Pgad424 and pgbt9 vectors

cDNAs were PCR-amplified using corresponding primers (Supplementary Table S3) and cloned into a modified pGEX4T1 vector (GE Healthcare) encoding the TEV protease cleavage site after GST and into the vector derived from pACYC and pET28a(+) (Novagen) bearing a p15A replication origin, kanamycin resistance gene, and pET28a(+) MCS. Apis mellifera cDNA was prepared using standard procedures from adult bees obtained from a local apiary. PCR-directed mutagenesis was used to create constructs expressing mutant proteins using mutagenic primers (Supplementary Table S3). For yeast two-hybrid assays, cDNAs were amplified using the corresponding primers (Supplementary Table S3) and fused with the DNA-binding or activation domain of GAL4 in the corresponding pGBT9 and pGAD424 vectors (Clontech). Details of assembling the constructs for expressing proteins in transgenic flies are available upon request.

To generate pSK-CP60 plasmid, CP60 cDNA was amplified with primers 5′-ataGAATTCatggcaatccaactggac-3′ (upstream, containing EcoRI site) and 5′-ataGGATCCctgctccagttcgtcg-3′ (downstream, containing BamHI site) from cDNA library. The PCR product was cleaved with BamHI and EcoRI, and the 1330 bp BamHI–EcoRI fragment was cloned into pBluescript II SK + vector cleaved with the same enzymes.
Plasmids for the yeast two-hybrid assay were prepared using the full-sized and truncated versions of CP60 protein, CP190, Mod(mdg4)-67.2, and Su(Hw) fused with pGAD424 and pGBT9 vectors (Clontech, Mountain View, CA, USA). Details of the cloning procedures are described in the Supplementary Materials.
Plasmid for expression CP60 protein in E. coli was generated by cloning the BamHI (filled in with Klenow fragment)–EcoRI fragment from pSK-CP60 into pET32a cleaved with NotI (filled in with Klenow fragment) and EcoRI. Expression constructs for Su(Hw) and CP190 proteins were described previously [58 (link),59 (link),60 (link),66 (link)]. Plasmid for expression Adf1 protein was kindly provided by Dr. Maxim Erokhin [80 (link)].

The pGAD424 and pGBT9 vectors (Clontech) were used to perform yeast two-hybrid (Y2H) assays. The pGAD424 vector contained a GAL4 activation domain, and the pGBT9 vector contained a GAL4 binding domain. The ORFs of the MdTCP46 and MdMYB1 sequences lacking the autonomously activated fragments were fused to the pGAD424 and pGBT9 vectors, respectively. MdTCP3-pGAD, MdTCP12-pGAD, MdTCP21-pGAD, MdTCP46-pGAD, MdMYB1^△-pGBD, MdBT2-pGBD, MdBT2-N-pGBD, and MdBT2-C-pGBD were constructed as described above. The genetic transformation of Y2H Gold yeast cells (Clontech) was accomplished by PEG induction. Transformed yeast cells were cultured on a selective medium for 3 d, and Y2H assays were performed as previously described (An et al., 2019b (link), 2020 (link)). The pGAD424 and pGBT9 empty vectors were used as negative controls.

To generate pSK-CP60 plasmid, CP60 cDNA was amplified with primers 5′-ataGAATTCatggcaatccaactggac-3′ (upstream, containing EcoRI site) and 5′-ataGGATCCctgctccagttcgtcg-3′ (downstream, containing BamHI site) from cDNA library. The PCR product was cleaved with BamHI and EcoRI, and the 1330 bp BamHI–EcoRI fragment was cloned into pBluescript II SK + vector cleaved with the same enzymes.
Plasmids for the yeast two-hybrid assay were prepared using the full-sized and truncated versions of CP60 protein, CP190, Mod(mdg4)-67.2, and Su(Hw) fused with pGAD424 and pGBT9 vectors (Clontech, Mountain View, CA, USA). Details of the cloning procedures are described in the Supplementary Materials.
Plasmid for expression CP60 protein in E. coli was generated by cloning the BamHI (filled in with Klenow fragment)–EcoRI fragment from pSK-CP60 into pET32a cleaved with NotI (filled in with Klenow fragment) and EcoRI. Expression constructs for Su(Hw) and CP190 proteins were described previously [58 (link),59 (link),60 (link),66 (link)]. Plasmid for expression Adf1 protein was kindly provided by Dr. Maxim Erokhin [80 (link)].