Virulence of the Phytopathogen Pseudomonas syringae pv. Maculicola Is rpoN Dependent

Abstract

ABSTRACT We cloned the rpoN ( ntrA and glnF ) gene encoding ς 54 from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa , Escherichia coli , and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the ς 54 protein encoded by the Pseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation, rpoN ::Km r . In contrast to wild-type ES4326, ES4326 rpoN ::Km r was nonmotile and could not utilize nitrate, urea, C 4 -dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326 rpoN ::Km r did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Arabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco ( Nicotiana tabacum ) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN ::Km r carrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326 rpoN ::Km r partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of hrpL in ES4326 rpoN ::Km r did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL .