crz1 regulator and calcium cooperatively modulate holocellulases gene expression … · 12...

1

CRZ1 regulator and calcium cooperatively modulate holocellulases gene 1

expression in Trichoderma reesei QM6a 2

3

Leonardo Martins-Santana1, Renato Graciano de Paula1, Adriano Gomes Silva2, Douglas 4 Christian Borges Lopes¹, Roberto do Nascimento Silva1 and Rafael Silva-Rocha2* 5

1 Department of Biochemistry and Immunology, University of São Paulo, Medical School 6 of Ribeirão Preto, São Paulo, Brazil 7

2Department of Cell and Molecular Biology and Pathogenic Agents, Systems and 8 Synthetic Biology Laboratory, University of São Paulo, Medical School of Ribeirão 9 Preto, São Paulo, Brazil 10

11

Keywords: Gene regulatory network, cellulase expression, transcriptional regulation, 12 calcium sensing 13

14

*Correspondence to: Rafael Silva-Rocha, [email protected]. 15

16

Abstract 17

Background: Trichoderma reesei is the main filamentous fungus used in industry to 18 produce cellulases. Over the last decades, there have been a strong increase in the 19 understanding of the regulatory network controlling cellulase-coding gene expression in 20 response to a number of inducers and environmental signals. In this sense, the role of 21 calcium and the Calcineurin-responsive protein (CRZ1) has been investigated in 22 industrially relevant strains of T. reesei RUT-C30, but this system has not been 23 investigated in wild-type reference strain of this fungus. 24

Results: Here, we investigated the role of CRZ1 and Ca2+ signaling in the fungus T. reesei 25 QM6a. For this, we first searched for potential CRZ1 binding sites in promoter regions 26 of key genes coding holocellulases, as well as transcriptional regulators and sugar and 27 calcium transporters. Using a nearly constructed T. reesei Δcrz1 strain, we demonstrated 28 that most of the genes expected to be regulated by CRZ1 were affected in the mutant 29 strain induced with sugarcane bagasse (SCB) and cellulose. In particular, our data 30 demonstrate that Ca2+ acts synergistically with CRZ1 to modulate gene expression, but 31 also exerts CRZ1-independent regulatory role in gene expression in T. reesei, suggesting 32 the existence of additional Ca2+ sensing mechanisms in this fungus. 33

Conclusions: This work presents new evidence on the regulatory role of CRZ1 and Ca2+ 34 sensing in the regulation of cellulolytic enzymes in T. reesei, evidencing significant and 35 previously unknown function of this Ca2+ sensing system in the control key 36 transcriptional regulators (XYR1 and CRE1) and on the expression of genes related to 37 sugar and Ca2+ transport. Taken together, the data obtained here provide new evidence on 38 the regulatory network of T. reesei related to plant biomass deconstruction. 39

.CC-BY-NC-ND 4.0 International licensenot certified by peer review) is the author/funder. It is made available under aThe copyright holder for this preprint (which wasthis version posted April 29, 2019. . https://doi.org/10.1101/622647doi: bioRxiv preprint

https://doi.org/10.1101/622647

http://creativecommons.org/licenses/by-nc-nd/4.0/

2

Background 40

Cellulases and hemicellulases are key enzymes for the production of second 41 generation ethanol production worldwide [1]. The promising utilization of agroindustrial 42 residues as energy source has triggered a tremendous interest for engineering 43 microorganisms for the low cost production of these enzymes aiming the conversion of 44 complex plant biomass into fermentable sugars, which in turn could be used for ethanol 45 production [2–4]. In this sense, the saprophyte organism T. reesei is one of the the main 46 filamentous fungus used in industry to produce these enzymes [2]. Despite the great 47 potential of this fungus for high performance cellulase and hemicellulase production, the 48 regulatory network controlling this system is a relevant field of study and not fully 49 understood yet. Additionally, the comprehension of transcriptional networks which 50 coordinate the transcription of holocellulases is one of the main focus to allow enzyme 51 production in a viable at industrial scale [5]. 52

T. reesei possess a great capability for enzyme secretion [6] but few cellulases and 53 hemicellulases genes. Even harboring this limited gene repertoire, its regulation is highly 54 precise and controlled at molecular levels by external signals [7]. Several transcription 55 factors (TFs) are now known for exert modulation on the expression levels of 56 holocellulases genes [8]. In this sense, the regulatory network which encompasses cell 57 wall deconstruction is under regulation of positive regulators, as XYR1 [9], ACEII [10], 58 LAE1 [11], BglR [12], VEL1 [13], HAP 2/3/5 complex [14] and other proteins which 59 play a negative control in such process, as CRE1 [15], ACEI [16] and RCE1[17]. 60 Additionally, it is already known that xylanases genes may also be exclusive targets of 61 regulation byTF such as Xpp1[18] and SxlR [19]. Interestingly, the same regulators are 62 not seem to be responsible for influencing cellulases expression in the same fungus. 63

The complex network that coordinates holocellulases expression in T. reesei is also 64 under regulation of nutritional variability. Therefore, differential gene expression was 65 discussed by Castro et al. [20] and Antonieto et al. [21] in studies which reported the 66 influence of carbon source on holocellulases transcription. Robust techniques as RNA 67 sequencing have already highlighted the importance of sugar availability on the 68 regulation mediated by transcription factors in T. reesei [22], evidencing the growing 69 need of elucidating deep layers of regulation in this fungus. 70

Nutritional requirements and pH stress conditions control are target of research among 71 a wide number of fungal species, such as Neurospora crassa [23], Trichoderma 72 harzianum [24] and T. reesei [25]. However, the influence of several stress conditions, 73 such as the effect of ions concentration exposure in transcription, still remains poorly 74 understood in the industrial workhorse T. reesei. Despite not completely elucidated, 75 recent studies have demonstrated the balance mediated by signaling pathways to keep 76 physiological homeostasis when T. reesei is exposed to Ca2+ stress through Calmodulin-77 Calcineurin downstream activation steps [26]. 78

CRZ1 (Calcineurin responsive protein) is a key target of Calcineurin (CN) in fungal 79 cells and is a well-conserved orthologue protein in several fungi species [27–29], being 80 responsible for many coordinated cellular functions. In Saccharomyces cerevisiae, a 81 deletion of this protein resulted in hypersensitivity to chloride and chitosan, as well as 82 mating and transcriptional response to alkaline and stress conditions defects [30]. In 83 another crz1 mutant yeast, Candida albicans, the knockout effect was correlated to 84 changes on the pattern of sensitiveness to Ca2+, Mn2+ and Li2+, as well as in 85 hypersensitiveness to azoles molecules [31]. The role of CRZ1 in filamentous fungi are 86 already described for some species, and in this context, for the pathogenic and 87


https://doi.org/10.1101/622647


3

opportunistic Aspergillus fumigatus, crz1 knockout resulted in virulence attenuation and 88 developmental defects in both hyphal morphology and conidiation [32]. Effects of CRZ1 89 in filamentous fungi were also mentioned for Cryptococcus neoformans, where growth at 90 temperatures higher than 39°C was compromised in crz1 knockout strains, as well as its 91 absence resulted in sensitiveness to cell wall perturbing agents [29,33]. 92

In T. reesei, the role of Ca2+-CaM/CN/CRZ1 pathway is poorly studied and established 93 in comparison to other fungal species. Yet, it is already known that Ca2+signaling pathway 94 may exert effects on the modulation of holocellulases gene expression in the industrial 95 strain of T. reesei RUT-C30 [26]. Therefore, Chen et al. [26] showed that the regulation 96 of relevant industrial genes, such as cbh1, eg1 and xyr1 genes were negatively modulated 97 in the absence of CRZ1 when calcium was added to a cellulose-supplemented medium. 98

Despite the evidence that CRZ1 and Ca2+ may improve holocellulases expression, 99 these results were obtained when the industrial strain of T. reesei RUT-C30 was evaluated 100 in different environmental conditions. Yet, this strain harbors a deletion of approximately 101 85 kilobases (kb) from T. reesei’s genome which includes the cre1 locus, resulting in 102 phenotypes that are independent on CRE1-mediated influence [34,35]. Regarding, the 103 role of CRZ1 and Ca2+ have not been investigated in wild type strain of T. reesei (QM6a), 104 which harbors the full set of regulatory components into its genome. In order to elucidate 105 this, in our study, we investigate the role of CRZ1/Ca2+ in a wild set genes coding for 106 transcriptional factors (TFs), enzyme and transporters related to holocellulose 107 deconstruction. In this study, we were able to identify CRZ1 transcription factor binding 108 sites (TFBS) in promoter regions of the differentially holocellulases expressed genes after 109 T. reesei QM6a exposure to SCB and cellulose as carbon source. Additionally, we 110 reported the fundamental role of CRZ1 on transcriptional regulation of such genes, as 111 well as we demonstrated that Ca2+ can cooperatively with CRZ1, induces or represses the 112 transcription of holocellulases genes. These findings bring evidences of Ca2+ to be 113 believed as a pivotal ion in the control of stimuli concerning expression of sugar and 114 calcium transporters in T. reesei QM6a cells in an CRZ1-independent but synergistic 115 mechanism. 116

117

Material and Methods 118

Analysis of promoters sequence 119

For the identification of potential CRZ1-binding elements, sequences of 1kb 120 upstream of the ATG from the Open Reading Frames (ORFs) of the promoter of TFs, 121 hollocellulases and transporter genes as well as for homologue of calcium and sugar 122 transporters were retrieved from the reference genome of T. reesei. Next, Position Weight 123 Matrix (PWM) from CRZ1 of S. cerevisiae were obtained from YeTFaSCo (The Yeast 124 Transcription Factor Specificity Compendium) collection [36] and used to calculate the 125 scores in all promoters of T. reesei. Scores greater than zero were selected and 126 normalized, revealing sequence with the higher probability of harboring a functional cis-127 regulatory element than a random motif. The normalization function was: 128 Score&'()*+,-./(Xij) = Score(Xij) MaxScore⁄ , being X the set of sense and antisense 129 upstream regulatory sequences; i the sequence index; j the initial position for score 130 calculation; and MaxScore the maximum score possible of the CRZ1 TF PWM. Through 131 the histogram analyses of the normalized score, we established a threshold of 0.8, using 132 a comparison between real sequences and a control dataset formed by random sequences. 133 Finaly, tis threshold was used to map potetinal cis-regulatory elements for CRZ1 in the 134 target of interest. 135


https://doi.org/10.1101/622647


4

Strains and media 136

T. reesei strain QM6aΔtmus53Δpyr4 was used as wild type (WT) strain in this 137 study and it was obtained from the Institute of Chemical Engineering (Viena University 138 of Technology, Viena, Austria) [37]. The strain was maintained in MEX medium (3% 139 (w/v) malt extract and 2% (w/v) agar)) at 30°C supplemented with 5 mM uridine for pyr4 140 auxotrophic selection [38]. T. reesei QM6aΔtmus53Δpyr4 wildtype and Δcrz1 141 (constructed as described below) strains were grown on MEX medium at 30°C for 7-10 142 days until complete sporulation. For gene expression assays, approximately 106 143 spores/mL from both strains were inoculated in Mandels Andreotti (MA) medium [39] 144 containing 1% (w/v) sugarcane bagasse, cellulose (Avicel, Sigma-Aldrich) or glycerol as 145 carbon source. For induction of gene expression in the presence of sugarcane bagasse, the 146 substrate in natura, gently donated by Nardini Agroindustrial Ltd., Vista Alegre do Alto, 147 São Paulo, Brazil, was previously prepared according to Souza et al. [40]. Mycelia from 148 both strains (WT and Δcrz1) were pre-grown in MA medium supplemented with 1% 149 (w/v) glycerol, in the presence and in the absence of 10 mM CaCl2, in orbital shaker at 150 30°C for 24 hours under 200 rpm. In these assays, MA medium presented a basal 151 concentration of 2.6 mM Ca2+ to ensure the maintenance of fungal cellular viability and 152 growth. The obtained mycelia were harvested and transferred to MA medium 153 supplemented with 1% (w/v) sugarcane bagasse or cellulose (Avicel, Sigma-Aldrich) in 154 orbital shaker at 200 rpm, 30°C for 8 h. All experimental conditions described were 155 performed with three biological replicates. The resulting mycelia were collected by 156 filtration, frozen in liquid nitrogen and stored at -80°C for RNA extraction procedures. 157

Construction of crz1 deletion cassette and T. reesei genetic transformation 158

Deletion of crz1 gene from T. reesei QM6aΔtmus53Δpyr4 was performed as 159 previously described [41]. For this, we design a deletion cassette containing the pyr4 gene 160 (orotidine-5¢-phosphate decarboxylase gene of T. reesei - Trire_74020) as auxotrophic 161 selection marker. All sequences of this cassette are available at the T. reesei genome 162 database (https://genome.jgi.doe.gov/Trire2/Trire2.home.html) and the primers used in 163 this study were designed to contain overlapping regions to the components sequence of 164 the cassette (Additional file 1: Table S1). The deletion cassette is composed by 1226 165 base pairs (bp) upstream to the 5’ crz1 CDS flanking region (Trire_36391) and 1465 bp 166 downstream to the crz1 ORF 3’ flanking region. These two sequences flanks pyr4 gene 167 sequence, which consists of 1100 bp upstream and 1000 bp downstream to the pyr4 CDS 168 sequence in T. reseei’s genome. Individual fragments were amplified at 60°C with 169 Phusion® High-Fidelity Polymerase (New England Biolabs) using T. reesei 170 QM6aΔtmus53Δpyr4 genomic DNA as template, according to manufacturer’s 171 instructions. The PCR fragments were purified using QIAquick PCR Purification Kit 172 (Qiagen). The assembly of the components was performed through homologous 173 recombination in S. cerevisiae, as previously reported [42–44]. For this purpose, we used 174 the pRS426 shuttle vector [45] previously treated with EcoRI and XhoI enzymes (New 175 England Biolabs). These treatment resulted on the occurrence of cohesive ends 176 correspondent to the external extremities in the primers used to obtain the 5’ and 3’ pyr4 177 ORF flanker regions of the deletion cassette. Yeast transformation procedures were 178 performed as described by Gietz and Schiestl, (2007) in the yeast S. cerevisiae strain 179 SC9721 (MATα his3-Δ200 URA3-52 leu2Δ1 lys2Δ202 trp1Δ63) (Fungal Genetic Stock 180 Center). The transformants were selected on YPD (1% (w/v) yeast extract, 2% (w/v) 181 peptone and 1% (w/v) glucose) medium supplemented with lysine, histidine, leucine, and 182 tryptophan and in the absence of uracil. 183


https://doi.org/10.1101/622647


5

To confirm the complete assembly of the cassette, genomic DNA of S. cerevisiae 184 was extracted according to Goldman et al. [46]. Primers 5’Pcrz1 and 3’Tcrz1 185 (Additional file 1:Table S1) were used to amplify the deletion cassette from the yeast 186 genome using Phusion® High-Fidelity Polymerase (New England Biolabs) according to 187 manufacturer’s instructions. The resultant amplicons were purified using QIAquick PCR 188 Purification Kit (Qiagen) and were stored at -20°C until T. reesei transformation. 189 Transformation of T. reesei was performed using 10 µg of the linearized deletion cassette 190 by protoplast fusion methodology [38]. Transformants were selected in MA medium 191 plates supplemented with 1% (w/v) glucose and 2% (w/v) agar (Sigma-Aldrich). 192 Selection was carried out in the absence of uridine. Genomic DNA was extracted to 193 confirm the cassette integration [46]. To verify the correct orientation of cassette 194 positioning in the positive transformants, it was performed two PCR reactions with 195 primers 5’Pcrz1 Check1/3’pyr4 Check 1 and 5’pyr4 Check 2/3’Tcrz1 Check 2 196 (Additional file 1: Table S1 , Additional File 2:Figure S1 A), which are specific for 197 pyr4 annealing regions and for external annealing regions into promoter and terminators 198 sequences. A PCR to confirm the deletion of the crz1 from T. reesei genome was 199 performed using the primers 5’crz1 ORF and 3’crz1 ORF (Additional file 1: Table S1 , 200 Additional File 2:Figure S1 B ). Null expression of crz1 gene was also investigated 201 with a quantitative PCR with total RNA using the primers PF crz1 qRT-PCR and PR crz1 202 qRT-PCR (Additional file 1: Table S1, Additional File 2:Figure S1 C ). 203

Gene expression analysis by Real-Time PCR 204

For gene expression analysis, 1 µg of total RNA of each culture condition was 205 treated with DNAse I (Sigma-Aldrich). cDNA samples were synthesized using 206 Maxima™ First Strand cDNA Synthesis kit (ThermoFisher Scientific) and posteriorly 207 diluted 1:50 in DEPC water. For gene expression levels detection, SsoFast™ EvaGreen® 208 Supermix (Bio-Rad) was used according to manufacturer’s instructions. A list of target 209 genes whose expression were evaluated are available in Additional file 3:Table S1. 210 Reactions were carried out at 95°C for 10 minutes, followed by 40 cycles at 95°C for 10 211 seconds and 60°C for 30 seconds in a Bio-Rad CFX96™ Real-Time System coupled to a 212 C1000™ Thermal Cycler (BioRad). Expression of target genes was normalized by the β-213 actin endogenous transcript levels for each RNA prevenient from the culture conditions 214 [47]. For sugarcane bagasse and cellulose expression quantification (for both in presence 215 or in the absence of 10 mM calcium chloride) the 2-ΔΔCt method was employed [48]. The 216 quantification was relative to transcript levels observed in the WT and Δcrz1 strains 217 grown in glycerol for 24 h (for both in the presence or in the absence of 10 mM calcium 218 chloride). 219

Enzymatic activity assays 220

The activity of β-glucosidases and β-xylosidases were measured as the capacity 221 of hydrolyzing pNPG and pNPX substrates as previously described [20,49,50]. 222 Endoglucanase activity was determined using Carboxymethylcellulose (CMC) (Sigma-223 Aldrich) as substrate in microplates, in a protocol adapted from Xiao et al. [51]. We 224 solubilize 1% (w/v) CMC in pH 4.8 sodium acetate buffer. After addition of 30 µL of 225 culture supernatant, reactions were incubated for 30 minutes at 50°C. After this time, 60 226 µL of DNS were added to the reaction and the mixture were submitted to incubation for 227 5 minutes at 95°C [52]. Absorbance at 540 nm were used for samples measurements. One 228 enzyme unit was defined as the amount of enzyme capable of releasing 1 µmol of 229 reducing sugar per minute. Endoxylanases activity was determined using xylan from 230 beechwood (Sigma-Aldrich) solubilized in pH 5.0 100 mM sodium acetate buffer as 231 substrate. Reactions were performed with 25 µL of the culture supernatant and 50 µL of 232


https://doi.org/10.1101/622647


6

substrate at 50°C for 30 minutes. After incubation, we mixed 75 µL of DNS and heated 233 the reactions at 95°C for 5 minutes to visualize the effects of reduction of dinitrosalicylic 234 acid. Absorbances were carried at 540 nm. One unit of enzyme was defined as the amount 235 of enzyme capable of releasing 1 µmol of reducing sugar per minute [53]. 236

Results 237

Identification of CRZ1 binding motifs in T. reesei promoters 238

We first investigated the existence of putative binding sites for CRZ1 in T. reesei 239 holocellulases and calcium and sugar transporter promoters using the defined sequence 240 for S. cerevisiae. For this, we selected 30 genes related to biomass degradation in plant 241 cell wall through proteins secreted by T. reesei, such as cellulases, hemicellulases and 242 sugar transporters, as well as calcium transporters and TFs which play a significant role 243 in this process. In this sense, we were able to identify potential cis-regulatory elements 244 for CRZ1 in 24 of these sequences, as represented in Figure 1A-B. Interestingly, the 245 major numbers of potential cis-regulatory elements for CRZ1 were observed in cre1 and 246 cel7a genes as well as for Trire_56440 and Trire_58952 calcium transporter proteins 247 promoters (Figure 1C). We also observed that the promoter of crz1 gene harbors 3 248 potential cis-regulatory elements in antisense orientation, suggesting that CRZ1 plays 249 autoregulation at transcriptional level (Figure 1C). Taken together, these results 250 evidenced the existence of putative binding sites for CRZ1 in several genes related to 251 gene regulation, biomass and calcium transport, highlighting the potential role of this TF 252 in the regulation of these genes. 253

CRZ1 and Ca2+ synergistically regulates TFs and cellobiohydrolase coding genes 254

Once we have identified putative binding sites for CRZ1 at promoter regions of 255 the genes of interest, we tried to understand how carbon source, as well as Ca2+ 256 supplementation, could modulate the transcription of these genes in the wild type and 257 Δcrz1 mutant background. In this sense, we observed that under exposure to SCB, the 258 expression of xyr1 was positively modulated in the wild type strain of T. reesei by the 259 supplementation of 10 mM Ca2+ to the medium (Figure 2A). However, this stimulatory 260 effect was lost in the T. reesei Δcrz1 strain, indicating that this process was completely 261 dependent on CRZ1. Additionally, the same effect was observed when Avicel was used 262 as inducer (Additional file 4: Figure S1 A). In the case of cre1, we observed only minor 263 changes in the expression of this TF under all conditions tested, with a small but 264 significant reduction in its expression in the wild type strain upon Ca2+ induction or 265 between wild type and Δcrz1 strain without the exposure to this ion (Figure 2B). Yet, 266 when its expression was assayed under Avicel exposure, no significant change was 267 observed between any condition analyzed (Additional file 4: Figure S1 B). It is worth 268 to notice that while putative CRZ1 binding sites were identified at both xyr1 and cre1 269 promoters (Figure 1C), only the former seems to be significantly regulated by this TF 270 under the conditions analyzed here. 271

Since we identified putative CRZ1 binding sites in crz1 promoter, we were also 272 interested in investigate how CRZ1-mediated autoregulation in T. reesei was related to 273 carbon source availability. In this sense, Figure 2C shows the result only for wild type 274 strain of T. reesei with or without Ca2+ exposure. As shown by the figure, under SCB 275 induction, the expression levels of crz1 were higher than those obtained after cellulose 276 induction. In addition, after supplementation of the medium with 10 mM Ca2+, the 277 expression of this gene decreased in the presence of SCB, suggesting that Ca2+ may exert 278 a modulatory effect on CRZ1 autoregulation. 279


https://doi.org/10.1101/622647


7

Since we observed that xyr1 expression was induced by Ca2+ in a CRZ1 dependent 280 manner, we next evaluated the role of these two components in the expression of cel6a 281 and cel7a, the two major cellobiohydrolases from T. reesei. We found that Ca2+ 282 supplementation resulted in a significant increase in cel6a and cel7a induction both under 283 exposure to SCB (Figure 2D-E) and Avicel (Additional file 4: Figure S1 C-D). 284 Additionally, deletion of crz1 resulted in a significant reduction in this stimulatory effect, 285 even though this was not completely abolished. Taken together, these results indicated 286 that CRZ1 and Ca2+ synergistically control the expression of celobiohydrolases coding 287 genes under SCB and Avicel exposure, and highlight that an additional Ca2+-dependent 288 and CRZ1-independent mechanism could take part in this process. 289

Other cellulases genes are also key of regulation by CRZ1 and Ca2+ 290

Since we observed that CRZ1 is capable to modulate xyr1 expression levels, we 291 next investigated how the expression of xylanase coding genes was modulated as a result 292 of such effect mediated by CRZ1. Thus, under exposure to SCB, we observed a significant 293 increase on the expression of these genes induced by the simple supplementation with 294 Ca2+ (Figure 3A-E). Additionally, the deletion of crz1 was not able to result in 295 differences on the expression of these genes, but we detected a loss in their expression in 296 the T. reesei Δcrz1 strain after Ca2+ supplementation. Taking in advantage of these 297 results, our findings suggest that the regulation of xylanase genes under SCB exposure is 298 modulated by Ca2+ in a synergistically CRZ1-dependent mechanism, highlighting the 299 relevance of this TF on a complete background concerning gene regulation in the wild 300 type strain of this fungus. Interestingly, no effects were observed when Avicel was used 301 as inducer, since none differential expression was detected under cellulose exposure 302 (Additional file 5: Figure S1 A-D). 303

The Ca2+- CRZ1-independent but synergistic mechanism was also observed when 304 we analyzed the expression of some β-glucosidases coding genes after SCB and cellulose 305 induction (Figure 4A-G, Additional file 6: Figure S1 A-F). Regarding β-glucosidases 306 regulation, such mechanism of cooperation was so remarkable that only one of the studied 307 genes (cebl3b) was modulated by CRZ1 alone (without Ca2+) (Figure 4D). Interestingly, 308 in opposite to the main cellulases of T. reesei (cel6a and cel7a), β-glucosidases gene 309 control tends to respond more effectively to cellulose stimuli, highlighting the role of 310 these enzymes on plant cell wall deconstruction. It is also worth to notice that β-311 glucosidases coding genes were the class of genes which we identified the major number 312 of putative CRZ1 binding sites in our in silico analysis, providing us an hypothesis of 313 why these genes are more responsible to cellulose than other known strongly expressed 314 genes (cellobiohydrolases). 315

The most heterogeneous effect on holocellulases expression was noticed for 316 endoglucanase coding genes expressed in SCB, as shown by Figure 5A-D. In this 317 condition, we observed a Ca2+-CRZ1-independent decrease on endoglucanases 318 expression (Figure 5A), a synergic Ca2+- CRZ1-independent effect which resulted in a 319 increase on expression (Figure 5B,D) and finally, a positive modulation exclusively 320 mediated by CRZ1 which loses its potential in a Ca2+-synergic mechanism (Figure 5C). 321 Interestingly, after cellulose induction, the Ca2+-CRZ1-independent but synergistic 322 mechanism was capable to potentialize the expression of only cel7b gene (Additional 323 File 7: Figure S1 B). 324

The effect of Ca2+ and the deletion of crz1 on accessory proteins expression was also 325 investigated in our study, as shown for the lytic polysaccharide monooxygenases (LPMO) 326 cel61a and swo (swollenin, an expansin-like protein) genes. In this way, we also observed 327


https://doi.org/10.1101/622647


8

the effect of a synergistic Ca2+- CRZ1-independent mechanism that may positively 328 regulate the expression of these genes in both SCB (Figure 5E-F) and cellulose induction 329 (Additional File 7: Figure S1 E-F), highlighting the role of both CRZ1 and Ca2+ on the 330 regulation of accessory proteins required for lignocellulose deconstruction. 331

Calcium and sugar transporters are regulated by CRZ1 in the presence of Ca2+ 332

Once the role of Ca2+ is clearly related to the CRZ1 regulatory role in T. reesei 333 [26], we decided to investigate whether calcium transporters were target of modulation 334 by CRZ1. After both SCB and cellulose growth, we observed that the deletion of crz1 335 elicited a loss in transcription of these genes in the presence of SCB (Figure 6B-C) while 336 it boosted the expression of the same genes after cellulose induction (Additional File 8: 337 Figure S1 B-D). In spite of CRZ1-independent modulator effect, it is also worth to notice 338 that Ca2+ synergistically to this TF was able to cause a decrease on the expression of such 339 genes predominantly in SCB (Figure 8A-C) in comparison to cellulose (Additional File 340 8: Figure S1 B). Interestingly, our in silico analysis also evidenced the occurrence of 341 putative CRZ1 binding sites in promoter regions of these genes, suggesting that calcium 342 homeostasis is precisely regulated at genomic levels by CRZ1, mainly under stress 343 conditions. 344

. 345

Finally, once we observed that carbon source could influence the transcription of 346 holocellulases genes in T. reesei in Δcrz1 strain, we studied the effects of crz1 knockout 347 on the expression of sugar transporter genes both after SCB and cellulose induction. 348 Interestingly, our results showed that CRZ1 itself plays a regulatory role on the expression 349 of these genes in the presence of complex substrates, since we detected a significant loss 350 on their expression in SCB (Figure 6D-F). At the same conditions, we also observed a 351 double effect on the synergic Ca2+- CRZ1-independent mechanism. In this sense, Ca2+ 352 allied to CRZ1, was able to significantly decrease the expression of Trire_50894 and 353 Trire_60945 genes, while the opposite effect was achieved by the increase of 354 Trire_79202 gene expression. In contrast, concerning cellulose-induced differential 355 regulation, we did not detect any differences on expression of the evaluated genes 356 (Additional file 9: Figure S1 C-E), suggesting a remarkable role of CRZ1 in the 357 regulation of genes involved in biomass transport. 358

Effect of crz1 deletion on enzymatic activity of holocellulases 359

The analysis of the culture supernatants where the QM6a wild type and Δcrz1 360 strains were grown showed that the activity of cellulases (β-glucosidases and 361 endoglucanases) was always superior in the supernatants from Δcrz1 strain in comparison 362 to the QM6a strain, regardless the carbon source that we evaluated (Figure 7A-B, 363 Additional file 10: Figure S1 A-B). In these assays, Ca2+ supplemenation was able to 364 increase the enzymatic activity of all tested supernatants, mainly for endoglucanases, as 365 we were not able to detect any enzymatic activity in the QM6a strain even in the presence 366 of 10 mM Ca2+ for both carbon sources that we investigated. 367

For xylanase activities (β-xylosidases and endoxylanases) in SCB and cellulose 368 (Figure 7C-D, Additional file 10: Figure S1 C-D), we observed that in the Δcrz1 369 supernatants the activities were higher compared to the QM6s strain except for the β-370 xylosidase in SCB (Figure 7C). Interestingly, the enzymatic activities of supernatants 371 whose QM6a strains were grown after Ca2+ addition were higher in presence of SCB 372 (Figure 7C-D) and presented a decrease in cellulose induction (Additional file 10: 373 Figure S1 C-D). For instance, the crz1 knockout resulted in supernatants with lower β-374


https://doi.org/10.1101/622647


9

xylosidase activity in SCB presence (Figure 7C) and higher xylanases activities measures 375 after cellulose induction (Additional file 10: Figure S1 C-D) when compared to the 376 QM6a strain at the same condition. 377

Discussion 378

In this study, we performed bioinformatics approaches to search for putative 379 CRZ1 binding sites in promoter regions of holocellulases, Ca2+ and sugar transporter 380 coding genes in T. reesei QM6a. Here we identified that differentially expressed 381 holocellulases genes of this fungus [22] harbored sense and antisense CRZ1 putative 382 binding sites at their ATG upstream sequences and it supports the evidence of the 383 regulation played by this TF independently on carbon source availability. To be clear that 384 CRZ1 was involved in gene regulation in the wild type strain of T. reesei, our study 385 reports that holocellulases genes were directly or either indirectly modulated by this 386 protein, corroborating the occurrences of putative CRZ1 binding sites sequences into T. 387 reesei genome. 388

Concerning these findings, we observed that distinct classes of cellulases and 389 xylanases were differentially expressed regardless the fungal growth condition. Although 390 cellulose was not capable to modulate a vast number of genes independently on CRZ1 391 presence, SCB provided the major number of differentially expressed genes after its 392 induction process (Figure 8A). This inducer capacity of SCB over the industrial strain 393 RUT-C30 of T. reesei was already reported [54]. In their study, Borin et al. [54] found 394 24 biomass degrading enzymes in T. reesei’s RUT-C30 secretome after SCB exposure, 395 showing that this carbon source is a potential activator of holocellulases production in 396 this fungus. Despite RUT-C30 genome does not harness a cre1 functional sequence, we 397 verified here that for T. reesei QM6a, SCB was either the carbon source which 398 preferentially elicited a higher holocellulases transcriptional response (Figure 8A).. 399

Interestingly, our results also show that CRZ1-mediated regulation was not 400 limited to lignocellulose degrading enzymes coding genes, but also intersected the 401 regulatory network that coordinates holocellulases expression in the T. reesei QM6a. 402 Therefore, we report that CRZ1 can modulate the transcript levels of relevant TFs such 403 as XYR1 and CRE1 (inactive in RUT-C30 background), which in turn, can modulate 404 cellulases and xylanases expression overall in this specie. This is specially interesting 405 when we consider CRE1 repressor role on cellulases transcription, suggesting that deeper 406 studies highlighting the interaction between CRZ1 and CRE1 may be a suitable strategy 407 to engineer T. reesei as an industrial chassis for cellulases production. 408

Taking these facts in advantage, our data suggest a mechanism of synergy between 409 the regulatory role of CRZ1 and the potential effect caused by Ca2+ upon the expression 410 of holocellulases genes. In summary, we observed a major number of differentially 411 expressed genes in presence of SCB supplemented with Ca2+ in comparison to cellulose 412 exposure at the same Ca2+ concentration (Figure 8A), endorsing the previously reported 413 effect of carbon source on modulation of T. reesei transcriptional profile [20]. In addition, 414 these evidences corroborate the suggested synergic mechanism between CRZ1 and Ca2+, 415 since we detected higher transcripts levels in all conditions supplemented with Ca2+ 416 regardless the carbon source availability. 417

Ca2+ plays a regulatory role on several mechanisms in T. reesei, e.g. on protein 418 secretion [55] and on events related to CRZ1-Calcineurin pathway [26]. In this sense, 419 Chen et al.[26] reported the influence of 5 mM Ca2+ on the transcription of relevant 420 industrial genes of T. reesei RUT-C30, such as cbh1 and xyr1. In our study, 10 mM Ca2+ 421 was able to potentialize the expression of xyr1 at the first 8 hours of induction. This result 422


https://doi.org/10.1101/622647


10

is in contrast to previous findings reported by Chen et al. [26], whose study showed the 423 first 6 hours of cellulose exposure as inefficient to increase expression of xyr1 in the 424 industrial strain of T. reesei. In conclusion, here we suggest that a higher Ca2+ 425 concentration could elicit a faster gene activation in the presence of cellulose. 426 Interestingly, we also observed a massive loss in transcription of xyr1 in our Δcrz1 strain 427 in the presence of Ca2+, in accordance with the results obtained by Chen et al. [26] when 428 authors investigated this effect in RUT-C30 strain, suggesting that CRZ1 is required for 429 the activation of this relevant TF in T. reesei. 430

In the face of the pioneer contributions provided by Chen et al.[26], this study was 431 performed using the RUT-C30 industrial strain of T. reesei, which does not harness 432 CRE1, the master regulator of carbon catabolite repression in this fungus. Such genomic 433 background could provide us an incomplete knowledge of the fully transcriptional CRZ1 434 pattern in these cells, mainly because in such transcriptional networks indirect regulation 435 mechanisms may occur. For this reason, understand the mechanisms which control 436 transcription in T. reesei QM6a could significantly contributes to the engineering 437 holocellulases secretion in T. reesei. 438

Ca2+/CRZ1-Calcineurin/Calmodulin is one of the best studied signaling pathways 439 which provides protection and sensibility for fungal cells against stress conditions. This 440 signaling pathway is already reported to confer Ca2+ and cell wall stress tolerance in 441 Aspergillus [56], pH responsiveness in S. cerevisiae [57] and it is either related to C. 442 neoformans viability [29,33]. In addition, it is also reported that Ca2+ supplementation 443 may influence distinct regulatory mechanisms that control holocellulases expression in 444 T. reesei [26]. Although this signaling pathway are well characterized among fungal 445 species, the effect of carbon source availability, Ca2+ concentration and CRZ1 446 dependence mechanisms together are still not completely clear in the field of cellulases 447 regulation for the wild type strain of T. reesei. 448

In this sense, our data emphasize that Ca2+ is crucial for the transcription of such 449 genes and this bivalent ion significantly contributed to the increase of transcript levels 450 analyzed in our work through stimuli on Calmodulin/Calcineurin pathway. Furthermore, 451 we were able to observe that Ca2+ seems to be critical for the expression of many 452 holocellulases, Ca2+ and sugar transporter coding genes more directly than the carbon 453 source whose fungal growth was previously established. Albeit the effect of SCB on 454 enzymes expression, Ca2+ and sugar transporter proteins had already been reported in a 455 T. reesei RUT-C30 gene co-expression network study by Borin et al. [5], here we firstly 456 described the seminal importance of Ca2+ on the expression of sugar transporters in a 457 CRZ1-dependent context in T. reesei QM6a strain, hypothesizing that under stress 458 conditions T. reesei QM6a may increase the transcription of sugar transporters to keep 459 metabolic homeostasis through Ca2+-CRZ1/Calcineurin pathway. 460

Finally, the effect of sense or antisense CRZ1 putative binding sites in promoter 461 regions is one of the main evidences that support regulation QM6a strain of T. reesei. The 462 effect of arrangements and repetitions of TFs binding sites in T. reesei was already 463 reported by Kiesenhofer et al. [58] as an important parameter for engineering 464 transcriptional regulation in this fungus. As observed in our work, through an in silico 465 analysis, from the total set of differentially expressed genes, only 6 of them did not 466 harness CRZ1 putative binding sites and only one of these was not modulated by Ca2+ 467 (Figure 8B). These evidences strongly suggest that the synergic mechanism played by 468 CRZ1 and Ca2+ is required for holocellulases and transporter coding genes expression in 469 T. reesei QM6a even not in the presence of stress conditions. In addition, our data also 470 assert that CRZ1 activation is pivotal for gene regulation, but Ca2+ is determinant for an 471


https://doi.org/10.1101/622647


11

increasingly modulation (positive or negative) on gene expression levels, claiming at the 472 necessity of deeper studies on indirect holocellulases regulation in T. reesei. 473

Conclusions 474

In this study, we highlight the major perspectives of bioinformatic approaches to 475 extend our knowledge and expertise to engineer fungi for industrial applications. In 476 summary, we describe the construction of a T. reesei crz1 knockout strain through 477 bioinformatics prediction and validation of the regulatory role of CRZ1 protein on 478 transcriptional regulation of T. reesei. Allied to Ca2+, an important modulator of the 479 regulatory effects of CRZ1, holocellulases and calcium/sugar transporter genes can be 480 positively or negatively modulated in a CRZ1-synergic mechanism, as well as the 481 enzymatic activity of such enzymes may direct or indirectly be affected by CRZ1 482 regulatory role. These phenomena are suitable attributes to biotechnological industry 483 applications, highlighting fungi engineering as a fundamental platform to enzyme 484 production at large scale. 485

486

References 487

1. Prasad S, Singh A, Joshi HC. Ethanol as an alternative fuel from agricultural, industrial 488 and urban residues. Resour Conserv Recycl. 2007;doi:10.1016/j.resconrec.2006.05.007. 489

2. Druzhinina IS, Kubicek CP. Genetic engineering of Trichoderma reesei cellulases and 490 their production. Microb Biotechnol. 2017;doi: 10.1111/1751-7915.12726. 491

3. Seppälä S, Wilken SE, Knop D, Solomon K V., O’Malley MA. The importance of 492 sourcing enzymes from non-conventional fungi for metabolic engineering and biomass 493 breakdown. Metab Eng. 2017; doi: 10.1016/j.ymben.2017.09.008. 494

4. Martins-Santana L, Nora LC, Sanches-Medeiros A, Lovate GL, Cassiano MHA, Silva-495 rocha R. Systems and Synthetic Biology Approaches to Engineer Fungi for Fine 496 Chemical Production. Front Bioeng Biotechnol. 2018; doi: 10.3389/fbioe.2018.00117. 497

5. Borin GP, Carazzolle MF, Augusto R, Riaño-pachón DM, Velasco J, Oliveira DC, et 498 al. Gene Co-expression Network Reveals Potential New Genes Related to Sugarcane 499 Bagasse Degradation in Trichoderma reesei RUT-30. Front Bioeng Biotechnol. 2018; 500 doi: 10.3389/fbioe.2018.00151. 501

6. Schmoll M, Dattenböck C, Carreras-villaseñor N, Mendoza-mendoza A, Tisch D, 502 Alemán MI, et al. The Genomes of Three Uneven Siblings : Footprints of the Lifestyles 503 of Three Trichoderma Species. Microbiol Mol Biol Rev. 2016; doi: 504 10.1128/MMBR.00040-15. 505

7. Gupta VK, Steindorff AS, Paula RG De, Silva-Rocha R, Mach-Aigner AR, Mach RL, 506 et al. The Post-genomic Era of Trichoderma reesei : What’ s Next ? Trends Biotechnol. 507 2016; doi: 10.1016/j.tibtech.2016.06.003. 508

8. Paula RG De, Cristina A, Antoniêto C, Fraga L, Ribeiro C, Carraro CB, et al. New 509 Genomic Approaches to Enhance Biomass Degradation by the Industrial Fungus 510 Trichoderma reesei. Int J Genomics. 2018; doi: 10.1155/2018/1974151. 511

9. Stricker AR, Grosstessner-hain K, Wu E, Mach RL. Xyr1 (Xylanase Regulator 1) 512 Regulates both the Hydrolytic Enzyme System and D -Xylose Metabolism in Hypocrea 513 jecorina. Eukaryot Cell. 2006; doi: 10.1128/EC.00211-06. 514


https://doi.org/10.1101/622647


12

10. Aro N, Saloheimo A, Ilme M, Penttila M. ACEII , a Novel Transcriptional Activator 515 Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei. J Biol 516 Chem. 2001; doi: 10.1074/jbc.M003624200. 517

11. Seiboth B, Karimi RA, Phatale PA, Linke R, Hartl L, Sauer DG, et al. The putative 518 protein methyltransferase LAE1 controls cellulase gene expression in Trichoderma 519 reesei. Mol Microbiol. 2012; doi: 10.1111/j.1365-2958.2012.08083.x. 520

12. Nitta M, Furukawa T, Shida Y, Mori K, Kuhara S, Morikawa Y. A new Zn ( II ) 2 521 Cys 6 -type transcription factor BglR regulates b -glucosidase expression in Trichoderma 522 reesei. Fungal Genet Biol. 2012; doi: 10.1016/j.fgb.2012.02.009. 523

13. Karimi RA, Németh Z, Atanasova L, Fekete E, Paholcsek M, Sándor E, et al. The 524 VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and 525 Is Essential for. PLoS One. 2014; doi: 10.1371/journal.pone.0112799. 526

14. Zeilinger S, Schmoll M, Pail M, Mach RL, Kubicek CP. Nucleosome transactions on 527 the Hypocrea jecorina ( Trichoderma reesei ) cellulase promoter cbh2 associated with 528 cellulase induction. Mol Gen Genomics. 2003; doi: 10.1007/s00438-003-0895-2. 529

15. Portnoy T, Margeot A, Linke R, Atanasova L, Fekete E, Sándor E, et al. The CRE1 530 carbon catabolite repressor of the fungus Trichoderma reesei : a master regulator of 531 carbon assimilation. BMC Genomics. 2011; doi: 10.1186/1471-2164-12-269. 532

16. Aro N, Ilme M, Saloheimo A, Penttila M. ACEI of Trichoderma reesei Is a Repressor 533 of Cellulase and Xylanase Expression. Appl Environ Microbiol. 2003; doi: 534 10.1128/AEM.69.1.56-65.2003. 535

17. Cao Y, Zheng F, Wang L, Zhao G. Rce1 , a novel transcriptional repressor , regulates 536 cellulase gene expression by antagonizing the transactivator Xyr1 in Trichoderma reesei. 537 Mol Microbiol. 2017; doi: 10.1111/mmi.13685. 538

18. Derntl C, Rassinger A, Srebotnik E, Mach RL, Aigner ARM. Xpp1 regulates the 539 expression of xylanases , but not of cellulases in Trichoderma reesei. Biotechnol Biofuels. 540 BioMed Central. 2015; doi: 10.1186/s13068-015-0298-8. 541

19. Liu R, Chen L, Jiang Y, Zou G, Zhou Z. A novel transcription factor specifically 542 regulates GH11 xylanase genes in Trichoderma reesei. Biotechnol Biofuels. BioMed 543 Central. 2017; doi: 10.1186/s13068-017-0878-x. 544

20. Castro S, Pedersoli WR, Antoniêto ACC, Steindorff AS, Silva-Rocha R, Martinez-545 rossi NM, et al. Comparative metabolism of cellulose , sophorose and glucose in 546 Trichoderma reesei using high-throughput genomic and proteomic analyses. Biotechnol 547 Biofuels. 2014; doi: 10.1186/1754-6834-7-41. 548

21. Antoniêto ACC, de Paula RG, Castro L dos S, Silva-Rocha R, Persinoti GF, Silva 549 RN. Trichoderma reesei CRE1-mediated Carbon Catabolite Repression in Response to 550 Sophorose Through RNA Sequencing Analysis. Curr Genomics. 2016; doi: 551 10.2174/1389202917666151116212901. 552

22. dos Santos Castro L, de Paula RG, Antoniêto ACC, Persinoti GF, Silva-Rocha R, 553 Silva RN. Understanding the Role of the Master Regulator XYR1 in Trichoderma reesei 554 by Global Transcriptional Analysis. Front Microbiol. 2016; doi: 555 10.3389/fmicb.2016.00175. 556

23. Virgilio S, Cupertino FB, Ambrosio DL, Bertolini MC. Regulation of the reserve 557


https://doi.org/10.1101/622647


13

carbohydrate metabolism by alkaline pH and calcium in Neurospora crassa reveals a 558 possible cross-regulation of both signaling pathways. BMC Genomics. 2017; doi: 559 10.1186/s12864-017-3832-1. 560

24. Moreno-Mateos MA, Codo AC. pH and Pac1 control development and antifungal 561 activity in Trichoderma harzianum. Fungal Genet Biol. 2007; doi: 562 10.1016/j.fgb.2007.07.012. 563

25. He R, Ma L, Li C, Jia W, Li D, Zhang D, et al. Trpac1 , a pH response transcription 564 regulator , is involved in cellulase gene expression in Trichoderma reesei. Enzyme 565 Microb Technol. 2014; doi: 10.1016/j.enzmictec.2014.08.013. 566

26. Chen L, Zou G, Wang J, Wang J, Liu R, Jiang Y, et al. Characterization of the Ca 2+ 567 -responsive signaling pathway in regulating the expression and secretion of cellulases in 568 Trichoderma reesei Rut-C30. Mol Microbiol. 2016; doi: 10.1111/mmi.13334. 569

27. Zhang T, Xu Q, Sun X, Li H. The calcineurin-responsive transcription factor Crz1 is 570 required for conidation, full virulence and DMI resistance in Penicillium digitatum. 571 Microbiol Res. 2013; doi: 10.1016/j.micres.2012.11.006. 572

28. Thewes S. Calcineurin-Crz1 Signaling in Lower Eukaryotes. Eukaryot Cell. 2014; 573 doi: 10.1128/EC.00038-14. 574

29. Chow EWL, Clancey SA, Billmyre RB, Averette AF, Granek JA, Mieczkowski P, et 575 al. Elucidation of the calcineurin-Crz1 stress response transcriptional network in the 576 human fungal pathogen Cryptococcus neoformans. PLoS Genet. 2017; doi: 577 10.1371/journal.pgen.1006667. 578

30. Zakrzewska A, Boorsma A, Brul S, Klaas J, Klis FM, Hellingwerf KJ. Transcriptional 579 Response of Saccharomyces cerevisiae to the Plasma Membrane-Perturbing Compound 580 Chitosan Transcriptional Response of Saccharomyces cerevisiae to the Plasma 581 Membrane-Perturbing Compound Chitosan. Eukaryot Cell. 2005; doi: 582 10.1128/EC.4.4.703-715.2005. 583

31. Santos M, de Larrinoa IF. Functional characterization of the Candida albicans CRZ1 584 gene encoding a calcineurin-regulated transcription factor. Curr Genet. 2005; doi: 585 10.1007/s00294-005-0003-8. 586

32. Soriani FM, Malavazi I, Da Silva Ferreira ME, Savoldi M, Von Zeska Kress MR, De 587 Souza Goldman MH, et al. Functional characterization of the Aspergillus fumigatus 588 CRZ1 homologue, CrzA. Mol Microbiol. 2008; doi: 10.1111/j.1365-2958.2008.06122.x. 589

33. Lev S, Desmarini D, Chayakulkeeree M, Sorrell TC, Djordjevic JT. The Crz1/Sp1 590 Transcription Factor of Cryptococcus neoformans Is Activated by Calcineurin and 591 Regulates Cell Wall Integrity. PLoS One. 2012; doi: 10.1371/journal.pone.0051403. 592

34. Seidl V, Seibel C, Kubicek CP, Schmoll M. Sexual development in the industrial 593 workhorse Trichoderma reesei. Proc Natl Acad Sci. 2009; doi: 594 10.1073/pnas.0904936106. 595

35. Mello-de-Sousa TM, Gorsche R, Rassinger A, Poc¸as-Fonseca MJ, Mach RL, Mach-596 Aigner AR. A truncated form of the Carbon catabolite repressor 1 increases cellulase 597 production in Trichoderma reesei. Biotechnol Biofuels. 2014; doi: 10.1186/s13068-014-598 0129-3. 599

36. De Boer CG, Hughes TR. YeTFaSCo: A database of evaluated yeast transcription 600


https://doi.org/10.1101/622647


14

factor sequence specificities. Nucleic Acids Res. 2012; doi: 10.1093/nar/gkr993. 601

37. Derntl C, Kiesenhofer DP, Mach RL, Mach-Aigner AR. Novel strategies for genomic 602 manipulation of Trichoderma reesei with the purpose of strain engineering. Appl Environ 603 Microbiol. 2015; doi: 10.1128/AEM.01545-15. 604

38. Gruber F, Visser J, Kubicek CP, de Graaff LH. The development of a heterologous 605 transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-606 negative mutant strain. Curr Genet. 1990; doi: 10.1007/BF00321118. 607

39. Schmoll M, Schuster A, Silva RDN, Kubicek CP. The G-alpha protein GNA3 of 608 Hypocrea jecorina (anamorph Trichoderma reesei) regulates cellulase gene expression in 609 the presence of light. Eukaryot Cell. 2009; doi: 10.1128/EC.00256-08. 610

40. Souza D, Souza WR De, Gouvea PF De, Savoldi M, Bernardes LADS. Transcriptome 611 analysis of Aspergillus niger grown on sugarcane bagasse Transcriptome analysis of 612 Aspergillus niger grown on sugarcane bagasse. Biotechnol Biofuels. 2011; doi: 613 10.1186/1754-6834-4-40. 614

41. Schuster A, Bruno KS, Collett JR, Baker SE, Seiboth B, Kubicek CP, et al. A versatile 615 toolkit for high throughput functional genomics with Trichoderma reesei. Biotechnol 616 Biofuels. 2012; doi: 10.1186/1754-6834-5-1. 617

42. Colot H V., Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, et al. A high-618 throughput gene knockout procedure for Neurospora reveals functions for multiple 619 transcription factors. Proc Natl Acad Sci. 2006; doi: 10.1073/pnas.0601456103. 620

43. Collopy PD, Colot H V, Park G, Ringelberg C, Christopher M, Borkovich KA, et al. 621 High-throughput construction of gene deletion cassetes for generation of Neurospora 622 crassa knockout strains. Methods Mol Biol. 2010; doi: 10.1007/978-1-60761-611-5_3. 623

44. Gietz RD, Schiestl RH. Yeast transformation by the LiAc/SS carrier DNA/PEG 624 method. Methods Mol Biol. 2007; doi: 10.1385/1-59259-958-3:107. 625

45. Christianson TW, Sikorski RS, Dante M, Shero JH, Hieter P. Multifunctional yeast 626 high-copy-number shuttle vectors. Gene. 1992; doi: 10.1016/0378-1119(92)90454-W. 627

46. Goldman GH, Savoldi M, Semighini CP, Oliveira RC De, Nunes LR, Travassos LR, 628 et al. Expressed Sequence Tag Analysis of the Human Pathogen Paracoccidioides 629 brasiliensis yeast phase: Identification of putative homologues of Candida albicans virulence 630 and pathogenicity genes. Eukaryot Cell. 2003; doi: 10.1128/EC.2.1.34-48.2003. 631

47. Verbeke J, Coutinho P, Mathis H, Quenot A, Record E, Asther M, et al. 632 Transcriptional profiling of cellulase and expansin-related genes in a hypercellulolytic 633 Trichoderma reesei. Biotechnol Lett. 2009; doi: 10.1007/s10529-009-0030-5. 634

48. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time 635 quantitative PCR and the 2-ΔΔCT method. Methods. 2001; doi: 636 10.1006/meth.2001.1262. 637

49. Hideno A, Inoue H, Tsukahara K, Yano S, Fang X, Endo T, et al. Production and 638 characterization of cellulases and hemicellulases by Acremonium cellulolyticus using rice 639 straw subjected to various pretreatments as the carbon source. Enzyme Microb Technol. 640 2011; doi: 10.1016/j.enzmictec.2010.10.005. 641

50. Lopes FAC, Steindorff AS, Geraldine AM, Brandão RS, Monteiro VN, Júnior ML, et 642


https://doi.org/10.1101/622647


15

al. Biochemical and metabolic profiles of Trichoderma strains isolated from common 643 bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia 644 sclerotiorum. Fungal Biol. 2012; doi: 10.1016/j.funbio.2012.04.015. 645

51. Xiao Z, Storms R, Tsang A. Microplate-based carboxymethylcellulose assay for 646 endoglucanase activity. Anal Biochem. 2005; doi: 10.1016/j.ab.2005.01.052. 647

52. Miller GL. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing 648 Sugar. Anal Chem. 1959;31:426–8. 649

53. Castro LDS, Antoniêto ACC, Pedersoli WR, Silva-Rocha R, Persinoti GF, Silva RN. 650 Expression pattern of cellulolytic and xylanolytic genes regulated by transcriptional 651 factors XYR1 and CRE1 are affected by carbon source in Trichoderma reesei. Gene Expr 652 Patterns. 2014; doi: 10.1016/j.gep.2014.01.003. 653

54. Borin GP, Sanchez CC, Souza AP De, Santana S De, Souza AT De, Franco A, et al. 654 Comparative Secretome Analysis of Trichoderma reesei and Aspergillus niger during 655 Growth on Sugarcane Biomass. PLoS One. 2015; doi: 10.1371/journal.pone.0129275. 656

55. Mach RL, Zeilinger S, Kristufek D, Kubicek CP. Ca2+-calmodulin antagonists 657 interfere with xylanase formation and secretion in Trichoderma reesei. Biochim Biophys 658 Acta - Mol Cell Res. 1998;1403:281–9. 659

56. Shwab EK, Juvvadi PR, Waitt G, Soderblom EJ, Barrington BC, Asfaw YG, et al. 660 Calcineurin-dependent dephosphorylation of the transcription factor CrzA at specific 661 sites control conidiation, stress tolerance, and virulence of Aspergillus fumigatus. Mol 662 Microbiol. 2019; doi: 10.1111/mmi.14254. 663

57. Roque A, Petrezsélyová S, Serra-Cardona A, Ariño J. Genome-wide recruitment 664 profiling of transcription factor Crz1 in response to high pH stress. BMC Genomics. 2016; 665 doi: 10.1186/s12864-016-3006-6. 666

58. Kiesenhofer DP, Mach RL, Mach-Aigner AR. Influence of cis element arrangement 667 on promoter strength in Trichoderma reesei. Appl Environ Microbiol. 2018; doi: 668 10.1128/AEM.01742-17. 669

670

671

672


https://doi.org/10.1101/622647


16

FIGURES 673

674

675

Figure 1. A – DNA Consensus sequence of CRZ1 binding in T. reesei obtained through 676 bioinformatics analysis. B – Representative scheme of genes which harness CRZ1 BS 677 (CRZ1 binding sites) at genomic level. C – in silico Identification of CRZ1 BS in 678 promoter regions of holocellulases and calcium and sugar transporter genes in T. reesei. 679 Blue spheres indicate that those sequences (previously shown in 1A) have been found in 680 sense orientation and orange spheres represent the antisense sequences occurrence. The 681 number of spheres represents the absolute frequency that these sequences were observed 682 in bioinformatics analysis. 683

684

Sense CRZ1 binding site Antisense CRZ1 binding site A C

-1000 -750 -500 -250 ATG

cel7b

cel1a

cel1bcel5bcel3e

xyn2

cel74a

cel6a

xyn1

swocel3b

cre1

crz1xyr1

Trire_74057

Trire_68169Trire_79202Trire_60945

Trire_56440Trire_55731

Trire_58952

cel3acel7a

cel45a

BCRZ1 BS

Absence ofCRZ1 BS

Trire_50894

cel3d

cel61a

cel3c

xyn4

xyl3b

bxl1

Trire_58952

Trire_55731

Trire_56440

Trire_68169


Trire_60945

xyr1

cel1bswo

xyn2

cel7a xyn1

cel74acel3e

cel45a

cel3b

cel1a

cel3a cel7b

cel6a

cel5b

cre1

crz1


https://doi.org/10.1101/622647


17

685

686

Figure 2. Analysis of differential expression of the transcription factors XYR1 (A), 687 CRE1(B), and CRZ1 (C) of T. reesei and of cellobiohydrolases CEL6A (D) and CEL7a 688 (E) from QM6a and Δcrz1 T. reesei strains after 8 h of growing in SCB and SCB 689 supplemented with 10 mM Ca2+. CRZ1 expression in the presence of cellulose and the 690 effect caused by calcium on this induction is also shown in panel C. Expression values 691 are represented as log10 means of three biological replicates with standard deviation 692 normalized by glycerol expression levels at the same condition. Statistical significance is 693 represented as asterisks, considering p-value as at least < 0.05 (* < 0.05 < ** < 0.005 < 694 *** < 0.0001 < ****). 695

A B

C

D E


https://doi.org/10.1101/622647


18

696

Figure 3. Relative expression of the β-xylosidase 1 and (A) the candidate xyl3b 697 xylosidase (B) and of the endoxylanases xyn1 (C), xyn2 (D) and xyn4 (E) genes from 698 QM6a and Δcrz1 T. reesei strains after 8 h of induction SCB and SCB supplemented with 699 10 mM Ca2+. Expression values are represented as log10 means of three biological 700 replicates with standard deviation normalized by glycerol expression levels at the same 701 condition. Statistical significance is represented as asterisks, considering p-value as at 702 least < 0.05 (* < 0.05 < ** < 0.005 < *** < 0.0001 < ****). 703

A B

C D

E


https://doi.org/10.1101/622647


19

704

Figure 4. A-G - qRT-PCR results for differential expression analysis of the β-705 glucosidases genes of T. reesei in the QM6a and Δcrz1 strains after 8 h of growing in 706 SCB supplemented or not with 10 mM Ca2+. Expression values are represented as log10 707 means of three biological replicates with standard deviation normalized by glycerol 708 expression levels at the same condition. Statistical significance is represented as asterisks, 709 considering p-value as at least < 0.05 (* < 0.05 < ** < 0.005 < *** < 0.0001 < ****). 710

A B

C D

E F

G


https://doi.org/10.1101/622647


20

711

Figure 5. Analysis of differential expression of the endoglucanases (A-D), LPMO 712 CEL61A (E) and swollenin (F) from QM6a and Δcrz1 T. reesei in the presence of SCB 713 and SCB supplemented with 10 mM Ca2+ after 8h of induction. Expression values are 714 represented as log10 means of three biological replicates with standard deviation 715 normalized by glycerol expression levels at the same condition. Statistical significance is 716 represented as asterisks, considering p-value as at least < 0.05 (* < 0.05 < ** < 0.005 < 717 *** < 0.0001 < ****). 718

A B

C D

E F


https://doi.org/10.1101/622647


21

719

Figure 6. Ca2+ transporter genes (A-C) and sugar transporter (D-F) differential genic 720 expression in the QM6a and Δcrz1 T. reesei strains after 8 h of growing in SCB and SCB 721 supplemented with 10 mM Ca2+. Results are expressed as log10 means of three biological 722 replicates with standard deviation normalized by glycerol expression levels at the same 723 condition. Statistical significance is represented as asterisks, considering p-value as at 724 least < 0.05 (* < 0.05 < ** < 0.005 < *** < 0.0001 < ****). Protein ID of the evaluated 725 genes are supported at T. reesei genome database. 726

A D

B E

C F


https://doi.org/10.1101/622647


22

727

Figure 7. Enzymatic activity of β-glucosidase (A), endoglucanase (B), β-xylosidase (C) 728 and endoxylanase (D) from QM6a and Δcrz1 T. reesei strains supernatants after 8 h of 729 growing in SCB and SCB supplemented with 10 mM Ca2+. Results are represented as 730 absolute values in unities per milliliter and are representative of a mean of three biological 731 replicates with standard deviation. Statistical significance is represented as asterisks, 732 considering p-value as at least < 0.05 (* < 0.05 < ** < 0.005 < *** < 0.0001 < ****). 733

734

A B

C D


https://doi.org/10.1101/622647


23

735

Figure 8. A – Heatmap of the differential expression profile of the holocellulases and 736 calcium and transporter sugars after 8 h of induction in the presence or in the absence of 737 10 mM Ca2+. Values are representative of the expression obtained with mutant crz1 strain 738 related to the QM6a strain. Genes presenting representative statistical significance are 739 show with asterisks. ID codes for calcium and sugar transporters are supported in T. reesei 740 genome database. B - Representative scheme of genes differentially expressed in the 741 absence of CRZ1 in T. reesei and its relation to the occurrence or absence of binding sites 742 (BS) for CRZ1 at genomic level. Bold characters represents the genes whose expression 743 was modulated by 10 mM Ca2+ addition. 744

-5.0 0.0 3.0

SC

B

Cel

lulo

se/C

a2+

Cel

lulo

se

SC

B/C

a2+

Trire_50894Trire_74057Trire_79202Trire_55731Trire_56440cel3dcel3ecel61acel1aswoxyr1cel6acel74axyl3bbxl1cel3bcre1cel45acel5bcel7bcel3cTrire_60945xyn1cel7axyn4cel1bTrire_58952Trire_68169

***

****

*

*

**

********

********

**

*

*****

*

cel1a

cel3acel3b

cel3e

cel6acel7a

cel7b cel45acel74a

xyn1 xyn2

xyr1cre1


Trire_68169

Trire_74057 Trire_60945swocel3c

cel61a

bxl1xyl3bxyn4

Trire_50984

cel3d

Genes differentiallyexpressed

CRZ1 BS

Absence ofCRZ1 BS

All genes evaluated

cel1bcel5b

Trire_56440

Trire_79202

A

B


https://doi.org/10.1101/622647


crz1 regulator and calcium cooperatively modulate holocellulases gene expression … · 12...

Documents