SUPPORTING INFORMATION
Appendix S1. Supporting experimental procedures
Plasmid construction and Arabidopsis transformation
The promoter region of LBD10 encompassing 1,808 bp from -1,809 to -1 bp relative to the
AUG initiation codon was isolated by PCR from the genomic DNA of Arabidopsis using Pfu
DNA polymerase, and subcloned into pENTRTM/SD/D-TOPO® (Invitrogen, CA, USA) using
the Gateway® BP recombination reaction, to yield pENTRTM/SD/D-TOPO®-ProLBD10. This
construct was then cloned into the pKGWFS7 vector (destination vector, VIB, Belgium) by
the Gateway® LR recombination reaction, yielding ProLBD10:GFP:GUS. To construct
Pro35S:LBD10:SRDX and Pro35S:EGFP:SRDX, the DNA fragment of LBD10 or EGFP was
amplified by PCR with Pfu DNA polymerase using the primers with the SRDX sequence
(NH2-LDLDLELRLGFA-COOH) (Hiratsu et al., 2003) and the Not I (N-terminus) and Asc I
(C-terminus) restriction sites and subcloned into the pENTRTM/SD/D-TOPO® (Invitrogen,
CA, USA) by the Gateway® BP recombination reaction, yielding pENTRTM/SD/D-TOPO®Pro35S:LBD10:SRDX and pENTRTM/SD/D-TOPO®-Pro35S:LBD10:SRDX, respectively. These
constructs were cloned into the pB7WG2 vector (destination vector, VIB, Belgium) by the
Gateway®
LR
recombination
reaction,
yielding
Pro35S:LBD10:SRDX
and
Pro35S:EGFP:SRDX. To construct a ProLBD10:LBD10:SRDX, the DNA fragment of
LBD10:SRDX was subcloned into pENTRTM/SD/D-TOPO®-ProLBD10 using the AscI
restriction site, yielding pENTRTM/SD/D-TOPO®-ProLBD10:LBD10:SRDX. This construct was
then subcloned into the pB7WG2 vector (destination vector, VIB, Belgium). To construct
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Pro35S:LBD10:EGFP and Pro35S:EGFP:LBD27, the DNA fragments of LBD10 and LBD27
were amplified by PCR using Pfu DNA polymerase, and subcloned into the pENTRTM/SD/DTOPO® (Invitrogen, California, USA) by the Gateway® BP recombination reaction, yielding
pENTRTM/SD/D-TOPO®-LBD10, which was then subcloned into the pK7FWG vector
(destination vector, VIB, Belgium) by the Gateway® LR recombination reaction. To
construct Pro35S:LBD10:GR, the DNA fragment of LBD10 was amplified by PCR using Pfu
DNA polymerase, and subcloned into the pBI-deltaGR vector (Lloyd et al., 1994) at the XbaI
(N-terminus)
and
XhoI
(C-terminus)
sites
as
a
translational
fusion,
yielding
Pro35S:LBD10:GR. All these plasmids were then introduced into either Arabidopsis,
Columbia-0
(Col-0)
or
lbd10
by
Agrobacterium-mediated
transformation.
The
Pro35S:LBD10:GR/lbd10 complementation line was generated by crossing Pro35S:LBD10:GR
with lbd10. For determining the subcelluar localization in Arabidopsis protoplasts, the DNA
fragment encoding the full-length LBD10 or LBD27 was amplified by PCR using Pfu DNA
polymerase (Stratagene, La Jolla, CA, USA) and inserted into the Pro35S:EGFP vector (Lee
et al., 2008) at the XhoI (N-terminus) and BamHI (C-terminus) sites as a translational fusion,
yielding Pro35S:EGFP:LBD10 and Pro35S:EGFP:LBD27. The IAA1 coding region was
inserted into the pPZP212:mRFP vector (Go et al., 2014) as the Sac1 (N-terminus) and
Xma1(C-terminus) sites as a translational fusion, yielding pPZP212:IAA1:mRFP. For the
BiFC assays, the DNA fragments of LBD10 and LBD27 were amplified by PCR using Pfu
DNA polymerase, and subcloned into pUC-SPYNE or pUC-SPYCE (Walter et al., 2004)
using the XbaI (N-terminus) and XhoI (C-terminus) restriction sites. All primers used for
plasmid constructs are described in Tables S1-S4.
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REFERENCES
Go, Y.S., Kim, H., Kim, H.J. and Suh, M.C. (2014) Arabidopsis cuticular wax biosynthesis
is negatively regulated by the DEWAX gene encoding an AP2/ERF-type transcription
factor. Plant Cell, 26, 1666-1680.
Lee, D.J., Kim, S., Ha, Y.M. and Kim, J. (2008) Phosphorylation of Arabidopsis response
regulator 7 (ARR7) at the putative phospho-accepting site is required for ARR7 to act
as a negative regulator of cytokinin signaling. Planta, 227, 577-587.
Lloyd, A.M., Schena, M., Walbot, V. and Davis, R.W. (1994) Epidermal cell fate
determination in Arabidopsis: patterns defined by a steroid-inducible regulator.
Science, 266, 436-439.
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