Organization of thehrpGene Cluster and Nucleotide Sequence of thehrpLGene fromPseudomonas syringaepv.morsprunorum

Abstract

Pseudomonas syringae pv. morsprunorum PM7 is pathogenic to cherry and induces the hypersensitive response (HR) in tobacco. Six of 1,300 Tn5 mutants from strain PM7 neither elicit the HR in tobacco nor produce necrotic lesions in cherry plantlets. Plasmid pPM419, isolated from a genomic DNA library of wild-type strain PM7, restored the ability to cause a HR in five of the mutants. Restriction enzyme analysis of pPM419 revealed a 37-kilobase (kb) insert of genomic DNA from P. s. morsprunorum PM7. Tnt-spice mutagenesis of pPM419 and a second plasmid clone, pPM41, followed by marker exchange into the genome of P. s. morsprunorum PM7, indicated a 22-kb DNA fragment from P. s. morsprunorum PM7 has genes for elicitation of necrotic lesions in cherry plantlets and HR in tobacco. Complementation studies revealed that the hrp region of P. s. morsprunorum PM7 is organized into eight putative transcriptional units. Units II, VI, and VII exhibit DNA homology with the hrpI, hrpH, and hrpZ genes, respectively, of P. s. syringae 61. The nucleotide sequence of hrpL, the first transcriptional unit in the hrp cluster of P. s. morsprunorum PM7, encodes a polypeptide of 185 amino acids that exhibits 92% identity and 96% similarity with HrpL of P. s. syringae 61. Two transcriptional start sites, P1 and P2, located 63 and 25 bp upstream of the hrpL start codon, were identified by primer extension analysis. The −12 and −24 regions of the putative P2 promoter resemble a 54 consensus sequence