DSPA, AN ESSENTIAL PATHOGENICITY FACTOR OF ERWINIA-AMYLOVORA SHOWING HOMOLOGY WITH AVRE OF PSEUDOMONAS-SYRINGAE, IS SECRETED VIA THE HRP SECRETION PATHWAY IN A DSPB-DEPENDENT WAY

In Erwinia amylovora, the dsp region, required for pathogenicity on th e host plant but not for hypersensitive elicitation on tobacco, is sep arated from the hrp region by 4 kb, The genetic analysis reported in t his paper showed that this 4 kb region is not required for pathogenici ty on pear seedlings, The environmental conditions allowing expression of a dsp::lacZ fusion were examined: expression was barely detected i n rich medium at 30 degrees C, and the highest expression was observed in M9 galactose minimal medium at 25 degrees C. A dsp::uidA fusion ap peared to be expressed only in a HrpL-proficient strain, indicating th at the dsp region, like the hrp region, is positively controlled via t he alternative sigma factor HrpL. Sequence analysis revealed that the dsp cluster encodes two genes, dspA (5517 bp) and dspB (420 bp), and t hat the insertions leading to the dsp::lacZ and the dsp::uidA fusions were within dspA, A HrpL-dependent promoter sequence (GGAACCN(15)-CAAC A) was identified upstream of dspA, and primer extension analysis dete cted four transcriptional starts 7, 8, 9 and 10 bp downstream of this sequence, A sigma(70) promoter sequence (TTGCCCN(16)-GATAAT) was obser ved upstream of dspB. The functionality of this second promoter was co nfirmed by complementation analysis, This promoter allowed constitutiv e expression of dspB, as measured by the expression of a dspB::uidA fu sion in rich medium. In M9 galactose medium, however, HrpL was shown t o activate dspB, as expression of the dspB::uidA fusion was twofold hi gher in a HrpL(+) background than in a HrpL(-) background, Transposon insertions in either dspA or dspB led to a non-pathogenic phenotype, T hus, both DspA and DspB were required for E. amylovora pathogenicity, as dspB could be expressed independently of dspA. DspA and DspB were v isualized as polypeptides with apparent sizes of 190 kDa and 15.5 kDa, respectively, when encoded in the T7 polymerase/promoter system, DspA , which showed homology with the protein predicted from the partial se quence of Pseudomonas syringae pv, tomato avrE transcriptional unit II I, was shown to be secreted into the external medium via the Hrp secre tion pathway, DspB was predicted to be acidic, like the Syc chaperone of Yersinia. A chaperone role for DspB was suggested further by the fa ct that DspA secretion required a functional DspB protein.