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Fig. 1. The cluster of Psy 61 genes carried on pHIR11 that enables nonphytopathogenic bacteria to elicit the HR in tobacco. hopPsyA (checkered) encodes an effector protein that apparently is delivered into plant cells. Other genes encode regulatory factors (shaded), Hrc components associated with export across the inner membrane (diagonal hatching) or outer membrane (cross hatching), extracellular Hrp proteins (stippled), or proteins with unknown function (open boxes). Squares on arrows denote the presence of HrpL-activated promoters (55).

divergent strain in our investigation of the evolution and function of Hrp systems.

In the last decade, research on the evolution and function of type III secretion systems in Salmonella and Yersinia spp. has yielded two revolutionary insights. First, genes associated with pathogenicity, such as those encoding type III secretion systems, are often clustered in horizontally acquired pathogenicity islands (Pais) that may enable the evolution of virulence in “quantum leaps ” (12, 13). Second, type III secretion systems have the remarkable ability to inject bacterial proteins into the cytoplasm of eukaryote host cells (14, 15). In this article, we will describe our progress in understanding how the P. syringae Hrp system expressed from pHIR11 enables a nonpathogen like E. coli to make a quantum leap in its ability to interact with plants by eliciting the HR, how hrp/hrc genes are arranged in Hrp Pais that also encode a variety of putative effectors, and how universal targeting signals and genetically dissectable secretion mechanisms underlie effector protein traffic through the pathway.

HopPsyA, pHIR11, and the Minimum Genetic Unit for Bacterial Elicitation of the Hypersensitive Response

Cosmid pHIR11 was seminal in establishing the minimum genetic requirements and relative role of the Hrp system and effectors in HR elicitation. pHIR11 was cloned from Psy 61 on the basis of its ability to complement several hrp::Tn5 mutations in that strain (7). It also enables P. fluorescens, P. putida, and E. coli (and probably many other Gram-negative bacteria) to elicit the HR in tobacco. However, pHIR11 does not enable nonpathogens to multiply or cause disease in any plants tested. For example, P. fluorescens (pHIR11) does not cause any symptoms in tobacco leaves unless inoculated at a very high level (≥5 × 106 cell/ml), such that enough individual plant cells undergo the HR to produce a confluent collapse. The DNA sequence of pHIR11 reveals a 25-kb cluster of hrp/hrc genes linked to an apparent operon encoding hopPsyA (hrmA) and ORF1 (16, 17, 18, 19, 20, 21 and 22) (Fig. 1). The hrp/hrc clusters of Psy B728a and Pto DC3000 are arranged similarly (further discussed below), but HopPsyA is unique to Psy 61 (18, 23). Three proteins, the HrpZ and HrpW harpins and HrpA pilin, are secreted by the P. syringae Hrp pathway in culture more abundantly than other Hrp-dependent proteins (24, 25, 26 and 27). Harpins are glycine-rich cysteine-lacking proteins that possess heat-stable HR elicitor activity when infiltrated at relatively high concentration into the intercellular leaf spaces of many plants ( 5, 28). However, in P. syringae their HR-elicitation activity does not correlate with bacterial host range, and these proteins appear to have an ancillary role in plant interactions (21). HrpA forms a Hrp-specific pilus that is 6–8 nm in diameter and is essential for all Hrp phenotypes (26).

Through a series of observations, HopPsyA was identified as the HR-triggering effector that is injected into plant cells by the pHIR11 Hrp system, and it was simultaneously shown to have salient characteristics of known Avr proteins: (i) Mutations in hopPsyA abolish the ability of pHIR11 to direct HR elicitation without affecting HrpZ production or secretion, indicating that the essential role of HopPsyA is not as a component of the Hrp secretion system (29). (ii) HopPsyA travels the Hrp pathway, as demonstrated by its secretion in culture (discussed below) (30). (iii) HopPsyA has no apparent effect when delivered exog-

Fig. 2. Summary of evidence that HopPsyA functions like an avirulence protein that interacts inside plant cells with the product of an R gene present in N. tabacum but not N. benthamiana. The upper squares, labeled “pHIR11,” indicate the responses in leaves of N. tabacum (N.t.) and N. benthamiana (N.b.) to P. fluorescens 55 carrying pHIR11 (+) or a hopPsyA::TnphoA derivative (−) after infiltration at a concentration of 5 × 107 cells/ml. “HR” indicates rapid confluent collapse of infiltrated tissue; “Null” indicates no visible response. The next two photographs, labeled “Agrobacterium,” show the effect in N.t. and N.b. of A. tumefaciens GV3101-mediated transient expression of hopPsyA via glucocorticoid-inducible expression vector pTA7002 (85). Plants receiving pTA7002 (−) or pTA7002::hopPsyA (+) were sprayed with the glucocorticoid dexamethasone 48 h after infiltration and then photographed 24 h later. Note that the confluent tissue collapse indicative of the HR is observed only when hopPsyA is expressed in the N.t. leaf. The lower two photographs, labeled “P. s. tabaci,” show the effect in N.t. leaves of P. syringae pv tabaci 11528 carrying empty vector pDSK519 (−) or pCPP2349 (hopPsyA+) (+) at 1 and 5 days after inoculation (23). The level of inoculum was 5 × 108 in the lowest sector on each side of each leaf and 5 × 106 cell/ml in the next sectors up. Note that the HR developed by the end of day 1 in the sector infiltrated with 5 × 108 cells/ml of P. syringae pv tabaci (pCPP2349), whereas disease symptoms caused by P. syringae pv tabaci(pDSK519) developed later, with a lower level of inoculum, and were uniquely marked with the bright yellow chlorosis characteristic of wildfire. [Reproduced with permission from ref. 23 (Copyright 1997, Mol. Plant–Microbe Interact.).]

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