HrpN (harpin) is a type III-secreted protein of the fire blight pathogen Erwinia amylovora that is essential for full virulence and is partially responsible for hypersensitive response (HR) elicitation in non-host plants. HrpN also elicits cell death and defense responses in plants when applied as purified protein or as a cell-free extract. This study represents a structure/function analysis of HrpN. The hrpN gene coding sequence was mutagenized using PCR. hrpN coding sequences with one or more point mutations were cloned into a hrpN expression plasmid. The ability of these hrpN mutant clones to complement a hrpN deletion mutant of E. amylovora was tested. Two nonsense mutations and one missense mutation abolished HrpN secretion by E. amylovora, virulence activity in immature pear and apple fruit and HrpN-dependent HR in tobacco. These mutations indicated that the C-terminal half of HrpN plays an essential role in its secretion. In addition, these three HrpN mutant proteins blocked the HrpN-independent HR in tobacco, suggesting that they may somehow block the secretion or translocation of other type III-secreted proteins. A HrpN protein with two missense mutations in the C-terminal half was secreted but had a reduced ability to induce HrpN-dependent HR and completely lacked virulence activity. A HrpN protein with three missense mutations in the C-terminal half was secreted but lacked virulence activity, did not induce HrpN-dependent HR, and blocked the HrpN-independent HR. Several single missense mutations attenuated the HrpN-dependent HR; however, none of the mutations examined affected the cell-free elicitor activity of HrpN. These findings point to the critical importance of the HrpN C-terminal half for its secretion, virulence and HR-eliciting activities. These findings also indicate that the cell-free elicitor activity of HrpN resides in the N-terminal half of the protein.
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