ISHS Acta Horticulturae 338: VI International Workshop on Fireblight
HARPIN, FROM ERWINIA AMYLOVORA, ELICITS THE HYPERSENSITIVE RESPONSE AND IS A DETERMINANT OF PATHOGENICITY |
| Authors: | Steven V. Beer, Z. Wei, Ron J. Laby, S. He, David W. Bauer, A. Collmer, Cathy H. Zumoff |
| Abstract:
The entire hrp gene cluster of E. amylovora strain Ea321 had been cloned earlier on a single cosmid designated pCPP430. This cosmid complements all hrp- mutants of E. amylovora and restores their ability to elicit the hypersensitive response (HR) in non-host plants like tobacco and their ability to cause Fire Blight in host plants like pear. It also bestows on Escherichia colii and other Gram-negative bacteria, the ability to elicit the HR in plants. Cell-free sonicates of E. coli DH5a(pCPP430) and Ea321 cuased collapse of infiltrated tobacco leaf sectors within 12 hours in a manner similar to that cuased by infiltration of metabolically active cells of the same bacteria. Harpin was isolated and purified from the sonicates by ion-exchange and reverse-phase chromatography. It is a heat-stable, glycine-rich, cell-surface-associated protein with a molecular size of 44 kDa and a pl of 4.3. The purified protein caused collapse of tobacco leaf tissue that was indistinguishable from that caused by whole bacteria. Harpin also elicited the K+/H+ exchange reaction (XR) in tobacco cell suspensions and reacted with anti-harpin antibodies raised in rabbits. The structural gene encoding harpin (hrpN) was identified by hybridization of a synthetic oligonucleotide, which corresponded to the ninth through fifteenth amino acids from the amino terminus of harpin, with the hrp gene cluster of Ea321. hrpN is 1155 base pairs long and it encodes a 385 amino acid protein, which corresponds well with actual analyses of harpin. hrpN was mutagenized with a nonpolar transposon and then marker-exchanged into pCPP430 and Ea321. The hrpN- strains failed to elicit the HR and to produce harpin. The hrpN- derivative of Ea321 failed to cause Fire Blight of immature pear fruit. | |
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