to the role of the interaction partner—initiation
of defense in the case of a host, and protection of
a virulence effector in the case of a pathogen.
It is interesting that NbGIP2 belongs to a different GIP clade than GmGIP1. Soybean (a rosid)
and N. benthamiana (an asterid) diverged around
107 to 117 million years ago (27), whereas the
genus Phytophthora is estimated to have radiated
into different species around 20 million years ago
(28). Thus, it is plausible that GmGIP1 and NbGIP2
evolved independently and convergently out of
existing GIP superfamilies to counter the XEG1
weaponry of emerging Phytophthora pathogens.
The substantial differences between GmGIP1 and
NbGIP2 may explain why PsXLP1 and PpXLP1
have also evolved substantial differences.
Hosts and pathogens are engaged in a conti-
nuous evolutionary struggle for physiological
dominance that determines the outcome of their
interaction (5, 8, 9). One example is Ecp6, an
apoplastic effector of Cladosporium fulvum, that
shields pathogen-derived chitin fragments from
recognition by host immune receptors (29, 30).
Here we have provided evidence that the essen-
tial apoplastic effector PsXEG1 and its homologs
constitute a focal point of this coevolutionary strug-
gle in diverse Phytophthora-plant pathosystems
(14) (figs. S14 and S24) and that, at least in the
P. sojae–soybean system, PsXEG1 is subject to two
layers of defense and counterdefense (Fig. 4C).
We previously showed (14) that recognition of
PsXEG1 by soybean’s PAMP recognition machin-
ery has the potential to block P. sojae infection,
but the PAMP-triggered immune response is in
turn suppressed by multiple intracellular RxLR
effectors. Here we have shown that PsXEG1 also
is targeted by a second defense layer, an apoplastic
inhibitor protein, GmGIP1, that can diminish the
virulence of P. sojae, but that PsXEG1 is protected
by its paralog, PsXLP1, which has evolved to bind
more tightly than PsXEG1 to GmGIP1.
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We thank W. Ma (University of California–Riverside) for helpful
suggestions. This work was supported in part by grants to Yu. W.
from the China National Funds for Distinguished Young Scientists
(31225022), the Key Program of the National Natural Science
Foundation of China (31430073), the Special Fund for Agro-scientific
Research in the Public Interest (201303018), and the China
Agriculture Research System (CARS-004-PS14); by a 111 International
Cooperation grant (B07030) to Nanjing Agricultural University
from the Chinese government; and by grants to B.M. T. from the
National Research Initiative of the U.S. Department of Agriculture
National Institute of Food and Agriculture (2011-68004-30104 and
2010-65110-20764). Y.F. is also supported by the Interdisciplinary Ph.
D. Program in Genetics, Bioinformatics, and Computational Biology
at Virginia Tech. Sequences have been deposited in GenBank
under the submission accession numbers provided in the supplementary
materials. The supplementary materials contain additional data.
Materials and Methods
Figs. S1 to S24
Tables S1 to S7
Data S1 to S6
13 August 2016; accepted 28 December 2016
Published online 12 January 2017
714 17 FEBRUARY 2017 • VOL 355 ISSUE 6326
Fig. 4. Neutralization of GmGIP1-mediated
defense by PsXLP1 is only observed in the
presence of PsXEG1 during P. sojae infection
of soybean hairy roots. (A) Overexpression of
PsXLP1 in P. sojae neutralizes the defense provided by overexpression of GmGIP1 in transgenic
soybean hairy roots. Shown is the relative biomass
of wild-type and transgenic P. sojae in infected
hairy roots expressing GmGIP1-GFP fusion protein, as measured by genomic DNA qPCR and
normalized to wild-type P. sojae infection of hairy
roots expressing GFP at 24 hpi. (B) PsXLP1 overexpression in hairy roots does not increase susceptibility to P. sojae PsXEG1-knockout lines. Shown
is the relative biomass of P. sojae strains infecting
transgenic hairy roots expressing GFP or PsXLP1-GFP
fusion, measured as in (A). T17, control; PsXEG1EE→DD,
PsXEG1 E136D, E222D double mutant. In (A) and (B),
experiments were replicated six times using 36
hairy roots from six different soybean cotyledons
per biological replicate. Different letters represent
significant differences (P < 0.01; Duncan’s multiple range test). Bars represent medians, and boxes
indicate the 25th and 75th percentiles. (C) Model
for counterdefense by P. sojae against two distinct
forms of PsXEG1-targeted plant defenses (PTI,