Are Harpins Universal Elicitors of the Hypersensitive Response of Phytopathogenic Bacteria?

Abstract

The entire hrp gene cluster of Erwinia amylovora strain Ea321 had been cloned previously on a single cosmid, designated pCPP430. This cosmid is well expressed in Escherichia coli and Gram-negative bacteria harboring it strongly elicit the hypersensitive response (HR) on non-host plants. Cell-free sonicates of E. coli DH5α(pCPP430) and Ea 321 caused collapse of tobacco leaf tissue within 12 hrs after infiltration of the intercellular spaces. From the HR-eliciting sonicates, we purified a 44 kD, heat-stable, cell-surface-associated protein (harpin) by ion-exchange chromatography and reverse-phase chromatography. Purified harpin caused collapse of leaf tissue that was indistinguishable from the collapse caused by DH5α(pCPP430) or wild-type E. amylovora. Harpin also elicited the K⁺/H⁺ exchange reaction in tobacco cell suspensions. A polyclonal antibody raised in rabbits in response to injections of harpin may prove useful in the diagnosis of the fire blight pathogen.

The structural gene encoding harpin (hrpN) was identified on pCPP430 with the aid of a synthesized oligonucleotide probe corresponding to the ninth to fifteenth amino acids of the NH₂- terminus of harpin. This gene hybridized with genomic DNA of E. amylovora, E. chtysanthemi, E. stewartii and Pseudomonas solanacearum. No consistent hybridization was observed with genomic DNA of P. syringae and Xanthomonas campestris. Evidence is accumulating that harpin-like proteins are the active products of hrp genes of Erwinia species. Based on compelling genetic evidence with respect to E. amylovora, harpin also plays a crucial role in disease.