provide a framework with which to understandthe many mutations
in the protein that lead todiseases.
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ACKNOWLEDGMENTS
We thank the beamline scientists at Diamond Light
Source,European Synchrotron Radiation Facility and SPring-8 for
helpwith data collection; L. Bruce at the Bristol Institute for
TransfusionSciences; National Health Service Blood and Transplant;
andA. Toye at the University of Bristol for useful discussion.S.
Weyand contributed to the refinement of the Fab fragment,and R.
Suno at Kyoto University contributed to the preparation ofthe
figures. The project was funded by the Biotechnology and
Biological Sciences Research Council (grants BB/G023425/1and
BB/D019516/1 to S.I.); the ERATO Human ReceptorCrystallography
Project of JST (to S.I.); the Targeted ProteinsResearch Program of
the Ministry of Education, Culture, Sports,Science, and Technology,
Japan (MEXT) (to S.I.); a Grant-in-Aidfor Scientific Research (B)
(grants 20370035 and 23370049 toT.K.); MEXT’s Platform for Drug
Discovery, Informatics, andStructural Life Science (to T.K. and
S.I.); and the European Union’sEuropean Drug Initiative for
Channels and Transporters (grant201924 to S.I. and A.C.). The
authors are grateful for the use of theMembrane Protein Laboratory,
funded by the Wellcome Trust(grant WT089809) at the Diamond Light
Source. The authorsdeclare no competing interests. The project was
conceived byS.I., D.K., and N.H. H.H., H.K., and N.H. purified the
AE1CTD protein.H.I. and T.Ha. raised the antibody using the
AE1CTD-expressingbaculovirus as an antigen. T.A., T.Hi., C.I.-S.,
and T.M. screenedthe antibody. T.K.-Y., T.K., T.A., Y.Ab., and M.I.
crystallized theAE1CTD protein. T.K., Y.Al., A.D.C., and S.I.
collected diffractiondata. The structure was solved and refined by
T.A., Y.Al. and A.D.C.The manuscript was written by T.K., A.D.C.,
Y.Al., and S.I. Thecoordinates and structure factors have been
deposited with theProtein Data Bank (4YZF, band 3 with Fab4201;
5a16, Fab4201).T.A., T.K., T.K.-Y., T.Hi., T.M., N.N., S.I., T.Ha.,
H.I., Y.M., and N.H.are authors on a patent filed by JST Agency (JP
2013-103881 A)that covers the monoclonal antibody to human band
3.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/350/6261/680/suppl/DC1Materials and
MethodsFigs. S1 to S15Tables S1 to S3References (63–84)
10 December 2014; accepted 25 September
201510.1126/science.aaa4335
PLANT EVOLUTION
The Papaver rhoeas S determinantsconfer self-incompatibility
toArabidopsis thaliana in plantaZongcheng Lin,* Deborah J. Eaves,
Eugenio Sanchez-Moran,F. Christopher H. Franklin, Vernonica E.
Franklin-Tong†
Self-incompatibility (SI) is a major genetically controlled
system used to prevent inbreeding inhigher plants. S determinants
regulate allele-specific rejection of “self” pollen by the
pistil.SI is an importantmodel system for cell-to-cell recognition
and signalingand could be potentiallyuseful for first-generation
(F1) hybrid breeding.To date, the transfer of S determinants
hasused the complementation of orthologs to “restore” SI in close
relatives.We expressed thePapaver rhoeas S determinants PrsS and
PrpS in Arabidopsis thaliana. This enabled pistils toreject pollen
expressing cognate PrpS. Moreover, plants coexpressing cognate PrpS
and PrsSexhibit robust SI.This demonstrates that PrsS and PrpS are
sufficient for a functionalsynthetic S locus in vivo.This transfer
of novel S determinants into a highly divergent species(>140
million years apart) with no orthologs suggests their potential
utility in crop production.
Many plants are hermaphrodites, withmaleand female organs in
close proximity.Because this risks self-fertilization
andundesirable inbreeding depression,manyplants use
self-incompatibility (SI) as a
mechanism to prevent selfing. SI is controlled byan S locus
allowing self/nonself recognition be-tween pistil and pollen (1,
2). SI in Papaver rhoeasis gametophytically controlled and
specified by apistil S determinant, PrsS [P. rhoeas stigma S
(3)],and a pollen Sdeterminant,PrpS [P. rhoeas pollen S(4)]. PrsS
and PrpS interact to trigger a signalingnetwork in incompatible
pollen, resulting inprogrammed cell death (PCD) (5–8).
Arabidopsisthaliana is a self-fertile member of the Brassica-ceae.
Self-compatibility in Arabidopsis originated
684 6 NOVEMBER 2015 • VOL 350 ISSUE 6261 sciencemag.org
SCIENCE
School of Biosciences, University of Birmingham,
Edgbaston,Birmingham B15 2TT, UK.*Present address: Department of
Plant Systems Biology, VlaamsInstituut voor Biotechnologie, 9052
Gent, Belgium.†Corresponding author. E-mail:
[email protected]
RESEARCH | REPORTS
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http://www.sciencemag.org/http://www.sciencemag.org/http://www.sciencemag.org/http://www.sciencemag.org/
in normal seed-set (50.6 ± 5, 50.0 ± 3.9, n = 10siliques), which
is significantly different fromthose with cognate combinations (P
< 0.001***,t test). Pollinations between Col-0 pistils and
At-PrpS1 or At-PrpS3 pollen gave normal seed-set(49.9 ± 3.7 and
47.6 ± 3.7, n = 10 siliques), so trans-genic stigmas and pollen are
fully functional.Thus, the Papaver S determinants function in
vivoin an S-specific manner, resulting in failure offertilization
with cognate, but not noncognate,pollen expressing PrpS.We
generated Col-0 lines coexpressing PrsS1
and PrpS1 (SI1 lines) by transforming homozy-gous At-PrpS1–GFP
plants with SLR1p::PrsS1.Lines coexpressing PrsS3 and PrpS1 (SC
lines)were also generated. Expression of PrsS1 andPrpS1 was
examined in three SI1 lines (Fig. 3A).Fluorescence microscopy of
pollen from theseSI1 lines confirmed the expression of
PrpS1-GFP(Fig. 3B). The SI1 and SC lines had a similar veg-etative
phenotype to Col-0, At-PrpS1, and At-PrsS1plants (fig. S7).
However, when left to set self-seednaturally, the SI1-line plants
had small siliques(Fig. 3C and fig. S7F), between 3 ± 0.5 and 7
±1.4 mm long (n = 470 siliques; fig. S8A), sig-nificantly shorter
than siliques of control plants(Fig. 3C and fig. S7F) Col-0 (15.5 ±
0.6 mm), At-PrpS1 (16.3 ± 1.0), At-PrsS1 (15.9 ± 0.5), and SCplants
(15.3 ± 0.5 mm; P < 0.001***, t test; n = 10siliques per plant).
Twelve of the SI1 lines set noseed; the remaining 35 plants had
between 0.1 ±0.3 and 7.0 ± 1.4 seeds per silique (n = 10
siliquesper plant; fig. S8B). This was significantly less(P <
0.001***) than the 58 ± 1.6 seeds per siliquein Col-0 plants,
At-PrpS1 plants (57.7 ± 2.8), At-PrsS1 plants (58.3 ± 1.6), and SC
lines (57.1 ± 1.7;n = 10). Total self seed-set from these SI1
linesgave between 0 and 680 seeds; ~60% had 8500 seeds per plant (n
= 12).This SI response is stronger than previously ob-tained using
the S determinants from A. lyrata(12, 18) and similar to that
achieved by (19). Linescoexpressing PrsS3 and PrpS3 (SI3 lines) had
asimilar vegetative phenotype to Col-0 plants, ex-cept for short
siliques (fig. S9). Self–seed-set anal-ysis revealed small siliques
(fig. S10, A and B) andno or very low seed-set (fig. S10, B and C),
whichwere similar to those for the SI1 lines. Analysis ofnaturally
self-pollinated pistils from SI linesrevealed that pollen tubes
were inhibited in theupper pistil, whereas comparable
self-pollinatedCol-0 pistils had pollen tubes extending throughthe
pistil (Fig. 3, E and F, and figs. S11 and S12).Together, these
data provide compelling evidencethat the SI lines are
self-incompatible.To confirm that SI lines were fully
functional,
pistils from representative SI1 lines (SI1-9, SI1-18,and SI1-32)
were pollinatedwithAt-PrpS3 or Col-0pollen (Fig. 3D, n = 9
pollinations per line).Siliques obtained were not significantly
differentfrom those pollinated using Col-0 stigmas (P =0.246, P =
0.703, ANOVA; n = 3 pollinations perline). Pollen from SI1 lines
was also pollinatedontoAt-PrpS1 stigmas. They produced siliques
andseed-set not significantly different from At-PrpS1stigmas
pollinatedwithCol-0pollen (P=0.931,P=
0.803, ANOVA; n = 3 pollinations per line; fig. S13,A and B).
Because pollen and pistils from these SIlines are functional, the
reason why these SI linesset no self-seed is not because they have
a fertilitydefect, but because they are self-incompatible.
Our data provide compelling evidence thatthe Papaver S
determinants coexpressed in A.thaliana make plants
self-incompatible andare the sole additional requirement to
establishSI in this highly diverged self-compatible species.
686 6 NOVEMBER 2015 • VOL 350 ISSUE 6261 sciencemag.org
SCIENCE
Fig. 2. At-PrpS pollen is inhibited on cognate At-PrsS pistils,
demonstrating S-specificity. (A) Anilineblue staining of
representative semi–in vivo pollinations of At-PrsS1 pistils with
At-PrpS1 or Col-0 pollen.(B) Quantitation of pollen tube lengths on
At-PrsS1 pistils using At-PrpS1 pollen (left) or Col-0
pollen(right); n = 4 stigmas per At-PrsS1 line. (C) At-PrsS1 and
At-PrsS3 pistils pollinated semi–in vivo withAt-PrpS3 or At-PrpS1
pollen. At-PrpS pollen tubes were inhibited on cognate At-PrsS
pistils (i and v),whereas controls were not. (D) Representative in
vivo pollination of an At-PrsS1 stigma with At-PrpS1pollen resulted
in a small empty silique. (E) Col-0 pollinated with Col-0 pollen
had a normal-lengthsilique and many seeds.
Table 1. In vivo pollination of At-PrsS stigmas with cognate
At-PrpS pollen resulted in shortersiliques and no seed set.
Pollination of emasculated At-PrsS1 stigmas with At-PrpS1 pollen
resultedin short siliques and reduced seed number, as did
pollination of At-PrsS3 with At-PrpS3 pollen. Other
control pollinations: Noncognate pollination of At-PrsS1 stigmas
with At-PrpS3 pollen; At-PrsS3stigmas with At-PrpS1 or Col-0
pollen; and Col-0 stigmas with Col-0, At-PrpS1, or At-PrpS3
pollen
gave normal silique length and seed number (mean +− SD, n = 10
siliques pollinated per cross).Numbers in bold indicate
incompatible combinations.
♀/♂ At-PrpS1 At-PrpS3 Col-0
At-PrsS1 (line 9)Silique lengths (mm) 6.2 T 1.4 16.1 T 0.8 16.4
T 0.7. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ...
.. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ...
... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... ..
... ... .. ... .. ... ... ..
Seeds per silique 0.5 T 1.0 50.6 T 5.1 49.3 T 5.3. ... .. ...
... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... ..
... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ...
.. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... ..
... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ...
.. ... .. ... ... .
At-PrsS3 (line 8)Silique lengths (mm) 16.4 T 0.8 6.3 T 1.7 16.5
T 0.5. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ...
.. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ...
... .. ... .. ... ... .. ... ... .. ... ... .. ... .. ... ... ..
... ... .. ... .. ... ... ..
Seeds per silique 50.0 T 3.9 1.2 T 1.8 50.0 T 3.2. ... .. ...
... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... ..
... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ...
.. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... ..
... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ...
.. ... .. ... ... .
Col-0Silique lengths (mm) 16.6 T 1.0 16.6 T 0.8 16.4 T 0.7. ...
... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ..
... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ...
.. ... ... .. ... ... .. ... ... .. ... .. ... ... .. ... ... ..
... .. ... ... ..
Seeds per silique 49.9 T 3.7 47.6 T 3.7 47.7 T 3.6. ... .. ...
... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ... ..
... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... .. ...
.. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ... ..
... .. ... ... .. ... ... .. ... .. ... ... .. ... ... .. ... ...
.. ... .. ... ... .
RESEARCH | REPORTS
This is a milestone, because the successful trans-fer of S
determinants to date has been betweenclose relatives sharing an
ancestral SI system,using complementation to “restore” SI (12,
13).Because the Papaveraceae and Brassicaceae areevolutionarily
separated by ~140 million years(8, 15), our finding that they
function in plantain Col-0 to display a robust SI rejection
responseis of considerable interest.We are not “restoring”a SI
system, because SI in Brassica andArabidopsishas genetically and
functionally distinct S deter-minants. Because we previously showed
thatrecombinantPrsS can trigger SI-PCD inArabidopsispollen
expressing PrpS (8), and there is no evidencethat Brassica and
Arabidopsis SI involves PCD, themost economical explanation is that
the Papaver Sdeterminants can interface with, and activate,
anetwork of common signaling components thatmediate PCD to induce a
“Papaver-like” SI re-sponse in Arabidopsis pollen. Papaver SI
usesCa2+, reactive oxygen species, and pH (7, 20),which have all
been described in Arabidopsissignaling networks achieving various
physio-logical responses, including PCD (21). We hypoth-esize that
these common signaling componentsare co-opted downstream of
PrsS-PrpS interac-tion to mediate SI. Our findings reinforce
pro-posals that SI may recruit preexisting signalingnetworks from
other biological processes (8, 22).This raises questions about how
SI systemsevolved, aswell as about recruitment and
functionaldiversification of preexisting components (23).Wide
transgenera functionality of the Papaver
SI system opens up the possibility that the trans-fer of these S
determinants may, in the longer
term, provide a tractable SI system for crop plants.Use of the
SLR1 promoter from Brassica (16, 17)allowsPrsS to be expressed
inmatureCol-0pistils,unlike older Col-0 pistils expressing
SCRb-SRKb(12, 18). The production of F1 hybrid plants innormally
self-compatible species typically useslaborious, expensive manual
emasculation toprevent self-fertilization. Because it is desir-able
to be able to control plant fertility for foodsecurity, this
demonstration of the transfer of aSI system into a self-compatible
species sug-gests an alternative method for the productionof F1
hybrids, a long-term goal of SI research.
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ACKNOWLEDGMENTS
We thank D. Goring for the Binary Ti vector pORE O3
containingthe SLR1 promoter and S. Price for technical assistance.
Z.L.held a Ph.D. studentship from the China Scholarship Council
(CSC).D.J.E. was funded by the Biotechnology and Biological
SciencesResearch Council (BBSRC). V.E.F-T. and F.C.H.F. are
co-inventorson a patent application (2691/KOLNP/2011) filed by the
Universityof Birmingham relating to PrsS and PrpS, to engineer
plants(ornamental or crop) to exhibit SI by using a PrsS and PrpS
S-allelepair, comprising a functional S locus with appropriate
tissue-specific promoters. Materials will be freely available upon
requestfor research purposes. The authors declare that they do
nothave other competing financial interests. The data reported
hereare available in the supplementary materials.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/350/6261/684/suppl/DC1Materials and
MethodsFigs. S1 to S14Table S1References (24–27)
24 August 2015; accepted 18 September
201510.1126/science.aad2983
SCIENCE sciencemag.org 6 NOVEMBER 2015 • VOL 350 ISSUE 6261
687
Fig. 3. A. thaliana coexpressing PrsS1 and PrpS1
areself-incompatible and set no seed. (A) RT-PCR of threeA.
thaliana SI1 lines coexpressing PrsS1 and PrpS1. (B) Pollenfrom SI1
lines exhibits GFP fluorescence (top); Col-0 pollenhas weak
autofluorescence. (C) Self-seed set: SI1 lines formedshort
siliques; controls, including an SC line coexpressingPrsS3 and
PrpS1-GFP, set normal siliques. (D) A selfed SI1plant gave small
siliques; pollinations with Col-0 or At-PrpS3pollen gave normal
siliques. (E) Aniline blue staining of aself-pollinated SI1-line
pistil; pollen tubes are inhibited in thestigma and style. (F) A
self-pollinated Col-0 pistil had longpollen tubes.
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