Pbridge

The Matchmaker two-hybrid system (Clontech) was used for the yeast (Saccharomyces cerevisiae) two-hybrid screen with yeast strain pJ69-2A. The StBEL constructs were amplified by PCR and cloned into the vector pACT-AD (Supplementary Table S1 available at JXB online), in frame with the GAL4 activation domain. The tobacco Knox cDNA constructs were amplified by PCR and cloned into pBridge (Clontech) in frame with the GAL4-binding domain. Sequencing of selected cDNAs and constructs was performed at the Iowa State University DNA Facility, Ames, IA, USA. Positive interactions were confirmed by co-transforming into pJ69-2A with each purified pAD and pBridge plasmid and plating on –Leu/–Trp (transformation control) and –Leu/–Trp/–His/–Ade (selection) nutrient medium. Knox/StBEL interactions were quantified for lacZ induction using a β-galactosidase assay (Pierce Chemical). The Knox cDNA clones from tobacco (NTH1, -15, -20, and -22) were graciously provided by M. Matsuoka (Nishimura et al., 2000 (link)).

For cloning of the full-length cDNAs of SmILK (Smp_079760), SmPINCH (Smp_020540.2), and SmNck2 (Smp_014850), total RNA was isolated from adult schistosomes using Trizol reagent (Invitrogen). Residual DNA was removed by DNase digestion (RNAeasy kit, Qiagen) following the manufacturer’s instruction. RNA quality was checked by Bioanalyzer microfluidic electrophoresis (Agilent Technologies). Starting RT-PCR the synthesis of cDNA was performed with 1 μg RNA using QuantiTect Reverse Transcription Kit (Qiagen). PCR reactions were performed in a final volume of 25 μl using primer end concentrations of 800 nM, denaturation at 95°C for 30 sec, annealing at 54°–64°C depending on the primer combinations (S1 Table), and elongation at 72°C for up to 2 min, and using FirePol-Taq (Solis biodyne). As vectors for cloning, pACT2 (Clontech), pcDNA3.1 (Invitrogen), or pBridge (Clontech) were used for directional cloning via restriction enzyme sites. Full-length SmILK cDNA was cloned via NotI and XbaI into pcDNA3.1, full-length SmPINCH cDNA via EcoRI/PstI into pBridge, and full-length SmNck2 via BamHI and XbaI into pcDNA3.1 (S1 Table). Primers designed for RT-PCRs to generate these cDNAs contained appropriate restriction sites for cloning. The sequence integrities of all cloned cDNAs were verified by sequencing (LGC Genomics, Berlin).

NF-YB1 CDS was cloned to fuse with GAL4 BD domain, and NF-YC12 was driven by a methionine-responsive promoter Met25 in pBRIDGE (Clontech, Dalian, China). NF-YB1-NF-YC12-pBRIDGE in strain Y2H Gold was mated with an AD domain-fused seed cDNA library in Y187 strain. The mated transformants were first selected on SD/-Leu/-Trp. Positive colonies were then transferred to SD/-Leu/-Trp/-His/-Ade/-Met/+X-a-Gal and SD/-Leu/-Trp/-His/-Ade/+X-a-Gal, respectively. The interaction was confirmed by the visualization of blue colonies on the medium.

The coding sequences of OsNF-YA8, OsNF-YB1,7,9, and OsNF-YC8,9,10,11,12 were cloned into pGBK-T7. We amplified the GAL4 activation domain from a pGAD-T7 empty vector and fused it with GAL4 DNA-binding domain using the ClonExpress® II One Step Cloning Kit (Vazyme). The construct was used as a positive control for transcriptional activation activity. We cloned OsNF-YB1 and OsNF-YC12 respectively into the multiple cloning site 1 (MCS1) and the MCS2 of the vector pBridge (Clontech). All of the constructs were then transformed into yeast strain Y2HGold to investigate the activity of transcriptional activation on SD–T/–H/–A medium. The primers used in the experiment are listed in Supplementary Table S1.

NF‐YB1 CDS was cloned to fuse with GAL4 BD domain, and NF‐YC12 was driven by a methionine responsive promoter Met25 in pBRIDGE (Clontech, Dalian, China). NF‐YB1‐NF‐YC12‐pBRIDGE in strain Y2HGold was mated with an AD domain‐fused seed cDNA library in Y187 strain. The mated transformants were first selected on SD/‐Leu/‐Trp/‐His/‐Ade/‐Met. Positive colonies were then transferred to SD/‐Leu/‐Trp/‐His/‐Ade/‐Met/+X‐α‐Gal and SD/‐Leu/‐Trp/‐His/‐Ade/+X‐α‐Gal respectively. The interaction was confirmed by the visualization of blue colonies on the medium.