growth deficits were observed relative to the
empty vector control (fig. S9). We also deter-
mined that residues required for effector func-
tion were also required for ETI elicitation by
our PsyTEC alleles for all newly discovered ETI
elicitors, indicating that effector activity is
being recognized to elicit ETI (fig. S10 and
tables S5 and S6).
We assessed what proportion of ETI-eliciting
alleles also caused a macroscopic HR, and dem-
onstrated that HR phenotypes were relatively
uncommon among ETI-eliciting effectors. No
visible macroscopic HR symptoms were ob-
served for 11 of 19 ETI-eliciting effectors (57.9%)
despite their impact on plant tissue chlorosis
and bacterial growth (Fig. 1B). The lack of HR
phenotypes was not due to effector-effector in-
teractions [so-called meta-effector effects ( 27 )],
as no additional HR responses were observed
when the 19 ETI-eliciting effector families were
screened in the effectorless PtoDC3000 D36E
background (table S7) ( 28 ). We also confirmed
that all HRs required the type III secretion
system by screening in a type III secretion sys-
tem mutant (PtoDC3000-DhrcC) background
(table S7). Interestingly, there was a significant
positive association (Studentttest,P=4.6×
10 –^23 ) between the strength of growth reduction
and the observation of a visible HR; effectors
that elicited a strong HR caused substantial
reductions in growth, whereas those that did
notelicitavisibleHRcausedmoremodestre-
ductions in bacterial growth (Fig. 1, B and C).
These data show that strong, macroscopic HRs
are a likely exception, not the rule, in this patho-
system, and are associated with greater reduc-tions in bacterial growth. The historic reliance
of HR phenotypes for identifying ETI-eliciting
effectors likely explains why relatively few of
these weak elicitors have been characterized
to date.
Finally, we assessed the theoretical preva-
lence of ETI among a global collection of 494
P. syringaestrains. We identified which strains
carried a putative ETI-eliciting effector and
mapped these strains onto theP. syringae
core genome phylogeny (Fig. 2). We found that
478 of 494 (96.8%)P. syringaestrains harbor
at least one highly similar ortholog of an ETI-
eliciting effector (>95% protein identity), whereas
349 (70.7%) strains harbor multiple orthologs
of ETI elicitors. This analysis did not change
substantially when we excluded putatively non-
functional truncated alleles (<75% length ofLaflammeet al.,Science 367 , 763–768 (2020) 14 February 2020 3of6
Fig. 1. Phenotypic effects of ETI-eliciting
effectors expressed in PtoDC3000.(A) Plant
disease scores for 529 PsyTEC effectors as
determined from percentage of yellow chlorotic
plant tissue ( 22 ), normalized to the negative
(virulent; 100% yellow) and positive (ETI-eliciting;
0% yellow) controls for each flat. A cutoff of 45%
yellow (dashed line) was used to distinguish ETI
elicitors from non–ETI elicitors. Red dots are
alleles that do not elicit ETI; blue dots are alleles
that elicit ETI with a hypersensitive response (HR);
yellow dots are alleles that elicit ETI without HR.
Stars highlight the representatives from each
ETI-eliciting family that were used for growth assay
verification and NLR screening. (B) Representative
ETI-eliciting effectors from each family result in
variable declines in bacterial growth in planta over
3 days. Box-and-whisker plots show data from a
single representative experiment (n= 8) for each
representative ETI-eliciting effector and the
corresponding empty-vector control (EV, gray).
Solid circles represent individual observations;
boxes show the first quartile, median, and third
quartile of treatment; whiskers extend to the
highest and lowest observations that are not
identified as outliers (>1.5× interquartile range).
Blue boxes indicate ETI-eliciting effectors
producing a macroscopic HR; yellow boxes indicate
effectors that did not show HR. All effectors
significantly reduced bacterial growth (Student
ttests with Holm-Bonferroni multiple test correction,
P< 0.01). Plant images show representative HR
results and the total number of HR responses
observed out of 60 leaves assayed. (C) ETI-eliciting
effectors producing a macroscopic HR (blue)
resulted in significant reductions in bacterial
growth relative to those that did not elicit HR
(yellow).P= 4.6 × 10–^23 (Studentttest).
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