Mn²⁺ modulates the expression of cellulase genes in Trichoderma reesei Rut-C30 via calcium signaling

Abstract

Background

The filamentous fungus Trichoderma reesei Rut-C30 is one of the most vital fungi for the production of cellulases, which can be used for biofuel production from lignocellulose. Nevertheless, the mechanism of transmission of external stimuli and signals in modulating cellulase production in T. reesei Rut-C30 remains unclear. Calcium is a known second messenger regulating cellulase gene expression in T. reesei.

Results

In this study, we found that a biologically relevant extracellular Mn²⁺ concentration markedly stimulates cellulase production, total protein secretion, and the intracellular Mn²⁺ concentration of Rut-C30, a cellulase hyper-producing strain of T. reesei. Furthermore, we identified two Mn²⁺ transport proteins, designated as TPHO84-1 and TPHO84-2, indicating that they are upstream in the signaling pathway that leads to cellulase upregulation. We also found that Mn²⁺ induced a significant increase in cytosolic Ca²⁺ concentration, and that this increased cytosolic Ca²⁺ might be a key step in the Mn²⁺-mediated regulation of cellulase gene transcription and production. The utilization of LaCl₃ to block plasma membrane Ca²⁺ channels, and deletion of crz1 (calcineurin-responsive zinc finger transcription factor 1) to interrupt calcium signaling, showed that Mn²⁺ exerts the induction of cellulase genes via calcium channels and calcium signaling. To substantiate this, we identified a Ca²⁺/Mn²⁺ P-type ATPase, TPMR1, which could play a pivotal role in Ca²⁺/Mn²⁺ homeostasis and Mn²⁺ induction of cellulase genes in T. reesei Rut-C30.

Conclusions

Taken together, our results revealed for the first time that Mn²⁺ stimulates cellulase production, and demonstrates that Mn²⁺ upregulates cellulase genes via calcium channels and calcium signaling. Our research also provides a direction to facilitate enhanced cellulase production by T. reesei.

Background

Lignocellulosic biomass, the most abundant renewable energy source, can be hydrolyzed to sugars for bioethanol production. A common host cited for the production of cellulases and hemicellulases is the saprotrophic, filamentous fungus Trichoderma reesei, which is well known for its excellent ability to secrete a broad range of cellulases at very high levels [1,2,3,4]. Due to its ability to degrade and thrive on cellulose-containing fabrics, T. reesei has attracted attention and was consequently studied in detail [5, 6]. However, compared with the energy-efficient production of cellulases and hemicellulases, the induction and regulation of the expression of genes enoding these enzymes in T. reesei are still not completely understood. Additionally, the induction of high-level cellulase production is dependent on inducers such as cellulose, d-xylose, lactose, or sophorose [3, 7,8,9], increasing the costs for the application of produced enzymes. Due to the extensive applications of cellulases and hemicellulases, the induction and regulation of the expression of genes encoding these enzymes have drawn significant attention.

Recent studies have demonstrated that cellulase production is regulated in response to environmental stress, such as light [10, 11], organic solvents [12], and metal ions [13,14,15]. A great example was shown by Chen and co-workers, who illustrated that Ca²⁺ plays an important role in the production of cellulase or hemicellulase in T. reesei Rut-C30 [13]. Chen et al. [13] suggested that external Ca²⁺ stimulated hyphal growth, growth-independent cellulase production, and total protein secretion of T. reesei Rut-C30 through the Ca²⁺ (/calmodulin)–calcineurin–CRZ1 signal transduction pathway.

Intracellular accumulation of Mn²⁺ can interfere with calcium metabolism [16]. In Ganoderma lucidum, one of the most well-known medicinal basidiomycetes producing many bioactive compounds such as ganoderic acids, Mn²⁺ is thought to enhance cytosolic Ca²⁺ to induce ganoderic acid biosynthesis through the calcineurin signal pathway, to upregulate its biosynthetic genes at the transcriptional level [17]. In Aspergillus nidulans, high levels of Mn²⁺ can induce an increase in intracellular Ca²⁺ levels, which leads to the nuclear accumulation of CrzA [18, 19]. These observations suggest that Ca²⁺ and Mn²⁺ have relevant impacts on the cellular physiology and metabolism of various organisms.

The ability to sense and respond to Mn²⁺ by the production of import and efflux systems to maintain Mn²⁺ homeostasis is critical for cells [20,21,22,23]. Such homeostasis factors include cell surface and intracellular Mn²⁺ transporters that collectively guide the metal through a designated trafficking pathway [20, 22]. PMR1, a P-type ATPase ion pump, is a transporter for both Ca²⁺ and Mn²⁺ and is also a homeostasis factor, associating with delivering both Mn²⁺ and Ca²⁺ to the secretory pathway [22, 24,25,26]. Cytosolic Mn²⁺ accumulates in yeast cells lacking the PMR1 transporter [22]. Although many studies have reported on Mn²⁺ homeostasis in various organisms, the detailed mechanisms are yet unclear. First, studies have focused mainly on yeast or bacterial species, and less work has been conducted with filamentous fungi. Second, the detailed roles of Mn²⁺ in the biological processes of filamentous fungi remain unclear and need further studies. Third, the conjunction between Mn²⁺ and calcium signaling in filamentous fungi is still not clear. Therefore, it is necessary to study the mechanism of Mn²⁺ stimulation in filamentous fungi.

In this study, the impact of Mn²⁺ on the growth and protein production of T. reesei Rut-C30 was investigated. The temporal dynamics of intracellular and extracellular Mn²⁺ were detected. Additionally, the function of Mn²⁺ transport proteins in T. reesei Rut-C30 was characterized. The conjunction between Mn²⁺ and Ca²⁺ was further investigated to elucidate how Mn²⁺ regulates the production of cellulase via calcium signaling in T. reesei Rut-C30. These results could be used for more efficient production of cellulase by T. reesei, and provide a new approach to understand the regulatory mechanisms that respond to environmental stimuli. This research may also offer the basis for the study of Mn²⁺-induced signal transduction in other fungi.

Results

Effects of the addition of Mn²⁺ on growth and cellulase production of T. reesei

To determine how Mn²⁺ influences the hyphal growth, T. reesei Rut-C30 strains were cultured on MM (minimal medium) plates supplemented with different concentrations of Mn²⁺ (0, 1, 10, 20, and 40 mM final concentration) and 2% glucose as the sole carbon source. The mycelium length of T. reesei Rut-C30 after addition Mn²⁺ is shown in Fig. 1a. There was no significant difference in the hyphal growth with 1–10 mM Mn²⁺. However, when the concentrations of Mn²⁺ increased to 20 mM, the strains grew more slowly and sparsely. As shown in Fig. 1b, treatment with 20 mM Mn²⁺ caused a 30% reduction in the colony diameter compared with that of the untreated strains. Moreover, after treatment with 40 mM Mn²⁺, the treated strains showed a severe reduction in the colony diameter (53%).

Fig. 1

Effects of Mn²⁺ on hyphal growth and protein production in T. reesei Rut-C30. a The hyphal growth of T. reesei Rut-C30 on plates. Mn²⁺ was added at a final concentration of 1, 10, 20, or 40 mM. b The measurement of colony diameter. The effects of different concentrations of Mn²⁺ (final concentration 0, 1, 10, 20, and 40 mM) on CMCase activity (c), pNPCase activity (d), and total protein concentrations (e) of T. reesei Rut-C30. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate a significant difference compared to the untreated strain (*p < 0.05, Student’s t test)

To evaluate the effects of Mn²⁺ on cellulase production and total protein secretion, the same weight of precultured mycelia of T. reesei Rut-C30 was transferred to liquid MM containing 1% Avicel as the sole carbon source and different concentrations of Mn²⁺ (0, 1, 10, 20 and 40 mM). As shown in Fig. 1c (Additional file 8: Figure S7A), addition of Mn²⁺ at a final concentration of 10 mM significantly stimulated CMCase activity (representing endo-β-glucanase activity), with an increase of approximately 140% compared to the controls without addition of Mn²⁺. However, 1, 20, and 40 mM of Mn²⁺ did not evidently affect CMCase activity. As shown in Fig. 1d (Additional file 8: Figure S7B) and e, the addition of Mn²⁺ at a final concentration of 10–40 mM significantly stimulated pNPCase activity (representing exo-β-glucanase activity) and increased total protein concentration after 1 day of fermentation, with an increase of approximately 327 and 55%, respectively.

The above results demonstrated that 10–40 mM Mn²⁺ could stimulate cellulase production and total protein secretion in T. reesei Rut-C30, and that 20–40 mM Mn²⁺ could delay hyphal growth. The optimal concentration of Mn²⁺ to enhance cellulase production was 10 mM, which was selected for further research in our study. CMCase or pNPCase activity was directly used to represent cellulase activity in our study.

To further determine the effects of Mn²⁺ supplementation on the synthesis of cellulases or total protein secretion, the expression levels of four main cellulase genes (cbh1 encoding cellobiohydrolase I, cbh2 encoding cellobiohydrolase II, egl1 encoding endoglucanase I, and egl2 encoding endoglucanase II), and a transcriptional regulator of cellulases, xyr1, were compared by quantitative reverse-transcription PCR (RT-qPCR) after induction for 24, 48, and 72 h in cultures with 0 or 10 mM Mn²⁺ supplementation. The primers used to detect transcriptional changes of these genes are listed in Additional file 1: Table S1. The transcriptional levels of four main cellulase genes significantly increased by almost 2- to 3.5-fold, after 24, 48, and 72 h of induction following supplementation with 10 mM Mn²⁺ (see Additional file 2: Figure S1A–D). In T. reesei, XYR1 is a global transcriptional activator of cellulose and hemicellulase genes [27]. In accordance with the transcription of cbh1, cbh2 and egl1, egl2, the expression level of xyr1 was also significantly stimulated after 72 h of induction (see Additional file 2: Figure S1E). These results were consistent with the upregulation of cellulase activity through the addition of 10 mM Mn²⁺. However, when compared to expression of other cellulase-related genes, the delayed upregulation of xyr1 expression implied that other putative regulators may participate in Mn²⁺ metabolism/regulation to directly induce cellulase gene expression, besides indirect induction through xyr1.

Variation in intracellular and extracellular Mn²⁺ concentration after the addition of Mn²⁺

Extracellular Mn²⁺ can significantly augment the intracellular Mn²⁺ content, and subsequently affect the physiology and metabolism of G. lucidum [17]. To investigate whether enhanced cellulase production is linked to intracellular Mn²⁺ in T. reesei Rut-C30, the intracellular and extracellular Mn²⁺ concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS) during cultivation. As illustrated in Fig. 2a, the levels of intracellular and extracellular Mn²⁺ were almost constant in the control sample without Mn²⁺ addition. On the contrary, upon Mn²⁺ addition (10 mM final concentration), the intracellular Mn²⁺ concentration initially markedly increased, reaching its maximum at 24 h and then declined gradually, while the extracellular Mn²⁺ concentration dropped initially, reaching its minimum at 24 h and then increased gradually, suggesting that Mn²⁺ was transported into cells initially in response to a higher extracellular Mn²⁺ concentration. Subsequently, intracellular Mn²⁺ gradually effused into the medium from 24 h. We hypothesized that a mechanism could pump Mn²⁺ in and out of the cell.

Fig. 2

Concentrations of Mn²⁺ in T. reesei Rut-C30 and its derivative mutants. a Filled circle, intracellular Mn²⁺ concentration after adding 10 mM MnCl₂; blank circle, extracellular Mn²⁺ concentration after adding 10 mM MnCl₂; filled square, intracellular Mn²⁺ concentration of control without MnCl₂ supplement; blank square, extracellular Mn²⁺ concentration of control without MnCl₂ supplement. b The concentrations of intracellular Mn²⁺ of T. reesei Rut-C30 and its derivative mutant strains were examined after cultured in medium containing 10 mM MnCl₂. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from parental strain Rut-C30 (*p < 0.05, Student’s t test)

Identification of Mn²⁺ transport proteins TPHO84-1 and TPHO84-2

Jensen et al. [16] suggested that PHO84, a low-affinity transporter of Mn²⁺ from Saccharomyces cerevisiae, transports Mn²⁺ when cells are exposed to higher Mn²⁺ concentrations. In our study, Mn²⁺ efficiently transported into T. reesei Rut-C30 cells at an Mn²⁺ concentration of 10 mM (Fig. 2a). To identify the protein(s) responsible for transporting Mn²⁺ in T. reesei, we conducted a homology search with the protein sequence of PHO84 (GenBank: KZV08715.1) in the T. reesei genome from the JGI database (http://genome.jgi.doe.gov/Trire2/Trire2.home.html). Five high score hits were obtained with the following protein identity matches: TRE77552, 59.6%, TRE81389, 51.8%, TRE45852, 49.0%, TRE45868, 40.1%, and TRE106118, 35.2%.

We hypothesized that Mn²⁺ transport proteins would be highly expressed during Mn²⁺ addition. To assess which proteins encoded putative Mn²⁺ transport activity, transcriptional levels of tre77552, tre81389, tre45852, tre45868 and tre106118 were monitored by RT-qPCR with 10 mM Mn²⁺ compared to no Mn²⁺ addition at different induction times. As illustrated in Additional file 3: Figure S2, expression of these genes was significantly upregulated in the samples treated with 10 mM Mn²⁺. Based on their induction at higher Mn²⁺ concentration, all five genes might be responsible for Mn²⁺ transport of T. reesei Rut-C30.

To further investigate whether any of these five proteins displayed a function similar to that of PHO84, involved in transport of Mn²⁺ in S. cerevisiae, the five deletion mutants, Δ77552, Δ81389, Δ45852, Δ45868, and Δ106118, respectively, were collected to measure intracellular Mn²⁺ concentrations by ICP-MS compared to that in the parental strain. As shown in Fig. 2b, Δ77552 and Δ45868 showed a marked decrease in steady-state levels of cellular Mn²⁺ when compared with the Rut-C30 strain, in which a 70–80% decrease in metal accumulation was obtained. However, under the same conditions, Δ81389, Δ45852, or Δ106118 had an Mn²⁺ concentration similar to that in Rut-C30 strain (see Additional file 4: Figure S3). The tre77552 and tre45868 double mutant strain Δtpho84-1/2 showed lower intracellular Mn²⁺ concentration than each single mutant (Fig. 2b). Δ77552 and Δ45868 were also complemented by transforming vectors ptpho84-1-rc and ptpho84-2-rc into them, respectively (see Additional file 5: Figure S4). Complementation strains (tpho84-1-rc and tpho84-2-rc) were obtained to demonstrate the restoration of Mn²⁺ transport with intracellular Mn²⁺ concentrations similar to those of the parent strain Rut-C30 (Fig. 2b). The results demonstrated that the intracellular Mn²⁺ concentration can be increased via putative Mn²⁺ transport proteins TRE77552 and TRE45868 in T. reesei Rut-C30.

As predicted by SMART (http://smart.embl-heidelberg.de/), both TRE77552 and TRE45868 are membrane proteins with an 11- and 9-transmembrane domain topology, respectively. To visualize the location of TRE77552 and TRE45868, we constructed two chimeric proteins, RFP-77552 and RFP-45868, by fusing red fluorescence protein to their N-terminus (see Additional file 6: Figure S5A). The two chimeric proteins were overexpressed via cbh1 promoter, which allowed us to confirm that TRE77552 and TRE45868 are located at the mycelial surface. Additional file 6: Figure S5B shows the in vivo epifluorescence analysis of the two chimeric protein (RFP-77552 and RFP-45868) transformants, rfp-tpho84-1 and rfp-tpho84-2, respectively, depicting strong and stable fluorescent signal at the mycelial surface, as expected for plasma membrane proteins. However, overexpression of genes encoding membrane bound proteins by a strong promoter might cause some uncertainty.

Based on these findings, we considered that both tre77552 and tre45868 encode Mn²⁺ transports with Mn²⁺ transport function located at the plasma membrane, and named the two genes as tpho84-1 and tpho84-2, respectively. Intracellular Mn²⁺ can be transported into T. reesei Rut-C30 cells via TPHO84-1 and TPHO84-2. It is presently unclear whether tre81389, tre45852, and tre106118 are inactive in the transport of Mn²⁺.

Role of TPHO84-1 and TPHO84-2 in cellulase production

Δ77552 (Δtpho84-1) and Δ45868 (Δtpho84-2), and their complementation strains tpho84-1-rc and tpho84-2-rc were used to determine the effect of TPHO84-1 and TPHO84-2 in mediating the growth and cellulase production in T. reesei Rut-C30. To determine the effect of TPHO84-1 and TPHO84-2 on mediating growth, Rut-C30, Δtpho84-1, Δtpho84-2, tpho84-1-rc, tpho84-2-rc and Δtpho84-1/2 strains were cultured on minimal medium plates adding 0 or 10 mM Mn²⁺ and 2% glucose as the sole carbon source. The mycelium morphology is shown in Fig. 3a. There was no significant effect on the hyphal growth of Rut-C30, Δtpho84-1, Δtpho84-2, tpho84-1-rc and tpho84-2-rc strains at 0 or 10 mM Mn²⁺. However, the hyphal growth of the Δtpho84-1/2 transformant was slightly repressed, compared with that of the parent strain at both 0 and 10 mM Mn²⁺ (Fig. 3b).

Fig. 3

TPHO84-1 and TPHO84-2 regulate cellulase production of T. reesei Rut-C30 with Mn²⁺ supplementation. a The hyphal growth of T. reesei Rut-C30 and its derivative mutant strains under 0 or 10 mM MnCl₂. b The measurement of colony diameter. The CMCase activity (c) and pNPCase activity (d) of T. reesei Rut-C30 and its derivative mutant strains were examined after culture in medium containing 0 or 10 mM MnCl₂. The transcriptional levels of cbh1 (e) and egl1 (f) in the parental strain Rut-C30 and its derivative mutant strains were analyzed after cultured in medium containing 0 or 10 mM MnCl₂. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences (*p < 0.05, Student’s t test)

To further test the effect of Mn²⁺ transport proteins on cellulase production, we investigated the effect of Rut-C30, Δtpho84-1, Δtpho84-2, tpho84-1-rc, tpho84-2-rc, and Δtpho84-1/2 strains on cellulase production after supplementation of 0 or 10 mM Mn²⁺. There was no obvious difference in CMCase (see Additional file 7: Figure S6A) and pNPCase (see Additional file 7: Figure S6B) activities between these six strains without Mn²⁺ addition. However, Δtpho84-1 and Δtpho84-2 strains showed approximately 30–40% reduction in CMCase activity, compared with the parental strain Rut-C30 with 10 mM Mn²⁺ addition. Furthermore, Δtpho84-1/2 strain had lower CMCase activity than each single mutant, at approximately 50% reduction compared with Rut-C30 (Fig. 3c and Additional file 8: Figure S8A). Similarly, Δtpho84-1 and Δtpho84-2 strains showed approximately 25 to 30% reduction in the pNPCase activity, compared with the parental strain Rut-C30, with 10 mM Mn²⁺ addition. Furthermore, the Δtpho84-1/2 strain showed lower pNPCase activity than each single mutant, and approximately 40% reduction compared with Rut-C30 (Fig. 3d and Additional file 8: Figure S8B). The cellulase production capabilities of tpho84-1-rc and tpho84-2-rc were complemented at 10 mM Mn²⁺ supplementation compared to parental strains Δtpho84-1 and Δtpho84-2, respectively, to a level similar to that of the original strain Rut-C30 (Fig. 3c, d and Additional file 8: Figure S8A, B).

Additionally, RT-qPCR was performed to determine the transcription levels of the important cellulase genes cbh1 and egl1 in the T. reesei Rut-C30, Δtpho84-1, Δtpho84-2, tpho84-1-rc, tpho84-2-rc, and Δtpho84-1/2 mutants. The primers used to detect transcriptional levels of these genes are listed in Additional file 1: Table S1. In agreement with the CMCase activity levels, no obvious difference in transcription levels of cbh1 and egl1 between above six strains was observed without Mn²⁺ addition (data not shown). However, the deletion strains Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 showed a marked reduction in cellulase transcription compared with the parental strain Rut-C30 upon 10 mM Mn²⁺ addition at all time points examined (Fig. 3e, f). Similarly, the expression levels of the cbh1 and egl1 genes of tpho84-1-rc and tpho84-2-rc were complemented under Mn²⁺ supplementation, to a level similar to that of the original strain Rut-C30.

These results indicate that TPHO84-1 and TPHO84-2 participate in inducing cellulase production of T. reesei Rut-C30 only under Mn²⁺ addition, which is in accordance with its function as Mn²⁺ transport protein.

Increase in cytosolic Ca²⁺ level and calcium signaling after Mn²⁺ addition

Next, we investigated how Mn²⁺ addition can upregulate cellulase gene expression via Mn²⁺ transport proteins TPHO84-1 and TPHO84-2. Using Fluo-3/AM fluorescent dye, a dye that only emits green fluorescence after crossing the cell membrane and binding with Ca²⁺ [28], whose intensity represents relative amounts of free intracellular Ca²⁺ [29], we found that the cytosolic Ca²⁺ concentration was increased after Mn²⁺ addition. As shown in Fig. 4A, a stronger green fluorescence intensity was observed in the Rut-C30 cells under 10 mM Mn²⁺ supplement on the second day than that observed with the control (no Mn²⁺ supplement), demonstrating that Mn²⁺ leads to an increase in the level of cytosolic Ca²⁺. The fluorescence level emitted by the Ca²⁺-activated fluorochrome reached a 2.23-fold increase under 10 mM Mn²⁺ addition compared to that in the control (Fig. 4B). These results indicate that Mn²⁺ induced an increase in the concentration level of cytosolic Ca²⁺. A similar phenomenon was reported in S. cerevisiae [30].

Fig. 4

Cytosolic Ca²⁺ levels and calcium signaling increase after Mn²⁺ addition. A The analysis of cytosolic Ca²⁺ levels via a Ca²⁺ fluorescent probe Fluo-3/AM. The T. reesei Rut-C30 and its derivative mutant strains were cultured in liquid minimal medium for 48–60 h with 0 or 10 mM final concentration of MnCl₂ (0 or 10 Mn, respectively). For detection, 50 μM Fluo-3/AM was used, and the intensity was monitored using Automatic Inverted Fluorescence Microscopy. Green fluorescence represents the free cytosolic Ca²⁺. DIC, differential interference contrast. B Comparative fluorescence ratio analysis of Mn²⁺ influence on cytosolic Ca²⁺ levels. The y-axis represents the Ca²⁺ fluorescence ratio measured by CLSM and the x-axis the different strains tested. The effects of Mn²⁺ on the expression levels of certain calcium signaling-related genes cam (C), cna1 (D) and crz1 (E) in T. reesei Rut-C30. 0 Mn, no MnCl₂ was added to the medium; 10 Mn, final concentration of 10 mM MnCl₂. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from untreated strains (*p < 0.05, **p < 0.01, Student’s t test). Different letters indicate significant differences between the columns (p < 0.05, according to Duncan’s multiple-range test)

We measured the level of cytosolic Ca²⁺ in the Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 strains with Mn²⁺ addition. As illustrated in Fig. 4A, B, the significant increase in intracellular Ca²⁺ induced by Mn²⁺, observed in parental strain Rut-C30, was absent in the Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 strains. The augmentation of cytosolic Ca²⁺ levels induced by Mn²⁺ was blocked in the Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 strains. These results suggest that rising cytosolic Ca²⁺ levels depend on Mn²⁺ transport into Rut-C30 cells via TPHO84-1 and TPHO84-2. This also indicates that Ca²⁺ influx in cells is closely associated with Mn²⁺ homeostasis.

Previous studies have demonstrated that the calcium signal transduction pathway can upregulate cellulase gene expression [13]. We investigated whether the increased levels of cytosolic Ca²⁺, induced by Mn²⁺, can trigger calcium signal transduction pathways in T. reesei. To test our hypothesis, RT-qPCR was carried out to analyze the transcriptional levels of calcium signaling-related genes, including calmodulin (cam, GenBank: ACZ26150.1), calcineurin (cna1, GenBank: EGR49476.1) [31], and calcineurin-responsive zinc finger transcription factor 1, crz1 [13], under Mn²⁺ addition. The primers used to detect transcription of these genes are listed in Additional file 1: Table S1. As shown in Fig. 4C–E, similar to the increase in the contents of cytosolic Ca²⁺, expression of these genes was significantly upregulated with Mn²⁺ addition. These results suggested that Mn²⁺ can increase the concentration of cytosolic Ca²⁺, thus stimulating the calcium signal transduction pathway to induce cellulase production in T. reesei Rut-C30.

Mn²⁺ induces cellulase production via cytosolic Ca²⁺

To investigate the assumption that Mn²⁺ induces cytosolic Ca²⁺ improvement and cellulase production via Ca²⁺ channels, we used LaCl₃, a plasma membrane Ca²⁺ channel blocker to prevent influx of external Ca²⁺ [32]. Figure 5A, B shows that the Fluo-3/AM fluorescence intensity of mycelia remarkably reduced almost 60% with LaCl₃ compared with no LaCl₃ addition, under 10 mM Mn²⁺. The increased content of cytosolic Ca²⁺ induced by Mn²⁺ could be effectively attenuated by adding LaCl₃ to T. reesei Rut-C30. Meanwhile, the increased expression of calcium signaling-related genes, cam, cna1, and crz1, which are induced by 10 mM Mn²⁺, was also effectively prevented by adding LaCl₃ (data not shown).

Fig. 5

Effects of Ca²⁺ channel inhibitor LaCl₃ on the cytosolic Ca²⁺ concentration and cellulase production. A Fluorescence analysis of LaCl₃ influence on cytosolic Ca²⁺ burst induced by Mn²⁺. The T. reesei Rut-C30 were cultured in liquid minimal medium for 48–60 h with 0 or 10 mM MnCl₂ (0 or 10 Mn, respectively), and then treated with 0 or 5 mM LaCl₃. For detection, 50 μM Fluo-3/AM was used, and the intensity was monitored using Automatic Inverted Fluorescence Microscopy. Green fluorescence represents the free cytosolic Ca²⁺. DIC, differential interference contrast, CK, not treated with LaCl₃. B Comparative fluorescence ratio analysis of LaCl₃ influence on the cytosolic Ca²⁺ burst induced by Mn²⁺. The y-axis represents the Ca²⁺ fluorescence ratio measured by CLSM and the x-axis the different treatments. The CMCase activity (C) and pNPCase activity (D) of T. reesei Rut-C30 were examined after culture in medium containing 0 or 10 mM MnCl₂ and with (−) or without (+) 5 mM LaCl₃. The expression levels of cbh1 (E) and egl1 (F) in T. reesei Rut-C30 were analyzed after culture in medium containing 0 or 10 mM MnCl₂ and with (−) or without (+) 5 mM LaCl₃. Values are the mean ± SD of the results from three independent experiments. Different letters indicate significant differences between the columns (p < 0.05, according to Duncan’s multiple-range test)

Ca²⁺ participates in regulating cellulase production in T. reesei [13]. To investigate whether a cellulase increase, induced by Mn²⁺, was blocked by LaCl₃, we analyzed CMCase and pNPCase activities and transcription of key cellulase genes cbh1 and egl1. As shown in Fig. 5C, D (Additional file 8: Figure S9A, B), under Mn²⁺ supplementation and with LaCl₃, the CMCase and pNPCase activities in T. reesei Rut-C30 decreased by 50 and 49%, respectively, compared with no LaCl₃ addition. Meanwhile, the expression levels of cbh1 and egl1 were reduced by 60% at 72 h (Fig. 5E, F). However, there was no obvious change to CMCase activities and transcriptional levels of cbh1 and egl1 with or without LaCl₃ when Mn²⁺ was not added (Fig. 5C–F, Additional file 8: Figure S9A, B). These results showed that LaCl₃ could significantly decrease the Mn²⁺-induced high expression levels of key cellulase genes in T. reesei Rut-C30.

These data indicated that Mn²⁺ induced cytosolic Ca²⁺ increase via the Ca²⁺ channel. When a Ca²⁺ channel blocker LaCl₃ was added, the increase in cytosolic Ca²⁺ concentration and cellulase production induced by Mn²⁺ were effectively attenuated.

To investigate whether a cellulase increase, induced by Mn²⁺, is associated with calcium signal transduction, we constructed a crz1 deletion mutant Δcrz1 as Chen et al. [13] to block the calcium signal transduction pathway. As shown in Fig. 6a, b (Additional file 8: Figure S10A, B), the remarkable increase of CMCase and pNPCase activities induced by Mn²⁺, observed in parental strain Rut-C30, was effectively attenuated by deleting crz1. Similarly, the transcriptional levels of cbh1 and egl1 were markedly reduced in the Δcrz1 mutant at all time points examined (Fig. 6c, d).

Fig. 6

Influence of CRZ1 on Mn²⁺-induced cellulase production. The CMCase activity (a) and pNPCase activity (b) of T. reesei Rut-C30 and Δcrz1 strains supplemented with 0 or 10 mM MnCl₂. The expression levels of cbh1 (c) and egl1 (d) in T. reesei Rut-C30 and Δcrz1 strains supplemented with 0 or 10 mM MnCl₂. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences (*p < 0.05, Student’s t test)

Taken together, these data indicated that Mn²⁺ induces cellulase improvement via calcium signal transduction in T. reesei Rut-C30. The improvement of cellulase production induced by Mn²⁺ is effectively prevented in a crz1 mutant, blocking the calcium signal transduction pathway.

TPMR1 functions in conjunction with channels of Mn²⁺ and Ca²⁺

Mn²⁺/Ca²⁺ homeostasis exists in other fungi [33,34,35,36]. PMR1 plays an important role in Mn²⁺/Ca²⁺ homeostasis, and may act as a Ca²⁺/Mn²⁺ exchanger to balance Mn²⁺ via Ca²⁺ [17]. To clarify how Mn²⁺ increased cytosolic Ca²⁺ concentration, using Neurospora crassa PMR1 (GenBank: CAB65296.1) as the query, we searched for a PMR1 homolog (TRE119592) in the T. reesei genome, named as TPMR1.

We constructed a tpmr1 deletion mutant Δtpmr1. To investigate whether TPMR1 is responsible for pumping in Ca²⁺ upon 10 mM Mn²⁺ addition in T. reesei, we measured the level of intracellular Ca²⁺ in the Δtpmr1 strain with or without Mn²⁺ addition. As shown in Fig. 7A and B, the increase in intracellular Ca²⁺ induced by Mn²⁺ in Rut-C30 was absent in the Δtpmr1 strain. The augmentation of Ca²⁺ levels induced by Mn²⁺ was blocked in the Δtpmr1 strain. These results suggested that TPMR1 is responsible for the increase of cytosolic Ca²⁺ under Mn²⁺ addition in T. reesei Rut-C30.

Fig. 7

Influence of TPMR1 on Mn²⁺-induced cytosolic Ca²⁺ burst and cellulase production. A Fluorescence analysis of the influence of TPMR1 on the cytosolic Ca²⁺ burst induced by Mn²⁺. The T. reesei Rut-C30 and Δtpmr1 strains were cultured in liquid minimal medium for 48–60 h with 0 or 10 mM MnCl₂ (0 or 10 Mn, respectively). For detection, 50 μM Fluo-3/AM was used, and the intensity was monitored using Automatic Inverted Fluorescence Microscopy. Green fluorescence represents the free cytosolic Ca²⁺. DIC, differential interference contrast. B Comparative fluorescence ratio analysis of TPMR1 influence on cytosolic Ca²⁺ burst induced by Mn²⁺. The y-axis represents the Ca²⁺ fluorescence ratio measured by CLSM and the x-axis the different treatments. The CMCase activity (C) and pNPCase activity (D) of T. reesei Rut-C30 and Δtpmr1 strains were examined after culture in medium containing 0 or 10 mM MnCl₂. The expression levels of cbh1 (E) and egl1 (F) in T. reesei Rut-C30 and Δtpmr1 strains were analyzed after culture in medium containing 0 or 10 mM MnCl₂. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from untreated strains (*p < 0.05, **p < 0.01, Student’s t test). Different letters indicate significant differences between the columns (p < 0.05, according to Duncan’s multiple-range test)

To further investigate the function of TPMR1 in conjunction with Mn²⁺ and Ca²⁺ channels, the intracellular and extracellular Mn²⁺ concentrations in the Δtpmr1 strain with or without Mn²⁺ addition were measured by ICP-MS during cultivation. As illustrated in Additional file 9: Figure S12, upon Mn²⁺ addition (10 mM final concentration), the intracellular Mn²⁺ concentration initially markedly increased, reaching and maintaining a high level after 24 h. The extracellular Mn²⁺ concentration dropped initially, reaching its minimum from 24 to 72 h. The intracellular and extracellular Mn²⁺ concentrations of the Δtpmr1 strain (Additional file 9: Figure S12) are quite different from that of Rut-C30 (Fig. 2a), whose intracellular Mn²⁺ gradually effused into the medium. These results suggested that TPMR1 is responsible for pumping Mn²⁺ out of the cell.

To further test the effect of TPMR1 on cellulase production under Mn²⁺ addition, we compared the cellulase production in parental strain Rut-C30 and Δtpmr1 mutant. Upon the addition of Mn²⁺, the Δtpmr1 strain caused about 26 and 51% reductions in the CMCase and pNPCase activities, respectively, compared with that of Rut-C30 (Fig. 7C, D and Additional file 8: Figure S11A, B). Moreover, the transcriptional levels of cbh1 and egl1 observed are in agreement with the CMCase and pNPCase activity levels, which were markedly reduced in the Δtpmr1 mutant at all time points examined (Fig. 7E, F). The increase in cellulase production induced by Mn²⁺ was remarkably weakened in the tpmr1 deletion mutant. These results demonstrated that TPMR1 functions in conjunction with channels of Mn²⁺ and Ca²⁺ in T. reesei Rut-C30.

Discussion

Metal ions are important in regulating cellular metabolism. For example, Ca²⁺ plays an important role in the regulation of cellulase or hemicellulase in T. reesei [13,14,15]. Paraszkiewicz et al. [37] reported Cd²⁺, Zn²⁺, and Pb²⁺ as environmental stress factors, which increased the biosynthesis of fungal emulsifier in Curvularia lunata. Addition of Ca²⁺, Na⁺, and Mn²⁺ enhanced ganoderic acid production in Ganoderma lucidum liquid cultures, through induction of the calcineurin signal pathway [17, 29, 38]. These results suggest that metal ions have significant impact on the cellular physiology and metabolism of various organisms. In this study, we first report that Mn²⁺ stimulates cellulase production and total protein secretion of T. reesei Rut-C30. However, whether other metal ions or stimuli have an impact on cellulase production of T. reesei will require further evidence for confirmation.

Diverse Mn²⁺ transports have been identified in the yeasts, plants, and bacteria [21, 22, 39,40,41,42]. Mn²⁺ can enter the cell through two different routes including high- and low-affinity transporters [21, 22, 41, 42]. SMF1, a plasma membrane NRAMP (Natural Resistance-Associated Macrophage Protein) family Mn²⁺ transporter, is a high-affinity transporter for Mn²⁺, contributing to Mn²⁺ accumulation under extreme Mn²⁺ starvation conditions [16, 22, 43, 44]. PHO84, a family of phosphate/proton symporters [45], can transport Mn²⁺ under excess Mn²⁺ stress [16, 25], indicating that PHO84 is a low-affinity transporter of Mn²⁺, contributing to Mn²⁺ accumulation only when cells are exposed to higher Mn²⁺ concentrations. T. reesei might contain both high- and low-affinity transporters of Mn²⁺, similar to what has been described for the metal transport systems for iron, copper, and zinc [46,47,48,49,50]. A BLAST search of the genome sequence of T. reesei identified a gene (tre79644) similar to SMF1, and this gene was named tsmf1. There was no significant difference on Mn²⁺ transport and cellulase production under 10 mM Mn²⁺ surplus using a tsmf1 deletion strain (data not shown). The alternative PHO84 homologs remain to be identified in T. reesei Rut-C30. Five candidates including TPHO84-1 and TPHO84-2 are similar to PHO84. We proved that extracellular Mn²⁺ is transported into Rut-C30 cells via TPHO84-1 and TPHO84-2, which are annotated as putative low-affinity transports of Mn²⁺ responding to higher Mn²⁺ concentration. Meanwhile, there were no significant differences between the cytosolic Ca²⁺ concentrations for the T. reesei Rut-C30, Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 mutants (see Additional file 10: Figure S13).

Calcium is widely used as a second messenger in prokaryotic and eukaryotic cells. It is known that a cytosolic Ca²⁺ burst and further induced calcium signaling regulate cellular responses when exposed to different external stimuli [51, 52]. For instance, Na⁺ induction enhances cytosolic Ca²⁺ to induce the ganoderic acid biosynthesis through calcineurin signal pathway, to upregulate its biosynthetic genes at the transcriptional level in Ganoderma lucidum [29]. High temperatures are known to trigger the generation of cytosolic Ca²⁺ in plants [53]. Alkaline pH triggers an immediate calcium burst in Candida albicans [54]. Our results indicated that Mn²⁺ induces a significantly increased cytosolic Ca²⁺ level in T. reesei Rut-C30 (Fig. 4A, B). Additionally, in our experiments, inhibition of cytosolic Ca²⁺ level by LaCl₃ effectively attenuated the cellulase increase induced by Mn²⁺ (Fig. 5C–E). The results showed that 10 mM Mn²⁺ stress led to a cytosolic Ca²⁺ burst in T. reesei Rut-C30. An earlier study demonstrated that Ca²⁺ (/calmodulin)–calcineurin–CRZ1 signaling could induce cellulase production at the transcription level by Ca²⁺ stimulation [13]. In our study, Mn²⁺ could also activate the expression of Ca²⁺ (/calmodulin)–calcineurin–CRZ1 signaling-related genes in T. reesei Rut-C30 (Fig. 4C–E). Furthermore, CRZ1 participated in regulating cellulase production in Mn²⁺-induced strains (Fig. 6a–d). The results presented here indicate that the regulation of cellulase gene expression and production by Mn²⁺ are dependent on cytosolic Ca²⁺ burst and further induce calcium signaling.

PMR1, encoding the Ca²⁺/Mn²⁺ P-type ATPase, is required to either scavenge trace amounts of Mn²⁺ and Ca²⁺ from the medium or maintain sufficient levels of Mn²⁺ and Ca²⁺ in an intracellular compartment [22, 24,25,26, 33]. Bowman et al. [33] reported that a pmr1 deletion strain accumulates 80% lesser Ca²⁺ than the wild type. Under higher levels of intracellular Mn²⁺, extracellular Ca²⁺ might transport into the cells through PMR1 to increase intracellular Ca²⁺ and then trigger calcium signaling to regulate cellular responses [35]. Our work found that the increase in Ca²⁺ levels induced by Mn²⁺ treatment was blocked in the tpmr1 deletion strain (Fig. 7A, B) and that the improvement of cellulase production induced by Mn²⁺ was remarkably weakened in the tpmr1 deletion mutant. These results demonstrated that TPMR1 functions as a channel of Mn²⁺ and Ca²⁺ in T. reesei Rut-C30. Cytosolic Ca²⁺ concentration was enhanced through TPMR1 under Mn²⁺ addition, thus inducing calcium signaling to upregulate cellulase genes. Additionally, PMR1 provides a major route for cellular sequestration of Mn²⁺ by pumping excess Mn²⁺ into the Golgi, from where the metal may exit the cell via the secretory pathway vesicles that merge with the cell surface and release the Mn²⁺ contents back into the extracellular environment [20, 22, 24, 34]. These data correlate well with our data indicating that intracellular Mn²⁺ gradually effused to the medium from 24 h (Fig. 2a). However, in the Δtpmr1 mutant, the intracellular Mn²⁺ concentration maintained its high level after 24 h, and the extracellular Mn²⁺ concentration reached its minimum from 24 to 72 h. These results implied that TPMR1 participates in pumping excess Mn²⁺ into the Golgi, and then releases it extracellularly, meanwhile accumulating cytosolic Ca²⁺ in T. reesei Rut-C30. However, the detailed role of TPMR1 in T. reesei needs further research.

We found that 10 mM Mn²⁺ could also stimulate cellulase production and increase total protein secretion from the T. reesei wild-type strain QM6a and the mutant strain Qm9414 (data not shown). However, the results from Rut-C30 may not be identical to those of the wild-type strain, because each strain likely has its own unique regulatory mechanism.

Conclusions

In summary, the putative mechanism of the extracellular Mn²⁺-induced stimulation of cellulase production was characterized in T. reesei Rut-C30 (Fig. 8). Mn²⁺ induces a significantly increased cytosolic Ca²⁺ level and triggers Ca²⁺-CRZ1 signaling to induce cellulase production at the transcription level. Moreover, we identified two Mn²⁺ transport proteins in T. reesei Rut-C30, and named TPHO84-1 and TPHO84-2. Furthermore, TPMR1 acts as a link between channels in Mn²⁺ and Ca²⁺ homeostasis in T. reesei. This study provides a successful approach to produce a higher yield of cellulase and to develop industrially applicable T. reesei strains, which is important for biofuel production from lignocelluloics. This study also provides a molecular basis for understanding the regulatory mechanism of divalent metal ions on the cellular metabolism of fungi.

Fig. 8

A mechanistic model of the Mn²⁺ stimulation of cellulase production in T. reesei. After addition of 10 mM MnCl₂, the intracellular Mn²⁺ content increases via Mn²⁺ transport proteins Tpho84-1 and Tpho84-2. Intracellular Mn²⁺ promotes a cytosolic Ca²⁺ burst that is required for cellulase gene transcription via Ca²⁺ signaling. After using LaCl₃ (plasma membrane Ca²⁺ channels blocker), we suggest that Ca²⁺ channels are responsible for the cytosolic Ca²⁺ burst and cellulase production induced by Mn²⁺. Furthermore, TPMR1 is one of the links between the channels of Mn²⁺ and Ca²⁺, which may function as a Mn²⁺/Ca²⁺ exchanger to regulate Mn²⁺ and Ca²⁺ homeostasis under Mn²⁺ stress. Mn²⁺ could also employ other as-yet-unidentified pathways to regulate cellulase production. The solid arrows indicate data supported by our own experiments; dashed arrows indicate undefined regulation

Methods

Strains and growth conditions

Escherichia coli DH5α was used for plasmid amplification. Agrobacterium tumefaciens strain AGL-1 was used as a T-DNA donor for fungal transformation [55]. T. reesei Rut-C30 (ATCC 56765) was used throughout the study and as the host for genetic transformation. Luria–Broth (LB) was used for culture of E. coli and A. tumefaciens. Mandels’ medium [56] was used for the general fungal culture. All strains were maintained on potato dextrose agar (PDA) plates at 28 °C. The fungal strains constructed in this study are summarized in Table 1. All strains were cultured in the dark.

Table 1 T. reesei strains constructed in this study

Minimal medium (MM, (NH₄)₂SO₄ 5 g/l; Urea 0.3 g/l; KH₂PO₄ 15 g/l; CaCl₂ 0.6 g/l; MgSO₄ 0.6 g/l; FeSO₄·7H₂O 5 mg/l; ZnSO₄·7H₂O 1.4 mg/l; CoCl₂·6H₂O 2 mg/l; pH 5.5) with 2% glucose was used to assess the effect of Mn²⁺ on hyphal growth. To analyze the effects of Mn²⁺ on cellulase activity, protein concentration, and gene expression levels, medium replacement experiments were performed. After growth in Mandels’ medium with 2% glucose for ~ 32 h at 28 °C and 220 rpm, approximately 0.1 g of mycelia were collected and washed thoroughly using 0.85% NaCl, then transferred to 100 ml MM containing 1% (w/v) Avicel (PH-101, Sigma-Aldrich) with the addition of MnCl₂ to final concentrations of 1, 10, 20, and 40 mM. Strains were induced for 1–5 days before being subjected to testing for enzymatic activity, protein concentration, or induced for 24, 48, or 72 h before being subjected to RNA extraction and RT-qPCR analyses, respectively. To assess the effect of plasma membrane Ca²⁺ channels on the regulation of cellulase production of T. reesei Rut-C30, 5 mM (final concentration) LaCl₃ (Aladdin, Shanghai, China) was added after 1 day of culture in MM.

Fungal growth, enzymatic activity, protein concentration, and biomass assays

For fungal hyphal growth assays, conidia were collected and diluted to 10⁷ ml⁻¹ in sterile water. An equal volume of the solution (2 μl) was inoculated onto the center of the MM plates as described above, and was grown for 3–5 days at 28 °C.

For enzymatic activity, protein concentration, and biomass assays, 1 ml of culture liquid was collected and subjected to 0.45-μm filtration. The culture supernatants were subjected to cellulase activity and protein concentration analysis. The mycelia were subjected to biomass measurement. Fungal CMCase and pNPCase activities were measured according to the method described by Wang et al. [57]. Protein concentrations were determined using the Bradford Protein Assay Kit (Generay, Shanghai, China). Biomass concentration was indirectly measured by calculating the amount of total intracellular proteins, with some modification [58]. Briefly, harvested mycelia were suspended in 1 ml 1 M NaOH in a reaction tube and the mixture was incubated for 2 h and frequently vortexed. Total protein was collected via centrifugation at 14,000×g at 4 °C for 10 min. Total protein concentration was determined by the Modified Lowry Protein Assay Kit (Sangon Biotech, Shanghai, China). The final protein content was furthermore corrected using a set of substrate controls where no inoculum was added to the medium. The biomass dry weight was then calculated assuming an average content of 0.32 g intracellular protein per gram of dry cell mass.

RNA isolation and quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR)

The levels of gene-specific mRNA were assessed using RT-qPCR, according to our previous study, with some modification [59]. In brief, the total RNA of 50 mg fresh weight cells was extracted using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA), according to the manufacturer’s instructions. Synthesis of cDNA from total RNA was performed using the PrimeScript RT Reagent Kit with gDNA eraser (TaKaRa, Japan) as per the manufacturer’s instructions. For RT-qPCR, the TransStart TipTop Green qPCR SuperMix (TransGen, Shanghai, China) was used with 200 nM of forward and reverse primers (see Additional file 1: Table S1). Gene transcription was analyzed using SYBR green assays. Transcription levels of target genes were normalized to that of the sar1 gene [60]. Thermocycling was performed in an ABI StepOne Plus thermocycler (Applied Biosystems, Foster City, CA, USA).

Determination of extracellular and intracellular Mn²⁺ concentration

Extracellular and intracellular Mn²⁺ concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS), as described for Ganoderma lucidum by Xu [17]. Five milliliter of culture liquid was collected and subjected to filtration. The culture supernatant and mycelium were subjected to extracellular and intracellular Mn²⁺ concentrations, respectively. The supernatant of the cultures was filtered through 0.22-μm membranes and then diluted with 1% HNO₃ for measuring the extracellular Mn²⁺ concentration. Mycelia were washed in distilled water to remove any nonspecifically bound Mn²⁺, and digested with 1 ml 68% HNO₃. The mixture was collected via centrifugation at 12,000×g for 5 min. The supernatant was then filtered through a 0.22-μm membrane and diluted with 1% HNO₃ for measuring intracellular Mn²⁺ concentration. The final intracellular Mn²⁺ concentration was shown as micromole per gram biomass.

Construction of plasmids and strains

To construct a tpho84-1 deletion mutant, the upstream (− 1 to − 762 bp) and downstream (+ 1917 to + 2681 bp) fragments of tpho84-1 were generated from the genome of T. reesei Rut-C30 using KOD-Plus-Neo (TOYOBO, Japan). All primers are indicated in Additional file 1: Table S1. First, the upstream fragment was ligated into the PacI- and XbaI-linearized LML2.0 [61] using the ClonExpressTM II One Step Cloning Kit (Vazyme, Nanjing, China) to form pFtpho84-1. Subsequently, the downstream fragment was inserted into SwaI-linearized pFtpho84-1 to form the binary vector pDtpho84-1 (see Additional file 5: Figure S4A) for the knockout of tpho84-1 in Rut-C30 using Agrobacterium-mediated transformation [59]. Strains were selected using hygromycin B and cefotaxime on Mandels’ medium. Then, the marker was excised using the method described by Zhang [61]. The putative tpho84-1 disruption mutants (Δtpho84-1/Δ77552) generated by double crossover were verified by diagnostic PCR using the primers tpho84-1-CF and tpho84-1-CR and tpho84-1-OF and tpho84-1-OR (see Additional file 5: Figure S4E).

Similarly, the 706-bp upstream and 710-bp downstream regions of tpho84-2 were amplified and then inserted into LML2.0 as described above to generate the binary vector pDtpho84-2 (see Additional file 5: Figure S4B). After transformation and selection, the putative tpho84-2 deletion mutant (Δtpho84-2/Δ45868) was verified by diagnostic PCR using the primers tpho84-2-CF and tpho84-2-CR, and tpho84-2-OF and tpho84-2-OR (see Additional file 5: Figure S4E). The tre81389, tre45852, tre106118, crz1, and tpmr1 disruption mutants (Δ81389, Δ45852, Δ106118, Δcrz1, and Δtpmr1) were constructed similarly (see Additional file 11: Figure S14).

To construct a tpho84-1/2 deletion mutant, the pDtpho84-2 cassette was transformed into Δtpho84-1 strain using Agrobacterium-mediated transformation. The tpho84-1/2 double deletion mutant (Δtpho84-1/2) was verified by diagnostic PCR.

For tpho84-1 and tpho84-2 re-complementation, 3916- and 4087-bp DNA fragments containing total tpho84-1 and tpho84-2 expression cassette were amplified from the Rut-C30 genome using tpho84-1-rc-1/tpho84-1-rc-2 and tpho84-2-rc-1/tpho84-2-rc-2, and then inserted into SwaI-linearized LML2.0 to generate vector ptpho84-1-rc and ptpho84-2-rc, respectively. Ptpho84-1-rc and ptpho84-2-rc were then transferred to the tpho84-1 and tpho84-2 deletion strain by Agrobacterium-mediated transformation (see Additional file 5: Figure S4C, D). tpho84-1 and tpho84-2 re-complementation strains (tpho84-1-rc and tpho84-2-rc) were selected and verified by PCR using tpho84-1-OF and tpho84-1-OR, and tpho84-2-OF and tpho84-2-OR (see Additional file 5: Figure S4F).

For the construction of N-terminal RFP-tagged translational fusion of tpho84-1 under the control of the cbh1 promoter, the upstream (− 1 to − 1038 bp) and downstream (+ 1 to + 1084 bp) fragments of tpho84-1 were generated from the genome of T. reesei Rut-C30 using KOD-Plus-Neo (TOYOBO, Japan). The promoter of cbh1 (P _cbh1 ) was obtained by PCR from the genome of T. reesei Rut-C30. Red fluorescent protein (rfp) was obtained by PCR using the plasmid pDsRed2-N1 (Clontech) as the template. First, the upstream fragment was ligated into the PacI- and XbaI-linearized LML2.0, using the ClonExpressTM II One Step Cloning Kit (Vazyme, Nanjing, China) to form pFrtpho84-1. Subsequently, the P _cbh1 , rfp, and downstream fragments were inserted into SwaI-linearized Frtpho84-1 to form the binary vector pRFP-TPHO84-1 for subcellular location of tpho84-1 using Agrobacterium-mediated transformation. Subsequently, the marker was excised following the method of Zhang [61]. The putative rfp-tpho84-1 mutants (rfp-tpho84-1) generated by double crossover were verified by diagnostic PCR using the primers rfp-tpho84-1-CF and rfp-tpho84-1-CR (see Additional file 12: Figure S15).

Similarly, the 997-bp upstream and 797-bp downstream regions of tpho84-2 were amplified and then inserted into LML2.0 as described above to generate the binary vector pRFP-TPHO84-2 (see Additional file 6: Figure S5A). After transformation and selection, the putative rfp-tpho84-2 mutants (rfp-tpho84-2) was verified by diagnostic PCR using the primers rfp-tpho84-2-CF and rfp-tpho84-2-CR (see Additional file 12: Figure S15).

The genome sequence of T. reesei is available at the US Department of Energy (DOE) Joint Genome Institute (http://genome.jgi-psf.org/Trire2/Trire2.home.html).

Fluorescence microscopy

To localize RFP-TPHO84-1/2 fusion proteins using microscopy, the rfp-tpho84-1 and rfp-tpho84-2 strains were inoculated into Mandels’ medium and grown for 48–60 h. The mycelia were then observed using an S Plan Fluor ELWD 100×, 1.3 numerical aperture (NA) objective on a Laser Scanning Confocal Microscope (A1R, Nikon, Japan) comprising a Texas Red filter (500–620 nm band-pass excitation filter and emission filter of 670 nm). Images were processed using the NIS elements software (Nikon).

Free cytosolic Ca²⁺ labeling and detection

Fluo-3/AM (Sigma) was used as a Ca²⁺-specific probe to assess the level of cytoplasmic Ca²⁺ in T. reesei Rut-C30 according to the manufacturer’s protocol. Fluo-3/AM (50 μM final concentration) was loaded into cells by incubation at 37 °C for 30 min, and the cells were then washed three times with phosphate-buffered saline. Images of Ca²⁺ green fluorescence were observed using an S Plan Fluor ELWD 20×, 0.5 numerical aperture (NA) objective and a digital sight camera on an Eclipse Ti inverted microscope system (Ti-E, Nikon, Japan), comprising an FITC filter (420–490 nm band-pass excitation filter, and emission filter of 535 nm). The intensity of green fluorescence was quantified using NIS-Elements F package software. To eliminate the contribution of background fluorescence, cells without Fluo-3/AM labeling were also imaged under identical conditions.

Statistical analysis

All experimental data shown in this paper were carried out at least three times with identical or similar results. For every experiment, three biological replicates were performed with three technical replicates. The error bars indicate the standard deviation (SD) from the mean of triplicates. Student’s t test was used to compare two samples. Duncan’s multiple-range test was used for multiple comparisons. p < 0.05 was considered to be significant.