The Alternative Sigma Factor RpoN Is Required for hrp Activity in Pseudomonas syringae pv. Maculicola and Acts at the Level of hrpL Transcription

Abstract

␤-Glucuronidase (uidA) reporter gene fusions were constructed for the hrpZ, hrpL, and hrpS genes from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. These reporters, as well as an avrRpt2-uidA fusion, were used to measure transcriptional activity in ES4326 and a ES4326 rpoN mutant. rpoN was required for the expression of avrRpt2, hrpZ, and hrpL in vitro in minimal media and in vivo when infiltrated into Arabidopsis thaliana leaves. In contrast, the expression of hrpS was essentially the same in wild-type and rpoN mutant strains. Constitutive expression of hrpL in an rpoN mutant restored hrpZ transcription to wild-type levels, restored the hypersensitive response when infiltrated into tobacco (Nicotiana tobacum), and partially restored the elicitation of virulence-related symptoms but not growth when infiltrated into Arabidopsis leaves. These data indicate that rpoN-mediated control of hrp gene expression acts at the level of hrpL and that in planta growth of P. syringae is not required for the elicitation of disease symptoms.In gram-negative bacteria, transcriptional activation in response to external stimuli often involves the alternative sigma factor 54 (1, 50). 54 , which is encoded by rpoN, works in conjunction with members of the NtrC superfamily of transcriptional activators (1, 50). Among the different enzymatic pathways under 54 control are those responsible for nitrogen utilization, dicarboxylate transport, xylene degradation, and hydrogen utilization (8,39,47,79).In the case of some phytopathogenic bacterial species, rpoN has been implicated indirectly as a regulator of a large cluster of pathogenicity-related genes known as the hrp gene cluster (17,27). For example, Pseudomonas syringae pv. syringae strain 61 contains a 25-kb hrp cluster consisting of 27 genes arranged as follows : hrpKL, hrpJ, hrcV, hrpQ, hrcN, hrpOP, hrcQRSTU, hrpVT, hrcC, hrpGF, hrpED, hrcJ, hrpBZA, and hrpSR (11,33). The acronym hrp stands for hypersensitive response and pathogenicity. The genes within the hrp cluster are required not only for pathogenicity but also for elicitation of the plant resistance reaction known as the hypersensitive response (HR) (44, 45). The HR involves rapid, localized plant cell death, which is triggered by a gene-for-gene interaction between a particular avirulence (avr) gene in the pathogen and a corresponding resistance gene in the host (21, 56). The widely conserved members of the hrp cluster, designated hrc, show significant homology to members of the Yersinia type III secretory pathway and in the new simplified nomenclature bear the letter designation of the corresponding Yersinia gene (9). (The exception is hrcV, which is homologous to the Yersinia lcrD genes.)Sequence analysis of the hrp cluster in P. syringae pv. phaseolicola suggested that 54 would be required for hrp gene expression in conjunction with the hrpRS genes, which are required for expression of the remaining hrp genes in the cluster (17, 27). hrpR and hrpS encode proteins that contain the domain conserved ...

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

“…In the accompanying study (23), we demonstrate that rpoN in ES4326 is required for the expression of hrpL, which encodes an alternative sigma factor and is required for expression of the ES4326 hrp genes and avrRpt2 (12,17,29,97). We also show that constitutive expression of hrpL on plasmid pHRPLC restores the ability of ES4326 rpoN::Km r to elicit disease symptoms in A. thaliana and an HR in tobacco.…”

“…When hrpL was constitutively expressed in ES4326 rpoN::Km r , both hrp gene expression (23) and defense gene induction (Fig. 4) were restored.…”

“…As seen in PR1, PR2 (BGL2), and PR5 genes in infiltrated plants, suggesting that defense gene activation and the elicitation of disease symptoms are a consequence of HrpL-dependent bacterial factors rather than growth of the pathogen per se. One difficulty encountered in this experiment was the instability of pHRPLC in wild-type ES4326 in planta (23). Thus, it was not possible to examine PR gene expression in response to ES4326 (pHRPLC).…”

“…Given the pleiotropic phenotype of rpoN mutants, it is not possible to state precisely why the ES4326 rpoN mutant failed to elicit disease symptoms and to grow in Arabidopsis leaves or to elicit an HR. In the accompanying study we show that the absence of a functional 54 in ES4326 blocks the transcription of hrp genes downstream of hrpRS (23), which would account for the nonpathogenic and HR-deficient phenotypes. However, we also report that the constitutive expression of hrpL in ES4326 Our experiments using a pseudorevertant of the ES4326 rpoN mutant that was able to utilize aspartate eliminated the possibility that the rpoN mutant is nonpathogenic solely due to its inability to utilize this amino acid.…”

“…For clarity, ES4326 carrying plasmid pLH12 (which carries the avirulence gene avrRpt2) is referred to as ES4326 (avrRpt2). Carbon and nitrogen source utilization tests for ES4326 rpoN mutants were performed in M9 salts minimal medium by providing a (23) carbon source at 10 mM and by replacing ammonium chloride with an alternative nitrogen source at 5 mM when required. Bacterial motility was tested on "swarm plates" consisting of 0.3% agar, 0.5% NaCl and 0.5% tryptone (38).…”