Identification and sequence analysis of the upstream genes of HOG pathway in T. reesei
HOG pathway has been reported to be regulated by two independent upstream branches, the Sho1 branch and the Sln1 branch, in S. cerevisiae [13, 14]. To investigate whether these two branches exist in T. reesei, blastP was performed using HOG signaling-associated proteins of S. cerevisiae as queries to search the T. reesei genomic sequence database (https://genome.jgi.doe.gov/Trire2/Trire2.home.html). For the Sho1 branch, orthologs of yeast Sho1 (YER118C) and Ste20 (YHL007C) were identified and named TrSho1 (Tr_5466) and TrSte20 (Tr_104364), respectively. Orthologous yeast Msb2 and Hkr1 were, however, not found in T. reesei genome. For the Sln1 branch, orthologs of yeast Sln1 (YIL147C) and Ypd1 (YDL235C) were identified and named TrSln1 (Tr_70943) and TrYpd1 (Tr_123344), respectively.
The Trsho1 encodes a predicted 298-amino acid protein, which showed a relatively lower identity (33%) to the Sho1 protein from S. cerevisiae, but a higher identity to its filamentous fungal orthologs such as that of N. crassa (58%), A. nidulans (52%), M. oryzae (68%) and F. oxysporum (70%). SMART analysis revealed that TrSho1 contains four predicted transmembrane domains near the N-terminus (amino acids 21–43, 58–77, 84–106 and 116–135, respectively), a linker domain and an SH3 domain at the C-terminus (amino acids 242–298) (Additional file 1: Fig. S2). A phylogram of the predicted Sho1 proteins further supports the close phylogenetic relationship among Sho1 orthologs in filamentous fungi (Additional file 1: Fig. S1A). Phylogenetic analysis also revealed that TrSte20 has a close evolutionary relationship with its filamentous fungal orthologs (Additional file 1: Fig. S1B), which displayed high-amino acid identity to that of N. crassa (60%), A. nidulans (81%), M. oryzae (59%) and F. oxysporum (66%), respectively. However, unlike the case of TrSho1, TrSte20 also displayed a high identity (70%) to the yeast Ste20. TrSte20 is an 821-amino acid protein containing a C-terminal catalytic domain and a PBD domain which is believed to interact with Cdc42 and Rho-like small GTPases [30] (Additional file 1: Fig. S2).
Similar to TrSho1, whereas a relatively lower identity exists between TrSln1 and ScSln1 (37%), TrSln1 shares considerable amino acid identity to the phylogenetically-related Sln1 proteins from filamentous fungi (Additional file 1: Fig. S1C), namely N. crassa (61%), A. nidulans (48%), M. oryzae (59%) and F. oxysporum (62%). TrSln1 encodes a relative large protein which contains 1170 amino acids and three predicted transmembrane domains near the N-terminus (amino acids 9–31, 233–255 and 405–427, respectively), a HisKA domain (amino acids 563–628), a HATPase histidine-like ATPase domain (705–903), and a REC signal receiver domain (999–1116) as revealed by SMART analysis (Additional file 1: Fig. S2). As the immediate phosphorelay protein downstream of Sln1, TrYpd1 encodes a relatively small protein (149 amino acids) with varied identities to the corresponding Ypd1 in S. cerevisiae, N. crassa, A. nidulans, C. albicans, M. oryzae and F. oxysporum of 39, 50, 55, 49, 58 and 69%, respectively. TrYpd1p is a putative phosphotransferase with a histidine-containing phosphotransfer domain (Additional file 1: Fig. S2). Phylogenetic analysis revealed that TrYpd1 has a close evolutionary relationship with other filamentous fungal orthologs as well (Additional file 1: Fig. S1D). Based on the above bioinformatics analyses, we believe that the upstream two branches of the Hog1-type Tmk3 exist in T. reesei.
Construction of mutant strains for the Sho1 and Sln1 branches in T. reesei and characterization of their vegetative growth and mycelia morphology
It has been reported that deletions of sln1 or ypd1 in S. cerevisiae are lethal [17]. To investigate the physiological roles of the Sho1 and Sln1 branches in T. reesei, particularly in hyperosmolarity response, cell wall integrity maintenance, and cellulase production that have not been studied in other fungal species, we constructed a series of promoter substitution strains of Trsho1, Trsln1 and Trypd1 using our recently developed copper-responsive promoter replacement system [31], resulting in P
tcu1
-sho1, P
tcu1
-sln1 and P
tcu1
-ypd1 strains, respectively (Fig. 1a). A Trste20 deletion mutant (Δste20) was also constructed by homologous recombination (Fig. 1b), allowing parallel comparison of the relevant properties of the different mutants. For the promoter substitution strains, target genes controlled by P
tcu1
were generally overexpressed when there’s no copper in the media. However, when copper was supplied in the media, the transcription of target genes were drastically repressed or even turned off [32], thus mimicking a knock-down/out status. The transcription level of the target genes in each promoter substitution strain was determined by qRT-PCR (Fig. 2). When copper was not added into the media, the transcription level of the target genes (Trsho1, Trsln1 and Trypd1) in the promoter substitution strains were significantly up-regulated compared with the parental strain (5.3- to 9.5-fold for Trsho1, 24.5- to 57.7-fold for Trsln1 and 74.5- to 86.9-fold for Trypd1). However, when 20 μM copper was added, the expression of the target genes was dramatically repressed (36.4- to 61.8-fold for Trsho1, 21.5- to 35.7-fold for Trsln1 and 12.5- to 13.1-fold for Trypd1 lower compared with the parental strain). As expected, the transcription of Trste20 could be hardly detected in the Δste20 strain (Fig. 2a). These data confirmed that the promoter substitution strains and the Trste20 deletion strain were successfully constructed.
We first analyzed the vegetative growth of mutant strains in the Sho1 branch (Δste20 and P
tcu1
-sho1) and in the Sln1 branch (P
tcu1
-sln1 and P
tcu1
-ypd1) on different carbon sources. For the Sho1 branch, deletion of ste20 displayed a slightly reduced growth on all the tested carbon sources on minimal medium but not on the malt extract agar plates. Notably, the Δste20 strain displayed a significantly reduced hydrolytic zone on Avicel-containing plates compared to the parental TU-6 strain (Fig. 3a, b), suggesting the involvement of ste20 in regulating cellulase production in T. reesei. We further examined the hyphal morphology of Δste20 and TU-6 strains. The mycelia of TU-6 were relatively highly branched and formed relatively compact clots in submerged culture, whereas the Δste20 strain displayed vimineous mycelia with significantly reduced branches resulting in much loose mycelia aggregates (Fig. 3c). In contrast to the absence of TrSte20, no obvious change could be detected in the growth, sporulation and hyphal morphology of P
tcu1
-sho1 (Additional file 1: Fig. S3 and data not shown), regardless the presence or absence of copper which has been proven to result in the repression or overexpression of the target gene placed under the control of P
tcu1
[31]. The copper ions had no significant effect on the growth and sporulation of the parent strain TU-6 (Fig. 4a).
For the Sln1 branch, the Trsln1 promoter-substituted strain P
tcu1
-sln1 showed no obvious difference in growth and sporulation no matter whether copper was added or not (Additional file 1: Fig. S3). In contrast, the growth and sporulation of the P
tcu1
-ypd1 strain were severely impaired under copper-repressing conditions, whereas no significant difference was observed compared to the parental strain when copper was excluded in the media (Fig. 4a, b). Microscopic examination revealed that highly branched hyphal morphology was observed with the repression of Trypd1, whereas overexpression of Trypd1 without copper resulted in a less-branched hyphal morphology (Fig. 4c).
Characterization of the functional roles of Trste20, Trsho1, Trsln1 and Trypd1 in different stress responses of T. reesei
Since the Hog1-related kinases have been shown to primarily participate in osmotic stress response [12], we examined the tolerance of the mutant and parental strains to different stresses. To investigate their roles in high osmolarity resistance, all the strains were grown on MM plates containing various concentrations of NaCl from 0.3 M to 1.2 M. Meanwhile, oxidative stress response was analyzed by including different concentrations of H2O2 from 15 to 60 mM. In addition, thermotolerance was also tested by growing all the strains at 37 °C. As expected, deletion of Trste20 in the Sho1 branch caused a clear growth defect under high salt stress (0.6 M NaCl and above) or at high temperature although hardly any effect was observed on the resistance to oxidative stress (Fig. 5 and Additional file 1: Fig. S4A). In contrast, when repressed with copper, the P
tcu1
-sho1 strain showed slightly enhanced growth under high salt (0.6 M NaCl and above) and oxidative stress (15 mM H2O2 and above) compared to the parental strain (Fig. 5 and Additional file 1: Fig. S4B). In corroborating the above results, the P
tcu1
-sho1 strain displayed decreased tolerance to high salt, H2O2 and higher temperature (37 °C) when overexpressed without copper.
The involvement of the Sln1 branch in stress responses was also investigated by growing the P
tcu1
-sln1 and P
tcu1
-ypd1 strains on MM plates under the respective stress conditions. TrSln1 exerted hardly any effect on growth under all the tested stress conditions compared to the parental strain no matter whether it was repressed or de-repressed (Fig. 5 and Additional file 1: Fig. S4C). However, the P
tcu1
-ypd1 strain appeared extremely sensitive to all the tested stresses when the expression of Trypd1 was repressed with copper. Specifically, the P
tcu1
-ypd1 strain could hardly grow on plates containing 0.6 M NaCl or 45 mM H2O2 under copper-repressed conditions (Fig. 5 and Additional file 1: Fig. S4D). When copper was not included, the P
tcu1
-ypd1 strain showed no significant growth difference on different stress plates compared to the parental TU-6 strain. The apparently highest sensitivity exhibited by the Trypd1-repressed strain is an indication of hampered tolerance to the above stress conditions, suggesting that aberrantly activated HOG-like MAP kinase cascade may compromise T. reesei tolerance to stresses.
The effect of Trste20, Trsho1, Trsln1 and Trypd1 on cell wall integrity maintenance of T. reesei
In addition to the involvement in different stress responses, Tmk3 was also found to participate in the cell wall integrity maintenance [8]. Besides, given the fact that the Sho1 and Sln1 branches differentially affect the response to various stresses, we analyzed their participation in cell wall integrity maintenance by testing the sensitivity of the mutant and parental strains to Calcofluor white (CFW) and Congo red (CR), which has been proven to be indicators of cell wall integrity [33]. For the Sho1 branch, contrary to our expectation, the Δste20 strain displayed no obvious difference in the sensitivity to both CFW and CR compared to the parental strain (Fig. 6 and Additional file 1: Fig. S5A). Similar to the case in tolerance to stresses, repression of Trsho1 with copper slightly increased in its resistance to CFW and CR while growth of the P
tcu1
-sho1 strain in the absence of copper was compromised compared to that of TU-6 when the concentration of CR or CFW raised to higher than 200 or 50 mg/l. Specifically, the P
tcu1
-sho1 strain could hardly grow at 400 mg/ml CR without copper (Fig. 6 and Additional file 1: Fig. S5B). Together these data suggested that, unlike TrSte20, Trsho1 plays a negative role in cell wall integrity maintenance, which may provide an explanation for its opposing role to that of TrSte20 in mediating stress responses.
In the case of the P
tcu1
-sln1 strain, no significant difference in the sensitivity to both CFW and CR was observed with and without copper between the P
tcu1
-sln1 and TU-6 parental strains (Fig. 6 and Additional file 1: Fig. S5C). In contrast, P
tcu1
-ypd1 showed severely compromised growth on plates with CR or CFW higher than 100 or 25 mg/l in the presence of copper, respectively (Fig. 6 and Additional file 1: Fig. S5D), indicating that repression of Trypd1 showed impaired cell wall integrity. When de-repressed without copper, P
tcu1
-ypd1 strain displayed a slightly increased tolerance to both CFW and CR. These results implicated that Trypd1 plays a critical role in the cell wall integrity maintenance in T. reesei.
Differential involvement of Trste20, Trsho1, Trsln1 and Trypd1 in T. reesei cellulase production
Previous studies revealed that although deletion of tmk2 increased the cellulase production, minor difference could be detected in the transcription level of cellulase genes, indicating that tmk2 was not directly involved in cellulase production [34]. The Hog1-type MAPK Tmk3 is the only MAPK involved in signal transduction for regulating cellulase expression on the transcriptional level in T. reesei [8]. Specifically, in submerged cultivation condition, deletion of tmk3 significantly down-regulated the transcription of the main cellulase genes, including cbh1, chb2, eg1, eg2 and bgl1 [8]. Characterizing the functional roles of the Tmk3 upstream pathways in cellulase production is thus important in the understanding of external signal sensing and signal transduction in T. reesei. Different cascade mutants and the parental strain were cultivated in Mandels–Andreotti medium supplied with 1% Avicel as the sole carbon source. The extracellular hydrolytic activities (pNPC and pNPG) and the filter paper activity (FPA) were analyzed. Furthermore, the mRNA levels of two major cellulase genes (cbh1 and eg1) and the major transcription activator xyr1 were analyzed by quantitative RT-PCR. As shown in Fig. 7a–c, the pNPC and pNPG hydrolytic activities and the filter paper activity (FPA) of the Δste20 strain were dramatically reduced compared with the parental strain, in accordance with the heavily reduced hydrolytic zone on the Avicel plates (Fig. 3a). Meanwhile, the total extracellular protein concentration was also significantly decreased (Fig. 7d). Moreover, the transcriptional level of chb1, eg1 and xyr1 in Δste20 was significantly lower than that of the TU-6 strain (Fig. 8a–c), suggesting that the reduced cellulase production resulted from the impaired cellulase gene transcription. These results indicated that Trste20 is critical for the efficient induction of cellulase gene expression in T. reesei.
In accordance with Δste20, when copper was added to a final concentration of 20 μM to repress the expression of Trsho1, the extracellular pNPC and pNPG hydrolytic activities decreased to 25–50% those of the parental TU-6 strain (Fig. 9a, b). FPA and the total extracellular protein concentration also showed a clear decline (Fig. 9c, d). Further transcriptional analysis revealed that repression of Trsho1 significantly reduced the transcription level of cbh1, eg1 and xyr1 (Fig. 8d–f). No significant differences were observed with these hydrolytic activities and the extracellular protein concentration when Trsho1 was not repressed with copper. The transcriptional level of chb1 and eg1 was even up-regulated compared to the parental strain in the absence of copper. Together, these data indicate that similar to Trste20, Trsho1 is critical for the transcription of cellulase genes, suggesting that the Sho1 branch is conductive to cellulase production.
Unlike the Sho1 branch, the two Sln1 branch components exhibited different effects on cellulase production by T. reesei. For the P
tcu1
-sln1 strain, the extracellular pPNC and pNPG hydrolytic activities maintained at a similar level to those of the parental strain regardless of the presence or absence of copper (Fig. 10a, b). Correspondingly, no significant changes were detected in the transcription of chb1 and eg1 under either copper-included or copper-free conditions (data not shown), indicating that Trsln1 plays a minor role in mediating cellulase gene expression. In accordance with TrSln1, repression of Trypd1 with copper exerted hardly any effect on the extracellular pPNC and pNPG hydrolytic activities (Fig. 10c, d). On the contrary, de-repression of Trypd1 in the absence of copper resulted in a significant reduction of the extracellular pNPC and pNPG hydrolytic activities of the P
tcu1
-ypd1 strain to about 25% those of the TU-6 strain. Further transcriptional analysis demonstrated that a significant decrease was observed in the transcription level of cbh1, eg1 and xyr1 in the P
tcu1
-ypd1 strain when copper was excluded in the medium (Fig. 8g–i). Together, these data suggested that, in contrast to its role in mediating stress tolerance, the hyperosmotic-response MAP kinase pathway fine-tuned by TrYpd1 may play a subtle role in the cellulolytic response in T. reesei.