Matchmaker library construction and screening kit

The Y2H experiments were carried out using Matchmaker library construction and screening kits following the manufacturer’s instructions (Clontech, Palo Alto, CA). First, the cDNA for HSFA9 and HSFA2 was inserted in frame into the GAL4 DNA-binding domain in vector pGBKT7 (BD) and the GAL4 activation domain in vector pGADT7 (AD), respectively. The resulting plasmids were co-transformed into Saccharomyces cerevisiae AH109 cells. Transformed yeasts containing both BD and AD vectors were selected on synthetic-defined (SD) medium lacking Trp and Leu. Protein interactions were assayed by growing the transformed yeasts on SC medium lacking adenine, His, Trp and Leu.

Following the manufacturer’s instructions, we used Match-maker Library Construction and Screening Kits (Clontech) to construct a rice cDNA library from leaves infected with Guy11 for use in this investigation.In 1:1 yeast two hybrid system, for the construction of the baited vector BD-OsMED16, BD-OsMED19 and BD-OsWRKY45, full-length CDS of OsMED16, OsMED19 and OsWRKY45 were amplified with corresponding primer pairs primers and cloned into the vector pGBKT7, and subsequently transformed into yeast strain Y2H. The full-length CDS of OsMED16, OsWRKY62-1 and OsWRKY62-2 were amplified with corresponding primer pairs and cloned them into pGADT7 and subsequently transformed into yeast strain Y187. The positive plasmid-transformed Y187 and Y2H strains were selected on SD-Leu-Trp, SD-Leu-Trp-His and SD-Leu-Trp-His-Ade media to confirm the interaction, according to the experimental objectives (Hu et al. 2018 (link)).

For bait construction, the full-length mouse Mib1 gene was amplified by PCR and cloned into the pGBKT7 vector containing the GAL4 DNA-binding domain. Bait construct did not show autonomous transcriptional activation and toxic effect for growth following transformation into the yeast strain, Y2H Gold. For library construction, the mouse myofiber cDNA library was constructed in a pGADT7-Rec vector containing a GAL4 activation domain using Matchmaker Library Construction and Screening Kits (Clontech, Kusatsu, Japan) and then transformed into the Y187 yeast strain. Yeast two-hybrid screening was performed using the Matchmaker Two-Hybrid system instruction (Clontech) according to the manufacturer’s instructions. Positive clones were selected on synthetic dropout medium/−Leu/−Trp/−His/−Ade/+AbA/+X-α-Gal (QDO/+X/+A) and cDNA inserts were identified by PCR amplification, sequencing, and BLAST alignment. The same positive and negative controls were included. The positive clones among primary transformants were PCR amplified, sequenced for clones over 700 bp PCR products, and aligned using the NCBI BLAST alignment search tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Wild-type Columbia (Col-0) plants were grown at 20 °C under short days (9h light/ 15h dark) for 30 d, and then transferred to long days (16h light/ 8h dark) for 14 d. Primary inflorescence tips with buds no older than stage 5 (Smyth et al., 1990 (link)) were dissected from surrounding leaves and the basal stem using a 26-gauge needle and snap frozen in liquid N₂. Total RNA was extracted using an RNeasy Plant Minikit (Qiagen) and treated with DNase I (Ambion). A cDNA library was generated using a Clontech Matchmaker Library Construction and Screening kit. Activation domain (AD)-tagged cDNA inserts in plasmid pGADT7-Rec were generated (~2.2×10⁷ clones) and amplified in yeast strain AH109.

A P. infestans cDNA library (prey) was used to construct a GAL4 activation domain (AD) fusion library using the Matchmaker library construction and screening kit (Clontech), using the pGADT7 vector and Saccharomyces cerevisiae strain AH109. For the bait, a DNA fragment for the catalytic domain of PKS1 was amplified by PCR and introduced in pGBKT7 as a fusion protein with the GAL4 DNA-binding domain (DNA-BD). After coculturing cells of strain Y187 (bait) and AH109 (prey) for 24 h at 30 °C, the mating mixture was spread on quadruple dropout plates (synthetic defined media/-leucine/-tryptophan/-histidine/-adenine/X-α-gal) for selection. Interactions were retested by co-transforming plasmids for the prey and bait into AH109.

For the yeast two-hybrid screen, a 550–1899 bp sequence of the Etv RDRP gene (GenBank: YP_009115500.1), which encodes the finger, palm and thumb domains, was cloned into pGBKT7 and used as bait. Using a Matchmaker Library Construction and Screening Kit (Clontech, Mountain View, USA), we constructed an E. tenella cDNA library using pGADT7 vector as prey. The yeast strain Y187 was transformed with the bait plasmid pGBKT7-Etv-RDRP, and a α-galactosidase assay was performed to examine autoactivation. The yeast strain Y187 was transformed with the preys, which contained a GAL4 activation domain. For interaction mating, the library and bait strains were co-cultured at 30 °C for 20 h. After mating, the culture was plated on selection media lacking histidine, leucine, tryptophan and adenine and containing 20 μg/ml X-α-gal (SD/-Ade/-His/-Leu/-Trp/X-α-gal). The resultant blue colonies were selected for further analysis. Co-cultured pGBKT7-53 and pGADT7-T plasmids were used as positive control, and co-cultured pGBKT7-Lam and pGADT7-T plasmids were used as negative controls. To reconfirm the hits from the above screen, Pp plasmid cloned into pGBKT7 from the initial screen was co-transformed into Y187 strain with the bait plasmid pGADT7-Etv-RDRP and plated on selection medium.

The Matchmaker Library Construction and Screening kit (Clontech Laboratories Inc., California) was used to construct Arabidopsis ecotype Col-0 cDNA libraries and screen them with bait vectors containing the DEM1 and DEM2 cDNAs. Further details are provided in S1 Text.