vector to generate p TAL1-TALE-NLS-GR, followed by exchanging the cassette of TALE-NLS-GR
into binary vector pMDC32 by LR reaction
(Invitrogen) to generate 35S::TAL-GR (fig. S21G).
35S::TAL- AD was constructed by using golden
gate reaction (Addgene) of the chosen 25-bp TALE
sequence into p TAL1 (Addgene) to generate
pTAL1-25bpTALE-NLS- AD, followed by exchanging the cassette of 25bp TALE-NLS- AD into
binary vector pMDC32 by LR reaction (Invitrogen)
to generate 35S::TAL- AD (fig. S21G).
7) 35S core promoter deletion (Dcore promoter) (fig. S21H) was created as follows: Both
pF3H::YFP and pF3H-KNU PRE 153bp::YFP
constructs were mutated by the KAPA PCR mutagenesis kit to delete 10 bp in the 35S core promoter sequence using the PSOLS257-35sdel
F/ PSOLS258-35sdelR primers. Subsequently,
the transgenes were recombined into the destination vector KGW.
All clones from 1) to 7) (fig. S21) were fully
sequenced for confirmation, and all the primers
used for construction are listed in table S4.
pEMF2::EMF2-VENUS and pFIE::FIE-VENUS
(fig. S21A) were introduced into ap1 cal 35S::AP1-GR.
All the GUS constructs from 2) and 3) (fig.
S21, B and C) were introduced into wild-type
Ler plants using the floral dipping method mediated by Agrobacterium. pEMF2::EMF2-VENUS
and pFIE::FIE-VENUS T1 transgenic plants
were selected on an MS solid medium plate with
the antibiotic kanamycin at a concentration of
50 mg/ml. All the GUS reporter transgenic plants
were selected on soil with the herbicide Basta
15 (Bayer; 0.2% of the commercial solution).
Constructs from 4) to 7) (fig. S21, D to H) were
introduced into T87 cultured cells, and transformation was performed as described below.
Arabidopsis Protoplast and Culture Cell Assay
The transient gene expression assay using Arabidopsis mesophyll protoplasts was performed as
previously described (37). The stable Arabidopsis
transgenic culture cells were established using
T87 suspensions cells (38). Briefly, the cells were
maintained in JPL3 medium under continuous
illumination at 22°C with rotary shaking at 120 rpm
as previously described (38). To generate transgenic lines, 2-week-old T87 cells were sieved
through a 500-mm stainless mesh and resuspended in B5 medium supplemented with 1 mM
1-naphthaleneacetic acid and sucrose (30 g/liter).
Cell suspension was cultured under continuous
illumination at 22°C with shaking at 120 rpm for
1 day. Then, 2.5 ml of overnight cultured Agrobacterium transformed with the appropriate vectors was added to the cell suspension and cultured
for a further 2 days. After cocultivation, the cell
suspension was washed twice with 10 ml of
JPL3 medium supplemented with carbenicillin
(200 mg/ml) by centrifugation at 100g for 2 min.
Finally, cells were resuspended in 3 ml of JPL3
medium and spread over a selection JPL3 agar
plate supplemented with carbenicillin (250 mg/ml)
and selection drugs. After 2 weeks of culture,
drug-resistant calli were transferred to fresh selection medium and maintained by subculture fortnightly. Kanamycin (30 mg/ml) and/or hygromycin B
(12 mg/ml) were used for single/cotransformation
on a plate. Kanamycin (25 mg/ml) and/or hygromycin
B (7.5 mg/ml) were used for single/cotransformation
in liquid medium. For reverse transcription PCR and
ChIP assays, we used 1- to 2-week-old transgenic
T87 suspension lines and collected ~0.2 ml of dry
weight pellet by centrifugation at 100g for 2 min.
For expression assay of the reporter lines, confocal
microscopy images were taken with a Leica SP5
(as described below).
Confocal Microscopy Imaging
For the observation of the reporter lines in Arabidopsis inflorescences, the transgenic seeds were
sowed on soil, and inflorescences were plucked
and mounted on slides. The older floral buds were
then carefully removed or spaced out to expose
the SAM and early-stage floral buds. Dissected
inflorescence was incubated with FM4-64 dye
(50 mg/ml) for ~45 min on slides. Plants were
imaged using a Zeiss LSM 510 upright (with
motorized stage) confocal microscope with EC
Plan-Neofluar 40×/1.30 oil differential interference contrast or Plan-Apochromat 20×/0.8 objective lens. GFP was stimulated with an argon
laser at 488 nm at 60 to 70% of its output, with
emission filtered using a 505- to 530-nm band-pass filter. VENUS was stimulated with an argon
laser at 514 nm at 65 to 80% of its output, with
emission filtered using a 530- to 600-nm band-pass filter. FM4-64 dye emission was filtered with
a 585-nm long-pass filter. The z-stack was acquired using a 512-by- 512–pixel frame, and the
three-dimensional projections of the obtained
z-stacks were then made with Zeiss LSM Image Brower version 4 and adjusted with Adobe
For the observation of T87 transgenic lines,
10 ml of cell suspension was mounted on slides
and imaged using a Leica SP5 inverted confocal
microscope with HCX PL APO 40×/1.25 objective lens. YFP was stimulated with an argon laser
at 514 nm at 80% of its output, with emission
filtered using a 520- to 550-nm band-pass filter. Images were acquired using 512 pixels by
512 pixels. Subsequently, images were made with
Leica Application Suite Advanced Fluorescence
v2.6.0 and adjusted with ImageJ v1.44 and Adobe
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