β galactosidase activity

The primers for transcriptional activation assays were as follows: pGBKT7-VvDREB2A-F, 5′-CCCATGGATGTCGTCCGGAGTCAT-3′, Nco I site underlined, pGBKT7-VvDREB2A-R, 5′-GCGGATCCTTAGAACCCCATATCTGATA-3′, BamH I site underlined. The construct, as well as pGBKT7 (the negative control) and ABD (the positive control), were transformed into yeast strain Y187 on SD/Trp- medium plates. The growth of the transformants on SD/Trp- medium plates was observed after 3 days. A β-galactosidase activity was detected based on the manufacturer’s instructions (Clontech, Palo Alto, CA, USA).

The reporter plasmids were constructed as described by Wang et al.75 (link). The PERE-like-element-containing fragment in TERF1 promoter was amplified with primer 5′-TCTTTAATTATAGATATTTTAAAC-3′ and 5′-CGTTAGTACTTATTTGAATGTATC-3′, and cloned as a trimer into the MCS upstream of HIS3 minimal promoter in pHISi-1 vector and that upstream of the lacZ minimal promoter in pLacZi vector (Clontech, USA). Then, these two plasmids were linearized and co-transformed into yeast stain YM4271 to obtain the reporter yeast. The cDNAs of LeEILs were cloned with following primers: 5′-TCATCCTGTGGAAGATGATGATGT-3′ and 5′-ACAACATGTCAACAGACTTCTGGC-3′ for LeEIL1, 5′-TGGCTGCCAAGATGATGATGTTTG-3′ and 5′-CTTGATGTTCATTTGAGTAATCGC-3′ LeEIL2, 5′-TGGTAAATGGGGATATTTGAAGAT-3′ and 5′-CAGTTTAATACTAGTACTAGTTCA-3′ for LeEIL3, and 5′-GAGTTTGTTCTTGTGAAGATGATG-3′ and 5′-ACGTTTCACCAATATCATGGCTAG-3′ for LeEIL4. Then, the coding sequences of LeEILs were fused into downstream of transcriptional activation domain of the yeast vector pB42AD (Clontech, USA), and introduced into the yeast reporter strain respectively. Transformants that could grow on the selective medium lacking histidine but containing 30 mM 3-AT were subjected to filter-lifted β-galactosidase activity according to the manufacturer’s protocol (Clontech, USA).

For yeast one-hybrid assay, the full-length CDS sequence of NtNAC053 transcription factor was inserted into pGAD424 to construct a pGAD424-NtNAC053 recombinant vector. Furthermore, the pGAD424-NtNAC053 recombinant vector was co-transformed with different LacZ reporter gene constructs containing three tandem reporter sequences into yeast strain YM4271. The β-galactosidase activity was assessed according to the manufacturer’s instruction (Clontech, United States).