Zhong-Min Wei

Erwinia chrysanthemi HarpinEch: an elicitor of the hypersensitive response that contributes to soft-rot pathogenesis

Mutants of the soft-rot pathogen Erwinia chrysanthemi EC16 that are deficient in the production of the pectate lyase isozymes PelABCE can elicit the hypersensitive response (HR) in tobacco leaves. The hrpNEch gene was identified in a collection of cosmids carrying E. chrysanthemi hrp genes by its hybridization with the Erwinia amylovora hrpNEa gene. hrpNEch appears to be in a monocistronic operon, and it encodes a predicted protein of 340 amino acids that is glycine-rich, lacking in cysteine, and highly similar to HrpNEa in its C-terminal half. Escherichia coli DH5 alpha cells expressing hrpNEch from the lac promoter of pBluescript II accumulated HrpNEch in inclusion bodies. The protein was readily purified from cell lysates carrying these inclusion bodies by solubilization in 4.5 M guanidine-HCl and reprecipitation upon dialysis against dilute buffer. HrpNEch suspensions elicited a typical HR in tobacco leaves, and elicitor activity was heat-stable. Tn5-gusA1 mutations were introduced into the cloned hrpNEch and then marker-exchanged into the genomes of E. chrysanthemi strains AC4150 (wild type), CUCPB5006 (delta pelABCE), and CUCPB5030 (delta pelABCE outD::TnphoA). hrpNEch::Tn5-gusA1 mutations in CUCPB5006 abolished the ability of the bacterium to elicit the HR in tobacco leaves unless complemented with an hrpNEch subclone. An hrpNEch::Tn5-gusA1 mutation also reduced the ability of AC4150 to incite infections in witloof chicory leaves, but it did not reduce the size of lesions that did develop. Purified HrpNEch and E. chrysanthemi strains CUCPB5006 and CUCPB5030 elicited HR-like necrosis in leaves of tomato, pepper, African violet, petunia, and pelargonium, whereas hrpNEch mutants did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of hrp genes and type III protein secretion in Erwinia amylovora by HrpX/HrpY, a novel two component system, and HrpS

Two novel regulatory components, hrpX and hrpY, of the hrp system of Erwinia amylovora were identified. The hrpXY operon is expressed in rich media, but its transcription is increased threefold by low pH, nutrient, and temperature levels--conditions that mimic the plant apoplast. hrpXY is autoregulated and directs the expression of hrpL; hrpL, in turn, activates transcription of other loci in the hrp gene cluster (Z.-M. Wei and S. V. Beer, J. Bacteriol. 177:6201-6210, 1995). The deduced amino -acid sequences of hrpX and hrpY are similar to bacterial two-component regulators including VsrA/VsrD of Pseudomonas (Ralstonia) solanacearum, DegS/DegU of Bacillus subtilis, and UhpB/UhpA and NarX/NarP, NarL of Escherichia coli. The N-terminal signal-input domain of HrpX contains PAS domain repeats. hrpS, located downstream of hrpXY, encodes a protein with homology to WtsA (HrpS) of Erwinia (Pantoea) stewartii, HrpR and HrpS of Pseudomonas syringae, and other delta54-dependent, enhancer-binding proteins. Transcription of hrpS also is induced under conditions that mimic the plant apoplast. However, hrpS is not autoregulated, and its expression is not affected by hrpXY. When hrpS or hrpL were provided on multicopy plasmids, both hrpX and hrpY mutants recovered the ability to elicit the hypersensitive reaction in tobacco. This confirms that hrpS and hrpL are not epistatic to hrpXY. A model of the regulatory cascades leading to the induction of the E. amylovora type III system is proposed.

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.