1.7.5S
PLANT-BACTERIAL INTERFACE: PSEUDOMONAS SYRINGAE EXTRACELLULAR PROTEINS CONTROLLED BY THE HRP SYSTEM

S HE1, J YUAN1, W WEI1, E ROINE2, M ROMANTSCHUK 2, W HU1, S GOPALAN1 and A JONES1

1Plant Research Laboratory, Michigan State University, East Lansing, USA; 2Department of Biosciences, University of Helsinki, Helsinki, Finland

Background and objectives
Hypersensitive reaction and pathogenicity (hrp) genes are essential for the majority of bacteria to elicit the hypersensitive reaction (HR) in resistant plants and to cause disease in susceptible plants. The Pseudomonas syringae hrp gene cluster consists of 27 open reading frames and is a core part of a pathogenicity island that spans over 35 kb in the bacterial genome. Gene homology suggests that many hrp genes encode components of a type-III secretion system, known as the Hrp system. We will discuss extracellular proteins secreted via the P. syringae pv. tomato (Pst) strain DC3000 Hrp system and an hrp-encoded bacterial surface appendage that is required for Pst to cause disease in Arabidopsis and to elicit HR in tobacco and tomato.

Results and conclusions
In hrp-inducing minimal medium, Pst DC3000 produces and secretes at least five proteins via the Hrp system, three of which have been shown to be encoded by hrp genes: HrpZ, HrpW and HrpA. Purified HrpZ protein of P. syringae pv. syringae has been shown to induce HR, systemic acquired resistance (SAR), and a novel plant defence gene, hinl. However, a non-polar hrpZ mutation of P. s. pv. syringae does not affect bacterial pathogenicity or HR elicitation. Results from Dr Alan Collmer's laboratory show that HrpW also elicits an HR in tobacco leaves and that it has several characteristics of harpin proteins. In addition, HrpW has a pectate lyase domain, although no pectate lyase activity has been detected. HrpA has been identified as a major structural component of a novel, hrp-dependent bacterial surface appendage named the Hrp pilus. Non-polar mutations in the hrpA gene eliminate the ability of Pst DC3000 to produce Hrp pili in vitro, to cause disease in Arabidopsis or to elicit HR in tobacco or tomato. Purified HrpA protein forms a pilus-like structure in vitro. A rabbit antibody against purified HrpA protein binds to the Hrp pilus.

It has been observed that injection of purified Avr proteins into the plant apoplast (outside the plant cell) does not result in HR. However, we showed that expression of the P. syringae pv. glycinea avrb gene directly in Arabidopsis cells containing the corresponding disease resistance gene, RPM1, resulted in HR, providing evidence that the action site of AvrB is inside the plant cell. Because the phenotypic expression of AvrB in bacteria is dependent on hrp genes, we argued that the AvrB protein must also be secreted via the Hrp secretion system. But unlike HrpZ or HrpA, AvrB must be secreted directly into the plant cell. The non-polar hrpA mutant apparently fails to deliver the AvrB signal to the plant cell, suggesting that formation of the Hrp pilus is important in the transport of AvrB protein from bacteria to plant cells.

Using a polyclonal antibody against partially purified Hrp pili, we have detected three additional proteins associated with Hrp pili: two of approximately 68 kDa in size and one of 49 kDa. These three proteins were detected in the Hrp pilus preparation from Pst DC3000, but not from the hrcC, hrpS or hrpA mutant. We are investigating whether the three proteins are additional subunits of the Hrp pilus.

References
1. Yuan J, He SY, 1996. Journal of Bacteriology 178, 6399-6402.
2. Roine E, Wei W, Yuan J et al., 1997. Proceedings of the National Academy of Sciences, USA 94, 3459-3464.