Science 14Feb2020

the reference sequence). If we assume that all
truncated alleles are unrecognized, 455 of 494
(92.1%)P. syringaestrains would still harbor
at least one full-length ortholog of an ETI-
eliciting effector (fig. S11). Furthermore, if we
focus exclusively on primary phylogroup strains,
which consist of most of the agricultural isolates
and type strains ( 9 ), all but six strains (470/476;
98.7%) would carry an ortholog of an ETI-
eliciting allele.
Note that these predictions of ETI prevalence
are made on the basis of the presence of an ETI-
eliciting effector or its ortholog (ETI potential)
and that the actual outcome of an interaction
could be influenced by three potential varia-
bles: (i) Meta-effector interactions within a
specific strain may modulate ETI responses
and the outcome of the interaction. (ii) Chro-
mosomally expressed effectors may not have
the same ETI-eliciting activity as effectors ex-
pressed from plasmids. This appears to be
the case for at least someAvrE effectors; how-
ever, even if we exclude all AvrE effectors
from our analysis, we still see that 78.8% of
primary phylogroup strains harbor an ETI-
eliciting effector. (iii) There is as much as 5%
amino acid divergence within each PsyTEC
clade (technically, up to 95% identity to the
cluster seed for each clade), and some of this
diversity may result in differential ETI out-
comes. Although we did not systematically
address this, we did look at 11 cases in which

we synthesized multiple alleles from the same

PsyTEC clade. In all of these cases, we observed

the same ETI response among alleles from the

same clade.

A. thalianaresistance toP. syringaeis

conferred by a small number of NLRs

We searched for NLRs associated with the

ETI-eliciting effectors by screening a represen-

tative effector from each of the 19 ETI-eliciting

families against a suite ofA. thalianaCol-0 NLR

mutants, including those from ourA. thaliana

R gene T-DNA insertion collection (table S8)

( 20 ). Specifically, PtoDC3000 strains bearing each

ETI-eliciting effector were spray-inoculated

on the collection of NLR mutant plants to iden-

tify loss-of-ETI mutants (Fig. 3A). We subse-

quently confirmed each NLR-effector pair via

quantitative measurements of plant chlorosis

(Fig. 3B) and in planta bacterial growth (Fig. 3C).

Finally, we confirmed that the same NLR was

required for all ETI-eliciting alleles within fam-

ilies (fig. S13).

Our screen confirmed all previously charac-

terized NLR-effector pairs ( 20 , 29 – 35 ) (Fig. 3,

A and B). We also identified two new NLRs in-

volved in the recognition of novel ETI-eliciting

effectors: The Toll/interleukin-1 receptor NLR

At5g18360 was requiredfor recognition of HopB

(HopB-Activated Resistance 1, BAR1), and the

coiled-coil NLR At1g50180 was required for re-

cognition of AvrE and HopAA1 (CEL-Activated

Resistance 1, CAR1). Both AvrE and HopAA1

are encoded in theP. syringaeconserved effector

locus, which is a highly conserved region that

flanks the genomic island encoding the type

III secretion system ( 36 ). We confirmed the

requirement of CAR1 for recognition of both

AvrE and HopAA1 using an independent CAR1

mutant generated by CRISPR-Cas9 mutagen-

esis (car1-2; fig. S14). The number of ZAR1-

dependent ETI responses identified in the screen

was also notable in that we identified novel

ZAR1-dependent ETI responses against the

HopO, HopX, and HopBA families, in addi-

tion to the previously characterized ZAR1-

dependent ETI responses against HopZ1a ( 20 )

and HopF2a ( 34 ). ZAR1 is also known to be re-

quired for the recognition of theXanthomonas

campestriseffector AvrAC ( 37 ). The recognition

of HopBA1 by ZAR1 was also surprising given

that its recognition in anotherA. thalianaac-

cession, Ag-0, is governed by the TIR-only pro-

tein RBA1 ( 23 ).

We predicted which, if any, NLRs would be

responsible for an ETI response against each

P. syringaestrain from that strain’s comple-

ment of putative ETI-eliciting effectors and

then mapped these“resistance NLRs”across

the phylogeny (Fig. 3). Remarkably, we found

thatA. thalianais predicted to have near-

complete immunity toP. syringae, mediated

by a very small number of resistance NLRs.

Indeed, as few as eight resistance NLRs are

Laflammeet al.,Science 367 , 763–768 (2020) 14 February 2020 4of6

Fig. 2. The potential forA. thalianaETI
againstP. syringaeis pervasive and often
multi-tiered.TheP. syringaecore-genome
phylogeny is shown at the bottom, with
designated phylogroups (P) indicated.
Color bars above the core-genome
phylogenetic tree illustrate whichP. syringae
strains harbor an ETI-eliciting variant of
each effector family (blue) and which
strains are thereby expected to be recognized
via each characterized plant NLR (green).
Effectors and NLRs are sorted according
to a hierarchical clustering analysis of the
plotted elicitation and recognition profiles,
respectively (left). Numbers to the right of
each color bar indicate the number of
P. syringaestrains that contain an ortholog of
an ETI-eliciting effector, or the number of
P. syringaestrains that are expected to be
recognized by each NLR based on the
complement of effectors carried by each
strain. HopF1r was formerly HopF2a.

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