Plasmid construction
Constructs for overexpression and RNAi assays: To construct theCOL13 overexpression construct, the predicted full-lengthCOL13 cDNA was cloned and inserted into the pCAMBIA1390 vector
between the Sal I and Eco RI restriction sites. To generate
the COL13 -RNAi transgenic plants, two fragments of theCOL13 coding sequence were amplified by PCR using primers
containing Pst I (5′ end) and Mlu I (3′ end) restriction
sites, and Hin dIII (5′ end) and Bam HI (3′ end) restriction
sites. The two fragments were inserted into the pRNAi-0 vector in
reverse orientation.
Constructs for GUS-staining assays: To construct the
pCOL13 -GUS-2000 construct, a region comprising the 2000-bp
promoter sequence of COL13 was cloned and inserted into the
pBI121 vector between the Hin dIII and Bam HI sites. To
construct the pCOL13 -GUS-2812 construct, a region comprising the
2812-bp promoter sequence of COL13 was cloned and inserted into
the “1301 vector” between the Sac I and Sal I sites. To
construct the pCOL3 -GUS construct, a region comprising the 967-bp
promoter sequence of COL3 was cloned and inserted into the pBI101
vector between the Hind III and Xba I sites.
Constructs for yeast assays: To make the COL3 -pGBKT7,COP1 -pGBKT7, COL13 -pGBKT7, COL3 -pGADT7,COP1 -pGADT7, and COL13 -pGADT7 constructs,
theCOL3 , COP1, and COL13 fragments were subcloned into
the pGBKT7 vector (Gal4 DNA binding domain, Cat. No. 630489, Clontech)
and pGADT7 (Gal4 activation domain, Cat. No. 630442, Clontech), as
appropriate. To construct the COL3-pBbidge and COL3-COL13-pBbidge
constructs, the COL3 and COL13 fragments were subcloned
into the pBbidgeTM vector (Cat. No. 630404, Clontech),
as appropriate. To construct the
GAD-HY5
or GAD-COL3 fusion protein in yeast, the HY5 or COL3 coding sequence was
subcloned into pJG4-5 with the EcoRI and XhoI sites. To
construct the COL3-LacZ reporter gene, the 1119-bp COL3 promoter region
was amplified from genomic DNA and cloned into the pLacZi2u vector with
the HindIII and XhoI sites (Lin et al., 2007). Other LacZ reporter gene
plasmids containing various truncated COL13 promoters were similarly
constructed using the primers listed in Supplementary Table S1.
Constructs for GFP, CFP, and YFP assays: To construct theCOL13 -GFP construct, the full-length COL13 coding region
was cloned and inserted into the pBEGFP vector between the Xba I
and Kpn I restriction sites. To construct the COL3-CFP, COP1-CFP,
COL13-CFP, COL3-YFP, COP1-YFP, and COL13-YFP constructs, the full-length
coding regions of COL3 , COP1, and COL13 were cloned
and inserted into the pBluescript II Phagemid vector (Y. Liu et al.,
2016), as appropriate.
Constructs for Co-IP assays: To construct the 35S:COL3- HA
construct, the full-length COL3 cDNA was cloned and inserted into
the pCAMBIA1390-HA vector (Fang et al.,
2019).
Constructs for dual-luciferase assays: Fragments of the COL3 or COL13
promoter were cloned into pGREEN0800-LUC to generate reporter vectors. A
modified pBluescript vector (pBS) was used as an effector (Han et al.,
2017).
The
primers used are listed in Supplementary Table 1.