School of Biological Sciences, 1.800 Stopford Building, University of Manchester, Manchester M13 9PT, UK

Background and objectives
Erwinia amylovora causes the commercially important disease fireblight, which affects apple, pear and several important ornamental plants. Infected tissue becomes brown or black in colour, giving a scorched appearance from which the disease takes its name. It has been suggested that the bacteria undergo nutrient stress on entering the host environment and that this signal coordinates the expression of genes required for survival and virulence. In an attempt to characterize the response of E. amylovora on entering the plant environment we have constructed a reporter fusion in the chromosomal copy of rpoS.

The rpoS gene encodes an alternative sigma factor, ss, which is involved in the expression of genes required for survival during stationary-phase growth. We describe the use of the reporter gene gusA, encoding beta-glucuronidase (GUS), to study the expression of the previously cloned rpoS gene from E. amylovora (Anderson M et al., unpublished data). The gusA reporter system has been widely used in bacteria and other organisms. The lack of detectable levels of beta-glucuronidase in plants and the wide availability of spectrophotometric substrates for the enzyme, makes the gusA system an ideal choice for studies involving E. amylovora.

Material and methods
The E. amylovora rpoS gene was previously cloned using a PCR probe from E. coli (Anderson M et al., unpublished data). Subcloning of the rpoS gene produced an 800 bp fragment which contained the promoter and a section of 5 sequence. This was cloned upstream of a promoterless E. coli gusA gene. The resultant fusion was then introduced into a suicide vector, pCVD422, and the construct transferred into E. amylovora OT1 by conjugation. The resulting single-crossover event produced the meridiploid strain, OTGUS. In vitro and in vivo experiments have been performed to determine the stability of the construct within the bacterial chromosome and to study transcription of the gene during different growth conditions.

Results and conclusions
The fusion construct was shown to be stable and active in vitro and in planta. In planta studies showed the population of OTGUS was at its maximum 7 days after inoculation, while the fusion reached a maximal rate of activity after 12 days. This may indicate expression of rpoS due to nutrient limitation as the disease process progresses. Growth-rate experiments with the OTGUS strain revealed that rpoS transcription increases rapidly during mid-log phase with a levelling-off of expression at the onset of stationary phase. This indicates that stationary-phase increases in ss levels in E. amylovora may be similar to those described for E. coli, where increased stabilization of the protein rather than increased expression of the rpoS gene result in the higher levels of protein. In vitro experiments have shown that E. amylovora rpoS transcription rapidly increases in response to osmotic shock and carbon starvation. As E. amylovora enters the plant via the nectaries, the bacterium would be expected to encounter conditions of high osmolarity. Active ss may therefore be vital to bacterial survival during the initial stages of infection.