Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice.

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.

Pathogenesis of the human opportunistic pathogen Pseudomonas aeruginosa PA14 in Arabidopsis.

The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 is a multihost pathogen that can infect Arabidopsis. We found that PA14 pathogenesis in Arabidopsis involves the following steps: attachment to the leaf surface, congregation of bacteria at and invasion through stomata or wounds, colonization of intercellular spaces, and concomitant disruption of plant cell wall and membrane structures, basipetal movement along the vascular parenchyma, and maceration and rotting of the petiole and central bud. Distinctive features of P. aeruginosa pathogenesis are that the surface of mesophyll cell walls adopt an unusual convoluted or undulated appearance, that PA14 cells orient themselves perpendicularly to the outer surface of mesophyll cell walls, and that PA14 cells make circular perforations, approximately equal to the diameter of P. aeruginosa, in mesophyll cell walls. Taken together, our data show that P. aeruginosa strain PA14 is a facultative pathogen of Arabidopsis that is capable of causing local and systemic infection, which can result in the death of the infected plant.

Plants and animals share functionally common bacterial virulence factors.

By exploiting the ability of Pseudomonas aeruginosa to infect a variety of vertebrate and nonvertebrate hosts, we have developed model systems that use plants and nematodes as adjuncts to mammalian models to help elucidate the molecular basis of P. aeruginosa pathogenesis. Our studies reveal a remarkable degree of conservation in the virulence mechanisms used by P. aeruginosa to infect hosts of divergent evolutionary origins.

Correlation of defense gene induction defects with powdery mildew susceptibility in Arabidopsis enhanced disease susceptibility mutants.

We investigated the relative importance of specific Arabidopsis thaliana genes in conferring resistance to bacterial versus fungal pathogens. We first developed a pathosystem involving the infection of Arabidopsis accession Columbia with a virulent isolate of the obligate biotrophic fungal pathogen Erysiphe orontii. E. orontii elicited the accumulation of mRNAs corresponding to the defense-related genes PR1, BGL2 (PR2), PR5 and GST1, but did not elicit production of the phytoalexin camalexin or the accumulation of defensin (PDF1.2) or thionin (THI2.1) mRNAs. We tested a set of 15 previously isolated Arabidopsis phytoalexin deficient (pad), non-expresser of PR (npr) and enhanced disease susceptibility (eds) mutants that are more susceptible to Pseudomonas syringae for their susceptibility to E. orontii. Four of these mutants (pad4-1, npr1-1, eds5-1 and a double npr1-1 eds5-1 mutant) as well as Arabidopsis lines carrying a nahG transgene exhibited enhanced susceptibility to E. orontii and reduced levels of PR gene expression. Comparison of the PR gene induction patterns in response to E. orontii in the various mutants and in the nahG transgenics suggests the existence of NPR1-independent salicylate-dependent and NPR1-independent salicylate-independent defense gene activation pathways. Eleven other eds and pad mutants did not show measurable enhanced susceptibility to E. orontii, suggesting that these mutants are defective in factors that are not important for the limitation of E. orontii growth.