Matchmaker gold yeast one hybrid system

Y1H assays were carried out using the Matchmaker Gold Yeast One-Hybrid System (Clontech, Fitchburg, WI, USA). The promoters of MaSGR1, MaGWD1, MaAMY3, MaPL8, MaEXP-A8, and MaXYL23-like were cloned into pAbAi, which was stored in our laboratory. It was then linearized and separately introduced into the Y1H Gold strain. The CDS of MaERF012 was ligated to the pGADT7 vector and subsequently introduced into the yeast strain. It was then transferred into the aforementioned bait reporter yeast strain containing the promoters of MaSGR1, MaGWD1, MaAMY3, MaPL8, MAEXP-A8, and MaXYL23-like. Their interaction was tested according to the method of Song et al. [12 (link)].

The Matchmaker™ Gold Yeast One-Hybrid System (Clontech, Cat. No. 630491) was used in this study for the Y1H assay. The promoters of MaFAD3-1, MaFAD3-4, MaFAD3-5, MaFAD6-2, MaFAD6-3, MaPAL-like, MaPAL-like1, MaC4H-like3, Ma4CL-like1, Ma4CL-like10, MaCHS6-4-like, and MaFLS were inserted into pAbAi as the bait vector. The constructed vectors were linearized and transformed into the Y1H Gold strain. MaABI5-like gene integrated into the pGADT7 vector was transferred into the aforementioned bait-reporter yeast strain [21 (link)]. The interactions between MaABI5-like and the promoters of MaFAD3-1, MaFAD3-4, MaFAD3-5, MaFAD6-2, MaFAD6-3, MaPAL-like, MaPAL-like1, MaC4H-like3, Ma4CL-like1, Ma4CL-like10, MaCHS6-4-like, and MaFLS were analyzed according to Song et al. [5 ].

Y1H assay was carried out using the Matchmaker™ Gold Yeast One-Hybrid System (Clontech). The LlCHS2 promoter was amplified by genome walking nested PCR method described previously for LIMYB3 promoter, and the fragment (−820 to −553) of LlCHS2 promoter containing four MYB binding sites was isolated and cloned into the pAbAi (bait) vector (shown in Figure 7b and Figure S3). Full-length LlMYB3 was inserted into pGADT7 (prey) vector yielding plasmid pGADT7-LlMYB3. The bait plasmids were linearized and transformed into the yeast strain Y1HGold. Positive yeast cells were then transformed with pGADT7-LlMYB3 plasmid. The DNA-protein interaction was determined based on the growth ability of the co-transformants on SD/-Leu medium with Aureobasidin A (AbA) according to the manual.

For ascertaining the interaction between MsMYB and MsGPPS.LSU promoter, a Matchmaker Gold yeast one‐hybrid library screening system (Clontech, USA) was used. As MsGPPS.LSU promoter encompasses two different MYB binding sites, two promoter regions approximately 40–45 bp in size harbouring the two sites, namely site 1 and site 2, were cloned into the MCS of pAbAi vector independently in addition to two mutant versions of these two sites. Furthermore, complete promoter was also cloned in pAbAi vector and used as bait for further confirmation. The MsMYB gene was cloned into the pGADT7 vector to be used as prey in the form of functional fusion protein along with GAL4 transcription activation domain (GAL4 AD). As a positive control, p53 bait and prey provided in the kit was used. Mutated site 1 and mutated site 2 were used as negative control. Yeast transformation and validation of positive interactions were implemented as described in the Matchmaker Gold Yeast one‐hybrid system user manual (www.clontech.com/xxclt_ibcGetAttachment.jsp?cItemId=17599). The primers used are listed in Table S3.

To screen upstream TFs for BnWRKY33 promoter‐binding capability, a yeast one‐hybrid assay was performed using the Matchmaker Gold Yeast One‐Hybrid System (Clontech) in accordance with the manufacturer's instructions. Briefly, the bait plasmid 33box‐pAbAi was constructed by inserting the 33box fragment of the BnWRKY33 promoter in front of the AUR1‐C gene, which is an antibiotic resistance gene in the pAbAi plasmid that confers resistance to AbA. In addition, reporter strains were generated by integrating linearized 33box‐pAbAi or null pAbAi plasmids into the genome of the yeast strain Y1HGold, and the appropriate inhibition concentration of AbA to the bait reporter strains was confirmed according to the manufacturer's instructions. RNA from B. napus (Ning RS‐1) leaves harvested at 24 and 48 h after S. sclerotiorum inoculation was used for reverse transcription, and the obtained cDNA was fused with the GAL4 activation domain of pGADT7‐REC to construct library for yeast one‐hybrid screen assay. The protein–DNA interaction is identified by activation of the AbA resistance gene when a prey protein from the library binds to the bait sequence. The library was screened on SD/‐Leu medium that contained 100 ng/mL AbA. The plasmid pGADT7‐Rec‐BnWRKY15 was rescued from positive yeast colonies and retransformed into bait reporter strains for interaction validation.

Yeast one-hybrid (Y1H) assays were performed using the Matchmaker Gold Yeast One-Hybrid System (Clontech, USA). The 1 kb region of the ScACS2 promoter (upstream from the translational start site) was cloned into the pAbAi vector and transformed into the yeast Y1HGold strain to generate the bait reporter strain. The minimal inhibitory concentration of aureobasidin A (AbA) and auto-activation tests were performed. To produce cDNA libraries, total RNA was extracted from leaf +1 (the highest unfolded leaf with a visible dewlap) and culm of sugarcane plants (cultivar RB855156) using an RNeasy Plant Mini kit (Qiagen, USA). Each cDNA library was prepared with a pool of RNA samples from five different plants according to the manufacturer’s instructions (Clontech, USA). Both libraries were screened, and after three rounds of re-streaking each colony, the positive clones were sequenced and the putative function of the proteins was determined by BLAST searches on the NCBI database.