Genetic organization of the hrp genes cluster in Erwinia pyrifoliae and characterization of HR active domains in HrpN∧Ep protein by mutational analysis

The disease-specific (dsp) region and the hypersensitive response and pathogenicity (hrp) genes, including the hrpW, hrpN∧(Ep, and hrpC operons have previously been sequenced in Erwinia pyrifoliae WT3 [Shrestha et al. (2005a)]. In this study, the remaining hrp genes, including the hrpC, hrpA, hrpS, hrpXY, hrpL and hrpJ operons, were determined. The hrp genes cluster (ca. 38 kb) was comprised of eight transcriptional units and contained nine hrc (hrp conserved) genes. 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 greater-than or equal to 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 protein 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. The HrpN∧Ep mutant proteins HNΔC187 (D1AIR), HNΔC187 (D2AIR) and HNΔC187 (DM41) retained similar HR activation to that of wildtype HrpN∧Ep. However, the HrpN∧Ep mutant protein HNΔC187 (D3AIR) lacking third amino acid insertion region (102 to 113 aa) reduced HR when compared to that of wild-type HrpN∧Ep. Reduction in HR elicitation could not be observed when single amino acids at different positions were substituted at third amino acids insertion region. But, substitution of amino acids at L103R, L106K and L110R showed reduction in HR activity on tobacco suggesting their importance in activation of HR faster in the HrpN∧Ep although it requires further detailed analysis.