X α gal

The yeast two-hybrid system was carried out to study the interaction between CgVDAC2 and CgBak using Clontech Matchmaker Gold Yeast Two-Hybrid System (TaKaRa, Shiga, Japan). pGADT7-CgVDAC2 and pGBKT7-CgBak were transformed into the Y187 and Gold yeast strains, respectively. Y187 cells were cultured onto selective plates with synthetically defined medium (SD) lacking leucine (SD/-Leu), while Gold cells were cultured on SD plates lacking tryptophan (SD/-Trp). After 3 d, positive yeast strains on SD/-Leu and SD/-Trp were hybridized in 2X yeast extract peptone dextrose (YPDA) medium and selected on double drop-out SD/-Leu/-Trp medium. The interaction between CgVDAC2 and CgBak was detected by the hybridized clones growing on quadruple drop-out SD/-Ade/-His/-Leu/-Trp medium supplemented with X-α-Gal and Aureobasidin A (TaKaRa, Shiga, Japan).

The initial yeast two-hybrid screen was performed using an embryonic eye mouse cDNA library (Kasioulis et al., 2014 (link)) with the JMJC domain of KDM3A as bait cloned through partial EcoRI digestion into the DNA-binding domain encoding plasmid pGBKT7. Bait and prey plasmids from blue colonies growing in quadruple selection (Leu/Trp/Ade/His) were rescued by cell lysis and bacterial transformation in ampicillin (pGADT7) or kanamycin (pGBKT7) plates and sequenced. Plasmids encoding CAPZB and ACTA2 were cotransformed back into the yeast two-hybrid Gold strain (Takara Bio Inc.) along with JMJC-pGKBT7, as described in the Matchmaker two-hybrid system protocol. Independent colonies grown in double selection (Leu/Trp) where resuspended, and a 1/100 dilution was replated in 0.2 µg/ml aureobasidin A double selection plates in the presence of X-α-Gal (Takara Bio Inc.).

To detect protein–protein interactions between Pikh-HMA and AVR-Pik effectors by Yeast Two-Hybrid, we used the Matchmaker Gold System (Takara Bio USA). We generated a plasmid encoding Pikh-HMA in pGBKT7 and co-transformed it into chemically competent Y2HGold cells (Takara Bio, USA) with the individual AVR-Pik variants in pGADT7 as described previously [20 (link),24 (link)]. Single colonies grown on selection plates were inoculated in 5 ml of SD^-Leu-Trp and grown overnight at 30°C. Saturated culture was then used to make serial dilutions of OD₆₀₀ 1, 10⁻¹, 10⁻², 10⁻³, respectively. 5 μl of each dilution was then spotted on a SD^-Leu-Trp plate as a growth control, and on a SD^{-Leu-Trp-Ade-His} plate containing X-α-gal (Takara Bio, USA). Plates were imaged after incubation for 60–72 hr at 30°C unless otherwise stated. Each experiment was repeated a minimum of 3 times, with similar results.
To confirm protein expression in yeast, total protein extracts from transformed colonies were produced by incubating the cells at 95°C for 10 minutes in LDS Runblue sample buffer. Samples were centrifuged and the supernatant was subjected to SDS-PAGE gels prior to western blotting. The membranes were probed with anti-GAL4 DNA-BD (Sigma) for the HMA domains in pGBKT7 and anti-GAL4 activation domain (Sigma) antibodies for the AVR-Pik effectors in pGADT7.

Screening of a Y2H library, consisting of Arabidopsis cDNA from various plant tissues and cloned into the appropriate prey vector, using Arabidopsis ERD7 [pGBKT7/ERD7 (see Molecular Cloning and Plasmid Construction section)] as “bait,” was carried out with the Matchmaker Gold Y2H System (Clontech Laboratories, Inc.), as described by the manufacturer and as we have done so in previously-published Y2H screens (Park et al., 2013 (link); Pyc et al., 2017a (link)). All yeast strains that grew on selective media [synthetic dextrose media lacking tryptophan and leucine but containing X-α-Gal and Aureobasidin A (Takara Bio United States Inc.)], were designated as either “strong,” “intermediate,” or “weak” interactors based on the relative color of the colony, which corresponds to the activation of the MEL1 reporter gene. Plasmids were extracted from yeast cells to determine the identity of encoded candidate ERD7-interacting (prey) proteins (listed in Supplementary Table 3) by automated DNA sequencing.

Yeast competent cells were prepared using the Yeast-maker Yeast Transformation System 2 Kit (Clontech, USA), and yeast two-hybrid assays were conducted according to the manufacturer’s instructions (Clontech). The ORFs of the tested genes were cloned by PCR and inserted into the plasmid pGADT7 (Mr-OPY2, Mero-BCK1, Mero-STE11 and Mero-SSK2) or pGBKT7 (Mr-STE50). The ORFs in the plasmids were confirmed by sequencing. The plasmid pGBKT7-Mr-STE50 was transformed into Y2HGold cells, and other plasmids (from pGADT7) were transformed into Y187 cells. After mating, the resulting strains were grown on the medium (SD-His-Ade-Leu-Trp) with X-α-gal and AbA (Aureobasidin A) (Takara). The autoactivation of Mr-STE50 in pGBKT7 was tested using Y2HGold cells. Yeast two-hybrid and autoactivation assays were repeated three times.

Yeast 2-hybrid assays were performed according to the manufacturer’s instructions (Clontech, Japan). The interactions between COH1 and 5 transcription factors were assayed. The coding sequence of each of the 5 transcription factors (MAA_08013, MAA_06777, MAA_07566, MAA_06937, and MAA_07838) was cloned by PCR and inserted into the plasmid pGADT7. The coding sequence of COH1 was also cloned by PCR and inserted into the plasmid pGBKT7 to produce the plasmid pGBKT7-COH1. All pGADT7-based plasmids were transformed into Y187 cells, whereas pGBKT7-COH1 was transformed into Y2HGold cells. The Yeastmaker Yeast Transformation System 2 (Takara Bio, Tokyo, Japan) was used to prepare yeast-competent cells. After mating, the resulting strains were grown on the medium SD/−Ade/−His/−Leu/−Trp with X-α-gal and AbA (Aureobasidin A; Takara Bio). The autoactivation of COH1 was tested by inoculating the strain containing the plasmid pGBKT7-COH1 on the medium SD/−Ade/−His/−Trp with X-α-gal. Yeast 2-hybrid and autoactivation assays were repeated 3 times.