These two authors contributed equally to this work.
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 confirmed 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 identified 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.
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Pseudomonas syringae is a host-specific 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 hrpconserved (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 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. fluorescens 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 confirmed effectors and several effector candidates (Buell et al., 2003; Collmer et al., 2002; Guttman et al., 2002; 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).
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 confirmed P. syringae tomato DC3000 effectors (designated as Hop or Avr proteins) for their ability to suppress the HR that is normally elicited in tobacco by P. fluorescens(pHIR11) and found that five 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 Agrobacterium-mediated transient expression experiments; and finally, 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.
pHIR11 assays identify five effectors capable of complete suppression of the HopPsyA-dependent HR
In the course of experiments with confirmed 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 identified DC3000 effectors (Collmer et al., 2002). To do this, we used the pHIR11 system, which allows non-pathogens such as E. coli and P. fluorescens to elicit the HR and secrete effectors in culture via the TTSS. This tool allowed us to test whether individual effectors suppressed the HopPsyA-dependent HR as depicted in Figure 1(a). We infiltrated P. fluorescens(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 confirmed effector genes into a broad-host-range plasmid and tested if the encoded effectors were able to suppress the HR elicited by P. fluorescens(pHIR11) (see Figure 1b for a list of the effectors tested).
We expressed each candidate suppressor gene in P. fluorescens(pHIR11) and infiltrated 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 identified suppressors, HopPtoF and AvrPtoB, were homologs of AvrPphF and VirPphA, 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 identified 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. fluorescens(pHIR11) to deliver the Avr protein HopPsyA into plant cells, we used a bacterial strain to deliver HopPsyA different from that used to deliver 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. fluorescens(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 first infiltrating into tobacco P. fluorescens(pLN18) with hopPtoE contained in a broad-host-range plasmid and, after 2 h, P. fluorescens(pHIR11) was infiltrated at an OD600 sufficient to cause HR elicitation. Figure 2(a) shows that P. fluorescens(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 identified suppressors were able to inhibit the pHIR11-dependent 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 (agroinfiltrations; Van den Ackerveken et al., 1996). In each case, the effector suppressed the HopPsyA-dependent HR (Figure 2b). We confirmed with immunoblots that the agroinfiltrations produced both HopPsyA and the specific suppressor tested (Figure 2c). These data complement the bacteria-delivered suppressor data shown above because agroinfiltrations demonstrate that the suppressor activity is dependent on suppressor action inside plant cells, whereas the experiments using P. fluorescens(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 relative-quantitative RT-PCR experiments using RNA isolated from tobacco plants challenged with P. fluorescens(pHIR11) strains carrying different suppressors. We infiltrated tobacco plants with P. fluorescens(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. fluorescens(pHIR11, pavrPpiB1Pto), which contained similar amounts of the PR1a product as control plants challenged with P. fluorescens(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 findings, 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 infiltrated 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 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 identified 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 identified as suppressors were more effective at eliciting an HR at lower concentrations, generally producing an HR at 10-fold higher dilution than wild-type DC3000 (Figure 4b). As an example, UNL109 (a DC3000 hopPtoF mutant) caused a confluent 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) confirming 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), confirmed that the observed differences between the strains were measurable (data not shown). Most effector gene mutants that were not identified 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 findings 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, 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 Bax-induced 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 characteristics and suggest that they are functioning by different mechanisms.
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 identified 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 identified 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 identified on the basis of Avr phenotypes are designated Avrs, whereas those identified 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 efficiently 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. fluorescens(pHIR11) in tobacco, proved to be simple and effective (Figure 1). Although there is the formal possibility that suppressors identified 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 HopPsyA-dependent 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 identified 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.
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 significant 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 (pssmE-value 0.0895) to heme-dependent peroxidases (Welinder, 1985). We find 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. fluorescens(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. fluorescens(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 identified to suppresses an HR elicited by avirulent P. syrinage strains (Espinosa et al., 2003), did not suppress the HR elicited by P. fluorescens(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 elicits 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. fluorescens(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 specific order (Thomas and Finlay, 2003). Such a hierarchy in delivery has been proposed to explain the deployment of effectors with conflicting activities, such as the Salmonella SopE and SptP proteins, in animal pathogens (Cornelis and van Gijsegem, 2000; Galán 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 artificially 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 identification 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 identified 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 final 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 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 identification 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.
Bacterial strains, plasmids, and media
Escherichia coli strains DH5α and DB3.1 (Invitrogen, Carlsbad, CA, USA) were used for general cloning and Gateway technology manipulations, respectively. P. syringae pv. tomato DC3000 and P. fluorescens strains were grown in King's B (KB) broth at 30°C (King et al., 1954). E. coli and A. tumefaciens C58C1 were grown in Luria-Bertani (LB) broth at 37 and 30°C, 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). Briefly, 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 42°C. When this strain was grown at 30°C, the ColE1 replicon replicated forcing it to recombine out of pHIR11. pHIR11 derivatives that lacked shcA and hopPsyA were identified with PCR. Antibiotics were used at the following concentrations (µg ml−1): rifampicin, 100; ampicillin, 100; gentamycin, 10; kanamycin, 50; tetracycline, 20; nalidixic acid, 20; and spectinomycin 50.
Table 1. Additional information on plasmid constructions
Primer nucleotide sequences and other relevant features
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 Petnicki-Ocwieja et al. (2002). pML123 constructs containing hopPtoF, hopPtoK, hopPtoT1, avrPtoB, avrPphEPto, avrPpiB1Pto, and avrPto are detailed in Table 1. P. fluorescens(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 infiltrated into N. tabacum cv. Xanthi, N. benthamiana, or A. thaliana Ws-0 leaves. For bacterial mixing experiments involving two different P. fluorescens strains, P. fluorescens(pLN18) and a pML123 effector construct, were infiltrated 2 h before P. fluorescens(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 infiltrating 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). Briefly, tobacco (N. tabacum cv. Xanthi) was infiltrated 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 floated 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 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 modified 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 infiltrating 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 infiltrated 4 h prior to infiltration 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 infiltrated zones, grinding leaf tissue with a mortar and pestle in the presence of liquid nitrogen, and re-suspending plant material in 50 µl of 1X SDS–PAGE tracking buffer. SDS–PAGE and immunoblot analysis were performed as described above using high affinity 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-Ω (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 confirmed with primers that flanked 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.
Pseudomonas fluorescens(pHIR11) and P. fluorescens(pHIR11) strains carrying different suppressors were adjusted to an OD600 of 0.2 and infiltrated into N. tabacum cv. Xanthi leaves. One gram leaf samples were taken 5 h after infiltration and made into a fine 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-free™ Dnase (Ambion, Austin, TX, USA) to remove any contaminating DNA. Relative quantitative RT-PCR was carried out using the Retroscript™ kit (Ambion) for first-strand synthesis and multiplex PCR was performed using the primers 5′-atgggatttgttctcttttcac-3′ (P915) and 5′-ttagtatggactttcgcctc-3′ (P916), which anneal within the tobacco PR1a-coding region, and with the QuantumRNA™ 18S Internal Standards kit (Ambion) according to manufacturer's instruction. The QuantumRNA™ kit contains primers to amplify 18S rRNA along with competimers that reduce the amplified 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 SC-His-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 µl of each dilution was dropped onto SC-His-Trp-L/galactose or SC-His-Trp-L/glucose plates. Cells were incubated at 30°C 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).
We thank K. Jaeger for the pKnockout-Ω 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. fluorescens(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.).