strains will likely continue to reveal novel ef-
fector diversity, our analysis has effectively sat-
urated the diversity of the broadly distributed
P. syringaeeffector families.
Widespread recognition ofP. syringae
effectors byA. thaliana
Our next objective was to identify allP. syringae
effectorsthatelicitanETIresponseinthe
A. thalianaaccession Col-0. We first confirmed
that spray inoculation of wild-typeP. syringae
pv.tomatoDC3000 (hereafter PtoDC3000) ( 17 )
ontoA. thalianaresulted in yellow chlorotic
disease symptoms within 7 days, whereas plants
inoculated with PtoDC3000 expressing the ETI-
eliciting effector HopZ1a ( 18 – 21 ) remained rel-
atively green and healthy (fig. S4, A and B). We
also confirmed this qualitative measurement of
overall plant health with quantitative measure-
ments of in planta bacterial growth (fig. S4C)
and plant disease symptoms (fig. S4D), as mea-
sured by plant immunity and disease image-based
quantification (PIDIQ) ( 22 ). After transform-
ing all 529 PsyTEC effectors into PtoDC3000,
we also assessed the hrp-induced protein ex-
pression of each effector via Western blot and
observed expression for 402 of the 529 (76.0%)
effectors (fig. S5 and table S3). When we ex-
cluded effectors with a length of less than half
the effector family average, 84.2% of effec-
tors were detected. A small number of the ex-
pressed effectors (26) had unexpected sizes,
which may reflect the use of alternative start
codons (table S3). Effectors whose expression
could not be confirmed included all or most
representatives from certain effector families
(e.g., HopAG1, HopAS1, HopAX1) as well as some
truncated effectors. All effectors were ultimately
screened, although it is possible that this subset
is not expressed.
We systematically screened the PsyTEC li-
brary onA. thalianaCol-0 by spray inoculation.
Disease symptoms induced by each PsyTEC ef-
fector were quantified by PIDIQ ( 22 ) in com-
parison to a virulent negative control (PtoDC3000
pBBR1-MCS2 empty vector) and one of two
ETI-eliciting positive controls (PtoDC3000
pBBR1-MCS2-HopZ1a or PtoDC3000 pBBR1-
MCS2-AvrRpm1). Effectors that reduced chlo-
rosis symptoms by a normalized percentage
of at least 55% (<45% yellow across all plants)
relative to the controls were classified as ETI
elicitors (fig. S6). This conservative cutoff cap-
tured effectors from eightP. syringaefamilies
that elicit ETI via knownA. thalianaCol-0
NLRs (figs. S6 and S7) ( 12 , 13 ), as well as two
ETI elicitors ofA. thalianaCol-0 that have
recently been identified but not character-
ized ( 23 , 24 ). The only previously identified
ETI-eliciting effectors we did not recover in
our screen were HopAS1 and HopZ5, both
of which elicit ETI only when expressed from
a heterologous promoter ( 25 , 26 ). HopAS1
was also one of the effector families for which
we could not confirm expression via West-
ern blot.
Our PsyTEC screen onA. thalianaCol-0
identified 59 ETI-eliciting effector alleles outof the total of 529 effector alleles (11.2%) from
19 of the 70 effector families (27.1%) (Table 1,
Fig. 1A, table S4, and fig. S6). These elicitors
included ninepreviously unidentified ETI-
eliciting effector families. We also found that
diversification within ETI-eliciting effector
families was critical for determining the out-
come of the interaction. Of the 19 effector fami-
lies carrying ETI-eliciting alleles, 18 also included
alleles that lacked the ability to elicit ETI, with
the lone exception being the two-member HopBJ1
family(fig.S3).ToshowthatournovelETIre-
sponses were not specific to our screening strain,
PtoDC3000, we tested all 59 ETI-eliciting alleles
in the phylogroup 5 radish isolateP. syringae
pv.maculicolaES4326 (PmaES4326), which is
divergent from the phylogroup 1 tomato isolate
PtoDC3000. Both of these strains are strong
pathogens ofArabidopsis. The PmaES4326
screen found that 91.5% (54 of 59) of ETI-
eliciting effectors found in the original PsyTEC
screen retained their ability to elicit ETI in this
new background (fig. S8). These data suggest
that immune elicitation phenotypes are largely
conserved across genetic backgrounds.
We then quantified in planta bacterial growth
ononerepresentativeETI-elicitingeffectorfrom
each of the 19 ETI-eliciting families. In planta
bacterial growth was reduced in allP. syringae
strains harboring an ETI-eliciting effector, al-
though the magnitude of these growth reduc-
tions varied among families (Fig. 1B). These
growth reductions were not due to general bac-
terial fitness reductions, because no in vitroLaflammeet al.,Science 367 , 763–768 (2020) 14 February 2020 2of6
Table 1. Summary of the PsyTEC ETI-eliciting effector families onA. thalianaCol-0.We could not identify HopAS1 and HopZ5 as ETI elicitors using their
native promoters ( 25 , 26 ) and therefore they are not listed among effector families.Effector family Synthesized PsyTEC effector alleles % ETI-eliciting PsyTEC effector alleles Cognate NLR Reference
AvrB............................................................................................................................................................................................................................................................................................................................................ 12 16.7% RPM1 ( 29 )
AvrRpm1............................................................................................................................................................................................................................................................................................................................................ 8 12.5% RPM1 ( 29 )
AvrRpt2............................................................................................................................................................................................................................................................................................................................................ 3 66.7% RPS2 ( 30 , 31 )
HopK (AvrRps4)............................................................................................................................................................................................................................................................................................................................................ 5 80.0% RPS4 ( 33 )
HopAR (AvrPphB)............................................................................................................................................................................................................................................................................................................................................ 8 75.0% RPS5 ( 35 )
HopA............................................................................................................................................................................................................................................................................................................................................ 16 25.0% RPS6 ( 32 )
HopF............................................................................................................................................................................................................................................................................................................................................ 26 15.4% ZAR1 ( 20 , 34 )
HopZ............................................................................................................................................................................................................................................................................................................................................ 17 29.4% ZAR1 ( 20 )
HopO............................................................................................................................................................................................................................................................................................................................................ 14 7.1% ZAR1 This study
HopX............................................................................................................................................................................................................................................................................................................................................ 11 18.2% ZAR1 This study
HopBA............................................................................................................................................................................................................................................................................................................................................ 3 33.3% ZAR1 This study
HopB............................................................................................................................................................................................................................................................................................................................................ 16 12.5% BAR1 This study
AvrE............................................................................................................................................................................................................................................................................................................................................ 22 59.1% CAR1 This study
HopAA............................................................................................................................................................................................................................................................................................................................................ 21 19.0% CAR1 This study
HopD............................................................................................................................................................................................................................................................................................................................................ 16 6.3% Unknown This study
HopI............................................................................................................................................................................................................................................................................................................................................ 19 5.3% Unknown This study
HopAX............................................................................................................................................................................................................................................................................................................................................ 7 28.6% Unknown This study
HopAZ............................................................................................................................................................................................................................................................................................................................................ 16 12.5% Unknown This study
HopBJ............................................................................................................................................................................................................................................................................................................................................ 2 100.0% Unknown ( 24 )
............................................................................................................................................................................................................................................................................................................................................RESEARCH | RESEARCH ARTICLE