3 amino 1 2 4 triazole 3 at

To test the interaction between AVR-Pii^CCH and OsExo70F3, Yeast-2-hybrid assays were performed as described previously [62 (link)]. Co-transformed yeast cells were prepared in a dilution series with OD₆₀₀ = 3.0 (x1), 0.3 (x10^-1) and 0.03 (x10^-2)] and spotted onto quadruple dropout medium (QDO); basal medium lacking Trp, Leu, Ade and His but containing 5-Bromo-4-Chloro-3- indolyl a-D-galactopyranoside (X-α-gal) (Clontech). To detect interactions, both QDO medium with and without 10 mM 3-amino-1,2,4-triazole (3AT) (Sigma) was used. Yeast cells were also spotted onto double dropout medium (DDO); basal medium lacking Trp, Leu to test cell viability.
To detect protein accumulation in yeast cells, cells were propagated in liquid DDO at 30°C overnight. 40 mg of yeast cells were collected and resuspended with 160 ul GTN + DC buffer [10% glycerol, 25mM Tris-HCl (pH 7.5), 150mM NaCl, 1 mM DTT and 1 tablet of cOmplete EDTA-free (Roche, Basel Switzerland)]. Then, 160 μl of 0.6 N NaOH was added, mixed gently, and incubated at room temperature for 10 min. Next, 160 μl of gel sample buffer [40%(w/v) glycerol, 240 mM Tris-HCl pH 6.8, 8% (w/v) SDS, 0.04% (w/v) bromophenol blue, 400 mM DTT] was added and incubated at 95°C for 5 min. After centrifugation at 20,000 g for 5 min, the supernatant was subjected to SDS-PAGE. Proteins expressed from bait and prey vectors were detected by using anti-Myc-tag mAb-HRP-DirectT (MBL, Nagoya, Japan) and anti-HA-Peroxidase 3F10 (Roche), respectively.
Yeast-2-hybrid assays of the interaction between ZiF effectors and the host target OsExo70F3 were performed as described previously [26 (link),37 (link),43 (link)]. In brief, pGADT7 plasmids encoding the effector domain of different ZiF were co-transformed into chemically competent Y2HGold cells (Takara Bio, USA) with a pGBKT7 plasmid encoding OsExo70F3 in using a Frozen-EZ Yeast Transformation Kit (Zymo research).
After growing in selection plates, single co-transformants were inoculated in liquid SD-Leu-Trp media overnight at 30°C. The saturated culture was used to make serial dilutions of OD₆₀₀ 1, 0.1, 0.01, and 0.001 and 5 μl of each dilution was spotted on a SD-Leu-Trp plate as a growth control, and on a SD-Leu-Trp-Ade-His plate containing X-α-gal and supplemented with 0.2 μg/ml Aureobasidin A (Takara Bio, USA). Plates were imaged after incubation for 60–72 hr at 30°C. Each experiment was repeated a minimum of three times, with similar results.
To assay the accumulation of protein in yeast cells, total yeast extracts were produced by harvesting cells from the liquid media and incubate them for 10 minutes at 95°C after resuspending them in LDS Runblue sample buffer. Samples were then centrifugated and the supernatant was subjected to SDS-PAGE and western blot. The membranes were probed with anti-GAL4 DNA Binding domain (Sigma) antibody for the OsExo70F3 protein in pGBKT7 and with the anti-GAL4 activation domain (Sigma) antibody for AVR-Pii and ZiF effectors in pGADT7.

The full-length open-reading frame of PtD14a, PtD14b, and PtMax2a45 (link) was each cloned into pENTR vector (Life technologies, CA). For the bait construct, the pENTR vector containing PtD14a or PtD14b was transferred into the pDEST32 destination vector by LR clonase-mediated reactions (Life technologies). For the prey construct, the pENTR vector containing PtMAX2a was transferred into the pDEST22 destination vector. One hundred ng of each plasmid of bait and prey construct was added into 100 μl of MaV203 competent yeast cells (Life technologies). For negative control, 100 ng of pDEST22 and pDEST32 empty vector was co-transformed with each other or with the counterpart of pDEST plasmid DNA. Co-transformation was performed by adding 600 μl of 40% PEG/1× LiAc to yeast cell and plasmid mixture followed by incubation at 30 °C for 30 min. After incubation, 35.5 μl of DMSO was added into the cell mixture to improve transformation efficiency. Then, the yeast cells were incubated for 20 min in a 42 °C water bath. Co-transformed yeast cell was centrifuged and the pellet was diluted in 1 ml of 0.9% NaCl. A total of 100 μl of diluted yeast cells was spread on SD plate deficient of Trytophane and Leucine (SD/-Trp/-Leu). Correctly co-transformed yeast cells were cultured in 2 ml of SD/-Trp/-Leu solution overnight at 28 °C. Cultured yeast cells were diluted up to 100 times with 0.9% NaCl. Fifteen μl of diluted yeast cells were dropped on SD plate deficient of Tryptophan, Leucine, and Histidine (SD/-Trp/-Leu/-His) supplemented with 5 mM 3-Amino-1,2,4-triazole (3AT; Sigma-Aldrich, MO) or 5 mM of 3AT plus 5 μM GR24. The plates were incubated for 3 days at 28 °C. Yeast cells grown on SD plate were imaged with Canon power shot SX210 IS digital camera (Canon USA Inc., NY).

Hybrigenics Services SAS (Paris, France) performed the initial yeast two-hybrid screen, using SAP54 (amino acids 34–124; lacking the signal peptide) cloned into pB27 bait plasmid, as a C-terminal fusion to LexA (N-LexA-SAP54-C). Preliminary testing revealed that SAP54 was not toxic to yeast and did not autoactivate the system. Two screens were performed against a random-primed Arabidopsis thaliana seedlings cDNA library constructed into pP6 prey plasmid. A total of 71.7 million clones (7-fold library coverage) were screened following a mating approach with Y187 (matα) and L40 Gal4 (mata) yeast strains as previously described [43] (link). Of the proteins identified in the Hybrigenics two-hybrid screen and listed in Table S1, we independently confirmed the interaction of each protein with SAP54 using yeast strain MaV203 (Invitrogen) with plasmids pDEST32 (GAL4–DNA-binding domain) and pDEST22 (GAL4-activation domain) as follows. MaV203 was transformed according to [44] (link), and transformants were selected and maintained by growth on minimal medium lacking leucine (to select for pDEST32) and tryptophan (to select for pDEST22). To examine protein–protein interactions, freshly transformed yeast colonies were resuspended in 1 mL sterile deionized water, and 10 µL aliquots were spotted upon medium lacking leucine and tryptophan (−L/−W) and medium lacking leucine, tryptophan, histidine, supplemented with 60 mM 3-Amino-1,2,4-triazole (3-AT; Sigma Aldrich) (−L/−W/−H). Growth was scored after 5 to 7 d of incubation at 28°C.
For the comprehensive MTF yeast two-hybrid assay, a matrix-based approach was followed as described previously [45] (link). The originally described GAL4-AD and GAL4-BD MTF collection [11] (link) was extended with a number of known MTF splicing variants [46] (link),[47] , making a total of 106 MTF proteins expressed from the pDEST22 and pDEST32 vector. The above-described pDEST32–SAP54 and a pDEST22–SAP54 construct were used as bait, respectively, in the pair-wise screening. Growth of yeast, and hence interaction events, was scored after 5 d of incubation at 20°C on synthetic dropout (SD) medium lacking leucine, tryptophan, histidine, supplemented with 1 mM 3-AT (−L/−W/−H). All identified positives were rescreened in a second experiment, in which the yeast was spotted onto selective medium lacking leucine, tryptophan, and adenine (−L/−W/−A).
For Western blot analysis, we followed a protocol established by Kushnirov [48] (link). Yeast strains were grown in liquid growth medium lacking leucine and tryptophan at 28°C overnight, and 2.5 OD₆₀₀ of yeast cells were harvested for each experiment.