1.6.17
ASSESSING THE ABILITY OF HETEROLOGOUSLY EXPRESSED HRP GENE CLUSTERS TO DELIVER AVR PROTEINS AND PROMOTE BACTERIAL PATHOGENESIS

DE FOUTS1, JR ALFANO2, AO CHARKOWSKI1, JH HAM1 and A COLLMER1

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

Background and objectives
The hrp gene clusters of phytopathogenic bacteria are ca 24 kb in size and encode the type III protein secretion machinery, regulators, and other accessory factors which are necessary for virulence. Pseudomonas syringae hrp mutants behave essentially like non-pathogens in plants, which indicates that proteins traveling the Hrp pathway must be essential for bacterial parasitism. The P. syringae Hrp system appears to secrete at least three classes of proteins: (i) harpins, which are glycine-rich, cysteine-lacking, heat-stable proteins with HR elicitor activity when introduced at high concentrations into the apoplast of certain plants; (ii) an Hrp pilus protein (HrpA); and (iii) Avr proteins/Hops (Hrp-dependent outer proteins) [1]. Analysis of hrp gene clusters and effector proteins from several distantly related phytopathogenic bacteria have indicated that the components encoding the type Ill secretion machinery are conserved while the avr genes are not conserved. It is the Avr proteins that confer host specificity at the race-cultivar level by interactions with specific R-gene products that are produced by certain non-host or resistant host plants. The cloned 24-kb hrp cluster from P. syringae pv. syringae 61 (pHIRI 1) can enable both P. fluorescens and E. coli to elicit the HR, but it fails to enable autonomous growth of these saprophytes [2]. This is presumably because these saprophytes lack the necessary effector proteins to enable pathogenesis. This supports a model whereby the primary function of the Hrp system is to deliver effector proteins to plant cells, and host specificity is determined by the collection of Avr proteins that are delivered and not by the protein components of the Hrp secretion system. To test this model, a complete deletion of the P. syringae pv. tomato DC3OOO (Pst DC3OOO)hrp gene cluster is being constructed, replacing all genes from hrpK through hrpR with chloramphenicol acetyl transferase (cat). Complementation of HR- and pathogenesis- phenotypes by pHIRI 1 and hrp gene clusters from other phytopathogenic bacteria will be attempted. The ability of these heterologous hrp gene clusters to secrete Avr proteins into the media and in planta is also being examined.

Results and conclusions
Sequence analysis of cosmid clones which contain the DNA flanking the Pst DC3OOO hrp cluster has enabled the engineering of plasmid constructs needed to generate a total deletion of the hrp gene cluster. A system for detecting the Hrp-dependent secretion of P. syringae Avr proteins into bacterial culture media has been developed: E. coli harbouring hrp genes encoding a functional type Ill secretion system from Erwinia chrysanthemi has been found to secrete AvrB and AvrPto into the culture media. The secretion event was detected using monoclonal antibodies against a FLAG epitope. To detect secretion of Avr proteins expressed under native conditions by heterologous hrp clusters, antibodies to AvrB, AvrPto and HrmA were generated. Experiments involving cloned functional hrp gene clusters operating heterologously in both saprophytes and pathogens are now being performed to address fundamental questions regarding the function of Hrp systems in Avr protein delivery and the determination of host specificity.

References
1. Alfano JR, Comer A, 1997. Journal of Bacteriology 179, 5655-5662.
2. Gopalan SD, Bauer DW, Alfano JR et al., 1996. Plant Cell 8,1095-1105.