Fig. S1 Cytological analysis of nodules formed in Lotus japonicus wild type (WT)
and rel3 mutants. (a, b)Transverse sections of mature nodules formed in WT (a) and
rel3 mutants (b). (c,d) Longitudinal sections of mature nodule formed in WT (c) and
rel3 mutants (d). Nodule vascular bundles indicated by arrows. Scale bars, 100μm.
Fig. S2 Root growth of Lotus japonicus wild type (WT) and rel3 mutants under
non-symbiotic conditions. One-week-old L. japonicus WT and rel3 seedlings were
transplanted onto 1/2 B5 plates (1.5% agar). The plants were grown on plates in a
vertical orientation that kept the roots along surface of agar medium. The primary root
length and lateral root numbers were measured for 4 weeks. (a) The average length of
primary root. (b) Numbers of lateral roots. Mean values ±SE are presented (n>16). *
indicates significant differences at P <0.05 according to a two-tailed t-test.
Fig. S3 qRT-PCR analysis of REL3 and TAS3 5’D7(+) in transgenic roots of rel3
mutants expressing 35S:LjREL3. Roots from one Lotus japonicus wild type (WT)
composite plant carrying 35S:GUS (WT-35S), one rel3 composite plant carrying
35S:GUS (rel3-35S) and three rel3 composite plants carrying 35S:REL3
(rel3-35S:LjREL3-1, -2,-3) were tested. REL3 mRNA quantification was normalized
against UBIQUITIN(Ubi) gene in each sample tested (relative units). Transcript levels
of TAS3 ta-siRNAs were normalized to U6 expression. Error bars indicate the range
of possible value based on SD of replicate Ct values.
Fig. S4 Nodulation phenotypes of rel1 and rel1rel3 mutants. One-week-old Lotus
japonicus wild type (WT), rel1, rel1rel3 seedlings were inoculated with
Mesorhizobium loti. Nodule formation was examined at 3 weeks post-inoculation. (a)
Visible nodule numbers per plant. (b) The ratio of the nodulation zone. The ratio of
the nodulation zone was calculated by dividing the length of the nodulation zone by
the main root length. Mean values ±SD are presented (n>7). **indicate significant
differences at P <0.01, according to a two-tailed t-test.
Fig. S5 Visualization of infection events in Lotus japonicus wild type (WT) and rel3
mutants. L. japonicus WT and rel3 seedlings were inoculated with Mesorhizobium loti
carrying a hemA:lacZ construct. Representative infection pictures of rel3 mutants and
WT at 7 day postinoculation were presented. (a,b) Rhizobia colonize the infection
pockets of curled root hair tips of WT(a) and rel3 (b). (c,d) Infection threads extend
within curled root hairs of WT(c) and rel3 (d). (e,f) Infection threads (arrowhead)
penetrate the cortex and ramify into fine networks (asterisk) in the developing nodule
primordia of WT(e) and rel3 (f). Bars, 1mm for a-d, 100µm for e and f.
Fig. S6 Expression of the REL3pro:GUS in nodule formation. Two-week-old Lotus
japonicus composite plants expressing the REL3pro:GUS fusion were planted into
pots and inoculated with Mesorhizobium loti containing a hemA:lacZ construct.
Double detection of REL3pro:GUS expression and LacZ activity of the bacteria was
carried out as described by Bersoult et al.(2005). Magenta-gluc was used for detecting
GUS activity (in magenta), X-Gal was used for detecting LacZ activity (in blue). (a)
REL3pro:GUS expression was observed only in central vascular bundle (VB) of
infected roots, and LacZ expression(Arrow) was detected in nodule primordium. (b)
Strong REL3pro:GUS expression in root central vascular bundle and at the nodule
base, in the region connecting the nodule to the root VB where pericycle cells (pe) are
located. Bars, 100µm.
Fig. S7 TAS3 ta-siRNA accumulation in the roots of Lotus japonicus wild type (WT)
in response to rhizobial infection. Quantitative real-time reverse transcription
PCR(qRT-PCR)for TAS3 ta-siRNA was performed according to the protocol described
by Varkonyi-Gasic et al.(2007). The stem-loop RT primer binding to the 3' portion of
5’D7(+)
TAS3
was
designed
(GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGAGGTC
), and the stem-loop RT primer was used to reverse transcribe small RNA molecules
using total RNA extracted from Lotus japonicus inoculated roots(IN) at 1 or 3 d
postinoculation and uninoculated roots(UN). qRT-PCR was conducted with Real-time
PCR Master Mix (SYBR® Green I) (TOYOBO) using a TAS3 5’D7(+)-specific
forward primer (GCGGCGGTTCTTGACCTTGTAA) and a universal reverse primer
(GTGCAGGGTCCGAGGT). The LjATP synthase gene was used as an internal
control
to
normalize
expression,
5’-CAATGTCGCCAAGGCCCATGGTG-3’
and
the
and
primers
LjATPs-FP:
LjATPs-RP:
5’-AACACCACTCTCGATCATTTCTCTG-3’ were used to amplify the LjATPs gene.
The relative expression amount was calculated by the ΔΔCt method according to the
manufacturer's protocol. PCR (95℃ for 5 min, 40 cycles at 95℃ for 5 s, 60℃for 10s,
72℃for 1s) was performed with Thermal Cycler Dice Real-Time System (Bio-Rad,
USA). Error bars indicate the range of possible value based on SD of replicate Ct
values.
Fig. S8 Nodule formation of Lotus japonicus wild type (WT) and rel3 mutants in the
presence of 1μM NPA and 1μM AVG. Four plants on the left were WT and four
plants on the right were rel3 mutants on agar plates. (a,b,c,d) Representative pictures
of nodule formation in WT and rel3 in the absence of 1μM NPA(-) and 1μM AVG(-)
(a), absence of AVG (-) and presence of NPA (+) (b), absence of NPA(-) and
presence of AVG (+)(c), presence of 1μM NPA(+) and 1μM AVG(+)(d). Red arrows
denote the agravitropic extent of root tips. Bars, 1cm.
Table S1 Primers used for Quantitative real-time reverse transcription
PCR(qRT-PCR) experiments
Gene
LjREL3
LjARF3a
LjARF3b
LjARF4
LjUBIQUITIN
TAS3 5’D7(+)
U6
Primers used for qRT-PCR
Sense
5’- TCCTCGGGAAACTGTCGGA -3’
Antisense 5’- CCACTGACGGTCATGGCGT -3’
Sense
5’- GAGTCCACCACCAAGGCTTAT -3’
Antisense 5’- ACTGCGAGCACCCTCAGTTA -3’
Sense
5’- CTGAAGTGAAAGGTGGAGCA -3’
Antisense 5’- CTGCCCAGCTGACTATGAAA -3’
Sense
5’- TTAATTACGCAGGCTTCACG -3’
Antisense 5’- ACATGCTGAATCCTTGACCA-3’
Sense
5’- TTCACCTTGTGCTCCGTCTTC -3’
Antisense 5’- AACAACAGCACACACAGCCAATCC -3’
5’-TTCTTGACCTTGTAAGACCTC-3’
5’-GAGAAGATTAGCATGGCCCCT-3’
Table S2 Complementation tests for nodulation by introducing a construct carrying
d35S:REL3 into rel3 mutants using hairy root transformation
Genotype
Transformed plasmid
rel3
rel3
Wild type
pCAMBIA-d35S:REL3
pCAMBIA-2301
pCAMBIA-2301
Full-length
REL3
cDNA
Nodule number per plant
Transformed GUS plants
18.6±3.1
7.0±0.8
16.7±1.5
was
30
20
22
amplified
using
primer:
5’-CATGCCATGGAAGAGACAGAGGA-3’
5’-CGGGATCCTCTAGAGATTCTAGCAG-3’.
and
The
amplified
product
was
sequenced to validate correction, and placed under the control of the double 35S
promoter, subsequently cloned into pCAMBIA-2301.The resulting construct harbored
a constitutively expressed GUS reporter gene for the identification of transgenic roots,
and introduced into rel3 mutants by hairy root transformation. Lotus japonicus wild
type and rel3 mutants transformed with the pCAMBIA-2301 were used as controls.
GUS staining was used for detecting transformed roots. Nodule number was scored at
3 wpi. Results are presented as means ± SE.
Table S3 Complementation tests for nodulation by introducing a construct carrying
REL3pro:REL3 into rel3 mutants using hairy root transformation
Genotype
Transformed plasmid
rel3
rel3
Wild type
pCAMBIA-REL3pro:REL3
pCAMBIA-1301
pCAMBIA-1301
The
REL3
promoter
was
Nodule number per plant
Transformed GUS plants
7.3±1.21
3.0±1.41
6.43±3.25
amplified
14
5
9
using
5’-GGGGTACCAGAATGGGCTGAATGCGAAG-3’
primers
LjREL3-F1:
and
LjREL3-R1:
5’-TTGTTTTTGTGGTGAGTTTCTGGGT-3’, REL3 cDNA was amplified using
primer LjREL3-F2: 5’-AAGAGACAGAGGATTCC-3’ and REL3-R2: LjREL3-R2:
5’-ACGCGTCGACTCTAGAGATTCTAGCAG-3’, respectively. The REL3 promoter
and REL3 cDNA PCR products were used templates for amplifying the fusion of
REL3 promoter and its cDNA by overlapping PCR using primers LjREL3-F1 and
LjREL3-R2.The resulting PCR products were sequenced, and inserted it between the
KpnI and SalI sites of the pCAMBIA1301vector(REL3pro:REL3). The resulting
construct harbored a constitutively expressed GUS reporter gene to facilitate the
identification of transgenic roots, and was introduced into rel3 mutants by hairy root
transformation. Lotus japonicus wild type and rel3 mutants transformed with the
pCAMBIA-1301 were used as controls. GUS staining was used for detecting the
transformed roots. Nodule number was scored at 2 wpi. Results are presented as
means ± SD.
Table S4 Nodulation kinetics in Lotus japonicus wild type and rel3 mutants
Genotype
Nodule number per plant
1 wpi
2 wpi
3 wpi
Wild type
5.3±0.20
13.84±0.43
19.68±0.65
rel3
4.71±0.30
9.47±0.53**
13.05±0.82**
The germinated seeds were grown in pots containing autoclaved vermiculite and
perlite mixture, and inoculated with Mesorhizobium loti NZP2235. Nodules that are
visible to the naked eyes on the roots of wild type and homogenous rel3 mutants were
scored at 1, 2 and 3 week postinoculation(wpi). At least 48 plants were measured.
Results are presented as means ±SE. **indicates significant level of difference
between rel3 mutants and wild type for P<0.01 using a two-tailed t-test.
Table S5 Transcript profiles of TAS3 and its target ARF genes in infected roots from
Lotus japonicus Gene Expression Atlas (LjGEA)
Transcript profiles of infected rootsa
Gene
Affymetrix Lotus probe ID
REL3
TAS3
ARF3a
ARF3b
ARF4
Ljwgs_040155.1_at
TC19658_at
TC12435_at
Ljwgs_009422.4_at
chr3.CM0111.13_at
Root-UN
135.6
478.8
328.9
164.1
306.3
Root-1dpi
215.8
700.5
252.6
140.0
241.1
Root-3dpi
135.4
660.6
284.7
157.9
218.2
a The
roots of three-week-old Lotus japonicus plants were collected at 1 and 3 d postinoculation
(dpi), and the corresponding uninoculated roots (UN) were used as a control.
References
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highly sensitive RT-PCR method for detection and quantification of microRNAs.
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