CHARACTERIZATION OF THE PROMOTER OF AVIRULENCE GENE-D FROM PSEUDOMONAS-SYRINGAE PV TOMATO

The avirulence gene D (avrD) from Pseudomonas syringae pv. tomato comprises the first open reading frame (ORF) of a putative operon consisting of at least five tandem ORFs. The promoter of the avrD operon was localized to a 150-bp DNA fragment occurring 5' to the avrD gene by using the Tn7-lux and gus reporter systems. The avrD promoter in P. syringae pv. tomato and P. syringae pv. glycinea was poorly expressed when bacteria were grown in complex culture media but was activated during bacterial growth in plants. The timing and level of induction were similar in compatible and incompatible plant-pathogen interactions. When bacteria were grown in minimal culture medium, promoter activity was repressed by certain carbon sources, high concentrations of nitrogen compounds, and pH values above 6.5. Primer extension experiments on RNA from bacteria grown in minimal medium identified two transcription initiation sites 87 and 41 nucleotides upstream from the translational start site. Only the -41 transcriptional start site was identified in bacteria grown in soybean leaves. A sigma54 promoter consensus sequence (GG-10 bp-GC) occurred 14 bp upstream of the -41 transcriptional start, and 3' deletions into this region completely abolished promoter activity. Little expression was observed when a gus fusion with the avrD promoter was introduced into an ntrA mutant strain of P. syringae pv. phaseolicola deficient in the sigma54 cofactor. Expression from the avrD promoter also required the hrp regulatory genes, hrpS and hrpL. Deletions from the 5' end of the promoter region and base substitution analyses also identified two upstream elements important for expression. Sequence comparison of these elements with other cloned avirulence genes revealed the presence of a conserved consensus sequence (GGAACC-N15/16-CCAC) in the promoters of nine different avirulence genes from P. syringae pathovars.