1.5.5S
CHARACTERIZATION OF THE HRP REGULON OF PSEUDOMONAS SYRINGAE

S HUTCHESON, JD WEAVER, N THAREJA, I ROZENBAUM and K PAK

University of Maryland, College Park, MD 20742, USA

Background and objectives
Pathogenesis by Pseudomonas syringae is predicted to involve adhesion of the bacteria to plant cells, induced hrp gene expression, assembly of a hrp-encoded protein translocation apparatus (PTA), and translocation of pathogenicity and host-range determinants into target plant cells [1]. A critical component of this model is the mechanism coordinating the assembly of the PTA and translocation of pathogenicity and host-range determinants, such as avr genes . Expression of the P. syringae hrp regulon has been reported to be affected by a variety of environmental conditions. By reconstituting a functional regulatory system in E. coli, we showed that the proteins HrpR, HrpS and HrpL mediate the regulation of the hrp regulon. HrpR and HrpS specifically activate the hrpL promoter, and HrpL is an alternative sigma factor that directs expression of hrp and avr transcriptional units by recognizing a conserved promoter sequence. The mechanism by which environmental stimuli affect expression of the hrp regulon genes in P. syringae strains is not obvious from the physical properties of HrpR, HrpS or HrpL.

Results and conclusions
Because an alternative mechanism for the activity of HrpR and HrpS has been proposed, we initially explored the role of HrpR and HrpS in the regulation of the hrpL promoter. As before, both proteins were required for maximal activity of the hrpL promoter. The interaction of HrpR with HrpS was confirmed by using a yeast two-hybrid assay. A yeast one-hybrid assay was then used to investigate the interaction of HrpR and HrpS with a putative enhancer-binding motif.

In an attempt to identify new regulatory factors controlling expression of the hrp regulon, we initiated a search for constitutive up mutants. A constitutive up phenotype is likely be caused by: (i) enhanced hrpL expression caused by a promoter mutation; (ii) altered expression or activity of presently unidentified positive transcriptional factors, such as HrpX and HrpY recently identified in Erwinia amylovora; (iii) nonsense mutations in a negative-acting determinant; or (iv) mutations affecting signal transduction cascade-mediating apparent contact-dependent regulation, as described below. Several apparent up mutants were detected in a chemically mutagenized population of cells. Plant responses to these mutants were more rapid than the wild type, as expected for the constitutive up phenotype. The properties of the mutated genes and their role in transducing environmental signals known or proposed to affect hrp expression will be discussed.

Finally, the size and complexity of the HrpL-dependent regulon has not been determined in any P. syringae strain. A unifying feature of the hrp-encoded PTA and the proteins secreted by this apparatus is their regulation by HrpL. For example, all avr genes characterized thus far from P. syringae strains and the dsp genes of E. amylovora are regulated by HrpL. This opens the possibility of using this property to identify novel pathogenicity and host-range determinants in P. syringae strains. For these experiments, we developed a simple plate assay to identify HrpL-regulated genes. The properties of this genetic screen and its application to identifying P. syringae pathogenicity factors will be presented.

References
1. Hutcheson SW, 1997. In Stacey G, Keen N, eds, Plant-Microbe Interactions, Vol. 3. Chapman & Hall, NewYork, pp. 145-179.