These two authors contributed equally to this work. SummaryThe Pseudomonas syringae pv. tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants. Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphE Pto , AvrPpiB1 Pto , AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Mixed inoculum and Agrobacterium-mediated transient expression experiments con®rmed that suppressor action occurred within plant cells. These suppressors, with the exception of AvrPpiB1 Pto , inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a. DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Additionally, HopPtoG was identi®ed as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.

The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants depends on the Hrp (hypersensitive response and pathogenicity) type III protein secretion system and the effector proteins it translocates into plant cells. Through iterative application of experimental and computational techniques, the DC3000 effector inventory has been substantially enlarged. Five homologs of known avirulence (Avr) proteins and five effector candidates, encoded by genes with putative Hrp promoters and signatures of horizontal acquisition, were demonstrated to be secreted in culture and͞or translocated into Arabidopsis in a Hrp-dependent manner. These 10 Hrp-dependent outer proteins (Hops) were designated HopPtoC (AvrPpiC2 homolog), HopPtoD1 and HopPtoD2 (AvrPphD homologs), HopPtoK (AvrRps4 homolog), HopPtoJ (AvrXv3 homolog), HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1 (an ADP-ribosyltransferase homolog). Analysis of the enlarged collection of proteins traveling the Hrp pathway in P. syringae revealed an export-associated pattern of equivalent solvent-exposed amino acids in the Nterminal five positions, a lack of Asp or Glu residues in the first 12 positions, and amphipathicity in the first 50 positions. These characteristics were used to search the unfinished DC3000 genome, yielding 32 additional candidate effector genes that predicted proteins with Hrp export signals and that also possessed signatures of horizontal acquisition. Among these were genes encoding additional ADPribosyltransferases, a homolog of SrfC (a candidate effector in Salmonella enterica), a catalase, and a glucokinase. One ADP-ribosyltransferase and the SrfC homolog were tested and shown to be secreted in a Hrp-dependent manner. These proteins, designated HopPtoS2 and HopPtoL, respectively, bring the DC3000 Hrp-secreted protein inventory to 22.

Pseudomonas syringae pv. tomato strain DC3000 is a pathogen of tomato and Arabidopsis. The hrp-hrcencoded type III secretion system (TTSS), which injects bacterial effector proteins (primarily called Hop or Avr proteins) into plant cells, is required for pathogenicity. In addition to being regulated by the HrpL alternative sigma factor, most avr or hop genes encode proteins with N termini that have several characteristic features, including (i) a high percentage of Ser residues, (ii) an aliphatic amino acid (Ile, Leu, or Val) or Pro at the third or fourth position, and (iii) a lack of negatively charged amino acids within the first 12 residues. Here, the well-studied effector AvrPto was used to optimize a calmodulin-dependent adenylate cyclase (Cya) reporter system for Hrp-mediated translocation of P. syringae TTSS effectors into plant cells. This system includes a cloned P. syringae hrp gene cluster and the model plant Nicotiana benthamiana. Analyses of truncated AvrPto proteins fused to Cya revealed that the N-terminal 16 amino acids and/or codons of AvrPto are sufficient to direct weak translocation into plant cells and that longer N-terminal fragments direct progressively stronger translocation. AvrB, tested because it is poorly secreted in cultures by the P. syringae Hrp system, was translocated into plant cells as effectively as AvrPto. The translocation of several DC3000 candidate Hop proteins was also examined by using Cya as a reporter, which led to identification of three new intact Hop proteins, designated HopPtoQ, HopPtoT1, and HopPtoV, as well as two truncated Hop proteins encoded by the naturally disrupted genes hopPtoS4::tnpA and hopPtoAG::tnpA. We also confirmed that HopPtoK, HopPtoC, and AvrPphE Pto are translocated into plant cells. These results increased the number of Hrp system-secreted proteins in DC3000 to 40. Although most of the newly identified Hop proteins possess N termini that have the same features as the N termini of previously described Hop proteins, HopPtoV has none of these characteristics. Our results indicate that Cya should be a useful reporter for exploring multiple aspects of the Hrp system in P. syringae.Pseudomonas syringae is a host-specific bacterial pathogen of plants whose various pathovars cause necrotic lesions on leaves or fruits of susceptible plants and elicit the hypersensitive response (HR), a defense-associated localized programmed cell death, in resistant plants. P. syringae interactions with plants depend on the hrp (for "hypersensitive response and pathogenicity") gene cluster, which encodes a type III secretion system (TTSS) or Hrp system that injects bacterial proteins into plant cells (1). Similar TTSSs exist in a variety of other gram-negative plant and animal pathogens (13, 28). The P. syringae proteins injected into host cells by the Hrp system are primarily known as effectors or Avr (avirulence) or Hop (Hrp-dependent outer protein) proteins. Hop proteins may contribute to P. syringae virulence by suppressing plant immunity. For example, AvrB, AvrRpm1...

The Pseudomonas syringae type III secretion system (TTSS) translocates effector proteins into plant cells. Several P. syringae effectors require accessory proteins called type III chaperones (TTCs) to be secreted via the TTSS. We characterized the hopO1-1, hopS1, and hopS2 operons in P. syringae pv. tomato DC3000; these operons encode three homologous TTCs, ShcO1, ShcS1, and ShcS2. ShcO1, ShcS1, and ShcS2 facilitated the type III secretion and/or translocation of their cognate effectors HopO1-1, HopS1, and HopS2, respectively. ShcO1 and HopO1-1 interacted with each other in yeast two-hybrid and coimmunoprecipitation assays. Interestingly, ShcS1 and ShcS2 were capable of substituting for ShcO1 in facilitating HopO1-1 secretion and translocation and each TTC was able to bind the other's cognate effectors in yeast two-hybrid assays. Moreover, ShcO1, ShcS1, and ShcS2 all bound to the middle-third region of HopO1-1. The HopS2 effector possessed atypical P. syringae TTSS N-terminal characteristics and was translocated in low amounts. A site-directed HopS2 mutation that introduced a common N-terminal characteristic from other P. syringae type III secreted substrates increased HopS2 translocation, supporting the idea that this characteristic functions as a secretion signal. Additionally, hopO1-2 and hopT1-2 were shown to encode effectors secreted via the DC3000 TTSS. Finally, a DC3000 hopO1-1 operon deletion mutant produced disease symptoms similar to those seen with wild-type DC3000 but was reduced in its ability to multiply in Arabidopsis thaliana. The existence of TTCs that can bind to dissimilar effectors and that can substitute for each other in effector secretion provides insights into the nature of how TTCs function.

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