Matchmaker gal4 two hybrid system

To confirm the interaction, the Matchmaker GAL4 two-hybrid system (Clontech, Palo Alto, CA) was used. pBD-PeWRKYs and pAD-PeVQs fusion constructs were generated from the full length of PeWRKY83 and PeVQ cDNAs, respectively. The prey and bait plasmids were transformed to yeast strain AH109. The transformed yeast cells were plated on selective SD/-Trp/-Leu, or SD/-Trp/-Leu/-Ade/-His plates, at 30 °C for 3–5 days to determine the protein-protein interaction.
The BiFC assays were carried out as previously described^{68 (link)}. In short, the cDNAs of PeWRKY83 and PeVQ14 were cloned into pSPYN(C) E-35S. The resulting constructs (PeWRKY83-cYFP and PeVQ14-nYFP) were transformed into Agrobacterium strain GV3101 and infiltrated into the 3-week-old leaves of tobacco (Nicotiana tabacum) plants, and analysed after infected 48 h. Fluorescence was observed under a confocal laser scanning microscope (Olympus, http://www.olympus-global.com).

The full-length CDS of SsYABBY2, SsYABBY5, and SsYABBY7 was cloned into the pGADT7 vector at the NdeI site for fusion with the GAL4 activation domain. The full-length CDS of SsMADS4, SsHOX32, and SsGAox6 were cloned into the pGBKT7 vector at the NdeI site for fusion with the GAL4 DNA-binding domain. Approximately 0.1 μg plasmids of bait and prey were co-transformed into the yeast strain AH109 using the Matchmaker™GAL4 Two-Hybrid System according to the manufacturer’s instructions (Clontech, USA). After growth at 28 °C for 3 days, yeast transformants were diluted and transferred to medium supplemented with SD/-Leu-Trp for growth. The yeast transformants on medium supplemented with SD/-Leu-Trp-His-Ade and 3-amino-1,2,4-triazole (3-AT) for protein interaction selection. The primers used to generate the constructs are listed in Table S7.

Yeast two-hybrid screening was carried out using the MATCHMAKER GAL4 Two-Hybrid System (Clontech) with all procedures following manufacturer protocols. Yeast strain AH109 (MATa, trp1-109, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2:GAL1_UAS-GAL1_TATA-HIS3, MEL1, GAL2_UAS-GAL2_TATA-ADE2, URA3:MEL1_UAS-MEL1_TATA-lacZ) was transformed first with BD:ROH1A or BD:ROH1D and then with AD:AtEXO70C2 wild type or its phospho-mimetic (PM) or phospho-dead (PD) variants. Transformed yeast single colonies were diluted in sterile water to obtain OD₆₀₀ = 2 and serial dilutions were made by repeated 30x dilutions. From each dilution, a 12 μl droplet was applied to plates containing -Trp-Leu, -Trp-Leu-His, or -Trp-Leu-His-Ade drop-out growth media and plates were incubated in 30°C for 3 days before scoring.

Matchmaker GAL4 Two-Hybrid System (Clontech) was used and the assays were performed according to the user manual. Briefly, the bait (in pGBKT7) and prey (in pGADT7) vectors were co-transformed into the yeast strain AH109. The protein interactions were determined by yeast growth on SD/-Leu/-Trp/-His/ medium. The empty vectors were used as negative controls.

Yeast two-hybrid screening was performed with the Matchmaker™ GAL4 two-hybrid system (Clontech, Palo Alto, CA, USA). The open reading frame (ORF) encoding C. parvum rhomboid 1 (CpROM1: GenBank FX115596.1) and C. parvum rhomboid 4 (CpROM4: Gene ID 3372282) were amplified by PCR with forward primer (CpROM1: 5′-ATG TCA AAT ATA CAC AG-3′; CpROM4: 5′-ATG TCT GAC AGA AAG ATT-3′) and reverse primer (CpROM1: 5′-TCA AGG ATT CAT AAG T-3′; CpROM4: 5′-TTA TCC ACA TCT TCT AAT CC-3′). The DNA fragments were inserted into the pGBKT7 vector digested with NcoI/SalI as the baits. The ORF encoding T. gondii rhomboid 2 (TgROM2: GenBank AY704176.1) was amplified by PCR (forward primer 5′-ATG GCC AAC ATT CGG AC-3′; reverse primer 5′-TCA GCA GCG AGG GAC CA-3′) from T. gondii cDNA and inserted into pGBKT7 digested with EcoRI/SalI.
Using LiAc-mediated yeast transformation, the bait plasmid pGBKT7-CpROM1, pGBKT7-CpROM4 or pGBKT7-TgROM2 was separately transformed into yeast strain AH109 and spread on the selection plate lacking histidine. Plasmids pCL1 and pGBKT7 were also transformed into AH109 as the positive control and negative control, respectively. The autonomous activity of the bait plasmids were detected by β-galactosidase activity assay (added X-α-gal to the plates at 20 μg/ml).