Strains growth conditions
N. crassa wild type strains, Δnca-2 and Δtrm-9 mutants (Table 1) were obtained from Fungal Genetics Stock Center (FGSC), University of Missouri, Kansas City MO 64110 (McCluskey 2003;McCluskey et al. 2010). The other cnb-1RIP mutant strains 599, 600 ,602, and Ptcu-1::crz-1::5xGly::V5::gfp strains were generated in the laboratory of Prof. Katherine A. Borkovich (University of California, Riverside). The Δtrm-9Δnca-2 double mutant was generated in our laboratory (Laxmi and Tamuli 2015) . For vegetative growth on solid medium, the strains were cultured in 1 X Vogel’s minimal medium N (VM; Vogel, 1956, 1964) containing 1.5% D-glucose as carbon source and 2% agar (VGM). The pantothenate auxotrophs were grown on media supplemented with 0.01 mg/ml pantothenic acid and bathocuproinedisulfonic acid (BCS). A potent inhibitor of calcineurin FK506 was supplemented to the media wherever mentioned at a concentration of 1 µg/ml.
RNA isolation from N. crassa strains and expression analysis using quantitative real time PCR (qRT-PCR)
For gene expression studies under the heat shock conditions, ~1 X 106 conidia were inoculated in two 250 ml conical flasks containing 25 ml VG liquid media and incubated at 28 ºC with shaking at180 rpm for 14 h. Then, one of the flasks was transferred to 48 ºC for about 1 h to induce heat shock proteins. In addition, for the transcriptional studies under Ca2+ stress conditions, the conidial suspension of ~1 X 106 conidia were grown in two 250 ml conical flasks containing 25 ml VG liquid media with or without supplementation with 0.2 M CaCl2 and incubated at 30 °C with shaking at 180 rpm for 16 h. The mycelia from these cultures were harvested by filtration and powdered using liquid nitrogen in a mortar and pestle. Then, RNA was isolated from the mycelial powders using Trizol reagent (Life Technologies, USA), and synthesis of cDNA from total RNA was done using VersoTM cDNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the manufacture’s protocol. To perform qRT- PCR, gene specific primers (Table 2), SYBR Select Master Mix (Life Technologies, USA) and 7500 Real Time PCR system (Applied Biosystems, USA) as per the manufacturer protocol. The expression of the above target genes was calculated by 2-ΔΔCT (Livak and Schmittgen 2001). The expression of β-tubulin was considered as the endogenous control.
In silico analysis of the promoter elements
Promoter analysis was performed for the hsp80 gene using an online database Genomatix MatInspector (Quandt et al. 1995; Cartharius et al. 2005).
Protein isolation and purification
For protein isolation of CRZ-1::5xGly::V5::GFP and 5xGly::V5::GFP, the Ptcu-1::crz-1 (559) and pccg-1_GFP (Table 1) strains were used. The strains were cultured in 50 ml of VG liquid medium using required supplements at 30 °C with shaking @ 180 rpm for 16 h. Mycelial mass was harvested using filter and was crushed into fine powder using liquid nitrogen in a mortar and pestle in 2 ml micofuge tubes and it was further suspended in 400 µl native protein extraction buffer [50 mM Tris-HCl (pH-7.5), 1 mM EDTA, 6mM MgCl2, 2.5 mM phenylmethylsulphonyl fluoride (PMSF) and 0.1% fungal protein inhibitor cocktail (FPIC, Sigma Aldrich, USA)]. Crude protein was extracted by centrifugation at 8000 X g for 8 min at 4 °C. Further to pull down the protein, 50 µg of DynabeadsTM Protein A magnetic beads (Life Technologies, USA) conjugated and crosslinked with mouse anti-V5 monoclonal antibody (Life Technologies, USA) was incubated along with the crude protein samples and kept overnight at 4 °C on a rocking platform. Protein was eluted from the beads by addition of 30 µl of elution buffer [50 mM of glycine (pH- 2.8)] followed by addition of 5 µl of 1M Tris-HCl (pH- 7.5) for neutralization of acidic pH. An aliquot of 5 µl of the protein sample was run on an 10% SDS-PAGE gel and stained with the Coomassie Brilliant Blue dye (Himedia, India) to check the purity. Quantification of the protein concentration was done using Bradford method (Bradford reagent, Himedia, India).
Chromatin immunoprecipitation and sequencing
Chromatin immunoprecipitation (ChIP) for heat shock conditions was performed using 15 h old germlings of N. crassa. 1 X 106 condia ml-1 was inoculated in two 250 ml conical flasks containing 50 ml VG liquid media and incubated at 28 ºC for 14 h in shaking condition at 180 rpm and then one of the flasks was subjected to heat shock at 48 ºC with 180 rpm in shaking for 1 h. For Ca2+ stress conditions 1 X 106 conidia were grown in two 250 ml conical flasks consisting of 50 ml VG liquid media with or without supplementation with 0.2 M CaCl2 at 30 °C with shaking @ 180 rpm for 5 h and with this 5 h old germlings ChIP analysis was performed. For both the above conditions, media was supplemented with 50 µM of BCS and 10 µg ml-1 Next, for fixing the cells about 1 % formaldehyde was added and kept incubated at 28 under shaking at 180 rpm for 1 h. For crosslinking about 125 mM glycine was added and kept for shaking under the similar condition for 30 mins. The culture was pelleted by centrifugation at 3000 g for 5 mins at 4. Then, the pellet was washed with 1X PBS at 3000 g for 5 mins in cold condition. Finally, the pellet was resuspended in 1.2 ml ChIP lysis buffer [50 mM of HEPES (pH- 7.5), 1 mM EDTA, 140 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM PMSF and 0.1% fungal protease inhibitor cocktail (FPIC, Sigma Aldrich, USA)] and samples were sheared in a sonicator (Vibra cell soncis, USA) using the parameters- 33 % amplitude, 8s ON pulse, 10s OFF pulse, 120 cycles and 20 mins time. Following that the lysate was centrifuged at 12000 g at 4 for 5 mins. The supernatant containing the DNA was quantified using spectrophotometer (BioSpectrometer kinetic, Eppendorf, Germany). Further immunoprecipitation of CRZ-1::5xGly::V5::GFP bound to hsp80 promoter, the sheared chromatin was incubated with anti GFP antibody (Life Technologies, USA) about 1 µg of antibody was used for 25 µg of DNA. Both the antibody treated and the control (without antibody) were incubated with 50 ul of pre-blocked Protein A magnetic beads (Life Technologies, USA) overnight on a rocking platform in cold room. RIPA buffer [2 mM EDTA, 50mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 1mM PMSF and 0.1% of FPIC (Sigma Aldrich, USA)] was used to wash the beads. Next, the beads were washed once with high salt, then with low salt and finally with LiCl wash buffer. Elution of the bound chromatin was done using elution buffer (100 mM NaHCO3, 1% SDS). De-crosslinking of the chromatin was done overnight at 65 using 5M NaCl in a circulating water bath. Then the decrosslinked chromatin was treated with RNAse A for 1 h at 65 followed by proteinase K for 1 h 45. Finally, the chromatin was purified using PCR purification kit (Qiagen, Germany). The DNA was then quantified in Nanophotometer. PCR primers 3F and 4R, 4F and 3R, 2F and 5R, 5F and 2R and 1F and 1R (Table 2) were used in appropriate pairs and using Phusion High Fidelity DNA Polymerase (New England Biolabs, USA) to perform the PCR and determine the binding position of CRZ-1 to hsp80 promoter. The PCRs were performed using reaction conditions 98 for 2 mins, 25 cycles of 98 for 10 s, 63 for 30 s and 72 for 18 s and then 72 for 10 min. The PCR products were run on 1.2 % agarose gel containing EtBr (0.5 µgml-1) and visualization was done using Gel Doc (Bio-Print ST4, Vilber Lourmat, France).
Electrophoretic mobility shift assay (EMSA)
Electrophoretic mobility shift assay was performed using Molecular Probes EMSA kit (Thermo Scientific, USA) following manufacturer’s protocol. DNA probes were amplified from N. crassa wild type genomic DNA as template with primers- 3F, 4R, 5F, EMSA _hsp80 1R, EMSA_hsp80 1F, 2R, Chip NCA-2 1F, Chip NCA-2 2R, Chip NCA-2 1F, EMSA NCA-2 Rv, EMSA NCA-2 Fw, and Chip NCA-2 2R (Table 2) in appropriate pairs using Phusion High Fidelity DNA Polymerase (New England Biolabs, USA). Further, the PCR products were analysed using 1.2 % agarose gel and then they were purified using QIAquick Gel Extraction kit (Qiagen, Germany). The 30 bp duplex DNA probes were prepared from the complementary primer pairs- Duplex Hsp80_1, Duplex Hsp80_1 comp, Duplex Hsp80_2, Duplex Hsp80_2 comp, Duplex Hsp80_1 Mut, Duplex Hsp80_1 Mut comp, Duplex Hsp80_2 Mut, Duplex Hsp80_2 Mut comp, Duplex NCA-2 _1 and Duplex NCA-2_1C, Duplex NCA-2_1, Duplex NCA-2_2C, Duplex NCA-2_ Mut 1 and Duplex NCA-2_ Mut 1C in appropriate pairs. These primers were initially denatured at 95 °C for 45 min, and then annealed at 55 °C for 50 min using a buffer [10 mM HEPES (pH 7.5), 0.1 mM EDTA, 0.1 M NaCl, and 5 mM MgCl2], in a thermal cycler (Arktik, Thermo scientific, USA). To detect the binding the individual DNA probes (5 µM) were incubated with the purified CRZ1::5xGly::V5::GFP protein (50 µM). To resolve protein-DNA complexes, 20 % non-denaturing polyacrylamide (30:0.8)/1 X TBE gel was used. Electrophoresis was performed at 200 volts for 1 h in 1X TBE running buffer. The gels were visualized in a gel documentation system (Bio-Print ST4, Vilber Lourmat, France).