Yeastmaker yeast transformation system 2 user manual

The full-length coding region of NRL3 without a termination codon was amplified by PCR and cloned into pGBKT7 vector. Two-Hybrid Library Screening Using Yeast Mating was used to find protein interactions with NRL3, applying the protocol described by the manufacturer with little adjustments (Matchmaker^® Gold Yeast Two-Hybrid System User Manual, PT4084-1 (092413), Clontech). After sequencing for identification of the interacting protein, full-length cDNA of VYL without a termination codon (also named NAL9, LOC_Os03g29810) was amplified using infusion primers for construction into PGADT7 vector. Yeast transformation and selection procedures were carried out according to the manufacturer’s instructions [Yeastmaker^TM Yeast Transformation system 2 User Manual, PT1172-1 (PR8Y2629), Clontech].

Yeast two-hybrid (Y2H) assays were performed according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). Each pair of indicated genes were cloned into pGBKT7-DEST for fusing with GAL4 DNA binding domain (BD) or pGADT7-DEST for fusing with GAL4 activation domain (AD). Yeast competent cells (Y2H Gold) was co-transformed with bait and prey constructs, followed by 10-fold serial dilution and plating onto synthetic defined (SD) yeast leucine and tryptophan dropout medium (SD/-Leu/-Trp) or leucine, tryptophan, histidine and adenine dropout medium (SD/-Leu/-Trp/-His/-Ade). The transformants were allowed by 4- to 6-day growth on the dropout mediums at 28°C. For immunoblot detection of protein expression in yeast, the co-transformed yeast cells were propagated in liquid SD/-Leu/-Trp medium, and the cultures were harvested at OD₆₀₀ of 0.5. The total proteins were extracted by using Yeast Protein Extraction Reagent (Takara), followed by immunoblot detection with anti-HA monoclonal or anti-Myc polyclonal antibody (Abcam). Membrane yeast two hybrid (MYTH) assays were exactly performed according to the user manual of DUALmembrane starter kits (Dualsystems Biotech). Yeast competent cells (NMY51) were co-transformed with each pair of the indicated constructs, and plated onto SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade mediums.

Y2H assay was performed according to the YEASTMAKER Yeast Transformation System 2 User Manual (Clontech). The cDNA of OsPID was cloned into pGADT7, and the cDNAs of LAX1, OsMADS16, LOC_Os02g27030, LOC_Os04g31910 and LOC_Os06g12580 were cloned into pGBKT7 with Gibson Assembly, respectively. The primers used here were listed in Table S10. The paired bait and prey plasmids were co‐transformed into yeast stain AH109. Protein interactions were tested under the growth conditions on synthetic dropout medium without Trp, Leu, Ade and His at 28°C.

Yeast two-hybrid interaction assays were performed according to the Yeast Maker Yeast Transformation System 2 User Manual (Clontech). The coding sequences of AtSCC3 and AtBMI1A were subcloned into pGBKT7 or pGADT7, respectively. The constructs were then co-transformed into yeast (AH109). The yeast cells containing the bait and the prey constructs were grown on selective plates (SD-Leu-Trp-His-Ala and SD-Leu-Trp) for analysis. The results were tested after 3–7 days of growth at 30˚C. The primers used are listed in Supplementary Data 8. Values came from three biological replicates.

To test autoactivation and toxicity of bait protein TgROP16 in yeasts, bait plasmids containing the above mentioned three fragments of ROP16 were individually transformed into Saccharomyces cerevisiae strain Y2H Gold according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech Laboratories, Inc., Mountain View, CA, United States). Transformants were then plated onto agar plates containing appropriate selection media, including SD/-Trp, SD/-Trp/X (40 μg/ml X-α-Gal) or SD/-Trp/X/A (40 μg/ml X-α-Gal and 125 ng/ml Aureobasidin A). The plates were incubated at 30°C for 3 days and the growth (color and size) of transformants were recorded. The empty vector pGBKT7 was included as a control.

Yeast transformation was performed according to the Yeastmaker Yeast Transformation System 2 User Manual (Clontech). Growth was determined as described in the yeast two-hybrid system user manual (Clontech).