3 amino 1 2 4 triazole 3 at

The CIDEB–NS5A interaction was determined by Y2H analysis48 (link). In brief, a panel of truncated mutants of CIDEB and NS5A was subcloned by PCR amplification. The mutants were inserted into pGBKT7 and pGADT7 by fusion with the DNA-binding and activation domains, respectively. Small-scale yeast mating was performed. AH109 yeast cells were pre-transformed with truncated NS5A in pGBKT7 and mated with Y187 yeast cells that were pre-transformed with truncated CIDEB in pGADT7. The mated yeast cells were then spread onto small SD agar plates, and positive clones were screened on SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His plates with 2 mM 3-amino-124-triazole (3-AT, Sigma) at 30 °C for 5 to 8 d. AH109 yeast expressing pGBKT7-53 was mated with Y187 yeast expressing pGADT7-SV40T; the resulting product was used as a positive control. Empty pGBKT7 and pGADT7 were used as negative controls.

pGBT9 and pGAD10 plasmids were co-transformed into AH109 cells (Clontech), as described previously^{31 (link)}. All selective media lacked leucine and tryptophan (-L-W) to ensure co-transformation of bait and prey plasmids was maintained. For screening of interactions, media were further deficient in histidine (-H-L-W) but contained or lacked additional reagents for detection of different affinity interactions. Selective media supplemented with 40 μg/mL X-α-gal (Progen), further supplemented with 1 mM 3-amino-1,2,4-triazole (3-AT; Sigma), or, additionally deficient in adenine, were used to probe for weak, moderate and high affinity interactions. Transformed yeast colonies were cultured in the appropriate media, adjusted to A_{600 nm} = 0.2, and two serial 1:10 dilution suspensions prepared designated as 10⁰, 10⁻¹, and 10⁻², respectively. 2-μL aliquots of all three dilutions were spotted onto plates and incubated at 30 °C for 72 h.

Fungal growth inhibition was assessed by measuring culture diameter extending from a 5 mm agar plug of each fungus on PDA (potato dextrose agar) medium supplemented with 1, 5, 10, 20, 50 or 100 mm H₂O₂; 100 μm methyl viologen (Sigma); 5 mm CaCl₂ (Sigma); 50 mm LiCl (Sigma); and 2000 U ml^–1 catalase (Sigma); or 5 mm of the irreversible catalase inhibitor 3-amino-1,2,4-triazole (3-AT; Sigma). As each fungus grew at a different rate, the effects of the oxidative stress-inducing agents on each pathogen were assessed at the following time points: R. collo-cygni (isolate Rcc09B4) at 42 d, Fusarium culmorum (isolate Fu42) at 5 d, Magnaporthe oryzae (isolate Guy11) and Oculimacula yallundae (isolate P149) at 21 d and Botrytis cinerea (isolate B05:WT) at 3 d. A minimum of three independent experiments were performed for all in vitro growth assays, with fungal growth measured on six replicate plates per experiment.

Yeast two-hybridization assays were performed using the Matchmaker^TM GAL4 Two-Hybrid System 3 (Clontech, USA), according to the manufacturer’s manual. The lithium acetate method was used to introduce pGADT7 (encoding the activation domain) and pGBKT7 (encoding the binding domain) plasmids into yeast (Saccharomyces cerevisiae strain AH109). The yeast were grown on the Yeast Minimal Media/Synthetic Defined (SD) media (Clontech, USA) without leucine and tryptophan, then transferred to selection media lacking leucine, tryptophan, and histidine but supplemented with 2 mM 3-amino-1,2,4-triazole (3-AT) (Sigma-Aldrich, USA). For the yeast spot assays, exponentially grown yeast cells were harvested and adjusted to OD₆₀₀ = 0.5 using sterilized water, and then further diluted (1:10 and 1:100). Yeast cells were spotted onto the SD medium without leucine and tryptophan, and the SD medium without leucine, tryptophan, or histidine. Their growth was observed after three days.

YTH screen was performed in Saccharomyces cerevisiae strain L40 (trp1-901, his3D200, leu2–3, ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lac GAL4) using MyoVa-GTD cloned into pBTM116 (LexA DNA-binding domain, DBD) as bait and a human fetal brain cDNA library (Clontech) cloned into pACT2 (Gal4 activation domain, AD) as prey. Yeast cells were transformed with pBTM116_MyoVa-GTD vector and the library as described by Alborghetti and co-workers47 (link). The screen was performed in solid Synthetic Defined Medium without tryptophan, leucine and histidine (SD-WLH) containing 5 mM 3-amino-1,2,4-triazole (3-AT) (Sigma-Aldrich, St. Louis, MO). To identify the preys, the pACT2 plasmids of positive clones were isolated and sequenced. The DNA sequences were then compared with those available in the NCBI data bank using the BLASTX program48 (link). The clone identified as encoding for the full-length RPGRIP1L isoform c (NP_001295263.1) was further selected for in vitro and in cell validation and characterization.

3-Amino-1,2,4-triazole (3AT), copper (II) sulfate pentahydrate (copper), 3,4-Dihydroxy-L-phenylalanine (L-DOPA), Bafilomycin A1, polybrene (hexadimetrine bromide), Hoechst 33258 and protease inhibitor cocktail tablets were from Sigma-Aldrich. Hygromycin B, Lipofectamine 2000 and all other tissue culture reagents were from Life Technologies (Invitrogen).

Tested proteins were fused to the Gal4 DNA-binding domain (BD), and the screening proteins were fused to the Gal4 activation domain (AD). The AD-fused proteins and BD-fused proteins were amplified using the primers listed in Table S6 and then cloned into the pGADT7 and pGBKT7 vectors, respectively. The vectors were subsequently cotransformed into yeast strain AH109. NdeI-SmaI was used to digest the tested protein, and EcoRI was used to digest the candidate screening protein. The freshly transformed yeast colonies were resuspended in 10 μL of sterile deionized water, and 0.5 μL aliquots were spotted onto four selective media: synthetically defined (SD) media lacking leucine and tryptophan (−LW); media lacking leucine, tryptophan and histidine (−LWH); −LWH media supplemented with 7 mM 3-amino-1,2,4-triazole (3-AT, Sigma Aldrich) (−LWH+3AT); and media lacking leucine, tryptophan, adenine, and histidine (−LWAH). The growth conditions of the above yeast colonies were monitored after 3 days of incubation at 28 °C^{47 (link)}.