Pgbkt7 vector

A yeast assay system (Takara Bio) was used to examine the transcriptional activity of CmJAZ1-like. An ORF of CmJAZ1-like lacking a termination codon was amplified using the primer pair CmJAZ1-BD-F/R (Table S2). Then, the amplicon and pGBKT7 vector (Invitrogen) were both digested with EcoRI and BamHI, and the PCR products were ligated using solution I ligase (Takara Bio), generating the construct pGBKT7-CmJAZ1-like. Following the manufacturer’s protocol, the plasmids pCL1 (positive control), pGBKT7 (negative control), and pGBKT7-CmJAZ1-like were transformed into the yeast strain Y2H. Transformants containing pGBKT7-CmJAZ1-like or pGBKT7 were cultured on SD/-Trp medium, whereas those containing the positive control pCL1 were cultured on SD/-Leu medium. SD medium (a minimal, synthetic, defined medium) includes carbon sources, yeast nitrogen sources without amino acids, and dropout supplements, which can be added to the minimal SD base to make a synthetic, defined medium lacking the specified nutrients. After 3 days at 30 °C, we selected single clones and transferred them onto SD/-Ade-His medium containing either 0 or 20 mg/mL X-α-gal. Similarly, after 3 days of growth, we assessed whether there were blue spots on the plates.

The transactivation experiment was conducted according to the manual of Yeast Protocols Handbook (Clontech). The full-length and truncated coding sequences of TaMYBs obtained by PCR amplification were fused in-frame to the pGBKT7 vector (Invitrogen), which contained the coding sequence of the GAL4 DNA-binding domain (BD) and Trp reporter gene. Eco RI restriction sites were introduced into the forward and the reverse gene-specific primers (Additional file 1: Table S1). The constructs, positive control pGBKT7–53 and negative control pGBKT7 were respectively transformed into the Saccharomyces cerevisiae AH109 strain (harboring the HIS3 reporter gene) following the manufacturer’s recommended procedures (Clontech). After incubation at 28 °C for two days, positive clones were identified by PCR and plated onto synthetic defined (SD)/−Trp and SD/−Trp/−His media. The AH109 strain could not grow on the SD medium lacking Trp unless a functional TRP1 gene was introduced, and the strain could not grow on the SD/−His medium without activation of a HIS3 gene. Therefore, the growth of yeast cells on SD/−Trp medium showed that the pGBKT7 constructs were successfully transformed into yeast, and the growth of yeast on SD/−Trp/−His medium demonstrated the ability of the MYB proteins to activate the transcription of a reporter gene.

A cDNA library from three-day-old etiolated seedlings, prepared in the pACT vector [77 (link)], was screened by Y2H using M5GAI and RG52 (the equivalent M5 truncated version of RGA) fused to the Gal4-DNA binding domain (DBD) in the pGBKT7 vector (Invitrogen) as bait. To test truncated versions of ARR1, all constructs were made by recombining entry clones to GATEWAY destination vectors via LR Clonase II (Invitrogen). Primers used for plasmid construction are listed in S5 Table. PCR products were cloned into pCR8/GW/TOPO (Invitrogen), then transferred into pDEST22 (Invitrogen) to create Gal4-AD fusion (ARR1-GAL4DBD versions display strong activation of the HIS3 reporter on their own). GAI deletions have been previously described [24 (link)]. Yeast AH109 cells were cotransformed with specific bait and prey constructs. All yeast transformants were grown on SD/-Trp/-Leu/-His/-Ade medium for selection or interaction tests, in the presence of different concentrations of 3-aminotriazol (3-AT) (Sigma).