polymorphic between the orthologous effectors
(fig. S12). Next, we modified the proteins to determine which of the polymorphic residues contribute to the difference in biochemical activity.
Protease inhibition assays with chimeric proteins
and with single-site mutants revealed that the
Gln-Arg polymorphism at position 111 is critical
for specificity (Fig. 3 and figs. S13 and S14). In
particular, EPIC1Q111R, carrying a Gln-to-Arg mutation, most closely recapitulated the function of
PmEPIC1, with more effective inhibition of the
M. jalapa protease MRP2 and less inhibition of
the Solanum proteases RCR3dms3 and RCR3lyc
(S. lycopersicum) (Fig. 3). Sequences of epiC1
or PmepiC1 alleles from 26 P. infestans isolates
and 9 P. mirabilis isolates indicated that the key
Gln or Arg residue is fixed in each population
(table S1 and fig. S11).
We also investigated which variant amino
acids determine specificity in the proteases. In-
spection of the tarocystatin-papain complex iden-
tified a protease region that interacts with a
tarocystatin residue equivalent to the EPIC1 or
PmEPIC1 key Gln or Arg residue (fig. S15A).
Structure-based sequence alignments of RCR3 or
MRP2 with papain indicated that the inhibitor-
binding region overlaps with a seven–amino acid
region that is polymorphic between RCR3 and
MRP2 (fig. S15, B and C). We constructed pro-
teases altered in this region by swapping the en-
tire seven–amino acid domain or by single–amino
acid changes. The results revealed the poly-
morphic residue His148 or Asn147 in RCR3 and
Asp152 in MRP2 as a key element of specificity
(figs. S16 and S17). Unlike wild-type RCR3
proteases, RCR3H148D and RCR3N147D mutants
with a single His to Asp or Asn to Asp muta-
tion could be inhibited by PmEPIC1 (fig. S17).
This suggests that effector PmEPIC1 adaptation
to host protease MRP2 was in part driven by the
occurrence of Asp152 in the M. jalapa protease.
Thus, in this case of oomycete infection of
potato and four o’clock flower, a single–amino
acid polymorphism in the host protease and a
reciprocal single–amino acid change in the path-
ogen effectors underpin the ecological diversi-
fication (fig. S18). The arginine substitution
found in the P. mirabilis effector may enhance
effector inhibition of the M. jalapa protease.
This same substitution would impair interaction
with Asn147 of RCR3dms3 and His148 of RCR3lyc
and so provide a molecular explanation for how
this effector works on one protease but not the
other (fig. S19).
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Acknowledgments: We thank D. G. O. Saunders for comments
on drafts of the manuscript and C. Taylor, M. D. Coffey,
and V. Vleeshouwers for providing biomaterial. This
project was funded by the Gatsby Charitable Foundation,
the U.K. Biotechnology and Biological Sciences Research
Council, the Ohio Agricultural Research and Development
Center at The Ohio State University, and a National
Research Initiative of the U.S. Department of Agriculture
grant OHO00963–SS. Sequences are deposited in
GenBank under the submission accession numbers
provided in (9).
Materials and Methods
Figs. S1 to S19
Tables S1 to S5
23 September 2013; accepted 11 December 2013