DOI: 10.1126/science.1167784
, 1485 (2009);323Science
et al.Martin Bayer,
Arabidopsis thaliana
Paternal Control of Embryonic Patterning in
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13. P. Cubas, N. Lauter, J. Doebley, E. Coen, Plant J. 18, 215
(1999).
14. S. Kosugi, Y. Ohashi, Plant J. 30, 337 (2002).
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21. P. Mas, D. Alabadi, M. J. Yanovsky, T. Oyama, S. A. Kay,
Plant Cell 15, 223 (2003).
22. We thank T. Imaizumi, E. Hamilton, E. Farre, B. Chow,
D. A. Nusinow, and M. Sawa for critical reading of the
manuscript; K. Yamada for providing the YM187 yeast
strain; Y. Zhao for microscopy assistance; and A. Schopke,
R. Sawaya, and S. Asbaghi for technical assistance. This
work was supported by NIH grants GM56006 and
GM67837 to S.A.K. and partially by postdoctoral
fellowships from Fundacion Antorchas to J.L.P.-P., the
National Sciences and Engineering Research
Council of Canada to G.B., and Wenner-Gren Stiftelserna
to A.P.
Supporting Online Material
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Materials and Methods
Figs. S1 to S13
Table S1
References
14 October 2008; accepted 7 January 2009
10.1126/science.1167206
Paternal Control of Embryonic
Patterning in Arabidopsis thaliana
Martin Bayer,1
* Tal Nawy,1
† Carmela Giglione,2
Mary Galli,1
‡
Thierry Meinnel,2
Wolfgang Lukowitz1,3
§
The YODA (YDA) mitogen-activated protein kinase pathway promotes elongation of the Arabidopsis
zygote and development of its basal daughter cell into the extra-embryonic suspensor. Here, we
show that the interleukin-1 receptor–associated kinase (IRAK)/Pelle-like kinase gene SHORT
SUSPENSOR (SSP) regulates this pathway through a previously unknown parent-of-origin effect.
SSP transcripts are produced in mature pollen but do not appear to be translated. Instead, they are
delivered via the sperm cells to the zygote and the endosperm, where SSP protein transiently
accumulates. Ectopic expression of SSP protein in the leaf epidermis is sufficient to activate
YDA-dependent signaling. We propose that SSP protein produced from paternal transcripts
upon fertilization triggers zygotic YDA activity, providing an essential temporal cue for the
regulation of the asymmetric first division.
M
ulticellular organisms rely on robust
spatial and temporal coordinates to
guide embryonic development. Little
is known about the cues used by plants to derive
such a framework. Growth of plant zygotes is
typically polar, and their division asymmetric (1).
In Arabidopsis, the zygote elongates about threefold
(Fig. 1A) before dividing into a small apical
cell, the founder of the spherical proembryo, and
a large basal cell, the founder of a filamentous,
largely extra-embryonic support structure called the
suspensor (Fig. 1E). This fundamental fate decision
is regulated by YODA (YDA)-dependent signaling.
Loss of the mitogen-activated protein kinase
kinase (MAPKK) kinase YDA (2) or the MAP
kinases MPK3 and MPK6 (3) suppress elongation
of the zygote (Fig. 1B) and suspensor formation.
Hyperactive variants of YDA promote extensive
elongation of the zygote and exaggerated suspensor
growth (2). Here, we report that the interleukin-1
receptor–associated kinase (IRAK)/Pelle-like
kinase gene SHORT SUSPENSOR (SSP) links
activation of the YDA MAP kinase cascade to fertilization
through a unique parent-of-origin effect
and propose a mechanistic basis for our finding.
Mutations in the SSP gene have no discernible
impact on adults but closely mimic the effect
of yda mutations on embryogenesis (2). Mutant
zygotes fail to elongate and generate basal cells
of diminished size (Fig. 1C) (23 T 5 mm, compared
with 61 T 7 mm for wild type). Reduced
growth and aberrant divisions in the basal cell
lineage subsequently result in a spectrum of defects,
ranging from the absence of a recognizable
suspensor (Fig. 1F) to cone-shaped suspensors
with multiple cell layers (Fig. 1G) and rudimentary
or short suspensors (Fig. 1, H and I). These
phenotypes imply that SSP promotes extraembryonic
or suspensor fates. In support of this
view, expression of a molecular marker for the
suspensor (4) is not detected in ssp mutants
(Fig. 1K).
Genetic analysis suggests that SSP acts upstream
of the YDA MAP kinase cascade in a common
pathway. Double mutant ssp-1 yda-1 embryos
are anatomically indistinguishable from yda-1
single mutants (table S1), arguing against an independent
function of the two genes. Hyperactive
variants of the YDA MAPKK kinase reverse the
suspensor phenotype caused by ssp mutations
(table S1), suggesting that SSP participates in activating
the YDA MAP kinase cascade.
Map-based cloning (5) revealed that SSP corresponds
to At2g17090, a member of the RLCK
II family of IRAK/Pelle-like kinases [also known
as receptor-like kinases (6)]. Four mutations, including
a premature stop (ssp-1) and an RNA
null (ssp-2), generate essentially indistinguishable
mutant phenotypes, which are complemented by
the introduction of a 9.8–kilobase pair genomic
DNA fragment spanning the locus (316 normal
embryos in 580 total) (7). Three motifs can be
identified in the predicted SSP protein (Fig. 2A):
an N-terminal consensus for myristoylation and
palmitoylation, a central protein kinase domain of
the Pelle/IRAK superfamily, and a C-terminal tetratricopeptide
repeat (TPR). Insertion of a yellow
fluorescent protein (YFP) moiety on either side
of the kinase domain does not impair SSP function
(Fig. 2A).
N-myristoylation at Gly2
and subsequent Spalmitoylation
at neighboring cysteines are predicted
to mediate stable plasma membrane association
(8). Consistent with this prediction, cells overexpressing
YFP-tagged SSP variants show robust
fluorescence at their surface (Fig. 2B). A point
mutation abolishing both N-myristoylation and
S-palmitoylation [G2→A2 (G2A) (9)] renders
the mutant protein cytoplasmic (Fig. 2C) and
unable to complement ssp mutants (n = 479 embryos).
Eliminating only palmitoylation [C3→S3
and C4→S4 (C3,4S) (9)] has a similar effect (n =
450 embryos). Peptides representing the N terminus
of wild-type SSP or the C3,4S mutant are
efficiently modified by Arabidopsis N-myristoyl
transferase NMT1 in vitro (10), whereas a peptide
representing the G2A mutant is not (Fig. 2D). We
conclude that diacylation-mediated membrane
association is essential for SSP function.
In contrast, protein kinase catalytic activity
may be dispensable. Alternative splicing of intron
2 generates a short SSP transcript harboring
an 18–amino acid in-frame deletion in the kinase
domain (Fig. 2A). This cDNA does not complement
the phenotype of ssp mutants when expressed
from the endogenous promoter (n = 311
embryos), whereas the long cDNA species does
(218 normal embryos in 449 total), suggesting
that gross structural changes in the kinase domain
are not tolerated. However, key residues of
the active site, most conspicuously the aspartate
of the canonical DFG (9) motif involved in Mg++
binding and phospho-transfer, are not found in
SSP. Moreover, a transition-state mutant predicted
to reduce catalytic activity to negligible
rates while leaving substrate binding unaffected
(11) fully complements the phenotype of ssp mutants
(Fig. 2A) [K78R (9); 211 normal embryos
in 442 total]. Although we cannot rule out that
SSP uses a noncanonical reaction mechanism
1
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
11724, USA. 2
Institut des Sciences du Végétal, Centre National
de la Recherche Scientifique, 91198 Gif-sur-Yvette,
cedex, France. 3
Department of Plant Biology, University of
Georgia, Athens, GA 30602, USA.
*Present address: Institute of Plant Biology, University of
Zürich, 8008 Zürich, Switzerland.
†Present address: Biology Department, New York University,
NY 10003, USA.
‡Present address: The Salk Institute, San Diego, CA 92186,
USA.
§To whom correspondence should be addressed. E-mail:
lukowitz@uga.edu
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(12), the primary role of the kinase domain may
lie in protein binding. Mammalian IRAK proteins
often lack kinase activity but rather contribute to
the assembly of receptor complexes (13). Similarly,
a number of plant IRAK/Pelle kinases does not
require catalytic activity for function (14).
The importance of protein-protein interactions
is further implied by similarity of the SSP
C terminus to TPR domains (Fig. 2A) (cluster
KOG0548) mediating the association of heat shock
protein 70 (HSP70) co-chaperone complexes
(15). Deletion of the entire TPR motif or only
the third, cryptic repeat unit completely inactivates
the protein (Fig. 2A) [for mutant Y300Z,
n = 408 embryos; for W431Z, n = 433 embryos
(9)]. These results argue that SSP acts as an
adaptor at the plasma membrane, possibly recruiting
a pathway activator.
In the course of our analysis, it became clear
that ssp mutations result in atypical segregation
of normal and mutant phenotypes. Self-fertilized
ssp plants hemizygous for a functional transgene
generate normal and mutant embryos in a ratio
of 1:1. The same 1:1 distribution is also seen in
the progeny of self-fertilized ssp-1/+ or ssp-2/+
plants (94 normal embryos in 194 total and 204
in 394, respectively). Our mapping data indicate
that transmission of the ssp-1 allele through the
haploid generation is not substantially distorted
(5), pointing to a parent-of-origin effect as a likely
cause for this phenomenon. Reciprocal crosses
indeed demonstrate that the phenotype of the
embryo is strictly dependent on the genotype of
the pollen. When homozygous ssp plants were
crossed with wild-type pollen, all embryos developed
normally (ssp-1 n = 104 and ssp-2 n =
185). Conversely, all embryos resulting from a
cross of wild-type plants with pollen from homozygous
ssp plants developed abnormally (ssp-1
n = 121 and ssp-2 n = 185). We conclude that
SSP exerts a male gametophytic, or paternal, effect
on embryonic patterning (16).
Parent-of-origin effects in plants and mammals
often arise from imprinting (17), in large
part mediated by differential DNA methylation
(18). However, SSP function is not sensitive to
global changes in DNA methylation (5), arguing
against epigenetic control. Alternatively, a paternal
effect may arise from pollen-specific expression.
A reverse transcription polymerase chain
reaction (RT-PCR) indicates that SSP mRNA
accumulates to readily detectable amounts only
in mature pollen (fig. S1A). Arabidopsis pollen
consists of a large vegetative cell that completely
encloses two small sperm cells. In situ hybridization
of germinated pollen reveals robust signals
associated with the sperm (Fig. 3A). In support of
this result, microarray profiling of isolated sperm
identified SSP as 1 of 74 genes preferentially
expressed in this cell type (19).
Surprisingly, the accumulation of SSP protein,
determined by using functional YFP-tagged
variants, does not coincide with RNA production.
No YFP fluorescence is apparent in germinated
pollen (fig. S1, C and D). SSP protein became
detectable only upon fertilization, when weak
fluorescence of the zygote was observed (Fig.
3D). This signal was transient and no longer
seen by the time of the first division. Reciprocal
crosses confirmed the absence of a specific signal
in zygotes produced by nontransgenic pollen. A
fluorescent reporter, consisting of the SSP upstream
region fused at the translational start site
to a nuclear-localized triple YFP, corroborates
these results. Again, no YFP fluorescence is apparent
in pollen, although YFP transcripts are
present (fig. S1B). When this construct was crossed
via the pollen, a transient fluorescent signal appeared
in the nuclei of the zygote and the micropylar
endosperm (Fig. 3E).
Our findings suggest a simple mechanistic
basis for the paternal effect: SSP transcripts are
produced but not translated in the sperm cells;
rather, they are delivered to the seed, where they
become translated and cause transient accumulation
of SSP protein in both products of double
fertilization, the zygote and central cell. In support
of this view, in situ hybridization reveals weak
signals in the zygote and the micropylar endosperm
(fig. S2). The presence and origin of SSP transcripts
after fertilization was independently confirmed
in RNA samples prepared from about
2000 immature seed dissected 24 hours after pollination
of Ws plants with Col pollen (5). RT-PCR
from this material detected only RNA transcribed
from the paternal Col allele (Fig. 3C).
We next sought to clarify whether SSP has a
role in the endosperm by examining fie seed
generated by fie; cdc2a pollen (5). In this genetic
combination, a relatively normal seed is
formed even though a single sperm fertilizes only
Fig. 1. Phenotype of ssp embryos. (A to D) Size of the apical (a) and basal (b) cell in wild-type (A),
yda (B), ssp (C), and fie cdc2a background (5) (D); average length and standard deviation (n > 20)
noted below. (E to I) Wild-type (E) and ssp-1 (F to I) globular embryos; frequency of phenotypic
classes listed below (n > 400); p indicates proembryo; s, suspensor. (J and K) Confocal microscopy
of suspensor-specific marker SUC3 in wild type (J) and ssp (K); green indicates reporter fluorescence;
red, propidium iodide counterstain; scale bars, 20 mm.
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the egg, leaving the endosperm without paternal
contribution (20). Despite this, zygote elongation
and suspensor development are similar to those
in wild type (Fig. 1D), suggesting that SSP mRNA
is not required in the endosperm.
The tight control of SSP expression implies
that YDA-dependent signaling may be sensitive
to the presence of SSP protein. Indeed, seedlings
expressing SSP from a strong, broadly active promoter
(5) exhibit a variety of dominant defects that
eventually become lethal. Perhaps most strikingly,
almost all primary transgenics completely lack
stomata on their embryonic leaves (Fig. 4A),
faithfully mimicking the phenotype caused by
hyperactive YDA variants (21). This effect is dependent
on the presence of a functional SSP variant
in the construct as well as an intact endogenous
YDA MAP kinase cascade (Fig. 4, B and C),
revealing that SSP protein is sufficient to activate
YDA-dependent signaling in the leaf epidermis.
We propose that, by analogy, SSP protein translated
from paternal mRNA triggers activation of
the YDA MAP kinase cascade in the zygote.
According to this model, SSP generates a temporal
cue linking the onset of YDA-dependent signaling
to fertilization. Turnover of the sperm-provided
mRNA may also limit the duration of this signaling
event. It remains to be determined through
which mechanism such a temporal signature contributes
to regulating the asymmetric first division
and thus embryo polarity.
Maternally provided factors are of key importance
for animal development because the
Fig. 2. Functional domains of SSP. (A) Gene model showing N-myristoylation
motif (orange), kinase domain (red), and two complete (blue) and cryptic (light
blue) TRP units; point mutations and insertion of mutant alleles are noted
above; alternative splice form and introduced mutations are noted below. (B and
C) Confocal images of root cells overexpressing YFP-tagged functional SSP (B)
and a variant with mutated myristoyl-acceptor (C); green, YFP fluorescence; red,
propidium iodide counterstain; arrow, cytoplasmic pocket containing nucleus;
scale bar, 40 mm. (D) In vitro myristoylation of peptides (9) representing the
N-termini of wild-type and mutant SSP variants by Arabidopsis NMT1; SOS3
peptides shown as control (5).
Fig. 3. Expression pattern of SSP transcripts and protein. (A and B) In situ
hybridization of germinated wild-type (A) and ssp-2 (B) pollen. (Inset) 4´,6´Diamidino-2-phenylindole
(DAPI) staining identifying sperm nuclei (arrows)
and the vegetative nucleus (asterisk). (C) Detection of paternal SSP mRNA in
developing seed 24 hours after pollination (5); samples of seed from Ws pistils
crossed with Col pollen (lane 1), Ws pollen (2), Col pollen (3), and unfertilized
Ws pistils (4). (Top) RT-PCR products restricted with BclI, cleaving the Ws (red
arrowhead) but not the Col (black arrowheads) allele; (middle) unrestricted
products; (bottom) control without reverse transcriptase. (D and E) Confocal
images of zygote-stage seed expressing a YFP-tagged functional SSP variant
(D) or no transgene (E); arrow, weak YFP fluorescence of the zygote. (F and G)
Confocal images of seed expressing a nuclear SSP reporter crossed via the pollen (F) or no transgene (G), about 24 hours after pollination; asterisks,
endosperm nuclei; arrow, nucleus of zygote; green, YFP fluorescence, red, propidium iodide counterstain; scale bars, 20 mm.
www.sciencemag.org SCIENCE VOL 323 13 MARCH 2009 1487
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zygotic genome typically does not become active
until some time after fertilization [maternal-zygotic
transition (22)]. Plant embryos apparently develop
with more autonomy (23), and it is open whether
an equivalent to the maternal-zygotic transition
of animals exists (24). The most prominent parentof-origin
effect in plants targets gene expression
in the endosperm and is thought to arise from a
parental conflict: Imprinting presumably enforces
maternal control over nutrient allocation to the
embryo (25, 26). Direct delivery of transcripts to
the seed would provide a general mechanism for
subverting such epigenetic regulation. Thus, paternal
control over growth of the suspensor, the
other organ mediating nutrient flux to the embryo,
perhaps evolved to antagonize maternal influences
in this conflict.
References and Notes
1. C. W. Wardlaw, Embryogenesis in Plants (Wiley,
New York, 1955).
2. W. Lukowitz, A. Roeder, D. Parmenter, C. Somerville, Cell
116, 109 (2004).
3. H. Wang, N. Ngwenyama, Y. Liu, J. C. Walker, S. Zhang,
Plant Cell 19, 63 (2007).
4. S. Meyer et al., Plant Physiol. 134, 684 (2004).
5. For details see materials and methods, available as
supporting material on Science Online.
6. S.-H. Shiu, A. B. Bleeker, Proc. Natl. Acad. Sci. U.S.A. 98,
10763 (2001).
7. For all molecular complementation experiments, the
frequency of normal and ssp embryos produced by five
or more independent primary transgenic lines was
determined; these results were confirmed in the progeny
of selected lines.
8. M. D. Resh, Biochim. Biophys. Acta 1451, 1 (1999).
9. Single-letter abbreviations for the amino acid residues
are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe;
G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro;
Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.
10. B. Boisson, T. Meinnel, Anal. Biochem. 322, 116 (2003).
11. J. A. Adams, Chem. Rev. 101, 2271 (2001).
12. K. Mukherjee et al., Cell 133, 328 (2008).
13. S. Janssens, R. Beyaert, Mol. Cell 11, 293 (2003).
14. E. Castells, J. M. Casacuberta, J. Exp. Bot. 58, 3503 (2007).
15. C. Scheufler et al., Cell 101, 199 (2000).
16. Both the diploid sporophyte and the haploid gametophyte
may affect embryo development; for simplicity, we refer to
“male gametophytic” effects as “paternal.”
17. R. Feil, F. Berger, Trends Genet. 23, 192 (2007).
18. J. H. Huh, M. J. Bauer, T.-F. Hsieh, R. Fischer, Curr. Opin.
Genet. Dev. 17, 480 (2007).
19. F. Borges et al., Plant Physiol. 148, 1168 (2008).
20. M. K. Nowack et al., Nature 447, 312 (2007).
21. D. C. Bergmann, W. Lukowitz, C. R. Somerville, Science
304, 1494 (2004).
22. A. F. Schier, Science 316, 406 (2007).
23. P. D. Jenik, C. S. Gillmor, W. Lukowitz, Annu. Rev. Cell
Dev. Biol. 23, 207 (2007).
24. S. Meyer, S. Scholten, Curr. Biol. 17, 1686 (2007).
25. R. J. Scott, M. Spielman, J. Bailey, H. G. Dickinson,
Development 125, 3329 (1998).
26. C. Spillane et al., Nature 448, 349 (2007).
27. This work was supported by the NSF (grant IOB-0446103
to W.L.), the Deutsche Forschungsgemeinschaft (M.B.),
the Cold Spring Harbor Laboratory Association (T.N.),
and the Association pour le Recherche sur le Cancer
(grant 4920 to T.M.). Seed of ssp-1 have been deposited
with the Arabidopsis stock center. Materials were kindly
made available by P. Das (Caltech), F. Besnard and
T. Vernoux (Ecole Normale Supérieure Lyon, pENTR triple
Venus), S. McCormick (University of California Berkeley,
GEX2 marker), N. Sauer (Erlangen University, SUC3
marker), A. Schnittger (Strasbourg University, cdc2a;fie1),
and K. Slotkin and R. Martienssen (Cold Spring Harbor
Laboratory, met1, ddm1, cmt3, drm2, and rdr1; rdr2;
rdr6). S. Alabaster and J. A. Traverso provided help in the
early phases of the project. We thank T. Berleth
(University of Toronto), C. S. Gillmor and M. Willman
(University of Pennsylvania), S. Jeong and C. Schulz
(University of Georgia), R. Schwab (Cold Spring Harbor
Laboratory), and D. Weijers (Wageningen University) for
comments on the manuscript.
Supporting Online Material
www.sciencemag.org/cgi/content/full/323/5920/1485/DC1
Materials and Methods
Figs. S1 and S2
Tables S1 to S3
28 October 2008; accepted 6 January 2009
10.1126/science.1167784
Preferential Generation of Follicular
B Helper T Cells from Foxp3+
T Cells in Gut Peyer’s Patches
Masayuki Tsuji,1
* Noriko Komatsu,2
* Shimpei Kawamoto,1,4
* Keiichiro Suzuki,1
Osami Kanagawa,3
Tasuku Honjo,4
Shohei Hori,2
† Sidonia Fagarasan1
†
Most of the immunoglobulin A (IgA) in the gut is generated by B cells in the germinal centers
of Peyer’s patches through a process that requires the presence of CD4+
follicular B helper
T (TFH) cells. The nature of these TFH cells in Peyer’s patches has been elusive. Here, we demonstrate
that suppressive Foxp3+
CD4+
T cells can differentiate into TFH cells in mouse Peyer’s patches. The
conversion of Foxp3+
T cells into TFH cells requires the loss of Foxp3 expression and subsequent
interaction with B cells. Thus, environmental cues present in gut Peyer’s patches promote the
selective differentiation of distinct helper T cell subsets, such as TFH cells.
T
he production and secretion of immunoglobulin
A (IgA) by the host is critical for
the maintenance of a vast community of
commensal bacteria in the intestinal lumen with
minimal penetration of the gut epithelium (1, 2).
Most of this IgA synthesis requires germinal
center (GC) formation in Peyer’s patches (PPs),
aggregations of lymphoid follicles in the gut. In
GCs, activated B cells express activation-induced
cytidine deaminase (AID) and switch from
making IgM to IgA (3–5). GC development in
PPs requires bacteria in the gut; germ-free mice
have small GCs, probably induced by bacterial
components in food (1). T cells, by providing
cytokines and costimulatory molecules to B cells,
are also required for GC induction. Mice that lack
Tcells are devoid of GCs, and GC formation can
be rescued by the adoptive transfer of CD4+
T
cells (1, 6, 7).
We investigated the origin of TFH in PPs by
examining the contribution of Foxp3+
CD4+
T
cells (8–10) obtained from Foxp3EGFP
reporter
mice (11), which express green fluorescent protein
(GFP) under the control of the Foxp3 promoter.
Total CD4+
T cells, Foxp3/GFP+
CD4+
T cells
(hereafter called Foxp3+
T cells) or Foxp3/GFP–
CD4+
(hereafter called Foxp3–
T cells) that were
isolated from the spleen and lymph nodes (LNs)
of Foxp3EGFP
reporter mice were adoptively
Fig. 4. Ectopic expression of SSP. (A to C) Confocal images of leaves producing a YFP-tagged
functional SSP variant (A), a myristoylation-deficient variant (B), and a functional variant in a yda
background (C); arrows, stomata; green, YFP fluorescence; red, chlorophyll fluorescence; scale bar,
20 mm. (Inset) Cluster of stomata progenitor cells, as typical for yda.
1
Laboratory for Mucosal Immunity, RIKEN, Yokohama 1-7-22,
Tsurumi, Yokohama, 230-0045, Japan. 2
Research Unit for
Immune Homeostasis, RIKEN, Yokohama 1-7-22, Tsurumi,
Yokohama, 230-0045, Japan. 3
Laboratory for Autoimmune
Regulation, Research Center for Allergy and Immunology, RIKEN,
Yokohama 1-7-22, Tsurumi, Yokohama, 230-0045, Japan.
4
Department of Immunology and Genomic Medicine, Kyoto
University, Graduate School of Medicine, Sakyo-ku, Kyoto
606-8501, Japan.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
shohei@rcai.riken.jp (S.H.); sidonia-f@rcai.riken.jp (S.F.)
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