The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei
Biotechnology for Biofuels and Bioproducts volume 16, Article number: 87 (2023)
Lignocellulose is the most abundant renewable resource in the world and has attracted widespread attention. It can be hydrolyzed into sugars with the help of cellulases and hemicellulases that are secreted by filamentous fungi. Several studies have revealed that the Ras small GTPase superfamily regulates important cellular physiological processes, including synthesis of metabolites, sporulation, and cell growth and differentiation. However, it remains unknown how and to what extent Ras small GTPases participate in cellulase production.
In this study, we found that the putative Ras small GTPase RSR1 negatively regulated the expression of cellulases and xylanases. Deletion of rsr1 (∆rsr1) significantly increased cellulase production and decreased the expression levels of ACY1-cAMP-protein kinase A (PKA) signaling pathway genes and the concentration of intracellular cyclic adenosine monophosphate (cAMP). Loss of acy1 based on ∆rsr1 (∆rsr1∆acy1) could further increase cellulase production and the expression levels of cellulase genes, while overexpression of acy1 based on ∆rsr1 (∆rsr1-OEacy1) significantly reduced cellulase production and transcriptional levels of cellulase genes. In addition, our results revealed that RSR1 negatively controlled cellulase production via the ACY1-cAMP-PKA pathway. Transcriptome analysis revealed significantly increased expression of three G-protein coupled receptors (GPCRs; tre62462, tre58767, and tre53238) and approximately two-fold higher expression of ACE3 and XYR1, which transcriptionally activated cellulases with the loss of rsr1. ∆rsr1∆ tre62462 exhibited a decrease in cellulase activity compared to ∆rsr1, while that of ∆rsr1∆tre58767 and ∆rsr1∆tre53238 showed a remarkable improvement compared to ∆rsr1. These findings revealed that GPCRs on the membrane may sense extracellular signals and transmit them to rsr1 and then to ACY1-cAMP-PKA, thereby negatively controlling the expression of the cellulase activators ACE3 and XYR1. These data indicate the crucial role of Ras small GTPases in regulating cellulase gene expression.
Here, we demonstrate that some GPCRs and Ras small GTPases play key roles in the regulation of cellulase genes in Trichoderma reesei. Understanding the roles of these components in the regulation of cellulase gene transcription and the signaling processes in T. reesei can lay the groundwork for understanding and transforming other filamentous fungi.
Trichoderma reesei abundantly produces extracellular cellulases and hemicellulases, which catalyze the degradation of lignocellulose to monosaccharides [1,2,3]. This fungus is a primary source of enzymes for the production of bioethanol [4,5,6,7,8]. A major issue associated with the cost-effective production of bioethanol is optimization of the fungal platform to increase enzyme production [9, 10]. Many transcription factors play a vital role in the process of cellulase production , including the main repressor, CRE1, which is related to carbon catabolite repression (CCR) [12,13,14], and the crucial transcription activators XYR1 [15,16,17] and ACE3 [18,19,20]. Notably, T. reesei only produces enzymes abundantly in the presence of inducing carbon sources, such as Avicel and lactose . The process of cellulase synthesis, stimulated by extracellular signals, is very complex . Understanding the signal-sensing pathways in T. reesei will have a major impact on enzyme preparation and fuel production.
protein-coupled receptors (GPCRs) receive extracellular signals and may transmit them through Ras small GTPases [22,23,24], which in turn, transmit them downstream to various effectors. Ras small GTPases are binary molecular switches between the active [guanosine triphosphate (GTP) binding] and inactive [guanosine diphosphate (GDP) binding] conformations [25,26,27]. The activity of Ras small GTPases is controlled by two proteins: guanine nucleotide exchange factors (GEFs) and GTPase activation proteins (GAPs) . When combined with GDP, the switch is closed, and an inactive structure exists . However, Ras GEFs control the exchange of GDP with GTP, switching to the activated state . The main functions of Ras GAPs are to hydrolyze GTP bound to Ras small GTPases to GDP and promote the inactivity of Ras small GTPases. In Saccharomyces cerevisiae and Candida albicans, activated Ras small GTPases transmit signals to the downstream signal-receiving kinase adenylate cyclase 1 (ACY1), which in turn, transmits them to protein kinase A (PKA) via cyclic adenosine monophosphate (cAMP), a vital second messenger [31, 32]. ACY1-cAMP-PKA is an important pathway that affects metabolism, enzyme production [12, 33], sporulation , vegetative growth [34, 35], and iron homeostasis .
In T. reesei, two Ras small GTPase genes, TrRas1 and TrRas2, are important for numerous cellular processes, such as hyphae branching, sporulation, and cAMP level adjustment . In S. cerevisiae, Ras1 and Ras2 homologs regulate cell cycle progression through the cAMP signaling pathway to sense extracellular glucose signals [38, 39]. Additionally, S. cerevisiae Ras2 controls pseudohyphal differentiation in cooperation with the cAMP pathways . In the human pathogen, C. albicans, Ras1 homologs control cellular morphology and virulence through the cAMP-PKA signaling pathway . Further, Ras1 reportedly controls filamentation, mating, and growth at elevated temperatures, which is necessary for virulence in Cryptococcus neoformans via the cAMP-dependent signaling cascade . In Fusarium graminearum, Ras GEF FgCdc25 regulates virulence and fungal development via the cAMP signaling pathway . Previous studies have revealed that the Ras small GTPase superfamily regulates important cellular physiological processes [37,38,39,40,41], including the synthesis of metabolites, sporulation, cell growth, and cell differentiation. However, there is a lack of research on the Ras small GTPase superfamily and their connection to the ACY1-cAMP-PKA pathway in T. reesei, which sparked our research interest.
In the present study, five Ras GTPases, four Ras GEFs, and two Ras GAPs from T. reesei  were knocked out to investigate the role of Ras small GTPases in cellulose-induced signal transduction and cellulase production. Intracellular cAMP concentration, expression of ACY1-cAMP-PKA pathway genes, and comparative transcriptome data were analyzed in the parent and deletion strain. This study provides new perspectives for improving cellulase production and insights into the mechanism of cellulase regulation by Ras small GTPases in T. reesei.
Ras small GTPase RSR1 negatively regulates cellulase and xylanase production
Using the BLAST online website in NCBI, 11 putative Ras subfamily genes were found in the genome of T. reesei (Table 1), including five Ras GTPases, four Ras GEFs, and two Ras GAPs. We constructed 11 putative knockout strains (Additional file 1: Table S1) by homologous recombination in the wild-type strain QM6a to explore the effect of these genes on cellulase production. Two of the Ras GTPases (tre107035 and tre66480) and one Ras GEF (tre70548) could not be knocked out. These three genes may play a decisive role in growth, leading to death by gene knockout. The loss of another three genes had a significant effect on enzyme activity (Table 1). Deletion of the TrRas2 gene led to a 60 ± 4.58% decrease in cellulase activity, which is consistent with the results obtained by Zhang et al. . The loss of tre107369 slightly increased cellulase activity by 10 ± 1.04% compared to the wild-type strain. The rsr1 deletion mutant (Δrsr1 strain) displayed significantly increased (100 ± 8.12%) cellulase activity compared to the wild-type strain QM6a in the presence of 1% (w/v) Avicel.
As shown in Additional file 2: Fig S1, when 2% (w/v) glucose was used as the sole carbon source, QM6a and Δrsr1 did not produce cellulases. Cellobiohydrolase (pNPCase), endoglucanase (CMCase), filter paper enzyme (FPase), and xylanase activities, and extracellular protein concentration were significantly improved in Δrsr1 compared to the wild-type strain QM6a in the presence of 1% (w/v) Avicel as the carbon source. The pNPCase, CMCase, and FPase activities of Δrsr1 were increased by approximately 100% compared with that of wild-type strain QM6a after 3–4 day of cultivation (Fig. 1A–C). The xylanase activity of Δrsr1 increased by 25–38% compared with that of QM6a (Fig. 1D). The extracellular protein concentration of the Δrsr1 strain was 34–60% higher than that of QM6a after 2 days of cultivation (Fig. 1E). We found that the deletion of rsr1 had almost no effect on the growth of T. reesei (Additional file 3: Fig S2), indicating that the enhancement of cellulase production was not achieved by controlling biomass production. Additionally, we constructed a complementation strain (RC-rsr1) based on the deletion strain Δrsr1 (Additional file 4: Fig S3). The enzyme activities in RC-rsr1 decreased to the same level as that in the wild-type strain QM6a (Fig. 1A–E). The results of the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (Fig. 1F) of the secretomes in supernatants of QM6a, RC-rsr1, and Δrsr1 were consistent with the cellulase activity data mentioned above. To investigate the effect of rsr1 deletion on cellulase expression, we used real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) to detect the transcriptional levels of the major cellulase and xylanase genes in the QM6a and Δrsr1 strains. As shown in Fig. 2A–F, deletion of rsr1 significantly increased the expression of four major cellulase-encoding genes (cbh1, cbh2, egl1, and egl2) and two major xylanase genes (xyn1 and xyn2). These results are consistent with the increased enzyme activity and extracellular protein production in the Δrsr1 strain (Fig. 1A–F). The results indicated that rsr1 acts as a negative regulator of cellulase, xylanase, and secreted protein production.
To study whether rsr1 deletion can enhance cellulase production in the cellulase hyper-producer T. reesei RUT-C30, an industrial mutant , we also constructed the rsr1 deletion strain C30-rsr1 from RUT-C30 using the same method. Consistent with previous results, the loss of rsr1 in RUT-C30 resulted in no significant differences in hyphal growth (Additional file 3: Fig S2). The pNPCase activity of C30- rsr1 increased by > 70% (Fig. 3A) and CMCase activity increased by > 50% (Fig. 3B) compared with that of the parental strain RUT-C30 after cultivation for 3–4 days. These results suggested that rsr1 also has a negative effect on cellulase production in the T. reesei mutant, RUT-C30, the deletion of which can effectively improve cellulase production. The data indicated that the rsr1 deletion strain could possibly be used as a suitable candidate for high-yield cellulase production.
Structure and phylogenetic analyses of RSR1
The RSR1 domain was predicted using the Pfam database (http://pfam.xfam.org). Only one GTPase catalytic domain was found at position 11–177 (Fig. 4A). Phylogenetic tree analysis (Fig. 4B) showed that RSR1 universally existed in some Ascomycota, including Sordariomycetes, Eurotiomycetes, Leotiomycetes, and Pezizomycetes, with amino acid similarity > 65%. The amino acid similarity of RSR1 homologs in Trichoderma exceeded 80%, and that in Fusarium and Neurospora ranged from 73 to 85%. Neurospora crassa is also an important filamentous fungus that breaks down lignocellulosic biomass and produces soluble sugars. The amino acid similarity of RSR1 homologs in this species was up to 85%. Although RSR1 is widely found in fungi, none of these putative RSR1 homologs were reported to regulate cellulase gene expression, which is worth studying.
RSR1 negatively regulates the ACY1-cAMP-PKA pathway
Ras small GTPase often transmits signals through the ACY1-cAMP-PKA signaling pathway [37, 40, 43,44,45]. Therefore, we detected the intracellular cAMP concentrations in the QM6a and Δrsr1 strains (Fig. 5A). The intracellular cAMP concentration in Δrsr1 was significantly decreased by 79.8% after 2 d of cultivation in the presence of 1% (w/v) Avicel compared to that observed in the original strain. The intracellular cAMP content in the complementation strain (RC-rsr1) was consistent with that of the original strain (Fig. 5A). However, the intracellular cAMP concentration in the Δrsr1 strain was unchanged compared with that in the wild-type and complementation strains, when glucose was used as the carbon source (Fig. 5B).
We also evaluated the transcription levels of the genes in the ACY1-cAMP-PKA signaling pathway in the QM6a and Δrsr1 strains. The transcription levels of the acy1, pkac1, pkac2, and pkar1 genes in the knockout strain were remarkably reduced compared to the wild-type and complementation strains in the presence of 1% (w/v) Avicel (Fig. 5C–F). The expression levels of ACY1-cAMP-PKA genes in RC-rsr1 recovered to the same levels as those in QM6a. However, their levels stayed consistent in Δrsr1, QM6a, and RC-rsr1 when glucose was used as the carbon source (data not shown). These results indicate that the deletion of rsr1 results in downregulated expression levels of acy1, pkac1, pkac2, and pkar1 genes and cAMP content at cellulase inducing conditions.
RSR1 negatively regulates cellulase production through the ACY1-cAMP-PKA pathway
To address if the decreased acy1 transcriptional level and intracellular cAMP content mediates cellulase enhancement in the rsr1 deletion strain, the deletion and overexpression of acy1 were constructed in QM6a and Δrsr1, and named T. reesei Δacy1, Δrsr1Δacy1, QM6a-OEacy1 and Δrsr1-OEacy1, respectively (Additional file 4: Fig S3). As shown in Fig. 6A–B, the further improvement of pNPCase and CMCase activities was observed in Δrsr1Δacy1 compared to Δrsr1 and QM6a. We found that the loss of acy1 remarkably stimulated pNPCase and CMCase activities after 3 days of fermentation in the Δrsr1Δacy1 strain, with an increase of approximately 174% and 128% compared with Δrsr1 and approximately 4.3- and 3.12-fold increase compared with QM6a, respectively. The enhanced cellulase production in the rsr1 deletion strain was partly attenuated by the overexpression of acy1 (Fig. 6A, B). The loss of acy1 in the wild-type strain led to a significant improvement in pNPCase and CMCase activities (Fig. 6A, B), which also implied that acy1 acted as a negative regulator of cellulase expression. Δrsr1Δacy1 showed a higher level of cellulase production capacity compared to Δacy1 (Fig. 6A, B). The results indicated that acy1 expression and intracellular cAMP concentration are involved in the negative regulation of cellulase production by RSR1.
To gain insight into how acy1 influences the rsr1-mediated cellulase high-yield process at the transcriptional level, RT-qPCR analysis was performed to detect the expression levels of four main cellulase-encoding genes (cbh1, cbh2, egl1, and egl2) in the QM6a, Δacy1, Δrsr1, Δrsr1Δacy1, QM6a-OEacy1, and Δrsr1-OEacy1 strains. As shown in Fig. 6C–F, and consistent with previous data regarding pNPCase and CMCase activities (Fig. 6A, B), the deletion of acy1 further enhanced the expression of cellulase-encoding genes in Δrsr1Δacy1, compared with that in Δrsr1, while the overexpression of acy1 partly abrogated the overexpression of cellulase-encoding genes in Δrsr1-OEacy1, compared with that in Δrsr1. These results indicated that the ACY1-cAMP-PKA pathway is required for rsr1 to negatively regulate cellulase production. For the first time, we proved that ACY1 acts as a negative regulator of cellulase in the RSR1 signal transduction pathway.
Transcriptional profile of the Δrsr1 mutant cultured in the presence of Avicel
To further understand the role of rsr1 in cellulase production, we compared the two transcriptomes of the deletion strain (Δrsr1) and the original strain (QM6a) using 1% (w/v) Avicel as the carbon source. Volcano analysis revealed significantly different expression of 172 genes (Log2fold change ≥ 1 or ≤ − 1 and adjusted P < 0.05; Fig. 7A), of which 133 were upregulated and 39 were downregulated (Δrsr1_vs_QM6a; Additional file 5: Table S2). Gene ontology (GO) annotation analysis revealed different expression genes on metabolic processes, binding, catalytic activity, membrane part, cellular process, and other life activities (Fig. 7B). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the participation of different expression genes in starch and sucrose metabolism, cyanoamino acid metabolism, the degradation of valine, leucine, and isoleucine, and protein processing in endoplasmic reticulum pathways (Fig. 7C). This suggested that rsr1 is a widely involved upstream signal that maintains the downstream switch ‘on’ because its deletion affects many pathways. This further suggested that rsr1 influences the entire physiological process, which is very important for cellulase production.
Comparison of cellulase and hemicellulase gene expression levels in Δrsr1 and QM6a (Additional file 6: Table S3) revealed that cellulase and xylanase gene expression levels were upregulated. Of these, the expression levels of endoglucanases (EGL1, EGL3, and EGL5), swollenin, and xylanase xyn4 were highly upregulated (approximately 4–eightfold), consistent with our RT-qPCR data (Fig. 7D). These data indicated that rsr1 was involved in the negative regulation of cellulase expression during induction. We then searched for the top 10 significantly upregulated and downregulated genes (Additional file 7: Table S4). The top 10 upregulated genes included endoglucanase (TRIREDRAFT_122081; egl1) and exo-1,4-beta-xylosidase bxlB (TRIREDRAFT_58450). The expression levels of the 12 transcription factors involved in cellulase expression are listed in Additional file 8: Table S5. The transcript abundance of the cellulase activator genes ace3 and xyr1 were significantly upregulated by 1.73- and 0.7-fold in Δrsr1, respectively (Table 2), compared to the parental strain QM6a. This indicates their positive effect on the improvement of cellulase production in Δrsr1. These results suggested that deletion of rsr1 greatly increases expression of cellulase-related genes.
Previous studies have shown that sugar transporters play an important role in the induction of cellulase [46, 47]. A total of 876 predicted transporter genes in T. reesei QM6a genome (https://mycocosm.jgi.doe.gov/mycocosm/annotations/browser/tcdb/summary;AtD9I7?p=Trire2) were selected (Additional file 9: Table S6), of which 22 were differentially expressed (adjusted P < 0.05) (Table 3) in Δrsr1 compared to QM6a. The MFS transporter (TRIREDRAFT_66228) and MFS general substrate transporter (TRIREDRAFT_70933) were significantly upregulated by 7.06- and 3.53-fold, respectively.
Connection between RSR1 and GPCRs
GPCRs receive extracellular signals that may be transmitted through the Ras small GTPases . Only three of 58 GPCR genes (Additional file 10: Table S7) showed significantly increased transcriptional levels in the RSR1 deletion strain compared to the wild-type strain (Table 4). The expression of VII GPCR (TRIREDRAFT_53238) was upregulated by 1.23-fold, while the expression of the two PTH11-like GPCRs (TRIREDRAFT_58767 and TRIREDRAFT_62462) was upregulated by 1.70- and 1.40- fold, respectively.
To further investigate the roles of three transcriptionally elevated GPCRs in rsr1 deletion-mediated cellulase overexpression, double knockout strains (Δrsr1Δtre62462, Δrsr1Δtre58767, and Δrsr1Δtre53238) were constructed. Figure 8A, B show that loss of tre58767 and tre53238 can lead to an even higher level of cellulase production than that of the parental strain, while the loss of tre62462 had the opposite effect. We then examined the transcript levels of cellulase-encoding genes of all strains by RT-qPCR (Fig. 8C–F), consistent with previous enzyme activity data (Fig. 8A, B). The Δrsr1Δtre58767 strain exhibited promotion of the transcript of cbh1 and egl2, while the Δrsr1Δtre53238 strain appeared to promote the expression of cbh1, egl1, and egl2. The expression levels of the main cellulase genes in Δrsr1Δtre62462 decreased by > 50% compared to Δrsr1 at 48 h. Moreover, we found that cellulase synthesis and gene regulation was not affected by the deletion of tre62462, tre58767, and tre53238 (Fig. 8A–F). The results suggest that RSR1 and GPCRs may be connected and that they are involved in a complex signal transmission pathway.
The Ras subfamily of the small GTPase family is one of the most highly researched protein families . In this study, 11 putative Ras subfamily genes were knocked out to investigate their involvement in cellulase signal transduction (Table 1). Three genes (tre107035, tre66480, and tre70548) could not be knocked out, indicating that they might be indispensable for growth. In our study, the deletion of TrRas1 had no effect on cellulase production, while the loss of TrRas2 showed a negative effect on cellulase production. These findings are consistent with those of a previous study by Zhang et al. . In the present study, the loss of rsr1 led to a remarkable increase in pNPCase, CMCase, FPase, and xylanase activities, and extracellular protein production (Fig. 1A–F). RT-qPCR analysis showed that the transcription levels of four major cellulases and two major xylanase genes (cbh1, cbh2, egl1, egl2, xyn1, and xyn2) were greatly improved (Fig. 2A–F), which is consistent with the enzyme activity data (Fig. 1A–F). The results illustrated that RSR1 has a negative effect on cellulase and xylanase production. Phylogenetic analysis revealed the widespread presence of rsr1 in Ascomycota (Fig. 4B). These findings indicated that RSR1 homologs perform conserved functions in these filamentous fungi. Rsr1 GTPase interacts with Cdc42 and its related proteins to direct the establishment of cell polarity at the proper bud site in S. cerevisiae . In Aspergillus fumigatus, the loss of rsrA (a rsr1 homolog in S. cerevisiae) leads to a modest growth rate reduction and delay in germ tube emergence . In Ashbya gossypii, rsr1 is involved in hyphal growth guidance . Moreover, Rsr1 was reported to participate in development regulation, aflatoxin biosynthesis and pathogenicity in Aspergillus flavus . RSR1 has been widely reported in other fungi, but unfortunately its function in T. reesei remains elusive.
The deletion of rsr1 significantly decreased the intracellular cAMP concentration (Fig. 5A) and transcription levels of ACY1-cAMP-PKA pathway genes (acy1, pkac1, pkac2, and pkar1) (Fig. 5C–F). Further, the deletion of acy1 was favorable for cellulase production, while the overexpression of acy1 was disadvantageous for cellulase production in Δrsr1, indicating that the ACY1-cAMP-PKA pathway is involved in the rsr1-related signal transduction pathway (Fig. 6A–F). In a previous study, Chen et al.  demonstrated that deletion of acy1 in T. reesei QM6a resulted in a significant decrease of enzymatic activity and the transcript abundance of the corresponding genes (cbh1 and egl1) during growth on Avicel. In their study, conidia were fed with 2% glucose for 36–48 h to form mycelia and then transferred to 1% (w/v) Avicel . When acy1 was deleted, the T. reesei strain failed to respond to a change of the input signal from repression to induction, resulting in less cellulase production, which also shows that acy1 may be a key signaling molecule in signal transmission. Schuster et al.  showed that cbh1/cbh2 transcript levels in the parental strain QM9414 are similar to those of the acy1-deleted strain in the early stage in darkness, whereas we found that the cbh1/cbh2 transcript levels in our ∆acy1 strain were enhanced. This divergence may be related to the different parental strains (QM6a in our study) and different carbon sources. Nonetheless, our finds agree with those of Schuster et al.  to what concerns the increased cbh1/cbh2 levels upon pkac1 loss in the dark. Nogueira et al.  showed that addition of 1 mM dibutyryl-cAMP in acy1-deleted strain leads to increased cellulase CBH1 secretion, whereas in the parental strain QM9414 has the opposite result. This finding illustrates that cAMP regulates the secretion of cellulolytic enzymes in a dual manner. In S. cerevisiae and C. albicans, the Ras/cAMP/PKA pathway plays an important role in regulating physiological metabolism [31, 32]. In F. graminearum, the RasGEF FgCdc25 modulates the cAMP signaling pathway to regulate fungal development and invasiveness . These studies suggest that the Ras/cAMP/PKA pathway is critical for fungi, including T. reesei.
The expression of cellulase genes needs to be stimulated by specific inducing carbon sources, such as Avicel or lactose. When these are recognized by the GPCRs or transporters on the surface of cells, the signals may be transmitted to the transcription factors by small GTPases through the ACY1-cAMP-PKA pathway [22, 23, 31, 32, 37, 55]. Here, transcriptomic analysis suggested that rsr1 might connect to some GPCRs and transporters and negatively regulate the expression of crucial cellulase transcription activators, such as ACE3 and XYR1 (Table 2). Three GPCRs (TRIREDRAFT_53238, TRIREDRAFT_58767, and TRIREDRAFT_62462) might be involved in RSR1 signaling because they showed significantly increased transcriptional levels in the rsr1 deletion strain (Table 4). A previous study reported that the TRIREDRAFT_53238 regulates secondary metabolism in T. reesei , indicating its regulatory role. Then, we used Δrsr1 as the starting strain to delete tre62462, tre58767, and tre53238. With the loss of tre62462, the cellulase enhancement mediated by the deletion of rsr1 was effectively attenuated (Fig. 8A–F). Based on Δrsr1, loss of tre58767 and tre53238 further promoted cellulase production (Fig. 8A–F). The results indicated that the three GPCRs may be involved in the rsr1-related signal transduction pathway in T. reesei. GPCR signaling can lead to the activation of Ras and Rho GTPases through heterotrimeric G-proteins [23, 55]; whether the extracellular signals received by GPCRs are transmitted directly or indirectly to Ras small GTPases in fungi has not been reported in detail. Therefore, further research is needed. A previous study showed that Ras-like small GTPases can also regulate GPCR anterograde trafficking, specifically the Rab and Sar1/ARF subfamilies which are well described as traffic cops to control cargo transport between various intracellular organelles . Whether rsr1 controls trafficking of GPCRs in T. reesei similar to other Ras GTPases remains unknown and should be investigated. Since the activity of Ras small GTPases is controlled by GEFs and GAPs , whether rsr1 interacts with these regulatory proteins should be explored.
Our study showed that the deletion of rsr1 can induce the expression of the crucial cellulase activators XYR1 and ACE3. We also found that the transcription level of the CCR repression transcription factor CRE1 decreased significantly (Additional file 2: Figure S1). The results implied that RSR1 may transmits signals from transporters (Table 3) or GPCRs (Table 4) to enhance acy1 expression, intracellular cAMP synthesis, and activation of PKA kinases, then to negatively control activators, such as XYR1 and ACE3, and to induce repressors, such as CRE1 (Fig. 9). In T. reesei, pkac1 is involved in negative modulation of the expression of cellulase genes by adjusting the expression of XYR1 . In Aspergillus nidulans, the lack of pkaA genes affects the localization of CRE1 in the nucleus, leading to increased secretion of hydrolase . How pkac1 phosphorylates transcription factors, such as ACE3, XYR1, and CRE1 in T. reesei remains unknown, which is also worthy of further study.
Our findings demonstrated that the Ras small GTPase RSR1 negatively regulates cellulase expression through the ACY1-cAMP-PKA pathway. Previous studies have shown that Ras2 promotes the expression of cellulase genes , while rac1 acts as a negative regulator , suggesting that the pathway of action of small GTPases is a multichannel circuit. In the future, overexpression of the Ras2 gene or knockout of rac1 in the RSR1 deletion strain will be conducted to determine whether there are interactions between the three small GTPases.
In summary, the deletion of Ras small GTPase RSR1 significantly enhanced cellulase production in the wild-type strain and cellulase hyper-producer RUT-C30. With the deletion of RSR1, we found the upregulation of the ACE3 and XYR1 transcriptional activators. Loss of RSR1 also significantly reduced the expression of ACY1-cAMP-PKA pathway genes and the content of intracellular cAMP. We conclude that RSR1 negatively regulates cellulase synthesis through the ACY1-cAMP-PKA pathway and that three upstream GPCRs may be involved in the rsr1-related signal transduction pathway. Our study revealed a putative mechanism for the regulation of cellulase production by Ras GTPase rsr1 in T. reesei (Fig. 9). Further, our results may be useful for the construction of cellulase hyper-producers to solve the energy crisis. This study also provides new perspectives for understanding the mechanism of Ras small GTPase in regulating cellulase production in fungi.
Strains and culture conditions
Escherichia coli strain DH5α was used for plasmid construction. T. reesei QM6a (ATCC 13,631) and RUT-C30 (ATCC 56,765) were used as parental strains, while Agrobacterium tumefaciens GV3101 was used to transform the T. reesei strains . The fungal strains were grown on potato dextrose agar with 2% (w/v) glucose as the carbon source at 28 °C for 6 d, and then the conidia were washed with 20% glycerol and stored at − 40 ℃. All fungal strains were cultivated in the dark.
To analyze enzyme production, the conidia (final concentration 106 mL−1) of T. reesei strains were placed in a 250-mL bottle with 40 mL MA liquid medium  containing 1% (w/v) Avicel (PH-101, Sigma-Aldrich, St. Louis, MO, USA), 2% (w/v) lactose, or 2% (w/v) glucose as the sole carbon source, and incubated at 28 °C in a dark incubator with a rotation speed of 220 rpm. Hyphae (3 mL) were collected at 36, 48, and 60 h, pressed dry with filter paper, quick-frozen in liquid nitrogen, and kept frozen at − 80 °C for RNA extraction. The culture medium (1 mL) was removed, centrifuged at 12,000 × g for 10 min, and the supernatant was used for enzyme activity determination.
Construction of plasmids and strains
To construct a rsr1 deletion mutant, the 758-bp upstream and 701-bp downstream fragments of the gene encoding RSR1 were generated from the genome of T. reesei QM6a using KOD-Plus-Neo (TOYOBO, Osaka, Japan) with the primer pairs rsr1-D1/rsr1-D2 and rsr1-D3/rsr1-D4, respectively. As shown in Additional file 4: Fig S3, the upstream fragment was ligated into the PacI and XbaI linearized LML2.1  using the ClonExpress™ II One Step Cloning Kit (Vazyme, Nanjing, China) to form pFrsr1. Subsequently, the downstream fragment was inserted into SwaI-linearized pFrsr1 to form the recombinant knockout plasmid pΔrsr1. The knockout plasmids for the other 10 genes were constructed similarly; the knockout verification primers are listed in Additional file 11: Table S8. The deletion cassettes were transformed into T. reesei by Agrobacterium-mediated transformation . The rsr1 deletion strain (Δrsr1) generated by double crossover was verified by diagnostic PCR using the primer pairs rsr1-CF/D70-4, Hg3.6/rsr1-CR, and rsr1-OF/ rsr1-OR with DNA sequencing (Additional file 4: Fig S3). Xylose-induced Cre recombinase was used to excise the hygromycin resistance cassettes prior to further analysis, as previously described . To complement the rsr1 in knockout strains, 3245-bp DNA fragments, including the 1.5-kb promoter, gene coding sequence, and 0.5-kb terminator, were amplified from the T. reesei genome using rc-rsr1-1/rc-rsr1-2 and rc-rsr1-3/rc-rsr1-4, and inserted into SwaI-linearized LML2.1 to generate the re-complementation vector pRrsr1 (Additional file 4: Fig S3). The complementation cassette was transformed into the Δrsr1 strain by Agrobacterium-mediated transformation. The complementation strain (RC-rsr1) was screened using PCR and DNA sequencing (Additional file 4: Fig S3). The deletion vectors (pΔacy1, pOEacy1, pΔtre62462, ptre58767, and pΔtre53238) were constructed following the principles above and subsequently transformed into the T. reesei QM6a and Δrsr1 strains using Agrobacterium-mediated transformation, respectively (Additional file 3: Fig S2). The verification method of transformants was the same as described above. The strains and primers used in this study are listed in Additional file 1: Table S1 and Additional file 11: Table S8, respectively.
The protein sequence of RSR1 was downloaded from the Joint Genome Institute of the Department of Energy (USDOE-JGI) website (http://genome.jgipsf.org/Trire2/). BLASTP (http://blast.ncbi.nlm.nih.gov/) was used to identify homologous proteins of RSR1 with an E value < 10−5 applied as a cutoff. MEGA7 software was used to perform phylogenetic analysis of proteins using a maximum likelihood tree with 1,000 bootstrap replicates .
Enzymatic activity assays
The cellobiohydrolase activity of pNPCase was determined using 5 mM p-nitrophenol-D-cellobioside (Sigma-Aldrich) as a substrate in 50 mM sodium acetate buffer (pH 5.0) at 50 °C for 30 min . The release of p-nitrophenol was assessed by measuring the absorbance at 405 nm. One unit of enzymatic activity was defined as 1 μmol of p-nitrophenol released from the substrate per min. CMCase, representing endoglucanase activity, was measured using 1% carboxymethylcellulose (CMC, Sigma-Aldrich) in 50 mM sodium acetate buffer (pH 5.0) at 50 °C for 30 min at an absorbance of 540 nm . One unit of CMCase activity was defined as the amount of enzyme producing 1 μmol of reducing sugar per minute. The total cellulase activity was characterized by FPase activity, which was measured using Whatman filter paper as a substrate in 0.2 mL of 50 mM sodium acetate buffer (pH 5.0) at 50 °C for 60 min at an absorbance of 540 nm . One unit of FPase activity was the amount of cellulase required to release 1 μmol of glucose within 1 min. Xylanase activity was measured using approximately 0.003 g xylan as a substrate in 0.2 mL of 50 mM sodium acetate buffer (pH 5.0) at 50 °C for 60 min at an absorbance of 540 nm . One unit of xylanase activity was defined as the production of 1 μmol of reducing sugar per min. Protein concentrations were determined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Biomass was measured indirectly by measuring intracellular protein concentration at an absorbance of 562 nm, and biomass weight was calculated as 0.32 g intracellular protein per gram cell dry weight . SDS-PAGE analysis of extracellular proteins was conducted on 12% Tris–HCl polyacrylamide gels using 20 μL of cell cultures after 3 d of cultivation.
RNA isolation and RT-qPCR
The levels of gene-specific mRNA were assessed using RT-qPCR based on our previous research with some modifications . Briefly, the total RNA from pre-frozen mycelia was extracted carefully using a FastRNA Pro Red Kit (MPbio, Irvine, CA, USA), according to the manufacturer’s instructions. The extracted RNA (500 ng) was reverse transcribed to cDNA using the PrimeScript RT Reagent Kit with gDNA eraser (TaKaRa Bio, Shiga, Japan), following the manufacturer’s instructions. Quantitative PCR was conducted using the TransStart TipTop Green qPCR SuperMix (TransGen, Shanghai, China) with 200 nM of forward and reverse primers (Additional file 11: Table S8). SYBR green assays along with ABI StepOne thermocycler (Applied Biosystems, Foster City, CA, USA) were used to analyze gene transcriptional levels using the 2−ΔΔCt method. Gene expression levels were normalized with the reference genes sar1 [64, 68, 69].
Determination of intracellular cAMP content
The cultured hyphae were collected, quickly chilled with liquid nitrogen, and frozen at − 80 °C. The extraction and determination of intracellular cAMP was conducted as described previously [53, 70], with some modifications. Briefly, the collected hyphae were ground into a powder with liquid nitrogen and dissolved in phosphate-buffered saline (PBS, pH 7.4). After centrifugation, the supernatant was diluted and cAMP content was evaluated with the microorganism cAMP ELISA Kit (mlbio, Shanghai, China), according to the manufacturer’s protocol. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to determine the total protein content of each sample. The intracellular cAMP content/intracellular protein concentrations were calculated.
The same amount of conidia (final concentration 106 mL−1) was cultivated at 28 °C (220 rpm) in 40 mL MA liquid medium supplemented with 1% (w/v) Avicel as the sole carbon source for 48 h. Three replicate samples of cultured hyphae were subsequently collected. The three parallel samples of QM6a and Δrsr1 were sent to Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) for preparation and RNA sequencing using the Illumina Novaseq 6000 sequencing platform (Illumina, San Diego, CA, USA). The original data after quality control, namely clean data (reads), were compared with the reference genome to obtain mapped data (reads) for subsequent transcript assembly and expression calculation. The transcriptome was sequenced simultaneously for comparison. Quality evaluation of the results mainly included sequencing saturation, gene coverage, distribution of reads in different regions of the reference genome, and analysis of the distribution of reads among different chromosomes. The following link (http://fungi.ensembl.org/Trichoderma_reesei/Info/Index) was used as the reference genome during the RNA sequencing process . The expression levels of transcripts were quantitatively analyzed by the software RSEM (http://deweylab.github.io/RSEM/)  with transcripts per million reads as the quantitative index. DESeq2 was used to analyze raw counts and for differential expression analysis. FDR correction with Benjamini/Hochberg was used in this study to control the probability or frequency of errors in the overall inference results and to correct the P value to adjusted P. Genes whose adjusted P was < 0.05 and whose |log2fold change|≥ 1 were selected as differentially expressed genes. Raw whole transcriptome shotgun sequencing data can be found on the NCBI SRA website (https://www.ncbi.nlm.nih.gov/sra/PRJNA888813).
Each independent sample was processed meticulously, with no significant difference between parallel samples (Additional file 12: Fig S4). The clean reads of each sample were compared with the designated reference genome. The comparison rate ranged from 96.49 to 97.03%. The correlation between the three biological replicates of the parent and deletion strains used in the transcriptional analysis was very high (Pearson correlation, r2 ≥ 0.8487; Additional file 12: Fig S4). To verify the reliability of transcriptome data, 14 genes were randomly selected, and their mRNA expression levels were detected by RT-qPCR. There was a significant Pearson correlation between the transcriptome and RT-qPCR data (R2 = 0.996), indicating that the transcriptome analysis was reliable (Fig. 7D).
All experimental data were obtained from at least three independent samples with the same or similar results. Standard deviations from the mean of triplicate determinations are indicated by error bars. Student’s t-test was used to evaluate differences between the sample groups. Multiple comparisons were performed using Duncan’s multiple-range test. The results were significantly different if P was < 0.05.
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Cyclic adenosine monophosphate
Carbon catabolite repression
Representing endo-β-glucanase activity
Filter paper activity, representing total extracellular cellulase activity
G-protein coupled receptor
Guanine nucleotide exchange factor
GTPase activation protein
Adenylate cyclase 1
Protein kinase A
Representing exo-β-glucanase activity
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Real-time quantitative polymerase chain reaction
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This research was supported by the National Key Research and Development Program of China (2022YFA0912300), National Natural Science Foundation of China (32000050), Natural Science Foundation of Shanghai (No. 22ZR1417600), Chenguang Program of the Shanghai Education Development Foundation and Shanghai Municipal Education Commission (grant no. 21CGA34).
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: Table S1. T. reesei strains constructed in this study
: Figure S1. The effect of rsr1 on CCR. pNPCase activity (A) of the three strains (QM6a, Δrsr1, and RC-rsr1) were measured, complementing with 2% (w/v) glucose as the sole carbon source. The cre1 transcriptional levels (B) of the three strains (QM6a, Δrsr1, and RC-rsr1) were measured, complementing with 1% (w/v) Avicel as the sole carbon source. Asterisks (*) indicate significant differences compared to parental strain (Student’s t-test, ***P < 0.001; ****P < 0.0001; ns means no significant difference)
: Figure S2. The effect of rsr1 on hyphal growth of T. reesei strains. A. Three strains (QM6a, Δrsr1, and RC-rsr1) were cultured on PDA medium with 2% glucose at 28 °C in the dark and then photos were taken at 3 days. B. RUT-C30 and C30-rsr1 were cultured on PDA medium with 2% glucose at 28 °C in the dark and then photos were taken at 3 days.
: Figure S3. Construction and verification of deletion mutants. A. To construct an rsr1 deletion mutant, the 758-bp upstream and 701-bp downstream fragments of rsr1 were amplified from T. reesei QM6a genomic DNA were amplified by PCR. Then, upstream and downstream fragments were attached to the PacI/XbaI and SwaI sites in an orderly manner, respectively, of linearized LML2.1 , to form the rsr1 deletion plasmid (p∆rsr1). The deletion cassettes were transformed into QM6a strain by Agrobacterium-mediated transformation. The primers rsr1-CF/D70-4, Hg3.6/rsr1-CR and rsr1-OF/ rsr1-OR were used to verify the putative rsr1 disruption mutants (Δrsr1) by diagnostic PCR using with DNA sequencing. Hyg casseette were used as screening markers. B. To construct an RSR1 complementation strain, the 1.5-kb promoter fragment, gene coding sequence fragment, and 0.5-kb terminator fragment together were obtained from the T. reesei genome QM6a, which constituted 3245-bp and inserted into SwaI-linearized LML2.1 to turn into the re-complementation vector pRrsr1 . The re-complementation cassettes were transformed into Δrsr1 strain by Agrobacterium-mediated transformation. Re-complementation strains (RC-rsr1) are screened by PCR using the primers rc-rsr1-CF and rc-rsr1-CR and DNA sequencing. The primers were used in this experiment in Table S3. C. Validated electrophoretic diagram to verify the knockout and complementation of rsr1. D. The skeleton schematic diagram to knockout acy1 in the knockout strain Δrsr1. E. The skeleton schematic diagram to overexpress acy1 in the knockout strain Δrsr1. F. The skeleton schematic diagram to knockout tre62462 in the knockout strain Δrsr1. G. The skeleton schematic diagram to knockout tre58767 in the knockout strain Δrsr1. H. The skeleton schematic diagram to knockout tre53238 in the knockout strain Δrsr1. I. Validated electrophoretic diagram to verify the deletion of tre62462, tre58767, tre53238, acy1 in the knockout strain Δrsr1, and the overexpression of acy1 in the knockout strain Δrsr1. 1: ∆rsr1∆tre62462-F; 2: ∆rsr1∆tre62462-R; 3: ∆rsr1∆tre58767-F; 4: ∆rsr1∆tre58767-R; 5: ∆rsr1∆tre53238-F; 6: ∆rsr1∆tre53238-R; 7: ∆rsr1∆acy1-F; 8: ∆rsr1∆acy1-R; 9: ∆rsr1-OEacy1-F; 10: ∆rsr1-OEacy1-R. J. Validated electrophoretic diagram to verify the deletion of acy1 in the original strain QM6a, and the overexpression of acy1 in the original strain QM6a. 1: ∆acy1-F; 2: ∆acy1-R; 3: ∆acy1-O; 4: QM6a-O; 5: QM6a-OEacy1-F; 6: QM6a-OEacy1-R. K. Validated electrophoretic diagram to verify the deletion of tre62462, tre58767, and tre53238 in the original strain QM6a. 1: ∆tre62462-F; 2: ∆tre62462-R; 3: ∆tre62462-O; 4: QM6a-O; 5: ∆tre58767-F; 6: ∆tre58767-R; 7: ∆tre58767-O; 8: QM6a-O; 9: ∆tre53238-F; 10: ∆tre53238-R; 11: ∆tre53238-O; 12: QM6a-O.
: Table S2. Transcriptional level of total DEGs in Δrsr1 and QM6a strains.
: Table S3. Comparison of cellulase and hemicellulase genes expression levels in Δrsr1 and QM6a strains
: Table S4. Log2 fold change (Log2fc) of the top 10 genes differentially expressed in Δrsr1 and QM6a strains
: Table S5. Log2fold change (Log2fc) of characterized transcriptional factors involved in the regulation of lignocellulase genes. NS represented not significant, P adjust > 0.05
Table S6 The changes of 876 predicted transporter genes in T. reesei QM6a genome were sorted out
: Table S7. The changes of 58 GPCR genes  in Δrsr1 and QM6a strains were sorted out, NS represented not significant, P adjust > 0.05
: Table S8 Primers used in this study
: Figure S4. The correlation between the biological replicates of each sample. The following heat map is used to show the correlation. Each value in the grid is the correlation coefficient between two samples. The larger the value, the greater the correlation between the two samples and the closer they are. These results indicate that the transcriptome data is more credible
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Li, N., Qiu, Z., Cai, W. et al. The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei. Biotechnol Biofuels 16, 87 (2023). https://doi.org/10.1186/s13068-023-02341-z