Ron J. Laby

The hrp Gene Cluster of Erwinia Amylovora

From a library of E. amylovora DNA, a clone, pCPP430, was identified that contains the entire hrp gene cluster. The cosmid complemented all (18) original Hrp- transposon-induced mutants of E. amylovora and bestows on Escherichia coli, and all other members of the Enterobacteriaceae tested, the ability to elicit the hypersensitive response in tobacco and other plants. Mutagenesis of pCPP430 revealed that the cluster spans ca. 40kb. Only the rightmost ca. 25 kb of the cluster is necessary to elicit the K+ efflux / H+ influx exchange reaction in tobacco cell suspension culture. This region hybridizes with a portion of the hrp cluster of Pseudomonas syringae, the wts (watersoaking) region of DNA of E. stewartii, and with genomic DNA of several other phytopathogenic bacteria. Studies of the regulation of hrp genes in E. amylovora, using the sglucuronidase reporter gene and through isolation of mRNA showed that the hrp genes are regulated in response to the nutritional status of the bacterium, rather than to compounds produced by plant cells or osmotic concentration. The predicted protein of one hrp locus is homologous to the conserved domain of several prokaryotic regulatory proteins including HrpS from P. syringae, NifA from Klebsiella pneumoniae and Rhizobium and NtrC from several bacteria.

Are Harpins Universal Elicitors of the Hypersensitive Response of Phytopathogenic Bacteria

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.