Fig. S1 Phylogenetic trees of OSCs (a),cytochrome P450s (b) and SDR-likeproteins (c)
constructed by the maximum likelihood method with a 1000 bootstrap replicates. The scale
bar indicates the number of amino acid substitutions per site. Cytochrome P450s from A.
thaliana, M. sativa, L. japonicus, G. uralensis and A. sativa adjacent to / highly coexpressed
with OSCs or previously found to participate in triterpene biosynthesis were used for the
phylogenetic analysis. SDR-like proteins from legumes L. japonicus, M. sativa, Glycine max,
from A. thaliana, A. lyrata, Theobroma cacao as well as the green algae Micromonas pusilla
are shown in (c). The open and black stars, open circles, open and black triangles indicate the
cytochrome P450s clustered together with the BARS1, AMY2, THAS1, AsBAS1 and MRN1,
respectively. The black box indicates the legume specific cytochrome P450 subfamily.
Fig. S2 Gene transcript levels of AMY2, LjCYP88D5 and LjCYP71D353, constituting the
AMY2 gene cluster, expressed relative to the level of each gene expression in seven-days-old
root tissues in both uninoculated (a) and inoculated with M. loti (b) L. japonicus roots, leaves
and nodules. Uninoculated and inoculated plants are of the same age at the stages of 7d old
(days old)-7dpi (days post infection), 14d old-14 dpi and 28d old-28 dpi, respectively, but are
analysed in different real-time PCR reactions. Total RNA was reverse transcribed, the
concentration was normalized between samples and then real-time PCR was performed.
Relative gene expression was measured with respect to UBQ transcripts. Mean values ± SE
are shown (n=3).
Fig. S3 In situ hybridization of AMY2 (a, b, c, d), LjCYP71D353 (e, f, g, h) and LjCYP88D5
(i, j, k, l) gene transcripts in mature 28dpi and developing 14dpi L. japonicus nodules. Eight
μm thin sections were hybridized with DIG-11rUTR labeled anti-sense RNA in vitro
transcribed from of PCR products of AMY2, LjCYP71D and LjCYP88D5. Hybridization
signal was visualized using an alkaline phosphate reaction product (blue-purple colour). For
all genes in mature and developing nodules the hybridization signal was detected in the inner
cortex (ic) and in vascular bundles (vb) (visible in panel K) and also in uninfected cells (not
shown). The hybridization signal was not detected in the infected cells of central tissue (ct) or
outer cortex parenchyma (oc). As a negative control, thin sections were hybridized with DIG11rUTR labeled sense RNA in vitro transcribed from PCR products of AMY2, LjCUP71D and
LjCYP88D5. Scale bar 100μm.
Fig. S4 Gene transcript levels of LjSDRt, present in the AMY2 gene cluster, were detected in
both uninoculated (a) and inoculated with M. loti (b) L. japonicus roots, leaves and nodules.
Uninoculated and inoculated plants are of the same age at the stages of 7d old (days old)-7dpi
(days post infection), 14d old-14 dpi and 28d old-28 dpi, respectively. Total RNA was reverse
transcribed, the concentration was normalized between samples and then real-time PCR was
performed. Relative gene expression was measured with respect to UBQ transcripts. Mean
values ± SD are shown (n=3). Gene transcript levels expressed relative to the level of gene
expression in seven-days-old root tissues in shown in uninoculated (c) and inoculated with M.
loti (d) L. japonicus roots, leaves and nodules.
Fig. S5 Gene transcript levels of AMY2, LjCYP88D5 and LjCYP71D353 in root tissues treated
with salt stress for 7 d. Total RNA from 14-d-old roots (20–50 seedlings per treatment) was
reverse transcribed, the concentration was normalized between samples and then real-time
PCR was performed. Relative gene expression was measured with respect to UBQ transcripts.
Data from a single representative experiment are presented, experimental repeats yielded
similar results.
Fig. S6 Main fragments in the mass spectrometry fragmentation patterns of the TMSderivatives of dihydrolupeol (a), 20-hydroxy-lupeol (b) and 20-hydroxy-betulinic acid (c).
Fig. S7 GC-MS analysis of saponified Nicotiana benthamiana leaf extracts after transient
expression of AsbAS1, LjCYP71D353 and/or LjCYP88D5. Arrows indicate the β-amyrin
peak.
Fig. S8 Syntenic analysis of multiple genomic regions encompassing OSC genes that
participate in metabolic gene clusters in L. japonicus (genes described in this study) and A.
thaliana (thalianol and marneral gene clusters), using the Gevo algorithm. Cytochrome P450
genes are indicated with red squares and OSC genes with yellow exons.
Table S1 Primers used for experimental procedures (restriction sites are in bold)
Procedure
Target
Primer
Sequence
In situ hybridization
AMY2
AMY2isF
AMY2isR
LjCYP88D5isF
LjCYP88D5R
LjCYP71DisF
LjCYP71DisR
AMY2-2F
5’-GGACTCGAGTCTAGACAACAGGATATTACTGGAGTATACG -3’
5’-TTAGGTACCAAGCTTGAACTTGTTTGATTATTTTATTTGCATG -3’
5’-TGGTCGGAAACTGGAGGATGG-3’
5’-AGTCTGCGAGGACACTCTTTAACC-3’
5’-AACGCTGGCTACTTGTGATTAG-3’
5’-CCTCAACATACCCTCTGCTACC-3’
5’-GGACTCGAG TCTAGACAACAGGATATTACTGGAGTATACG-3’
AMY2-2R
AMY2-3F
AMY2-3R
LjCYP88D5-1F
LjCYP88D5-1R
LjCYP88D5-3F
LjCYP88D5-3R
UBQF
UBQR
35S-F
5’-TTAGGTACC AAGCTTGAACTTGTTTGATTATTTTATTTGCATG-3’
5’-GGACTCGAGTCTAGAGTACAGAAATAATTTTTTTACAACGATGG-3’
5’-AAGGTACCAAGCTTGCAACAAACCGACACTAAATAC-3’
5’-AAGACTCGAGTCTAGATGGTCGGAAACTGGAGGATGG-3’
5’-TTAGGTACCAAGCTTAAGTCTGCGAGGACACTCTTACC-3’
5’-CCGACTCGAGTCTAGAACCCACACATCTTGAATAAAGC-3’
5’-AAAGGTACCAAGCTTTATTGGCACACCGCAACG-3’
5’-ATGCAGATCTTTTGTGAAGAC-3’
5’-ACCACCACGGAAGACGGAG-3’
5’-TGTGATAACATGGTGGAGCA-3’
35S-R
Hyg-F
Hyg-R
UBQrtF
UBQrtR
AMY2rtF
5’-GGTGATTTCAGCGTGTCCTC-3’
5’-GACCAATGCGGAGCATATACG-3’
5’-CAGCTTCGATGTAGGAGGGC-3’
5’-TTCACCTTGTGCTCCGTCTTC-3’
5’-AACAACAGCACACACAGCCAATCC-3’
5’-GCAGTTTAACTTGTAAAGATAGC-3’
AMY2rtR
LjCYP88D5rtF
LjCYP88D5rtR
LjCYP71DrtF
LjCYP71DrtR
LjCYP88D5Fl-F
5’-GGCAACAAACCGACACTAAATAC-3’
5’- TAGTGTTCTGGAAGTCAATGATG-3’
5’- AGATGTGTGGGTGTTGTGTAAG-3’
5’- ACATTAAAGCCGTTCTTCAGGAC-3’
5’- CCTCAACATACCCTCTGCTACC-3’
5’-ATGGAACTATACTGGGCTTGG-3’
LjCYP88D5Fl-R
LjCYP71DFl-F
LjCYP71DFl-R
LjAMY2FlBstBi
5’-TAATTACATGAAACCTTTATCACC-3’
5’-TGCCCTTTTGCTAATGATGG-3’
5’-TTATTCAACAGAAACAGGATTGTAAG-3’
5’-AATATTCGAACATGTGGAAGCTGAAGGTAG-3’
LjAMY2Fl-StuI
LjCYP88D5FlBsu36i
LjCYP88D5FlStuI
LjCYP71DFlBsu36i
LjCYP71DFlStuI
LjCYP71D-Bs-F
LjCYP71D-Bs-R
LjSDRt-Bs-F
LjSDRt-Bs-R
5’-TTATAGGCCTTGCACACAGCTATCTTTACAAG-3’
5’-TACACCTGAGGAATGGAACTATACTGGGCTTGG-3’
LjCYP88D5
LjCYP71D
Plant transformation
AMY2
AMY2
LjCYP88D5
LjCYP88D5
RT-PCR
Ubiquitin
35S
promoter
Hygromycin
Q-PCR
Ubiquitin
AMY2
LjCYP88D5
LjCYP71D
Cloning of full-length
genes
LjCYP88D5
LjCYP71D
Expression of
heterologous proteins in
N. benthamiana
AMY2
LjCYP88D5
LjCYP71D
Bisulfite sequencing
LjCYP71D
LjSDRt
5’-TTAGAGGCCTTTAATTACATGAAACCTTTATCACC-3’
5’-TACACCTGAGGTGCCCTTTTGCTAATGATGG-3’
5’-TTATAGGCCTTTTTATTCAACAGAAACAGGATTGTAAG-3’
5’-TAATCACTGCTCTCCCTCCC-3’
5’-CAACACCTCTTTGGCAATTTC-3’
5’- CCCGAAAACTACTTTTTGCA-3’
5’-CGATTAGTATTCGCTTAAACC-3’