Identification ofPseudomonas syringaetype III effectors that can

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,
Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, NE 68588-0660, USA,
Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203, USA, and
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]).
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
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.
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
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.
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
(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
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
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
Gateway entry
Gateway entry
Gateway entry
Gateway entry
Gateway entry
Gateway entry
Gateway entry
Gateway entry
Gateway entry
Gateway recombination
Gateway recombination
Gateway recombination
Gateway recombination
Gateway recombination
Gateway recombination
Gateway recombination
Gateway recombination
shcA‡ hopPsyA
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
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
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.
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).
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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