The Plant Journal (2004) 37, 554±565 doi: 10.1046/j.1365-313X.2003.01982.x Identi®cation of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast Yashitola Jamir1,y, Ming Guo1,y, Hye-Sook Oh2, Tanja Petnicki-Ocwieja1, Shaorong Chen1, Xiaoyang Tang3, Martin B. Dickman1, Alan Collmer2 and James R. Alfano1, 1 Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, NE 68588-0660, USA, 2 Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203, USA, and 3 Department of Plant Pathology, Kansas State University, Manhattan, KS 66502-5502, USA Received 20 September 2003; revised 21 October 2003; accepted 4 November 2003. For correspondence (fax 1 402 472 3139; e-mail [email protected]). y These two authors contributed equally to this work. Summary The 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 AvrPphEPto, AvrPpiB1Pto, 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 AvrPpiB1Pto, 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. Keywords: type III effectors, Avr proteins, bacterial plant pathogens, plant defense, innate immunity, programmed cell death. Introduction Pseudomonas syringae is a host-speci®c plant pathogen whose interactions with plants can take two strikingly different courses. In virulent interactions with susceptible plants, the bacteria multiply for several days before producing visible symptoms (typically necrotic lesions on leaves and fruit), whereas in avirulent interactions with resistant plants, the bacteria trigger a rapid, localized, defense-associated, programmed cell death (PCD) known as the hypersensitive response (HR; Alfano and Collmer, 1996). Both outcomes are controlled by effector proteins that are injected into plant cells by a type III secretion system (TTSS) encoded by HR and pathogenicity (hrp) and hrp conserved (hrc) genes (Alfano and Collmer, 2001). The HR can be triggered in a gene-for-gene manner if the product of an effector gene is recognized by the product of a matching 554 resistance (R) gene in the plant (Keen, 1990). Plant hypersensitivity is normally triggered only by pathogens, but the ability to elicit the HR can be conferred on non-pathogens such as P. ¯uorescens and Escherichia coli if the bacteria carry a cloned cluster of P. syringae hrp/hrc genes and a gene encoding an effector that is recognized by the test plant (Gopalan et al., 1996; Huang et al., 1988; Pirhonen et al., 1996). Relatively less is known about virulent interactions in susceptible plants, but unlike elicitation of the HR, successful parasitism appears to require multiple TTSS effectors. Indeed, genomic searches for TTSS effector genes in genome of P. syringae tomato DC3000 has revealed 33 con®rmed effectors and several effector candidates (Buell et al., 2003; Collmer et al., 2002; Guttman et al., 2002; ß 2004 Blackwell Publishing Ltd Pseudomonas type III effectors that suppress PCD 555 Petnicki-Ocwieja et al., 2002; Zwiesler-Vollick et al., 2002). This plethora of effector genes presents both puzzles and problems in current research. Puzzling is the function for so many effectors and the means by which these effectors can evade recognition by the R gene surveillance systems of tomato and Arabidopsis, the hosts of DC3000. Problematic is the apparent redundancy of effectors, as indicated by the observation that mutations in individual effector genes typically have little or no phenotype in plant interactions (White et al., 2000). Although many effectors have strong, gain-of-function, avirulence (Avr) phenotypes when heterologously expressed in a P. syringae strain whose normal host carries an appropriate R gene, this reveals little about the function of such effectors in virulence (White et al., 2000). Important clues to effector function in virulence were gained when Jackson et al. (1999) discovered that some effectors, such as the P. syringae phaseolicola VirPphA, can block the ability of other `masked' effectors to trigger the HR, which suggested that VirPphA `may allow subversion of the HR and lead to disease development'. Additional effectors AvrPphC and AvrPphF were found to have a similar ability (Tsiamis et al., 2000), and a previous report of the ability of heterologously expressed avrRpt2 to be epistatic over avrRpm1 in the elicitation of characteristic HRs (Reuber and Ausubel, 1996; Ritter and Dangl, 1996) provides further support for the concept of effector interference in planta. Indeed, several P. syringae effectors were recently shown to suppress plant defenses (Abramovitch et al., 2003; Axtell and Staskawicz, 2003; Bretz et al., 2003; Espinosa et al., 2003; Mackey et al., 2003). The concept of suppressive effectors encourages the development of new tools to support functional genomic investigation of the large (and growing) inventory of P. syringae tomato DC3000 TTSS effectors is important to determine their effects inside plant cells. One promising resource is cosmid pHIR11, which carries a 25-kbp section of the P. syringae pv. syringae 61 Hrp pathogenicity island expressing a functional TTSS and, HopPsyA, an effector that is recognized hypersensitively by tobacco and Arabidopsis Ws-0 and is secreted in a TTSS- and ShcA chaperone-dependent manner (Alfano et al., 1997; Huang et al., 1988; van Dijk et al., 2002). pHIR11 is a particularly useful tool for Hrp TTSS effector research because it enables: (i) non-pathogens to elicit the HR in experimentally amenable tobacco plants; (ii) the action in planta of one or a few effectors to be studied in the absence of other effectors and virulence factors; and (iii) effector actions to be studied without the confounding effects of overexpression that may occur when effectors are transgenically or transiently expressed via Agrobacterium tumefaciens or viral vectors. Here, we used pHIR11 to systematically test 19 con®rmed P. syringae tomato DC3000 effectors (designated as Hop or Avr proteins) for their ability to suppress the HR that is ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 normally elicited in tobacco by P. ¯uorescens(pHIR11) and found that ®ve effectors suppressed the pHIR11-dependent HR. Our results show that these effectors exert suppression by acting inside plant cells. First, in planta bacterial mixing experiments that deliver an Avr protein (HopPsyA) from one bacterial strain and type III effector from a different bacterial strain retain HR suppression ability; second, certain P. syringae tomato DC3000 effector mutants have increased ability to elicit the HR in tobacco suggesting that they lack an HR suppressor; third, each suppressor functions when expressed in plants via Agrobacteriummediated transient expression experiments; and ®nally, several of the effectors that suppress the HR can also suppress Bax-triggered PCD in yeast and plants. Our study indicates that AvrPphEPto, AvrPpiB1Pto, HopPtoE, AvrPtoB, HopPtoF, and HopPtoG effectors possess suppressor activity, which provides a global picture of the capacity of this bacterium to regulate PCD pathways in plants. Results pHIR11 assays identify five effectors capable of complete suppression of the HopPsyA-dependent HR In the course of experiments with con®rmed DC3000 type III effectors, we observed that the effector HopPtoD2 was capable of suppressing the HR elicited by P. syringae phaseolicola on Nicotiana benthamiana plants (Espinosa et al., 2003). These results prompted us to expand our screen for HR suppressors to include many of the recently identi®ed DC3000 effectors (Collmer et al., 2002). To do this, we used the pHIR11 system, which allows non-pathogens such as E. coli and P. ¯uorescens to elicit the HR and secrete effectors in culture via the TTSS. This tool allowed us to test whether individual effectors suppressed the HopPsyAdependent HR as depicted in Figure 1(a). We in®ltrated P. ¯uorescens(pHIR11) strains carrying a number of different effector constructs into tobacco (N. tabacum cv. Xanthi). Interestingly, HopPtoD2, the effector that suppressed an HR elicited by P. syringae phaseolicola, did not suppress the HopPsyA-dependent HR (Figure 1b). We cloned 19 con®rmed effector genes into a broad-host-range plasmid and tested if the encoded effectors were able to suppress the HR elicited by P. ¯uorescens(pHIR11) (see Figure 1b for a list of the effectors tested). We expressed each candidate suppressor gene in P. ¯uorescens(pHIR11) and in®ltrated these strains into tobacco and Arabidopsis Ws-0, two plants that produce an HR in response to pHIR11-containing bacteria. Surprisingly, several of the effectors tested were able to suppress the pHIR11-dependent HR on both Arabidopsis and tobacco (Figure 1c,d). Two of the identi®ed suppressors, HopPtoF and AvrPtoB, were homologs of AvrPphF and VirPphA, 556 Yashitola Jamir et al. Figure 1. Identi®cation of P. syringae tomato DC3000 effectors that suppress the HR on tobacco and Arabidopsis. (a) Schematic representation of the pHIR11based suppression assay in P. ¯uorescens (Pf) 55. When DC3000 effectors are individually expressed in trans in Pf(pHIR11), they can potentially suppress the HopPsyA-dependent HR. (b) List of DC3000 effectors that were tested in the pHIR11 assay. `y' indicates that the effector inhibited the HR, `n' indicates that it did not, and `y' indicates that it partially suppressed the HR. Refer to Experimental procedure for information regarding effector constructs. (c) Nicotiana tabacum cv. Xanthi leaves were in®ltrated with Pf(pHIR11) with different effector constructs (noted above each picture). Complete suppression of the HR is denoted by `N'. (d) The same strains described in panel (c) were in®ltrated into Arabidopsis Ws-0, producing identical results. (c,d) The fraction underneath each picture indicates the number of times the results shown were observed over the number of times the experiment was performed. respectively, two Avr proteins able to `block' the HR produced by P. syringae phaseolicola (Jackson et al., 1999; Tsiamis et al., 2000). The VirPphA homolog AvrPtoB was recently reported to suppress the HR elicited by AvrPto (Abramovitch et al., 2003). The other HR suppressors identi®ed were AvrPphEPto, AvrPpiB1Pto, HopPtoD1, HopPtoE, and HopPtoK. The HR suppression observed for HopPtoD1 and HopPtoK was not complete (i.e. the HR was present, although much reduced). Because their phenotypes were different from the proteins that completely suppressed the production of the HopPsyA-dependent HR, we decided not to pursue further the apparent suppression activity of HopPtoK and HopPtoD1 in the experiments described below. One possible explanation for the observed phenotypes was that the type III effectors were blocking the type III secretion of other type III substrates, including Avr proteins. There is actually a precedent for type III substrates, such as HrpZ and HrpW, to block the type III secretion of proteins from P. syringae (Alfano et al., 1996; Charkowski et al., 1998). To eliminate the possibility that HopPtoE affected the ability of P. ¯uorescens(pHIR11) to deliver the Avr protein HopPsyA into plant cells, we used a bacterial strain to deliver HopPsyA different from that used to deliver Figure 2. Evidence that type III suppressors function inside plant cells. (a) The suppressors retain their activity when delivered by different bacterial cells from those that deliver HopPsyA. P. ¯uorescens(pHIR11) (Pf(pHIR11)) mixing experiments in N. tabacum cv. Xanthi show that HR suppression can occur when HopPtoE and HopPsyA are TTSS delivered by different bacteria. pLN18 is a pHIR11 derivative that lacks hopPsyA, but encodes a functional TTSS. pCPP2089 (Huang et al., 1991) is a pHIR11 derivative encoding a defective TTSS. (b) The HopPsyA-dependent HR can be suppressed via Agrobacteriumtransient expression of effectors. N. tabacum cv. Xanthi leaves were coin®ltrated with A. tumefaciens (At) C58C1 carrying phopPsyA and another strain carrying each candidate suppressor. All of the suppressive effectors identi®ed in the pHIR11 screen also suppressed the HR elicited by HopPsyA in this test. (c) Immunoblot of plant tissues with different agroin®ltrations shows that each HA epitope-tagged effector was made in planta. The asterisks indicates a protein of the predicted size of the effector in that lane. ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 Pseudomonas type III effectors that suppress PCD 557 HopPtoE into plant cells. To accomplish this, we constructed a pHIR11 derivative, pLN18, which lacks hopPsyA and shcA, a gene that encodes a chaperone for HopPsyA (van Dijk et al., 2002). P. ¯uorescens(pLN18) does not elicit an HR on tobacco because it lacks HopPsyA (Figure 2a), while maintaining the ability to secrete proteins via its functional TTSS (data not shown). We performed in planta mixed-inoculum experiments by ®rst in®ltrating into tobacco P. ¯uorescens(pLN18) with hopPtoE contained in a broad-host-range plasmid and, after 2 h, P. ¯uorescens(pHIR11) was in®ltrated at an OD600 suf®cient to cause HR elicitation. Figure 2(a) shows that P. ¯uorescens(pLN18) retained the ability to suppress the pHIR11-dependent HR. This indicates that the HR suppression activity does not occur in the bacterial cell. Other in planta mixed-inoculum experiments similar to those described in Figure 2(a) were carried out on effectors that showed suppressor activity, and these experiments demonstrated that all of the identi®ed suppressors were able to inhibit the pHIR11dependent HR (data not shown). These results indicate that the site of suppressor activity was outside of the bacteria. Agrobacterium transient assays that co-deliver HopPsyA and individual HR suppressors confirm that each effector suppresses the HopPsyA-dependent HR inside plant cells To determine if the HR suppression is solely because of the suppressor proteins, we co-delivered and transiently expressed each suppressor separately with HopPsyA using Agrobacterium-mediated transient assays (agroin®ltrations; Van den Ackerveken et al., 1996). In each case, the effector suppressed the HopPsyA-dependent HR (Figure 2b). We con®rmed with immunoblots that the agroin®ltrations produced both HopPsyA and the speci®c suppressor tested (Figure 2c). These data complement the bacteria-delivered suppressor data shown above because agroin®ltrations demonstrate that the suppressor activity is dependent on suppressor action inside plant cells, whereas the experiments using P. ¯uorescens(pHIR11) more closely resemble what happens in nature, and protein levels are closer to the levels that the pathogen `injects' into plant cells. All the suppressors, with the exception of AvrPpiB1, were capable of suppressing expression of the tobacco pathogenesis-related (PR) gene PR1a induced during plant defense To determine the effect the HR suppressors have on other common hallmarks of the plant defense response, we determined whether the suppressors inhibited the induction of the PR transcript PR1a by performing relativequantitative RT-PCR experiments using RNA isolated from tobacco plants challenged with P. ¯uorescens(pHIR11) strains carrying different suppressors. We in®ltrated ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 Figure 3. Type III HR suppressors also suppress the expression of the PR gene PR1a. RT-PCR results from samples of tobacco plants that were in®ltrated with P. ¯uorescens(pHIR11) (Pf(pHIR11)) with different effector constructs as shown in Figure 1(c) show that all the suppressors, except AvrPpiB1Pto, reduce the induction of PR1a. The PCR reaction was a multiplex with two primer sets. The ®rst annealed to a 500-bp region within the coding region of the tobacco PR gene PR1a and the second set was to 18S rRNA gene. Competimers to the 18S rRNA gene were used to reduce the ampli®ed product within a linear range of PR1a (See Experimental procedure for more details). This experiment was repeated 6 times with similar results. tobacco plants with P. ¯uorescens(pHIR11) strains, and after 5 h, harvested plant tissue and isolated RNA. Using these RNA samples as templates, we performed multiplex RT-PCR experiments using primers that recognize the tobacco PR1a gene and the 18S rRNA. PR1a transcript was reduced or absent from samples isolated from plants challenged with most of the bacterial strains containing a suppressor compared to samples from control plants (Figure 3). The only exception was observed for samples isolated from plants challenged with P. ¯uorescens(pHIR11, pavrPpiB1Pto), which contained similar amounts of the PR1a product as control plants challenged with P. ¯uorescens(pHIR11) alone. Thus, the HR suppressors AvrPphEPto, AvrPtoB, HopPtoE, and HopPtoF also suppressed the induction of the PR transcript PR1a. DC3000 suppressor mutants display an enhanced ability to elicit an HR on non-host plants consistent with reduction of HR suppression activity in the pathogen Based on our ®ndings, we were cognizant that a pathogen may encode multiple HR suppressors, each contributing, perhaps incrementally, to the suppression of the HR and/or plant defenses. To analyze these proteins in more detail, we made mutants defective in each effector corresponding to the effector list shown in Figure 1(b). As effectors are likely to have functionally redundant roles, which may partially mask a phenotype, we performed a more sensitive HR assay where we in®ltrated 10-fold serially diluted bacterial strains into tobacco leaf panels to detect any subtle difference in the ability of different strains of bacteria to elicit an HR. When the DC3000 hopPtoE mutant UNL139 was tested in this assay, we found that it was more effective than DC3000 at HR elicitation at lower cell density (Figure 4a). Interestingly, when hopPtoE was provided in trans to UNL139, the mutant strain was less effective at HR elicitation than DC3000 (Figure 4a). Thus, the enhanced HR 558 Yashitola Jamir et al. phenotype of the hopPtoE mutant was complemented by hopPtoE provided in trans. These observations are consistent with HopPtoE acting as an HR suppressor and suggest that HopPtoE contributes incrementally to the ability of the pathogen to suppress the HR. We tested other DC3000 mutants defective in the effectors listed in Figure 1(b), which includes many effector genes that were not identi®ed as suppressor genes in our other assays, for their ability to produce an enhanced HR phenotype. Interestingly, all the mutants defective in effectors that were identi®ed as suppressors were more effective at eliciting an HR at lower concentrations, generally producing an HR at 10-fold higher dilution than wildtype DC3000 (Figure 4b). As an example, UNL109 (a DC3000 hopPtoF mutant) caused a con¯uent HR at a titer of 106 cells ml 1, whereas DC3000 only produced a spotty HR or no HR at this titer. It is important to note that DC3000 produced a typical HR at dilutions of 107 cells ml 1 or higher. When each was supplied in trans, the HR-eliciting ability returned to a DC3000-like HR (Figure 4) con®rming that the enhanced HR phenotype produced by each suppressor mutant resulted from the absence of the effector. Ion leakage measurements, which correlate strongly with the HR (Baker et al., 1991), con®rmed that the observed differences between the strains were measurable (data not shown). Most effector gene mutants that were not identi®ed as suppressors in our earlier assays did not exhibit any detectable difference with DC3000 in their ability to elicit an HR in our assay. The lone exception was the hopPtoG mutant UNL124, which caused an enhanced HR phenotype typical of an HR suppressor mutant. Moreover, additional assays shown below suggest that HopPtoG does function as an HR suppressor. Thus, our ®ndings show the phenotype of potential suppressor mutants on non-host plants is consistent with these effector genes encoding HR suppressors and complement our HR suppression data. Additionally, this assay proved sensitive enough to identify a suppressor independent of other assays. P. syringae HR suppressors inhibit PCD induced by the pro-apoptotic protein Bax in both plants and yeast The pro-apoptotic mouse protein Bax has been shown to induce PCD in plants that resembles the HR (Kawai-Yamada et al., 2001; Lacomme and Santa Cruz, 1999). Bax is a member of the Bcl-2 family of pro-apoptotic proteins and is thought to initiate PCD by localizing to the mitochondria and causing the release of pro-apoptotic factors, including cytochrome c (Jurgensmeier et al., 1998). Recently, Figure 4. Pseudomonas syringae tomato DC3000 suppressor mutants display an enhanced ability to elicit the HR. (a) Quantitative differences in the ability of DC3000 wild-type (WT), hopPtoE mutant UNL139, and complemented mutant UNL139(phopPtoE) to elicit the HR in N. tabacum cv. Xanthi leaves. Different dilutions of bacterial cells per ml (1, 108 cells ml 1; 2, 107 cells ml 1; 3, 106 cells ml 1; and 4, 105 cells ml 1) were in®ltrated into leaves, and leaves were photographed after 24 h. (b) N. tabacum cv. Xanthi leaves were in®ltrated with P. syringae strains that were 10-fold serially diluted from 108 cells ml 1. The last dilution (106 cells ml 1) that resulted in an HR is shown. In all cases, the mutants exhibit more HR at this dilution than the WT, and this phenotype was complemented when the suppressors were provided in trans. The following strains were in®ltrated: DC3000 wild type, WT; avrPphEPto mutant, UNL113; avrPpiB1Pto mutant, UNL114; avrPtoB mutant, UNL127; hopPtoF mutant, UNL109; and hopPtoG mutant, UNL124. HR was scored for each sample: spotty HR (HR ); strong HR (HR); or no HR. (a,b) The fraction underneath each picture indicates the number of times the results shown were observed over the number of times the experiment was performed. ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 Pseudomonas type III effectors that suppress PCD 559 teristics and suggest that they are functioning by different mechanisms. Discussion Figure 5. Pseudomonas syringae tomato DC3000 HR suppressors inhibit the PCD initiated by Bax in plants and yeast. (a) Agrobacterium C58C1 strains carrying binary vectors that encode Bax or a speci®c effector were co-in®ltrated into N. benthamiana leaves. Leaves were photographed after 7 days. Effector constructs were the same as that in Figure 4. (b) Yeast strain EGY48 carrying plasmids that encoded for Bax (pJG4-5-Bax) and a speci®c effector were spotted on plates at ®vefold dilutions. Expression of Bax was induced by galactose, whereas effector expression was constitutive. Only AvrPpiB1 was unable to suppress Bax-induced killing. BclxL (pGilda-Bcl-xL), an animal protein known to inhibit Bax-induced PCD, was used as a positive control. Abramovitch et al. (2003) reported that AvrPtoB suppresses Bax-induced PCD in plants. We tested the suppressors isolated in our screen in their ability to suppress Baxinduced PCD in plants. With the exception of AvrPpiB1Pto, all of them suppressed Bax-induced cell death in plants (Figure 5a). Interestingly, AvrPphEPto, HopPtoG, HopPtoF, and HopPtoE also suppressed Bax-induced PCD in yeast (Figure 5b). In contrast, AvrPtoB was reported not to be capable of suppressing the Bax-induced PCD in yeast (Abramovitch et al., 2003), which highlights a difference between the activity of these suppressors. Moreover, AvrPphEPto, HopPtoG, HopPtoF, and HopPtoE were unable to suppress the PCD initiated in yeast by H2O2 (data not shown), while AvrPtoB did suppress PCD in this assay (Abramovitch et al., 2003). This further demonstrates that the suppressors display different PCD-suppressing characß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 Effectors delivered by the Hrp TTSS appear central to P. syringae pathogenesis, but the anti-host functions of these proteins remain obscure. Here, we have subjected many of the newly identi®ed TTSS effectors from P. syringae tomato DC3000 to several novel bioassays and obtained evidence that many of these proteins appear to suppress one or more broadly conserved eucaryote PCD pathways. To understand these results, we must consider the collection of effectors that were assayed, the utility and limitations of the bioassays, and the role of plant cell death in disease and defense. The 19 effectors considered here were recently identi®ed in DC3000 on the basis of their homology with known effectors and/or their ability to be secreted and/or translocated by the DC3000 TTSS (Collmer et al., 2002). In general, P. syringae TTSS effectors identi®ed on the basis of Avr phenotypes are designated Avrs, whereas those identi®ed through secretion assays are designated Hops. However, our working assumption is that all of the `Avrs' are injected into plant cells by the TTSS and many of the `Hops' will confer Avr phenotypes to bacteria if tested in hosts that happen to carry a corresponding R gene; that is, effectors, Avrs, and Hops are often synonymous terms. It must be noted that these effectors do not represent the entire inventory of DC3000 effectors. However, the set of effectors analyzed here suggests that many DC3000 effectors have HR suppression activity. The bioassays that we used were designed to ef®ciently detect HR suppressor activity, determine whether suppressor action occurs in plant cells rather than in bacteria, and determine if the test effectors could also suppress PCD in other plants and the model eucaryote, yeast. The primary screen, based on suppression of the HR elicited by P. ¯uorescens(pHIR11) in tobacco, proved to be simple and effective (Figure 1). Although there is the formal possibility that suppressors identi®ed with this bioassay could be merely interfering with the delivery of HopPsyA, multiple lines of evidence indicate that they act after delivery into plant cells. For example, HopPtoE suppressed HopPsyAdependent HR elicitation when delivered by a functional TTSS in a different strain (Figure 2a) or when transiently expressed in plant cells following inoculation with A. tumefaciens (Figure 2b). It is also noteworthy that the suppressors we identi®ed functioned when delivered via the TTSS, a natural route that is thought to yield relatively low levels of effectors within plant cells. Agrobacterium-mediated transient expression, in contrast, can produce far higher levels of effectors within plant cells potentially leading to artifactual responses. 560 Yashitola Jamir et al. In an attempt to identify plant targets or sites of action of the suppressors in plants, we subjected HopPtoE, AvrPphEPto, AvrPpiB1Pto, AvrPtoB, HopPtoF, and HopPtoG to cursory bioinformatic analyses. BLASTP and PSI-BLAST searches (Altschul et al., 1997) did not identify any proteins (other than clear Avr homologs) that shared signi®cant similarity with any of the suppressors. However, 3D-PSSM analyses, a method that uses protein-fold recognition to identify proteins with similar folding patterns (Kelley et al., 2000), indicated that AvrPtoB had similarity (PSSM E-value 0.0895) to heme-dependent peroxidases (Welinder, 1985). We ®nd this result intriguing because of the clear involvement of reactive oxygen species (ROS) in plant defense (Mittler, 2002) and the potential of peroxidases to modulate ROS. Furthermore, there have been reports of peroxidases rescuing Bax-induced cell death in yeasts (Kampranis et al., 2000; Moon et al., 2002), and transgenic antisense tobacco plants with reduced amounts of ascorbate peroxidase were `hyperresponsive' to P. syringae (Mittler et al., 1999) causing a phenotype reminiscent of the enhanced HR phenotypes produced by the suppressor mutants reported here. Whether these similarities are biologically important awaits further experimentation. We found that AvrPphEPto, HopPtoG, HopPtoF, and HopPtoE suppress Bax-induced yeast PCD, indicating that the targets are likely to be broadly conserved and not unique to plants. Interestingly, AvrPpiB1Pto (Figure 5b) and AvrPtoB (Abramovitch et al., 2003) failed to do so, even though both suppressed the HR elicited by P. ¯uorescens(pHIR11) in both tobacco and Arabidopsis, and DC3000 avrPtoB and avrPpiB1Pto mutants produced enhanced HRs. It is also puzzling that HopPtoG failed to suppress the HR elicited by P. ¯uorescens(pHIR11), although a DC3000 hopPtoG mutant had enhanced HR activity and HopPtoG suppressed Bax-induced yeast PCD. Moreover, it is also noteworthy that HopPtoD2, a protein tyrosine phosphatase effector that was recently identi®ed to suppresses an HR elicited by avirulent P. syrinage strains (Espinosa et al., 2003), did not suppress the HR elicited by P. ¯uorescens(pHIR11). AvrPto was also unable to suppress the pHIR11-dependent HR (Figure 1b); however, recently, it was shown that it suppresses callose deposition in the plant cell wall that normally occurs during the defense response (Hauck et al., 2003). These exceptions suggest that multiple bioassays will be required to identify all the DC3000 effectors with some ability to suppress PCD and/or other plant defense responses. A general model of suppressor function must also reconcile several behaviors of bacterium±plant interactions that involve multiple effectors. Expression in P. syringae of a heterologous effector typically results in HR elicitation in test plants that carry a corresponding R gene despite the presence of resident suppressor effectors. For example, DC3000 heterologously expressing avrRpt2 or avrRps4 eli- cits the HR in Arabidopsis plants carrying the corresponding R genes (Hinsch and Staskawicz, 1996; Kunkel et al., 1993). On the other hand, suppressors can block HR elicitation by resident effectors, as revealed by the original discovery of suppressors like VirPphA and effectors with masked Avr activity in P. syringae phaseolicola (Jackson et al., 1999), and by our observations here that several effectors can block HR elicitation by HopPsyA in the heterologous P. ¯uorescens(pHIR11) system. A reasonable question to ask is why is the plant defense response ever successful if the pathogen is armed with multiple defense suppressors? Recent evidence suggests that type III secretion systems translocate proteins into host cells in a speci®c order (Thomas and Finlay, 2003). Such a hierarchy in delivery has been proposed to explain the deployment of effectors with con¯icting activities, such as the Salmonella SopE and SptP proteins, in animal pathogens (Cornelis and van Gijsegem, 2000; GalaÂn and Zhou, 2000). Perhaps, if any one effector is overexpressed, it can override the effector hierarchy allowing the Avr protein to act inside the plant cell before the suppressors can function or vice versa. One must also remember that the macroscopic HR assay used in the laboratory is arti®cially exposing plant cells to an extremely high number of bacterial cells. HR elicitation and its suppression may occur differently when bacterial cells come into contact with plant cells at lower cell densities as they do in nature. The global identi®cation of a set of suppressors in P. syringae tomato DC3000 should facilitate systematic investigation of the underlying functions of TTSS effectors in P. syringae pathogenesis. Currently, there are several P. syringae effectors known to suppress defense responses: AvrPtoB suppresses the AvrPto-dependent HR, Bax-dependent PCD in plants, and stress-induced PCD in yeast (Abramovitch et al., 2003); AvrRpt2 eliminates the RIN4 protein in Arabidopsis interfering with the ability to recognize the Avr protein AvrRpm1 (Axtell and Staskawicz, 2003; Mackey et al., 2003); AvrPto suppresses cell wall-based defenses (Hauck et al., 2003); and HopPtoD2 suppresses several plant defenses, including PR gene expression, the oxidative burst, and the HR (Bretz et al., 2003; Espinosa et al., 2003). Here, we show that in addition to suppressing PCD in plants and yeast, many of the effectors we have identi®ed also suppress the induction of PR genes. Thus, the type III suppressors are likely to suppress many of the hallmarks of plant defense in addition to the HR. It will be important to determine which plant defenses are suppressed and what mechanisms are used to exert these effects. A ®nal aspect of PCD and pathogenesis is that the ability to elicit host cell death appears to be a general characteristic of TTSS-dependent pathogens like P. syringae despite the fact that these bacteria typically rely upon living host cells as sites of multiplication (Alfano and Collmer, 1997; Knodler and Finlay, 2001). This is ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 Pseudomonas type III effectors that suppress PCD 561 Table 1 Additional information on plasmid constructions Gene name Primer nucleotide sequences and other relevant features Parent plasmid Plasmid construct shcA and hopPsyA pHIR11 pLN18 pBBR1 MCS2 pLN526 pML123 pLN256 pTA7002 pLN555 Gateway entry pCPP5057 Gateway entry pCPP5052 Gateway entry pLN323 Gateway entry pLN458 Gateway entry pLN714 Gateway entry pLN324 Gateway entry pCPP5070 Gateway entry pLN520 Gateway entry pCPP5100 avrPphEPto P21: 50 -GTAAAACGACGGCCAGT-30 P23: 50 -ATGAGAATTCGCATCTCCATGCATCTT-30 (EcoRI) P227: 50 -CGGACTCGAGCTCAGGGCGCGAAACTGA-30 (XhoI) P228: 50 -GTATGGTACCCCGACCTGGCAACCGCAG-30 (KpnI) P792: 50 -AGTCCTCGAGACTAAAGAGGGTATACGAATGGGAAATATA-30 (XhoI) P793: 50 -AGTCGATATCTCATTGCCAGTTACGGTACGGGC-30 (EcoRV) P582: 50 -GATGGATCCAAGTAACCGGTCTGCACA-30 (BamHI) P583: 50 -ATATCTAGATCATTTATCATCATCATCTTTATATGACTTTTGAGCCGCCTG-30 (XbaI) P0942: 50 -GGCCTCGAGATGGACGGGTCCGGGGAGCAGCTT-30 (XhoI) P0943: 50 -GGCACTAGTTCAGCCCATCTTCTTCCAGATGGTG-30 (SpeI) P683: 50 -CACCTATTTAATTCGTTGAGAAACAATGAAAATA-30 P68: 50 -GACATCTCGTCTCGCCAAGCC-30 P685: 50 -CACCAAGCAACGTCTGGAGGCAACAATGCA-30 P686: 50 -GTCGCCTAGGAAATTATTTAGTTCCCATGA-30 P693: 50 -CACCAAGATCGGAGAGGATCAGAATATGGCG-30 P694: 50 -GGGGACTATTCTAAAAGCATACTTGGC-30 P787: 50 -CACCTTAGCGTAAGGAGCTAACAATGAACCC-30 P788: 50 -GTTTCGCGCCCTGAGCGC-30 P1082: 50 -CACCCACCCGACAAATCCACAG-30 P788: 50 -GTTTCGCGCCCTGAGCGC-30 P695: 50 -CACCCATAGGGGTGCAATAACAATGAATAGA-30 P696: 50 -GTCAATCACATGCGCTTGGCC-30 P900: 50 -AAAAAGCAGGCTTCGAAGGAGATAGAACCATGTATAGCCCATCC-30 P901: 50 -AGAAAGCTGGGTAACAGACCCTTTCGAC-30 P0904: 50 -CACCCACATAGGATATGTAAACAATGCAAATAAAGAAC-30 P0905: 50 -GCCGTTGTAAAACTGCTTAGAGGC-30 P940: 50 -CACCACAAAGAGGTTTTCAAACAATGAATC-30 P941: 50 -GCAGTAGAGCGTGTCGCGAC-30 Gateway recombination avrPpiB1Pto Gateway recombination avrPtoB Gateway recombination hopPsyA hopPtoE hopPtoF Gateway recombination Gateway recombination Gateway recombination hopPtoG hopPtoK avrPphEPto Gateway recombination Gateway recombination P166: 50 -ATACATAACGCTGGCCTA-30 P167: 50 -CGGATCCATGACAATCGT-30 P168: 50 -GCAAATCCTTTAAGCTCT-30 P169: 50 -TGTTTCGCTAAGCCACTG-30 P304: 50 -TCG CGCCAAACCAGGGAG-30 P305: 50 -TCCCACATTCTGCAACGC-30 P188: 50 -AACCCCATTCAGTCACGC-30 P189: 50 -TTTGCCATGCGTGATTGC-30 P160: 50 -CCTCTACGATCTATTCAA-30 P161: 50 -GGCAATGCTCGCGGCCTG-30 P913: 50 -TCCGGTAGCTCGTCAGCG-30 P914: 50 -GTGGATGACCACATAGTTATG-30 P179: 50 -AGCCCATCCCATACACAA-30 P180: 50 -CAC TTT CTG TCC TTT GGG-30 P256: 50 -TATTCAGCTTCAAGAATG-30 P257: 50 -ACCCGCATAGACCTGTCTG-30 P194: 50 -ATCACTCCGTCTCGATATC-30 P195: 50 -TGCCCTGTACTTCATGCG-30 pML123 pPZP212 pML123 pPZP212 pML123 pPZP212 pPZP212 pPZP212 pML123 pPZP212 pPZP212 pML123 pKnockout-V pCPP5068 pLN535 pCPP5063 pLN503 pLN347 pLN502 pLN474 pLN524 pCPP5070 pLN525 pLN530 pCPP5100 pLN15 pKnockout-V pLN16 pKnockout-V pLN42 pKnockout-V pLN23 pKnockout-V pLN4 pKnockout-V pLN543 pKnockout-V pLN7 pKnockout-V pLN29 pKnockout-V pLN27 avrPto hopPtoT Mouse a-Bax avrPphEPto avrPpiB1Pto avrPtoB hopPsyA shcA hopPsyA hopPtoE hopPtoF hopPtoG hopPtoK avrPpiB1Pto avrPtoB hopPsyAPto hopPtoD1 hopPtoE hopPtoF hopPtoG hopPtoH ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 562 Yashitola Jamir et al. Table 1 continued Gene name Primer nucleotide sequences and other relevant features hopPtoJ P173: P174: P171: P172: P190: P191: P192: P193: P854: P855: P860: P861: P858: P859: P856: P857: P862: P863: hopPtoK hopPtoS1 hopPtoT1 avrPphEPto avrPpiB1Pto hopPtoE hopPtoF hopPtoG 50 -CTATGTATTTCAAAACAC-30 50 -ATCACCCTCTGTAATTCCC-30 50 -CGCATTTCAACCAGCTCA-30 50 -CAGCACCGGAAGCCCTTC-30 50 -GGTAATATTTGTGGTACTTC-30 50 -CAGATGTAACGTGACATC-30 50 -ACAGTCAGCAATCACTCG-30 50 -TACACTCCATACACTGCTG-30 50 -TTGAATTCATGAAAATACATAACGCTGG-30 (EcoRI) 50 -TTCTCGAGTCAGACATCTCGTCTCGC-30 (XhoI) 50 -TTGGATCCgtATGCACGCAAATCCTTTAAGCTC-30 (BamHI) 50 -TTCTCGAGTcAGTCGCCTAGGAAATTATTTAGTTCC-30 (XhoI) 50 -TTGAATTCATGAATAGAGTTTCCGGTAGCTC-30 (EcoRI) 50 -TTCTCGAGTCAGTCAATCACATGCGCTTGG-30 (XhoI) 50 -TTGAATTCATgGGTAATATTTGCGGCACCTC-30 (EcoRI) 50 -TTCTCGAGTCAGACCCTTTCGACCGG-30 (XhoI) 50 -TTGAATTCATGCAAATAAAGAACAGTCATCTC-30 (EcoRI) 50 -TTCTCGAGTcAGCCGTTGTAAAACTGCTTAGAG-30 (XhoI) particularly puzzling with P. syringae because late-stage infections with most strains produce necrotic lesions, but the symptomless growth of P. syringae gacS mutants suggests that such cell killing may be gratuitous (Willis et al., 1990). Similarly, puzzling are recent observations suggesting that plants compromised in PCD pathways are unexpectedly more resistant to P. syringae (Stone et al., 2000; Lincoln et al., 2002). Thus, rapid and delayed host cell death are associated with defense and disease, respectively, and pathogen manipulation of cell death pathways may be a central process in pathogenesis. Therefore, the identi®cation of type III effectors that modulate PCD and/or defense responses and the assays used to identify them appear critical if we are to better understand the bacterial pathogenicity of plants. Experimental procedures Bacterial strains, plasmids, and media Escherichia coli strains DH5a and DB3.1 (Invitrogen, Carlsbad, CA, USA) were used for general cloning and Gateway technology manipulations, respectively. P. syringae pv. tomato DC3000 and P. ¯uorescens strains were grown in King's B (KB) broth at 308C (King et al., 1954). E. coli and A. tumefaciens C58C1 were grown in Luria-Bertani (LB) broth at 37 and 308C, respectively. Unless otherwise noted, constructs used were made by PCR, and Table 1 includes a list of nucleotide primer sequences that were used. The pHIR11 derivative pLN18, which lacks shcA and hopPsyA, was generated as described previously by van Dijk et al. (2002). Brie¯y, 2-kbp regions upstream and downstream of shcA and hopPsyA were PCR cloned into pBluescript-II KS on either side of an nptII antibiotic marker. When transformed into the E. coli strain C2110 (Kahn and Hanawalt, 1979) containing pHIR11, this construct recombined into pHIR11 because ColE1 plasmids, such as pBluescript-II KS , cannot replicate in this polA mutant at 428C. When Parent plasmid Plasmid construct pKnockout-V pLN8 pKnockout-V pLN9 pKnockout-V pLN41 pKnockout-V pLN25 pGilda pLN508 pGilda pLN507 pGilda pLN504 pGilda pLN505 pGilda pLN506 this strain was grown at 308C, the ColE1 replicon replicated forcing it to recombine out of pHIR11. pHIR11 derivatives that lacked shcA and hopPsyA were identi®ed with PCR. Antibiotics were used at the following concentrations (mg ml 1): rifampicin, 100; ampicillin, 100; gentamycin, 10; kanamycin, 50; tetracycline, 20; nalidixic acid, 20; and spectinomycin 50. Pseudomonas plant bioassays The broad-host-range vector pML123 was used to express effector genes in Pseudomonas strains (Labes et al., 1990). The pML123 constructs containing hopPtoB, hopPtoC, hopPtoD1, hopPtoD2, hopPtoE, hopPtoG, hopPtoH, hopPtoI, hopPtoJ, hopPtoL, hopPtoS1, and hopPtoS2 were described previously by PetnickiOcwieja et al. (2002). pML123 constructs containing hopPtoF, hopPtoK, hopPtoT1, avrPtoB, avrPphEPto, avrPpiB1Pto, and avrPto are detailed in Table 1. P. ¯uorescens(pHIR11) or DC3000 strains carrying pML123 constructs with effector genes or vector controls with an OD600 of 0.2 (c. 108 cells ml 1) in 5 mM MES (pH 5.6) and in®ltrated into N. tabacum cv. Xanthi, N. benthamiana, or A. thaliana Ws-0 leaves. For bacterial mixing experiments involving two different P. ¯uorescens strains, P. ¯uorescens(pLN18) and a pML123 effector construct, were in®ltrated 2 h before P. ¯uorescens(pHIR11). The plants were scored for the production of an HR after 12 h. DC3000 strains were tested for their ability to elicit an HR on N. tabacum cv. Xanthi by in®ltrating strains with an OD600 of 0.2 along with 10-fold serially diluted samples with a needleless syringe. To determine the amount of ion leakage occurring in plant tissue challenged with DC3000 suppressor mutants, we followed a protocol recently described (Mackey et al., 2002). Brie¯y, tobacco (N. tabacum cv. Xanthi) was in®ltrated with bacterial strains containing 107 cells ml 1. At time points of 0, 3, and 6 h, six 8-mM diameter leaf discs were removed and ¯oated in 50 ml of water. After 30 min, the wash water was removed and replaced with 10 ml of fresh water. Conductance of this water was then measured using a conductivity meter. These experiments were repeated four times. Pathogenicity assays were performed by dip-inoculation into bacterial suspensions that were adjusted to an OD600 of 0.2 as described by Espinosa et al. (2003). Bacteria were enumerated from leaf tissue by plating dilutions on KB plates ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 Pseudomonas type III effectors that suppress PCD 563 with the appropriate antibiotics as described by Alfano et al. (2000). Agrobacterium-mediated transient assays The avr gene hopPsyA was recombined into a derivative of pPZP212 (Hajdukiewicz et al., 1994), pLN462, which was modi®ed to be a Gateway destination vector, resulting in pLN474. The bax gene was PCR cloned into pTA7002, creating pLN531, and expression of bax was induced with dexamethasome as previously described by Aoyama and Chua (1997). The effector genes carried on Gateway entry vectors avrPphEPto, avrPpiB1Pto, avrPtoB, hopPtoE, hopPtoF, and hopPtoG were recombined into pLN462 (which fused each gene to a hemagglutinin epitope) creating constructs pLN535, pLN503, pLN502, pLN524, pLN525, and pLN530, respectively. Agrobacterium-mediated transient expression experiments were performed by in®ltrating A. tumefaciens C58C1 (Van Larebeke et al., 1974) harboring the disabled Ti plasmid pMP90 (Koncz and Schell, 1986) at an OD600 of 0.4 into N. benthamiana and N. tabacum cv. Xanthi plants using a needleless syringe as described by Van den Ackerveken et al. (1996). For co-expression experiments, Agrobacterium strains carrying pPZP212 binary plasmids with different effector genes were in®ltrated 4 h prior to in®ltration of strains expressing either Bax or HopPsyA. Evidence of production of effectors from transient assays was acquired by harvesting 1-cm diameter leaf disks from in®ltrated zones, grinding leaf tissue with a mortar and pestle in the presence of liquid nitrogen, and re-suspending plant material in 50 ml of 1X SDS± PAGE tracking buffer. SDS±PAGE and immunoblot analysis were performed as described above using high af®nity anti-hemagglutinin antibodies (Roche, Indianapolis, IN, USA). Construction of DC3000 effector mutants In-frame internal fragments of the effector genes were PCR cloned into XcmI-digested pKnockout-V (Windgassen et al., 2000) using primer sets listed in Table 1. The resulting constructs were conjugated separately into DC3000 by triparental mating using spectinomycin as selection for the plasmid marker. The following effector mutants were con®rmed with primers that ¯anked each coding region: hopPtoD1, UNL104; hopPtoC, UNL106; hopPtoE, UNL139; hopPtoK, UNL107; hopPtoJ, UNL108; hopPtoF, UNL109; avrPhEPto, UNL113; avrPpiB1Pto, UNL114; hopPtoH, UNL118; hopPtoT1, UNL122; hopPtoG, UNL124; hopPtoS1, UNL126; and avrPtoB, UNL127. RT-PCR experiments Pseudomonas ¯uorescens(pHIR11) and P. ¯uorescens(pHIR11) strains carrying different suppressors were adjusted to an OD600 of 0.2 and in®ltrated into N. tabacum cv. Xanthi leaves. One gram leaf samples were taken 5 h after in®ltration and made into a ®ne powder in liquid nitrogen. RNA was isolated using a Trizol (Sigma, St Louis, MO, USA) protocol as recommended by the manufacturer and treated with DNA-freeTM Dnase (Ambion, Austin, TX, USA) to remove any contaminating DNA. Relative quantitative RTPCR was carried out using the RetroscriptTM kit (Ambion) for ®rst-strand synthesis and multiplex PCR was performed using the primers 50 -atgggatttgttctcttttcac-30 (P915) and 50 -ttagtatggactttcgcctc-30 (P916), which anneal within the tobacco PR1a-coding region, and with the QuantumRNATM 18S Internal Standards kit (Ambion) according to manufacturer's instruction. The QuantumRNATM kit contains primers to amplify 18S rRNA along with ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 554±565 competimers that reduce the ampli®ed 18S rRNA product within in the range to allow it to be used as endogenous standard. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. Yeast viability assays To determine whether type III effector-encoding plasmids rescued yeast from Bax-induced lethality, the effector genes avrPphEPto, avrPpiB1Pto, hopPtoG, hopPtoF, and hopPtoE were PCR cloned into the yeast expression vector pGilda (Clontech, Palo Alto, CA, USA), resulting in constructs pLN508, pLN507, pLN506, pLN505, and pLN504, respectively. Table 1 contains information for the nucleotide primers used to make these constructs. Saccharomyces cerevisiae EGY48 strain containing pJG4-5-Bax (kindly provided by J.C. Reed, Burnham Institute, La Jolla, CA, USA) and various pGilda plasmids containing effector genes were grown in SCHis-Trp-L/glucose media overnight. The chicken Bcl-xL cloned in pGilda was kindly provided by C. Thompson (University of Chicago, Chicago, IL, USA), which acted as a positive control for PCD suppression in these experiments. The yeast cultures were then serially 10-fold diluted into SC medium, and 5 ml of each dilution was dropped onto SC-His-Trp-L/galactose or SC-His-Trp-L/glucose plates. Cells were incubated at 308C for 5 days and photographed. For oxidative stress experiments, EGY48 strains containing pGilda effector constructs were grown in SC-His media overnight and treated as described by Abramovitch et al. (2003). Acknowledgements We thank K. Jaeger for the pKnockout-V plasmid, J. C. Reed for pJG4-5-Bax, C. Thompson for pGilda-Bcl-xL, K. van Dijk for constructing pLN18, and W. Gassmann for informing us that P. ¯uorescens(pHIR11) elicits an HR on A. thaliana Ws-0. We also thank S. T. Chancey and A. Espinosa for reviewing the manuscript, and G. B. Martin for helpful discussions regarding AvrPtoB. This work was supported by the National Science Foundation (NSF) Plant Genome Research Program Cooperative Agreement DBI-0077622 (A.C., J.R.A.) and NSF Grant IBN-0096348 (J.R.A.). 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