CRZ1 regulator and calcium cooperatively modulate holocellulases gene expression in Trichoderma reesei QM6a

Abstract Trichoderma reesei is the main filamentous fungus used in industry to produce cellulases. Here we investigated the role of CRZ1 and Ca2+signaling in the fungus T. reesei QM6a concerning holocellulases production. For this, we first searched for potential CRZ1 binding sites in promoter regions of key genes coding holocellulases, as well as transcriptional regulators and sugar and calcium transporters. Using a nearly constructed T. reeseiAcrz1 strain, we demonstrated that most of the genes expected to be regulated by CRZ1 were affected in the mutant strain induced with sugarcane bagasse (SCB) and cellulose. In particular, our data demonstrate that Ca2+ acts synergistically with CRZ1 to modulate gene expression, but also exerts CRZ1-independent regulatory role in gene expression in T. reesei, highlighting the role of the major regulator Ca2+ on the signaling for holocellulases transcriptional control in the most part of cellulases genes here investigated. This work presents new evidence on the regulatory role of CRZ1 and Ca2+ sensing in the regulation of cellulolytic enzymes in T. reesei, evidencing significant and previously unknown function of this Ca2+sensing system in the control key transcriptional regulators (XYR1 and CRE1) and on the expression of genes related to sugar and Ca2+ transport.

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  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 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 Ca 2+ -CaM/CN/CRZ1 pathway is poorly studied and established 93 in comparison to other fungal species. Yet, it is already known that Ca 2+ 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. (Additional file 1: Table S1 , Additional File 2: Figure S1 A), which are specific for  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

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Identification of CRZ1 binding motifs in T. reesei promoters 238 We first investigated the existence of putative binding sites for CRZ1 in T. reesei 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

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Since we observed that xyr1 expression was induced by Ca 2+ 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 Ca 2+ 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 Ca 2+ synergistically control the expression of celobiohydrolases coding 287 genes under SCB and Avicel exposure, and highlight that an additional Ca 2+ -dependent 288 and CRZ1-independent mechanism could take part in this process.

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Other cellulases genes are also key of regulation by CRZ1 and Ca 2+

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Since we observed that CRZ1 is capable to modulate xyr1 expression levels, we

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The Ca 2+ -CRZ1-independent but synergistic mechanism was also observed when increase on expression (Figure 5B,D) and finally, a positive modulation exclusively 320 mediated by CRZ1 which loses its potential in a Ca 2+ -synergic mechanism ( Figure 5C).

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Interestingly, after cellulose induction, the Ca 2+ -CRZ1-independent but synergistic 322 mechanism was capable to potentialize the expression of only cel7b gene (Additional 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, Ca 2+ 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 Ca 2+ for both carbon sources that we investigated.  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.

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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.