanisms will promote the development of novel strategies for engineered plant defence. The study of gene expression in bacteria is constrained by the lack of polyadenylation at the 3' end of mRNA. Recently, a method for differential display (DD) RT-PCR of Enterobacteriaceae has been reported [1] which circumvents this problem. They describe the design of small oligonucleotide primers which anneal to conserved regions flanking Escherichia coli genes. We have used these primers for comparing the profiles of gene expression between Eca and Ecc and, in addition, assessed their use for in planta detection of genes from these bacteria induced early in the infection proccess.

Materials and methods
Fislage et al. [1] have designed a set of 20 short primers (11-mers and 10-mers) which match sequences in enterobacterial coding regions available in the databases. The 11-mers are complementary to the 3-end of the bacterial RNA and used for reverse transcription. The 10-mers are complementary to the 5'-end of the RNA. We have used these primers for reverse transcription of RNA extracted from Eca and Ecc cultures grown on a variety of media, and from both healthy potato leaves and potato leaves 1 h after infection. Differential display has been optimized and specific PCR reactions have been performed with the bacterial cDNA thus synthesized.

Results and conclusions
Single-stranded cDNA synthesised from both bacterial cultures using the 11-mer primers was of high quality, up to 2000 bases. Conditions for DD RT-PCR were optimized and revealed similar results between Eca, Ecc and E. coli, from which the primers were designed. Different profiles were observed according to the growth medium and the bacterial strain used. The results suggest that the primers allow the synthesis of a large and representative population of cDNA molecules. Using primers specific to pectate lyase genes, strong amplification was obtained from cDNAs of erwinias grown in the presence of polygalacturonic acid, a pel (pectate lyase gene) inducer. The same specific band was obtained from plant-inoculated cDNA but not from healthy plants. These results show that good quality cDNAs can be obtained from bacterial RNAs for use with differential display RT-PCR for in planta expression studies. A number of differentially expressed sequences are currently being characterized. The production of high molecular-weight bacterial cDNA opens up horizons for new applications that have only been possible with eukaryotic cDNA, e.g. cDNA library construction, cDNA-AFLP analysis and subtractive hybridization.

References
1. Fislage R, Berceneanu M, Humboldt Y et al., 1997. Nucleic Acids Research 25,1830-1835.