The possible relationships between identified proteins PcTOR and PcDUF1630 protein and secretion

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Fungi have to adapt to their environment

Fungi are known to be the largest group of eukaryotic organisms on the planet, with an estimate of 3.5 to 5.1 million species, most of them being unknown (O’Brien et al., 2005). The most basic feature of fungal growth and development is the production of spores, which can be distributed in the air or mobilized to all parts of the planet by water (Bayram and Braus, 2012). Therefore, the ability to adapt to new ecological niches is the most important dynamic for their evolution. Yeasts are fungi that exist mainly as unicellular organisms. They account for approximately 1% of the described fungal species. They have been evolved into several taxonomic groups (Kurtzman and Piškur, 2015), as shown in Fig. 1A. The fundamental feature of the yeast’s developmental program is the transition from the round single-cell yeast form to the filamentous growth mode. The switch between these two modes is known as fungal dimorphism and depends on environmental conditions. The dimorphism can generate pseudohyphae and true hyphae (Fig. 1A). Pseudohyphae are the form with interconnected cellular units produced from adhesion of elongated cells. They are typical forms of diploid budding yeasts.
A-C. Cellular forms and life cycle of the filamentous model fungus Aspergillus nidulans. A. Yeast form: unicellular fungal growth mode; pseudohyphae: filamentous growth form with individual cells; true hyphae: filaments, often separated by permeable septae; conidiophore: composed asexual structure; cleistothecium: spherical closed sexual fruiting body (common in Aspergillus species). S, stalk; V, vesicle; M, metulae; P, phialides; C, conidia; HC, Hülle cells. B. Life cycle from vegetative growth to asexual or sexual alternatives of development (Bayram and Braus, 2012). C. Environmental factors in soil and at the surface. A conidiophore and a cleistothecium of A. nidulans are produced on the surface and in the substrate, respectively. ROS represents for reactive oxygen species (Rodríguez-Romero et al., 2010).
The specific fungal growth mode is the formation of multicellular hyphae, as described in Fig. 1A. Hyphae have a tube-like structures that are formed from the germination of a fungal spore. They are the basic growth units of most filamentous fungi and expand at the apex of the tip cell. Polar tip growth is due to plasma membrane expansion combined with the biosynthesis of cell wall components (Steinberg, 2007).
Filamentous fungi comprise fungi without dimorphic forms. The fundamental feature of their developmental program is the formation of vegetative hyphae before moving on to other development programs. Vegetative growth starts from spore germination with the production of a fungal hypha. The differentiation capability of the latter is determined by its susceptibility to environmental signals, as shown in Fig. 1B. The range of time from spore germination to the fungal hypha, depends on the considered fungal species and is called the competent time, which is linked to the growth rate. For example, the competent time of Aspergillus nidulans is from 12 to 20 hours (Fig. 1B).
For filamentous fungi, developmental programs include the transition to asexual spore formation and to sexual fruiting bodies, as described in Fig. 1B. The transition from asexual to sexual development programs depends on environmental interactions and signals sensing, including nutrients, fungal pheromones, stressors, surface, oxygen, or light (Bayram and Braus, 2012). These interactions also modify the production of secondary metabolites that could play a role in fungal protection against competitive interactions in their ecosystems (Rohlfs et al., 2007).
To adapt to their environment, fungi have to sense their chemical environment and react appropriately. Saprophytic fungi use organic matters as nutritional resources. A. nidulans, a soil-living organism, has for instance to adapt to the low oxygen concentration in underground conditions. Such anoxic environment can induce the generation of cleistothecia resulting from the sexual reproductive system (Fig. 1C). At the soil surface or above the ground, environmental conditions change rapidly and drastically. For instance, A. nidulans can be confronted to a large range of temperature, humidity, and light in a short period of daytime (night-day cycle for instance). In these conditions, the fungal mycelium may desiccate quickly leading to osmotic stress. Besides, light exposure through ultraviolet irradiation could induce damages to fungal DNA or production of harmful reactive oxygen species (ROS) (Fig. 1C). Light affects the fungal metabolism and possibly induce the production of secondary metabolites either potentially toxic for human or animals or industrially valuable (Rodríguez-Romero et al., 2010).

Carbon nutrient sensing and responses in lignocellulose degradation

The lignocellulose degrading process is mainly mediated by extracellular enzymes involved in the cleavage of polysaccharides. Nutrient sensing pathways, especially pathways enabling the use of preferred carbon sources are very important for the survival strategy used of the fungi. In filamentous fungi, those pathways function as the upstream of the direct activation of genes encoding CAZymes (Fig. 5). This activation results in the inhibition of the energy-consuming production of lignocellulose degrading enzymes (Huberman et al., 2016). Up to date, the best-studied pathway is carbon catabolite repression (CCR).
The utilization of a diverse array of carbon sources derived from lignocellulose degradation requires the coordination of the cellular metabolism and the preferential consumption of glucose prior to other carbon sources, a phenomenon known as carbon catabolite repression (CCR). CCR mechanism represses transcription of secreted and intracellular metabolic enzymes and has been described in both yeasts and filamentous fungi (Brown et al., 2014). The transcription of these genes is tightly regulated by a Cys2-His2 type DNA-binding zinc finger factor, named Mig1 in S. cerevisiae, and CreA/Cre1 in filamentous fungi (Fig. 5, 6).
In S. cerevisiae, the repressor protein ScMig1 is imported into the nucleus when glucose is added to derepressed cells (Fig. 6A). In the nucleus, ScMig1 binds to upstream regulatory elements (URE) of genes coding for transcription factors. This leads to inhibit the expression of those transcription factors which transcriptionally regulate alternative carbon usage. In addition to ScMig1, CCR is also regulated by nuclear chromatin structure which can be linked to intrinsic cellular program and environmental factors (Brosch et al., 2008). In yeast, ScMig1 reacts with the corepressors ScTup1 and ScSsn6 (Fig. 6A). The obtained complex binds to promoters of alternative carbon usage genes leading to their repression through the modulation of nucleosome positioning (Li et al., 2007a; Treitel and Carlson, 1995).
In filamentous fungi, the carbon nutrient-dependent nuclear localization of CreA/Cre1 (Fig. 6B) is similar to ScMig1 in S. cerevisiae. However, this repressor is not only regulated by glucose but also by other sugars resulting from carbon nutrient sources (Brown et al., 2013; Sun and Glass, 2011). The role of CreA nuclear localization in regulation of CCR has been confirmed in A. nidulans, and N. crassa by use of GFP fusions (Brown et al., 2013; Sun and Glass, 2011).
Orthologues of the S. cerevisiae corepressors ScTup1 and ScSsn6 have also been widely identified in filamentous fungi (García et al., 2008). In A. nidulans, the homolog of ScTup1 is AnRcoA (Fig. 6B). This corepressor is dispensable for CCR and affects the repressed chromatin structures in the alcR promoter during repression (García et al., 2008). alcR is a trans-acting gene containing a specific site allowing the binding of the repressor CreA (Kulmburg et al., 1993). In comparison with S. cerevisiae, the regulatory role of the CreA/Cre1 complex to CCR is less well-known in detail in filamentous fungi. The role of CreA in nucleosome positioning has been only shown in T. reesei. In particular, TrCreA is essential for correct nucleosome positioning in the cellulase promoter cbh2 under repressing and inducing conditions (Zeilinger et al., 2003).

G – protein-coupled receptors (GPCRs) in nitrogen sensing of yeast and filamentous fungi

There are no GPCRs established as nitrogen receptors in S. cerevisiae while orthologues of ScGpr1 known as glucose sensor (Fig. 4) were reported to sense methionine in pathogenic C. albicans and C. neoformans (Maidan et al., 2005; Xue et al., 2005). Methionine sensing is important for the hyphal transition of C. albicans on agar medium, and it can induce a transient cAMP production, as well as mating hyphal elongation in C. neoformans (Maidan et al., 2005; Xue et al., 2005).
In filamentous fungi, amino acids appear to be sensed by the cAMP receptor-like GPCR named GprH which is the glucose sensor in A. nidulans (Fig. 3) (Li et al., 2007b). Orthologues of GprH have been found in other filamentous fungi such as NcGpr4 in N. crassa, and AfGprC/D in Aspergillus flavus (Fig. 3) (Affeldt et al., 2014; Li and Borkovich, 2006). Additionally, the function of cAMP receptor-like GPCRs in the regulation of sexual development is conserved in fungi. GPCRs therefore may have a dual role in sensing both sugars and amino acids.
There is very little literature dealing with GPCRs requirement in ammonium sensing of filamentous fungi. To date, ammonium sensing via GPCRs has been only shown in Aspergillus flavus (Affeldt et al., 2014). In this fungus, mutations in GprC/D receptors impair growth on ammonium chloride and proline (Affeldt et al., 2014).
In summary, in addition to cAMP/PKA signaling pathways, the TOR signaling pathway may play an important role in regulating a wide range of functions in response to both carbon and nitrogen nutrient sources in the filamentous fungi. In the next part, TOR signaling will be described and discussed in these fungi.


Target Of Rapamycin (TOR) signaling as a key regulator of growth in the adaptation of filamentous fungi

All living organisms sense and respond to nutrient signals by regulating growth and developmental programs for survival. In eukaryotic organisms, a role for the TOR signaling pathway has emerged as a global regulator of cell growth. The central component of this signaling pathway is Target Of Rapamycin (TOR), a conserved serine/threonine kinase which belongs to the family of phosphatidylinositol kinase-related kinases (PIKK) (Wullschleger et al., 2006).
TOR was first found in S. cerevisiae via the characterization of strains resistant to rapamycin. Rapamycin is a metabolite produced by Streptomyces hygroscopicus (Heitman et al., 1991). Rapamycin binds to the intracellular receptor FKBP12 (FK506 binding protein 12) to form a complex which then inhibits TOR kinase activity resulting in cellular toxicity (Wullschleger et al., 2006). In S. cerevisiae, two genes encoding two proteins named TOR1 and TOR2 are present. The resulting proteins are the main components of two complexes named TORC1 and TORC2. Up to date, TOR characterization in fungi has been mainly carried out in S. cerevisiae wherein TOR has been demonstrated as the central controller of growth, proliferation, and survival in response to nutrients and stress (Loewith and Hall, 2011). Up to date, TOR has been identified in all tested eukaryotes, controlling cellular processes via regulating networks named TOR signaling pathways. The TOR pathway components in S. cerevisiae (A) and S. pombe (B). The functional homologs between the two species are shown in the same shape and color (Shertz et al., 2010). In the fungal kingdom, TOR signaling pathways have been best-characterized in model yeasts S. cerevisiae and S. pombe as presented in Fig. 7 (Shertz et al., 2010). In S. cerevisiae, only ScTORC1 is sensitive to rapamycin. It comprises ScTOR1 or ScTOR2, ScKog1, ScTco89, and ScLst8 (Fig. 7A). ScTORC1 controls protein synthesis, mRNA synthesis and degradation, ribosome biogenesis and autophagy. ScTORC2 is composed of ScTOR2, ScLst8, ScAvo1, ScAvo2, ScAvo3 (Fig. 7A), it is insensitive to rapamycin and is involved in the control of actin polarization and cell wall integrity (Loewith and Hall, 2011). The downstream effectors of ScTORC1 are the ScSit4, one PP2A-like phosphatase, and the AGC kinase ScSch9. ScSch9 has been deeply studied due to its involvement in stress response. Under stress conditions including nutrient starvation, high salt, redox stress, temperature, ScSch9 phosphorylation is strongly reduced (Urban et al., 2007). Due to this property, ScSch9 has been used to quantify ScTORC1 activity through its phosphorylation in various nutrient and stress conditions (González et al., 2015). In comparison to S. cerevisiae, there is an extension or expansion in upstream components of the TOR pathway in S. pombe, with the presence of ScTsc1 and ScTsc2 (Fig. 7B). In this fungus, the TOR pathway has functional roles in nutrient and stress signaling, cell growth and differentiation, and sexual development (Alvarez and Moreno, 2006; Otsubo and Yamamoto, 2010; Weisman and Choder, 2001).
In addition to S. cerevisiae and S. pombe, TOR signaling has been also well-studied in human pathogenic yeasts such as C. albicans and C. neoformans (Rutherford et al., 2019). C. albicans is the first fungus found to be inhibited by rapamycin in 1975 which opened the story of TOR signaling researches (Sehgal et al., 1975). In contrast to yeasts, TOR signaling pathways have been less investigated in filamentous fungi. In these organisms, TOR and TOR signaling pathways have been only identified and partially functionally characterized (focusing on genetics, development, and pathogenicity) in several fungal models such as A. nidulans (Fitzgibbon et al., 2005), N. crassa (Park et al., 2011), Podospora anserina (Pinan-Lucarré et al., 2006), A. fumigatus (Castro et al., 2016), C. neoformans (Lee et al., 2012), Fusarium fujikuroi (Teichert et al., 2006), Fusarium graminearum (Yu et al., 2014), Fusarium oxysporum (López-Berges et al., 2010), Magnaporthe oryzae (Qian et al., 2018), and Verticillium dahliae (Li et al., 2019). In all these fungi, TOR signaling is functionally conserved and involved in the regulation of cellular growth, metabolism, virulence and vegetative development. In addition to the TOR identification, the components of the TOR signaling pathway have also been identified in the focus on downstream effectors of TOR. Indeed, homologs of ScSit4 (AnSit4) and ScSch9 (AnSchA and NcSch9) were identified in A. nidulans (Fitzgibbon et al., 2005) and in N. crassa (Park et al., 2011). Functional characterization of these components showed similar roles in response to stress and in autophagy regulation under rapamycin treatment or nutrient-starvation. For these fungi, to date, only one upstream component of TOR signaling pathways has been identified in N. crassa: the protein VTA for Vacuolar TOR-Associated protein (Ratnayake et al., 2018). This is the homolog of ScEgo1 in S. cerevisiae (Fig. 6) and Lamtor1 in mammals. In N crassa, it has a role in maintaining circadian daily rhythmicity (Ratnayake et al., 2018).

Table of contents :

I. Fungal adaptation to environment
I.1 Fungi have to adapt to their environment
I.2 Fungal sensing of the environment
I.2.1 Nutrient sensing
Carbon sensing
Carbon nutrient sensing and responses in lignocellulose degradation
Nitrogen sensing
Target Of Rapamycin (TOR) signaling as a key regulator of growth in the adaptation of filamentous fungi
I.2.2 Stress sensing and responses for adaptation to environment
II. Wood and wood degrading fungi
II.1 Wood components
II.1.1 Structural compositions
II.1.1 Cellulose
II.1.2 Hemicellulose
II.1.3 Lignin
II.I.2 Wood extractives
II.I.2.1 Phenolic compounds
II.I.2.1.1 Flavonoids
II.I.2.1.2 Tannins
II.I.2.1.3 Stilbenes
II.I.2.1.4 Lignans
II.1. 2.1.5 Quinones
II.1.2.2 Terpenoids
II.2 Wood decaying fungi
II.2.1 Brown rot fungi
II.2.2 White rot fungi
II.2. 3 General biological characteristics of Phanerochaete chrysosporium
II. 3 Antifungal mechanisms of wood extractives and fungal adaptation
Article I
Article II Supplementary results
Experiment protocols
Discussion and conclusions (Article I & II)
Article III Supplementary results
Mutagenesis and screening mutant resistant to CTWE
Phenotypes of chy mutants
Identification of the causal mutation(s) leading to CTWE resistance
Involvement of AGR57_10098 in resistance against CTWE
Wood extractives as tools to characterize wood decaying fungi
The possible relationships between identified proteins PcTOR and PcDUF1630 protein and secretion
The possible relationships between PcTOR and PcNACHT protein
Understanding the functions of TOR signaling in P. chrysosporium
Proteomic analysis could be used to characterize deeply the role of TOR in regulating the secretome response of P. chrysosporium
The possible relationship between TOR and the intracellular detoxification system
Future perspectives
General conclusions


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