1.6.4S
CHARACTERIZATION OF HRP and AVR GENES ON THE FLANKS OF THE PSEUDOMONAS SYRINGAE HRP CLUSTER

DE FOUTS1, AO CHARKOWSKI1, JR ALFANO2 and A COLLMER1

1Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA; 2Department of Biological Sciences, University of Nevada, Las Vegas, NV 89154, USA

Background and objectives
Pseudomonas syringae is a plant pathogen whose individual strains are classified into pathovars largely on the basis of host specificity. As is characteristic of the common Gram-negative plant pathogenic bacteria, elicitation of the hypersensitive response (HR) in non-hosts or pathogenesis in hosts is dependent on hrp/c genes [1]. The hrp/c genes encode the type III protein secretion pathway that Pseudomonas syringae uses to secrete at least two classes of protein, harpins and Avrs. Harpins are glycine-rich, cysteine-lacking proteins secreted in culture when the type III system is expressed, which possess heat-stable HR elicitor activity when infiltrated into the leaf intercellular spaces of tobacco and several other plants. Avr proteins are not secreted in culture and have no apparent effect when infiltrated into the intercellular spaces of leaves. There is now strong, but indirect, evidence that many Avr proteins are transferred to the interior of plant cells by the Hrp systems of Pseudomonas and Xanthomonas spp., and that at least one pair of avr-R gene products (AvrPto-Pto) physically interact within the plant cell cytoplasm. According to a current model for P. syringae-plant interactions, harpins may aid transfer of multiple 'Avr' proteins across the plant cell wall and into plant cells, where the Avrs either collectively promote parasitism or individually betray the parasite to the host R gene surveillance system. Genes encoding the type III pathway are clustered on plasmids, or in pathogenicity islands containing related virulence functions including genes for proteins that travel the type III secretion pathway. To identify all the proteins secreted by the type III pathway in P. syringae, we have begun characterization of genes flanking two P. syringae hrp/c clusters.

Results and conclusions
Cosmids that contain DNA flanking one side of the hrp/c genes encoding the type III secretion pathway were isolated from P. syringae pv. tomato DC3000 and P. syringae pv. syringae B728a genomic libraries. Sequence analysis of the cosmids identified five operons, four of which were partially characterized by Lorang et al. [2], in the hrplc regulon between housekeeping genes and the core hrplc cluster. These operons are conserved between the two pathovars, and DNA hybridization analysis indicated that the operons are widespread among P. syringae pathovars. Complementation group 11 (sensu Lorang et al. [2] encodes a lysozyme homologue, groups III and IV encode the avrE locus, and group V encodes a single gene, hrpW, which encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. Like other harpins, HrpW is heat-stable, glycine-rich, cysteine-lacking, secreted by the Hrp system, and able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1-186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187-425) is similar to Pels from Nectria haematococca and Erwinia carotovora, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A230 assays under a variety of reaction conditions, but the Pel fragment binds to calcium pectate. AhrpZ::nptil or hrpW::0Sp' P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in an hrpz hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is the plant cell wall. The hrpW gene is flanked by a putative avr gene, which is flanked by several genes which are apparently not part of the hrplc regulon, suggesting that this putative avr gene is one edge of the P. syringae hrp region. The contribution of the putative avr gene and of these five operons collectively to HR elicitation and pathogenicity is being tested.

References
1. Alfano JR, Colimer A, 1997. Journal of Bacteriology 179, 5655-5662.
2. Lorang JM, Keen NT, 1995. Molecular Plant-Microbe Interactions 8, 49-57.