To elucidate the role of harpins produced by Pseudomonas syringae, the corresponding hrpZ gene was isolated from P. s. pv. tabaci. The sequence information revealed that this gene carries a serious mutation with 326 bp lacking in the central region and potentially encodes only 140 N-terminal amino acids because of a frame shift. The investigation of biological properties using recombinant harpin indicated harpin,,t, was incapable of inducing HR in both host and nonhost plants. Based on an immunoblot analysis to detect harpin from P. s. pathovars in hrp-inducing medium, the truncated harpinpsta was neither expressed nor secreted into the culture medium. These results suggest that harpin is not the sole determinant of the host-parasite specificity in P. s. pv. tabaci.

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The hrp gene locus of Pseudomonas syringae pv. pisi (P. s. pisi) race 1 was cloned in SuperCosl vector. The nucleotide sequences of hrpA, hrpB and hrpZ were determined, and the deduced amino acid sequences were compared with different races of P. s. pisi and corresponding genes in other bacteria. In particular, over 99% of the similarity in HrpZ (harpin) amino acid sequences was observed among the different races of P. s. pisi. Among P. syringae pathovars, very high similarity was observed, but the level of similarity was considerably lower than that of Erwinia. The HrpZ protein is rich in glycine but lacks cysteine. It is also heat tolerant as has been reported with other bacterial harpins. The carboxyl terminus of harpin also contains two directly repeated sequences of GGGLGTP and QTGT commonly found in P. syringae pv. syringae. It is very interesting to note that a tetrapeptide sequence, GDET, homologous to the hexapeptide Mycosphaerella pinodes-supprescin B (SSGDET) was found in HrpZ of P. s. pisi.

To investigate the role of the proteinaceous elicitor, harpin, on host and nonhost plants, we isolated the harpin-coding gene, hrpZ, from Pseudomonas syringae pvs. pisi, glycinea, tabaci and tomato. Effects of the recombinant harpin proteins on pea plants were analyzed and compared with the effects of the corresponding bacterial treatment. After inoculation of pea with pea pathogen P. syringae pv, p h i , the bacterial population increased and the accumulation of PAL-mRNA and pisatin was inhibited. The nonpathogenic pathovars, glycinea, tabaci and tomato induced both defense responses in pea. However, none of the harpins induced the hypersensitive reaction or accumulation of PAL-mRNA and pisatin in pea. Harpins from P. syringae pvs. glycinea, tomato and p h i did induce these defense responses in tobacco, however, suggesting that externally applied harpins either are not recognized or are nonfunctional in pea plants.The hypersensitive reaction (HR) is one of the most important plant defense responses against avirulent pathogens and/or nonpathogens and is characterized by rapid, localized cell death at the site of pathogen invasion. Many Gram-negative phytopathogenic bacteria elicit the HR in nonhost or resistant plants. Molecular and genetic investigations revealed that hrp genes regulate the hypersensitive zeaction and pathogenicity in several pathovars of Pseudomonas syringae 2 8 5 8 I Q 3 11-13) and other phytopathogenic bacteria, such as X a n t~~~~ campes~ris pathovars, X . oryzae pv. oryzae, Ralstonia solanacearum, Erwinia amybvora, E. chrysanthemi and E. carotovor#". Hrp is known as a gene cluster about 12-25kb long comprising six to eight transcriptional units that encode for 19-26 unique protein products. Sequencing and functional analyses have suggested that Hrp proteins have various functions as components of the type I11 secretion system and regulatory proteins, as well as the proteinaceous HR elicitor, harp~3,~1*-i0,13,14). Harpins are glycine-rich, cysteine-lacking, heat-stable extracellular proteins of ca. 35-kDa having HR-elicitor activity'O).To elucidate the role of harpins in host-parasite interactions, we have recently isolated a hrp cluster from P. syringae pv. pisi 299A (race 1) and the hrpZ gene that encodes the harpin from other races of P. syringae pvs. pki12' , glycinea (race 4), tabaci 6605 and toma~o DC3000 (race 0) using PCR with two primers corresponding to the hrpA and hrpB genes15). Sequencing analysis of the hrpZ gene from race 1 and race 3 of P. syr~ngae pv. pisi revealed that they share a completely identical amino acid sequence, suggesting that harpin is not involved in race-cultivar specificity1z). In this study, we investigated the role of harpins from P. syringae in basic compatibility (species specificity) and the effects of recombinant harpin proteins on the pea defense response.To evaluate host responses after inoculation with four pathovars of P. syringae, we first measured the propagation of each bacterium in inoculated pea leaves (Pisum sativum cv. Midoriusui), Inoculat...

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