Download Pseudomonas syringae type III effector repertoires: last words in

Review
Feature Review
Pseudomonas syringae type III effector
repertoires: last words in endless
arguments
Magdalen Lindeberg1, Sébastien Cunnac2 and Alan Collmer1
1
2
Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY, USA
UMR Résistance des Plantes aux Bioagresseurs, Institut de Recherche pour le Développement, Montpellier, France
Many plant pathogens subvert host immunity by injecting compositionally diverse but functionally similar
repertoires of cytoplasmic effector proteins. The bacterial pathogen Pseudomonas syringae is a model for
exploring the functional structure of such repertoires.
The pangenome of P. syringae encodes 57 families of
effectors injected by the type III secretion system. Distribution of effector genes among phylogenetically diverse strains reveals a small set of core effectors
targeting antimicrobial vesicle trafficking and a much
larger set of variable effectors targeting kinase-based
recognition processes. Complete disassembly of the 28effector repertoire of a model strain and reassembly of a
minimal functional repertoire reveals the importance of
simultaneously attacking both processes. These observations, coupled with growing knowledge of effector
targets in plants, support a model for coevolving molecular dialogs between effector repertoires and plant immune systems that emphasizes mutually-driven
expansion of the components governing recognition.
The bacterial plant pathogen P. syringae is a model for
studying cytoplasmic effector repertoires as dynamic
systems
Many microbial pathogens deliver effector proteins into
the cytoplasm of their eukaryotic hosts to subvert immunity, remodel cellular functions and promote pathogen
proliferation. Pathogens of animals or plants typically
deploy cytoplasmic effectors as components of complex
repertoires that feature internal redundancy, effector interplay and varying degrees of inter-strain diversity. Understanding the molecular functions and host targets of
cytoplasmic effectors has been a dominant theme in pathogenic microbiology over the past two decades [1,2], and
more recently, comprehensive identification of candidate
effectors has been the motivation and primary product of
many pathogen genome sequencing projects [3–5]. The
cytoplasmic effector repertoires of the bacteria, fungi
and oomycetes that attack plants have similar properties
in being commonly large, highly diverse among related
strains, and composed of proteins that are collectively
Corresponding author: Collmer, A. ([email protected]).
Keywords: effector-triggered immunity; PAMP-triggered immunity; nonhost resistance; hypersensitive response; pathogenicity.
essential but individually dispensable for pathogenesis.
These properties appear to have arisen independently in
the coevolution of plant immune systems and biotrophic
Glossary
Avirulence (Avr): designation applied to effectors discovered on the basis of
their ability to cause otherwise virulent pathogens to elicit an avirulent
response on plants carrying the corresponding R gene; some Avr proteins also
have Hop designations, as explained below.
Biotroph: a pathogen that has a sustained parasitic nutritional relationship with
living plant tissue. Many biotrophs cause host cell death later in pathogenesis
and are referred to as hemibiotrophs.
Effector: for convenience in this review the term is used synonymously with
cytoplasmic effector, Hop and Avr protein to denote virulence-related proteins
delivered into host cells by pathogens; however, the term is often used more
broadly to include other pathogen molecules such as phytotoxins.
Effector-triggered immunity (ETI): based typically on surveillance of effectors
or their activities within plant cells by nucleotide-binding site leucine-rich
repeat R proteins.
Host resistance: the resistance of cultivars or other subspecific groups of a
plant species against races of an otherwise virulent pathogen. Races within P.
syringae pathovars are typically determined by R-protein mediated recognition
of an individual effector in a cultivar-race and ‘gene-for-gene’ manner resulting
in host resistance.
Hrp/Hrc: hypersensitive response and pathogenicity/conserved; designation
for components of the P. syringae type III secretion system; Hrc refers to
secretion machinery components conserved also in animal pathogens.
Hrp outer protein (Hop): a generic designation for P. syringae type III effectors.
Some effector families are known only by their original Avr designations (e.g.
AvrE), whereas others are described by both Hop and Avr designations where
extensive literature is associated with the Avr designation (e.g. HopAB2/AvrPtoB).
Hypersensitive response (HR): a localized programmed cell death associated
with ETI.
Nonhost resistance: the resistance of plant species to pathogen species or
pathotypes (such as P. syringae pathovars) that normally cause disease in
other plant species.
Nucleotide-binding site and leucine-rich repeat protein (NB-LRR): most of the
known resistance (R) proteins involved in direct or indirect recognition of
pathogen effectors are in this class of proteins.
PAMP-triggered immunity (PTI): based on detection of microbial PAMPs by
PRRs.
Pathogen-associated molecular pattern (PAMP): highly conserved features of
bacterial cells that elicit an innate immune response known as PAMP-triggered
immunity; PAMPs are also known as microbe-associated molecular patterns
(MAMPs).
Pathovar: a subspecific group in P. syringae defined largely by host specificity;
some pathovars are polyphyletic, and some strains are virulent on divergent
hosts, such as P. syringae pv. tomato DC3000 on tomato and Arabidopsis.
Pattern recognition receptor (PRR): plant proteins that perceive molecular
patterns (PAMPs) at the cell surface, initiating PTI.
Redundant effector group (REG): effectors that interfere with a common
defense pathway or process.
R protein: plant resistance proteins that perceive type III effectors, initiating
ETI; typically NB-LRR proteins.
Type III secretion system (T3SS): injects effectors from bacterial pathogens of
plants and animals into host cells.
0966-842X/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2012.01.003 Trends in Microbiology, April 2012, Vol. 20, No. 4
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pathogens, which grow in living plant tissues and are often
host specific.
The proteobacterium P. syringae has become a model for
systems-level exploration of effector repertoires and their
operation in biotrophic pathogenesis. P. syringae typically
grows for several days in the intercellular spaces of living
plant tissues and then (as is characteristic of ‘hemi’ biotrophs) triggers host cell death and disease lesions. P.
syringae growth in planta is dependent on the injection
of effectors by the type III secretion system (T3SS, see
Glossary) [6]. The species P. syringae is composed of
pathovars and races differing in host range among crop
species and cultivars, respectively [7]. For example, strains
of P. syringae pv. tomato cause bacterial speck of tomato, P.
syringae pv. glycinea causes bacterial blight of soybean,
and both pathovars contain multiple races distinguished
by their interactions with cultivars of the host. In addition
to being representative of pathogens with an effector-dependent biotrophic lifestyle, P. syringae is now a model for
systems analysis because of its experimental amenability,
the recent identification of virtually all effector genes in the
pangenome [8], the development of a model strain, P.
syringae pv. tomato DC3000 (Pto DC3000), whose complete
repertoire of effector genes has been deleted and experimentally reassembled [9], and the recent large-scale identification of candidate effector interactors in the
Arabidopsis proteome [10].
Also importantly, effector functions can now be analyzed in the context of a unified model for a two-layered
immune system in plants [11]. According to this model, the
primary function of cytoplasmic effectors is to suppress
pathogen-associated molecular pattern (PAMP)-triggered
immunity (PTI) or microbe-associated molecular pattern
(MAMP)-triggered immunity (MTI), which is elicited by
common bacterial features such as flagellin, lipopolysaccharide (LPS), peptidoglycan and elongation factor Tu [12].
PAMPs are perceived by pattern recognition receptors
(PRRs) at the surface of plant cells. A second layer of
immunity involves detection of injected effectors or their
activity by resistance (R) proteins, typically nucleotidebinding site leucine-rich repeat (NB-LRR) proteins, which
results in effector-triggered immunity (ETI) and often a
localized programmed cell death known as the hypersensitive response (HR).
Plant breeders exploit this second layer of immunity by
introgressing into elite but susceptible cultivars of a crop a
gene encoding an R protein that recognizes one of a pathogen’s effectors, thereby producing a new cultivar that is
strongly resistant to the pathogen. Unfortunately, selection for pathogen variants in the field results in the breakdown of resistance through the emergence of a new race
that lacks the ETI-eliciting ‘avirulence’ effector. The subsequent breeding of a new cultivar with yet another introgressed R gene provides new but unstable resistance. This
process leads to multiple boom–bust cycles in crop production and the proliferation of differential cultivars and
pathogen races [11]. Although such R gene-based ‘host
resistance’ is often rapidly defeated, resistance against a
pathovar that is adapted to another plant species (for
example, the resistance of soybean to P. syringae pv.
tomato) is more durable, and the basis for this ‘nonhost
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Trends in Microbiology April 2012, Vol. 20, No. 4
resistance’ is an active area of research that we will
address. Importantly, laboratory studies of P. syringae
effector repertoires can be informed by many decades of
agricultural ‘experiments’ involving crop breeding and
pathogen evolution in the field.
This review will discuss P. syringae effectors as virulence system components that collectively subvert the host
immune system. We will discuss the structure of P. syringae effector repertoires and genetic experiments that
reveal functional groups of effectors, interplay among
effectors and their targets in planta, and a model for
repertoire coevolution with the plant immune system.
We will use selected examples from the increasingly vast
literature on P. syringae effectors, and for more comprehensive coverage, readers are referred to recent reviews on
P. syringae genomics [7], effector functions [6,13] and plant
PTI and ETI systems [14,15].
The P. syringae pangenome effector super-repertoire
includes core and variable effectors
The super-repertoire of effectors in the pangenome of the P.
syringae species complex comprises 57 families (Figure 1),
with individual strains typically expressing circa 15–30
effectors [8]. Progress in identifying effectors and determining phylogenetic relationships among and within
pathovars provides a firm foundation for exploring repertoire evolution within the P. syringae complex. Multilocus
sequence typing (MLST) has revealed that P. syringae
pathovars fall into at least six groups [16,17]. Gold-standard reference genome sequences exist for representatives
of the three best-studied major groups: Pto DC3000 (Group
I, causing bacterial speck of tomato and Arabidopsis), P.
syringae pv. syringae B728a (Group II, brown spot of bean),
and P. syringae pv. phaseolicola 1448A (Group III, halo
blight of bean), and these strains have been used by several
independent research groups to hone methods for comprehensive identification of effector genes [18–20]. Importantly, all active P. syringae effector genes appear to be
preceded by promoter sequences responsive to the HrpL
alternative sigma factor, and the N-terminal regions of the
proteins carry T3SS targeting signals capable of directing
the translocation of effector–reporter fusions into plant
cells [8].
P. syringae effectors are generically designated Hop
(Hrp outer protein) based on their ability to travel through
the T3SS, which is encoded by hrp/hrc (hypersensitive
response and pathogenicity/conserved) genes, although
several effectors are designated ‘Avr’ because they were
discovered in the pre-genomics era by their avirulence
phenotype on resistant cultivars of various crops
(Figure 1) [21]. Systematic study of P. syringae effector
repertories is fostered by community standards and databases at the Pseudomonas-Plant Interaction website for
designating new Hop families or subfamilies and their
functional properties and by PAMDB, a multilocus sequence typing (MLST) database and website for plantassociated microbes [17,21,22]. Next-generation methods
have enabled the sequencing of additional strains, yielding insights into pathogenesis on important hosts such
as rice (a monocot) and olive (a woody plant) [23,24], as
well as a comprehensive picture of effector repertoire
Review
Trends in Microbiology April 2012, Vol. 20, No. 4
Occurrence
Effector
family
Frequency within group
Group 3
AvrE
HopI
HopM
HopAA
HopX
HopAE
HopAF
HopR
HopAS
HopAB
HopQ
HopT
HopD
HopO
HopW
HopF
HopV
HopAZ
AvrPto
HopG
HopAU
HopAW
HopAO
HopAV
HopAT
HopZ
HopA
HopH
HopAI
HopAG
HopC
HopE
HopBD
AvrB
HopN
HopY
HopS
HopAR
AvrRpm
AvrRps4
HopAD
HopAM
HopAY
AvrA
HopB
HopAX
HopBA
HopBB
HopBG
HopBF
HopAQ
AvrRpt2
HopU
HopBC
HopBE
HopK
HopAL
Group 2
Group 1
Function
+/–
Cell death
Avr
DC3000 Plant ac. N. benth.
*
12
30
45
1
22
3
Variable
25
1
6
1
28
Variable
24
27
3
19
25
3
3
6
3
43
1
26
7
12
16
4
20
11
TRENDS in Microbiology
Figure 1. The phylogenetic distribution of effector families in the Pseudomonas
syringae effector super-repertoire and functional structure of the P. syringae pv.
tomato DC3000 repertoire in the context of Nicotiana benthamiana pathogenesis.
Effector families are displayed in order of descending overall frequency according
to Figure 3 in Baltrus et al. [8]; pale green denotes effectors encoded in the Hrp
pathogenicity island conserved effector locus, and pale blue denotes effectors that
have high pairwise amino acid diversity and appear to be commonly lost and
regained by P. syringae strains [8]. Occurrence frequency of effector families in the
six strains in each group is indicated: 0 (white), 1–2 (yellow), 3 (orange), 4–5 (pink),
6 (red) (P. syringae pv. maculicola ES4326 is included in the Group 1 tally). Pto
DC3000, a Group 1 strain, produces 28 highly expressed effectors (in brown and
other colors), with additional properties indicated: vesicle trafficking redundant
effector group (REG; green), pathogen-associated molecular pattern (PAMP)
perception REG (blue) and minimal repertoire for virulence in N. benthamiana
(boxed). Cell death in plant accessions (Plant ac.) following Agrobacteriummediated transient expression of effectors from various strains is indicated by pale
pink boxes, which also show numbers of positive responses out of 59 accessions
tested (gray, no cell death observed; white, untested) [31]. N. benthamiana
(N. benth.) responds with cell death to several of the 28 highly expressed Pto
DC3000 effectors following delivery by P. fluorescens expressing a cloned
P. syringae T3SS (red; the boxed effector is the sole avirulence determinant).
The avirulence (Avr) phenotype resulting from transgenic expression in an
composition among 19 strains spanning the diversity
within P. syringae [8].
Here, we present a few of the important lessons from the
latter comprehensive repertoire study [8] (Figure 1). First,
the 14 new genome sequences yielded only nine new
effector families and most of these were present in multiple
strains. Thus, the super-repertoire is likely to be nearly
complete with 57 effector families. (We are excluding
HopAH from this tally because it possesses several properties of harpins, which appear to be T3SS substrates that
help the translocation of true effectors [25]). Second, only
four effector families are core members of the super-repertoire. Three of these are encoded in the conserved effector
locus of the Hrp pathogenicity island, whose acquisition
along with the hrp/hrc genes by horizontal gene transfer
preceded the radiation of P. syringae into its pathovars
[26]. A third important finding was that MLST Group II
strains have markedly smaller effector repertoires than
the other groups and lack many effector families beyond
the core set. As noted [8], Group II strains also differ from
the other groups in producing three toxins with particularly disruptive effects on plant cells: syringomycin, syringopeptin and syringolin. The first two have cytotoxic
membrane pore-forming activity, and syringolin is a proteasome inhibitor [27,28]. A fourth striking finding is that
most of the effectors found on the basis of their avirulence
phenotypes are among the rarer of the variable effectors
[19]. (AvrE is atypical of Avr effectors in not eliciting any
known R gene-mediated resistance in differential cultivars
[29,30].) Phylogenetic analyses of the variable effectors
suggest that several have been acquired, lost and reacquired repeatedly and in diverse pathovars [8]. ETI surveillance of these effectors may be widespread in plants, as
indicated by the many plant accessions that respond with
cell death to Agrobacterium-mediated transgenic expression of them (Figure 1) [31]. A final notable feature of P.
syringae effector repertoires is that divergent repertoires
can be found in strains that are pathogenic on the same
host [7,8,19,32,33].
Although this review is focused on P. syringae, it
should be noted here that a recent study of Xanthomonas
(which independently acquired a T3SS and effector genes)
suggests convergent evolution of effector repertoires in
strains adapted for the same host [34], a pattern not
observed in P. syringae [8]. Regarding the question of
effector adaptation for different hosts, it is noteworthy
that transgenically expressed P. syringae effectors that do
not elicit ETI in test pathosystems often stimulate pathogen growth, regardless of the source strain or its host
[35–37]. Collectively, these observations suggest that
most P. syringae effectors can function as ‘generalists’
in a broad range of plants and that immune surveillance
of effectors is the primary driver of effector repertoire
diversification [13,38]. Importantly, effector genes are
part of the flexible genome in P. syringae strains and
are frequently associated with mobile genetic elements
[39]. In a particularly striking example of effector gene
dynamics in the determination of race-cultivar specificity,
otherwise virulent P. syringae strain was used in many pre-genomics era studies
to discover effectors (purple; *AvrE is atypical as discussed in the text).
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Trends in Microbiology April 2012, Vol. 20, No. 4
alternative subsets of the 57 effector families encoded
by the pangenome.
infection by P. syringae pv. phaseolicola of bean cultivars
carrying resistance to AvrPphB (or HopAR1) was found to
trigger excision, as an episome, of a 106-kb region carrying the effector gene [40]. The episome poorly expresses
avrPphB and can be lost, later integrated into the genome, or transformed into other P. syringae strains coinfecting leaf tissue [40–42].
Micro-evolutionary studies based on MLST analysis
and genomics of P. syringae strains isolated from diseased
tomato plants in the field provide a global perspective on
the dynamic nature of effector repertoires. Pto DC3000
and Pto T1 both cause bacterial speck of tomato, but they
share only 14 effector genes, with 15 others being present
only in DC3000 and another 11 only in T1 [32]. Analysis of
strains isolated over time from diseased tomato plants
around the world reveals that DC3000-like strains were
prevalent until 1961 when T1-like strains appeared and
then rapidly predominated [43,44]. These observations
of repertoire variability among and within pathovars
indicate that P. syringae can defeat plants with many
Negative regulation by symbiont of pathogen-associated molecular
pattern-induced host innate immunity (GO:0052034)
AvrRpt2
Pto
Fen
Prf
EFR
FLS2 BAK1
AvrRpm1
RIN4
RPS2
AvrB1
BIK1
MPK4
RAR1
HopZ1
GmHID1
HopF2
MKK5
HopAI1
MPK3,6
HopU1
GRP7
HopM1
AtMIN7
HopI1
Hsp70
PTI
Key:
red
RPM1
PBS1
RPS5
HopD
HopE
HopF
HopK
HopX
HopZ
HopAM
AvrRps4
CERK
AvrPtoB
AvrPphB
AvrB1 AvrPphB
Interactions impeding defense
Interactions triggering defense
Confirmed physical interaction
ETI
HopF2
Negative regulation by
symbiont of host R-gene
dependent defense
response (GO:0033660)
HopD
HopE
HopF
HopG
HopN
HopS
HopU
HopX
HopZ
HopAB
HopAM
HopAO
AvrB
AvrRpm1
AvrRpt2
Negative regulation by symbiont of host
defense-related programmed cell death
(GO:0034054)
HopA
HopC
HopS
HopT
HopAA
HopAB
HopAF
HopAM
HopAO
HopAR
AvrB
AvrE
AvrPto
AvrRpm1
AvrRps4
AvrRpt2
AvrPtoB
Positive regulation by
symbiont of host resistance
gene-dependent defense
response (GO:0052527)
AvrPto
Disassembly and reassembly of effector repertoires
reveals jumps in host range, redundant effector groups
and a minimal functional repertoire
P. syringae strains typically carry multiple effectors that
can elicit ETI in nonhost plants [8,38]. Genetically disrupting recognition of these avirulence determinants can
enable compatible growth in nonhosts. For example, deletion of hopQ1-1 from Pto DC3000 allows the bacterium to
grow to high levels and cause disease in nonhost Nicotiana
benthamiana [33,45]. Compromising tomato ETI surveillance of AvrPto and AvrPtoB (or HopAB2) enables several
P. syringae pathovars that are adapted to other plant
species to cause disease in tomato [46]. Arabidopsis ETI
surveillance of HopAS1 appears to play a similarly broad
role in nonhost resistance: a P. syringae pv. tomato T1
mutant lacking HopAS1 and AvrRpt2, another effector
recognized by Arabidopsis Col-0, grows significantly better
Kinase or phosphorylation-dependent
NB-LRR
ARF-GEF regulating vesicle trafficking
TRENDS in Microbiology
Figure 2. Overview of Pseudomonas syringae effector targets and interplay in the context of the two levels of plant immunity and Gene Ontology (GO) terms. Effectors are
shown suppressing pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) or eliciting and/or suppressing effector-triggered immunity (ETI), with
effector–target interactions superimposed on the two levels of immunity [6,36,68]. Notably, the vast majority of known interactions involve kinases or act in a
phosphorylation-dependent manner in recognitional battles at both levels of immunity. Two types of effector interplay involving shared targets are evident: redundant
interaction with PAMP perception pathway components and interfering interaction with ETI system components. Detailed GO annotation is available for all of these
effectors (http://pseudomonas-syringae.org), and only high-level terms are shown here. Effector subfamilies, such as AvrPtoB (or HopAB2), are designated for specific
effector–target interactions, whereas effector family designations, such as HopAB, are used for broader activities.
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in planta than the wild type, although not nearly as well as
Pto DC3000 [47].
To systematically explore the functional structure of an
effector repertoire in the context of pathogenic growth in a
single plant, mutants containing progressive deletions in
the 28 well-expressed effector genes in Pto DC3000
(Figure 1) were constructed and analyzed for changes in
growth in N. benthamiana, an experimentally amenable
wild tobacco species (with all strains lacking the hopQ1-1
avirulence determinant) [9,33,48,49]. Deleting 15 of the
effector genes produced remarkably little reduction in
pathogen growth [48]. However, strong reductions in
growth accompanied loss of two redundant effector
groups (REGs): AvrPto/AvrPtoB and AvrE/HopM1/HopR1
(Figure 1; the numerical suffix indicates the effector subfamily). These REGs contain internally redundant effectors that appear to target two PTI processes, PAMP
perception and vesicle trafficking, respectively (Figure 2)
[6,48]. As an indicator of the distinctness of these processes, deleting fliC, which encodes the major PAMP flagellin,
rescued growth of an avrPto/avrPtoB mutant but not of an
avrE/hopM1/hopR1 mutant [48]. These observations predict that repertoires structured to redundantly target important processes in PTI would continue to function
robustly despite loss of any effector due to selection pressure from immune surveillance.
A minimal set of Pto DC3000 effectors sufficient to
promote bacterial growth and disease in N. benthamiana
was identified by constructing polymutant DC3000D28E,
which lacks all 28 of the well expressed effector genes, and
then restoring effector genes in small combinatorial sets
(Figure 1) [9]. DC3000D28E is functionally effectorless and
grows 4 logs worse than DC3000DhopQ1-1 in planta. The
minimal effector repertoire was constructed in three
stages, each yielding insights into effector interplay. First,
various components of the two known REGs were introduced. This revealed that hopM1 or avrE and the entire
conserved effector locus were unable to promote growth
unless either avrPto or avrPtoB was present. Each of the
later two effectors could independently promote growth.
Second, a DC3000D28E derivative carrying avrPto and
hopM1 (growing almost 2 logs better than DC3000D28E)
was used as a recipient for randomly assembled sets of
three or five effector genes from a collection of 17 effectors
not associated with the known REGs. Effector genes were
shuffled, concatenated and integrated into the exchangeable effector locus in the Hrp pathogenicity island of
DC3000D28E. Many of the resulting strains grew better
than the DC3000D28E derivative carrying just avrPto and
hopM1. This revealed the frequent occurrence of several
genes, including hopE1, hopG1 and hopAM1, in the combinatorial sets that promoted further growth. In the third
stage, information from the first two stages was used to
construct a minimal functional set of eight effectors (AvrPtoB, HopE1, HopG1, HopAM1, AvrE, HopM1, HopAA1 and
HopN1), which enhanced the growth of DC3000D28E in N.
benthamiana by more than 3 logs to near wild type levels
and enabled production of necrotic/chlorotic disease
lesions.
The two REGs in the Pto DC3000 repertoire are clearly
important, but it is noteworthy that effectors other than
Trends in Microbiology April 2012, Vol. 20, No. 4
HopE1, HopG1 and HopAM1 can also promote growth of
DC3000D28E expressing just AvrPto and HopM1 and that
P. syringae pv. syringae B728a and P. syringae pv. tabaci
11528 also cause disease in N. benthamiana although their
genomes lack these three effectors [50,51]. Furthermore,
mutations affecting single or multiple effector genes in a
given P. syringae strain can produce significantly different
phenotypes in alternative hosts [33,50,52]. In summary,
genomic comparison of multiple effector repertoires or
genetic manipulation of a model repertoire suggests that
P. syringae defeats plant immunity with a few core effectors and many interchangeable effectors.
Effector interactions with the plant immune system
involve two phases of PTI and both weak and strong ETI
The known interactions of P. syringae effectors with components of the PTI and ETI subsystems of immunity are
summarized in Figure 2. PTI can be conceived as involving
a perception phase and a response phase (Figure 3a). The
first extends from PAMP perception by PRRs to the activation of defense gene expression [14]. The response phase
is posttranscriptional and culminates in highly localized
vesicle trafficking of various antimicrobial compounds to
the point of pathogen contact [12,53,54].
The perception phase of PTI is dependent on a kinase
signaling system characterized by limited polymorphism
and high redundancy. For example, FLS2 is an ancient
PRR that recognizes flagellin and is widespread in higher
plants but not functional in a subset of Arabidopsis ecotypes [14]. However, plants recognize multiple bacterial
PAMPs, and FLS2-deficient Arabidopsis plants are only
partially impaired in immunity [55]. FLS2 is a receptorlike kinase, and downstream kinase signaling involves a
network of mitogen-activated protein kinase (MAPK) and
calcium-dependent protein kinase (CDPKs) cascades that
function robustly unless multiple components are disrupted [12,56]. Thus, the perception system is sufficiently
redundant that pathogens such as P. syringae cannot
evade it but must suppress it, and they must do so with
multiple effectors. However, it is noteworthy that PAMPPRR polymorphism may, itself, significantly affect P. syringae–plant interactions. For example, transfer from Arabidopsis to tomato of a PRR that recognizes bacterial
elongation factor Tu (naturally present in cruciferous
plants but lacking in solanaceous plants) resulted in tomato plants with increased resistance to multiple bacterial
pathogens [57]. Also, strains of P. syringae pv. tomato
isolated from diseased tomato plants in recent years differ
from previously isolated strains in producing a flagellin
variant that lacks a second PAMP epitope and therefore
elicits weaker PTI [44].
The response phase of PTI is likely to involve similar
polymorphisms and redundancies regarding antimicrobial
factors, but it also employs vesicle trafficking for antimicrobial deployment and receptor recycling [54]. These processes, largely regulated by small GTPases, are likely to be
bottlenecks in immunity. The core conserved effector locus
effectors AvrE and HopM1 appear to attack this phase, as
discussed below, whereas the variable effectors more commonly attack kinase complexes associated with the perception phase (Figure 2).
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(a)
Vesicle trafficking of antimicrobials +
PAMP perception
PTI
PP
VT
PTI
Nucleus
(b)
Suppression of vesicle trafficking +
PAMP perception
PTI
PP
VT
Susceptibility
(c)
Suppression of vesicle trafficking +
PAMP perception
PTI
ETI
PP
VT
PTI/ETI?
(d)
Suppression of vesicle trafficking +
Suppression of PAMP perception
PTI
PP
VT
Susceptibility
(e)
(f)
PTI
PTI
PP2
ETI
ETI
PP
PP
VT
sVT + sPP + PP2
sVT + sPP + sPP2
R
VT
PTI/ETI?
Susc.
sVT + sPP + R
sVT + sPP + sR
ETI
Susc.
TRENDS in Microbiology
Figure 3. The proposed two major stages, based on core-effector protection, in the
coevolution of Pseudomonas syringae type III effect repertoires and host immune
systems. In the first stage (a), P. syringae progenitors were unable to block
pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) by
suppressing either PAMP perception (PP) or vesicle trafficking of antimicrobials
(VT), until (b) acquisition of the type III secretion system (T3SS)-encoding Hrp
pathogenicity island and ancient effectors, such as HopM, enabled suppression of
vesicle trafficking (sVT), a bottleneck vulnerability in the immune system. (c) We
postulate that subsequently evolved immune detection of sVT elicited effectortriggered immunity (ETI; green font) when PAMPs were simultaneously detected.
(d) Pathogens countered this ancient ETI by suppressing components of the
kinase-based PAMP perception system (sPP). In the second stage of coevolution
(e,f), proliferation and horizontal transfer of variable effector genes was
accompanied by increasing redundancies in the PP system (e.g. PP2) and by ETI
(pink font) elicited by R protein surveillance of effector actions on PP system
components or their decoys. Extant repertoires are the product of ongoing
coevolutionary recognition/evasion skirmishes. A functional repertoire renders a
host plant susceptible because it is able to: (e) suppress redundantly robust PP
204
The ETI system appears largely directed at the latter
attack. ETI recognition of P. syringae effectors is typically
indirect and dependent on NB-LRR recognition of effectorcatalyzed changes to targets that are considered guardees
if they have a demonstrable role in immunity or decoys if
they mimic a susceptibility target [6,58,59]. Guarding a
few vulnerable susceptibility targets may enable a smaller
number of NB-LRRs to provide ETI protection against a
potentially enormous number of rapidly evolving effectors,
and using decoys may disengage evolution of decoy and
susceptibility target sensitivities to effector action [11,58].
PTI and ETI are defined on the basis of distinct classes
of elicitors, but pathogens present complex mixtures of
both classes during infection, and overlap between PTI
and ETI signaling systems and responses makes it difficult
to distinguish them [60–62]. Although HR cell death has
been a workhorse marker for ETI, some effectors trigger
weak ETI characterized by increased resistance without
cell death [47,50,63]. Further confounding a simple model
for collective effector action, some effectors suppress ETI
elicited by other effectors or by other domains within the
same effector (Figure 2) [64–68]. Furthermore, as model
effectors, such as AvrPto, AvrPtoB, AvrB and HopF, receive further scrutiny, additional domains, activities and
targets are discovered [6,35]. Importantly, those activities
demonstrate the possibility of interplay with other effectors on common targets, and a given effector may suppress
PTI, elicit weak or strong ETI, suppress ETI or do some or
all of the above depending on the effector repertoire and
immune system context (Figure 2). It is noteworthy here
that P. syringae pv. tabaci was able to grow well in nonhost
Arabidopsis when PTI and ETI were simultaneously compromised by mutation of fliC in the bacterium and rar1
(compromising NB-LRR stability) in the plant [69]. Collectively, these observations highlight the utility of considering the perception phases of PTI and ETI as integrated
processes important in both host and nonhost resistance,
and they suggest a broad ability of P. syringae to grow in
nonhost plants if pathogen perception is defeated.
The ancient effectors AvrE and HopM1 differ from
variable effectors in their interactions with the immune
system
As discussed above, AvrE and HopM1 are part of the
vesicle trafficking REG in Pto DC3000. HopM1 destabilizes, through the host 26S proteasome, the Arabidopsis
AtMIN7 protein, a plant ADP ribosylation factor (ARF)
guanine nucleotide exchange factor (GEF) protein regulating vesicle trafficking [70]. Both AtMIN7 and HopM1 are
localized to the trans-Golgi network/early endosome [71].
Arabidopsis plants lacking AtMIN7 through HopM1 action
or through mutation by T-DNA insertion are more susceptible to a Pto DC3000 conserved effector locus deletion
mutant [70], demonstrating the importance of AtMIN7
to immunity. However, ETI elicited by AvrRpt2 or two
(sPP2), and (f) suppress ETI elicited by any variable effectors that are recognized
(sR). Also, variable effectors that elicit unsuppressed ETI in a plant can be
eliminated with little cost because of redundancies in the repertoire. ETI-level
incompatibilities (f) have been observed to limit the host range of multiple P.
syringae pathovars, but PTI-level specializations and other adaptations to plant
species (e), even if only quantitatively beneficial, may obviate the selective
advantage of maintaining or evolving ETI-level compatibility with additional hosts.
Review
other effectors stabilizes AtMIN7 in HopM1 transgenic
plants [71]. Although the target of AvrE is unknown, the
effector carries a WxxxE motif and a putative C-terminal
endoplasmic reticulum membrane retention/retrieval signal, which are required for all AvrE biological activities
and suggest G-protein mimicking activity [72].
AvrE and HopM1 have a general ability to elicit cell
death in plants [73], but several observations suggest that
cell death elicited by AvrE/HopM1 is fundamentally different than ETI elicited by variable effectors attacking the
kinase system. Elicitation by the former is not dependent
on any known R gene, and HopM1 does not appear to elicit
cell death through AtMIN7 destabilization because Arabidopsis Atmin7 mutants are viable [70]. In striking contrast, AvrRpt2 cysteine protease-mediated destruction of
RPM1 interacting protein 4 (RIN4) in Arabidopsis elicits
ETI when RIN4 is guarded by the NB-LRR RPS2 protein,
and rin4 mutants are not viable in an RPS2 background
[6]. AvrE and HopM1 also differ from the variable effectors
in contributing significantly to the cell death associated
with disease symptoms [73].
We postulate that ETI may be triggered by AvrE or
HopM1 damage to a process (vesicle trafficking) rather
than a specific protein (AtMIN7). We further postulate that
plants reduce spurious cell death responses resulting from
vesicle trafficking perturbations by requiring a second
signal, namely PAMP perception, or associated increases
in salicylic acid (SA), a defense hormone important in plant
immunity to biotrophic pathogens [74]. It is noteworthy
that two-signal models involving simultaneous perception
of PAMPs and pathogen-associated activities appear to
underlie animal innate immune responses that have a
potential for self damage [75].
A two-signal model would explain why AvrE and
HopM1 cannot promote DC3000D28E growth in N.
benthamiana unless AvrPto or AvrPtoB is present [9]. This
model for ETI elicitation by conserved effector locus effectors is also consistent with the ancient core-effector status
of HopI, which suppresses generation of SA [76,77]. Interestingly, plant cell death is observed in Arabidopsis bip2
(ER-resident chaperone) mutants in the presence of SA
analogs, an effect attributed to an extreme unfolded protein response [78]. Also, SA-dependent cell death is observed when components of the PAMP perception system
that also function in plant development, such as BAK1 and
BIK1, are mutated, and Arabidopsis pmr4 (callose
synthase) mutants constitutively produce higher levels
of SA [14]. Thus, plants appear to default to death and
defense when multipurpose or bottleneck immunity-associated proteins and processes are disrupted.
Establishing the interactome of P. syringae effectors
and plant targets is an ongoing challenge
Figure 2 categorizes P. syringae effector impacts on the two
levels of plant immunity in the context of Gene Ontology
(GO) process terms, and it also depicts the consequential
physical interactions of effectors with immunity-associated
plant proteins [6,79]. Effectors modify these immunity
proteins by a variety of enzymatic activities, which have
recently been reviewed [6]. One lesson from Figure 2 is that
progress has been slow in the search for plant proteins that
Trends in Microbiology April 2012, Vol. 20, No. 4
interact with effectors, and only a handful of the effectors
in the super-repertoire have established targets [6,79]. A
recently reported Herculean effort based on stringent yeast
two-hybrid screening addressed comprehensive identification of the plant pathogen protein–protein interactome
network in Arabidopsis [10]. The screen involved 101 P.
syringae effectors representing diverse effector families
and subfamilies, a collection of effectors from Hyaloperonospora arabidopsidis (an obligate biotroph oomycete), and
a collection of immune-related and circa 8000 full-length
Arabidopsis proteins (a subset of the proteome). Interacting plant proteins were analyzed for their connectivity with
other Arabidopsis proteins that had previously been assembled into an interactome using the same methods in a
companion study [80]. Fifty nine Arabidopsis proteins
interacted directly with at least one member of 30 P.
syringae effector subfamilies, and several of these plant
proteins also interacted with H. arabidopsidis effectors.
Many of the interacting plant proteins interact with highly
connected cellular hubs, leading to the conclusion that
decoys are a rarity.
However, it is possible that stringent yeast two-hybrid
screening may yield misleading results for effectors such as
AvrPto, which have low structural stability [81], and that
many effectors may act promiscuously by targeting host
molecules with low specificity and affinity [13]. Indeed,
previous yeast two-hybrid screens involving AvrPto have
produced candidates that were not validated by subsequent tests for interaction in planta [82,83]. Of the previously studied interactors for all P. syringae effectors, only
one was identified in this stringent screen of Arabidopsis
proteins [10], and AvrPto failed to interact with FLS2 or
BAK1, but instead interacted with 13 new proteins. One of
these, PFD6, was shown to contribute to flagellin epitope
flg22-elicited immunity, but it is not clear that AvrPto
interacts with this protein physically in planta or functionally during infection. It is noteworthy that AvrPto delivered via the T3SS only slightly promotes growth of
effectorless Pto DC3000D28E in N. benthamiana [9],
whereas AvrPto transgenically expressed in Arabidopsis
confers strong susceptibility to a Pto DC3000 T3SS-deficient mutant [84]. Perhaps AvrPto interacts with additional plant proteins when present at high levels or suppresses
PTI more effectively by its presence in advance of pathogen
contact. In summary, the yeast two-hybrid interactome
screen has produced a plethora of leads for further investigation of effector functions and brings to the fore the
question of whether pathogen effectors preferentially target cellular hubs in the plant interactome [10].
A protected-core model for effector repertoire
expansion and host specialization suggests two stages
of coevolution
A model for the coevolution of P. syringae effector repertoires and the plant immune system is presented in
Figure 3. Three points addressing the structure of effector
repertoires warrant discussion. First, an essential property of a functional repertoire appears to be its ability to
suppress ETI elicitation by its own members. In this
regard, we suggest that the core and variable effectors
have different evolutionary paths and present different
205
Review
challenges for maintaining compatibility with host ETI
systems. Second, the expansion of variable effectors and
PTI/ETI perception components may have been mutually
driven and then tolerated in the plant by the relative
independence of recognition systems from basic cellular
functions. For example, RIN4 is a hub in the realm of NBLRRs, effectors and other immunity factors (Figure 2)
[85,86], but it is not highly connected to other Arabidopsis
proteins [10], and can be eliminated with little consequence beyond its immunity functions [85,87]. Third, investigation of field-based interactions of P. syringae pv.
phaseolicola races with differential cultivars of its host
bean has yielded multiple examples of the loss of a variable
effector unmasking the avirulence activity of another variable effector in the same repertoire [64,65,88], and such
ETI suppressors are now considered to be prevalent components of effector repertoires [68].
The underlying basis for host specialization (and
reciprocally, nonhost resistance) remains unresolved. Adaptation to a host clearly necessitates coevolutionary
maintenance of a compatible effector repertoire, analogous
to the maintenance of a set of rotating passwords for entry
into a high-security facility. It is not surprising that this
process has the collateral effect of allowing accumulation of
multiple ETI-level incompatibilities with other plant species. However, even when these incompatibilities are genetically corrected by removal of corresponding avr or R
genes (as discussed above), the pathogen usually is significantly less virulent on a nonhost plant than are strains
that normally cause disease [46,47]. Unfortunately, we
cannot determine whether weaker growth in the nonhost
results from insufficient suppression of PTI, residual elicitation of weak ETI or other factors. Promising approaches
to this problem include reassembling minimal functional
repertories with effectors from various repertoires and the
use of different plant species and immunity mutants that
reduce redundancy and enable probing of different immune system sectors. Indeed, the latter approach has
already revealed that in Arabidopsis, PTI signaling components appear to function synergistically for signal amplification, whereas ETI signaling components function in
a compensatory manner [89].
It is also worth noting here that pathogens also may be
adapted to nutritional and antimicrobial differences
among plants. For example, Pto DC3000 infection induces
in a T3SS-dependent manner a set of Arabidopsis sugar
transporter SWEET genes that differs from that induced
by powdery mildew fungus infection [90], and Pto DC3000
and other P. syringae strains pathogenic on Arabidopsis
carry sax (sensitivity to Arabidopsis extracts) genes that
confer tolerance to an Arabidopsis aliphatic isothiocyanate
[91]. The apparent capacity for catabolism of aromatic
compounds in P. syringae pathovars pathogenic in woody
hosts likewise supports the potential importance of nutrient acquisition in host adaptation [24,92].
Newly identified biotrophic pathogen effector
repertoires raise new questions for the P. syringae
system
Recent genome sequence-based reports of candidate effector repertoires in closely related groups of fungi and
206
Trends in Microbiology April 2012, Vol. 20, No. 4
oomycetes that are specialized for different plant species
has provided a similar picture of remarkably divergent and
large repertoires, with some pathogens having hundreds of
candidate effector genes [93–95]. A model for repertoire
evolution and host adaptation based on these eukaryotic
pathogens proposes that the adaptation of effector repertoires to diverging PTI components drives host specialization and that as evolutionary distance between host and
non-host plant species increases so does the relative contribution of PTI over ETI [96]. It is possible that the
functional divergence of HopM1 provides an example of
PTI-level differences affecting host range. However, observations that many P. syringae effectors can promote virulence in a broad range of plants and that several variable
effector families seem to recycle among strains adapted to
divergent hosts suggests that PTI targets are phylogenetically widespread and that ETI is a pervasively important
component of nonhost resistance [8]. It is possible that the
potential for P. syringae to horizontally exchange effectors
and to be widely disseminated as part of the global water
cycle affects the dynamics of effector repertoire evolution
[97]. More progress in identifying plant proteins and processes targeted by the cytoplasmic effectors of all classes of
pathogens will be key in resolving this issue. Pto DC3000
may be a widely useful tool in this effort and has recently
been used to probe 64 candidate effectors from the oomycete H. arabidopsidis (engineered for T3SS delivery) for
their ability to suppress plant immunity [98].
A satisfying and agriculturally useful explanation for
the patterns of effector distribution and the plant targets
presented in Figures 1 and 2 will probably require the
perspectives of systems biology [99,100]. The P. syringae
community now has key tools to support this goal, such as
cloned effector genes representing the complete superrepertoire, a facile system for reassembling alternative
repertoires in an effectorless strain, a large collection of
Arabidopsis candidate effector interactors, and experimental and bioinformatic pipelines to use with next-generation
sequencing for an increasingly detailed picture of natural
variation in the field. A community website (http://pseudomonas-syringae.org) provides extensive resources for data
aggregation and interpretation, including literature-based
annotation of effector functions, interactors and plant
responses using GO terms that are machine-readable
and applicable to effectors from all pathogen classes. Thus,
insights into effector function can be efficiently compared
as we explore better ways to prevent all pathogens from
getting the last word in their interactions with crop plants.
Acknowledgments
Community web resources and laboratory work are supported by
National Science Foundation grant IOS-1025642.
References
1 Finlay, B. and Falkow, S. (1997) Common themes in microbial
pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61, 136–169
2 Kenny, B. and Valdivia, R. (2009) Host-microbe interactions: bacteria.
Curr. Opin. Microbiol. 12, 1–3
3 Pallen, M.J. and Wren, B.W. (2007) Bacterial pathogenomics. Nature
449, 835–842
4 Vinatzer, B.A. and Yan, S. (2008) Mining the genomes of plant
pathogenic bacteria: how not to drown in gigabases of sequence.
Mol. Plant Pathol. 9, 105–118
Review
5 Dodds, P.N. (2010) Genome evolution in plant pathogens. Science 330,
1486–1487
6 Block, A. and Alfano, J.R. (2011) Plant targets for Pseudomonas
syringae type III effectors: virulence targets or guarded decoys?
Curr. Opin. Microbiol. 14, 39–46
7 O’Brien, H.E. et al. (2011) Evolution of plant pathogenesis in
Pseudomonas syringae: a genomics perspective. Annu. Rev.
Phytopathol. 49, 269–289
8 Baltrus, D.A. et al. (2011) Dynamic evolution of pathogenicity
revealed by sequencing and comparative genomics of 19
Pseudomonas syringae isolates. PLoS Pathog. 7, e1002132
9 Cunnac, S. et al. (2011) Genetic disassembly and combinatorial
reassembly identify a minimal functional repertoire of type III
effectors in Pseudomonas syringae. Proc. Natl. Acad. Sci. U.S.A.
108, 2975–2980
10 Mukhtar, M.S. et al. (2011) Independently evolved virulence effectors
converge onto hubs in a plant immune system network. Science 333,
596–601
11 Jones, J.D. and Dangl, J.L. (2006) The plant immune system. Nature
444, 323–329
12 Boller, T. and Felix, G. (2009) A renaissance of elicitors: perception of
microbe-associated molecular patterns and danger signals by patternrecognition receptors. Annu. Rev. Plant Biol. 60, 379–406
13 Hann, D.R. and Rathjen, J.P. (2010) The long and winding road:
virulence effector proteins of plant pathogenic bacteria. Cell. Mol. Life
Sci. 67, 3425–3434
14 Segonzac, C. and Zipfel, C. (2011) Activation of plant patternrecognition receptors by bacteria. Curr. Opin. Microbiol. 14, 54–61
15 Oh, C.S. and Martin, G.B. (2011) Effector-triggered immunity
mediated by the Pto kinase. Trends Plant Sci. 16, 132–140
16 Sarkar, S.F. and Guttman, D.S. (2004) Evolution of the core genome of
Pseudomonas syringae, a highly clonal, endemic plant pathogen.
Appl. Environ. Microbiol. 70, 1999–2012
17 Bull, C.T. et al. (2011) Multilocus sequence typing of Pseudomonas
syringae sensu lato confirms previously described genomospecies and
permits rapid identification of P. syringae pv. coriandricola and P.
syringae pv. apii causing bacterial leaf spot on parsley.
Phytopathology 101, 847–858
18 Vinatzer, B.A. et al. (2005) Bioinformatics correctly identifies many
type III secretion substrates in the plant pathogen Pseudomonas
syringae and the biocontrol isolate P. fluorescens SBW25. Mol.
Plant Microbe Interact. 18, 877–888
19 Lindeberg, M. et al. (2006) Closing the circle on the discovery of genes
encoding Hrp regulon members and type III secretion system effectors
in the genomes of three model Pseudomonas syringae strains. Mol.
Plant Microbe Interact. 19, 1151–1158
20 Chang, J.H. et al. (2005) A high-throughput, near-saturating screen
for type III effector genes from Pseudomonas syringae. Proc. Natl.
Acad. Sci. U.S.A. 102, 2549–2554
21 Lindeberg, M. et al. (2005) Proposed guidelines for a unified
nomenclature and phylogenetic analysis of type III Hop effector
proteins in the plant pathogen Pseudomonas syringae. Mol. Plant
Microbe Interact. 18, 275–282
22 Almeida, N.F. et al. (2010) PAMDB, a multilocus sequence typing and
analysis database and website for plant-associated microbes.
Phytopathology 100, 208–215
23 Reinhardt, J.A. et al. (2009) De novo assembly using low-coverage
short read sequence data from the rice pathogen Pseudomonas
syringae pv. oryzae. Genome Res. 19, 294–305
24 Rodriguez-Palenzuela, P. et al. (2010) Annotation and overview of the
Pseudomonas savastanoi pv. savastanoi NCPPB 3335 draft genome
reveals the virulence gene complement of a tumour-inducing
pathogen of woody hosts. Environ. Microbiol. 12, 1604–1620
25 Kvitko, B.H. et al. (2007) Identification of harpins in Pseudomonas
syringae pv. tomato DC3000, which are functionally similar to HrpK1
in promoting translocation of type III secretion system effectors. J.
Bacteriol. 189, 8059–8072
26 Alfano, J.R. et al. (2000) The Pseudomonas syringae Hrp
pathogenicity island has a tripartite mosaic structure composed
of a cluster of type III secretion genes bounded by exchangeable
effector and conserved effector loci that contribute to parasitic
fitness and pathogenicity in plants. Proc. Natl. Acad. Sci. U.S.A.
97, 4856–4861
Trends in Microbiology April 2012, Vol. 20, No. 4
27 Groll, M. et al. (2008) A plant pathogen virulence factor inhibits the
eukaryotic proteasome by a novel mechanism. Nature 452, 755–758
28 Bender, C.L. et al. (1999) Pseudomonas syringae phytotoxins: mode of
action, regulation, and biosynthesis by peptide and polyketide
synthetases. Microbiol. Mol. Biol. Rev. 63, 266–292
29 Lorang, J.M. et al. (1994) avrA and avrE in Pseudomonas syringae pv.
tomato PT23 play a role in virulence on tomato plants. Mol. Plant
Microbe Interact. 7, 508–515
30 Rohmer, L. et al. (2004) Diverse evolutionary mechanisms shape the
type III effector virulence factor repertoire in the plant pathogen
Pseudomonas syringae. Genetics 167, 1341–1360
31 Wroblewski, T. et al. (2009) Comparative large-scale analysis of
interactions between several crop species and the effector
repertoires from multiple pathovars of Pseudomonas and
Ralstonia. Plant Physiol. 150, 1733–1749
32 Almeida, N.F. et al. (2009) A draft genome sequence of
Pseudomonas syringae pv. tomato strain T1 reveals a repertoire of
type III related genes significantly divergent from that of
Pseudomonas syringae pv. tomato strain DC3000. Mol. Plant
Microbe Interact. 22, 52–62
33 Wei, C-F. et al. (2007) A Pseudomonas syringae pv. tomato DC3000
mutant lacking the type III effector HopQ1-1 is able to cause disease
in the model plant Nicotiana benthamiana. Plant J. 51, 32–46
34 Hajri, A. et al. (2009) A ‘repertoire for repertoire’ hypothesis:
repertoires of type three effectors are candidate determinants of
host specificity in Xanthomonas. PLoS ONE 4, e6632
35 Nguyen, H.P. et al. (2010) Two virulence determinants of type III
effector AvrPto are functionally conserved in diverse Pseudomonas
syringae pathovars. New Phytol. 187, 969–982
36 Cunnac, S. et al. (2009) Pseudomonas syringae type III secretion
system effectors: repertoires in search of functions. Curr. Opin.
Microbiol. 12, 53–60
37 Lin, N.C. et al. (2006) Diverse AvrPtoB homologs from several
Pseudomonas syringae pathovars elicit Pto-dependent resistance
and have similar virulence activities. Appl. Environ. Microbiol. 72,
702–712
38 Lindeberg, M. et al. (2009) The evolution of Pseudomonas syringae
host specificity and type III effector repertoires. Mol. Plant Pathol. 10,
767–775
39 Lindeberg, M. et al. (2008) Roadmap to new virulence determinants in
Pseudomonas syringae: insights from comparative genomics and
genome organization. Mol. Plant Microbe Interact. 21, 685–700
40 Pitman, A.R. et al. (2005) Exposure to host resistance mechanisms
drives evolution of bacterial virulence in plants. Curr. Biol. 15, 2230–
2235
41 Godfrey, S.A. et al. (2011) The stealth episome: suppression of gene
expression on the excised genomic island PPHGI-1 from Pseudomonas
syringae pv. phaseolicola. PLoS Pathog. 7, e1002010
42 Lovell, H.C. et al. (2009) Bacterial evolution by genomic island
transfer occurs via DNA transformation in planta. Curr. Biol. 19,
1586–1590
43 Yan, S. et al. (2008) Role of recombination in the evolution of the model
plant pathogen Pseudomonas syringae pv. tomato DC3000, a very
atypical tomato strain. Appl. Environ. Microbiol. 74, 3171–3181
44 Cai, R. et al. (2011) The plant pathogen Pseudomonas syringae pv.
tomato is genetically monomorphic and under strong selection to
evade tomato immunity. PLoS Pathog. 7, e1002130
45 Ferrante, P. et al. (2009) Contributions of the effector gene hopQ1-1 to
differences in host range between Pseudomonas syringae pv.
phaseolicola and P. syringae pv. tabaci. Mol. Plant Pathol. 10, 837–842
46 Lin, N-C. and Martin, G.B. (2007) Pto/Prf-mediated recognition of
AvrPto and AvrPtoB restricts the ability of diverse Pseudomonas
syringae pathovars to infect tomato. Mol. Plant Microbe Interact.
20, 806–815
47 Sohn, K.H. et al. (2011) HopAS1 recognition significantly contributes
to Arabidopsis nonhost resistance to Pseudomonas syringae
pathogens. New Phytol. 193, 58–66
48 Kvitko, B.H. et al. (2009) Deletions in the repertoire of Pseudomonas
syringae pv. tomato DC3000 type III secretion effector genes reveal
functional overlap among effectors. PLoS Pathog. 5, e1000388
49 Goodin, M.M. et al. (2008) Nicotiana benthamiana: its history and
future as a model for plant–pathogen interactions. Mol. Plant Microbe
Interact. 21, 1015–1026
207
Review
50 Vinatzer, B.A. et al. (2006) The type III effector repertoire of
Pseudomonas syringae pv. syringae B728a and its role in survival
and disease on host and non-host plants. Mol. Microbiol. 62, 26–44
51 Studholme, D.J. et al. (2009) A draft genome sequence and functional
screen reveals the repertoire of type III secreted proteins of
Pseudomonas syringae pathovar tabaci 11528. BMC Genomics 10, 395
52 Crabill, E. et al. (2010) Plant immunity directly or indirectly restricts
the injection of type III effectors by the Pseudomonas syringae type III
secretion system. Plant Physiol. 154, 233–244
53 Forcat, S. et al. (2010) Rapid linkage of indole carboxylic acid to the
plant cell wall identified as a component of basal defence in
Arabidopsis against hrp mutant bacteria. Phytochemistry 71, 870–876
54 Frei dit Frey, N. and Robatzek, S. (2009) Trafficking vesicles: pro or
contra pathogens? Curr. Opin. Plant Biol. 12, 437–443
55 Zipfel, C. et al. (2004) Bacterial disease resistance in Arabidopsis
through flagellin perception. Nature 428, 764–767
56 Boudsocq, M. et al. (2010) Differential innate immune signalling via
Ca(2+) sensor protein kinases. Nature 464, 418–422
57 Lacombe, S. et al. (2010) Interfamily transfer of a plant patternrecognition receptor confers broad-spectrum bacterial resistance.
Nat. Biotechnol. 28, 365–369
58 Hogenhout, S.A. et al. (2009) Emerging concepts in effector biology of
plant-associated organisms. Mol. Plant Microbe Interact. 22, 115–122
59 Zhou, J.M. and Chai, J. (2008) Plant pathogenic bacterial type III
effectors subdue host responses. Curr. Opin. Microbiol. 11, 179–185
60 Katagiri, F. and Tsuda, K. (2010) Understanding the plant immune
system. Mol. Plant Microbe Interact. 23, 1531–1536
61 Thomma, B.P. et al. (2011) Of PAMPs and effectors: the blurred PTIETI dichotomy. Plant Cell 23, 4–15
62 Qi, Y. et al. (2011) Physical association of pattern-triggered immunity
(PTI) and effector-triggered immunity (ETI) immune receptors in
Arabidopsis. Mol. Plant Pathol. 12, 702–708
63 Gassmann, W. (2005) Natural variation in the Arabidopsis response
to the avirulence gene hopPsyA uncouples the hypersensitive
response from disease resistance. Mol. Plant Microbe Interact. 18,
1054–1060
64 Jackson, R.W. et al. (1999) Identification of a pathogenicity island,
which contains genes for virulence and avirulence, on a large native
plasmid in the bean pathogen Pseudomonas syringae pathovar
phaseolicola. Proc. Natl. Acad. Sci. U.S.A. 96, 10875–10880
65 Tsiamis, G. et al. (2000) Cultivar-specific avirulence and virulence
functions assigned to avrPphF in Pseudomonas syringae pv.
phaseolicola, the cause of bean halo-blight disease. EMBO J. 19,
3204–3214
66 Rosebrock, T.R. et al. (2007) A bacterial E3 ubiquitin ligase targets a
host protein kinase to disrupt plant immunity. Nature 448, 370–374
67 Jamir, Y. et al. (2004) Identification of Pseudomonas syringae type III
secreted effectors that suppress programmed cell death in plants and
yeast. Plant J. 37, 554–565
68 Guo, M. et al. (2009) The majority of the type III effector inventory of
Pseudomonas syringae pv. tomato DC3000 can suppress plant
immunity. Mol. Plant Microbe Interact. 22, 1069–1080
69 Zhang, J. et al. (2010) Effector-triggered and pathogen-associated
molecular pattern-triggered immunity differentially contribute to
basal resistance to Pseudomonas syringae. Mol. Plant Microbe
Interact. 23, 940–948
70 Nomura, K. et al. (2006) A bacterial virulence protein suppresses host
innate immunity to cause plant disease. Science 313, 220–223
71 Nomura, K. et al. (2011) Effector-triggered immunity blocks pathogen
degradation of an immunity-associated vesicle traffic regulator in
Arabidopsis. Proc. Natl. Acad. Sci. U.S.A. 108, 10774–10779
72 Ham, J.H. et al. (2009) Multiple activities of the plant pathogen type
III effector proteins WtsE and AvrE require WxxxE motifs. Mol. Plant
Microbe Interact. 22, 703–712
73 Badel, J.L. et al. (2006) A Pseudomonas syringae pv. tomato avrE1/
hopM1 mutant is severely reduced in growth and lesion formation in
tomato. Mol. Plant Microbe Interact. 19, 99–111
74 Robert-Seilaniantz, A. et al. (2011) Hormone crosstalk in plant
disease and defense: more than just JASMONATE-SALICYLATE
antagonism. Annu. Rev. Phytopathol. 49, 317–343
75 Fontana, M.F. and Vance, R.E. (2011) Two signal models in innate
immunity. Immunol. Rev. 243, 26–39
208
Trends in Microbiology April 2012, Vol. 20, No. 4
76 Jelenska, J. et al. (2007) A J domain virulence effector of Pseudomonas
syringae remodels host chloroplasts and suppresses defenses. Curr.
Biol. 17, 499–508
77 Jelenska, J. et al. (2010) Pseudomonas syringae hijacks plant stress
chaperone machinery for virulence. Proc. Natl. Acad. Sci. U.S.A. 107,
13177–13182
78 Wang, D. et al. (2005) Induction of protein secretory pathway is
required for systemic acquired resistance. Science 308, 1036–1040
79 Zhou, H. et al. (2011) Pseudomonas syringae type III effector HopZ1
targets a host enzyme to suppress isoflavone biosynthesis and
promote infection in soybean. Cell Host Microbe 9, 177–186
80 Arabidopsis-Interactome-Mapping-Consortium (2011) Evidence for
network evolution in an Arabidopsis interactome map. Science 333,
601–607
81 Wulf, J. et al. (2004) The solution structure of type III effector protein
AvrPto reveals conformational and dynamic features important for
plant pathogenesis. Structure 12, 1257–1268
82 Bogdanove, A.J. and Martin, G.B. (2000) AvrPto-dependent Ptointeracting proteins and AvrPto-interacting proteins in tomato.
Proc. Natl. Acad. Sci. U.S.A. 97, 8836–8842
83 Speth, E.B. et al. (2009) Subcellular localization and functional
analysis of the Arabidopsis GTPase RabE. Plant Physiol. 149,
1824–1837
84 Hauck, P. et al. (2003) A Pseudomonas syringae type III effector
suppresses cell wall-based extracellular defense in susceptible
Arabidopsis plants. Proc. Natl. Acad. Sci. U.S.A. 100, 8577–8582
85 Liu, J. et al. (2009) RIN4 functions with plasma membrane H+ATPases to regulate stomatal apertures during pathogen attack.
PLoS Biol. 7, e1000139
86 Chung, E.H. et al. (2011) Specific threonine phosphorylation of a host
target by two unrelated type III effectors activates a host innate
immune receptor in plants. Cell Host Microbe 9, 125–136
87 Belkhadir, Y. et al. (2004) Arabidopsis RIN4 negatively regulates
disease resistance mediated by RPS2 and RPM1 downstream or
independent of the NDR1 signal modulator and is not required for
the virulence functions of bacterial type III effectors AvrRpt2 or
AvrRpm1. Plant Cell 16, 2822–2835
88 Arnold, D.L. et al. (2011) Pseudomonas syringae pv. phaseolicola: from
‘has bean’ to supermodel. Mol. Plant Pathol. 12, 617–627
89 Tsuda, K. et al. (2009) Network properties of robust immunity in
plants. PLoS Genet. 5, e1000772
90 Chen, L.Q. et al. (2010) Sugar transporters for intercellular exchange
and nutrition of pathogens. Nature 468, 527–532
91 Fan, J. et al. (2011) Pseudomonas sax genes overcome aliphatic
isothiocyanate-mediated non-host resistance in Arabidopsis.
Science 331, 1185–1188
92 Green, S. et al. (2010) Comparative genome analysis provides insights
into the evolution and adaptation of Pseudomonas syringae pv. aesculi
on Aesculus hippocastanum. PLoS ONE 5, e10224
93 Baxter, L. et al. (2010) Signatures of adaptation to obligate
biotrophy in the Hyaloperonospora arabidopsidis genome.
Science 330, 1549–1551
94 Spanu, P.D. et al. (2010) Genome expansion and gene loss in powdery
mildew fungi reveal tradeoffs in extreme parasitism. Science 330,
1543–1546
95 Raffaele, S. et al. (2010) Genome evolution following host jumps in the
Irish potato famine pathogen lineage. Science 330, 1540–1543
96 Schulze-Lefert, P. and Panstruga, R. (2011) A molecular evolutionary
concept connecting nonhost resistance, pathogen host range, and
pathogen speciation. Trends Plant Sci. 16, 117–125
97 Morris, C.E. et al. (2008) The life history of the plant pathogen
Pseudomonas syringae is linked to the water cycle. ISME J. 2,
321–334
98 Fabro, G. et al. (2011) Multiple candidate effectors from the oomycete
pathogen Hyaloperonospora arabidopsidis suppress host plant
immunity. PLoS Pathog. 7, e1002348
99 Pritchard, L. and Birch, P. (2011) A systems biology perspective on
plant–microbe interactions: biochemical and structural targets of
pathogen effectors. Plant Sci. 180, 584–603
100 Schneider, D.J. and Collmer, A. (2010) Studying plant–pathogen
interactions in the genomics era: beyond molecular Koch’s
postulates to systems biology. Annu. Rev. Phytopathol. 48, 457–479