Plasmid construction
Constructs for overexpression and RNAi assays: To make the COL13overexpression construct, the predicted full-length COL13 cDNA
was cloned and inserted into the pCAMBIA1390 vector between theSal I and Eco RI restriction sites. To generate theCOL13 -RNAi transgenic plants, two fragments of the COL13coding sequence were amplified by PCR using primers containingPst I (5’ end) and Mlu I (3’ end) restriction sites, andHin dIII (5’ end) and Bam HI (3’ end) restriction sites. The
two fragments were inserted into the pRNAi-0 vector in the reverse
orientation.
Constructs for GUS staining assays: To make the pCOL13 -GUS-2000
construct, a region comprising the 2000 bp promoter sequence ofCOL13 was cloned and inserted into the pBI121 vector between theHin dIII and Bam HI sites. To make 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 make 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, the COL3 ,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
make the COL3-pBbidge and COL3-COL13-pBbidge constructs, the COL3and COL13 fragments were subcloned into the
pBbidgeTM vector (Cat. No. 630404, Clontech) as
appropriate.
Constructs for GFP, CFP and YFP assays: To make the COL13 -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 make the COL3-CFP, COP1-CFP, COL13-CFP, COL3-YFP,
COP1-YFP and COL13-YFP constructs, the full-length coding regions ofCOL3 , 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 make the 35S:COL3- HA construct,
the full-length COL3 cDNA was cloned and inserted into the
pCAMBIA1390-HA vector.
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.