THE hrp GENES CLUSTER IN ERWINIA PYRIFOLIAE AND DETERMINATION OF HR ACTIVE DOMAIN IN HrpN_Ep PROTEIN

Previously, the disease-specific (dsp) region and the hypersensitive response and pathogenicity (hrp) genes including the hrpW, hrpN_Ep and hrpC operons were sequenced in Erwinia pyrifoliae WT3. In this study, we therefore, determined the remaining hrp genes including the hrpC, hrpA, hrpS, hrpXY, hrpL and hrpJ operons and sequenced completely. The genetic organization of the hrp/hrc genes and their orientation for the transcriptions were also similar to and collinear with those of E. amylovora, showing ≥80% homologies. However, ORFU1 and ORFU2 of unknown functions, present between the hrpA and hrpS operons of E. amylovora, were absent in E. pyrifoliae. To determine the HR active domains, several proteins were prepared from truncated fragments of the N-terminal and the C-terminal regions of HrpN_Ep of E. pyrifoliae. The proteins prepared from the N-terminal region elicited HR but not from those of the C-terminal region indicating that HR active domains are located in only N-terminal region of the HrpN_Ep protein. Two synthetic oligopeptides produced HR on tobacco confirming presence of two HR active domains in the HrpN_Ep. The HR positive N-terminal fragment (HNΔC187) was further narrowed down by deleting C-terminal amino acids and internal amino acids to investigate whether amino acid insertion region have role in faster and stronger HR activity in HrpN_Ep than HrpN_Ea. Deletion of third amino acid insertion region showed reduction in HR activation when compared to wild type. However, deletion of other amino acid insertion regions did not cause any change in HR elicitation speculating the importance of third amino acid insertion region in HrpN_Ep for causing faster HR activity.