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Legends for all Supporting Information
Figure S1. The multiple alignment of expansin protein sequences by CLUSTAL W.
(a) The amino acid sequence between 55 (as “1” in the illustration) and 181 (as “127” in
the illustration) of AtEXPA7 and the equivalent regions of each expansin protein
sequence were aligned by CLUSTAL W. Residues that are identical among all amino
acid sequences (black) and conserved substitutions (gray) are indicated. (b)
Phylogenetic tree based on the amino acid sequences shown in (a).
Figure S2. Root hair-specific expression analysis of RH101 and RH102 in response
to infection by M. loti.
Real-time RT-PCR analysis of RH101 and RH102 transcripts in root hair cells, stripped
roots or whole roots 2 days after mock-inoculation (Control) or inoculation with M. loti
(+M. loti). The data are indicated as means and SD of 3 or 4 biological replications.
Means with different letters are significantly different (Tukey-Kramer multiple
comparison test, P<0.05).
Figure S3. Promoter sequences of LjEXP7 and LjEXPA8.
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The promoter sequences of LjEXPA7 and LjEXPA8 from -557 to +3 are shown in (a)
and (b), respectively. The transcriptional start site is designated +1. Red underlined
nucleotide sequences represent putative root hair-specific cis element (RHE) cores
located within the promoter sequences of LjEXPA7 and LjEXPA8. Highly conserved
nucleotides within the RHE core are denoted by asterisks. An “r” of E2r and E3r
indicates a reverse orientation of the RHE. Bold nucleotide sequences below the arrows
show sequences of three types of RHEs-containing promoters, pEpi168 (from -168 to
-1), pEpi308 (from -308 to -1) and pEpi400 (from -400 to -1).
Figure S4. Histochemical localization of GUS activity in wt roots transformed with
pEpi::GUS+ vector.
(a-f) Bright-field images of X-Gluc incubated wt roots transformed with an empty
vector control (Empty vector) (a, b), pEpi::GUS+ (pEpi::GUS+) (c, d) and p35S::GUS+
(e, f) vectors. Expression patterns of GUS+ gene from transformed roots were observed
2 weeks after mock-inoculation (Control) (a, c, e) or inoculation with DsRed-labeled M.
loti (b, d, f). (g, h) Root hairs of wt/pEpi::GUS+ roots observed 7 days after
mock-inoculation (g) or inoculation with DsRed-labeled M. loti (h). Bright-field and red
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fluorescence images were merged into single images. (h) ITs can be seen inside the
curled and GUS-stained root hairs. Scale bars are 1 mm (a-f) and 100 μm (g, h).
Figure S5. Rhizobial infection and nodule organogenesis phenotypes of wt roots
transformed with pEpi::GUS+ vector.
(a-l) Symbiotic phenotypes of transformed wt roots were observed 4 weeks after
inoculation with DsRed-labeled M. loti. Root phenotypes of wt/Empty vector (a-d),
wt/pEpi::GUS+ (e-h), and wt/p35S:: GUS+ (i-l). (a, e, i) ITs can be seen inside the
curled root hairs, shown as merged images of bright-field and red fluorescence images.
Bright-field images (b, f, j) and their corresponding red (c, g, k) and green (d, h, l)
fluorescence images of nodulation phenotypes are shown. Nodules formed on the roots
of wt/Empty vector (b-d), wt/pEpi::GUS+ (f-h) and wt/p35S::GUS+ (j-l). (c, g, k)
Infection of DsRed-labeled M. loti was observed as red fluorescence in the central zone
of nodules on those roots. (d, h, l) Green fluorescence as a transgenic marker is shown.
Scale bars are 100 μm (a, e, i) and 1 mm (b-d, f-h, j-l).
Figure S6. Expression analysis of symbiosis genes in root hairs, stripped roots and
whole roots of L. japonicus.
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Real-time RT-PCR analysis of NFR1, NFR5, NUP85, NUP133, CASTOR, POLLUX,
CCaMK, CYCLOPS, SYMRK, NSP1, NSP2 and NIN transcripts in root hairs, stripped
roots or whole roots 2 days after mock-inoculation (Control) or inoculation with M. loti
(+M. loti). The data are indicated as means and SD of 3 or 4 biological replications.
Means with different letters are significantly different (Tukey-Kramer multiple
comparison test, P<0.05).
Figure S7. Rhizobial infection and nodule organogenesis phenotypes of nup85-2,
nup133-3, castor-4, pollux-3 and ccamk-3 mutant roots transformed with an empty
vector control.
(a-o) Symbiotic phenotypes of transformed non-nodulating mutant roots were observed
4 weeks after inoculation with DsRed-labeled M. loti. Root phenotypes of
nup85-2/Empty vector (a-c), nup133-3/Empty vector (d-f), castor-4/Empty vector (g-i),
pollux-3/Empty vector (j-l) and ccamk-3/Empty vector (m-o). (a, d, g, j, m) Neither
bacterial colonization nor IT formation was observed; however, we show bright-field
and red fluorescence images merged into single images. Bright-field images (b, e, h, k,
n) and their corresponding red fluorescence images (c, f, i, l, o) of nodulation
phenotypes are shown. Cortical cell division did not occur in the roots of
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nup85-2/Empty vector (b, c), nup133-3/Empty vector (e, f), castor-4/Empty vector (h, i),
pollux-3/Empty vector (k, l) and ccamk-3/Empty vector (n, o). Scale bars are 100 μm (a,
d, g, j, m) and 1 mm (b, c, e, f, h, i, k, l, n, o).
Figure S8. Rhizobial infection and nodule organogenesis phenotypes of nfr1-4,
nfr5-2, nsp1-1 and nsp2-1 mutant roots transformed with an empty vector control.
(a-l) Symbiotic phenotypes of transformed non-nodulating mutant roots were observed
4 weeks after inoculation with DsRed-labeled M. loti. (a, d, g, j) Bright-field and red
fluorescence images merged into single images. Neither bacterial colonization nor IT
formation was observed on the roots of nfr1-4/Empty vector (a), nfr5-2/Empty vector
(d), nsp1-1/Empty vector (g) and nsp2-1/Empty vector (j). Bright-field images (b, e, h,
k) and their corresponding red fluorescence images (c, f, i, l) of nodulation phenotypes
are shown. Cortical cell division did not occur in the roots of nfr1-4/Empty vector (b, c),
nfr5-2/Empty vector (e, f), nsp1-1/Empty vector (h, i) and nsp2-1/Empty vector (k, l).
Scale bars are 100 μm (a, d, g, j) and 1 mm (b, c, e, f, h, i, k, l).
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Figure S9. Stacked histograms comparing frequency counts in terms of the number
of nodules or bumps per mutant plants transformed with CCaMK, CASTOR,
POLLUX, NUP85 and NUP133 driven by pEpi or p35S.
Black bar, positioned at x=0, represents the frequency of non-nodulating plants per total
plants examined (Nod-). X-axis; the number of nodules or bumps/plant. Y-axis; frequency
of non-nodulation (black), bump-formed (pink) or nodulated (red) plants.
Figure S10. Spontaneous nodulation phenotypes of wt roots transformed with
CCaMKT265D driven by pEpi or p35S.
(a-h) Introduction of gain-of-function CCaMKT265D (CCaMKTD) under the control of
p35S could induce spontaneous nodulation in the absence of rhizobia (g, h). However,
on the roots of wt/pEpi308::CCaMKT265D (wt/pEpi::CCaMKTD) (c, d) as well as those
of wt/pEpi308::CCaMKT265T (wt/pEpi::CCaMKTT) (a, b) and wt/p35S::CCaMKT265T
(wt/p35S::CCaMKTT) (e, f), spontaneous nodulation did not occur even 6 weeks after
transplantation. Green fluorescence as a transgenic marker is shown (b, d, f, h). Scale
bars are 1 mm (a-h).
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Figure S11. Complementation tests of rhizobial infection and nodule organogenesis
phenotypes of nup85-2, nup133-3, castor-4 and pollux-3 mutants transformed with
the corresponding genes driven by p35S.
(a-l) Symbiotic phenotypes of transformed roots were observed 4 weeks after
inoculation with DsRed-labeled M. loti. Root phenotypes of nup85-2/pEpi::NUP85
(a-c),
nup133-3/pEpi::NUP133
(d-f),
castor-4/p35S::CASTOR
(g-i)
and
pollux-3/p35S::POLLUX (j-l). (a, d, g, j) ITs can be seen inside the curled root hairs,
shown as merged images of bright-field and red fluorescence images. Bright-field
images (b, e, h, k) and their corresponding red fluorescence images (c, f, i, l) of
nodulation phenotypes are shown. (c, f, i, l) Infection of DsRed-labeled M. loti was
observed as red fluorescence in the central zone of nodules. Scale bars are 100 μm (a, d,
g, j) and 1 mm (b, c, e, f, h, i, k, l).
Figure S12. Stacked histograms comparing frequency counts in terms of the
number of nodules or bumps per mutant plant by co-transformation with CCaMK
under the control of pEpi and truncated CCaMK1-314 or CCaMK1-340 under the
control of p35S.
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Black bar, positioned at x=0, represents the frequency of non-nodulating plants per total
plants examined (Nod-). X-axis; the number of nodules or bumps/plant. Y-axis; frequency
of non-nodulation (black), bump-formed (pink) or nodulated (red) plants.
Figure S13.
Spontaneous
nodule-like
structure
of
ccamk-3
mutant
by
co-transformation with CCaMK under the control of pEpi and a truncated
CCaMK1-314 under the control of p35S.
(a, b) Nodulation phenotypes of transformed roots were observed 4 weeks after
inoculation with DsRed-labeled M. loti. Bright-field image (a) and the corresponding
red fluorescence image (b) of nodulation phenotypes are shown. In addition to infected
nodules as shown in Figure 4, spontaneous nodule-like structures without rhizobial
infection were also formed on the roots of ccamk-3/pEpi::CCaMK-p35S::CCaMK1-314
(a, b). Scale bars are 1 mm (a, b).
Figure S14. Complementation tests of rhizobial infection and nodule organogenesis
phenotypes of cyclops-3 mutants transformed with CYCLOPS driven by pEpi or
p35S.
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(a-i) Symbiotic phenotypes of transformed roots were observed 4 weeks after
inoculation with DsRed-labeled M. loti. Root phenotypes of cyclops-3/Empty vector
(a-c),
cyclops-3/pEpi::CYCLOPS
(d-f)
and
cyclops-3/p35S::CYCLOPS
(g-i).
Micro-colony (a) or ITs can be seen inside the curled root hairs (d, g), shown as merged
images of bright-field and red fluorescence images. Bright-field images (b, e, h) and
their corresponding red fluorescence images (c, f, i) of nodulation phenotypes are shown.
On the roots of cyclops-3/Empty vector, though deformation of root hairs and
colonization of bacteria in curled root hairs were observed, development of ITs within
the root hairs did not occur (a). Cortical cell division arrested at bump structure (arrows
in b). On the roots of cyclops-3/pEpi::CYCLOPS, epidermal IT formation was
recovered (d), while cortical cell division was not coupled with IT penetration, resulting
in the formation of bumps (arrows in e). (h, i) Nodules formed on the roots of
cyclops-3/p35S::CYCLOPS. Infection of DsRed-labeled M. loti was observed as red
fluorescence in the central zone of nodules (i). Scale bars are 100 μm (a, d, g) and 1 mm
(b, c, e, f, h, i).
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Figure S15. Stacked histograms comparing frequency counts in terms of the
number of nodules or bumps per mutant plant transformed with CYCLOPS, NSP1,
NSP2, NFR1 and NFR5 driven by pEpi or p35S.
Black bar, positioned at x=0, represents the frequency of non-nodulating plant per total
plants examined (Nod-). X-axis; the number of nodules or bumps/plant. Y-axis; frequency
of non-nodulation (black), bump-formed (pink) or nodulated (red) plants.
Figure S16. Complementation tests of rhizobial infection and nodule organogenesis
phenotypes of nsp1-1 and nsp2-1 mutants transformed with the corresponding
genes driven by pEpi or p35S.
(a-l) Symbiotic phenotypes of transformed roots were observed 4 weeks after
inoculation with DsRed-labeled M. loti. Root phenotypes of nsp1-1/pEpi::NSP1 (a-c),
nsp2-1/pEpi::NSP2 (d-f), nsp1-1/p35S::NSP1 (g-i) and nsp2-1/p35S::NSP2 (j-l). (a, d,
g, j) ITs can be seen inside the curled root hairs, shown as merged images of bright-field
and red fluorescence images. Bright-field images (b, e, h, k) and their corresponding
red fluorescence images (c, f, i, l) of nodulation phenotypes are shown. Nodules formed
on the roots of nsp1-1/p35S::NSP1 (h, i) and nsp2-1/p35S::NSP2 (k, l). (i, l) Infection
of DsRed-labeled M. loti was observed as red fluorescence in the central zone of
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nodules. Nodule organogenesis was not induced on the roots of nsp1-1/pEpi::NSP1 (b,
c) and nsp2-1/pEpi::NSP2 (e, f). Scale bars are 100 μm (a, d, g, j) and 1 mm (b, c, e, f,
h, i, k, l).
Table S1. Spontaneous nodulation phenotypes.
The numbers indicate the ratio of the number of plants that showed spontaneous
nodulation events, i.e., formation of small bump-like structures (Bump) or spontaneous
nodules (SpN) in the cortex, to the total number of plants 6 weeks after transplantation.
Numerals in parentheses indicate the numbers of spontaneous nodules (Sp nodules) as
averages (±SE) of nodulated plants. Data were compiled from more than two
independent experiments. wt, Gifu B-129; pEpi, promoter Epi308; p35S, promoter
CaMV 35S; CCaMKTT, wt-CCaMK; CCaMKTD, CCaMKT265D
Table S2. Primer sequences used for construction.
Lower case characters indicate engineered sequences including restriction sites or
D-TOPO recognition site.
Table S3. Primer sequences used in the expression analysis.
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Methods S1. Plasmid construction.
For promoter-GUS analysis and complementation tests, Gateway compatible destination
vectors, pEpi168::GW-p35S::GFP and pEpi308::GW-p35S::GFP, were constructed as
follows: Genomic sequences of the LjEXPA7 promoter regions (168 and 308 bp) were
amplified by PCR. Each fragment was digested with DraI and SpeI and then inserted
into the HindIII (blunt)-SpeI site of p35S::GW-p35S::GFP (P35S:GFP-gw; Banba et al.,
2008). For the construction of the pEpi400::GW-p35S::GFP vector, the genomic
sequence of the LjEXPA8 promoter region (400 bp) was amplified by PCR. The
fragment
was
digested
with
HindIII
and
SpeI
and
then
ligated
with
p35S::GW-p35S::GFP. For co-transformation with CCaMK under the control of
pEpi308 and an inactive truncated CCaMK under the control of p35S, a Gateway
compatible destination vector, pEpi308::CCaMK-p35S::GW, was constructed by
introducing the reading frame cassette C.1 (RfC.1) of the Gateway vector conversion
system (Invitrogen, Carlsbad, CA, USA) into the XhoI (blunt)-XhoI (blunt) site of
pEpi308::CCaMK-p35S::GFP. All primer sets used for construction are listed in Table
S2.
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Information of entry clones of symbiotic genes and GUSplus (GUS+) and
conversion of these entry vectors into pEpi destination vectors are described below.
cDNA sequences of NFR5 and NUP85 were amplified by PCR and cloned into
pENTR/D-TOPO (Invitrogen, Carlsbad, CA, USA). cDNA sequence of NUP133 was
amplified by PCR and digested with SacII and AscI and then inserted into the
SacII-AscI site of pENTR/D-TOPO. The entry clones constructed in this study, and
entry clones of GUS+ (Yano et al., 2008), NFR1 (Nakagawa et al., 2011), CASTOR,
POLLUX, CCaMK, CCaMKT265D (Banba et al., 2008), an inactive truncated CCaMK
(CCaMK1-340) (Shimoda et al., 2012), CYCLOPS (Yano et al., 2008), NSP1 (Yokota et
al., 2010) and NSP2 (Yokota et al., 2010), were converted with the destination vector of
pEpi308::GW-p35S::GFP by the LR reaction (Gateway LR clonase II Enzyme Mix;
Invitrogen, Carlsbad, CA, USA).
Methods S2. RNA isolation from root hairs.
Surface-sterilized seeds were germinated on sterilized filter paper on 1.5% agar
containing half-strength B&D medium (Broughton and Dilworth 1971) in square plastic
plates (Eiken Chemical Co., Tokyo, Japan, http://www.eiken.co.jp/). The plastic plates
were incubated vertically for 2 days in a growth cabinet with a 16 h-day/8 h-night cycle
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at 24°C, and then the seedlings were inoculated with a suspension of DsRed-labeled M.
loti. The plants were grown for an additional 2 days with the roots shielded from light.
The roots were then collected and dropped immediately into a 100-ml stainless steel
beaker filled with liquid nitrogen. The samples were stirred gently with a magnetic
stirrer for 20 min. In this step, the root hairs were cut off from the roots. The liquid
nitrogen containing root hairs was then filtered through a stainless testing sieve with a
mesh aperture of 500 μm. Liquid nitrogen was evaporated in a 50-mL sample tube and
root hairs were collected. The roots trapped by the testing sieve were also collected, as
“stripped roots”. Total RNAs were isolated by the CTAB method (Chang et al., 1993)
with some modifications. Briefly, the collected root hairs or stripped roots were ground
to a fine powder in liquid nitrogen, immediately dissolved in the extraction solution (2%
CTAB, 100 mM Tris pH 9.5, 20 mM EDTA, 1.4 M NaCl, 1% 2-mercaptoethanol), and
then incubated at 65°C for 10 min. After two successive extractions with chloroform,
RNAs were precipitated using LiCl (final concentration 2.5 M) at -20°C overnight. The
precipitated RNAs were collected by centrifugation, and were dissolved in distilled
water. As necessary, the samples were further purified using an RNeasy Mini Kit
(Qiagen, http://www.qiagen.com/) according to the manufacturer’s instructions.
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