THE SECRETION OF EOPB FROM ERWINIA AMYLOVORA

Some proteins that are secreted or translocated by the type III secretion system (TTSS) require chaperones. In general, chaperones of the TTSS are small proteins that aid in the efficient secretion or translocation of other proteins. EopB (formerly known as OrfB) was previously shown to be secreted by Erwinia amylovora in minimal media in a TTSS-dependent manner. Whether this secreted protein utilizes chaperones was examined. Transposon-induced mutants of E. amylovora with insertions in eopB and two putative chaperone genes, orfA and orfC, were tested for pathogenicity and for the secretion of FLAG-tagged EopB. These two putative chaperones and DspF, chaperone to the E. amylovora protein DspE, were screened for interaction with EopB using yeast two-hybrid assays. Strong interaction occurred between EopB and OrfA, and weaker interaction occurred between EopB and DspF. This suggests that OrfA is the more likely chaperone for EopB. The orfA and eopB mutants were not compromised in pathogenicity in an immature pear fruit assay. In an in vitro secretion assay, EopB-FLAG was secreted from an orfA mutant as well as from the wild-type strain.