Virulence of the phytopathogen Pseudomonas syringae pv. maculicola is rpoNdependent

We cloned the rpoN (ntrA and glnF) gene encoding sigma(54) from the phytopa thogen Pseudomonas syringae pv. maculicola strain ES4326. The P, syringae E 4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and K lebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, i ncluding the inability to utilize nitrate as sole nitrogen source. DNA sequ ence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the sigma(54) protein encoded by the Pseudomonas putida rpoN gene. A marker ex change protocol was used to construct an ES4326 rpoN insertional mutation, rpoN::Km(r). In contrast to wild-type ES4326, ES4326 rpoN::Km(r) was nonmot ile and could not utilize nitrate, urea, C-4-dicarboxylic acids, several am ino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpo N was essential for production of the phytotoxin coronatine and for express ion of the structural genes encoding coronamic acid. In addition, ES4326 rp oN::Km(r) did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Ar abidopsis defense-related mRNAs, and did not elicit a hypersensitive respon se (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermo re, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicite d an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 r poN::Km(r) carrying avrRpt2 elicited no response. Constitutive expression o f ES4326 hrpL in ES4326 rpoN::Km(r) partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense ge ne induction in a compatible host-pathogen interaction However, constitutiv e expression of hrpL in ES4326 rpoN::Km(r) did not restore coronatine produ ction, showing that coronatine biosynthesis requires factors other than hrp L.