The CRZ-1 Transcription Factor Regulates Hsp 80 Involves in the Acquisition of Thermotolerance, and NCA-2 for Calcium Stress Tolerance in Neurospora Crassa

Heat shock proteins (Hsps) are molecular chaperones and required for survival of organisms under heat stress conditions. In this study, we studied Hsp80, a member of the Hsp90 family, in Neurospora crassa. The expression of hsp80 was severely reduced in the N. crassa calcineurin B subunit RIP-mutant (cnb-1 RIP ) strains under the heat shock conditions. Furthermore, the expression levels of cnb-1, hsp60, hsp80, and the calcineurin-regulated transcription factor crz-1 were increased, but expression levels were reduced in the presence of the calcineurin inhibitor FK506 under the heat shock stress in the N. crassa wild type. Therefore, the calcineurin-crz-1 signaling pathway transcriptionally regulates hsp60 and hsp80 under the heat shock stress condition in N. crassa. In addition, the transcript levels of trm-9 and nca-2, a Ca 2+ sensor and a Ca 2+ ATPase, respectively, were increased under the heat shock stress condition. Moreover, the expression of the hsp80, but not the hsp60, was reduced in the Δtrm-9, Δnca-2, and the Δtrm-9 Δnca-2 double mutants. These results suggested that hsp80, trm-9, and nca-2 play a role in coping the heat shock stress in N. crassa. We found that CRZ-1 binds to 5 (cid:0) -CCTTCACA-3 (cid:0) and 5 (cid:0) -AGCGGAGC-3 (cid:0) 8 bp nucleotide sequences, located about 1075 bp and 679 bp upstream of the ATG start codon, respectively, of hsp80. We also found that CRZ-1 binds to an 8 bp nucleotide sequence 5 (cid:0) -ACCGCGCC-3 (cid:0) , located 234 bp upstream of the ATG start codon of nca-2 under Ca 2+ stress condition. Thus, cnb-1, hsp60, hsp80, and crz-1 are involved in the heat shock stress response in N. crassa.

Imai and Yahara, 2000). In fungi, calcineurin activates CRZ-1, also known as nuclear factor of activated T cells (NFAT) in mammals, an important transcription factor (Thewes 2014). CRZ-1 is required for the expression of a number of stress response genes such as PMC1, PMR1, PMR2A, and FKS2, necessary for the Ca 2+ , Mn 2+ , Na + , stress responses, respectively, in S. cerevisiae (Matheos et al. 1997). In A. fumigatus, the ΔcrzA mutant showed sensitivity to heat shock stress (Soriani et al. 2008). In the lamentous fungus Neurospora crassa, CRZ-1 upregulates the expression of ncs-1 under the high Ca 2+

Materials And Methods
Strains growth conditions N. crassa wild type strains, Δnca-2 and Δtrm-9 mutants (Table 1)  ). The other cnb-1 RIP mutant strains 599, 600 ,602, and P tcu-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, 1956Vogel, , 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 10 6 conidia were inoculated in two 250 ml conical asks containing 25 ml VG liquid media and incubated at 28 ºC with shaking at180 rpm for 14 h. Then, one of the asks was transferred to 48 ºC for about 1 h to induce heat shock proteins. In addition, for the transcriptional studies under Ca 2+ stress conditions, the conidial suspension of ~1 X 10 6 conidia were grown in two 250 ml conical asks containing 25 ml VG liquid media with or without supplementation with 0.2 M CaCl 2 and incubated at 30 °C with shaking at 180 rpm for 16 h. The mycelia from these cultures were harvested by ltration 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 Verso TM cDNA Synthesis Kit (Thermo Fisher Scienti c, USA) according to the manufacture's protocol. To perform qRT-PCR, gene speci c primers (
Further to pull down the protein, 50 µg of Dynabeads TM 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. Quanti cation 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 10 6 condia ml -1 was inoculated in two 250 ml conical asks 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 asks was subjected to heat shock at 48 ºC with 180 rpm in shaking for 1 h. For Ca 2+ stress conditions 1 X 10 6 conidia were grown in two 250 ml conical asks consisting of 50 ml VG liquid media with or without supplementation with 0.2 M CaCl 2 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 xing 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 quanti ed 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 nally with LiCl wash buffer. Elution of the bound chromatin was done using elution buffer (100 mM NaHCO 3 , 1% SDS). Decrosslinking 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 puri ed using PCR puri cation kit (Qiagen, Germany). The DNA was then quanti ed 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).

Results
Hsp 80 is necessary for acquisition of thermotolerance in N. crassa To test if Hsp80, belonging to the family of Hsp90 hsps, is involved in the heat shock stress tolerance, we analysed the expression of hsp80 and hsp60 in the cnb-1 RIP mutant strains under the heat shock conditions. The expression of hsp80, but not hsp60, was reduced by almost ~0.4, 0.3, and 0.2-fold in the 599, 600 and 602 mutant strains in comparison with the wild type under the heat shock conditions ( Fig.   1B and 1A). Furthermore, the expression levels of cnb-1, hsp60, hsp80, and crz-1 were studied in the wild type strain in the VG liquid media with or without supplementation with FK506 both with and without heat shock conditions. The results indicated that expression levels of cnb-1, hsp60, hsp80, and crz-1 were increased by ~1.7, 2.1, 1.9-fold, but expression levels were reduced by about ~0.8, 0.2, 0.6, and 0.8-fold on supplementation with FK506 under the heat shock stress (Fig. 1C). Therefore, the above results suggested that hsp60 and hsp80 are transcriptionally regulated by the calcineurin-crz-1 signaling pathway under the heat shock stress condition.
Previous studies in our laboratory revealed that double mutants of trm-9 and nca-2 results in decreased cell survival on exposure to heat shock stress condition as compared to the wild type (Laxmi and Tamuli 2015). Therefore, to analyse the involvement of trm-9 and nca-2 genes in the regulation of the heat shock stress conditions, expressions of trm-9 and nca-2 were determined in the wild type strain under het shock conditions. The transcript levels of trm-9 and nca-2 were increased by ~2 and 3-fold (Fig. 2C), suggesting a requirement of trm-9 and nca-2 for the heat shock stress tolerance, and further support the phenotypic results observed previously (Laxmi and Tamuli 2015). Furthermore, to test if the hsp80 was involved in the trm-9 and nca-2 mediated heat shock response, we determined the expressions of both hsp60 and hsp80 in the trm-9 and nca-2 mutant strains under the heat shock conditions. The expression of the hsp80, but not the hsp60, in the Dtrm-9, Dnca-2, and the Dtrm-9 Dnca-2 double mutant was reduced by 0.2, 0.15 and 0.6-fold ( Fig. 2B) compared to the wild type strain . Therefore, Hsp80 is necessary for coping up the heat shock stress in N. crassa.

CRZ-1 binds to the promoter of hsp80 under thermotolerance conditions
To identify the consensus sequences for transcriptional regulation, hsp80 promoter analysis was performed using an online database Genomatix MatInspector (Quandt et al. 1995; Cartharius et al. 2005). This analysis identi ed a putative 8 bp sequence for CRZ-1 binding with consensus 5'-GTGGCTG-3' (Table 3), located at about ~1208 bp upstream of the start codon.
The expression of the crz-1 was increased by ~2.5-fold under heat stress conditions and also the decrease in the fold change of hsp80 in the wild type strain supplemented with FK506 (Fig. 1C). To determine CRZ-1 binding to the hsp80 promoter, chromatin immunoprecipitation (ChIP) assay was performed using a tagged version of crz-1 N. crassa strain ( Table 1) grown in VG liquid media with and without heat shock conditions. To map the CRZ-1 binding region, ve pairs of overlapping primers were designed covering 1263 bp of the hsp80 promoter region. Binding of the CRZ-1 to the hsp80 was found at two regions, a 174 bp fragment ampli ed using primers 3F and 4R, and another fragment of 331 bp ampli ed using 5F and 2R primers (Fig. 4A). The binding intensity of CRZ-1 to the promoter of hsp80 was increased under the heat shock conditions (Fig. 4B). To further unravel the binding site for CRZ-1 in the promoter of hsp80, electrophoretic mobility shift assay (EMSA) was performed. The P tcu-1 ::crz-1::5xGly::V5::gfp (559) strain was grown on VG liquid media to isolate CRZ-1::5xGly::V5::GFP protein and EMSA was performed using four different PCR ampli ed fragments covering the 174 bp and 331 bp candidate regions, identi ed in the ChIP assay, in the promoter of hsp80 (  (Table S1), two 30 bp DNA duplex probes were designed consisting of the rst two predicted nucleotide sequences having the highest support vector machine (SVM) score and lowest P-value. EMSA was performed using the DNA probes and it was observed that the recombinant CRZ-1 proteins bind to two probes containing 5 -CCTTCACA-3 and 5 -AGCGGAGC-3 nucleotide sequences ( Figure 5C). However, no gel shift was observed when the mutated version of 5 -CCTTCACA-3 and 5 -AGCGGAGC-3 nucleotide sequences were used (Fig. 5C), con rming that CRZ-1 speci cally binds to the two different 8 bp nucleotide sequences. Therefore, CRZ-1 binds to 5 -CCTTCACA-3 and 5 -AGCGGAGC-3 8 bp nucleotide sequences located about 1075 bp and 679 bp upstream of the ATG start codon, and upregulates the expression of hsp80 under the heat shock stress condition in N. crassa.
Calcineurin-CRZ-1 signaling pathway regulates Ca 2+ stress tolerance in N. crassa The cnb-1 RIP mutants showed sensitivity to Ca 2+ stress when grown in VG agar media supplemented with higher concentration of CaCl 2 about 0.4 M (Kumar et al. 2019). The previous work in our laboratory showed that single and double mutants of the Ca 2+ sensor ncs-1 and a Ca 2+ ATPase nca-2 were sensitive to Ca 2+ stress (Deka and Tamuli 2013) in N. crassa. Furthermore, for the tolerance to high Ca 2+ , CRZ-1 upregulates NCS-1, but CRZ-1 mediated regulation of NCA-2 was not known (Gohain and Tamuli 2019). To test if CRZ-1 binds to the nca-2 promoter, a pair of primers covering 400 bp of the nca-2 promoter were used for ChIP analysis (Fig. 6A). In the ChIP assay, a distinct band of about 400 bp was observed, and intensity of CRZ-1 binding to the nca-2 promoter was increased on addition of 0.2 M CaCl 2 (Fig. 6B). To further unravel the binding site for CRZ-1 in the promoter of nca-2, EMSA was performed using the P tcu-1 ::crz-1::5xGly::V5::gfp (559) strain grown on VG liquid media to isolate CRZ-1::5xGly::V5::GFP protein, and three different PCR ampli ed fragments covering the 400 bp candidate region in the nca-2 promoter (Fig. 7A). The EMSA results suggested the binding of CRZ-1 fusion protein to a 220 bp region of the 400 bp fragment in the nca-2 promoter (Fig. 7B). Furthermore, to determine the exact nucleotide sequences in the 220 bp region that renders the binding of CRZ-1 to nca-2, putative binding sites were predicted using online prediction tool for DNA binding sites in the Cys 2 His 2 zinc nger proteins (http:://zf.princeton.edu; Persikov et al. 2009; Persikov and Singh 2014). From the predicted results (Table S2) two 30 bp DNA duplex probes were designed consisting of the rst two predicted nucleotide sequences having the highest support vector machine (SVM) score and lowest P-value. EMSA was performed using the DNA probes and it was observed that the CRZ-1 fusion protein binds to the probe containing 5 -ACCGCGCC-3 nucleotide sequence (Fig. 7C). However, no gel shift was observed either for the mutated version of 5 -ACCGCGCC-3 or the DNA duplex having 5 -TGCGCAGC-3 nucleotide sequence (Fig. 7C), located in the 180 bp region (Fig. 7A) where no binding for CRZ-1 has been observed when EMSA was performed for the binding of CRZ-1 to the promoter of nca-2. Therefore, CRZ-1 speci cally binds to an 8 bp nucleotide sequence 5 -ACCGCGCC-3 , located 234 bp upstream of the ATG start codon, and upregulates the expression of nca-2 under the Ca 2+ stress condition in N. crassa.

Discussion
Earlier studies from our laboratory have shown the role of cnb-1 RIP mutants in acquisition to thermotolerance (Kumar et al. 2019). Since, different families of Hsps are there so it will be important to decipher overexpression of which hsp family responsible for the acquisition to thermotolerance in N.
crassa. Expression studies of hsp60 and hsp80 in the wild type strain along with cnb-1 RIP mutants and in the trm-9, nca-2 and their double mutants suggested that hsp80 has a role in the survival of N. crassa under heat shock conditions. Till date, the interaction of hsp90 with cna-1 have been established in C. albicans and this pathway have been targeted for antifungal drug development (Gong et al. 2017). Still, the mechanism behind the regulation of hsp80 is limited. Therefore, to unravel this molecular mechanism, analysis of the hsp80 promoter sequence revealed the presence of a 5'-GTGGCTG-3' 8 bp consensus sequence responsible for CRZ-1 binding. Next, the expression studies depicted the reduced expression of crz-1 under heat shock condition in the wild type strain when the growth medium was supplemented with FK506, a potent inhibitor of calcineurin. Also, the expression of cnb-1 and hsp80 was reduced in the wild type strain in the presence of FK506 under the heat shock stress. The above observations along with the promoter analysis indicates that CRZ-1 might have a role in the regulation of hsp80 under thermotolerance conditions. Henceforth, to con rm the molecular mechanism behind the calcineurin-CRZ-1 mediated activation of hsp80, we have designed primers to map the promoter of hsp80. The ChIP assay using the CRZ-1 tagged strain under the heat shock condition con rmed the binding of CRZ-1 to the promoter of hsp80. CRZ-1 probably, binds to two speci c regions of about 174 bp and 331 bp upstream of hsp80, which are ampli ed by the primers 3F and 4R, and 5F and 2R, respectively. Thus, to further con rm the exact nucleotide sequences responsible for the binding of CRZ-1, EMSA was performed that revealed that 5´-CCTTCACA-3´ and 5 -AGCGGAGC-3 8 bp sequences results in the binding of CRZ-1 to the promoter of under heat shock stress conditions in N. crassa. In general, transcription factors may have multiple binding sites within the promoter of speci c target genes, thereby regulates different cell functions (Harbison et al. 2004). In organisms such as S. cerevisiae, computational studies revealed that a single transcription factor can have more than one binding sites in the promoter of the target gene and further, sometimes the speci c pattern of the consensus is not followed across the different binding sites (Harbison et al. 2004). In S. cerevisiae, the Reb1 transcription factor binds to many genes with a deviation in the consensus sequence, and more the number of binding sites on the same promoter, greater is the deviation (Bilu and Barkai 2005). A similar trend was observed in CRZ-1 binding sites on the promoter of hsp80, and altogether the binding sequences differed from GTGGCTG, the consensus sequence found in the promoter analysis of hsp80. Also, no calcineurin dependent response element (CDRE) sequence − 5 -AGCCTC-3 speci c for N. crassa (Kumar et al. 2006) was observed in the promoter of hsp80. Therefore, it indicates that the binding sites for the same transcription factor may vary across the genes within the same species.
Previous work from our laboratory showed how the calcineurin activated CRZ-1 binds to the promoter of ncs-1 to upregulate its expression in response to the high concentrations of Ca 2+ in the growth medium Cunningham and Fink 1994Fink , 1996Matheos et al. 1997), NCA-2 is transcriptionally regulated via calcineurin signaling pathway. In S. cerevisiae, regulation of PMC1 by CRZ-1 is still not known; however, our study con rmed that CRZ-1 binds to a sequence different from that of the calcineurin-dependent response element (CDRE) sequence 5'-AGCCTC-3' in the nca-2 promoter (Kumar et al. 2006). Therefore, there could be some unique binding sequence for CRZ1 other than CDRE present in the upstream of nca-2 which remains unique to N. crassa. We proposed a model governing the regulation of hsp80 and nca-2 via calcineurin-CRZ-1 pathway (Fig. 8). During stress conditions, the in ux of Ca 2+ ions increases in the cytoplasm via the Ca 2+ -ATPase transporters and Ca 2+ channel proteins such as NCA-2. When the [Ca 2+ ] c concentration rises above the threshold level of about 1 mM, then the Ca 2+ signaling machinery is activated and the Ca 2+ ion binds to one of the Ca 2+ sensor CaM which thereafter activates calcineurin complex. The activated calcineurin dephosphorylates its downstream target CRZ-1, which shuttles into the nucleus from the cytoplasm to bind the promoter of both hsp80 and nca-2. The HSP80 protein is involved in the survival of cell against the thermotolerance condition, while NCA-2 transports the excess Ca 2+ ions into the internal stores, including vacuoles and also exports excess Ca 2+ to extracellular environment to maintain the Ca 2+ homeostasis in the cell. Also, when the growth medium is supplemented with FK506 that inhibits calcineurin, the downstream signaling cascade is disrupted and results in reduced expressions of cnb-1, hsp80 and crz-1 (Fig. 1C). In C. albicans, A. fumigatus, and Cryptococcus neoformans, hsp90 have been reported to be important among different hsps to confer antifungal drug resistance . Therefore, our study shade light on the regulation of hsp80 in N. crassa. Therefore, targeting the calcineurin-CRZ-1 pathway, which activates hsp80 in N. crassa, in different pathogenic fungal model systems might unravel new strategies to combat the increasing drug resistance a big challenge in future.

Declarations
Availability of data and materials (data transparency) All data generated or analysed during this study are included in this published article (and its supplementary information les). Additional datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests/Con ict of interest (data transparency)
The authors have no con icts of interest.
Funding (information that explains whether and by whom the research was supported).
The RT laboratory is supported by a DBT-NER twinning grant BT/PR24473/NER/95/737/2017, from DBT, Govt. of India. However, the funder has no role in this publication.

Authors' contributions
All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Avishek Roy and Ranjan Tamuli. The rst draft of the manuscript was written by Avishek Roy and all authors commented on previous versions of the manuscript. All authors read and approved the nal manuscript.      and nca-2 in the wild type strains being subjected to thermal stress. Fold changes were calculated using 2-ΔΔCT method (Livak and Schmittgen 2001) by taking as wild type (without heat shock) control as calibrator and β-tubulin as endogenous control. The standard deviation was calculated from the data of three individual experiments each with three triplicates (n = 9). The Standard deviation was calculated from the data of three individual experiments each with three triplicates (n = 9) with P values <0.05 (*), <0.01 (**), <0.001 (***) and otherwise non-signi cant (ns) as measured by one-way ANOVA test.   region. The DNA probes 1and 2 were used alone as controls (lanes 1 and 3, respectively) or together with the CRZ-1::5xGly::V5::GFP protein (lanes 2 and 4, respectively). Shift was observed when probes 1 and 2 were used together with the protein (lanes 2 and 4), showing that CRZ-1::5xGly::V5::GFP binds to probes 1 and 2 that contain the predicted CRZ-1 binding sequence sites 5´-CCTTCACA-3´and 5 -AGCGGAGC-3 . The shift was not observed for probes 3 and 4 containing mutations in the predicted CRZ-1 binding sites (lanes 6 and 8). In lanes 9 and 10, 5xGly::V5::GFP protein with probes 1 and 2 was used as a negative control, and no shift was observed. perform PCR for the nca-2 promoter region using the DNA template obtained in the ChIP are located between the 5 UTR and ORF of the nca-2 gene. The primers Chip NCA-2 1F and Chip NCA-2 1R were used to PCR amplify a fragment of 400 bp size as indicated in the diagram. (B) The PCR products were resolved using a 1.2% agarose gel to identify the CRZ-1 binding region in the nca-2 promoter sequence.
The PCR ampli cation for the fragment is indicated in the gel. The analysis of the PCR revealed a positive product for the fragment of 400 bp in size, and the band intensity was further enhanced when the 559 strain was grown in the presence of 0.2 M CaCl2 stress condition. The antibody control (Ab-) indicates the control PCR for the sample, where only beads were used for immunoprecipitation. The 100 bp DNA ladder (New England Biolabs, USA) was used as marker.

Figure 7
Electrophoretic mobility shift assay to identify the CRZ-1 binding nucleotide sequence in the promoter of nca-2 (A) Schematic representation for the position of the PCR primers to map the CRZ-1 binding sequence in the nca-2 promoter shown using a bar. The primers, indicated using arrows, used for PCR region. The DNA probes A, B, C, and D were used alone as controls (lanes 1, 3, 5, and 7, respectively) or together with the CRZ-1::5xGly::V5::GFP protein (lanes 2, 4, 6, and 8, respectively). A shift was observed when probe A was used together with the protein (lane 2), showing that CRZ-1::5xGly::V5::GFP binds to probe A that contains the predicted CRZ-1 binding sequence site 5 -ACCGCGCC-3 . The shift was not observed either for the probe B having the sequence 5 -TGCGCAGC-3 or probe C containing mutations in the predicted CRZ-1 binding site as mentioned in probe A (lanes 4 and 6). No binding was observed in lane 8 containing probe D, which is the mutated version of probe B. In the lane 9, 5xGly::V5::GFP protein with probe A and was used as a negative control, and no shift was observed. Similarly, in lane 10, 5xGly::V5::GFP protein with probe B was used, and there was no shift. activation of calcineurin and CRZ-1. Activated CRZ-1 then nuclear localized and binds the promoter of its target genes, including hsp80 and nca-2, and upregulates their expression to cope the heat shock and Ca2+ stress responses in N. crassa. Supplementation of FK506, an inhibitor calcineurin, prevents calcineurin activation thereby downregulates the expressions of crz-1, hsp80, and nca-2 ensuing decreased viability of the cells in response to stress conditions. Also, another probable mechanism for regulation of hsp80 which indicates there could be a possible interaction of Hsp80 with CNA, which can further drive the calcineurin-CRZ-1 signaling pathway in response to stress conditions, is shown in the model.

Supplementary Files
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