PLANT PATHOLOGY
The pan-genome effector-triggered immunity
landscape of a host-pathogen interaction
Bradley Laflamme^1 , Marcus M. Dillon^1 , Alexandre Martel^1 *, Renan N. D. Almeida^1 ,
Darrell Desveaux^1 †‡, David S. Guttman1,2†‡
Effector-triggered immunity (ETI), induced by host immune receptors in response to
microbial effectors, protects plants against virulent pathogens. However, a systematic
study of ETI prevalence against species-wide pathogen diversity is lacking. We constructed the
Pseudomonas syringaeType III Effector Compendium (PsyTEC) to reduce the pan-genome
complexity of 5127 unique effector proteins, distributed among 70 families from 494 strains,
to 529 representative alleles. We screened PsyTEC on the model plantArabidopsis thaliana
and identified 59 ETI-eliciting alleles (11.2%) from 19 families (27.1%), with orthologs
distributed among 96.8% ofP. syringaestrains. We also identifiedtwo previously undescribed
host immune receptors, including CAR1, which recognizes the conserved effectors AvrE and
HopAA1, and found that 94.7% of strains harbor alleles predicted to be recognized by
either CAR1 or ZAR1.
A
ll microbial pathogens face highly adapted
andmultifacetedhostimmunesystems
that constrain their host range. The first
layer of plant defense against pathogens
is governed by preformed barriers and
pattern recognition receptors (PRRs) located
on plant cell surfaces. The perception of con-
served microbe-associated molecular patterns
(MAMPs) by PRRs elicits pattern-triggered im-
munity (PTI), which can suppress the growth
of invading microbes ( 1 , 2 ). However, many
microbes have evolved mechanisms to inject
effector proteins into host cells that can sup-
press PTI or otherwise facilitate pathogen
growth ( 3 ). Plants have in turn evolved a second
layer of immunity, termed effector-triggered
immunity (ETI), to respond to this challenge.
ETI is of greater amplitude than PTI and is
elicited when intracellular nucleotide-binding
leucine-rich repeat (NLR) receptor proteins
detect the presence or activity of microbial
effectors ( 2 , 4 ). The ETI response is associated
with the gene-for-gene model of resistance and
a localized, programmed cell death that limits
pathogen growth, called the hypersensitive re-
sponse (HR) ( 5 , 6 ).
ETI plays a dominant role in protecting
specific plant genotypes from specific path-
ogen races. PTI, in contrast, provides broad-
spectrum resistance from the recognition of
evolutionarily conserved pathogen epitopes
( 2 , 4 , 7 ). Given the genotype-specific nature
of ETI, it is not clear whether it is ubiquitous
and broadly effective against a diverse bacte-
rial pathogen species carrying a dynamic suite
of effectors ( 8 – 10 ). Although the firstP. syringae
“avirulence”gene (i.e., ETI-eliciting effector) was
cloned in 1984 ( 11 ), only nineP. syringaeeffector
alleles that elicit ETI throughA. thalianaNLRs
have been identified to date ( 12 , 13 ), and we still
know relatively little about the genetic diversity
of effectors across theP. syringaespecies com-
plex, the range of ETI interactions that they
mediate, and whether ETI can contribute to
broad-spectrum plant resistance.
To better understand the frequency of ETI
interactions between a host plant and the di-
verse effector repertoire of a pathogenic species
complex, we focused on type III secreted effectors
that mediate interactions betweenP. syringae
andA. thaliana( 14 ). The diverseP. syringae
species complex carries 70 distinct effector
families and infects nearly every major agri-
cultural crop, although individual strains
only cause disease on a small subset of hosts
( 9 , 13 , 15 ). Wehypothesized that surveying
the species-wide diversity of effectors would
uncover novel ETI responses and provide in-
sight into the role of ETI in determining host
specificity.ThePseudomonas syringaeType III Effector
Compendium (PsyTEC)
We created theP. syringaeType III Effector
Compendium (PsyTEC) to study global effector
diversity. We queried the genome sequences
of 494P. syringaestrains isolated from more
than 100 plant hosts from 28 countries with
P. syringaetype III effector protein sequences
assembled from public databases using BLASTP.
From this we identified 14,613 sequences, of
which 4636 were unique at the amino acid
level and 5127 were unique at the nucleotide
level. We delimited these effectors into 70 homol-
ogy families and 89 subfamilies using stringent
homology criteria, which reflected acceptedfamily designations of the sequences in the
majority of cases (table S1) ( 9 ). Finally, we
clustered the diversity within each effector
family into PsyTEC clades of highly similar
sequences using UCLUST with a percent iden-
tity cutoff of 95%. After discarding the 271 single-
ton PsyTEC clades to avoid screening potential
pseudogenes, we were left with a total of 622
multisequence PsyTEC clades spanning the
70 families (table S2).
We constructed PsyTEC by identifying and
synthesizing a single representative effector for
each multisequence PsyTEC clade on the basis
of the following criteria: (i) The effector se-
quence contained the conserved upstream hrp
box promoter sequence within 10 kbp of the
start codon, (ii) the effector sequence and up-
stream region leading up to the hrp box con-
tained no ambiguous bases, and (iii) the effector
hrp box and the 25 bp upstream of the hrp box
contained no ambiguous bases and did not run
into the end of a contig. The presence of hrp
boxes and the absence of ambiguous sequences
were crucial for accurate expression and syn-
thesis of the representative effectors, respectively.
We were able to identify a suitable effector in
529 of the 622 multisequence effector clades.
Each PsyTEC representative allele shared at
least 95% amino acid identity with the cluster
seed for its clade. We synthesized each repre-
sentative effector with its corresponding native
hrp box, including all intergenic sequences
between the hrp box and the effector that did
not contain other coding regions. Desired prim-
ing sites, cloning sites, a unique barcode, and a
hemagglutinin epitope tag were also included
with each synthesized representative (fig. S1A).
Each fragment was then cloned into the pBBR1-
MCS2 vector to be mated intoP. syringaerecip-
ient strains for phenotypic screening ( 16 ). This
subset of 529 representative effectors spans the
P. syringaepan-genome effector diversity and
allows us to screen for ETI responses induced
by effectors from both host-adapted and non–
host-adapted strains (table S1 and figs. S2
and S3).
Given the nonuniform distribution and diver-
sity of most effector families acrossP. syringae
strains ( 8 ), we assessed the level of completeness
of the PsyTEC library. Analysis of all protein
sequences showed that most genomes carry a
number of singleton effectors (i.e., effector found
in only one strain), with the rarefaction analysis
identifying an average of 5.63 singleton effectors
per additional genome and a decay parame-
ter (a) of 0.33 (fig. S1B). In contrast, when we
clustered effectors sharing≥95% protein iden-
tity, we found an average of only 0.60 new
clades per genome (a= 0.68; fig. S1C). This
rarefaction plateau was amplified when we con-
sidered only multisequence effector clades, yield-
ing an average of only 0.04 new multi-effector
clades per additional genome (a=0.76;fig.S1D).
Thus, although further sampling ofP. syringaeRESEARCH
Laflammeet al.,Science 367 , 763–768 (2020) 14 February 2020 1of6
(^1) Department of Cell and Systems Biology, University of
Toronto, Toronto, ON M5S 3B2, Canada.^2 Center for the
Analysis of Genome Evolution and Function, University of
Toronto, Toronto, ON M5S 3B2, Canada.
*These authors contributed equally to this work.
†These authors contributed equally to this work.
‡Corresponding author. Email: [email protected] (D.D.);
[email protected] (D.S.G.)