Journal of Microbiological Methods 108 (2015) 70–73
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Construction of an efficient RNAi system in the cellulolytic fungus Trichoderma reesei Ronglin He a,b,c, Wei Guo a, Lixian Wang a, Dongyuan Zhang a,⁎ a b c
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, PR China Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, PR China Tianjin Key Lab of Industrial Microbiology, Tianjin University of Science and Technology, Tianjin 300457, PR China
a r t i c l e
i n f o
Article history: Received 22 September 2014 Received in revised form 6 November 2014 Accepted 14 November 2014 Available online 20 November 2014
a b s t r a c t An improved RNA interference method was developed in Trichoderma reesei, using convergent dual promoters for efficient and high-throughput RNA silencing. This new vector allowed for the silencing of the eGFP gene and target genes to occur simultaneously, significantly facilitating the rapid screening of the transformants using eGFP as a reporter. © 2014 Published by Elsevier B.V.
Keywords: RNAi Dual promoter Trichoderma reesei eGFP
Trichoderma reesei (teleomorph Hypocrea jecorina) is a famous filamentous fungus that is widely used for the production of hydrolytic enzymes, and is recognized as a potential candidate for the conversion of renewable lignocellulosic biomass to biofuels (Wilson, 2009). As one of the most important cellulase producing strains, the yield and the performance of T. reesei have been remarkably improved by random mutagenesis which has contributed a lot to the modification of industrial strains. However, the classical strain improvement method has many limitations, such as labor- and time-consuming, lack of inherent stability and so on. The publication of the genome sequence of T. reesei has made it possible to introduce state-of-the-art gene manipulation methods into the strain improvement programs (Martinez et al., 2008). Therefore, developing efficient molecular tools is a feasible way to further study the mechanisms of cellulase or hemicellulase gene regulation and improve protein secretion in T. reesei. RNA silencing is an efficient tool for knocking down the expression of a target gene in cells. In T. reesei, the highly expressed genes cel6a and cbh1 were reported to be suppressed by means of RNAi based on a hairpin design (Brody and Maiyuran, 2009; Qin et al., 2012). Generally, RNA silencing is performed using silencing vectors which produce hairpin RNA in filamentous fungi (Nakayashiki et al., 2005). When constructing an hpRNA silencing vector, the target sequence needs to be cloned twice in opposite directions to form the hpRNA needed to produce double-stranded RNA and trigger the RNA silencing machinery
⁎ Corresponding author. Tel./fax: +86 22 84861932. E-mail address: [email protected] (D. Zhang).
http://dx.doi.org/10.1016/j.mimet.2014.11.010 0167-7012/© 2014 Published by Elsevier B.V.
(Nakayashiki, 2005). Therefore, application of the hpRNA expression vector is relatively limited. In this study, an improved RNA silencing system with dual promoters was constructed in T. reesei. A trpC promoter from Aspergillus nidulans (Sakai et al., 2008), and an rp2 promoter from T. reesei (He et al., 2013) were subsequently cloned into the SacI/KpnI and SmaI/ BamHI sites of pCAMBIA1300-1 to generate pCAMBIA1300-1-dual (Fig. 1a). For eGFP gene silencing, a 500 bp eGFP fragment was inserted into the SmaI site in pCAMBIA1300-1-dual to generate pCAMBIA13001Sgfp (Fig. 1b). For co-silencing of the target genes and the eGFP gene, pCAMBIA1300-1-dual-based vectors were constructed by inserting of a coding fragment of the target gene (Fig. 1c). A transformant named GFP-11 with strong green fluorescence was acquired in a previous study (He et al., 2013). To evaluate the applicability of pCAMBIA1300-1dual vector for inducing RNA silencing, the enhanced green fluorescence protein gene (eGFP) was used as the reporter. The obtained transformants were screened under fluorescence microscopy. Green fluorescence appeared to be reduced in 11 of the total 20 transformants, compared with GFP-11 (data not shown). Transformants T13, T14, and T2, which appeared to have different intensities of green fluorescence, were selected and cultivated on PDA for 3 days. In comparison with GFP-11, green fluorescence was scarcely observed in T13 and T14, whereas T2 showed strong green fluorescence in both the hyphae and conidia (Fig. 2a). The expression level of gfp mRNA was examined by quantitative RT-PCR of T13, T14, T2, and GFP-11. As expected, the expression level of gfp was significantly reduced in T13 and T14 while no significant reduction was detected in the non-silenced transformant T2 (Fig. 2b). The results demonstrated
R. He et al. / Journal of Microbiological Methods 108 (2015) 70–73
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Fig. 1. Schematic of the RNA silencing vectors pCAMBIA1300-1dual (a), pCAMBIA1300-1Sgfp (b), and pCAMBIA1300-1SgfpT (c) with two dual promoters; PtrpC, trpC promoter from Aspergillus nidulans and Prp2, rp2 promoter from Trichoderma reesei.
that the eGFP gene was successfully silenced using the pCAMBIA13001-dual vector. To investigate the effect of co-silencing using the pCAMBIA1300-1dual vector, three genes (RhoA, Cla4, and Ras2) were targeted for cosilencing. To facilitate the screening of transformants, the eGFP gene was used as a reporter for co-silencing in the pCAMBIA1300-1-dual vector. The target genes were fused with the eGFP gene in pCAMBIA13001SgfpT and then transformed into T. reesei GFP-11 through AMT method. The green fluorescence intensity of the obtained transformants decreased
by varying levels compared with the parent strain GFP-11 (data not shown). For each gene, three transformants with different levels of reduction in their green fluorescence intensity were selected for further quantitative PCR analysis. As shown in Fig. 3a–c, the relative expression levels of RhoA in RhoT11/17/19, Ras2 in RasT6/9/13 and Cla4 in ClaT3/8/21 were significantly decreased, suggesting that the pCAMBIA1300-1-dual based vector can induce gene silencing of RhoA, Ras2 and Cla4 to different degrees. In a previously study, these three genes were reported to be involved in fungal morphology, especially in hyphae branching. The gene
Fig. 2. Green florescence and mRNA expression level of the eGFP gene in the silenced strains and GFP-11. (a). GFP-11, the parent strain; T13 and T14, strongly silenced transformants; T2, non-silenced transformants. Left column, images of green fluorescence; right column, images of bright fields. Bars, 10 μm. (b). Expression levels of eGFP gene in the silenced strains and GFP-11. The expression level of the eGFP gene was analyzed by qRT-PCR and normalized with an endogenous control gpd1. The respective ratio in GFP-11 was set to 1. The experiment was repeated three times. The error bars represent the deviation from three independent experiments.
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R. He et al. / Journal of Microbiological Methods 108 (2015) 70–73
Fig. 3. RNA silencing of genes involved in hyphal morphology. mRNA expression levels of T. reesei RNA silencing transformants of genes involved in hyphal morphology (a–c). (a). mRNA expression level of the RhoA gene in RhoT11/17/19, RhoT11, strongly silenced transformant, RhoT17/19, moderately silenced transformant. (b). mRNA expression level of the Ras2 gene in RasT6/9/13, RasT6, strongly silenced transformant; RasT9/13, moderately silenced transformants. (c). mRNA expression level of the Cla4 gene in ClaT3/8/21, ClaT3, strongly silenced transformants, ClaT8/21, moderately silenced transformant. The expression levels of RhoA, Cla4 and Ras2 were analyzed by qRT-PCR and the respective ratio of each gene in GFP-11 was set to 1. The experiment was repeated three times. The error bars represent the deviation from three independent experiments. Hyphal morphology of the most strongly silenced transformants (d–g). (d). The parent strain GFP-11. (e). The silenced transformant RhoT11 of RhoA. (f). The silenced transformant RasT6 of Ras2. (g). The silenced transformant ClaT3 of Cla4. Bars, 20 μm.
null mutants of these three genes showed more branches in their hyphae (Ayad-Durieux et al., 2000; Guest et al., 2004; Zhang et al., 2012). Therefore, the branching phenotypes were further examined in the most
strongly silenced transformants of these genes, RhoT11, RasT6 and ClaT3. As compared to GFP-11, the silencing transformants of RhoT11, RasT6 and ClaT3 appeared highly branched hyphae (Fig. 3e–g). These
R. He et al. / Journal of Microbiological Methods 108 (2015) 70–73
changes in the hyphae branching changing suggest that RhoA, Cla4 and Ras2 were silenced in the transformants at an extremely high level, similar to the gene null mutants. Our results demonstrate that the pCAMBIA1300-1dual-based vector for RNA silencing could be used as a powerful genetic tool in T. reesei. The greatest advantage of this RNA silencing system is that the vector construction can be completed in one step, without considering the direction of the target gene. This advantage makes the construction of multiple silencing vectors in a single transformation experiment possible. However, an RNA silencing vector with opposite promoters was previously reported to have relatively lower silencing efficiency compared with hpRNA silencing vectors (Nguyen et al., 2008). This reduced efficiency might be due to the different formation of dsRNA in the RNA silencing machinery. In this study, this disadvantage was overcome by fusing an eGFP gene as a reporter with a target gene for cotranscription in the transformants. This facilitated the observation of green fluorescence for screening the transformants and compensated for the relatively low efficiency of this RNA silencing system. In conclusion, an improved RNA silencing system was established in T. reesei. The silencing vector pCAMBIA1300-1Sgfp, carrying dual opposing promoters and an eGFP reporter gene, facilitated the screening of transformants and made the silencing of target gene highly convenient and effective. Using this vector, three genes involved in hyphal branching were successfully silenced and the strongly silenced transformants of these three genes displayed a highly branched morphology compared with the parent strain. The results confirm that this improved RNA silencing vector is an efficient tool for gene manipulation in T. reesei. Acknowledgments This work was supported by the National Natural Science Foundation of China for the Youth (No. 21406259), the National High Technology Research and Development Program of China (No. 2012AA023202), the Key Technology R&D Program of Tianjin Municipal Science and Technology Commission (No. 13ZCZDSY05500) and the Foundation of
73
Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Lab of Industrial Microbiology (Tianjin University of Science & Technology) (No. 2014IM004). References Ayad-Durieux, Y., Knechtle, P., Goff, S., Dietrich, F., Philippsen, P., 2000. A PAK-like protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii. J. Cell Sci. 113, 4563–4575. Brody, H., Maiyuran, S., 2009. RNAi-mediated gene silencing of highly expressed genes in the industrial fungi Trichoderma reesei and Aspergillus niger. Ind. Biotechnol. 5, 53–60. Guest, G.M., Lin, X., Momany, M., 2004. Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis. Fungal Genet. Biol. 41, 13–22. He, R., Zhang, C., Guo, W., Wang, L., Zhang, D., Chen, S., 2013. Construction of a plasmid for heterologous protein expression with a constitutive promoter in Trichoderma reesei. Plasmid 70, 425–429. Martinez, D., Berka, R.M., Henrissat, B., Saloheimo, M., Arvas, M., Baker, S.E., Chapman, J., Chertkov, O., Coutinho, P.M., Cullen, D., Danchin, E.G., Grigoriev, I.V., Harris, P., Jackson, M., Kubicek, C.P., Han, C.S., Ho, I., Larrondo, L.F., de Leon, A.L., Magnuson, J.K., Merino, S., Misra, M., Nelson, B., Putnam, N., Robbertse, B., Salamov, A.A., Schmoll, M., Terry, A., Thayer, N., Westerholm-Parvinen, A., Schoch, C.L., Yao, J., Barabote, R., Nelson, M.A., Detter, C., Bruce, D., Kuske, C.R., Xie, G., Richardson, P., Rokhsar, D.S., Lucas, S.M., Rubin, E.M., Dunn-Coleman, N., Ward, M., Brettin, T.S., 2008. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat. Biotechnol. 26, 553–560. Nakayashiki, H., 2005. RNA silencing in fungi: mechanisms and applications. FEBS Lett. 26, 5950–5957. Nakayashiki, H., Hanada, S., Nguyen, B.Q., Kadotani, N., Tosa, Y., Mayama, S., 2005. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet. Biol. 42, 275–283. Nguyen, Q.B., Kadotani, N., Kasahara, S., Tosa, Y., Mayama, S., Nakayashiki, H., 2008. Systematic functional analysis of calcium-signalling proteins in the genome of the rice-blast fungus, Magnaporthe oryzae, using a high-throughput RNA-silencing system. Mol. Microbiol. 68, 1348–1365. Qin, L.N., Cai, F.R., Dong, X.R., Huang, Z.B., Tao, Y., Huang, J.Z., Dong, Z.Y., 2012. Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene. Bioresour. Technol. 109, 116–122. Sakai, K., Kinoshita, H., Shimizu, T., Nihira, T., 2008. Construction of a citrinin gene cluster expression system in heterologous Aspergillus oryzae. J. Biosci. Bioeng. 106, 466–472. Wilson, D.B., 2009. Cellulases and biofuels. Curr. Opin. Biotechnol. 20, 295–299. Zhang, J., Zhang, Y., Zhong, Y., Qu, Y., Wang, T., 2012. Ras GTPases modulate morphogenesis, sporulation and cellulase gene expression in the cellulolytic fungus Trichoderma reesei. PLoS One 7, e48786.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Construction of an efficient RNAi system in the cellulolytic fungus Trichoderma reesei Ronglin He a,b,c, Wei Guo a, Lixian Wang a, Dongyuan Zhang a,⁎ a b c
Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin 300308, PR China Key Laboratory of Industrial Fermentation Microbiology (Tianjin University of Science & Technology), Ministry of Education, Tianjin 300457, PR China Tianjin Key Lab of Industrial Microbiology, Tianjin University of Science and Technology, Tianjin 300457, PR China
a r t i c l e
i n f o
Article history: Received 22 September 2014 Received in revised form 6 November 2014 Accepted 14 November 2014 Available online 20 November 2014
a b s t r a c t An improved RNA interference method was developed in Trichoderma reesei, using convergent dual promoters for efficient and high-throughput RNA silencing. This new vector allowed for the silencing of the eGFP gene and target genes to occur simultaneously, significantly facilitating the rapid screening of the transformants using eGFP as a reporter. © 2014 Published by Elsevier B.V.
Keywords: RNAi Dual promoter Trichoderma reesei eGFP
Trichoderma reesei (teleomorph Hypocrea jecorina) is a famous filamentous fungus that is widely used for the production of hydrolytic enzymes, and is recognized as a potential candidate for the conversion of renewable lignocellulosic biomass to biofuels (Wilson, 2009). As one of the most important cellulase producing strains, the yield and the performance of T. reesei have been remarkably improved by random mutagenesis which has contributed a lot to the modification of industrial strains. However, the classical strain improvement method has many limitations, such as labor- and time-consuming, lack of inherent stability and so on. The publication of the genome sequence of T. reesei has made it possible to introduce state-of-the-art gene manipulation methods into the strain improvement programs (Martinez et al., 2008). Therefore, developing efficient molecular tools is a feasible way to further study the mechanisms of cellulase or hemicellulase gene regulation and improve protein secretion in T. reesei. RNA silencing is an efficient tool for knocking down the expression of a target gene in cells. In T. reesei, the highly expressed genes cel6a and cbh1 were reported to be suppressed by means of RNAi based on a hairpin design (Brody and Maiyuran, 2009; Qin et al., 2012). Generally, RNA silencing is performed using silencing vectors which produce hairpin RNA in filamentous fungi (Nakayashiki et al., 2005). When constructing an hpRNA silencing vector, the target sequence needs to be cloned twice in opposite directions to form the hpRNA needed to produce double-stranded RNA and trigger the RNA silencing machinery
⁎ Corresponding author. Tel./fax: +86 22 84861932. E-mail address: [email protected] (D. Zhang).
http://dx.doi.org/10.1016/j.mimet.2014.11.010 0167-7012/© 2014 Published by Elsevier B.V.
(Nakayashiki, 2005). Therefore, application of the hpRNA expression vector is relatively limited. In this study, an improved RNA silencing system with dual promoters was constructed in T. reesei. A trpC promoter from Aspergillus nidulans (Sakai et al., 2008), and an rp2 promoter from T. reesei (He et al., 2013) were subsequently cloned into the SacI/KpnI and SmaI/ BamHI sites of pCAMBIA1300-1 to generate pCAMBIA1300-1-dual (Fig. 1a). For eGFP gene silencing, a 500 bp eGFP fragment was inserted into the SmaI site in pCAMBIA1300-1-dual to generate pCAMBIA13001Sgfp (Fig. 1b). For co-silencing of the target genes and the eGFP gene, pCAMBIA1300-1-dual-based vectors were constructed by inserting of a coding fragment of the target gene (Fig. 1c). A transformant named GFP-11 with strong green fluorescence was acquired in a previous study (He et al., 2013). To evaluate the applicability of pCAMBIA1300-1dual vector for inducing RNA silencing, the enhanced green fluorescence protein gene (eGFP) was used as the reporter. The obtained transformants were screened under fluorescence microscopy. Green fluorescence appeared to be reduced in 11 of the total 20 transformants, compared with GFP-11 (data not shown). Transformants T13, T14, and T2, which appeared to have different intensities of green fluorescence, were selected and cultivated on PDA for 3 days. In comparison with GFP-11, green fluorescence was scarcely observed in T13 and T14, whereas T2 showed strong green fluorescence in both the hyphae and conidia (Fig. 2a). The expression level of gfp mRNA was examined by quantitative RT-PCR of T13, T14, T2, and GFP-11. As expected, the expression level of gfp was significantly reduced in T13 and T14 while no significant reduction was detected in the non-silenced transformant T2 (Fig. 2b). The results demonstrated
R. He et al. / Journal of Microbiological Methods 108 (2015) 70–73
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Fig. 1. Schematic of the RNA silencing vectors pCAMBIA1300-1dual (a), pCAMBIA1300-1Sgfp (b), and pCAMBIA1300-1SgfpT (c) with two dual promoters; PtrpC, trpC promoter from Aspergillus nidulans and Prp2, rp2 promoter from Trichoderma reesei.
that the eGFP gene was successfully silenced using the pCAMBIA13001-dual vector. To investigate the effect of co-silencing using the pCAMBIA1300-1dual vector, three genes (RhoA, Cla4, and Ras2) were targeted for cosilencing. To facilitate the screening of transformants, the eGFP gene was used as a reporter for co-silencing in the pCAMBIA1300-1-dual vector. The target genes were fused with the eGFP gene in pCAMBIA13001SgfpT and then transformed into T. reesei GFP-11 through AMT method. The green fluorescence intensity of the obtained transformants decreased
by varying levels compared with the parent strain GFP-11 (data not shown). For each gene, three transformants with different levels of reduction in their green fluorescence intensity were selected for further quantitative PCR analysis. As shown in Fig. 3a–c, the relative expression levels of RhoA in RhoT11/17/19, Ras2 in RasT6/9/13 and Cla4 in ClaT3/8/21 were significantly decreased, suggesting that the pCAMBIA1300-1-dual based vector can induce gene silencing of RhoA, Ras2 and Cla4 to different degrees. In a previously study, these three genes were reported to be involved in fungal morphology, especially in hyphae branching. The gene
Fig. 2. Green florescence and mRNA expression level of the eGFP gene in the silenced strains and GFP-11. (a). GFP-11, the parent strain; T13 and T14, strongly silenced transformants; T2, non-silenced transformants. Left column, images of green fluorescence; right column, images of bright fields. Bars, 10 μm. (b). Expression levels of eGFP gene in the silenced strains and GFP-11. The expression level of the eGFP gene was analyzed by qRT-PCR and normalized with an endogenous control gpd1. The respective ratio in GFP-11 was set to 1. The experiment was repeated three times. The error bars represent the deviation from three independent experiments.
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R. He et al. / Journal of Microbiological Methods 108 (2015) 70–73
Fig. 3. RNA silencing of genes involved in hyphal morphology. mRNA expression levels of T. reesei RNA silencing transformants of genes involved in hyphal morphology (a–c). (a). mRNA expression level of the RhoA gene in RhoT11/17/19, RhoT11, strongly silenced transformant, RhoT17/19, moderately silenced transformant. (b). mRNA expression level of the Ras2 gene in RasT6/9/13, RasT6, strongly silenced transformant; RasT9/13, moderately silenced transformants. (c). mRNA expression level of the Cla4 gene in ClaT3/8/21, ClaT3, strongly silenced transformants, ClaT8/21, moderately silenced transformant. The expression levels of RhoA, Cla4 and Ras2 were analyzed by qRT-PCR and the respective ratio of each gene in GFP-11 was set to 1. The experiment was repeated three times. The error bars represent the deviation from three independent experiments. Hyphal morphology of the most strongly silenced transformants (d–g). (d). The parent strain GFP-11. (e). The silenced transformant RhoT11 of RhoA. (f). The silenced transformant RasT6 of Ras2. (g). The silenced transformant ClaT3 of Cla4. Bars, 20 μm.
null mutants of these three genes showed more branches in their hyphae (Ayad-Durieux et al., 2000; Guest et al., 2004; Zhang et al., 2012). Therefore, the branching phenotypes were further examined in the most
strongly silenced transformants of these genes, RhoT11, RasT6 and ClaT3. As compared to GFP-11, the silencing transformants of RhoT11, RasT6 and ClaT3 appeared highly branched hyphae (Fig. 3e–g). These
R. He et al. / Journal of Microbiological Methods 108 (2015) 70–73
changes in the hyphae branching changing suggest that RhoA, Cla4 and Ras2 were silenced in the transformants at an extremely high level, similar to the gene null mutants. Our results demonstrate that the pCAMBIA1300-1dual-based vector for RNA silencing could be used as a powerful genetic tool in T. reesei. The greatest advantage of this RNA silencing system is that the vector construction can be completed in one step, without considering the direction of the target gene. This advantage makes the construction of multiple silencing vectors in a single transformation experiment possible. However, an RNA silencing vector with opposite promoters was previously reported to have relatively lower silencing efficiency compared with hpRNA silencing vectors (Nguyen et al., 2008). This reduced efficiency might be due to the different formation of dsRNA in the RNA silencing machinery. In this study, this disadvantage was overcome by fusing an eGFP gene as a reporter with a target gene for cotranscription in the transformants. This facilitated the observation of green fluorescence for screening the transformants and compensated for the relatively low efficiency of this RNA silencing system. In conclusion, an improved RNA silencing system was established in T. reesei. The silencing vector pCAMBIA1300-1Sgfp, carrying dual opposing promoters and an eGFP reporter gene, facilitated the screening of transformants and made the silencing of target gene highly convenient and effective. Using this vector, three genes involved in hyphal branching were successfully silenced and the strongly silenced transformants of these three genes displayed a highly branched morphology compared with the parent strain. The results confirm that this improved RNA silencing vector is an efficient tool for gene manipulation in T. reesei. Acknowledgments This work was supported by the National Natural Science Foundation of China for the Youth (No. 21406259), the National High Technology Research and Development Program of China (No. 2012AA023202), the Key Technology R&D Program of Tianjin Municipal Science and Technology Commission (No. 13ZCZDSY05500) and the Foundation of
73
Key Laboratory of Industrial Fermentation Microbiology of Ministry of Education and Tianjin Key Lab of Industrial Microbiology (Tianjin University of Science & Technology) (No. 2014IM004). References Ayad-Durieux, Y., Knechtle, P., Goff, S., Dietrich, F., Philippsen, P., 2000. A PAK-like protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii. J. Cell Sci. 113, 4563–4575. Brody, H., Maiyuran, S., 2009. RNAi-mediated gene silencing of highly expressed genes in the industrial fungi Trichoderma reesei and Aspergillus niger. Ind. Biotechnol. 5, 53–60. Guest, G.M., Lin, X., Momany, M., 2004. Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis. Fungal Genet. Biol. 41, 13–22. He, R., Zhang, C., Guo, W., Wang, L., Zhang, D., Chen, S., 2013. Construction of a plasmid for heterologous protein expression with a constitutive promoter in Trichoderma reesei. Plasmid 70, 425–429. Martinez, D., Berka, R.M., Henrissat, B., Saloheimo, M., Arvas, M., Baker, S.E., Chapman, J., Chertkov, O., Coutinho, P.M., Cullen, D., Danchin, E.G., Grigoriev, I.V., Harris, P., Jackson, M., Kubicek, C.P., Han, C.S., Ho, I., Larrondo, L.F., de Leon, A.L., Magnuson, J.K., Merino, S., Misra, M., Nelson, B., Putnam, N., Robbertse, B., Salamov, A.A., Schmoll, M., Terry, A., Thayer, N., Westerholm-Parvinen, A., Schoch, C.L., Yao, J., Barabote, R., Nelson, M.A., Detter, C., Bruce, D., Kuske, C.R., Xie, G., Richardson, P., Rokhsar, D.S., Lucas, S.M., Rubin, E.M., Dunn-Coleman, N., Ward, M., Brettin, T.S., 2008. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat. Biotechnol. 26, 553–560. Nakayashiki, H., 2005. RNA silencing in fungi: mechanisms and applications. FEBS Lett. 26, 5950–5957. Nakayashiki, H., Hanada, S., Nguyen, B.Q., Kadotani, N., Tosa, Y., Mayama, S., 2005. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet. Biol. 42, 275–283. Nguyen, Q.B., Kadotani, N., Kasahara, S., Tosa, Y., Mayama, S., Nakayashiki, H., 2008. Systematic functional analysis of calcium-signalling proteins in the genome of the rice-blast fungus, Magnaporthe oryzae, using a high-throughput RNA-silencing system. Mol. Microbiol. 68, 1348–1365. Qin, L.N., Cai, F.R., Dong, X.R., Huang, Z.B., Tao, Y., Huang, J.Z., Dong, Z.Y., 2012. Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gene. Bioresour. Technol. 109, 116–122. Sakai, K., Kinoshita, H., Shimizu, T., Nihira, T., 2008. Construction of a citrinin gene cluster expression system in heterologous Aspergillus oryzae. J. Biosci. Bioeng. 106, 466–472. Wilson, D.B., 2009. Cellulases and biofuels. Curr. Opin. Biotechnol. 20, 295–299. Zhang, J., Zhang, Y., Zhong, Y., Qu, Y., Wang, T., 2012. Ras GTPases modulate morphogenesis, sporulation and cellulase gene expression in the cellulolytic fungus Trichoderma reesei. PLoS One 7, e48786.
