Plant Molecular Biology
Supplementary material
Tomato FRUITFULL homologues act in fruit ripening via forming MADS-box
transcription factor complexes with RIN
Yoko Shima,Mamiko Kitagawa, Masaki Fujisawa, Toshitugu Nakano, Hiroki Kato,
JunjiKimbara, Takafumi Kasumi and Yasuhiro Ito*
*Corresponding author:National Food Research Institute, NARO, 2-1-12 Kannondai,
Tsukuba, Ibaraki 305-8642, Japan
Email: yasuito@affrc.go.jp
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Sup. Table 1Primers used for this study
Gene
Primer name
Primer sequence (5’ to 3’)
LeFUL1F
AAACCATGGGAAGAGGAAGAG
LeFUL1R
AATGAGCTCTTTTAATTATTAAGATGACG
LeFUL2F
TTACCATGGGTAGAGGAAGAG
LeFUL2R
AATGAGCTCTTTAACCGTTGAGATGGCG
LeFUL1R3-SacI
CGAGCTCAGTCCTTTTTTTGAAGC
FUL1-F3NcoI
GACCATGGAGCACCAGCTTGATTC
Analysis
FUL1
FUL2
FUL1/
FUL2
of
theFUL1proteinexpressing vector
Construction
of
theFUL2proteinexpressing vector
Construction
of
theFUL1
RevpEU3aEcoRI
GTGAATTCTATACAAAACCAC
CAC-F
CCTCCGTTGTGATGTAACTGG
CAC-R
ATTGGTGGAAAGTAACATCATCG
Constructionof
the
FUL1-F
CAACAACTGGACTCTCCTCACCTT
FUL1-R
TCCTTCCACTTCCCCATTATCTATT
FUL2-F
CACACCCCTTTAACAATCTTCACA
FUL2-R
GCGATGATCCTTCTACTTCTCCAT
RIN-F
ATGGCATTGTGGTGAGCAAAG
RIN-R
GTTGATGGTGCTGCATTTTCG
F
ATCATACGATCACAAACGAGCTATTC
R
ATTCACACCACCTTTAAACCCTTAC
F
AGAATAGCGTAGAATTCATATAAGTAAAAG
R
AACTTTCGGGGGAAAACAAA
F
CTCCGCATACGACTAGCGACT
R
CACTTTTTGGGGGTAATGGATG
F
ACGAGGAGTAAGGTTTGCAACGAC
qRT-PCR
FUL2
qRT-PCR
RIN
qRT-PCR
ACS21)
ChIP-PCR
ACS41)
ChIP-PCR
PSY1-a1)
ChIP-PCR
2
FUL1
expression
vectorfor Y2H experiment
Reference forqRT-PCR
FUL1
or
FUL2proteinexpressing vector
orFUL2protein
CAC
PSY1-b2)
Construction
ChIP-PCR
R
TGACTTGTCCAATAATTTAGGGCG
F
ATTTTAAACTTATTCTATAACTGGATTTCA
R
ATTCCAACTCTAATTTGTTATAGTCAAGA
F
CAATAGGCTTCGAAACTATGTAGAGG
R
AAAAATATTAAAAGGAAATGCTTCACT
F
CCATCAATGATCGGAATGGAAG
R
TCAGCAATACCAGGGAACATTG
F
CCTAGTATTGTGGGACGTCC
R
TAGATCCTCCGATCCAGACA
F
TAGACATGAACAGCCTTCTC
R
GGAGATCAATCCTAGCCTTG
RIN1)
ChIP-PCR
FUL11)
ChIP-PCR
Actin1)
ChIP-PCR
Actin3)
Control for RT-PCR
rin3)
RT-PCR
1)
The primers forripening-induced genes were used in a previous study(Ito et al. 2008;
Fujisawa et al. 2011; Fujisawa et al. 2012).
2)
The primersforPSY1-bwere used in a previous study (Martel et al. 2011).
3)
The primers were used for RT-PCR in Supp. Fig. 3.
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Supplemental Figure 1Homodimerization of the RIN protein in yeast cells.
a.Schematic of the full-length RIN and two types of truncatedproteins expressed in
yeast cells.b.Identification of a domain required for the homodimerization of RIN by
yeast two-hybrid assay. As shown in the left panel, either RIN-MIK(bMIK) or
RIN-MIKC215 (bMIKC215)was expressed in yeast cells as a bait protein, and
eitherRIN-MIK (pMIK) orRIN-MIKC (pMIKC) wasexpressed as a prey protein.Three
independentcolonies of yeast expressing each combination of the bait and
preyproteinswere spread on SD/-Leu/-Trp medium (control medium)
orSD/-Leu/-Trp/-His/-Ade medium (selective medium).We did not examine the
RIN-MIKC protein as a bait because RIN-MIKC has a transcription activating activity
and the yeast cells expressing RIN-MIKC as a bait protein grew on the selective
medium without interaction with a prey protein (Ito et al. 2008). The yeast cells grew
only when expressing bMIKC215 and pMIKC, demonstrating that the C-domain is
indispensable to form the homodimer.
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Supplemental Figure 2 Two hypotheses for complex formation of FUL1 with RIN.
The figure shows a schematic of a gel retardation assay with FUL1, RIN and RIN-MIK
(tRIN). The gel retardation assay with RIN and tRIN gives three signals, RIN/RIN,
RIN/tRIN, and tRIN/tRIN (center, Fig, 4a lane 6). Two possible hypotheses for
complex formation between FUL1 and RIN are predicted: In hypothesis I, FUL1
would bind to the RIN homodimer; thus in the experiment in the left panel, three types
of RIN dimersexhibited in the center panel would be super-shifted by FUL1. A
super-shifted signal composed of FUL1, RIN and tRIN is indicated by a red arrow. The
super-shifted signalwas, however,never found in the actual experiment (Fig. 4a, lanes3
and 7). In hypothesis II,FUL1 would form a heterodimer with RIN or tRIN. The two
DNA binding signals generated by each heterodimer would correspond to the
experiments with RIN and FUL1 (Fig. 4a, lanes1 and 5) or tRIN and FUL1 (Fig. 4a,
lanes4 and 8), but no novel super-shifted signal would be found.
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Supplemental Figure 3Fruitsof rinmutants used for the ChIP analyses in this study.
a.The rinmutant fruits used in this study. The fruits were harvested at ages identical to
the green and pink coloring stages of wild type fruits. The latter stage rinfruits
exhibited a yellow color; thus we referred to the fruits as yellow stage fruits in the text.
b. Expression of the rinmutant gene in the rin mutant fruits used for the ChIP assay in
this study. A fraction of the fruits used for the ChIP assay was used for the expression
analysis. The mRNA was extracted and analyzed by RT-PCR with a primer pair
specific to the rinmutant allele (Supp. Table 1). Green fruits did not express the mutant
gene but yellow fruits did. The Actin gene was used as an internal control.
.
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Supplemental Figure 4Gene structures of FUL1 and FUL2. Both FUL1 and FUL2
consist of eight exons and seven introns, and the lengths of the exons1 to 6 are
conserved between the two paralogs.
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Supplemental Text
Construction of the plasmids used in this study
Primers for gene amplification are listed in Supp. Table1. Theplasmids expressing
FUL1 or FUL2 for in vitro protein synthesis system were constructed as follows. The
coding regions of FUL1 and FUL2 were amplified with oligonucleotide primers: for
FUL1, LeFUL1F and LeFUL1R; for FUL2, LeFUL2F and LeFUL2R. The resulting
fragments were digested with NcoI and SacI and then were inserted into the same
restriction sites of pEU-3a vector (Ito et al. 2002). The resultingplasmids were
designated as pEU-FUL1 and pEU-FUL2, respectively. The DNA fragment encoding
the C-domain truncated FUL1 was amplified from pEU-FUL1 with oligonucleotide
primers LeFUL1F and LeFUL1R3-SacI. The amplified fragment was digested with
NcoI and SacI and then was inserted into pEU-3a, resulting in pEU-FUL1-MIK. The
DNA fragments encoding the C-domain truncated FUL2 were amplified from
pEU-FUL2 with oligonucleotide primers LeFUL2F and LeFUL1R3-SacI, which is the
same primer used for FUL1 because the priming site shows only a single-base
substitution between FUL1 and FUL2 without amino-acid substitution for the
encoding proteins. The amplified fragment was inserted into pEU3a in the same
manner as the construction of pEU-FUL1-MIK, resulting in pEU-FUL2-MIK. The tKC
region of FUL1 and FUL2 within pGBK-FUL1-tKC and pGBK-FUL2-tKC (described
below) were excised by digestion with NcoI and SacI and cloned into pEU3a, resulting
in pEU-FUL1-tKC and pEU-FUL2-tKC.The plasmid vector expressing whole RIN
protein (pEU-RIN) and the C-terminal domain truncated RIN protein (pEU-MIK) was
prepared previously (Ito et al. 2008).
To construct bait vectors for yeast two hybrid experiments, pGBKT7 (Takara)
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was used. The DNA fragments including the coding region of FUL1 or FUL2 were
excised from pEU-FUL1 or pEU-FUL2 by digestion with NcoI and EcoRI. The
excised fragments were inserted into pGBKT7 at the same restriction sites, and the
resulting plasmids were designated pGBK-FUL1 and pGBK-FUL2. Similarly,
pEU-FUL1-MIK and pEU-FUL2-MIK were digested with NcoI and EcoRI and the
C-domain truncated FUL1 and FUL2 (FUL1-MIK and FUL2-MIK) were cloned into
pGBKT7, resulting in pGBK-FUL1-MIK and pGBK-FUL2-MIK. To clone a partial
FUL1 fragment composed of the truncated K-domain and whole C-domain
(FUL1-tKC), the DNA fragment was amplified from pEU-FUL1 with the
oligonucleotide primers FUL1-F3NcoI and RevpEU3aEcoRI. The amplified fragment
was digested with NcoI and EcoRI and inserted into pGBKT7, resulting in
pGBK-FUL1-tKC. Because only a few nucleotide substitutions and no predicted
amino acid substitutionswerefound between FUL1 and FUL2 at the site of the primer
FUL1-F3NcoI, the FUL2-tKC fragment was amplified with FUL1-F3NcoI and
RevpEU3aEcoRI. The amplified fragment was inserted into pGBKT7 after digestion
with NcoI and EcoRI, resulting in pGBK-FUL2-tKC. The bait vectors expressing the
full length or part sequence of RIN (pGBK-RIN, pGBK-MIK, pGBK-MIKC215 and
pGBK-KC) were constructed previously (Ito, et al. 2008).
To construct prey vectors, pGADT7 (Takara) was used. The DNA fragments
including full-length or parts of FUL1 or FUL2 were excised from pGBK-FUL1,
pGBK-FUL1-MIK, pGBK-FUL1-tKC, pGBK-FUL2, pGBK-FUL2-MIK and
pGBK-FUL2-tKC by digestion with NcoI and BamHI and cloned into pGADT7 at the
same restriction sites, resulting in pGAD-FUL1, pGAD-FUL1-MIK,
pGAD-FUL1-tKC, pGAD-FUL2, pGAD-FUL2-MIK and pGAD-FUL2-tKC,
respectively. The RIN coding region within pEU-RIN (Ito, et al. 2008) was excised by
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digestion with NcoIand SacI and cloned into pGADT7, resulting in pGAD-RIN. The
DNA fragments encoding the C-domain truncated RIN and the KC domain of RIN
were excised from pGBK-MIK and pGBK-KC by digestion with NcoI and BamHI and
cloned into pGADT7, resulting in pGAD-RIN-MIK and pGAD-RIN-KC, respectively.
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