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HABILITATION THESIS

Environmental signalling in

Trichoderma reesei (Hypocrea jecorina)

In the fulfilment of the requirements

for the venia docendi in the field of

Molecular Genetics and Genomics

DI Dr. techn. Monika Schmoll

Institute of Chemical Engineering

Vienna University of Technology

Vienna (Austria)

December 2011

Habilitation thesis Monika Schmoll 2

Dream big and dare to fail. It is possible to make a dream come true. (James Hetfield)


Abstract

Life on earth is dominated by the need to deal with the consequences of the rotation of earth – light and darkness,

the availability of appropriate nutrients and the quest for reproduction. Successful competition in nature is dependent on efficient sensing and interpretation of environmental signals aimed at an optimal reaction to changing environmental conditions with minimal consumption of cellular resources. Filamentous fungi, such as Trichoderma reesei (anamorph of Hypocrea jecorina) have to deal with countless challenges to succeed in the battle for nutrients, space and reproduction in the rich habitat of a tropical rain forest. Due to its efficient enzymes for decomposition of plant cell walls, T. reesei has become a paradigm for industrial cellulase production and is nowadays the main industrial cellulase producer as well as subject to basic research. Hence, an efficient machinery for perception and interpretation of environmental signals enables T. reesei to adjust to such different environments as rotting trees in the tropics and a shake flask with chemically exactly defined minimal medium in the lab. In this work initial steps towards elucidation of the interplay between the light response machinery, nutrient signaling and sexual development are described (Figure 1). As with most fungi, light causes considerable adaptations in the physiology of T. reesei. Besides early phenomena such as conidiation and growth, more recent studies showed an influence of light on expression of glycoside hydrolase genes, including cellulase encoding genes. The photoreceptors of T. reesei, BLR1 and BLR2, as well as the light regulatory protein ENV1 play an important role in this regulation, which is conserved also in the fungus Neurospora crassa. This obvious interplay between the light response pathway and nutrient signaling was further investigated focused on the heterotrimeric G-protein pathway with cellulase gene expression as a model output pathway. These studies revealed intriguing crosstalk between these signaling pathways and showed that different extracellular signals as transmitted by the G-protein alpha subunits GNA1 and GNA3 enhance cellulase gene expression in a light dependent way. Thereby, ENV1 was found to represent a crucial node between these pathways. ENV1 is on the one hand induced by the

Figure 1. Overview on components involved in regulation of cellulase gene expression and/or signal transduction in T. reesei investigated and described in this thesis.


photoreceptors BLR1 and BLR2 and on the other hand impacts abundance of gna3 and interferes with the positive transcriptional feedback of gna1. One important function of ENV1 in this pathway is suggested to be a negative effect on the activity of phosphodiesterases and hence a regulatory function in the cAMP pathway, which targets cellulase gene expression. While the nutrient signals transmitted by GNA1 and GNA3 remain to be determined, methionine was identified as an extracellular signal important for cellulase regulation in a light dependent way. Accordingly, sulphur metabolism also plays a light dependent role in this process. Although previous studies postulated that expression of cellulase genes is regulated at the transcriptional level, evidence was obtained that dependent on the light conditions, also posttranscriptional regulatory steps must be involved in cellulase regulation. Not only metabolism, but also reproduction is adjusted to environmental conditions and light as well as nutrients are important signals for the decision whether to initiate sexual or asexual development. G-protein coupled receptors are thereby responsible for perception of the pheromone signal transmitted by a mating partner. Although the sexually competent species Hypocrea jecorina was found to be indistinguishable from T. reesei, the capability of this fungus to reproduce sexually was only recently detected (Figure 2). T. reesei was confirmed to be heterothallic and the parental strain of all strains used in research of industry, QM6a, showed mating type MAT1-2. However, QM6a is female fertile and needs a wild isolate to form fruiting bodies. Another peculiarity of T.

reesei is the presence of a novel type of peptide pheromones, the h-type, with HPP1 as first representative. This peptide pheromone shares characteristics of a- and alpha type pheromones and assumed a-type function in T. reesei. In summary, we could elucidate the function of the light response machinery, as represented by BLR1, BLR2 and ENV1, in cellulase gene expression and we provide first insights into the interplay of these components with nutrient signaling. Our studies with N. crassa revealed that the effect of light on cellulase gene expression is conserved in fungi and we postulate that cellulase are also regulated at the posttranscriptional level. With methionine we identified a nutrient signal with light dependent effect. We showed sexual development in Trichoderma reesei and discovered a novel type of peptide pheromone precursors.

Figure 2. Fruiting body

formation with T. reesei


Contents

Abstract 3 Contents 5

Concept of this thesis 6 List of publications of the habilitation 7

Preface – Development and background of the research focus 13

1- An industrial workhorse and its evolutionary heritage 15

1.1. Trichoderma reesei – the fungus 15 1.2. Cellulose, cellulases and Trichoderma reesei 16 1.3. Regulation of cellulase gene expression 18

1.4. Second generation biofuels – sustainable and environmentally safe 20

2- Dealing with a changing environment 21

2.1. Adaptation to the rotation of earth – light and darkness 22 2.1.1. Circadian rhythms and light response 22 2.1.2. Light response and cellulase gene expression in Trichoderma 23

2.1.3. Crosstalk between light response and cellulase gene expression in Neurospora 25

2.2. Integrating nutrient signaling with light response 27

2.2.1. The heterotrimeric G-protein pathway 27 2.2.2. Heterotrimeric G-protein signaling in T. reesei 27 2.2.3. Phosducins as light dependent regulators of

heterotrimeric G-protein signalling 29 2.3. Elucidating the mechanism of crosstalk 30 2.4. cAMP signaling, light response and cellulase gene expression 32

3- The quest for reproduction – sensing and reacting to partners 33

3.1. Sexual development in fungi 33

3.2. The special case of Trichoderma 34 3.3. Causes for female sterility in fungi 35 3.4. Determinants of sexual development 36

3.4.1. Pheromones in Trichoderma 36 3.4.2. Pheromone receptors – sensing a partner and reacting to signals 37 3.4.3. The influence of light on sexual development of Trichoderma 37

3.5. Importance of sexual development in research and industry 38 4- Exploring genomes – the treasury of research and industry 40

4.1. A genome wide view on plant cell wall degradation by fungi 41 4.2. The genomes of three unequal siblings – Trichoderma species and their characteristics 42

4.3. A genome wide view on environmental signaling pathways 42 4.3.1. The signaling highways of Trichoderma 42 4.3.2. A glimpse on signaling mechanisms in basidiomycetes 43

5- Conclusions and outlook 45

6- Literature cited 47 Curriculum vitae 59

Acknowledgements 63 Appendix I – Publications of this habilitation thesis 65

Appendix II – Further scientific work (see Addendum)


Concept of this thesis The following introduction starts with an outline of current issues, which formed the basis for the research described in this thesis. Thereafter background on the history and relevance of Trichoderma reesei for research and biotechnology is provided. The core of this thesis describes the interplay of light signaling and nutrient signaling using cellulase gene expression as output pathway as well as current knowledge on sexual development in T. reesei/H. jecorina and hence summarizes my work on these topics. In these chapters also results of ongoing projects, which are to be published in the near future, are discussed. The last chapter comprises a summary of the relevance of the presented work along with an outlook to further research in the field. Cited publications to which I contributed as author are written in bold. The publications summarized in the following can be found in the appendix of this habilitation thesis and further scientific work is provided in the Addendum (Appendix II).


List of publications in this habilitation thesis

A. Fungi and light

A. Tisch, D. & Schmoll, M. (2010) Light regulation of metabolic pathways in fungi (invited review). Applied Microbiology and Biotechnology, 85 (5): 1259 - 1277.

My contribution: Conception of the review and writing of the manuscript.

B. Light response in Trichoderma reesei

Schmoll, M., Franchi, L., & Kubicek, C. P. (2005) Envoy, a PAS/LOV domain protein of Hypocrea jecorina (anamorph Trichoderma reesei), modulates cellulase gene transcription in response to light. Eukaryotic Cell 4, 1998-2007.

My contribution: All experimental work, data analysis and interpretation. Writing of the manuscript was done in collaboration with CPK.

Schuster, A., Kubicek, C. P., Friedl, M. A., Druzhinina, I. S., & Schmoll, M. (2007) Impact of light on Hypocrea jecorina and the multiple cellular roles of ENVOY in this process. BMC Genomics 8, 449.

My contribution: Design of the study, experiments were done under my supervision except BIOLOG analysis, interpretation of results. Writing of the manuscript was done in collaboration with CPK.

Castellanos, F.*, Schmoll, M.*, Martínez, P., Tisch, D., Kubicek, C. P., Herrera-Estrella, A., Esquivel-Naranjo, E. U. (2010) Crucial factors of the light perception machinery and their impact on growth and cellulase gene transcription in Trichoderma reesei. Fungal Genet Biol, May; 47 (5): 468 - 76 *These authors contributed equally to this work

My contribution: Participation in design of the study, construction of one mutant strain, supervision of analysis of cellulase gene expression in all described strains (performed by DT). Writing of the manuscript was done in collaboration with AHE and EUEN.

Gyalai-Korpos M., Nagy G., Mareczky Z., Schuster A., Réczey K. and Schmoll M. (2010) Relevance of the light signaling machinery for cellulase expression in Trichoderma reesei (Hypocrea jecorina), BMC Research Notes, 3:330

My contribution: Design of the study, supervision of experimental work of MGK together with KR, data interpretation and writing of the final version of the manuscript.


C. Transmission of nutrient signals in Trichoderma reesei Gremel, G., Dorrer, M., & Schmoll, M. (2008) Sulphur metabolism and cellulase gene expression are connected processes in the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei). BMC Microbiology 8, 174.

My contribution: Design of the study, experimental work using the Yeast One Hybrid system, EMSA analysis, transcript analysis (partial), supervision of experimental work done by GG and MD, interpretation of data and writing the manuscript.

Schmoll, M. (2008) The information highways of a biotechnological workhorse--signal transduction in Hypocrea jecorina. BMC Genomics 9, 430. Schmoll, M., Schuster, A., Silva Rdo, N., & Kubicek, C. P. (2009) The G-alpha protein GNA3 of Hypocrea jecorina (Anamorph Trichoderma reesei) regulates cellulase gene expression in the presence of light. Eukaryotic Cell 8, 410-420.

My contribution: Participation in design of the study, supervision of experimental work done by AS and RNS and data interpretation. Writing of the manuscript was done in collaboration with CPK.

Schuster, A. & Schmoll, M. (2009) Heterotrimeric G-protein signaling and light response: Two signaling pathways coordinated for optimal adjustment to nature. Communicative & Integrative Biology 2, 308-310.

My contribution: Design of the study supervision of experimental work done by AS, interpretation of data and writing of the manuscript.

Seibel, C., Gremel, G., do Nascimento Silva, R., Schuster, A., Kubicek, C.P. & Schmoll, M. (2009) Light-dependent roles of the G-protein alpha subunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei). BMC Biology 7: 58.

My contribution: Design of the study together with CPK, supervision of experimental work done by CS, GG, RNS and AS, interpretation of data. Writing of the manuscript was done in collaboration with CPK

Tisch, D., Kubicek, C. P. and Schmoll, M. (2011) New insights into the mechanism of light modulated signaling by heterotrimeric G-proteins: ENVOY acts on gna1 and gna3 and adjusts cAMP levels in Trichoderma reesei (Hypocrea jecorina), Fungal Genet Biol, 48 (6): 631 – 40

My contribution: Design of the study, supervision of experimental work, interpretation of data together with DT and writing of the manuscript.

Schuster, A., Kubicek, C. P. and Schmoll, M. (2011) The dehydrogenase GRD1 represents a novel component of the cellulase regulon in Trichoderma reesei (Hypocrea jecorina). Appl Environ Microbiol, 77(13):4553-63

My contribution: Design of the study, supervision of experimental work, interpretation of data and writing of the manuscript.


Tisch, D., Kubicek C. P. and Schmoll, M. (2012) The phosducin-like protein PhLP1 impacts regulation of glycoside hydrolases and light response in Trichoderma reesei. BMC Genomics, manuscript accepted for publication

My contribution: Design of the study, supervision of experimental work and interpretation of data together with DT, writing of the manuscript.

D. Sexual development in Trichoderma reesei

Seidl, V., Seibel, C., Kubicek, C. P., & Schmoll, M. (2009) Sexual development in the industrial workhorse Trichoderma reesei. Proc Natl Acad Sci 106, 13909-13914.

My contribution: Discovery of mating, design of the study, supervision of experimental work done by VS and CS, interpretation of data together with VS. Writing of the manuscript was done in collaboration with VS and CPK.

Schmoll M., Seibel C., Tisch D., Dorrer M. and Kubicek C. P. (2010) A Novel Class of Peptide Pheromone Precursors in Ascomycetous Fungi, Molecular Microbiology, 2010 Sep. 77(6): 1483-501

My contribution: Design of the study, participation in experimental work, supervision of experimental work done by CS, DT and MD, genomic and phylogenetic sequence analyses, data interpretation and writing of the manuscript.


Additional scientific work – Appendix II

I. Reviews

Schmoll, M. (2011) Assessing the relevance of light for fungi – implications and insights into

the network of signal transmission, Invited review, Advances in Applied Microbiology, 76: 27 - 78 Tisch, D. and Schmoll, M. (2011) Novel approaches to improve cellulase biosynthesis for

biofuel production – adjusting signal transduction pathways in the biotechnological workhorse Trichoderma reesei (Hypocrea jecorina), in: Biofuel production – recent developments and prospects, Ed: MA dos Santos Bernardes, Intech, Rijeka, Croatia,199-224 Schmoll, M., Esquivel-Naranjo, E. U. and Herrera-Estrella, A. (2010) Trichoderma in the light

of day – physiology and development. Invited review, Fungal Genetics and Biology, Nov. 47(11): 909 - 916. Schuster, A. and Schmoll M. (2010) Biology and Biotechnology of Trichoderma. invited

review, Applied Microbiology and Biotechnology, Jul;87(3):787-99 Kubicek, C. P., Mikus, M., Schuster, A., Schmoll, M., & Seiboth, B. (2009) Metabolic

engineering strategies for the improvement of cellulase production by Hypocrea jecorina. Biotechnology for biofuels 2, 19. Schmoll, M. & Kubicek, C. P. (2003) Regulation of Trichoderma cellulase formation: lessons

in molecular biology from an industrial fungus. A review. Acta microbiologica et immunologica Hungarica 50, 125-145.

II. Genome analyses

Pel, H.J., de Winde, J.H., Archer, D.B., Dyer, P.S., Hofmann, G., Schaap, P.J., Turner, G., de Vries, R.P., Albang, R., Albermann, K., Andersen, M.R., Bendtsen, J.D., Benen, J.A., van den Berg, M., Breestraat, S., Caddick, M.X., Contreras, R., Cornell, M., Coutinho, P.M., Danchin, E.G., Debets, A.J., Dekker, P., van Dijck, P.W., van Dijk, A., Dijkhuizen, L., Driessen, A.J., d'Enfert, C., Geysens, S., Goosen, C., Groot, G.S., de Groot, P.W., Guillemette, T., Henrissat, B., Herweijer, M., van den Hombergh, J.P., van den Hondel, C.A., van der Heijden, R.T., van der Kaaij, R.M., Klis, F.M., Kools, H.J., Kubicek, C.P., van Kuyk, P.A., Lauber, J., Lu, X., van der Maarel, M.J., Meulenberg, R., Menke, H., Mortimer, M.A., Nielsen, J., Oliver, S.G., Olsthoorn, M., Pal, K., van Peij, N.N., Ram, A.F., Rinas, U., Roubos, J.A., Sagt, C.M., Schmoll, M., Sun, J., Ussery, D., Varga, J., Vervecken, W., van de Vondervoort, P.J., Wedler, H., Wosten, H.A., Zeng, A.P., van Ooyen, A.J., Visser, J., & Stam, H. (2007) Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nature Biotechnology 25: 221-231. 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., and Brettin, T.S. (2008) Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nature Biotechnology 26, 553-560.


Martinez, D., Challacombe, J., Morgenstern, I., Hibbett, D., Schmoll, M., Kubicek, C.P.,

Ferreira, P., Ruiz-Duenas, F.J., Martinez, A.T., Kersten, P., Hammel, K.E., Vanden Wymelenberg, A., Gaskell, J., Lindquist, E., Sabat, G., Bondurant, S.S., Larrondo, L.F., Canessa, P., Vicuna, R., Yadav, J., Doddapaneni, H., Subramanian, V., Pisabarro, A.G., Lavin, J.L., Oguiza, J.A., Master, E., Henrissat, B., Coutinho, P.M., Harris, P., Magnuson, J.K., Baker, S.E., Bruno, K., Kenealy, W., Hoegger, P.J., Kues, U., Ramaiya, P., Lucas, S., Salamov, A., Shapiro, H., Tu, H., Chee, C.L., Misra, M., Xie, G., Teter, S., Yaver, D., James, T., Mokrejs, M., Pospisek, M., Grigoriev, I.V., Brettin, T., Rokhsar, D., Berka, R., & Cullen, D. (2009) Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion. Proc Natl Acad Sci 106, 1954-1959. Kubicek CP, Herrera-Estrella A, Seidl-Seiboth V, Martinez DA, Druzhinina IS, Thon M, Zeilinger S, Casas-Flores S, Horwitz BA, Mukherjee PK, Mukherjee M, Kredics L, Alcaraz LD, Aerts A, Antal Z, Atanasova L, Cervantes-Badillo MG, Challacombe J, Chertkov O, McCluskey K, Coulpier F, Deshpande N, von Doehren H, Ebbole DJ, Esquivel-Naranjo EU, Fekete E, Flipphi M, Glaser F, Gomez-Rodriguez EY, Gruber S, Han C, Henrissat B, Hermosa R, Hernandez-Onate M, Karaffa L, Kosti I, Le Crom S, Lindquist E, Lucas S, Lubeck M, Lubeck PS, Margeot A, Metz B, Misra M, Nevalainen H, Omann M, Packer N, Perrone G, Uresti-Rivera EE, Salamov A, Schmoll M, Seiboth B, Shapiro H, Sukno S,

Tamayo-Ramos JA, Tisch D, Wiest A, Wilkinson HH, Zhang M, Coutinho PM, Kenerley CM, Monte E, Baker SE, Grigoriev IV (2011) Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma. Genome Biol. 2011 Apr 18;12(4):R40.

III. Biocontrol

Seidl, V., Schmoll, M., Scherm, B., Balmas, V., Seiboth, B., Migheli, Q., and Kubicek, C.P. (2006) Antagonism of Pythium blight of zucchini by Hypocrea jecorina does not require cellulase gene expression but is improved by carbon catabolite derepression. FEMS Microbiology Letters 257, 145-151. Scherm, B.*, Schmoll, M.*, Balmas, V., Kubicek, C. P., & Migheli, Q. (2009) Identification of

potential marker genes for Trichoderma harzianum strains with high antagonistic potential against Rhizoctonia solani by a rapid subtraction hybridization approach. Current Genetics 55, 81-91. *These two authors contributed equally Seidl, V., Song, L., Lindquist, E. A., Gruber, S., Koptchinskiy, A., Zeilinger, S., Schmoll, M.,

Martinez, P., Sun, J., Grigoriev, I., Herrera-Estrella, A., Baker, S. E & Kubicek C. P. (2009) Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the close presence of a fungal prey. BMC Genomics 10: 567

IV. Tools for Trichoderma

Schmoll, M., Zeilinger, S., Mach, R. L., & Kubicek, C. P. (2004) Cloning of genes expressed

early during cellulase induction in Hypocrea jecorina by a rapid subtraction hybridization approach. Fungal Genetics and Biology 41, 877-887. Guangtao, Z., Hartl, L., Schuster, A., Polak, S., Schmoll, M., Wang, T., Seidl, V., and Seiboth, B. (2009) Gene targeting in a nonhomologous end joining deficient Hypocrea jecorina. Journal of Biotechnology 139, 146-151.


Druzhinina, I. S., Schmoll, M., Seiboth, B., & Kubicek, C. P. (2006) Global carbon utilization profiles of wild-type, mutant, and transformant strains of Hypocrea jecorina. Applied and Environmental Microbiology 72, 2126-2133.

V. Physiological aspects and industrial use of Trichoderma

Zeilinger, S., Schmoll, M., Pail, M., Mach, R. L., & Kubicek, C. P. (2003) Nucleosome transactions on the Hypocrea jecorina (Trichoderma reesei) cellulase promoter cbh2 associated with cellulase induction. Molecular Genetics and Genomics 270, 46-55. Schmoll, M. & Kubicek, C. P. (2005) ooc1, a unique gene expressed only during growth of

Hypocrea jecorina (anamorph: Trichoderma reesei) on cellulose. Current Genetics 48, 126-133. Kratzer, C., Tobudic, S., Schmoll, M., Graninger, W., & Georgopoulos, A. (2006) In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp. The Journal of Antimicrobial Chemotherapy 58, 1058-1061. Friedl, M. A., Schmoll, M., Kubicek, C. P., & Druzhinina, I. S. (2008) Photostimulation of

Hypocrea atroviridis growth occurs due to a cross-talk of carbon metabolism, blue light receptors and response to oxidative stress. Microbiology (Reading, England) 154, 1229-1241. Schmoll M., Kotlowski, C., Seibel, C., Liebmann B., Kubicek, C. P. (2010) Recombinant

production of an Aspergillus nidulans class I hydrophobin, (DewA) in Hypocrea jecorina (Trichoderma reesei) is promoter-dependent, Applied Microbiology and Biotechnology, 88(1):95-103.


Preface

Development and background of the research focus

Rising energy costs and environmental issues due to the use of fossil fuels have caused enhanced awareness for the need of alternative energy sources. Among numerous promising approaches, also the production of ethanol from natural sources was promoted. However, recent years showed that the well-meaning initiatives to use this bioethanol raised considerable ethical and environmental problems. Utilization of food crops for production of fuels causes competition with food supply and land use for food production as well as rising costs in developing countries. Additionally, novel plantations of now profitable energy crops, such as palm trees or sugar cane pose a serious threat to “non profit” natural rain forest areas. Research and technology, however, have in the meantime developed far beyond these early and easy approaches to produce the so-called biofuels from natural resources. Second generation biofuels produced from agricultural waste material offer environmental sustainability. Cellulosic plant material is degraded to small building blocks, which can then be utilized for production of Ethanol in a biological process using yeast. However, this degradation process poses a severe challenge. Cellulose represents a high molecular, insoluble and recalcitrant material, which enabled evolutionary success to plants and is also mainly responsible for the durability of wood – as exemplified by the ubiquitous presence of wood and its products in our daily life. Nevertheless, there are specialists in nature, which are able to use cellulosic plant material as substrate and which by degradation of this material provide a crucial contribution to the cycle of matter on earth – fungi.

Figure 3. Understanding physiology represents a central requirement for efficient utilization of natural resources by fungi


Numerous fungi are able to break down cellulose into small building blocks which then can also be utilized as substrate by other organisms. The enzymes fungi produce for this task are cellulases. Since their production requires considerable cellular resources, cellulases are only produced by the fungus if absolutely necessary and if not enough other more easily digestible substrates are available. This implies on the one hand, that expression of cellulase genes is tightly regulated. On the other hand, such a tight regulation also requires a sophisticated screening system, which enables the fungus to detect extracellular resources. At the same time an efficient system for transmission and correct interpretation of such extracellular signals is needed in order to adjust the physiology of the fungus to the quality of its current environment. In short words: the life of the fungus is optimized to enable maximal competitive success with minimal input of resources. For efficient production of cellulases and other valuable products using fungi as cell factories, this evolutionarily developed machinery has to be adjusted in a way, which maximizes output of the desired product. Therefore, detailed understanding of regulatory and signalling processes is of utmost importance. The quest is to understand the fungus as a whole, its physiology as well as the mechanisms and signals triggering or enhancing enzyme production (Figure 3). While in Trichoderma reesei the application of such knowledge to industrial fermentations may be a primary interest, research in signal transduction processes will also yield intriguing and crucial insights into the physiology of fungi in general, which enables improvement of countless further applications of fungi. Ultimately, this quest should lead to the possibility to reach beyond trial and error and optimize a production organism in a knowledge based process. Additionally, understanding the physiology of fungi will also further our understanding of natural processes in which fungi are involved – from recycling of plant material to plant protection or human pathogenesis and last but not least the global carbon cycle.


1

An industrial workhorse and its evolutionary heritage

Talent is an accident of genes - and a responsibility. Alan Rickman

Fungi are of high importance for our society. They serve mankind with their metabolic capabilities to utilize a plethora of substrates. The enzymes they produce for this purpose are of high value, because these catalysts enable conversions often not possible or economically feasible by chemical means. Additionally, fungi can be used as production host of performance proteins, including human enzymes (Adrio & Demain, 2003). Therefore one important reason for research with fungi, especially with those which can be used as so called cell factories, is their application in industry.

Nevertheless, fungi are important in many other areas of our daily life. For example investigation of fungi is warranted concerning the increasing number of immunocompromised patients, who are threatened by pathogenic fungi (Walsh et al, 2004). Fungi are also producers of peptaiboles, which are discussed as an alternative to classical antibiotics (Duclohier, 2007). Moreover, especially several Trichoderma spp. play an important role in biocontrol of plant pathogens and can thus serve as biological fungicide.

In all these processes, signal transduction proteins – reflecting the reaction of fungi to their current environment - are key regulators of their efficiency. Hence a more detailed understanding of signalling in fungi will provide insights into mechanisms of signal transduction not only relevant to industrial applications, but also to elucidate further mechanisms of importance for health and economy (Bahn et al, 2007; Schmoll, 2011).

1.1. Trichoderma reesei – the fungus

Members of the genus Trichoderma are saprophytic fungi belonging to the phylum of

ascomycetes and the class of sordariomycetes. They can be isolated from soils, decaying wood and decomposing plant matter in various zones and habitats with organic matter (Jaklitsch, 2009; Jaklitsch, 2011). The capability of Trichoderma to survive under various conditions in numerous environments makes it easily culturable and therefore these fungi are predestined for the use in technological processes (Schuster & Schmoll, 2010).

The taxonomic history of Trichoderma is highly complex although the anamorph genus Trichoderma was discovered at the beginning of the 19th century and was associated with its


teleomorph Hypocrea in 1865 by the Tulasne brothers (Kubicek & Penttila, 1998). This association of Trichoderma with the teleomorph genus Hypocrea was supported by the finding that Trichoderma reesei and Hypocrea jecorina are phylogenetically indistinguishable (Kuhls et al, 1996). Only recently, the discovery of sexual development in Trichoderma reesei with Hypocrea jecorina finally confirmed this relationship (Seidl et al, 2009). Although the

“holomorph concept” would require using the teleomorph for every species, in case of T. reesei, the name of the anamorph species is still predominantly used in most publications. In this theses we will use the name of the well known anamorph Trichoderma reesei for studies and results with the original isolate QM6a and its derivatives and we will refer to the sexually competent wild-type strain CBS999.97 using the teleomorph name Hypocrea jecorina in order to avoid confusion. In recent years taxonomic methods like DNA Oligonucleotide barcodes at molecular level were applied to identify different species (Druzhinina et al, 2005). DNA Sequences of ITS1 and ITS2 (internal transcribed spacer regions), D1 and D2 regions of the 28S rDNA, the translation elongation factor 1-alpha and parts of the gene ech42 are recruited for taxonomic analysis (Lieckfeldt et al, 2000; Samuels et al, 2006). The number of newly-discovered species increases permanently and with them metabolic and physiological capabilities awaiting discovery. Trichoderma reesei QM6a has been isolated during the world war II because it degraded army equipment made of cotton (Reese, 1976). Later on, the impressive amounts of cellulases this strain secrets, which led to its isolation in the first place, became the focus of research programs (Reese, 1956). In the decades since, Trichoderma reesei has become one of the most important cellulase producers in industry and its efficient cellulase promoters also serve for production of heterologous proteins (Keranen & Penttila, 1995; Mach & Zeilinger, 2003). The case of T. reesei is special in so far as both research as well as industrial strain improvement is solely based on the single isolate QM6a and its progeny. Strain development in this fungus is currently a major focus of industrial research due to the need of large amounts of enzymes for biofuel production from cellulosic biomass (Somerville, 2007). Only few organisms are able to degrade the recalcitrant material in lignocellulosic plant material. While several basidiomycetes are known to be very efficient in this task, T. reesei is most frequently used in industry therefore (Merino & Cherry, 2007). One previously less well known characteristic of Trichoderma spp. is their reaction to light. Even a brief light pulse triggers conidiation in these fungi and therefore Trichoderma spp. became important model organisms in the early days of investigation of light response in fungi (reviewed in (Schmoll et al, 2010a)). Thereafter, research efforts on this topic

decreased and Neurospora and Phycomyces took the lead as model organisms in photomorphogenesis. Only recently, the discovery that cellulase gene expression is modulated by light (Schmoll et al, 2005) led to a combination of research topics and

investigation of the influence of light on metabolic processes in T. reesei (reviewed in (Schmoll, 2011)).

1.2. Cellulose, cellulases and Trichoderma reesei Cellulose is the most abundant biological material found on earth. Although the exceptional efficiency of T. reesei in degrading cellulosic material was initially considered chemical warfare, the industrial potential of the enzymes responsible for this characteristic was soon recognized and elaborate investigation of substrates triggering cellulase gene expression as well as induction mechanisms was initiated (Schmoll & Kubicek, 2003). Over the years,

cellulases have become the third largest industrial enzyme worldwide and are about to become the most important one due to the applicability for biofuel production (Wilson, 2009). Cellulose is a simple polymer consisting of glucose monomers, which form a very recalcitrant and insoluble material additionally strengthened by lignin and hemicellulose in the natural plant cell wall. Decomposition of this polymer initially requires detection of its presence in the


environment, which is accomplished by sensing of small degradation products , because the polymer of cellulose cannot enter a cell. T. reesei carries cellulases (predominantly CBH2/CEL6a) on its spore surface for this purpose (Kubicek et al, 1988; Messner et al, 1991) and produces very low constitutive levels of cellulases for an initial attack (Carle-Urioste et al, 1997; El-Gogary et al, 1989). However, not just a small oligomeric component derived from cellulose is considered the natural inducer, but the transglycosylation product sophorose (Sternberg & Mandels, 1979). Detection of cellulosic material outside the cell results in production of a powerful enzymatic machinery for degradation of plant cell walls, the natural substrate of T. reesei. Nevertheless, despite strong hints, the identity of the natural inducer(s) remains to be conclusively proven and the sensing mechanism for cellulosic substrates is still not known. Interestingly, T. reesei has no homologue of the G-protein coupled receptor shown to sense carbon sources in N. crassa (Li & Borkovich, 2006; Schmoll, 2008), which of course does

not mean that no other GPCR could assume this task. Additionally, recent studies in the model fungus Neurospora crassa revealed two transporters to be important for growth on cellulose, which facilitated cellodextrin utilization in S. cerevisiae (Galazka et al, 2010). Consequently also a “transceptor” could assume the task of signal transmission from outside the cell. Besides the insoluble substrate cellulose, numerous other carbon sources induce cellulase gene expression (Mandels & Reese, 1957). Also cellobiose (Mandels & Reese, 1960), the transglycosylation product sophorose (Mandels et al, 1962; Sternberg & Mandels, 1979; Vaheri et al, 1979), lactose, a byproduct of the dairy industry (Mandels & Reese, 1957; Seiboth et al, 2007), L-sorbose (Kawamori et al, 1986), L-arabitol and several additional substrates (Margolles-Clark et al, 1996) induce cellulase gene expression in T. reesei. Thereby, besides cellulosic plant material, the soluble and relatively cheap lactose is frequently used in cellulase fermentation and for production of heterologous proteins, i. e. especially for processes in which a soluble carbon source is crucial for efficient downstream processing (Kubicek et al, 2009; Seiboth et al, 2007). Intriguingly, however, the set of cellulolytic enzymes produced and their regulators on these various substrates differs depending on the carbon source used and also from that secreted into the medium upon growth on cellulose (Messner et al, 1988; Schmoll & Kubicek, 2005a; Sternberg & Mandels, 1980). With respect to interpretation of environmental signals, these differences in production of hydrolytic enzymes reflects a sophisticated interpretation of nutrient signals from outside the cell, which in every case leads to a fine tuned output, which is similar in terms of the potential to degrade cellulose, but still distinctly adjusted to the respective carbon source. Already in the early days of research with T. reesei QM6a, its cellulolytic capacity was exploited and investigations focused on improvement of the efficiency of the secreted enzyme mixture. Mutation programs resulted in countless mutant strains, some of which showed exceptional degradative potential such as QM9414 or the cellulase hyperproducer strain RutC30 (Seidl & Seiboth, 2010), both of which are still used in research. In addition, numerous strains were created, which had lost the capability to produce cellulases (Torigoi et al, 1996; Zeilinger et al, 2000). Despite considerable efforts, the mutations that led to increased or abolished cellulase gene expression could not be unequivocally determined in the pregenomic era (Schmoll et al, 2004; Zeilinger et al, 2000) and only the deletion in the

carbon catabolite repressor gene encoding CRE1 was determined to be responsible for the strongly increased cellulase gene expression of RutC30 (Ilmen et al, 1996). In recent years, the genomes of some of these mutants have been characterized and although numerous single nucleotide polymorphisms and mutations were detected, these elaborate studies could still only provide hints as to the mechanisms causing cellulase overproduction (Le Crom et al, 2009; Vitikainen et al, 2010).


1.3. Regulation of cellulase gene expression

The mechanism triggering and modulating cellulase gene expression has been investigated for decades (Figure 4). At the molecular level, cloning of the major cellulases gene of T. reesei, cbh1 (encoding cellobiohydrolase 1, today known as CEL7A) and cbh2 (also known as CEL6A) represent early mile stones in cellulase research (Saitou & Nei, 1987; Shoemaker et al, 1983a; Shoemaker et al, 1983b; Teeri et al, 1983; Teeri et al, 1987). Subsequent development of antibodies against these cellulases provided an important basis for analysis of regulatory mechanisms in T. reesei (Mischak et al, 1989). Among the first environmental cues found to be important for efficient cellulase expression besides the carbon source were pH and temperature (Sternberg & Mandels, 1979).

The following studies revealed that T. reesei uses at least three different types of enzymes for cellulose degradation: exoglucanases (cellobiohydrolases EC 3.2.1.91), endoglucanases (EC 3.2.1.4) and ß-glucosidase (EC 3.2.1.21), which occur in various isozymic forms (Schmoll & Kubicek, 2003; Seiboth et al, 2011). Besides these enzymes directly needed for

degradation of cellulose, also related enzymes, such as hemicellulases or pectinases are essential for efficient attack of the complex polymer mixture present in a plant cell wall (Seiboth et al, 2011). In this array of enzyms secreted by T. reesei, synergistic action was detected for some of them (Kumar et al, 2008; Medve et al, 1998; Medve et al, 1994; Valjamae et al, 1999) and also occurred with cellulolytic enzymes from other fungi (Ng et al, 2011).

Figure 4. Schematic representation of regulation of expression on plant cell wall degrading enzymes. Cellulosic plant material as well as several inducing soluble carbon sources induce cellulases, while carbon catabolite repression (CCR) is caused by easily metabolizable carbon sources. Cellulases and many other plant cell wall degrading enzymes are coregulated in T. reesei. Light, nutrients, temperature and pH are known to modulate cellulase expression. Received environmental signals are interpreted resulting in a defined output in terms of cellulases, which leads to a fine tuned attack on cellulosic biomass.

The major cellobiohydrolase genes cbh1/cel7a and cbh2/cel6a as well as the endoglucanase genes egl1/cel7b, egl2/cel5a and egl5/cel45a are coordinately transcribed, with cbh1/cel7a being the most highly expressed cellulase gene (Ilmen et al, 1997). Additionally, most of the


genes known or assumed to contribute to plant cell wall degradation are coregulated (Foreman et al, 2003).

In nature, efficient utilization of available resources is crucial to competitive success. Being a prosperous competitor in its natural habitat, T. reesei uses the best carbon source present in a given mixture in the environment and produces a tailored enzyme mixture to utilize this substrate. The underlying regulatory mechanism is called carbon catabolite repression (CCR) (Ilmen et al, 1996; Ruijter & Visser, 1997). Consequently, the recalcitrant substrate of lignocellulosic plant cell walls will only be efficiently degraded if no easily metabolizable carbon source such as glucose is available and production of cellulases is tightly regulated.

Regulation of biomass degrading genes is mainly accomplished by five transcription factors, which are assumed to bind to their target promotors not only in the presence of an inducer such as cellulose or sophorose, but also under non inducing conditions (Rauscher et al, 2006; Zeilinger et al, 1998). Thereby, ACE1 (activator of cellulase expression 1) and CRE1 (carbon catabolite repressor 1) are negative regulators and ACE2 (activator of cellulase gene expression 2), XYR1 (Xylanase regulator 1) and the HAP2/3/5 complex are positive ones (Kubicek et al, 2009). Their constitutive binding to the cellulase promotors implies that additional factors modulate the activity of these transcription factors and hence achieve fine tuning of cellulase gene expression in response to environmental conditions. One of the most important binding motifs known in cellulase promotors is the CAE (cellulase activating element) within the CBH2 promotor (Zeilinger et al, 1998) and ACE1 and ACE2 were found to bind to a similar sequence in the CBH1 promotor (Saloheimo et al, 2000). Consequently, these factors are likely to be the targets of signal transduction pathways transmitting the cellulose signal along with signals important for modulation of cellulase gene expression (Tisch & Schmoll, 2011). At the promotor level, also chromatin remodelling mechanisms are

crucial for regulation of cellulase gene expression. Induction of cellulase gene expression is accompanied by a loss of positioning of two nucleosomes downstream of the CAE. While the transcription factors binding to CAE are not affected by nucleosome positioning, CRE1 is essential for strict nucleosome positioning upstream of CAE (Zeilinger et al, 2003).

Accordingly, crucial promotor binding sites were also identified within the promotors of the xylanases XYN1 and XYN2 and XYR1 as well as ACE1 were shown to bind these sequences (Rauscher et al, 2006). Besides several transcription factors also a number of metabolic genes, especially those acting upon growth on lactose are known to impact cellulase gene expression (reviewed in (Seiboth et al, 2007)). Recently, also a novel dehydrogenase, GRD1, was found to be part of the regulatory machinery of cellulase production, albeit the precise role in the complex network of cellulase regulation remains to be established (Schuster et al, 2011).

Early studies on transcript levels, secreted protein levels and cellulase activity in the culture filtrate in several strains of T. reesei and under various conditions led to the conclusion that regulation of cellulase gene expression happens at the pretranslational level (El-Gogary et al, 1989; Messner & Kubicek, 1991; Morawetz et al, 1992). However, in recent years, investigation of cellulase gene expression under controlled light conditions and the function of light dependent regulators strongly suggests that at least under some conditions, the respective regulatory mechanisms also target posttranscriptional processes acting on cellulases (Gyalai-Korpos et al, 2010; Schuster et al, 2011).

For decades, research on cellulase production with Trichoderma reesei was largely focused on gene regulation at the promotor level, on novel or modified cellulose degrading enzymes and on amending enzyme mixtures to enhance the efficiency of biomass degradation (Martinez et al, 2008; Schmoll & Kubicek, 2003). Although cellulase inducing compounds

are well studied, only little is known on additional signals modulating cellulase gene expression and which factors are essential for transmission of these signals to the transcription factors triggering cellulase gene expression and how they impact cellulase regulation. One of these important signals is light, which has been known to considerably influence metabolic processes in fungi for decades (Tisch & Schmoll, 2010). After the initial discovery that light modulates cellulase gene expression (Schmoll et al, 2005), the


involvement of the light response machinery as well as the crosstalk with nutrient sensing processes was studied in more detail (Schmoll et al, 2010a). Chapter 2 summarizes our

contributions to elucidation of these important phenomena. 1.4. Second generation biofuels – sustainable and environmentally safe

For millions of years plants captured atmospheric carbon, which was stored as crude oil and coal until only few decades ago mankind started consuming this energy resource. It took only 150 years to increase atmospheric carbon dioxide levels by more than one third, which nowadays leads to the greenhouse effect and climate changes. Despite millions of years of accumulation, we only have a small amount of fossil fuel left. With the recent rise of energy costs and the increasing awareness of the ecological problems caused by fossil fuels, research toward development of biofuels has again received attention. In this respect the production of biofuels has come into focus (Farrell et al, 2006). However, the current use of food crops for this purpose is a highly controversial issue. An interesting alternative would be the use of cellulosic waste materials from agriculture and even municipal waste (Patel-Predd, 2006; Sticklen, 2008). For this task, filamentous fungi provide a broad array for tools, which allow for efficient conversion of lignocellulosic substrates to small builiding blocks utilizable by microorganisms producing fuels (Dashtban et al, 2009). As one of the most prolific cellulase producers, H. jecorina (T. reesei) represents an ideal model system to study regulation of plant cell wall degrading enzymes, which play an important role in conversion of cellulosic waste into bioethanol by yeast. However, the efficiency of the respective enzyme mixture still needs considerable improvement to render this process economically feasible. (Sticklen, 2008) Despite considerable efforts during past decades, the cost efficiency of approaches using cellulases and cellulosic plant materials in the process of biofuel production has still not reached the cost efficiency of enzymatic treatment of food crops. Although reductions in enzyme costs per litre bioethanol have been achieved, these costs are still 20 times higher than for the enzymes used for production of corn ethanol (Somerville, 2007) and hence pose a serious challenge to research. Strain improvement of organisms producing plant cell wall degrading enzyms as well as of those utilizing the substrate converted to small building blocks for production of biofuels is of utmost importance for industry to reach this goal. Research towards more efficient production of cellulases in fungi, and especially in T. reesei therefore represents a very timely strategy and currently covers topics from transcriptional regulation at the promotor level over adjustment of metabolic pathways related to cellulase expression to signal transduction pathways modulating this process. Only recently, an important additional tool became available for T. reesei. The discovery of sexual development and hence the capability of mating represents a milestone for research with this fungus (Seidl et al, 2009). The possibility of mating not only accelerates

combination of beneficial properties of different production strains. By mating also recombinant marker genes used during strain improvement can be removed and deleterious mutations not associated with enhanced cellulase production can be eliminated while retaining high production capacity. Consequently, the possibility of crossing of industrial strains also offers a way of using engineered strains on a broad basis, while still adhering to guidelines for application of genetically altered organisms, which will significantly increase the usefulness of the technology to be developed for small scale production plants. Our contributions to this topic are surveyed in Chapters 2 and 3.


2

Dealing with a changing environment

Chance favors only the prepared mind (Louis Pasteur)

Life of most organisms is dominated by nutrient availability, the need for reproduction and the rotation of the earth which results in daily changes between light and darkness. Signal transduction in any organism is aimed at an optimal reaction to changing environmental conditions with minimal consumption of cellular resources (Figure 5). Efficient tools for this purpose enable an organism to survive in a hostile environment and to successfully compete for natural resources. One crucial requirement for this efficiency is, to interpret and rate the received signals and to convert these data into an appropriate response. The pathways responsible for the necessary adaptations in T. reesei have been studied with respect to nutrient signaling and light response.

Figure 5 Schematic representation of signal transduction and its consequences in fungi. Examples for environmental signals and possible adaptive reactions are shown. Taken from (Tisch & Schmoll, 2011)

Filamentous fungi, such as T. reesei have to deal with countless challenges to succeed in the battle for nutrients, space and reproduction in the rich habitat of a tropical rain forest. However, due to its efficient enzymes for decomposition of plant cell walls, T. reesei has also


become a paradigm for industrial cellulase production and is nowadays the main industrial cellulase producer as well as subject to basic research. Hence, an efficient machinery for perception and interpretation of environmental signals enables T. reesei to adjust to such different environments as rotting trees in the tropics and a shake flask with chemically exactly defined minimal medium in the lab. 2.1. Adaptation to the rotation of earth – light and darkness Life in light is fundamentally different than life in darkness (Figure 6). At least that’s what is reflected in numerous studies of light response in fungi, be it transcriptional analysis or investigation of metabolic genes, the tendency to reproduce sexually or asexually or simply mycelial growth (reviewed in (Schmoll, 2011)). Although the most logical reaction to light is

initiation of protective measures against the harmful effects of UV-light during the day, investigations of numerous fungi revealed that virtually every metabolic pathway can be regulated in a light dependent manner in fungi (Tisch & Schmoll, 2010). Therefore, elucidation of the rationale behind the often drastic responses to illumination and their regulation at the molecular level will provide intriguing insights into physiology of fungi.

Figure 6. Environmental changes in day and night as perceived by the fungus also as light and darkness.

2.1.1. Circadian rhythms and light response Most living organisms do not only react to light as adaptation to the rotation of earth – they anticipate daylight and prepare. The mechanism for this adaptation is called circadian rhythmicity and is best studied in Neurospora crassa (Heintzen et al, 2001). In this fungus, oscillating pathways were found to trigger circadian expression of numerous genes, with FRQ (Frequency) and the photoreceptor WC-1 (White Collar-1) being among the most crucial components for this purpose (Figure 7). Complex regulatory circuits of phosphorylation and dephosphorylation are crucial for appropriate function of circadian rhythmicity (Diernfellner & Schafmeier, 2011). In many cases, the function of these phosphorylation steps is regulation of stability and / or capability to interact with other proteins. In case of FRQ, phosphorylation also acts as a degradation signal and destines it to degradation via the ubiquitin proteasome pathway with FWD-1 being the crucial E3-ubiquitin ligase (He & Liu, 2005). Although circadian rhythms continue oscillation for some time also in darkness and hence establish the internal clock of every organism, light is the most important “Zeitgeber”, which is


necessary for adjustment to actual environmental conditions (Schafmeier & Diernfellner, 2011). Light can thereby advance or delay the phase of the clock, depending on the time in subjective night when the light pulse is received (Devlin, 2002). Additionally, the response to light signals during circadian time is gated. That means that the same signal not necessarily has the same effect at every time, because only a window of responsiveness allows reaction (Devlin, 2002; Loros & Dunlap, 2001). The third known photoreceptor of N. crassa, VIVID, regulates gating and acts negatively on the White collar complex (WCC) (Chen et al, 2010; Heintzen et al, 2001; Hunt et al, 2010). VIVID is of crucial importance for photoadaptation and acts as a universal brake for gene expression after the initial photoresponse (Chen et al, 2009). The physiological function of VIVID besides photoadaptation also lies in sensing of different light intensities, which allows N. crassa to distinguish between day and night, despite the naturally occuring periods of moonlight (Malzahn et al, 2010).

Figure 7. Schematic representation of the regulation and function of WC-1, WC-2 and VVD. Taken from (Schmoll, 2011).

2.1.2. Light response and cellulase gene expression in Trichoderma

Species of the genus Trichoderma were besides Phycomyces among the first fungi to be investigated for light responses as a model organism (Schuster & Schmoll, 2010). However

after numerous basic studies, the focus of research with Trichoderma reesei shifted to cellulase gene expression (Schmoll & Kubicek, 2003). In recent years, the homologues of the N. crassa photoreceptors WC-1 and WC-2 were described in Trichoderma atroviride (Casas-Flores et al, 2004) and crosstalk with the cAMP pathway was shown (Casas-Flores et al, 2006). When cellulase gene expression was found to be influenced by light (Schmoll et al, 2005), also light response and its physiological consequences in T. reesei again

received attention. Interestingly however, this finding resulted from a screening aimed at


genes responsible for high level cellulase gene expression, which revealed a light regulatory protein, ENVOY, a homologue of N. crassa VIVID, to be involved in regulation of cellulase transcription (Schmoll et al, 2004). In the following it was shown that not only light, but also

the blue light photoreceptors BLR1 and BLR2 and the blue light regulatory protein ENVOY (encoded by env1) influence growth and cellulase gene expression in T. reesei (Castellanos et al, 2010; Schmoll et al, 2005). An even stronger effect of BLR1 and BLR2 on cellulase

gene expression was observed in T. atroviride (M. Friedl and M. Schmoll, unpublished data). In T. reesei, transcription of env1 is dependent on the presence of BLR1 and BLR2, which likely act as a complex for induction of env1 transcription. Nevertheless, the function of the BLR-complex (BLRC; (Carreras-Villasenor et al, 2011)) is not exclusively mediated by ENV1, because these factors all have a distinct impact on gene expression and phenotype (Castellanos et al, 2010). These initial data on the influence of the light response machinery in cellulase gene transcription raised interest in the applicability of this phenomenon for improvement of industrial fermentations aimed at production of plant cell wall degrading enzymes. Indeed, BLR2 and ENV1 were found to impact cellulase gene expression also under fermentation conditions and lack of blr2 and env1 resulted in clearly increased efficiency of the secreted enzyme mixture (Gyalai-Korpos et al, 2010). Intriguingly, this study confirmed earlier reports

indicating that in contrast to the previous postulation, cellulase gene expression is not exclusively regulated at the pretranslational level, because despite decreased cellulase gene transcription (Castellanos et al, 2010), increased specific cellulase activity levels were

observed. Besides the role of the light response machinery in cellulase gene expression, also the functions and mechanism of action of ENVOY was subject to further investigations. More detailed analyses revealed that ENVOY impacts numerous cellular processes including carbon source utilization, some of them even in darkness (Schuster et al, 2007). Moreover, complex formation within the EUM1 motif (Envoy upstream motif 1; (Schmoll et al, 2005)) of the env1 promotor is induced by light (Schuster & Schmoll, 2009).

Studies with the N. crassa homologue of ENVOY, the photoreceptor VIVID revealed a photoinduced N-terminal conformational change, which is essential for its signal transmitting function. This mechanism appears to be conserved in fungi and is likely to be operational also with ENVOY (Zoltowski et al, 2007). Conformational switching is thereby of crucial importance for dimerization of VIVID with partner domains with the light-state homodimer showing an increased tendency for aggregation compared to the dark state (Peter et al, 2011). The postulated interconnection between the light response pathway and carbon source signaling (Schmoll et al, 2005) was supported by studies with T. atroviride showing that BLR1 is responsible for carbon source selectivity, while the intensity of the initiated response required both BLR1 and BLR2 (Friedl et al, 2008b). The important phenotypic trait of

conidiation, which was previously believed to be primarily triggered by light, however, was found to be significantly regulated by the carbon source with BLR1 and BLR2 being important determinants in this process (Friedl et al, 2008a). Genome wide transcriptome analysis of T. reesei in light and darkness revealed a proportion of 2.7 % of total genes to be at least two fold differentially regulated by light (Tisch et al, 2012), which is in good correlation with data from T. atroviride (Rosales-Saavedra et al,

2006). Besides protective measures reflected by induction of photolyase activity, cultivation in light caused significant enrichment in transcription of genes involved in carbohydrate metabolic processes, cellulase activity and cellulose binding as well as regulation of oxidoreductase activity. Also genes involved in sexual development were found to be up-regulated in light, which is in accordance with the finding that sexual development in T. reesei occurs preferentially in light. Additionally, an unexpectedly high number of glycoside hydrolase genes was observed to be regulated in response to light. Nevertheless, regulation patterns of glycoside hydrolases are not unequivocally consistent with biomass formation


and consequently, growth rate is not exclusively regulated at the level of substrate utilization (Tisch et al, 2012).

2.1.3. Crosstalk between light response and cellulase gene expression in Neurospora

Neurospora crassa is one of the best studied filamentous ascomycetes and was even termed a model of model microbes (Davis & Perkins, 2002). This fungus has a long history as laboratory model organism for genetics and biochemistry. The natural habitat of N. crassa are predominantly burned trees and shrubs which turn bright orange due to the growth of this fungus (Turner et al, 2001) Several initial studies on the cellulase system of N. crassa are available (Eberhart et al, 1977; Myers & Eberhart, 1966). However, with the success of T. reesei as biotechnological workhorse for cellulase production, the hydrolytic enzyme machinery of N. crassa was obviously hardly studied anymore. Only recently, due to the need for new ideas and approaches for biofuel production from environmentally safe sources such as agricultural waste products and fast growing grasses, the cellulase system of N. crassa came into focus again and was investigated in detail on a genome wide scale (Tian et al, 2009). Transcriptome and secretome analysis highlighted the value of N. crassa as a potential model system also for cellulase regulation: Screening of several mutants showed a considerable potential for optimization of the plant cell wall degrading enzyme mixture secreted by this fungus. Therefore N. crassa can become a useful model system in combination with the workhorse T. reesei, but additionally its versatile enzyme machinery warrants further investigations and could render N. crassa a promising complement to T. reesei in industry. Although both T. reesei and N. crassa are able to efficiently degrade cellulose, their enzymatic machineries for this tasks vary considerably. In contrast to T. reesei, which has only 10 cellulases, the genome of N. crassa comprises 23 predicted cellulase genes and also the number of hemicellulases of this fungus is higher (Borkovich et al, 2004; Martinez et al, 2008; Tian et al, 2009). Its history makes N. crassa the ideal candidate to study the interrelationship of light response and cellulase gene expression, which has been discovered in T. reesei (Schmoll et al, 2005). Using the light response model system N. crassa for a genome wide study of the light-

dependent regulation of cellulase gene expression as found in T. reesei the advantages of the two most important model organisms of both physiological phenomena can be combined. Moreover comparison of this phenomenon in N. crassa and T. reesei allows for evaluation, whether conserved regulatory processes related to plant cell wall degradation are adjusted to the light status in ascomycetes. The preferred natural habitats of both Trichoderma and Neurospora are humid tropical and subtropical forests (Schuster & Schmoll, 2010; Turner et al, 2001) and hence utilization of

cellulosic substrates is crucial in their life style. Using the resources available for N. crassa together with the extensive background provided by research in cellulose degradation with T. reesei we aimed to obtain a more detailed understanding of the physiology of cellulase gene expression in fungi. Especially the detailed investigation of the once unexpected influence of light on cellulase gene expression is likely to provide novel insights into regulatory pathways that can be exploited for industrial cellulase fermentations. Investigation of the interrelationship of production of plant cell wall degrading enzymes with the light response pathway revealed intriguing insights into the mechanism governing light modulated cellulase gene expression. As already described for T. reesei (Castellanos et al, 2010; Gyalai-Korpos et al, 2010), the components of this pathway, white collar-1, white

collar-2 and vivid influence cellulase gene expression and secreted cellulase activity (Schmoll et al., manuscript submitted). Hence light modulation of cellulase gene expression appears to be a conserved phenomenon in fungi. Cellulase gene expression thereby seems to be subject to photoadaptation by VIVID and its role in regulation of White collar complex (WCC) formation (Figure 8). Genome wide transcriptional analysis revealed that the ability to


properly respond to amino acid starvation is important for regulation of cellulase gene expression. Additionally, a contribution of oxidative depolymerization to plant cell wall degradation is suggested for N. crassa.

amino acid

metabolismoxidative

depolymerization

specific

ribosomes

UPR

specific

ribosomes

photoadaptation

carbohydrate degradation

energy supply

TR

AN

SC

RIP

TIO

NA

L E

FF

EC

TS

CRE-1

carbon

catabolite

repression

Figure 8. Model for regulation of plant cell wall degrading capacity in N. crassa by WC-1/WC-2/WCC and VVD. VVD, WC-1 and WC-2 negatively affect efficiency of cellulose degradation during early stages of growth. Regulation of cellulolytic enzymes and energy supply is subject to photoadaptation by VVD, while carbon catabolite repression as mediated by CRE-1 is regulated by the WCC via VVD. Independent regulatory functions of VVD influencing cellulolytic efficiency include regulation of oxidative depolymerization, while for WC -1 and WC-2 these functions are most obvious in regulation of specific ribosomes. The WCC moreover specifically targets amino acid metabolism. Since the effect of the light response machinery is likely to be indirect, their output may involve additional target transcription factors. Adapted from Schmoll et al., 2012, manuscript submitted.


2.2. Integrating nutrient signaling with light response

2.2.1. The heterotrimeric G-protein pathway

The pathway of heterotrimeric G-protein signaling is of crucial importance for signal perception and transmission also in fungi (Li et al, 2007), especially for sensing extracellular nutrients (Figure 9).

Figure 9. Binding of a ligand to a G protein coupled receptor (GPCR) leads to a conformational change of the G protein alpha subunit and thereby exchanging GDP (guanosine diphosphate) to GTP (guanosine triphosphate). The GTP bound G alpha subunit is activated and releases the G protein beta and gamma subunits. G beta and gamma work as a dimer to affect further targets. The intrinsic GTPase activity of G alpha hydrolyzes GTP to GDP to terminate the signalling cycle and enables the G beta gamma dimer to reassociate with the G alpha subunit. As a heterotrimeric G protein it binds to the GPCR the circle of G protein signalling is closed (Hamm, 1998). Taken from (Tisch & Schmoll, 2011)

The heterotrimeric G-protein pathway uses G-protein alpha-, beta- and gamma-subunits for signal transmission in eukaryotic cells. Signals received by the heptahelical G-protein coupled receptors (GPCRs) from outside the cell are relayed to impact numerous regulatory pathways via their respective effectors, which in turn effect activity of secondary messengers (Hamm, 1998). The G-alpha subunit is tightly associated with the respective G-beta and -gamma subunit and binds GDP in its inactive state. Upon activation following binding of a ligand to its corresponding GPCR, GDP is exchanged for GTP and the G-alpha subunit dissociates from the G-beta-gamma heterodimer. Both parts are then free to activate their downstream targets (Cabrera-Vera et al, 2003). Regulators of G-protein signaling (RGS) have an enhancing effect on the intrinsic GTPase activity of G-alpha subunits and thus can cause termination of the mediated signal (Hollinger & Hepler, 2002). Also phosducins and phosducin-like proteins are known to be important for G-protein function by acting as co-chaperones for G-beta gamma folding (Willardson & Howlett, 2007). An extracellular nutrient source is likely to be sensed by a membrane bound receptor, with G-protein coupled receptors being among the best studied examples. Activation of such a receptor then initiates a signaling cascade which leads to expression and secretion of the enzymes needed to degrade and exploit the detected substrate. Heterotrimeric G-protein signaling is reported to be involved in sexual and asexual development, biocontrol, pathogenicity and carbon source signaling in fungi (Lafon et al, 2006; Li et al, 2007; Omann & Zeilinger, 2010). 2.2.2. Heterotrimeric G-protein signaling in T. reesei Like with many ascomycetes (Li et al, 2007), the genome of T. reesei comprises three G-alpha subunits, one G-beta subunit and one G-gamma subunit (Schmoll, 2008).

The nutrient sensing function of heterotrimeric G-protein pathways made it a primary candidate for a sensing mechanism detecting cellulosic substrates in the environment of T. reesei. Therefore also the G-protein alpha subunits represent likely candidates for a function


in cellulase regulation in response to carbon availability. We analyzed whether the G-alpha subunits GNA1 and GNA3 would transmit the cellulose related signal and thus influence cellulase transcription. Because of our findings on the connection of light sensing and cellulase transcription, all analyses were performed under controlled light conditions. Intriguingly, we found that the significance of both GNA1 and GNA3 for cellulase regulation is dependent on the light status. The most dramatic effect was observed for GNA3: While constitutive activation did not have any effect in darkness, in light this constant transmission of the GNA3 associated signal caused 10fold increase of the cel7a transcript on cellulose. (Schmoll et al, 2009; Seibel et al, 2009). A regulation of G-alpha subunits in response to

light or a light dependent effect of these proteins has not been reported for fungi so far. Nevertheless, it came as a surprise that despite this regulation, GNA1 and GNA3 obviously do not transmit the signal associated with the presence of cellulose outside the cell, because their constitutive activation did not lead to constitutive cellulase gene expression in the absence of an inducer (Schmoll et al, 2009; Seibel et al, 2009). Consequently, GNA1 and

GNA3 both transmit signals of light dependent significance for T. reesei, which lead to adjustment of cellulase gene expression. In accordance with a light-dependent function of GNA3, light was shown to cause protein complex formation within the EUM1 motif (Schmoll et al, 2005) of the gna3-promotor. This

EUM1 (ENVOY-upstream motif 1) motif was first found within the env1-promotor and also shows light-dependent binding of potential transcription factors, which might also target the gna3-promotor (Schuster & Schmoll, 2009).

The light-dependent function of both G-alpha subunits as well as the light dependent complex formation within the gna3 promotor prompted further studies on the crosstalk between heterotrimeric G-protein signaling and light response. In an initial attempt to elucidate the molecular basis of light dependent signaling by G-protein alpha subunits we found that deletion of env1 in strains expressing constitutively activated GNA1 or GNA3 led to the env1-deletion phenotype in light, while in darkness the effect of constitutive G-alpha activation was observed. (Tisch et al, 2011). We found that ENV1 impacts growth,

conidiation, cellulase gene expression and cAMP levels in light, while in darnkess, the effect of constitutive activation of GNA1 or GNA3 respectively prevailed. A further interesting and novel finding was, that activation of GNA1 and GNA3 resulted in drastically enhanced transcript levels of gna1 and gna3, reflecting the action of positive feedback upon activation of these G-protein alpha subunits. ENV1 is obviously involved in regulation of this feedback in case of gna3, but not with gna1 and acts negatively on phosphodiesterase activity (Figure 10).

Figure 10. Model for the regulatory interaction of ENVOY with the signaling pathways of heterotrimeric G-proteins and cAMP signaling (Taken from (Schmoll, 2011)).

Although so far the ligands of G-protein coupled receptors in T. reesei have not been elucidated, work on proteins binding to the cbh2/cel6a promotor provides an interesting hint


that methionine, which can be utilized as sulphur source by fungi, might be sensed by a heterotrimeric G-protein cascade (Gremel et al, 2008).

Many sulphur compounds, especially cysteine, methionine and S-adenosylmethionine are essential for the viability of most cells. Thus, many organisms have developed a complex regulatory circuit that governs the expression of enzymes involved in sulphur assimilation and metabolism. Appropriate mechanisms have been analyzed in the filamentous fungi Aspergillus nidulans and Neurospora crassa as well as the yeast Saccharomyces cerevisiae (reviewed by (Marzluf, 1997)). Searching for transcription factors binding to the cellulase activating motif (CAE) within the cellobiohydrolase 2 promotor, we unexpectedly isolated an E3 ubiquitin ligase putatively involved in regulation of sulphur metabolism. Investigation of the influence of the availability and nature of the sulphur source revealed that these factors are indeed important for regulation of cellulase gene expression. Also the organic sulphur source methionine impacts cellulase transcription: In darkness, addition of methionine to cellulose grown cultures enhances cellulase transcription, while in light no cellulases could be detected anymore under otherwise equal conditions. An influence of light on sulphur metabolism had not been studied before. Therefore the presence and nature of the sulphur source must have a light dependent significance beyond just the availability of a nutrient (Gremel et al, 2008). In C.

neoformans, methionine is sensed by a G-protein coupled receptor (GPCR), which binds the orthologue of the H. jecorina G-alpha subunit GNA3 (Xue et al, 2006). It will be interesting to learn whether the cellulase influencing G-alpha subunit GNA3 indeed transmits the presence of methionine via this GPCR.

2.2.3. Phosducins as light dependent regulators of heterotrimeric G-protein signalling Besides crosstalk with the light signal transduction machinery, we also investigated the role of phosducin like proteins, for which a function in light signaling was shown in higher eukaryotes (Lee et al, 1984). Acting as co-chaperons for G-protein beta and gamma folding they regulate efficiency of G-protein signaling (Willardson & Howlett, 2007). For fungi, functions in regulation of the sterigmatocystin biosynthesis pathway, vegetative growth or pathogenicity (Kasahara et al, 2000; Salamon et al, 2010; Seo & Yu, 2006), but light dependent phenomena related to the phosducin function were not studied. T. reesei possesses one class I phosducin like protein, which is related to those involved in light signaling and one class II phosducin like protein (Schmoll, 2008). Characterization of the regulatory functions of GNB1 (G-protein beta subunit 1), GNG1 (G-protein gamma subunit 1) and PhLP1 (Phosducin like protein 1) revealed that numerous glycoside hydrolases are among their regulatory targets and that their output is modulated by light (Tisch et al, 2012). Accordingly, the class I phosducin like protein PhLP1, which acts as

a cochaperone for G-protein beta and gamma folding (Willardson & Howlett, 2007) shows a closely related phenotype and regulatory function, hence confirming its function as a cochaperone also in T. reesei. Interestingly, these presumable nutrient signaling components obviously influence light responsiveness of gene expression, because the number of genes differentially regulated between light and darkness is considerably higher in the mutants than in the wild-type (Figure 11) (Tisch et al, 2012). Additionally, functions in conidiation and sexual development were detected.


Figure 11. Model for the function of GNB1, GNG1 and PhLP1. GNB1, GNG1 and PhLP1 integrate signals received from the light response pathway and the heterotrimeric G-protein pathway, which predominantly transmits nutrient signals. Thereby, a clear negative effect of ENV1 on phlp1 was observed, but this effect is not due to the regulation of ENV1 by BLR1 and BLR2, which in contrast to ENV1 do not significantly alter transcription of gnb1, gng1 or phlp1. Phenotypic effects include conidiation and cellulase production, while sexual development was only influenced by GNB1 and PhLP1. GNB1, GNG1 and PhLP1 dampen light responsiveness (differential transcription between light and darkness, both positive and negative) of numerous genes, including 99 glycoside hydrolase (GH) encoding genes. This effect is likely to be due to a positive effect of GNB1, GNG1 and PhLP1 on transmission of a light signal enhancing transcription of these genes. Accordingly, the genes differentially regulated in the strains lacking GNB1, GNG1 or PhLP1 compared to the wild-type, i. e. the targets of GNB1, GNG1 and PhLP1, are predominantly found upon cultivation in light (13 genes upregulated, 628 genes downregulated) in contrast to a considerably smaller number in darkness (42 genes upregulated, 7 genes downregulated).

In the course of microarray data analyses we found two further targets of PhLP1 – ste6 and hpp1, both of which are involved in sexual development. hpp1 is an h-type pheromone precursor and was shown to be necessary for male fertility in T. reesei QM9414 (Schmoll et al, 2010b). ste6 is a homologue of the yeast pheromone transporter gene and most likely involved in the secretion process of an a-type pheromone (Fowler et al, 1999). Both genes were transcribed in a pronounced way in light in QM9414 and to a basal level in darkness, what confirms the requirement of light for mating (Seidl et al, 2009). In summary these

findings show for the first time in that phosducins have a light dependent role in fungi, independently of the available carbon source and that their expression increases with time of illumination. 2.3. Elucidating the mechanism of crosstalk

Having shown the intriguing light dependent function of a phosducin like protein, we tackled the issue at which stage in the signaling cascade the light signal might be integrated with the G-protein signal. Genome wide transcriptional analysis along with detailed analysis of single genes in mutants of several components of the heterotrimeric G-protein pathway provided first insights into crosstalk between nutrient and light signaling (Tisch and Schmoll, manuscript in preparation). Transcription analysis indicated that PhLP1, GNB1 and GNG1 are more likely to mediate crosstalk than the G-protein alpha subunits GNA1 and GNA3. Therefore microarray analysis


was performed with deletion strains in the photoreceptors BLR1 and BLR2, in ENV1 as well as PhLP1, GNB1 and GNG1 in light and darkness.

Figure 12. Hierarchical cluster analysis of the transcriptomes of deletion mutants in env1, blr1, blr2, gnb1, gng1, phlp1 and the wild-type strain QM9414.

Clustering of transcript profiles in all mutants showed that in darkness, strains lacking components of the light signaling machinery cluster with the wild-type and those of the heterotrimeric G-protein pathway for a separate clade. In light the situation is more complex. While the wild-type QM9414 still clusters with all strains in darkness, all other strains are separated, with ∆env1 being most distinctly different (Figure 12). Analysis of gene expression in mutants in genes of the light response machinery or heterotrimeric G-protein signaling suggests a model in which after induction of ENV1 expression, its negative effect on blr1 and blr2 transcription leads to a steady state level of transcription of these three genes. ENV1 has a clearly negative effect on transcription of phlp1 and PhLP1 on the other hand acted positively on early light response of env1 transcription. Our findings suggest that ENV1 acts negatively on G-protein signaling during early light response by decreasing transcription of phlp1, gnb1 and gng1 in order to provide resources for protective measures. The positive action of PhLP1 on env1 transcription appears to enhance this effect. Subsequently the positive action of PhLP1 on complex formation and hence G-protein signaling via the beta and gamma subunit – which are likely to transmit nutritional signals - might be important for metabolic adaptation to light. Comparison of the positive PhLP1-GNB1-GNG1 targets with those of ENV1 - which showed the most severe impact on gene expression in light - showed that 77 % (483 genes) of the positive targets of PhLP1-GNB1-GNG1 to overlap with those of ENV1 in light (Tisch and Schmoll, manuscript in preparation).

We conclude that the nutrient signals transmitted via PhLP1-GNB1-GNG1 are closely interrelated with light signaling via ENV1. Lack of one of these four components causes the system mediating the respective positive signaling output to shut down. The data confirm the key function of ENV1 and PhLP1-GNB1-GNG1 in interconnecting nutrient- and light signaling (Figure 13).


Figure 13. Schematic representation of the mechanism of crosstalk between light response and heterotrimeric G-protein signaling. Our analyses shows that regulation of G protein signaling is at least in part accomplished by ENV1 and PhLP1 in response to light.

2.4. cAMP signaling, light response and cellulase gene expression

Being a second messenger, levels of intracellular cAMP are likely targets of heterotrimeric G-protein cascades in fungi (D'Souza & Heitman, 2001; Li et al, 2007). Therefore the cAMP pathway with its most important members ACY1 (Adenylate cyclase 1) and PKAC1 (Protein kinase A, catalytic subunit 1) was studied as one possible output pathway for light dependent modulation of cellulase gene expression in response to nutrient signals and light (Schuster et al., 2012 manuscript in revision). The adenylate cyclase (ACY1) generates cAMP from ATP and the protein kinase A catalytic subunit 1 is cAMP dependent which regulates a lot of different downstream pathways (D'Souza & Heitman, 2001). The protein kinase A (PKA) is a holoenzyme composed of one regulatory subunit and two catalytic subunits in T. reesei (Schmoll, 2008). Previous studies

revealed that cAMP levels positively influence cellulase gene expression (Sestak & Farkas, 1993). These data are in agreement with our findings showing an influence of GNA1 and GNA3 on cAMP levels (Schmoll et al, 2009; Seibel et al, 2009).

Our investigation of the cAMP pathway indicated that ACY1 has a positive effect on cellulase gene transcription while PKAC1 has a negative effect. Altered complex formation within the cbh2/cel6a promotor in light and darkness and also in the strain lacking pkac1 suggested that PKAC1 influences formation or stability of transcription factors binding to cellulase promotors. Transcript analysis of ace1, ace2 and xyr1, three important transcription factors known to be involved in cellulase gene expression (Mach & Zeilinger, 2003) led to the conclusion that PKAC1 is involved in regulation of XYR1 abundance or stability.


3

The quest for reproduction – sensing and reacting to partners

The very existence of sex implies its advantage

R. A. Fisher

3.1. Sexual development in fungi Sexual development is one of the most important achievements in evolution. Not only does it contribute to improvement of the fitness and adaptation of a given organism to its ecological niche, the fact that sexual development is induced if environmental conditions deteriorate, also indicates that this process is of utmost importance for survival of a species (Aanen & Hoekstra, 2007). However, aside from the evolutionary significance of sex in nature, this process can also be exploited for research and improvement of industrial processes. Sex and recombination ensure that genotypes – either beneficial or detrimental – become altered (Goddard, 2007) and hence combination of preferred properties of different strains is possible. Sexual development occurs between strains of compatible mating types. Filamentous ascomycete fungi can have two mating types, MAT1-1 and MAT1-2, and these MAT loci occupy the same chromosomal location but lack sequence similarity and are thus termed “idiomorphs” rather than alleles (Debuchy et al, 2010; Debuchy & Turgeon, 2006; Metzenberg & Glass, 1990). The genes necessary for signal transduction and the formation of sexual reproduction structures for both mating types are present in each genome, but are strictly regulated by the respective MAT locus. Ascomycete fungi are haploid during their vegetative life cycle and can either have a heterothallic or a homothallic sexual cycle. Heterothallic fungi need a compatible strain carrying the opposite MAT idiomorph for sex (Poggeler, 2001), while homothallic fungi are self-fertile. In heterothallic fungi, mating occurs only between haploid cells of opposite mating types and mating is initiated by cell specific pheromone and receptor combinations (Bolker & Kahmann, 1993). It underlies a complex signal transduction pathway, the pheromone response system. The specific recognition launches a complex mitogen-activated protein kinase cascade (MAPK) cascade, the pheromone response pathway. To date, two types of pheromone


precursor genes have been identified in fungi; the MFa and MFα genes of Saccharomyces cerevisiae are the best-studied examples (Kurjan, 1993). Fungi are usually hermaphroditic and can form both male and female reproductive structures, which is strongly dependent on environmental factors. At the molecular level, heterotrimeric G-protein cascades are important for initiation of sexual development (Bolker, 1998). In most cases G-protein coupled pheromone receptors are crucial for female fertility (Kim & Borkovich, 2004), while peptide pheromone precursors are reported to be essential for male fertility (Coppin et al, 2005; Kim & Borkovich, 2006; Schmoll et al, 2010b) (Figure 14).

Figure 14. Schematic representation of pheromone signaling and signal reception in fungi. Peptide pheromone precursor genes (a-type or alpha type) are mostly expressed in a mating type dependent manner, pheromone receptors are not. While pheromones are important for male fertility in a mating type dependent manner, they are dispensable for female functions. Pheromone receptors are important for female fertility, again in a mating type dependent manner in most fungi. G-protein coupled signal transduction cascades often play important roles in signal transmission and sexual development.

3.2. The special case of Trichoderma

Trichoderma reesei QM6a has been isolated during the world war II in the South Pacific, because it degraded army equipment made of cotton (Reese, 1976). Later on, the impressive amounts of cellulases this strain secrets, which led to its isolation in the first place, became the focus of research programs. In the decades since, T. reesei has become one of the most important cellulase producers in industry and its efficient cellulase promoters also serve for production of heterologous proteins (Mach & Zeilinger, 2003). The case of T. reesei is special in so far as both research as well as industrial strain improvement is solely based on the single isolate QM6a and its progeny. As with all other filamentous fungi applied in industry at a larger scale, the possibility of crossing was not available for T. reesei for decades, although in silico data identified Hypocrea jecorina as its teleomorph more than a decade ago (Kuhls et al, 1996). Recently, we were for the first time able to induce sexual reproduction of T. reesei QM6a (MAT1-2) in crossings with a MAT1-1 wild-type isolate of H. jecorina (CBS999.97), and we obtained fertilized stromata and mature ascospores (Seidl et al, 2009) (Figure 15). Interestingly, we

found that QM6a is female sterile, which may have been one reason why attempts to achieve sexual development with T. reesei remained unsuccessful for decades. The presence of a functional sexual cycle in T. reesei expands the arsenal for manipulation of this organism and will aid in boosting research towards efficient and economically feasible biofuel production.


Knowledge on mating type loci in the order Hypocreales will moreover provide a basis for investigation of sexual development in other Trichoderma spp. of importance in agriculture and human health. External factors significantly affect regulation of sexual development. This process necessitates proper conditions during growth of the mycelium and also up to and including maturation of ascospores. Major environmental factors influencing sexual development are nutrients, pH, atmospheric conditions and light (Debuchy et al, 2010). Consequently, regulation of sexual development is intimately connected to the various signal transduction pathways perceiving and transmitting environmental signals such as the light response pathway and the heterotrimeric G-protein pathway, which are central topics of this thesis. In the following the name of the anamorph T. reesei will be used with experiments involving strain QM6a and its progeny, which had been considered an asexual clonal line and the name of the teleomorph H. jecorina for strains known to propagate sexually. 3.3. Causes for female sterility in fungi

Several mutations causing female sterility have been reported. For example in N. crassa, mutants lacking the G-protein alpha subunit GNA1 are male fertile, but female sterile. During our work with the T. reesei orthologue of this gene we found the respective gene to be functional and we could confirm a role in regulation of cellulase gene expression (Seidl et al, 2009). In preliminary experiments we could confirm that male fertility has been retained in

strains lacking or overexpressing gna1. In many cases the mutations causing female sterility would not be implicated in sexual reproduction (Hornok et al, 2007). While we cannot exclude that decades of cultivation and maintenance of T. reesei QM6a under laboratory conditions caused the loss of female fertility, the theory of Leslie and Klein (Leslie & Klein, 1996) provides an alternative explanation: They suggest that in a heterothallic haploid ascomycete capable of sexual as well as asexual reproduction, loss-of-meiosis mutations accumulate during vegetative reproduction which result in female sterile mutants. For these mutants the advantage not to spend metabolic resources for production of female structures, which may never be fertilized, can even be beneficial during asexual reproduction (Hornok et al, 2007). Based on the relative frequency of female fertile strains an estimation of the relative amounts of sexual and asexual reproduction occurring in a given population is possible (Leslie & Klein, 1996). The extent of female sterility within a population varies widely. However, it is tempting to speculate that female sterility of T. reesei QM6a led to efficient asexual growth, prevalence in its natural habitat and eventually to its isolation. Female sterility may be a reason why sexual development has not been achieved in other industrially important fungi. Knowledge on its causes in T. reesei will thus benefit research with fungi in general. Efforts to elucidate the defects of QM6a causing female fertility are in progress in our group and so far indicate that more than one gene is altered compared to the sexually competent nature isolate CBS999.97.

Figure 15. Sexual development of H.

jecorina strains – fruiting body formation. Taken from (Seidl et al, 2009)


3.4. Determinants of sexual development

3.4.1. Pheromones in Trichoderma

In most ascomycetes, one of the peptide pheromone precursor genes encodes a polypeptide containing multiple repeats of a putative pheromone sequence bordered by protease processing sites. These resemble the alpha factor precursor gene of Saccharomyces cerevisiae and the P-factor precursor gene of Schizosaccharomyces pombe, respectively (Imai & Yamamoto, 1994; Singh et al, 1983). The other gene encodes a short polypeptide with a c-terminal CaaX motif expected to produce a mature pheromone with a C-terminal carboxy methyl isoprenylated cysteine. In the heterothallic fungus N. crassa, both pheromone precursor genes are expressed specifically dependent on the mating type and these genes are highly expressed in conidia. Furthermore, both pheromone precursor genes are subject to regulation by the circadian clock, implying a role for the endogenous clock in the regulation of mating (Bobrowicz et al, 2002). For N. crassa it has also been shown that short illumination with blue light induces the development of protoperithecia, the female reproductive structures of N. crassa (Innocenti et al, 1983). Upon encounter of a potential mating partner, such peptide pheromones are sensed by G-protein coupled receptors, which transmit the signal via the heterotrimeric G-protein pathway (Bolker, 1998; Bolker & Kahmann, 1993). Searching for signaling factors, we found a small mRNA encoding a 48 amino acid polypeptide (HPP1, hybrid peptide pheromone precursor 1) being differentially regulated between a wild-type strain and the cellulase non inducible mutant strain (Schmoll et al, 2004). The encoded protein contains a C-terminal CAAX domain, characterizing a-type

peptide pheromone precursors, but additionally two Kex2-protease sites, a threefold repeated motif and a phosphorylation site – features not found in any other a-type peptide pheromone precursor, but rather in alpha-type peptide pheromone precursors. Also its genomic locus does not correspond to the locus of these genes in other related fungi. While initially considered a fortunate but random gene product recovered after loss of the original a-type peptide pheromone precursor, HPP1 turned out to represent the first member of a novel class of peptide pheromones – the h-type peptide pheromone precursors (Schmoll et al, 2010b).

Similarly unique structures as found in HPP1 also occurred in other fungi, such as Fusarium spp., and T. virens. The most important characteristic of h-type peptide pheromone precursors was identified as the threefold appearance of the conserved motif (Figure 16). The genomic locus of the genes encoding h-type peptide pheromone precursors, however, is not syntenic in these fungi. HPP1 assumes the function of a-type peptide pheromones in H. jecorina and is essential for male fertility. Transcription of hpp1 is regulated by light and accordingly by the light regulatory protein ENV1 (Schmoll et al, 2010b). However, the mating-type dependent

regulation of a-type (h-type) and alpha-type peptide pheromone precursors is not as tightly regulated in H. jecorina as in other fungi (Bobrowicz et al, 2002; Shen et al, 1999; Zhang et al, 1998)

Figure 16. Consensus sequence of h-type peptide pheromone pre-cursors. Adapted from Schmoll et al., 2010b


3.4.2. Pheromone receptors - sensing a partner and reacting to signals

Sensing of a potential mating partner in the surroundings of a fungus is a crucial prerequisite for initiation of sexual development. Therefore both potential mating partners need to assume their distinct roles in this interplay. Signals are sent in the form of the small peptide pheromone precursors and received by the aid of G-protein coupled pheromone receptors (Figure 14). We investigated the role of the two pheromone-receptors HPR1 (STE3-type) and HPR2 (STE2-type) in the unusual Hypocrea jecorina pheromone-system. Thereby, an unexpectedly high sequence variability among pheromone receptors between sexually compatible strains of H. jecorina was observed. HPR1 and HPR2 are not needed for normal growth, conidiation or female fertility in T. reesei QM9414 or H. jecorina CBS999.97. However, we found that compatible pheromone – pheromone receptor pairs in the mating partners (i. e. the receptor in one partner and its cognate pheromone in the other) are essential for mating in T. reesei / H. jecorina. These analyses moreover confirmed that the h-type peptide pheromone precursor HPP1 (Schmoll et al, 2010b) has assumed a-type function in H. jecorina.

Interestingly, while strains lacking their mating type specific pheromone receptor (HPR1/STE3 in MAT1-1 and HPR2/STE2 in MAT1-2) are able to produce fruiting bodies, in these crosses no ascospores are formed. Consequently we could show that pheromone receptors not only are involved in signal reception and transmission, but additionally impact ascosporogenesis. Investigation of transcript levels of pheromone precursor genes hpp1 and ppg1 as well as pheromone receptor genes hpr1 and hpr2 upon asexual growth and onset of sexual development revealed that hpp1 and hpr2 are enhanced in MAT1-2 in strains growing in the presence of a mating partner compared to asexual growth. Conversely, ppg1 and hpr1 are enhanced in MAT1-1 strains. Nevertheless, transcription of the respective other pheromone – receptor pair is not repressed and hence indicates rather loose control of mating type specific gene expression (Seibel et al., 2012a manuscript in preparation). 3.4.3. The influence of light on sexual development of Trichoderma Sexual development in Hypocrea jecorina occurs preferentially in light. We therefore investigated whether photoreceptors with known function in H. jecorina (Castellanos et al, 2010) play a role in this process (Figure 17) (Seibel et al., 2012b, mansucript submitted).

The most interesting finding of this study was that the light regulatory protein ENV1 is essential for female fertility under daylight conditions. Lack of ENV1 causes strong overexpression of pheromone genes and the mating type gene mat1-2-1. This deregulation of genes, which are crucial for sexual development can be compensated by a wild-type mating partner or at least one that does not lack env1. This finding is also in agreement with the fact that lack of env1 results in earlier and more vigorous fruiting body formation (Schmoll et al, 2010b). Consequently, deregulation of pheromone genes and mat1-2-1 upon lack of env1 is concluded to cause signal strength exceeding saturation levels and thereby prevents controlled operation of mechanisms triggering sexual development. A contribution of ENV1 to determination of sexual identity is therefore likely (Seibel et al., 2012b, manuscript

submitted). Strains lacking env1 exhibit a severe growth defect in light (Castellanos et al, 2010; Schmoll et al, 2005; Tisch et al, 2011). Since pheromones were shown to induce growth

arrest in S. cerevisiae we assumed that overexpression of hpp1 might be the reason for this growth defect. Surprisingly, a double mutant in env1 and hpp1 did not show any alteration in growth compared to the single mutant in env1 and hence the severe phenotype was not rescued. Strong up-regulation of mating type genes was shown to result in suppression of vegetative growth (Paoletti et al, 2007). The fact that ENV1 also regulates transcription of mat1-2-1, therefore represents an interesting finding to pursue.


Figure 17. Schematic representation of the involvement of the light signaling machinery in regulation of sexual development. BLR1 and BLR2 exert their function predominantly via ENV1. The effect of the light signaling machinery acts negatively on gene expression of pheromones and their receptors. ENV1 likely integrates nutrient signals into the cascade.

BLR1 and BLR2 were found not to be essential for fertility. Nevertheless, in strains lacking BLR1 or BLR2 fruiting body formation starts earlier and proceeds more vigorously. Despite certain effects on transcription of pheromone precursor and receptor genes, the regulatory effects of BLR1 and BLR2 are considerably weaker than those of ENV1. BLR1 and BLR2 are essential for induction of env1 transcription, however, despite their absence, env1 is still transcribed at low levels. The findings of this study therefore suggest that the function of BLR1 and BLR2 is predominantly exerted via ENV1. Since ENV1 is known to represent an important node between nutrient signaling and light response, it is likely that also in its function in regulation of sexual development, ENV1 integrates nutrient signals with light signals and consequently its effect is stronger than that of the photoreceptors receiving and transmitting predominantly light signals. In summary, our study showed that BLR1, BLR2 and ENV1 exert a negative effect on sexual development and thereby contribute to ordered sequence of regulatory processes in its regulation. 3.5. Importance of sexual development in research and industry Sexual development in the industrial work horse Trichoderma reesei (Hypocrea jecorina) was an important achievement, especially for strain improvement for industrial fermentations. Identification of the gene(s) relevant for female fertility will even increase the utility of sexual development in H. jecorina, because then production strains could be crossed with less manipulation effort and without the risk to loose the high production phenotype. Essentially the same applies for application of sexual development in fundamental research with T. reesei (H. jecorina). Creating double mutants by crossing requires much less hands on time and is much more efficient than using the conventional method of transformation. Thereby methods used in other model fungi such as Aspergillus nidulans or N. crassa for decades now also become feasible for T. reesei, which will strongly boost research with this fungus. We consequently applied a streamlined procedure for high throughput vector construction for gene deletion and transformation into a strain defective in the non


homologous endjoining pathway (∆tku70; (Guangtao et al, 2009)) to Trichoderma reesei. Rescue of the tku70 deletion was done by backcrossing with a female fertile derivative of QM9414 (Schuster et al., 2012, manuscript in revision). Especially in the important area of

biofuels research, this contribution will be of importance. Despite the importance of sexual development for industrial applications and as a tool in research, analysis of this process will also yield new insights into physiology of T. reesei. Investigation of this phenomenon in T. reesei is only at the very beginning. So far we could show intriguing particularities of T. reesei, such as a novel type of peptide pheromone precursors. We also showed that the function of the light response machinery is crucial for fertility of T. reesei. Our ongoing research on this topic in the course of this project can thus be expected to elucidate further intriguing aspects of sexual developments in fungi.


4

Exploring genomes – the treasury of

research and industry

Character, I am sure, lies in the genes. Taylor Caldwell

The genome of an organisms defines its physiology and hence its capabilities to deal with a changing environment. This ability to adapt to a given situation is crucial for competitive success of an organism in its habitat. Sophisticated enzyme systems have evolved for utilization of diverse substrates, but also mechanisms for all kinds of climate conditions including resistance to toxic compounds and the harmful effects of UV light. At the same time, a complex network of regulatory mechanisms ensures that this adaptation is achieved with minimal consumption of cellular resources. Studying genomes reveals fascinating insights into the physiology of an organism. Nevertheless, for most sequenced organisms it was found that for only about 50 % of genes a function could be assigned, albeit the number of genes for which this function is actually proven, is very small. Consequently, investigation of the role of a gene in the regulatory network of an organism can only provide a spotlight on a certain process. Therefore, besides knowledge on single genes present in a genome, the challenge for research with genomes lies in the development of hypotheses how this gene might be integrated into the complex regulatory network. Evaluation of these hypotheses will finally lead to a detailed understanding of the physiology and biological role of an organism. However, this knowledge at the same time can provide a basis for targeted modification by genetic engineering hence enabling knowledge based process design instead of trial and error in industrial applications.


4.1. A genome wide view on plant cell wall degradation by fungi

T. reesei is currently the most prolific producer of plant cell wall degrading enzymes used in industry. Interestingly, the recently sequenced genome of T. reesei (anamorph of H. jecorina) (Martinez et al, 2008) revealed that it has a surprisingly limited set of plant cell wall

degrading enzymes. Nevertheless, despite this limitation it successfully competes with other fungi growing on plant material and has gained significant industrial importance. The inability to rationalize this discrepancy underscores our limited understanding of the underlying regulatory principles of plant cell wall degradation and regulation of the necessary enzymes. In fact, since the isolation of T. reesei QM6a (anamorph of H. jecorina) on the Solomon Islands in the South Pacific during world war II, decades of research on its highly productive cellulase machinery could not provide us with a comprehensive model for regulation of cellulase gene expression (Schmoll & Kubicek, 2003).

Only few organisms are able to efficiently degrade the recalcitrant blend of polymers in lignocellulose. Among fungi, basidiomycetes are the most potent organisms in this respect, which attack the complex plant material applying both hydrolytic and oxidative enzymes. White rot fungi can decompose cellulose and hemicellulose as well as lignin. Although industrially not as efficiently utilized than T. reesei, basidiomycetous fungi are among the most potent degraders of cellulosic material in nature (Baldrian & Valaskova, 2008). Their enzyme systems however, were in some cases found to differ considerably from those of ascomycetes. The various roles as litter-decomposing, wood-rotting, plant pathogenic or mycorrhizal fungi correspond with the relevance of cellulose degradation for their life style and ecological niche. Connecting the characteristics of their enzyme systems – especially of brown rot and white rot fungi – allows for elucidation of genes relevant for specific degradation of cellulose and lignin, respectively. The white rot fungus Phanerochaete chrysosporium was one of the first basidiomycetes to be sequenced (Martinez et al, 2004). It secretes diverse extracellular glycosyl hydrolases to simultaneously attack cellulose and hemicelluloses (Van den Wymelenberg et al, 2010), but also oxidases and peroxidases. Among basidiomycetes, the brown rot fungus Postia placenta revealed a very interesting degradation mechanism. In contrast to P. chrysosporium, P. placenta possesses only two putative cellulases (1,4-ß glucanases) and completely lacks genes encoding cellulose binding domains. It attacks a cellulosic substrate by secretion of predominantly hemicellulases and an extracellular Fenton system (Martinez et al, 2009; Van den

Wymelenberg et al, 2010). The overall number of glycoside hydrolases is similar to the ectomycorrhizal symbiont Laccaria bicolor, the human pathogen Cryptococcus neoformans and the plant pathogen Ustilago maydis. Comparison to P. chrysosporium indicates that during evolution the capability to degrade lignin was lost and a shift from white rot to brown rot occurred, which is also reflected in a decreased number of glycoside hydrolases and copper radical oxidases in the genome (Martinez et al, 2009).

While P. chrysosporium simultaneously degrades cellulose, hemicellulose and lignin, Ceriporiopsis subvermispora, also a white rot fungus, decomposes lignin before utilization of cellulose and hemicellulose (Fernandez et al., 2012 manuscript submitted). Differences in

their strategies to degrade plant cell walls are also reflected in their inventory of certain glycoside hydrolases. C. subvermispora possesses less exocellobiohydrolases (GH7), cellulose binding modules (CBMs) and beta glucosidases (GH3) than P. chrysosporium. In contrast, expression of important oxidoreductases is considerably higher in C. subvermispora than in P. chrysosporium and may explain the different modes of attacking the cellulosic substrate in these two fungi. It is particularly interesting, that the ectomycorrhizal Laccaria bicolor lacks enzymes involved in the degradation of plant cell wall components, which may prevent the symbiont from


degrading host cells. In contrast, gene families involved in hydrolysis of bacterial and microfauna components are expanded. Consequently, this fungus has developed an enzyme system, which enables symbiosis and growth in living plant cells, but at the same time utilization of non-plant cell wall polysaccharides allows for survival also in soil (Martin et al, 2008; Martin & Selosse, 2008). 4.2. The genomes of three unequal siblings – Trichoderma species and their

characteristics

Comparative genomic analysis of three Trichoderma species, the two mycoparasites T. atroviride and T. virens, and the industrial workhorse T. reesei provided intriguing insights in to evolution of these fungi and their lifestyle (Druzhinina et al, 2011; Kubicek et al, 2011).

The ancestors of Trichoderma obviously were mycoparasites on wood-degrading basidiomycetes. In order to follow their prey they are assumed to have acquired the ability to live as saprotrophs. T. atroviride turned out to be the ancestor of T. virens and T. reesei. However, only one of them, T. virens, has retained mycoparasitic competence, while T. reesei largely lost the genes required for this process. Interestingly, despite its widespread use in industry because of its potent cellulase system, glycoside hydrolase genes are not significantly enriched among the genes unique to T. reesei. Genome wide transcriptional analysis of Trichoderma atroviride in the presence of plant pathogenic fungi revealed increased expression of genes involved in cell motility, amino acid metabolism and posttranslational events, but somewhat decreased expression of carbohydrate metabolic genes compared to mycelial growth and light/sporulation conditions (Seidl et al, 2009). Accordingly, the cross pathway control protein encoding gene cpc1, a

major regulator of amino acid metabolism was also enhanced under mycoparasitic conditions. Consequently, mycoparasitism is apparently correlated with a state of amino acid starvation. 4.3. A genome wide view on environmental signaling pathways

4.3.1. The signaling highways of Trichoderma

In recent years several reports revealed an important role of signal transduction pathways in regulation of cellulase gene expression (Kubicek et al, 2009; Schuster & Schmoll, 2010; Tisch & Schmoll, 2011) and thereby opened up a novel approach towards improvement of cellulase gene expression for industrial applications. The signal transduction machinery of T. reesei is in general comparable to that of other fungi (Schmoll, 2008). Nevertheless in several pathways interesting expansions or shortcomings

are apparent. For an industrial workhorse like T. reesei maybe the most surprising particularity of its genome is the lack of the orthologue of the carbon sensing G-protein coupled receptor (GPCR) characterized in N. crassa (Li & Borkovich, 2006; Schmoll, 2008). However, due to the presence of several genes encoding closely related GPCRs (Brunner et al, 2008; Schmoll, 2008), which are not characterized yet, it cannot be excluded that another

receptor has assumed the task of carbon sensing in T. reesei. Interestingly, with 4 RGS (regulator of G-protein signaling) proteins and 3 GprK-type GPCRs also comprising RGS domains has the highest number of genes encoding these regulator proteins known in Sordariomycetes. These proteins impact the activity of G-protein alpha subunits, which represent a crucial bottleneck between reception of signals by GPCRs and output of the regulatory cascade initiated thereafter. Hence RGS-proteins modulate strength and duration signal transmission within the cell after reception. Consequently, sensing of extracellular compounds as well as transmission of the resulting signal are distinctly regulated in T. reesei.


A second characteristic in T. reesei concerns regulation of circadian rhythmicity of T. reesei. Although not conclusively proven, evidence for circadian conidiation as provided for Trichoderma pleuroticola (Steyaert et al, 2010) suggests the presence of a circadian rhythm also in Trichoderma spp. Nevertheless, the characteristic, rhythmic conidiation upon growth in constant darkness after entrainment was not observed in T. reesei (Schmoll, 2008). T.

reesei possesses homologues of FRQ, WC-1, WC-2, VVD, FWD-1 and FRH, representing the most crucial components for circadian rhythmicity (Brunner & Kaldi, 2008). Interestingly, the transcript of the MAPK tmk3 (related to S. cerevisiae HOG1) is responsive to light and regulated by ENVOY (Schuster et al, 2007). Also, this gene was found in the genome

adjacent to the G-protein alpha subunit 3 (gna3) gene, which influences cellulase gene expression in a light dependent manner, as a constituent of the MGG cluster (Schmoll et al, 2009).

Signal transduction is mainly established by components responsible for signal perception, most prominently represented by the G-protein coupled receptors (Li et al, 2007), and transmission of the received signal to the promotors of target genes, for which kinases and phosphatases (Dickman & Yarden, 1999), are of crucial importance. Regulation of protein function by phosphorylation and dephosphorylation is well studied in eukaryotes and was found to impact virtually every cellular activity. This ubiquitous signaling mechanism can alter protein behaviour in various ways from modulation of biological activity, alteration of localization to half life and interaction with other proteins.(Cohen, 2000; Miranda-Saavedra & Barton, 2007) Although T. reesei protein kinase C (PKC) was among the first PKCs to be characterized in detail in filamentous fungi (Lendenfeld & Kubicek, 1998; Morawetz et al, 1996) this topic did not become a research focus in this fungus and information on characteristics and regulatory targets of protein kinases and phosphatases of T. reesei is still scarce. Nevertheless, the availability of the sequenced genome (Martinez et al, 2008) enabled an initial evaluation of the signaling inventory of this fungus (Schmoll, 2008), which for example revealed

interesting differences to other ascomycetes in the number of available two component histidine kinases and casein kinases. Since casein kinases are important for regulation of photoreceptor function in N. crassa (He et al, 2006; Huang et al, 2007) and light signaling is important for cellulase gene expression (see above), the function of casein kinases in T. reesei deserves further attention. A detailed comparative in silico study on fungal protein kinases revealed that the kinome density of T. reesei is comparable to other ascomycetes and that the number of its kinases lies around the average of 85 genes (Kosti et al, 2010). 4.3.2. A glimpse on signaling mechanisms in basidiomycetes The different strategies to attack cellulosic plant materials – brown rot and white rot – each represent a specific reaction to extracellular nutrients in a given habitat. Signal transduction pathways have been analyzed in the basidiomyetous genomes of Postia placenta (Martinez et al, 2009), Phanerochaete chrysosporium and Ceriporiopsis subvermispora (Fernandez et al., manuscript submitted) and compared to the potent cellulose degrader Trichoderma

reesei. With respect to heterotrimeric G-protein signaling one of the major findings was, that these fungi not only contain the three G-protein alpha subunits common in most ascomycetes (Li et al, 2007), but numerous additional ones (for example 6 in C. subvermispora) which cluster with Ustilago maydis GBA4 (Regenfelder et al, 1997). Unfortunately, the precise function of GBA4 is not known and therefore the relevance of the considerable expansion of this group in all three basidiomycetes studies is hard to assess. Screening for G-protein coupled receptors revealed a most interesting situation in P. placenta. The genome of this fungus only comprises 8 GPCRs belonging to one class of G-protein coupled receptors (STE3-type pheromone receptors), 6 of which are located in the putative B-mating type locus (Figure 18). Clustering of this type of receptors has also been


observed in Laccaria bicolor, Phanerochaete chrysosporium and Coprinopsis cinerea. These GPCRs are suggested to represent ancestral duplications of proteins with demonstrated functions in mating (Martinez et al, 2009).

Within this proposed B-mating type locus also numerous genes for lipopeptide pheromone precursors were detected (Figure 18).

Figure 18. Analysis of the P. placenta B-mating locus (adapted from Martinez et al., 2009). The genomic locus is given along with the location of pheromone receptors (grey) and pheromone precursor genes.

The major question which arises from investigation of the important sensing mechanism of heterotrimeric G-protein signaling is whether the respective lifestyle of P. placenta, C. subvermispora and P. chrysosporium i.e. brown rot or white rot is reflected in their genomic inventory for environmental sensing. However, no conclusive evidence was found to support such a hypothesis (Fernandez et al., manuscript submitted).


5

Conclusions and outlook

Genes that underlie the capacity to receive, use and transmit information are the evolving properties.

Peter R. Grant

The work summarized in this thesis contributed to a better understanding of the light response in Trichoderma reesei and its influence on cellulase gene expression by providing a comprehensive overview of gene regulation upon growth on cellulose and the influence of light under these conditions. Given that cellulases are useful in industry for production of second generation biofuels, the understanding of molecular mechanisms underlying the cellulase gene expression are therefore very important. In particular the knowledge of the influence of light on the expression mechanisms can serve for improvement of the cellulase production processes. The evolutionary heritage of T. reesei does not only influence its physiology in its natural habitat. Several findings described here show that also in the artificial environments of a laboratory incubator and a defined minimal medium the reaction to environmental conditions such as light has to be considered for meaningful and reproducible results. The enzymes used in industry are mostly produced in darkness in big steel fermenters, while strain improvement and screening assays are often performed in light or under uncontrolled conditions. Our results show that many physiological processes are adjusted in response to light. Therefore, effects seen under these conditions are likely to be altered in the darkness of a steel fermenter. Accordingly, potential positive effects of a certain mutation may only be obvious upon growth in darkness and missed when the screening is done in light. Consequently, costly drawbacks due to different conditions during screening and up-scaling can be avoided by working under controlled and comparable light conditions. Moreover the positive effect of light is currently not exploited. Work on further steps towards elucidation of the mechanism of light response and signaling, which can be used to predict environmental effects in nature and in industrial settings are in progress. In particular, our aim is to reveal the signal transduction pathway from the cellulase promotor(s) up to the signal perception mechanism, with the regulatory components described in this thesis as central anchors. Both nutritional conditions and light are crucial determinants of the decision of T. reesei whether to reproduce sexually and asexually and consequently connect the pathways of nutrient and light signaling with regulation of sexual development. Research with sexual


development in T. reesei is still at its very beginning. Nevertheless, initial findings such as female fertility of the most commonly used strain of the genus as well as the discovery of a novel type of peptide pheromone precursors provide intriguing insights into physiology of T. reesei as well as a fascinating subject for further research. Also here the industial application deserves attention, predominantly because of the significance of T. reesei for biofuel production and heterologous protein production. Using sexual development as a tool, combination of beneficial characteristics of nature isolates and industrial or laboratory strains can be achieved. Additionally, construction of double and multiple mutants has become considerably more efficient with the possibility to use a crossing approach as routinely used with Aspergillus or Neurospora for decades. Our current work on this topic is focused on elucidation of the defects of QM6a which led to its female sterility along with analysis of the relevance of the mating type and capability for sexual development in carbon source utilization and enzyme production.


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Curriculum vitae

Address: Vienna University of Technology, Gumpendorfer Strasse 1a/1665, 1060 Vienna,

Austria Email: [email protected] Homepage: www.vt.tuwien.ac.at/pg/schmoll

Telephone: +43 1 58801 166552

Professional Experience

2012 Member of the Editorial board of Applied and Environmental Microbiology

2011 Member of the Editorial board of Frontiers in Genetic Architecture

2011 Management committee member of COST action FP0602 “Biotechnology for lignocellulose biorefineries”

2011, July Chairperson of the session “Fungal Genetics and Genomics” at the Annual World congress of Microbes, Beijing, China

2008, Sep.-Oct. Visiting scholar with Prof. N. Louise Glass, Plant and Microbial Biology Department, University of California, Berkeley

2008, April Chairperson of Symposium 6, Regulation of Gene expression at the 9th European

Conference on Fungal Genetics, Edinburgh, UK (together with Mark Caddick, University of Liverpool, UK)

since 2007 Group leader in the Research Area Gene Technology and Applied Biochemistry, Vienna University of Technology.

April 2006 Member of the local organizing committee for the 8th

European Conference on Fungal Genetics (ECFG8), April 8 – 11, Vienna, Austria

Member of the local organizing committee for the First European Neurospora

Meeting (Satellite Meeting of the ECFG8), April 8th

,Vienna, Austria Chairperson of this Meeting (together with L. Corrochano, University of Sevilla)

2005, Sept. Guest researcher at the Dipartimento di Protezione delle Piante, University degli Studi di Sassari, Sassari, Italy

2005, Sept. Guest researcher at the Hungarian Academy of Sciences and University of Szeged, Microbiological Research Group, Szeged, Hungary

2003, Sept. Guest researcher at the Dipartimento di Biotecnologie, Cellulari Ed Ematologia, Sezione di Genetica Moleculare, Universita’ di Roma “La Sapienzia”, Italy

Education

2003 Ph. D. (with distinction) at the Vienna University of Technology on “Regulation of cellulase expression and signal transduction in the filamentous fungus Hypocrea jecorina (Trichoderma reesei)”; date: march 25

th

1999 Master degree on „Analysis of a cis-acting element of cellulase induction in

Trichoderma reesei“ at the Vienna University of Technology; date: june 28th

mailto:[email protected]

http://www.vt.tuwien.ac.at/pg/schmoll


Fellowships

2004 – 2006 Postdoctoral fellow with FWF (Austrian Science Fund) -Project P17325-B12 as

principal scientist

2006, May - Dec Project with industry partner BASF, Ludwigshafen, Germany “Production and

secretion of heterologous proteins with Trichoderma reesei”

Teaching activities and Supervision

Since 2004 Principal advisor of 1 Bachelor student, 5 Master students and 4 PhD students

from Austria, Germany, Hungary and Brazil in the Research Area Gene Technology and Applied Biochemistry

Supervisor of guest students from Taiwan, Hungary and Italy

Various lab courses for undergraduate students (2 – 6 weeks) dealing with

selected topics of the ongoing research projects

Lectures “Molecular physiology of fungi“ and “Genetics and industrial genomics”

Lab courses “Protein chemistry“, “Biochemistry 2“ and “Biotechnology 2“

since 2009 Member of the female junior faculty for the “AB-Tech” PhD-program at the Vienna University of Technology

2007, Feb. Member of the board of examiners for graduation as master at the Technische

Universität Dresden, Germany

2011, July Member of the board of examiners for a PhD defence at the University of Alicante,

Spain

Funding

2008 - 2010 Supervising scientist of a recipient of a DOC fFORTE fellowship (PhD-

programme for women in research and technology, Austrian Academy of Sciences) “Analysis of the role of the class I phosducin like protein in Hypocrea jecorina (anamorph Trichoderma reesei)” (project sum: 90000 EUR)

2008 - 2011 Project leader of FWF (Austrian Science Fund) stand alone project P21703-B03

“Analysis of the role of the class I phosducin like protein in Hypocrea jecorina (anamorph

Trichoderma reesei)” (project sum: 51269 EUR)

2007 - 2011 Project leader of FWF (Austrian Science Fund) stand alone project

P20004-B17 “Functions of peptide pheromones in H. jecorina” (project sum: 279468 EUR)

2007 - 2009 Principal investigator and project leader with APART (Austrian Programme for Advanced Research and Technology of the Austrian Academy of Sciences) fellowship No. 11212 “Interacting signal transduction pathways in H. jecorina”

(project sum: 201500 EUR)

2010 - Principal investigator and project leader with Elise Richter fellowship (FWF)

V152-B20 “Sexual development in H. jecorina”(project sum: 315 240 EUR)

2010 - Project leader with FWF stand alone project “Light dependent signaling by

G-proteins in H. jecorina” P22511 (project sum: 363290 EUR)


International Collaborations

N. Louise Glass

(University of California, Berkeley, USA)

Regulation of cellulase gene expression and signaling

in H. jecorina and N. crassa

Scott E. Baker (Pacific Northwest National

Laboratories, Richland, USA)

Sexual development and female sterility in Hypocrea

jecorina (Trichoderma reesei)

Michael Freitag (Center for Genome

Research and Biocomputing, Oregon State University, USA)

Sexual development and female sterility in Hypocrea

jecorina (Trichoderma reesei)

Alfredo Herrera-Estrella (CINVESTAV, Mexico)

Influence of light on Hypocrea jecorina (Trichoderma reesei)

Ting-Fang Wang (Academia Sinica, Taipei, Taiwan)

Molecular mechanisms in sexual development of Hypocrea jecorina (Trichoderma reesei)

Kati Réczey (Budapest University of Technology and Economics, Hungary)

Cellulase gene expression in Trichoderma reesei

Laszlo Kredics (University of Szeged, Hungary)

Differential gene expression during pathogenesis of Trichoderma longibrachiatum

Participation in the EC-projects EUROFUNG and EUROFUNGBASE which involved leading research groups in the field of fungal genetics (2004 – 2008).

Scientific achievements

Publications in peer reviewed journals 34 Book chapters 2 First author papers 9

Articles in group leader position 15 Papers without supervisor co-authorship 9 Total impact factor of publications 184,2

Impact factor of first/last author papers 78,2 h-index (Scopus) 13 Sum of acquired grants 1 300 766 EUR

Reviewer assignments for Journals

PLoS ONE Current Genetics Applied and Environmental Microbiology Journal of Basic Microbiology

Fungal Genetics and Biology Acta Biochimica et Biophysica Sinica FEMS Microbiology Letters Letters in Applied Microbiology BMC Genomics Electronic Journal of Biotechnology

Applied Microbiology and Biotechnology Microbiological Research Gene Mycological Progress

Reviewer assignments for funding bodies

NSF, National Science Fund, USA BSF, Israel Binational Science Foundation STW, Technology Foundation, The Netherlands


Acknowledgements I want to thank Christian Kubicek for his continued guidance and support, and also for the numerous arguments we had, which prepared me to withstand the often harsh conditions in science. He also gave me the opportunity to build my own research topic and develop into an independent scientist, for which I am very grateful. My fellow groupleaders, Verena and Susanne deserve a loud “Thank you!” for sharing excitement as well as frustration and for those small comments that help a lot sometimes. Also the positive atmosphere and collaboration in our groups is a precious achievement. And last but not least: Thanks go to Verenas “Positive weeks” for the many smiles that they caused. However, my greatest thanks go to all current and previous, long-term or short term members of my group. Your work, enthusiasm and persistence formed the basis of our scientific success. Especially I want to thank André and Doris, who in recent years developed from struggling beginners to promising and reliable scientists and also to dear friends. Special thanks go to those friends, who were there for me when nobody else was. I will never forget that. My gratitude also goes to the Austrian Science Fund (FWF) and the Austrian Academy of Sciences (ÖAW), who funded my work and my group and enabled me to become a recognized researcher. I also have to thank our former Dean, Prof. Hannes Fröhlich, who provided the financial resources to buy a new light/dark shaker after we had lost our cultivation room due to the reconstruction work. Without that we could not have fulfilled our project tasks. Thank you! My time at this institute became enriched by many colleagues and students, from whom I could learn and gain valuable experience. I appreciate this shared time and hope that I could give them as much as they gave me. Life changing experiences are rare and mostly recognized only quite some time after they happen, but by now I am sure I was lucky enough to have a few of them. Over the years I had the pleasure to meet many colleagues from all over the world, whose names I once knew only from publications, in person. Some of them became partners, some even friends. With others I only shares a few hours of exciting discussions or small but valuable comments on my work. This input was crucial to my research and often sparked many more ideas than I could realize in a lifetime – nevertheless, some of them are part of this thesis as published work. Especially the time I spent with other work groups abroad was extremely precious for my work and my life, when I found my work to be appreciated and where I got to know people who became my role model. I am very grateful for this experience and despite the less than optimal situation of science this experience makes up for the many challenges in keeping my job and my group up and running. Last but not least I want to thank my family, my husband, my parents, my sisters and brother, who at many occasions unknowingly were like an island for me, where I could rest and restore my energy.


APPENDIX I

A. Fungi and light Tisch, D. & Schmoll, M. (2010) Light regulation of metabolic pathways in fungi (invited review). 67

B. Light response in Trichoderma reesei

Schmoll et al., (2005) Envoy, a PAS/LOV domain protein of Hypocrea 86 jecorina (anamorph Trichoderma reesei), modulates cellulase gene transcription in response to light.

Schuster et al., (2007) Impact of light on Hypocrea jecorina and the multiple cellular 96 roles of ENVOY in this process.

Castellanos et al., (2010) Crucial factors of the light perception machinery and their 113 impact on growth and cellulase gene transcription in Trichoderma reesei.

Gyalai-Korpos et al., (2010) Relevance of the light signaling machinery for 122 cellulase expression in Trichoderma reesei (Hypocrea jecorina)

C. Transmission of nutrient signals in Trichoderma reesei

Gremel et al., (2008) Sulphur metabolism and cellulase gene expression are connected 132 processes in the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei).

Schmoll (2008) The information highways of a biotechnological workhorse- 150 signal transduction in Hypocrea jecorina.

Schmoll et al., (2009) The G-alpha protein GNA3 of Hypocrea jecorina (Anamorph 175 Trichoderma reesei) regulates cellulase gene expression in the presence of light.

Schuster & Schmoll (2009) Heterotrimeric G-protein signaling and light response: 186 Two signaling pathways coordinated for optimal adjustment to nature.

Seibel et al., (2009) Light-dependent roles of the G-protein alpha subunit GNA1 of 189 Hypocrea jecorina (anamorph Trichoderma reesei).

Tisch et al., (2011) New insights into the mechanism of light modulated signaling 204 by heterotrimeric G-proteins: ENVOY acts on gna1 and gna3 and adjusts cAMP levels in Trichoderma reesei (Hypocrea jecorina)

Schuster et al., (2011) The dehydrogenase GRD1 represents a novel component 214 of the cellulase regulon in Trichoderma reesei (Hypocrea jecorina).

Tisch et al., (2012) The phosducin-like protein PhLP1 impacts regulation of 225 glycoside hydrolases and light response in Trichoderma reesei.

D. Sexual development in Trichoderma reesei

Seidl et al., (2009) Sexual development in the industrial workhorse Trichoderma reesei. 253 Schmoll et al., (2010) A Novel Class of Peptide Pheromone Precursors 259

in Ascomycetous Fungi

trichoderma reesei hypocrea jecorina › c7c9 › e49cc67a5dcc... · dream big and dare to fail. it...

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