Role of regulatory proteins in carotenoid biosynthesis and metabolism in Fusarium fujikuroi

University of Seville Department of Genetics Role of regulatory proteins in carotenoid biosynthesis and metabolism in Fusarium fujikuroi Report prepared by Julia Natividad Marente Bernal to be qualified for the degree of PhD in Biology. This work has been carried out in the Department of Genetics of the Faculty of Biology of the University of Seville under the direction of Dr. Francisco Javier Ávalos Cordero and Dr. María del Carmen Limón Mirón. Sevilla, 2024 Dr. Francisco Javier Dr. María del Carmen Julia Natividad Ávalos Cordero Limón Mirón Marente Bernal Mi interés se tornó bien pronto analítico. Cansado de maravillarme quise saber; he ahí el invariable y funesto fin de toda aventura. Julio Cortázar A Freddie Index Index ................................................................................................ 1 Abbreviations .................................................................................... 1 Summary .......................................................................................... 5 Introduction ....................................................................................... 9 1 Fusarium species ......................................................................... 9 1.1 Characteristics and life cycle of F. fujikuroi .................................................. 10 2 Light and photoreception in fungi ............................................... 11 2.1 Photoreception model in fungi: White-Collar complex ................................. 12 2.2 Light regulation in F. fujikuroi ..................................................................... 14 2.2.1 Blue-light photoreceptors: White-collar complex and cryptochromes .. 14 2.2.2 Green-light photoreceptors: Rhodopsins ............................................... 17 2.2.3 Red-light photoreceptors: Phytochromes .............................................. 18 2.3 Relation of light with other regulatory systems ........................................... 19 3 Regulation of nitrogen metabolism ............................................ 21 3.1 Major nitrogen regulators .......................................................................... 22 3.2 Other nitrogen regulators........................................................................... 24 4 Secondary metabolism ............................................................... 26 4.1 Main secondary metabolites of F. fujikuroi .................................................. 26 4.2 Carotenoids ............................................................................................... 29 4.3 Regulation of secondary metabolism .......................................................... 31 4.3.1 Carotenoid biosynthesis regulation ........................................................ 31 4.3.2 Role of global regulators and histone modifications in secondary metabolism ............................................................................................................ 37 Aims ............................................................................................... 43 Chapter I: Comparative analysis of wcoA and wcoB deletions in Fusarium fujikuroi: insights into widespread functions as a complex . 47 1 Introduction .............................................................................. 47 2 Results ...................................................................................... 49 2.1 Generation of wcoA and wcoB deletion mutants ......................................... 49 2.2 Influence of wcoA and wcoB deletions on environmental signal responses ... 52 2.2.1 Influence of wcoA and wcoB deletions on expression of photoreceptor genes……………………..………………………………………………………………………………………….53 2.2.2 Influence of wcoA and wcoB deletions on two key genes involved in HOG response and conidiation ...................................................................................... 54 2.2.3 Influence of wcoA and wcoB deletions on mycelial hydrophobicity ...... 57 2.3 Functions of wcoA and wcoB genes in secondary metabolism ....................... 58 2.4 Effect of wcoA and wcoB deletions on secondary metabolism under nitrogen starvation ............................................................................................................. 60 2.5 Retarded carotenoid biosynthesis in wco mutants ....................................... 63 3 Discussion ................................................................................. 68 Chapter II: Controlled transcription of regulator gene carS by Tet-on or by a strong promoter confirms its role as a repressor of carotenoid biosynthesis in Fusarium fujikuroi .................................................... 81 1 Introduction .............................................................................. 81 2 Results ...................................................................................... 84 2.1 Improvement of the gene expression control by the Tet-on system in F. fujikuroi using a reporter gene ............................................................................................ 84 2.2 Use of the Tet-on system to control carS expression .................................... 87 2.3 Effect of control of carS expression on carotenogenesis in the carS mutant... 90 2.4 Effect of constitutive carS overexpression on the carotenoid biosynthesis pathway ............................................................................................................... 94 3 Discussion ................................................................................. 97 Chapter III: Influence of light and CarS regulation in Fusarium fujikuroi metabolome 103 1 Introduction ............................................................................ 103 2 Results .................................................................................... 105 2.1 Multivariate analysis of metabolic profiles of F. fujikuroi strains affected in carotenoid production ........................................................................................ 105 2.2 Identification of metabolic changes in response to carS expression and light in F. fujikuroi .......................................................................................................... 107 2.3 Effect of CarS in the F. fujikuroi metabolic profiling .................................... 110 2.4 Comparative analysis between metabolites accumulated and related biosynthetic pathways ........................................................................................ 114 3 Discussion ............................................................................... 118 Chapter IV: Functional analysis of nutZ, a regulatory gene adjacent to the car cluster ................................................................................129 1 Introduction .............................................................................129 2 Results .....................................................................................132 2.1 Sequence features and regulation of gene FFUJ_11800. ............................ 132 2.2 Generation of nutZ deletion mutants ........................................................ 133 2.3 Complementation of nutZ deletion mutants and generation of nutZ overexpressing strains ........................................................................................ 135 2.4 Effect of nutZ deletion or overexpression on carotenoids biosynthesis ....... 138 2.5 In vitro NutZ binding assays on car gene promoters ................................... 140 2.6 Genes differentially expressed in nutZ overexpressing strains compared to wildtype and ΔnutZ strains ....................................................................................... 143 2.7 Genes affected in both OEnutZ strains and identification of predominant GO functional categories .......................................................................................... 146 2.8 Effect of nutZ overexpression on transcription of genes related to nitrogen transport ........................................................................................................... 148 2.9 Effect of nutZ overexpression on nitrogen regulation ................................ 153 2.9.1 Repressor effect of nutZ overexpression in nitrate assimilation .......... 154 2.9.2 Effect of nutZ overexpression on genes for amino acids metabolism . 154 2.9.3 Influence of nutZ overexpression on secondary metabolism .............. 159 2.10 Comparative analysis of gene expression changes induced by nutZ overexpression and oxidative stress .................................................................... 161 3 Discussion ................................................................................165 General discussion .........................................................................175 Conclusions ....................................................................................185 Material & methods .......................................................................189 1 Strains and plasmids used in this work ......................................189 1.1 Plasmid constructions .............................................................................. 193 2 Media and culture conditions ....................................................194 2.1 Media and culture conditions for Fusarium fujikuroi ................................ 194 2.1.1 Solid media ............................................................................................ 194 2.1.2 Liquid media and buffers ...................................................................... 197 2.2 Media and culture conditions for Escherichia coli ..................................... 201 2.3 Media and culture conditions for Saccharomyces cerevisiae ..................... 202 3 Genetic manipulation, transformation, and analysis ................. 203 3.1 Nucleic acid quantification .............................................................................. 203 3.1.1 Nanodrop spectrophotometer ............................................................. 203 3.1.2 Electrophoresis on agarose gel ............................................................. 203 3.2 Polymerase Chain Reaction (PCR) ................................................................... 204 3.2.1 DNA polymerases and standard PCR protocol ..................................... 204 3.2.2 Overlap extension PCR protocol ........................................................... 205 3.2.3 Primers used in this study ..................................................................... 206 3.3 Purification of PCR product ............................................................................. 212 3.4 DNA precipitation ............................................................................................ 212 3.5 Plasmid construction: design, cloning and isolation ....................................... 212 3.5.1 Plasmid design and cloning ................................................................... 212 3.5.2 E. coli transformation ............................................................................ 214 3.5.3 S. cerevisiae transformation ................................................................. 217 3.5.3.1. Plasmid isolation from S. cerevisiae ....................................................... 218 3.6 F. fujikuroi transformation .............................................................................. 219 3.6.1 Preparation of source culture ............................................................... 219 3.6.2 Protoplast formation ............................................................................ 219 3.6.3 Protoplast transformation .................................................................... 220 3.7 Genomic DNA isolation ................................................................................... 221 3.8 Southern blot analysis ..................................................................................... 221 4 RNA isolation and gene expression analysis .............................. 223 4.1 RNA isolation from F. fujikuroi ........................................................................ 223 4.1.1 Samples from liquid media ................................................................... 223 4.1.2 Samples from fungal colonies grown on solid media ........................... 224 4.2 Analysis by quantitative Real time PCR ........................................................... 225 4.2.1 DNase treatment................................................................................... 225 4.2.2 cDNA synthesis ...................................................................................... 225 4.2.3 Quantitative RT-PCR (qRT-PCR) ............................................................ 226 4.2.4 Analysis of qRT-PCR results ................................................................... 229 4.3 Analysis by RNA-sequencing ........................................................................... 229 5 Luminescence assay to check controlled gene expression .......... 230 6 Recombinant Protein Production in E. coli for in vitro Protein-DNA interaction assays ...........................................................................231 6.1 Plasmid design and cloning of the target gene ............................................... 231 6.2 Expression of recombinant proteins in E. coli ................................................. 232 6.2.1 Pre-culture conditions .......................................................................... 233 6.2.2 Induction of NutZ protein expression ................................................... 233 6.2.3 Purification by tag affinity chromatography ......................................... 234 6.3 Protein analysis ............................................................................................... 235 6.3.1 Total protein extraction ........................................................................ 235 6.3.2 Polyacrylamide gel electrophoresis ...................................................... 235 6.3.3 Coomassie blue staining ....................................................................... 236 6.4 Protein-DNA interaction: Electrophoretic Mobility Shift Assay (EMSA) ........ 236 6.4.1 DNA-protein binding reaction ............................................................... 237 6.4.2 Electrophoresis of binding reaction products and transfer to a nylon membrane ........................................................................................................... 238 6.4.3 Detection of biotin-labelled DNA by chemiluminescence .................... 239 7 Secondary metabolites extraction and analysis ..........................239 7.1 Carotenoids extraction and analysis ............................................................... 239 7.1.1 Quantification by spectrophotometry .................................................. 240 7.1.2 Qualitative and quantitative analysis by HPLC ..................................... 240 7.2 Bikaverins extraction and analysis .................................................................. 241 7.3 Gibberellins quantification .............................................................................. 241 7.4 Metabolomic analysis by GC/MS .................................................................... 242 7.4.1 Metabolites extraction ......................................................................... 242 7.4.2 GC-MS analysis ...................................................................................... 243 7.4.3 Data analysis ......................................................................................... 243 8 Bioinformatic analysis ...............................................................244 8.1 Statistics analysis ............................................................................................. 244 8.2 Sequence analysis ........................................................................................... 244 8.3 Integration of metabolic data ......................................................................... 245 Annex ............................................................................................247 References .....................................................................................249 6 a comparative with the knowledge of WcoA function in F. fujikuroi FKMC1995 strain. The results confirmed the action of WcoA as a heterodimer with the WcoB protein, as indicates the same phenotype exhibited by mutants for both genes in most of the affected traits investigated. In addition, it showed regulatory differences between the functions of WcoA in IMI58289 and those formerly described in FKMC1995, suggesting a functional diversification of this class of regulatory protein. The following two chapters are focused on the regulatory roles of protein CarS. First, the overexpression of the encoding carS gene is described using two strategies: with the use of a constitutive promoter from the gpdA gene of A. nidulans, and with the Teton system, which allows external modulation of the expression of a target gene by using a promoter inducible by the addition of an antibiotic. In both cases, carS overexpression resulted in a total repression of the carotenoid pathway, indicating a fine modulation of carotenoid synthesis by the concentration of CarS in the cell and confirming its role as a repressor of the biosynthetic pathway. Former data indicated that the CarS protein influences the transcription of hundreds of F. fujikuroi genes, many of them involved in metabolism. For this reason, metabolomic techniques were employed in the third chapter to extend our understanding of CarS function through the study of the metabolic consequences of changing carS expression. This approach was based on GC-MS data revealed that the metabolome is not deeply impacted by light or carS mutation, but it is influenced by carotenoid production. This evidence was supported by correlation among carotenoid phenotype and the metabolic profile among the different mutants, revealing metabolic similarities between carS mutants and carS-overexpressing albino strains. In the last chapter, the function of a gene encoding a predicted transcription factor adjacent to the genes of the car cluster, and therefore suspected to be involved in the regulation of carotenoid biosynthesis, was investigated. The results indicated a lack of role of this gene in the regulation on carotenogenesis, but nutZ overexpression resulted in deregulation of genes related to transport or catabolic enzymes involved in nitrogen assimilation. This result, together with the low expression of nutZ in culture conditions with nitrate as the only N source, is consistent with a role of NutZ on nitrogen metabolism dependent on the presence of certain nitrogen sources, with implications in nitrogenregulated secondary metabolism. 7 Introduction 8 9 Introduction Introduction 1 Fusarium species Fusarium is a genus of filamentous ascomycete fungi, widely distributed in nature, which includes pathogenic and non-pathogenic strains. Opportunistic Fusarium infections in humans and animals (fusarioses) were commonly unusual, but some Fusarium species have been categorized as emergent pathogens, producing invasive infections especially in immunosuppressed patients (Nucci and Anaissie 2002; Muraosa et al., 2017). However, Fusarium species are mostly considered as plant pathogens and special attention has been paid on several species recognized as the most economically destructive and mycotoxigenic fungi, causing major diseases in many economically important crops and resulting in billion dollars in losses in agriculture worldwide. Because of their mycotoxin production, not only represent a risk to the health of plants, but also to animals and humans through consumption of contaminated feeds and foods (Aoki et al., 2014). The first description of Fusarium was reported in 1809 by Link and since then over a thousand species have been identified, of which 70 are well-known. At present, the genus Fusarium includes about 300 phylogenetically diagnosable species, distributed in 23 evolutionary lineages referred to as species complexes (O’Donnell et al., 2020). The first taxonomic classifications were created based on morphological characteristics of the species and their life cycles, establishing a “dual nomenclature” based on sexual or asexual stages (Leslie and Summerell, 2006). This approach was later changed to conform to the International Code of Nomenclature for Algae, Fungi, and Plants, which required a single scientific name for each fungal species, and after 2012 they all were clustered in the genus name of Fusarium (Summerell, 2019), although an enduring debate stills remains in the scientific community (Geiser et al., 2013; O’Donnell et al., 2013; Summerell, 2019). Fusarium species have the genetic potential to produce several structurally diverse secondary metabolites. Despite many of them are barely known, others have been investigated in detail and found to play a role in pathogenicity. In some cases, they have beneficial properties and have biotechnological interest (Bakker et al., 2018; Pessôa et al., 2017; Stępień et al., 2018). Whether for its detrimental or beneficial effects, secondary 10 Introduction metabolism has long captured scientific attention and is currently an active area of research. 1.1 Characteristics and life cycle of F. fujikuroi F. fujikuroi is a rice pathogen. Infection of the plant by this fungus results in a disease known as bakanae (which means foolish seedling in Japanese) and was reported more than 100 years ago by Japanese scientists (Sawada, 1917). The causing fungus, first described as Lisea fujikuroi (Tudzynski 2005; Bömke and Tudzynski, 2009) and later as Gibberella fujikuroi (Nelson, 1983), was one of the first fungal pathogens to be described. Plant pathologist Sawanda identified it as the causal agent of the bakanae in 1917 (Sawada, 1917). It was later determined that the abnormal elongation of plants with bakanae was due to the ability of this pathogen to produce gibberellins (GAs), and most especially, gibberellin GA3, also named gibberellic acid. The biotechnological interest of the growth-promoting properties of the gibberellins explains the extensive attention received by this fungal biosynthetic process. Asexual spores play a crucial role promoting dispersal and colonization by fungi of new habitats, including their pathogenic hosts, acting as a primary source of plant infection (Ajmal et al., 2023). Fusarium species can produce three types of asexual spores (mitotic): macroconidia, microconidia, and chlamydospores (Figure 1.A). Not all Fusarium species produce all forms of spores (Ma et al., 2013; Ajmal et al., 2023), and despite their common characteristics, there is considerable morphological diversity, even among different F. fujikuroi isolates. Macroconidia, for example, are very unusual in cultures of the wild-type strain investigated in this thesis, IMI58289, which normally produces only microconidia (Figure 1.B), while they are frequent in cultures of FKMC1995 and many other isolates (Niehaus et al., 2017a; Pardo-Medina et al., 2023). Many aspects of fungal life, such as spore germination, hyphal development, and primary or secondary metabolism (Yu and Fischer, 2019), are controlled by complex regulatory mechanisms, able to respond to diverse environmental signals, such as nutrients sources, light, or abiotic stresses. The capacity of F. fujikuroi to generate very 11 Introduction diverse secondary metabolites, alongside its pathogenicity, has focused scientific attention in this fungus. Understanding regulatory systems able to sense and transduce environmental signals, such as light, stressing conditions, or the presence of certain nutrient sources, has been a major goal in modern fungal research and has achieved notable advances in the last decades. Figure 1. Asexual spores of Fusarium species. A. Illustration of the three kinds of spores in Fusarium. Modified from Ajmal et al., 2023. B. Microscopy image of microconidia from a F. fujikuroi IMI58289 strain culture. 2 Light and photoreception in fungi Light plays a regulatory role in the expression of a significant number of genes involved in different aspects of fungal life, including circadian rhythms, morphogenesis, tropism, and pigmentation, among others (Fischer et al., 2016; Yu and Fischer, 2019). Fungi have developed photoreceptor systems to perceive light. Photoreceptor proteins contain a cofactor, known as a chromophore, which determines the type of light absorbed. This is usually a flavin in diverse types of receptors that sense blue light, with the additional participation of either methyltetrahydrofolate or methenyltetrahydrofolate (MTHF) chromophore in cryptochromes, a retinal in rhodopsins that detect green light, and a 12 Introduction linear tetrapyrrole in phytochromes that absorb red light. Most fungi contain at least two types of these photoreceptors, and some even all three (Yu and Fischer, 2019). 2.1 Photoreception model in fungi: White-Collar complex Flavoprotein photoreceptors are a diverse group of blue light-sensing proteins, achieved through the absorption of a flavin chromophore, bound to a LOV domain (from Light, Oxygen, and Voltage), including the well-known WC-1 proteins of the White-Collar system and Vivid proteins (Pardo-Medina et al., 2023). The White-Collar system was discovered in the fungus Neurospora crassa (Harding and Turner, 1981) and consists of a complex formed by the blue-light photoreceptor WC- 1 and the transcription factor WC-2. The White-Collar complex (WCC) controls all known photoresponses in this fungus, including carotenogenesis, conidiation, circadian rhythm, and the orientation of its perithecial beaks (Ballario et al., 1996; Linden and Macino, 1997; Degli-Innocenti and Russo, 1984). WC-1, in turn, the first LOV-domain protein identified in fungi, has three different domains. (1) A LOV domain to which a flavin chromophore, either flavin mononucleotide (FMN) or flavin dinucleotide (FAD), is non-covalently attached via hydrogen bonds, Van der Waals forces, and electrostatic interactions. Flavin excitation by light absorption results in the formation of a transient covalent bond with a conserved cysteine of the LOV domain (Crosson and Moffat, 2002; Pfeifer et al., 2009). This link induces changes in WC-1 structure resulting in a cascade of signals in response to light. When light excitation stops, this link is slowly broken and the initial fully oxidized state of the protein is restored (Pardo-Medina et al., 2023). (2) A PAS domain for interaction with other proteins. (3) A zinc finger domain to bind to DNA (Taylor and Zhulin, 1999; Briggs, 2007). Meanwhile, WC-2, smaller than WC-1, just contains a PAS domain and a zinc finger domain, playing a role of partner transcription factor (Pardo-Medina et al., 2023). The mechanism of action of the N. crassa WCC has been investigated in detail (Figure 2). The WCC binds to the promoters of light-regulated genes, but it is unable to activate their transcription. Light induces the formation of active WCC dimers through the 13 Introduction interaction of two WC-1 proteins via their LOV domains, enabling transcriptional activation (Wu et al., 2014; Sancar et al., 2015). The dimer mediates chromatin remodelling via H3K14 acetylation by the histone acetyl transferase NGF-1 (Brenna et al., 2012; Grimaldi et al., 2006), facilitating RNA polymerase accessibility and transcription of early light responsive genes. These include transcription factors that activate late lightresponsive genes, and the flavoprotein Vivid (VVD) (Figure 2.A). Subsequently, VVD promotes degradation of WCC by leading to phosphorylation of WC proteins by protein kinase C (PKC). WCC phosphorylation decreases its DNA binding capacity, resulting in an attenuation of the expression of light-inducible genes, a phenomenon known as photoadaptation (Schwerdtfeger and Linden, 2000; He and Liu, 2005; Schafmeier et al., 2005). This attenuation process also involves chromatin modifications by DIM-5 methyltransferase, resulting in the trimethylation of histone H3 at lysine 9 (H3K9me3) (Ruesch et al., 2015) (Figure 2.C). WCC stability and inactivation also depend on the FRQ protein, which rhythmically inhibits the activity of the WCC (Franchi et al., 2005; Dunlap and Loros, 2017). Both WCC and FRQ are central elements of the circadian clock. Figure 2. Simplified model of action of the white-collar complex in N. crassa. A. White-collar proteins (WC-1 and WC-2) bind as a complex (WCC) to the promoter of light-inducible genes in the dark. B. Blue-light induces interaction of two WC-1 proteins, conforming a WCC dimer, which induces transcription of many target genes, including vvd, through histone modifications (H3K14ac, small green circles) by the histone acetyltransferase NFG-1. C. VVD 14 Introduction interacts with WC-1 in the WCC dimer, while methyltransferase DIM-5 contributes to stop the WCC transcription activation through repressive histone modifications (H3K9me3, small red circles). D. WCC dissociates from promoters, while VVD leads phosphorylation of WCC through PKC activity (not shown), promoting partial WCC degradation. After dephosphorylation or synthesis of new WC proteins in the dark, the cycle restarts. Modified from Pardo-Medina et al., 2023, previously adapted from Fischer et al., 2016. 2.2 Light regulation in F. fujikuroi 2.2.1 Blue-light photoreceptors: White-collar complex and cryptochromes Fusarium genomes contain several genes involved in blue-light photoreception (Figure 3), among them orthologues for N. crassa wc-1 and wc-2, wcoA and wcoB. Study of wcoA gene in F. fujikuroi unveiled functional differences with its wc-1 counterpart. In N. crassa, wc-1 is strongly induced by light, while wcoA in F. fujikuroi did not show such photoinduction. Figure 3. Structure of photoreceptors and regulators involved in blue-light photoreception in F. fujikuroi and their chromophores. The White-collar proteins (WcoA, encoded by FFUJ_13691, and WcoB, encoded by FFUJ_00530) and Vivid (VvdA, encoded by FFUJ_06055) contain LOV domains where an FAD is predicted to bind covalently to a cysteine upon illumination (Castrillo and Avalos, 2014; Estrada and Avalos, 2008). The WcoA protein, as WC- 15 Introduction 1 in N. crassa, contains two PAS domains and a zinc-finger domain for DNA binding. The cryptochrome/photolyase proteins (CryD, encoded by FFUJ_05732, CryP, encoded by FFUJ_03105, and Phr, encoded by FFUJ_00436) contain a flavin and a MTHF (Castrillo et al., 2015). The sizes of the photoreceptors are indicated in amino acids. Adapted from (Pardo- Medina, 2021), modified from (Yu and Fischer, 2019). Additionally, induction of carotenoids biosynthesis by light is WCC-dependent in the mycelium of N. crassa (Yu and Fischer, 2019), whereas wcoA deletion mutants in F. fujikuroi and F. oxysporum (called wc1 in the latter) produce more carotenoids under continuous light than in the dark (Estrada and Avalos, 2008; Ruiz-Roldán et al., 2008). Another class of blue-light photoreceptors in Fusarium species are the proteins of the cryptochrome-photolyase family, which have two chromophores, FAD and MTHF, that allow to absorb radiation from the UV-A/blue spectral range. Fusarium genomes contain two cryptochromes genes: one coding for a plant-type cryptochrome (CryP) and another for a DASH cryptochrome (CryD) (Castrillo et al., 2015; Pardo-Medina et al., 2023), and transcription of cryP and cryD genes is induced by light through WcoA in F. fujikuroi (Pardo-Medina et al., 2021). An RNA-seq study has shown that the WcoA protein is a master regulator in F. fujikuroi strain FKMC1995. The wcoA mutation affects transcript levels of more than one thousand genes in this strain, and most transcriptional responses to light were lost in the mutant. This includes loss of photoinduction of most of the identified photoreceptor genes, including CryD and CryP cryptochromes, VvdA flavoprotein, CarO rhodopsin, and PhrA photolyase, supporting that WcoA plays a central role in Fusarium photobiology (Figure 4). Furthermore, WCC has been associated with roles in virulence in F. asiaticum and F. oxysporum, although it was not observed in F. graminearum (Tang et al., 2020; Ruiz- Roldán et al., 2008; Kim et al., 2015). In addition, a decrease in mycelium hydrophobicity was reported in wcoA mutants in F. fujikuroi and wc1 mutants of F. oxysporum (Estrada and Avalos, 2008; Ruiz-Roldán et al., 2008). Gene deletion experiments were extended to the wc2 gene in F. graminearum and F. asiaticum, observing the same mutant phenotypes than with the wc1 deletion, 22 Introduction 3.1 Major nitrogen regulators AreA/NIT-2 can be regulated in different ways, but a most conserved mechanism among different filamentous fungi is its inactivation by NmrA/NMR-1 (Figure 8) (Lamb et al., 2004; Xiao et al.,1995). NmrA/NMR-1 can bind directly to AreA/NIT-2, playing a repressor role in the presence of preferred nitrogen sources, blocking the ability of AreA to bind DNA and activate target genes (Kotaka et al., 2008; Pan et al., 1997; Xiao et al., 1995). Regulation of NmrA/NMR-1 is dependent on nitrogen availability through the control of its expression by the bZIP transcription factor MeaB. In addition, a posttranslational mechanism has been described in A. nidulans through protein degradation by proteases such as PnmB under nitrogen starvation (Li et al., 2021). However, no PnmB homologue was found in N. crassa, suggesting that this regulation may be specific to some filamentous fungi (Kerkaert and Huberman, 2023). Figure 8. Regulatory model of Nitrogen Catabolite Repression (NCR) in filamentous fungi. In A. nidulans and N. crassa, in the presence of a nitrogen preferred source, such as ammonium (NH4+) or glutamine (Gln), AreA/NIT-2 activity is repressed by NmrA/NMR-1 and the transcription of genes involved in transport and assimilation of non-preferred nitrogen source is repressed. AreA/NIT-2 activity could also be modulated by other mechanisms. In F. fujikuroi 23 Introduction Nmr1 (orthologue of NmrA/NMR-1) has a minor repressor effect in the regulation of nitrogen catabolite repression by AreA, thus other factors must be more relevant in modulating AreA activity in this fungus. Figure adapted from Kerkaert and Huberman, 2023. The role of NmrA has diverged between A. nidulans and F. fujikuroi. Although the NmrA homologue of in F. fujikuroi (Ffnmr) interacts with AreA and can complement N. crassa and A. nidulans nmr/nmrA mutants, NmrA plays a minor role repressing AreA activity in F. Fujikuroi and is expressed under nitrogen-limiting conditions (Mihlan et al. 2003; Schönig et al. 2008; Wagner et al., 2010), indicating that in this fungus Nmr is not a strong inhibitor of AreA as postulated in N. crassa and A. nidulans (Wong et al., 2007). AreA is also regulated by other mechanisms, such as increasing mRNA degradation in an RrmA-dependent manner under nitrogen availability, as observed in A. nidulans, or by changing AreA/NIT-2 localization in response to nitrogen starvation, which induced translocation to nucleus; or by swift exportation in response to nitrogen (Todd et al. 2005; Bernardes et al., 2017). In F. graminearum, three nuclear localization signals required for AreA nuclear localization, were identified, including a non-canonical arginine-based bipartite (NLS) sequence that is important for its nuclear localization, which is conserved in most fungi (Hou et al., 2015). The second relevant regulator is the GATA transcription factor AreB. In F. fujikuroi AreA and AreB regulate a large number of genes, not all of them dependent on nitrogen availability. About 80% of all nitrogen-regulated genes in F. fujikuroi were affected by one or both TFs (Pfannmüller et al., 2017b). The expression of the areB gene is regulated by AreA, and AreB can interact directly with AreA during nitrogen starvation (Michielse et al., 2014), but despite synergistic function, AreB can also regulate specific AreA-independent targets. AreB can play a positive or negative role and, unlike AreA, regulates some genes also under nitrogen-sufficient conditions (Pfannmüller et al., 2017b). In A. nidulans, AreB controls transcription factors involved in carbon and nitrogen metabolism, besides asexual development, and conidial germination (Chudzicka-Ormaniec et al., 2019; Wong et al., 2009). However, the AreB homologue in N. crassa, ASD-4, regulates sexual development but does not appear to play a role in nitrogen regulation (Feng et al., 2000). 24 Introduction Genes encoding catabolic enzymes involved in nitrogen assimilation are usually directly regulated by pathway-specific transcription factors, while those encoding nitrogen transporters are mainly regulated by NIT-2/AreA (Huberman et al., 2021). 3.2 Other nitrogen regulators The Zn(II)2Cys6-type transcription factor TamA was identified as a coactivator of AreA in the regulation of relevant nitrogen metabolic genes following ammonium deprivation in A. nidulans (Small et al., 1999; Small et al., 2001). However, its regulatory effect regulating on AreA, and therefore on nitrogen catabolite repression in this fungus, is minor (Kerkaert and Huberman, 2023). Recently, it was observed in T. reesei that the TamA orthologue, Tam1, plays a role in ammonium assimilation and contributes to promoting induced cellulase gene expression (Yang et al., 2023). However, this factor has not been studied in more detail in many fungi. An additional factor in nitrogen regulation in fungi is the cross-pathway transcription factor Cpc1, which mediates transcriptional upregulation of genes involved in amino acid metabolism under conditions of amino acid imbalance (Barthelmess and Kolanus, 1990; Tian et al., 2007). In A. nidulans Cpc1 may regulate prnB gene, regulated by AreA and encoding a proline-specific permease, because a binding sequence motif for Cpc1 was identified in the prnB promoter (Tazebay et al., 1997). In F. fujikuroi, cpc1 expression and Cpc1 accumulation in the nucleus is repressed by nitrogen starvation and induced by amino acids imbalance, either due to lack or excess of amino acids (Schönig et al., 2009). Apparently, cpc1 transcription is repressed under nitrogen starvation in an AreA-dependent manner. However, because the cpc1 promoter containing only one GATA box, it is unlikely that AreA can regulate it directly, because in several fungi it usually binds preferentially to double GATA elements (Marzluf, 1997), although the possibility of binding to a single GATA element cannot be excluded. The bZIP transcription factor MeaB has a repressor role on certain genes related to nitrogen metabolism, and it seems to be down-regulated by AreA in N. crassa, Fusarium spp., and Aspergillus spp. (Pan et al., 1997; Wong et al., 2007; López-Berges et al., 2010; 25 Introduction Wagner et al., 2010; Amaike et al., 2013). However, there are differences among species in this protein and its regulation. In F. fujikuroi, MeaB plays a moderate repressive role for some nitrogen-regulated AreA target genes, although insufficient to overcome the regulation by AreA. Notably, MeaB has an AreA-independent repressive role on the bikaverin cluster during nitrogen starvation (Wagner et al., 2010). In this fungus MeaB does not appear to participate in the same regulatory cascade as Nmr, and meaB deletion does not impact nmr expression (Wagner et al., 2010). In A. nidulans the weak impact on nmrA expression resulting from meaB deletion mutant (Wagner et al., 2010) contrasts with the MeaB-mediated induction of nmrA gene expression under nitrogen-sufficient conditions (Wong et al., 2007). However, it must be noted again the divergent Nmr functions observed between A. nidulans and F. fujikuroi, that would explain the low ability of MeaB to regulate NmrA in F. fujikuroi (Mihlan et al., 2003; Schönig et al., 2008). Furthermore, the postulated binding site for MeaB in the A. nidulans nmrA promoter is not conserved in the promoters of nmrA homologues in other fungal species (Wagner et al., 2010). In F. oxysporum, additionally to the reported repressor effect of MeaB and TOR (from Target of Rapamycin) kinase over some nitrogen-regulated genes (niaD, nitrate reductase; niiA, nitrite reductase; and mepB, ammonium permease), both play a repressive role on virulence-related genes, as evidenced by the derepression of cellophane penetration in the presence of ammonium, in meaB deletion mutants or by TOR kinase inhibition (López-Berges et al., 2010). A repressor effect of TOR on AreA-controlled genes, including those related to secondary metabolism, such as bikaverin and gibberellins, was also reported in F. fujikuroi. Furthermore, TOR influences the expression of a set of genes similar to those of S. cerevisiae and mammals, which include genes involved in transcriptional and translational regulation, ribosome biogenesis, carbon metabolism, and autophagy (Teichert et al., 2006). Nitrogen uptake and sensing by specific permease play crucial roles in nitrogen metabolism, which has been extensively studied in S. cerevisiae. Orthologues of some yeast nitrogen permeases have been characterized in F. fujikuroi, such as the ammonium permeases MepA, MepB, and MepC (Teichert et al., 2008), or the general amino acids transporter FfGap1 (Pfannmüller et al., 2015). While in S. cerevisiae Mep2 ammonium 26 Introduction permeases and Gap1 general amino acid permease have been proposed to play nitrogen signalling roles (Boeckstaens et al., 2007; Rutherford et al., 2008; Donaton et al., 2003), F. fujikuroi mepB gene restored signalling in a S. cerevisiae strain lacking the Mep2 permease, indicating a nitrogen sensing role also in this filamentous fungus, but only transporter roles have been attributed to the F. fujikuroi Gap1 orthologue, FfGap1 (Pfannmüller et al., 2015). In addition, there is evidence associating glutamine synthetase (GS) with a role in ammonium sensing and signal transduction, targeting NCR-responsive transcription factors and their subsequent target genes (Wagner et al., 2013). In contrast, no sensing role was attributed to GS of A. nidulans. Nitrogen also plays a significant role in the regulation of secondary metabolism, and several described regulators involved in nitrogen utilization have been associated to the control of secondary metabolite biosynthesis. Indeed, a genome-wide microarray analysis under high and low nitrogen conditions revealed that half of the secondary metabolite gene clusters in F. fujikuroi depend on nitrogen availability (Wiemann et al., 2013). This topic will be covered in more detail in following sections. 4 Secondary metabolism 4.1 Main secondary metabolites of F. fujikuroi Fusarium species can produce a wide variety of secondary metabolites. Although they are not essential, their persistence in evolution implies a competitive benefit in nature, and, depending on the environmental circumstances, they must be beneficial for fungal life (Avalos and Limón, 2022; Tudzynski, 2014). The numerous compounds identified usually belong to very diverse chemical families, mainly polyketides, terpenoids, nonribosomal peptides (NRPs), and hybrid non-ribosomal peptide/polyketides (NRP/PKs). Consequently, the F. fujikuroi genomes contain many genes encoding key enzymes for secondary metabolite biosynthesis, frequently organised as pathway-specific gene clusters, among which polyketide synthases (PKSs), non-ribosomal peptide synthetases (NRPSs), dimethylallyltryptophan synthases (DMATSs) and terpene cyclases (TCs) stand 27 Introduction out for their major role (Janevska and Tudzynski, 2018). Special mention deserves those producing the gibberellin phytohormones, responsible for the elongation symptoms of bakanae disease in rice. Figure 9. Gene clusters for representative secondary metabolites in F. fujikuroi. Coloured panels represent different chemical families: polyketides (PKs), non-ribosomal peptides (NRPs), hybrid polyketide-nonribosomal peptides, and terpenoids. Secondary metabolite 28 Introduction clusters are grouped according to their chemical family. Specific-cluster transcription factors are highlighted in pink and key pathway starting enzymes in blue. Genes that do not belong to the clusters are indicated in grey. Adapted from Janevska and Tudzynski, 2018. Due to this rich secondary metabolism, F. Fujikuroi has become a model for the production of secondary metabolites, including harmful mycotoxins, such as fusaric acid, moniliformin, fusarins, fumonisin, and beauvericin (Díaz-Sánchez et al., 2012; Niehaus et al., 2013, Niehaus et al., 2016a; Niehaus et al., 2014b; Rösler et al., 2016b), to pigments, such as carotenoids, bikaverin, and fusarubin (Avalos et al., 2012; Studt et al., 2012; Wiemann et al., 2009), whose biosynthetic clusters genes has been described (Figure 9). Many well-known mycotoxins serve as virulence factors in the development of plant diseases and are often toxic to humans and animals due to consumption of contaminated agricultural crop products (Stępień et al., 2018). A well-known example of toxins are fumonisins, whose biosynthetic gene cluster (fum) was first described in F. verticillioides (Brown et al., 2007), belonging to the F. fujikuroi species complex (Rösler et al., 2016b). The cluster contains 17 genes encoding biosynthetic enzymes, a transcription factor, and an ABC transporter, which provides self-protection by exporting the toxin from the cell and reducing its cellular concentration (Stępień et al., 2018). In contrast, other mycotoxins, such as fusaric acid, have phytotoxic properties but low toxicity to animals and humans (Janevska and Tudzynski, 2018). Genome sequencing of some F. fujikuroi strains led to the identification of many secondary metabolite gene clusters and linking them to their products (Brown et al., 2007; Chiara et al., 2015; Niehaus et al., 2016b; Wiemann et al., 2013), among them those for fusaric acid, fumonisins, fusarins, beauvericin, trichosetin, fusarubins, or apicidin F (Janevska and Tudzynski, 2018). Gene clusters for the biosynthesis of gibberellins, bikaverin, and carotenoids were identified and experimentally characterized before genome sequencing in 2013 (Janevska and Tudzynski, 2018). Gibberellins and carotenoids belong to the terpenoid family, derived from a fivecarbon (5-C) molecule, isopentenyl diphosphate (IPP), produced in the mevalonate pathway. The synthesis of all terpenoids begins with the fusion of IPP with dimethylallyl diphosphate (DMAPP), an isomer of IPP (Kellogg and Poulter, 1997), that results in 10-C 29 Introduction geranyl diphosphate (GPP), precursor of monoterpenoids. Further additions of IPP produce 15-C farnesyl diphosphate (FPP), the origin of sesquiterpenoids, and 20-C geranylgeranyl diphosphate, which gives rise to diterpenoids, such as gibberellins. The union of two FPP molecules gives rise to triterpenoids, and the union of two GGPP to tetraterpenoids, such as carotenoids. 4.2 Carotenoids Carotenoids are found in all major fungal groups, where they have different functions, although they have commonly been associated with a role as antioxidants (Avalos and Limón, 2015; Sandmann, 2022). However, unlike plants, carotenoids are not essential in fungi, and not all species can produce them. Carotenoid biosynthesis has been studied in detail in some model fungi, one of them F. fujikuroi. The main carotenoid product of this fungus is an unusual carboxylic xanthophyll called neurosporaxanthin (NX), whose name derives from its original discovery in Neurospora (Zalokar, 1957). NX shows a high antioxidant capacity in vitro (Parra-Rivero, Paes de Barros, et al., 2020), and a high bioavailability and provitamin A activity in mice (Miller et al., 2023), which gives this xanthophyll a considerable biotechnological interest as a potential food or feed additive. The carotenoid biosynthetic pathway in F. fujikuroi has been completely elucidated, and all genes involved have been described (Figure 10). Three of them are clustered in the genome (Figure 10.B): carRA, which encodes a bifunctional phytoene synthase/cyclase, carB, which encodes the desaturase, and carX, which encodes a β-carotene-cleaving oxygenase (Prado-Cabrero et al., 2007). The cluster also includes the carO gene, which encodes a proton-pumping rhodopsin using retinal as a chromophore (García-Martínez et al., 2015). Two key enzymes in the pathway are CarRA, which catalyses the condensation of two GGPP units to produce the first carotene molecule, phytoene, and CarB, responsible for the five desaturation steps of NX biosynthesis. CarRA is also responsible for the two cyclization steps, the first giving rise to β-zeacarotene, required to produce, through two desaturations, γ-carotene and the NX precursor torulene (Prado-Cabrero et al., 2009); the 30 Introduction second cyclization, achieved on γ-carotene, is optional and leads to a side branch to produce β-carotene. CarX oxygenase symmetrically cleaves β-carotene to produce retinal, which is used as a chromophore by rhodopsins. This provides functional meaning to car cluster coregulation: carRA, carB, and carX are required to produce retinal, and presumably used by the rhodopsin encoded by the coregulated carO gene (Avalos, Nordzieke, et al., 2017). Figure 10. Carotenoid biosynthesis pathway and genomic organization of car genes in Fusarium. A. Chemical modifications in each step are highlighted in the same colour as the gene for the enzyme involved. The dashed arrow summarises three sequential desaturations. The orange area corresponds to the chemical steps performed by the enzymes encoded in the car cluster. B. Genomic map of the four genes of the car cluster: carX, carRA, carB, and carO; and the genes not linked to the cluster: carT and carD. Gaps in the genes represent introns. Modified from (Ruger-Herreros et al., 2022). 31 Introduction Retinal could also be used for a second rhodopsin, encoded by the opsA gene. Retinal could also be a substrate for other enzymes. One suspected candidate is the enzyme encoded by gene carY, which has sequence similarity with RALDH enzymes that convert retinal to retinoic acid in mammals. This enzyme was investigated in F. verticillioides, and it was found to exhibit retinoic acid forming activity in vitro (Díaz- Sánchez et al., 2016). Mutation of the carY gene in F. verticillioides had no detectable effect on carotenogenesis but resulted in some developmental alterations. However, the biochemical function of the CarY enzyme in Fusarium remains uncertain because retinoic acid could not be detected in the wild-type strain (Díaz-Sánchez et al., 2016). The two genes located outside the cluster, carT, and carD, are specifically involved in NX production. Regarding the encoded enzymes, CarT is a torulene-cleaving dioxygenase (Prado-Cabrero, Estrada, et al., 2007) and CarD is an aldehyde dehydrogenase responsible for the formation of the carboxylic group of NX (Díaz-Sánchez et al., 2011). 4.3 Regulation of secondary metabolism 4.3.1 Carotenoid biosynthesis regulation Carotenoid biosynthesis in F. fujikuroi is modulated by environmental signals, with light as a major stimulatory factor (Avalos and Estrada, 2010). It is also affected by other environmental conditions such as nitrogen starvation, heat shock, and oxidative stress (Ruger-Herreros et al., 2022). Light induction of carotenoid biosynthesis of Fusarium was first described in F. aquaeductuum (Rau, 1980). In F. fujikuroi, transcription of car genes is induced by light, reaching maximal levels approximately 1 hour after illumination (Prado et al., 2004). This photoresponse is controlled by at least three photoreceptors, the white-collar protein WcoA, the DASH cryptochrome CryD, and the VVD-like protein VvdA (Castrillo and Avalos, 2015). According to the effect of the wcoA mutation on the transcriptome, analysed by RNA-seq in F. fujikuroi strain FKMC1995 (Pardo-Medina et al., 2021a), WcoA is a positive regulator of most car genes, as well as the ggs1 gene (Figure 11.A), responsible for the formation of 38 Introduction nitrogen, the mycotoxins fusaric acid and fusarins are only produced under nitrogen-rich conditions in F. fujikuroi (Studt and Tudzynski, 2014). Nitrogen starvation is essential for gibberellin, bikaverin, and fusarubin production and, as mentioned in the previous section, also induces carotenoid biosynthesis (Rodríguez-Ortiz et al., 2009; Ruger- Herreros et al., 2022; Studt and Tudzynski, 2014; Tudzynski, 2014). In F. fujikuroi, AreA mediates nitrogen starvation activation of gibberellin cluster genes by binding directly to their promoters to induce transcription. Thus, deletion of AreA results in repression of gibberellin biosynthesis (Mihlan et al., 2003; Tudzynski et al., 1999; Janevska and Tudzynski, 2018). Moreover, AreA and AreB are required for chromatin remodelling processes in the gibberellin gene cluster (Janevska and Tudzynski, 2018; Pfannmüller et al., 2017b). In addition, AreB is needed for the expression of apicidin F, fusaric C, and fusaric acid genes under high nitrogen conditions, as well as for gibberellins and fumonisins biosynthetic genes under low nitrogen conditions and has a repressor role on bikaverin biosynthesis (Michielse et al., 2014; Pfannmüller et al., 2017b). A model for nitrogen regulation of secondary metabolism has been proposed in F. fujikuroi, that includes the effects of nitrogen sensor proteins, such as Glutamine synthetase (GS), ammonium permease MepB, and TOR kinase, in an interplay with the GATA TFs AreA and AreB (Tudzynski, 2014). GS plays a regulatory role in nitrogen metabolism by regulating intracellular glutamine levels. It has been proposed that GS acts by sensing nitrogen status, required to modulate the response of nitrogen regulators (Tudzynski, 2014; Wagner et al., 2013). This hypothesis contradicts the effect of deletion of the gln1 gene (encoding GS), which results in a repression of the biosynthesis of gibberellins, bikaverin, fusarins, and apicidin F, even under nitrogen-limiting conditions (Niehaus et al., 2013; Niehaus et al., 2014a; Teichert et al., 2004), however, other evidence suggested that it is an indirect effect due to the impact of gln1 deletion, because of a less efficient glucose utilization and energy generation (Wagner et al., 2013). Furthermore, MepB, identified as the main ammonium permease in F. fujikuroi, has been attributed a regulatory role in the detection of the ammonium signal. Deletion of the mepB gene resulted in deregulation of certain nitrogen-repressed secondary metabolism genes, such as those for gibberellins and bikaverin, and was detrimental to mycelium growth (Teichert et al., 2008). The impact on secondary metabolism is a direct result of the regulatory role 39 Introduction of MepB and not just a consequence of the lack of ammonium intake, because overexpression of mepC, which encodes another ammonium transporter in F. fujikuroi, resulted in significantly improved growth due to ammonium incorporation but did not prevent the deregulated expression pattern of secondary metabolism genes. Other internal nitrogen sensors, such as those of the TOR signalling pathway, may also be involved in this regulation. In F. fujikuroi, inhibition of TOR kinase resulted in partial deregulation of gibberellin and bikaverin biosynthesis genes. However, TOR inhibition was not enough to suppress the inhibitory effect of high nitrogen availability on the production of these secondary metabolites (Teichert et al., 2006). The interconnections between these regulatory elements are potentially complex, and the molecular mechanisms and possible interactions between regulators are still poorly understood. Sge1 is another global regulator involved in Fusarium secondary metabolism (Janevska and Tudzynski, 2018). In F. fujikuroi Sge1 was found to be a positive regulator of several secondary metabolite cluster genes, such as those for gibberellins, fumonisins, fusaric acid, fusarins, and apicidin F (Michielse et al., 2015). The molecular mechanism by which FfSge1 exerts this regulation is currently unknown, but its permanent nuclear localization may be associated with a transcriptional regulator role, not necessarily directly on the secondary metabolite cluster genes. Deletion of Ffsge1 affected the expression of several histonemodifying enzyme genes in F. fujikuroi and other Fusarium species, including F. graminearum, F. oxysporum, and F. verticillioides, suggesting that FfSge1 may also play a global role in gene regulation at the chromatin level. Currently, it is unknown whether FfSge1 has DNA-binding capacity to regulate secondary metabolite genes, whether it affects the expression of these genes through histone modifications, or whether both mechanisms operate simultaneously (Janevska and Tudzynski, 2018; Michielse et al., 2015). In F. fujikuroi, the disruption of the velvet complex genes (vel1, vel2, and lae1) represses the biosynthesis of gibberellins, fumonisins, fusarins and fusaric acid, being considered that this complex is a positive regulator of these secondary metabolite clusters (Niehaus et al., 2014b; Wiemann et al., 2010). Lae1 and Vel1 also play a repressive role in bikaverin biosynthesis under high nitrogen conditions (Janevska and Tudzynski, 2018; Wiemann et al., 2010), and in PKS13, responsible for gibepyrone biosynthesis (Janevska 40 Introduction et al., 2016). In F. oxysporum, AreA plays a role in influencing the chromatin accessibility and expression of NRPS gene cluster regulated by Velvet, which is responsible for the biosynthesis of the mycotoxin beauvericin and the siderophore ferricrocin, and both regulators were essential for the expression of catabolic genes involved in nitrate assimilation, suggesting a interplay between Velvet complex and AreA in regulation of nitrogen utilization and secondary metabolism (López-Berges et al., 2014). Chromatin structure plays a crucial role in regulating the biosynthesis of secondary metabolites, and certain histone marks, such as those achieved by Kmt6 (H3K27me3) or GCN5 (H3K4, H3K9, H3K18, H3K27, and H3K36ac), appear to be very relevant in the expression of these genes (Atanasoff-Kardjalieff and Studt, 2022). Kmt6 is lysine methyltransferase, responsible for histone H3 lysine 27 tri-methylation (H3K27me3), a mark of repressive chromatin domains, which has been associated with secondary metabolism regulation in Fusarium species (Connolly et al., 2013; Studt et al., 2016b). Deletion of KMT6 gene had an impact on secondary metabolism in F. graminearum (Connolly et al., 2013). In F. fujikuroi, deletion of kmt6 gene was not possible, suggesting an essential role in this species, but generation of a kmt6 knockdown strain resulted in a reduction of H3K27me3 marks at a genome-wide level (Studt et al., 2016b). Reduced expression or deletion of this gene in both Fusarium species led to derepression of multiple secondary metabolite genes (Atanasoff-Kardjalieff and Studt, 2022; Janevska and Tudzynski, 2018). In F. fujikuroi Kmt6 may regulate the carotenoid gene carRA gene, other genes related to the biosynthesis of beauvericin, trichosetin, gibberellins, fujikurins and fumonisins, as well as several as yet uncharacterized secondary metabolite genes, such as those encoding various non-ribosomal peptide synthetase and sesquiterpene cyclase enzymes (Atanasoff-Kardjalieff and Studt, 2022; Studt et al., 2016b). The histone acetyltransferase (HAT) Gcn5, a member of the SAGA complex, is responsible for the acetylation of several H3 lysines in F. fujikuroi. Deletion of this gene has a considerable impact on the transcriptomes of F. fujikuroi, F. graminearum, and F. oxysporum, including many genes involved in primary and secondary metabolism (Atanasoff-Kardjalieff and Studt, 2022; Liu et al., 2022; Rösler et al., 2016a). In F. fujikuroi, this deletion deregulates about 61% of genes encoding secondary metabolite key enzymes, most of them downregulated in at least one condition (Rösler et al., 2016a). There are other many chromatin 41 Introduction remodelling proteins associated with secondary metabolism in Fusarium. However, further investigation is still needed to comprehend the interplay between histone marks and this regulatory process. (Atanasoff-Kardjalieff and Studt, 2022). 42 43 Aims The general purpose of this thesis is to deepen the knowledge of the regulatory mechanisms involved in carotenoids biosynthesis and its connections with other secondary metabolite pathways. In particular, four specific objectives were addressed: 1. To study the regulatory roles and the interdependence of the two white-collar proteins in secondary metabolism and light-inducible genes. 2. To analyse the effect of carS over-expression on the regulation of carotenoid biosynthesis. 3. To investigate the influence of light and CarS protein in the F. fujikuroi metabolome. 4. To determine the role of gene encoding a transcription factor with a zinc finger domain, linked to the car cluster. 44 45 Chapter I: Comparative analysis of wcoA and wcoB deletions in Fusarium fujikuroi: Insights into widespread functions as a complex 46 47 Chapter I Chapter I: Comparative analysis of wcoA and wcoB deletions in Fusarium fujikuroi: insights into widespread functions as a complex 1 Introduction Light is a source of information about the environment for organisms. In most investigated fungi, light influences the regulation of hundreds of genes and impact a wide array of biological processes, including sporulation and primary and secondary metabolic pathways (Yu and Fischer, 2019). The remarkable responsiveness of fungi to light is exemplified in A. nidulans, where exposure to light for only 15 minutes results in significant changes in the expression of over 1,100 genes, which represent approximately 10% of the genes in the genome (Yu et al., 2021). Similarly, the transcriptome of T. harzianum is highly sensitive to light, with 460 genes under the control of blue light (Cetz-Chel et al., 2016). In N. crassa, microarray data revealed that approximately 300 genes were regulated by light (Chen et al., 2009), and this number raised to approximately 500 genes in RNA-seq analysis; of the latter 58 corresponded to transcription factors (TFs) which represented 23% of the total number of TFs, 252, identified in this fungus (Wu et al., 2014; Sancar et al., 2015). Fungi have developed complex photosensory systems consisting of different photoreceptor proteins capable of sensing UV, blue/UV, green, or red light. The sophistication of these photosensory systems led to the conception of fungi as models to better understand light-dependent regulatory networks in eukaryotic cells. The whitecollar complex (WCC) was the first photoreceptor system studied in N. crassa (Harding and Turner, 1981), which is involved in most light responses in this fungus. This includes the clock system (Arpaia et al., 1993; Lee et al., 2000), whose study in this species contributed to understanding circadian rhythms in many organisms. In N. crassa about 41% of the TFs regulated by light are direct targets of the WCC (Smith et al., 2010). This complex is composed of a blue-photoreceptor WC-1, which contains a LOV (Light-Oxygen- Voltage) domain that binds to flavin chromophores, and the transcription factor WC-2; both proteins have a zinc finger domain to interact with DNA sequences. The flavin bound to the WC-1 LOV domain undergoes structural changes upon light detection, resulting in 54 Chapter I In the case of wco genes, wcoA showed photoinduction in the wild type, which was not observed in previous works with FKMC1995 (Estrada and Avalos 2008, Pardo-Medina et al, 2021), and it was lost in the wcoB mutants, consistent with autoregulation of the WCC. Nevertheless, no photoinduction was detected for the wcoB gene in the wild type, and the apparent photoinduction observed in the mutant lacking wcoA was not statistically significant. 2.2.2 Influence of wcoA and wcoB deletions on two key genes involved in HOG response and conidiation The hogA/hog1 gene, which encodes a high osmolarity glycerol (HOG) mitogenactivated protein kinase (MAPK), plays a central role in responding to osmotic and oxidative stress (Tang et al., 2023). This gene was found to be stimulated by light in F. fujikuroi FKMC1995 (García Martínez et al. 2014), a result that was confirmed by RNA-seq analysis (Figure CI.4.A). Figure CI.4. Expression analysis of hog1 and csp1 genes from RNA-seq data of F. fujikuroi FKMC1995. Relative mRNA levels of hog1 and csp1 genes in the wild-type and wcoA mutant strain of F. fujikuroi FKMC1995 grown for 3 days in 30 ºC in darkness and after 15, 60, or 240 minutes of light exposure. The graphs show mean and standard error of the mean of three independent experiments (data retrieved from original datasets of RNA-seq analyses described by Pardo-Medina et al. 2021). Correspondence of gene codes between F. fujikuroi strains FKMC1995 (FFC1) and IMI58289 (FFUJ) are FFC1_09974/FFUJ_04484 for hog1 gene and FFC1_01589/FFUJ_01494 for csp1 gene. 55 Chapter I Our results confirmed photoinduction of hogA/hog1 transcription also in the wildtype strain IMI58289 (Figure CI.5A), which was totally lost in the wcoA and wcoB mutants. However, the impaired photoinduction did not affect the ability of the mutants to grow under hyperosmotic conditions, as revealed by the wild-type aspect of their colonies on media with either 1.2 M sorbitol or 0.68 M NaCl (Figure CI.5.B). In fact, the growth of the mutants in 1.2 M sorbitol was even higher than the observed in the wild type. The lack of sensitivity of the wcoA and wcoB mutants to hyperosmotic conditions suggests that hog1 is efficiently activated by other regulatory signals by a WCC-independent mechanism, presumably the HOG MAP kinases pathway. Figure CI.5. Effect of wcoA and wcoB deletions on light-activated genes involved in conidiation and osmotic stress. A. Transcript levels of csp1 and hog1 genes after 3 days growth in DG medium at 30 °C, with or without one hour illumination, in the wild-type strain (WT), and wco mutants ΔwcoA SG300 and SG301, and ΔwcoB SG299 and SG297. B. Effect of 56 Chapter I osmotic stress on growth on agar of wild-type strain (WT) and wco mutants, ΔwcoA SG300 and SG301, and ΔwcoB SG299 and SG297. The strains were grown for 8 days on control conditions (DG solid medium), or DG supplemented with 1.2 M sorbitol or 0.68 M NaCl (hyperosmotic conditions). C. Colony diameter of strains cultured under the conditions described above. Bars show the mean and standard error of the mean of three independent experiments. A two-way ANOVA statistical analysis was performed on the data from the three biological replicates and asterisks correspond to the level of significance (* p < 0.05). The changes in conidia production in the wcoA mutants of F. fujikuroi FKMC1995 (Estrada and Avalos 2008) led us to focus our attention to regulatory genes predictably involved in conidiation. For this purpose, we chose the csp1 gene, found to be activated by light in the previous RNA-seq study in FKMC1995 (Figure CI.4.B). The gene csp1 is ortholog of the conidial separation csp-1 gene of N. crassa (Selitrennikoff et al. 1974), encoding a repressor also involved in other biological processes in this fungus, as azole resistance (Chen et al., 2016) or circadian cycle (Cemel et al.,2023). We confirmed a notable photoinduction of csp1 gene in the wild-type IMI58289 strain, which disappeared in both wco mutants (Figure CI.5.A). In contrast to hog1, the csp1 gene appears to be regulated by WCC also in the dark. Because of the influence of WcoA and WcoB on csp1 regulation, experiments were performed to test the effect of their mutations on conidiation. After several experimental replicates, differences were observed between the wild type and the mutants, but in some cases conidiation stimulation was found and in others suppression. The unreliability of these data prevents their representation since the results underwent strong variations even in the wild type. It is concluded that conidiation in these strains is influenced by variables difficult to control in our experiments. It should also be considered that the IMI58289 strain sporulates much less than the FKMC1995 strain, and that in the latter the data are much more reliable, which facilitated its study in a previous work (Estrada and Avalos, 2008). 57 Chapter I 2.2.3 Influence of wcoA and wcoB deletions on mycelial hydrophobicity Mycelial surface of the mutants of the wcoA gene in the FKMC1995 background were found to be less hydrophobic than those of the wild type, as judged by the ability to rapidly absorb water drops (Estrada and Avalos, 2008). So, this trait was also investigated in our wcoA and wcoB mutants. In this case, no differences were visible in the capacity to absorb water by mycelia of both kinds of mutants grown on minimal DG medium. However, assays carried out on mycelia grown on sporulation agar showed the opposite pattern: hydrophobicity was higher in the mutants compared to the wild type; so, while the mycelial surface of the wild-type strain rapidly absorbed the water droplets, in wcoA and wcoB mutants they persisted without being absorbed by the mycelium for at least 1 hour, suggesting a different regulation of the hydrophobins by WCC (Figure CI.6.B). It must be noted that, because to their higher sporulation capacity, the hydrophobicity of the wcoA mutant obtained from FKMC1995 was not checked in sporulation agar. Figure CI.6. Effect of wcoA and wcoB mutations on hydrophobicity of aerial mycelium of F. fujikuroi. A. Transcript levels of the hyd3 gene after growth for 3 days in DG medium at 30 °C, with or without one hour illumination, in the wild-type strain (WT), and in the wco mutants 58 Chapter I ΔwcoA, SG300 and SG301, and ΔwcoB, SG299 and SG297. Bars show the mean and standard error of the mean of three independent experiments. A two-way ANOVA statistical analysis was performed with data from the three biological replicates and no significance differences were determined. B. Pictures of colonies of wild-type and ΔwcoA and ΔwcoB mutant strains after 7 days of growth on EG medium under continuous illumination. Gentle addition of distilled water droplets to the mycelial surface revealed qualitative differences in hydrophobicity. Among the five hydrophobins described in Fusarium graminearum (Quarantin et al., 2019), hyd3 was the one more predominantly expressed in F. fujikuroi FKMC1995 in DG medium (Pardo-Medina et al., 2021a). Relative hyd3 mRNA levels were measured from samples grown for 3 days in DG medium in light and dark. Transcription of hyd3 was not affected by light and no significant differences were detected between wild type and wco mutants either after illumination or in the dark (Figure CI.6.A). Growth on sporulation medium is very faint compared to DG medium. Experiments with RNA from samples grown on sporulation agar were discarded because of the technical difficulties to obtain sufficient RNA for the analysis. 2.3 Functions of wcoA and wcoB genes in secondary metabolism To analyze the effect of wcoA and wcoB mutations on the expression of secondary metabolism genes, RT-PCR assays were carried out with RNA samples obtained from cultures grown in DG minimal medium in the dark or after 1 h illumination. We investigated key genes from six pathways whose expression was severely affected in the wcoA mutant obtained from the FKMC1995 strain, down-regulated in the cases of beauvericin (bea1), fusarin (fus1), fusaric acid (fub1), and gibberellins (DES), and upregulated in the cases of gibepyrone (gpy) and bikaverin (bik1). The results revealed in different cases regulatory alterations in the absence of WcoA or WcoB. Usually, the changes in the expression patterns were similar in both mutant strains, consistent with the participation of the WCC in the control of the synthesis of these secondary metabolites, although not necessarily as the main regulator. 59 Chapter I Figure CI.7. Expression analysis of some genes involved in secondary metabolism. B. Relative transcript levels of the biosynthetic genes bea1, fus1, gpy, DES, fub1 and bik1, involved in beauvericin, fusarin, gibepyrone, gibberellins, fusaric acid and bikaverin, respectively, and sge1 gene. Data of A and B graphs show mean and standard error of the mean of three independent experiments, analysed by quantitative RT-PCR. Wild-type (WT) and both mutant strains, ΔwcoA SG300 and SG301, and ΔwcoB SG299 and SG297, were cultured in DG medium in agitation for 3 days at 30 °C in the dark, after which they were incubated 4 hours in a Petri dish in the dark and exposed for 1 hour under illumination or kept in the dark. A two-way ANOVA statistical analysis was performed with data from the three biological replicates. Asterisks indicate the level of significance: *, p <0.05, **, p <0.01, ***, p < 0.001, ****, p <0.0001. The strongest effect, a 5-fold induction, was observed for bea1, whose mRNA levels were severely decreased in the four wco mutants (Figure CI.7). In contrast, the transcript levels of fus1, gpy, fub1, bik1 and DES, involved in fusarin, bikaverin and gibberellin productions, were barely affected by illumination (Figure CI.7.B). However, in the case of fus1, in overall, there was a clear decrease in mRNA levels irrespective of illumination, indicating again a light-independent regulatory role of the WCC in the control of this 60 Chapter I pathway. Whereas the data on bik1 mRNA levels revealed a high experimental variation, with a tendency in three of the four mutants to accumulate higher amounts of transcript for this gene. However, the strong oscillations in the mRNA data did not allow a reliable statistical analysis. Finally, transcript levels of the DES gene showed non-relevant variations in the light in the wco mutants but a noticeable reduction in the dark, indicating an unexpected dark-specific regulatory effect. The sge1 gene, which encodes a global regulator involved in the regulation of several secondary metabolite pathways (Michielse et al., 2015), was analysed in the same cultures. A noticeable decrease of sge1 transcripts was observed in the four wco mutants in DG medium in the light, but not in the dark (Figure 4), an effect that seems insufficient to explain the different alteration patterns in the SM gene transcript levels. 2.4 Effect of wcoA and wcoB deletions on secondary metabolism under nitrogen starvation Bikaverin and gibberellin biosynthesis in F. fujikuroi are up-regulated by nitrogen starvation (Rodríguez-Ortiz et al. 2009). Therefore, additional analyses were performed cultivating the strains in low-nitrogen 10% ICI medium for at least 7 days in light and dark conditions. Gibberellins do not absorb visible light, but bikaverin absorb light at about 500 nm, resulting in a reddish pigmentation. Interestingly, incubation of the wild type and the mutants under nitrogen starvation resulted in cultures of the four wco mutants with a deeper pigmentation than the wild type, very apparent in the filtrates which are shown below (Figure CI.8.A). As could be foreseen by the reddish colour of their filtrates, the cultures of the wco mutants produced more bikaverin than those of the wild type, either in darkness or under illumination (Figure CI.8). Considering that this fungus produces other pigmented metabolites, the absorption spectrum, and the behaviour on TLC were analysed to confirm the bikaverin identity using a bikaverin sample as a positive control. The maximal absorption peak observed for these pigments at 521 nm corresponded to bikaverin, and the pigmented band in the mutant run on the TLC at the same position that reference bikaverin (Figure CI.8.C). The concentration of bikaverin in the cultures of the mutants 61 Chapter I exhibited more variability in the dark than in the light between different experiments, but the four mutants coincided in the tendency for a higher bikaverin production in both culture conditions, indicating a repressor role for WCC (Figure CI.8.D). However, mRNA levels of the PKS gene bik1 were not very affected in the samples grown in the light (Figure CI.8.E). Figure CI.8. Extraction and quantification of bikaverin in cultures grown in low-nitrogen medium. A. Reddish pigmentation of filtrates of wco mutant cultures in low nitrogen medium. Filtrates of wild strain (WT), the wcoA mutants SG300 and SG301, and the wcoB mutants SG299 and SG297 grown in 10% ICI medium at 30 ºC for 7 days under continuous illumination. B. Key steps in the extraction of bikaverin from culture filtrates of wild-type and wco mutants, grown in low nitrogen 10% ICI medium for 7 days in the dark or under illumination. The samples were acidified by adding HCl, 1 volume of chloroform was added to each sample, mixed in vortex, and centrifuged to separate bikaverin from the remaining culture supernatant (right picture). Bikaverin, located at the bottom of the tube (chloroform phase), were collected for quantification. C. Thin-layer chromatography (TLC) of samples extracted from the filtrates of the wild-type (WT), ΔwcoA mutants SG300 and SG301, and ΔwcoB mutants SG299 and SG297. A bikaverin standard was used as a positive control (C). Mobile phase in the TLC consisted of chloroform:acetone:formic acid (84:15:1). The extracts were resuspended in chloroform before loading them in the TLC. D. Bikaverin concentrations in the filtrates of the cultures from the same strains under the culture conditions described in A. Data from the of three different experiments, each with two experimental quantification replicates. E. Transcript levels of bik1 gene determined by qRT-PCR from the wild-type strain 62 Chapter I (WT), the ΔwcoA mutants SG300 and SG301, and the ΔwcoB mutants, grown in low nitrogen 10% ICI medium at 30 °C for 7 days under continuous illumination. A one-way ANOVA statistical analysis was performed with data from three biological replicates and asterisks correspond to the level of significance (* p< 0.05, ** p<0.01, *** p<0.001). In the case of gibberellins, production by the wco mutants was similar to that of the wild type in low N medium, in the light nor in the dark, even with an extended incubation time of 14 days (Figure CI.9.A.) This indicates that the WCC does not play an important role in the regulation of gibberellin biosynthesis. Figure CI.9. Effect of light and nitrogen availability on DES expression and gibberellin production in ICI medium. A. Gibberellins in the filtrates of the cultures from wild-type strains and the four wco strains, cultured under the conditions described above for 7 days in dark or light conditions, or for 14 days in dark. A. Relative DES and sge1 gene transcript levels quantified by qRT-PCR from the wild-type and the four wco mutant strains cultivated in 10% 63 Chapter I ICI low nitrogen medium at 30 ºC for 7 days under continuous illumination. Data show mean and standard error of the mean of three independent experiments. A one-way ANOVA statistical analysis was performed on data from the three biological replicates and asterisks correspond to significance levels: * p< 0.05, ** p<0.01. However, a modest decrease in transcript levels of gene des, involved in a final desaturation step of the gibberellin biosynthetic pathway, was observed in wcoA and wcoB mutants under light (Figure CI.9.B). This is not explained by lower expression of the sge1 gene (Figure 6B), which was found to be required for induction of the gibberellin genes under nitrogen starvation (Michielse et al., 2015). In conclusion, our data revealed that WCC positively regulates the mRNA levels of the key genes for carotenoid, fusarins, and beauvericin biosynthesis, while it has only minor effects on key genes for bikaverin and gibberellin biosynthesis. In the case of bikaverin, a notable effect was observed, however, at the level of synthesis of these pigments, which was not mirrored by its effect on bik1 mRNA levels. In addition, the data showed regulatory roles for WCC in some of these secondary metabolite biosynthetic pathways also in the dark, suggesting a light-independent regulatory activity. 2.5 Retarded carotenoid biosynthesis in wco mutants In the case of the carotenoid pathway, as formerly described for WcoA, the mutants of both genes exhibited a notable loss of photoinduction in the structural genes carRA and carB (Figure CI.10), supporting an activator role of WCC in photoactivation of carotenoid biosynthetic genes. The down-regulation effect was also observed in the dark, as formerly described for the wcoA mutant in the FKMC1995 background (Pardo-Medina et al., 2021a). Here, the same effect was also exhibited by the wcoB mutant, indicating that the up-regulation of carotenogenesis in the dark is carried out by the WCC and not by WcoA alone. Although the wcoA and wcoB mutants exhibited a deficient transcriptional photoinduction of car genes in liquid medium, both mutants responded with a carotenoid 70 Chapter I regulation of sge1 under high nitrogen conditions in the light could be related to the observed down-regulation of the fus1 gene, which would be consistent with the positive regulator role of Sge1 in the fusarin cluster (Michielse et al., 2015). However, the repressor effect of light on sge1 expression fits with the lower mRNA levels of fusA (fus1) in FKMC1995 and with the lower fusarin production by this strain in the light, although at fusarin level this effect could be partially explained by the photosensitivity of these compounds (Díaz-Sánchez et al. 2012). In the case of bikaverin biosynthesis, the minor effect of wco mutations on bik1 mRNA levels in mycelia grown in sufficient nitrogen can be explained by the low expression of the bik cluster expected under these conditions, as it is strongly stimulated by nitrogen starvation. However, the effect of the wco mutations at low nitrogen conditions was also moderate, possibly due an adaptation of bik1 expression after the long incubation time, 7 days, considering that nitrogen is exhausted during the first three days (Candau et al., 1992). This is consistent with former evidence that bik1 (called before pks4) transcripts decay after the first 100 hours of culture incubation (Rodríguez-Ortiz et al., 2009). However, bikaverin biosynthesis is clearly up regulated in both wco mutants, strongly suggesting that this biosynthetic pathway is regulated by WCC in F. fujikuroi IMI58289. Concerning gibberellin biosynthesis, the wco mutants are barely affected in the production of these phytohormones. The DES gene, formerly termed ORF3, is subject to the characteristic induction under nitrogen starvation of the gibberellin pathway. So, its mRNA levels appear rapidly upon nitrogen depletion and are kept hight for at least 60 hours in the low-nitrogen 10% ICI medium (Mihlan et al., 2003; Tudzynski et al., 2003), but longer incubations times were not tested. Here we found that despite a tendency for slightly lower DES mRNA levels after seven days of growth in the light in the wco mutants, the gibberellin production was not affected, indicating a lack of role for WCC in gibberellin biosynthesis. However, a noticeable decrease of DES transcripts was quite patent in our standard minimal medium in the four wco mutants, in which because of its high N/C ratio no activation of the genes of the cluster is expected. Overall, we have found several instances where the production of secondary metabolites or the expression of their key genes is similarly affected in the wcoA and wcoB mutants of the IMI58289 background, pointing to WcoA and WcoB acting as a complex. 71 Chapter I This action is in some cases repressive, as in the case of bikaverin production, or stimulatory, as in the case of beauvericins and fusarins. A stimulating role is also played in carotenoid biosynthesis, which was investigated in this Thesis in deeper, and requires more detailed discussion. Role of Wco proteins in photocarotenogenesis Carotenogenesis is a preferred model in fungal photoresponse by visual detection of carotenoids. Two experimental approaches were used to investigate the role of the Wco proteins in the regulation of carotenoid biosynthesis by light. The first one was the experimental design used to investigate regulation by light in the short term, based in the illumination of young mycelia under submerged conditions. The results used with this approach confirmed previous data with FKMC1995, showing that the strong induction after one hour light exposure exhibited by the wild type is totally lost in the wcoA and wcoB mutants. On the other hand, it confirmed regulatory role in the dark, resulting in a ten-fold decrease of the carRA and carB mRNA levels. The novelty here is that both regulations, those of the dark and the fast induction by light, are mediated by the WCC, as indicates the same result obtained with the wcoA and wcoB mutants. It remains to be verified if such regulation is mediated by the direct binding of the WCC to the carRA and carB promoters, or if it is achieved through the activation/repression of other regulatory protein. The second experimental approach was used to investigate the kinetics of lightinduced carotenoid accumulation on agar cultures. Carotenoid production in F. fujikuroi is more efficient on surface than under submerged conditions, presumably because of the higher aeration. Thus, 24 h incubation in the light in the experimental set described above results in a 2-fold increase in carotenoid content (Ruger-Herreros et al., 2022) while in our experiments the mycelia grown on surface in the dark increased more than ten-fold their carotenoid content 24 hours after light onset. The use of these experimental conditions confirmed the slower accumulation of carotenoids upon illumination of the four wcoA and wcoB mutants, as indicated the lower carotenoid content after 6 hours. However, the four wco mutants exhibited a more sustained increase in carotenoid synthesis over longer 72 Chapter I periods of light exposure, even exceeding the carotenoid content of the wild type. This result differs from that obtained with the wcoA mutant in the FKMC1995 background, in which carotenoid levels remained much lower than those of the wild type during the same period of incubation under light in very similar culture conditions (Castrillo and Avalos, 2015). Even more unexpected was the result of the analysis of the mRNA levels of the pathway genes during the kinetics of carotenoid production. Contrary to what might be expected, the wco mutants contained more mRNA of the carRA and carB genes than the wild-type strain after 6 and 24 hours of illumination. It should be considered that in the wild type there must have been a strong increase in the amount of these transcripts during the first hour of light exposure, which must not have occurred in the wco mutants, since this increase is mediated by this photoreceptor system. Thus, the lower mRNA levels in the wild type are possibly due to the photoadaptation mechanism mediated by the vivid protein VvdA (Castrillo and Avalos 2014; 2015). However, given that this photoadaptation acts by overriding the WCC, we would not expect the transcript levels to be higher in the wco mutants, unless the wco system also activates an mRNA degradation mechanism to reinforce the photoadaptation, in which VvdA could be involved. It should be noted that photoinduction of the vvdA gene is mediated by the WC system itself, and therefore, a decrease in VvdA protein is also expected in the wco mutants. The behaviour of the transcripts of the carT and carD genes, involved in the late steps of the pathway, differed from those of the carRA and carB genes. In this case there were no important differences between the wco mutants and the wild-type strain, even more so considering the high experimental variability of the mRNA data, although there seems to be a tendency to increase their levels throughout the 48 h of exposure to light. It was previously known that light exerts a more moderate inducing effect on the transcript levels of the carT and carD genes, especially the latter (Avalos et al., 2017). Furthermore, these two genes are not found in the car cluster, with carRA, carB, carX, and carO, nor even in the same genomic region since they are located on other chromosomes of the F. fujikuroi genome. Indeed, the function of the cluster is to produce retinal and photoactive rhodopsin CarO, while carT and carD are specifically involved in the synthesis of neurosporaxanthin. For their part, the carRA and carB genes have overlapping functions 73 Chapter I with both processes, and this may be the reason why their expression levels are particularly high in light. The HPLC analyses on the carotenoids accumulated by the wild type and the wco mutants after 24 hours exposure to light revealed no relevant qualitative differences. At a first glimpse, it could be unexpected considering that in the wco mutants there is a strong increase in carRA and carB transcripts in the first 6 hours, and the values remain elevated until 48 hours, whereas the wild-type strain takes this time to equal these levels. In contrast, the transcripts of the carT and carD genes show values comparable to those of the wild-type strain during the whole time of exposure to light, so that their enzymes could be in lower proportion to CarRA and CarB in the wco mutants. According to the pathway, a relative increase in CarRA and CarB activity could result in higher torulene and β-carotene proportions in the mutants, but this was not observed. Neurosporaxanthin was equally predominant in all the strains tested, indicating that the oxygenase CarT and the aldehyde dehydrogenase CarD are not bottlenecks in the biosynthetic pathway. It must be taken also into account that γ-carotene is a crossroads in the pathway, as CarB desaturase and CarRA cyclase activities compete for this molecule as substrate to convert it to torulene or β-carotene, respectively. However, carB and carRA transcripts change in parallel, and so no differences are expected in this competition step. Finally, β-carotene content depends also on CarX activity. The carX gene has not been investigated in this thesis, but because of its location in the cluster it exhibits a similar expression pattern than carRA and carB (Avalos et al, 2017), and the expression of the three genes is expected to be encompassed, providing further support to the lack of changes in the accumulated carotenoid mixtures. The results in this chapter confirm the differences in regulation of carotenogenesis by light between F. fujikuroi and N. crassa. Referred to the regulation in the N. crassa mycelium, since in spores the pathway has a light-independent activation, induction of carotenoid biosynthesis (Degli-Innocenti and Russo, 1984) and transcription of structural genes (de Nadal and Posas, 2022) by light is completely lost in wc-1 and wc-2 mutants. However, in F. fujikuroi photoinduction persists in the absence of WCC, either at carotenoid or mRNA levels, although the induction patterns are in both cases affected. Thus, taking together the results of this chapter with former data of wc mutants in F. 74 Chapter I fujikuroi FKMC1995 and other Fusarium species (Estrada and Avalos, 2008; Kim et al., 2015; Pardo-Medina et al., 2021a, 2023b; Ruiz-Roldán et al., 2008; Tang et al., 2020), the available information unequivocally indicate the involvement of at least another photoreceptor in the activation by light of the carotenoid pathway. Kinetics studies of photocarotenogenesis in mutants of the photoreceptors WcoA, CryD and VvdA in FKMC1995 led to the postulation of cryptochrome CryD as the second photoreceptor (Castrillo and Avalos, 2015). However, according to our data for 1 hour illumination, and as formerly observed in FKMC1995 in light-induction kinetics up to 4 hours (Castrillo et al, 2013; Pardo-Medina et al., 2021), expression of cryD is strongly stimulated by light in a WC-dependent manner, and so the participation of other photoreceptor in addition to WcoA and CryD is not discarded. Early studies on the action spectrum of photocarotenogenesis in F. aqueductuum are consistent with the mediation of a flavoprotein as a photoreceptor (Rau, 1967). The second Fusarium cryptochrome, CryP, has not been investigated, but unpublished data from the group of B. Tudzynski (University of Münster, Germany) suggest a possible participation, and it will be object of future attention. Transcript levels of cryP gene are less strongly influenced by light, although also dependent on WcoA activity (Pardo-Medina et al., 2021). Finally, genomic analyses found two additional LOV-domain predicted flavoproteins, termed LovA and LovB (Pardo-Medina et al., 2023b), whose functions remain to be investigated. Therefore, the identification of the photoreceptors involved in photocarotenogenesis in Fusarium is a subject that is not yet closed. Role of Wco proteins in photosensory response and development A former study revealed that WcoA upregulates the expression of several genes for photoreceptors in F. fujikuroi FKMC1995 (Pardo-Medina et al, 2021), indicating that it plays a central role in light regulation. Here we have confirmed the total loss of photoinduction of the genes for the cryptochrome CryD and the rhodopsin OpsA both in the wcoA and wcoB mutants, suggesting that the control of other photoreceptors is also achieved by the WCC. Similarly, in Trichoderma ghizhouense the genes for the photoreceptors Cry1 and cry-DASH (cryptochromes), Env1 (Vivid) and Phr (photolyase), 75 Chapter I are induced by light, and such inductions are lost in the mutants of the wc gene blr1 (Y. Li et al., 2021). Our data show that mRNA levels of gene hog1 increase upon illumination, a result formerly observed in FKMC1995 (García-Martínez et al., 2014), and that induction requires both wcoA and wcoB proteins. Hog1 orthologues have been investigated in detail in other fungi, most especially in S. cerevisiae. This protein is well known as the MAP kinase effector of the high-osmolarity glycerol (HOG) pathway, which is activated under hyperosmotic stress to trigger a complex response, that includes the accumulation of glycerol as osmolyte to compensate the high external osmotic pressure (de Nadal and Posas, 2022), also linked to light. In A. nidulans, the hog1 gene (hogA/sakA) is not only activated by light, but also executes the transcriptional induction by light of many genes through the reception of the signal from the phytochrome FphA (Yu et al., 2016). The hog1 gene relationship with light has also been investigated in T. ghizhouense, whose hog1 mRNA levels increase significantly after light exposure in the wild type but not in the blr1 (wcoA orthologue) mutant. However, transcriptome analyses of the effect of blr1 or hog1 mutations on light regulation revealed very important roles, since in both mutants the expression of a large proportion of the light-regulated genes is altered. Therefore, as in Aspergillus, Hog1 is an important protein in the transmission from the light signal to the transcription of photoregulated genes. Since Hog1 is not a photoreceptor, it must receive the signal from a photoreceptor protein, which must include the Blr (Wco) system in this fungus. Given the phylogenetic proximity between the genera Trichoderma and Fusarium, it is likely that Hog1 plays a similar role in F. fujikuroi. This Hog1 function has been confirmed in F. asiaticum, in which its relationship with light and Wc1 has been studied, and the effect of hog1 mutation has been analyzed (Tang et al., 2023). The activation of Hog1 by the HOG pathway consists of its phosphorylation and entry into the nucleus to activate the transcription of target genes. In F. asiaticum, light significantly increases the presence of Hog1 in the nucleus, as do osmotic stress or oxidative stress. Consistently, light increases the degree of Hog1 phosphorylation, and expression of its gene is mediated by Wc1. The relationship of hog1 to oxidative stress in this fungus is also evidenced by the increased sensitivity to H2O2 exhibited by the hog1 mutant. Interestingly, hog1 mutants of F. asiaticum show less orange pigmentation in 76 Chapter I flasks exposed to light, and overexpression of hog1 results in increased pigmentation, although it has not been shown at the biochemical level the orange color to be due to carotenoids. The hog1 gene can be activated by more than one signal, including at least light through the WC system and osmotic stress (Tang et al., 2023) through the HOG MAP kinase cascade. Thus, in F. asiaticum hog1 photoactivation requires an active WC1 protein, but not its activation by osmotic stress. However, in F. fujikuroi, the presumed activation of hog1 by osmotic stress does not seem to affect carotenogenesis, as judged from the lack of pigmentation of wild-type strains under these conditions. So, Hog1 activation seems to be insufficient to stimulate carotenogenesis by itself. The different types of activation could result in different regulatory effects of Hog1 depending on the synergies with other regulatory proteins, which may explain the lack of effect of hyperosmotic conditions on carotenogenesis. The previous study of the wcoA mutation in F. fujikuroi FKMC1995 revealed effects in conidia production (Estrada and Avalos, 2008), either in liquid or in solid cultures. Many experiments with Fusarium typically start with the production of conidia for subsequent inoculation steps. It is well known to researchers who routinely obtain conidia of F. fujikuroi IMI58289 that this strain does not produce conidia in liquid cultures, and that conidial production on solid cultures is poorly reproducible no matter how much effort is made to maintain the same experimental conditions. An explanation for this variability is the possible influence of circadian rhythms on conidiation. In N. crassa, conidiation is also regulated by the circadian clock (Dunlap et al., 2007) and a similarly effect was observed in Trichoderma pleuroticola (Steyaert et al., 2010). Whatever the cause, this variability prevented us from obtaining reproducible results on the effect of wco mutations on conidiation, although disparate behaviour was always observed between wco mutants and the wild type. Other Fusarium species sporulate much more abundantly and are more prone to these studies. This is the case of F. graminearum, in which no difference was found between Δwc-1 and Δwc-2 mutants and wild-type strain in conidia production in liquid medium with carboxymethyl cellulose (CMC), in turn optimal conditions for such production of this species, but conidiation was strongly enhanced in the wc mutants on medium without CMC, but only in the light, indicating a light-mediated conidiation repressing role for the WC system in this fungus (Kim et al., 2014). 77 Chapter I The global transcriptome analysis found that several genes predictably involved in conidiation are affected by the wcoA mutation in F. fujikuroi FKMC1995 (Pardo-Medina et al., 2021). One of them was gene FFUJ_01494, ortholog of the N. crassa csp-1 gene, encoding a transcription factor required for conidial separation (Selitrennikoff et al., 1974) and whose transcription is activated by the WCC (Smith et al., 2010). However, the process of conidial formation in N. crassa has different characteristics than in Fusarium, since spores are formed by a strangling process of precursor hyphae, so the functions of its orthologue in Fusarium could be different. CSP-1 is not only involved in conidiation in N. crassa, since it plays an important role in the circadian metabolic regulation (Hurley et al., 2018), with 298 clock-controlled genes found to be repressed by this protein (Smith et al., 2010; Sancar et al., 2015). In Fusarium, only 30% of the 483 orthologous genes were affected by WcoA, whose mutation resulted in a down-regulation of csp-1 (Pardo-Medina et al., 2021). Consistently, we observed that the absence of WcoA or WcoB lead to a reduced accumulation of mRNA levels for csp-1 gene, indicating that WCC mediates its photoactivation in the F. fujikuroi IMI58289. However, it is uncertain whether this gene plays any function in the regulation of conidiation in F. fujikuroi. Expression analyses of the five hydrophobins genes encoded in Fusarium genomes show very different mRNA levels under laboratory culture conditions, and the patterns change between species, as observed in F. graminearum and F. asiaticum (Shin et al., 2022). Similarly, in F. fujikuroi FKMC1995 mRNA levels for hyd2, hyd4, and hyd5 were low, no mRNA could be detected for hyd1, and higher mRNA levels were detected for hyd3 (Pardo-Medina et al., 2021a). In accordance with the lower hydrophobicity shown by the wcoA mutants in this genetic background the amount of hyd3 mRNA was strongly reduced in these mutants. Another example of variability in hydrophobin regulation, no differences in mycelial hydrophobicity between wild type IMI58289 and wco mutants were observed in the conditions in which such differences were notorious in the FKMC1995 strain. And no differences in hyd3 mRNA levels were observed in the mutants. Hydrophobins production is associated not only with aerial development of hyphae, but also with conidiation (Cai et al., 2020), so it seems plausible that the differences in regulation observed between IMI58289 and FKMC1995 are associated with their different conidiation capacities in the culture media tested. In any case, the differences in 78 Chapter I hydrophobicity exhibited by the wcoA and wcoB mutants compared to the IMI58289 wildtype strain in sporulation medium confirm the participation of the WC system in hydrophobins regulation in Fusarium. Conidiation and hydrophobins are also regulated by Hog1. Conidiation of the Δhog1 mutant was reduced to 27% in F. graminearum (Zhang et al., 2023). The relationship of the HOG pathway with light regulation and the WC system opens new avenues of research in the study of the regulation of carotenogenesis by light and its connection with responses to different light-related environmental stresses. 79 Chapter II: Controlled transcription of regulator gene carS by Tet-on or by a strong promoter confirms its role as a repressor of carotenoid biosynthesis in Fusarium fujikuroi 86 Chapter II Figure CII.3. Luminescence emission of TETluc transformants induced with doxycycline (Dox) and compared to the wild strain: (A) The TETluc transformants (SG253 and SG255) and the wild strain (WT) were grown in DGpep medium for 16 hours in 96-well plates; then, 0.2 mM luciferin and 20 μg/ml of Dox were added to the wells and incubated in the dark at 30 °C for the time indicated. Data are average and standard deviation for two independent experiments. As a negative control, strains were grown without Dox. (B) Luminescence emitted by the TETluc transformant SG255 in DGpep medium in 96-well plates. Dox was added to get desired final concentrations (0, 2.5, 5, 10, and 20 μg/ml) after mycelia was grown for 18 hours. (C) Growth of SG255 in the 96-well plates described in panel B, measured by their absorbance at 600 nm. Data are average and standard deviations for three independent experiments. 87 Chapter II 2.2 Use of the Tet-on system to control carS expression As stated above, mutations of the carS gene provoke an overproduction of carotenoids in F. fujikuroi, indicating a role of CarS as a negative regulator of carotenogenesis in this fungus. To deepen in the knowledge on this role, carS expression was controlled using the Tet-on system. Figure CII.4. Molecular analysis of transformants TETcarS of wild F. fujikuroi: (A) Map of genome with integrated pPO5 plasmid in TETcarS candidate transformants. Green continuous 88 Chapter II line represents amplicon after PCR with primer set PC2.2-PC24. Primers are represented by arrowheads at the end of the lines. B. Electrophoresis after PCR amplification using primer set PC2.2-PC24 of genomic DNA from wild strain (WT) and transformant candidates: T1, T2, T3, T4, T5, T6, and T7. Those showing a 1.6-kb band were considered positives. Plasmid pPO5 was used as positive control. C. Genomic region of WT and transformants TETcarS with integration of plasmid pPO5. Enzyme restriction sites are indicated with triangles. Discontinuous lines represent DNA fragments after digestion with enzymes ScaI and EcoRV. Sizes are indicated below the lines. Code of colours: Red for EcoRV and blue for ScaI. D. Southern blot of WT and transformants TETcarS genomic DNA digested with EcoRV and ScaI and hybridized with a 1.4- kb probe amplified with primers PC2.1 and PC2.4. Transformant T1 is SG260. T5 is SG261. The wild strain and carS mutant SG39 were transformed with plasmid pPO5, derived from pVG3 (Marente et al., 2020). Four transformants obtained from the wild strain were confirmed to contain the construct by PCR, but only two, T1 and T5, showed the correct band pattern in Southern blot analysis (Figure CII.4). When these two strains were grown on DG agar medium with 20 μg/ml Dox, only T1, hereafter SG260, exhibited an albino phenotype in the presence of Dox in the light. On the other hand, three out of nine transformants derived from the carS mutant SG39 were found to contain the construct by PCR (Figure CII.5). Figure CII.5. Molecular analysis of transformants TETcarS derived from SG39 carS mutant. Map of genomic region with integrated pPO5 plasmid. Green continuous line represents the 89 Chapter II 1.6-kb amplicon expected in positive transformants after PCR amplification with primers rtTA- 1F and CarS-9R. Green arrowheads at the end of the line represent the primers; PCR results of the amplification of genomic DNA from SG39 mutant and transformant candidates T6, T7, T8, and T9. DNA from pPO5 plasmid was used as positive control. T6 was named SG262 and chosen as representative TETcarS transformant derived from SG39 for future analysis. As observed with SG260, the SG39-derived transformant T6, named SG262, showed an albino phenotype on DG agar with Dox addition (Figure CII.6.A). The effect of Dox addition on transcript levels of the carS gene was analyzed by qRT-PCR in SG260 and SG262 (Figure CII.6.B). As a result, higher levels of carS mRNAs were observed when cultured with Dox compared to their corresponding parental strains (wild strain and SG39, respectively). Figure CII.6. Control of expression of the carS gene using the Tet-on system in F. fujikuroi. A. Phenotypic characterization of TETcarS transformant (SG260 and SG262) and parental strains, wild type (WT) and SG39 (carS mutant), grown in DG agar medium (control) and in DG with 20 μg/ml Dox (20 Dox) at 30 °C for five days in the dark. B. Transcripts levels of the carS 90 Chapter II gene in the strains grown in minimal DG medium for 18 hours and induced with 20 μg/ml of Dox (20 Dox) or grown without Dox (control) and then incubated for up to three days in darkness. Values in the left graph refer to those of WT without Dox, and values in the right graph refer to those of SG39 without Dox. The qRT-PCR data are the average of two independent experiments and bars represent the standard deviation. * Indicates p < 0.05. The carS mRNA increased in SG260 in the absence of Dox, probably due to the basal expression of the Tet-on construct, which is added to that of the native carS gene. Unexpectedly, carS mRNA levels were particularly high in the SG39-derived transformant SG262 in the absence of Dox, and the strain had less pigmentation, suggesting that the increase in carS mRNA reduces the pigment synthesis. We conclude that the albino phenotype shown by SG262 in the presence of Dox is due to the enhanced levels of CarS protein under these conditions. 2.3 Effect of control of carS expression on carotenogenesis in the carS mutant Because of its high carotenoid content and the absence of a wild-type carS allele, the effect of controlling carS expression on carotenogenesis is more amenable to follow in the carS mutant SG39. For this reason, we analyzed the carotenoid content and the expression of two structural genes of carotenogenesis in mycelia of the carS mutant SG39 and its transformant SG262 grown in darkness. Because of their strong regulation by CarS, the carB gene, which encodes the enzyme phytoene desaturase, and the carRA gene, which encodes the cyclase in NX biosynthesis, were chosen to study the effect of the increase in carS mRNA. As expected, high carB and carRA transcript levels were found in the carS mutant SG39 regardless of Dox addition (Figure CII.7.A). Induction of carS mRNA by Dox in the TETcarS transformant SG262 caused a decrease in the expression of carB and carRA (Figure CII.6.A). Accordingly, the transformant SG262 did not produce carotenoids due to the strong activation of carS expression in the dark (Figure CII.7.B). The carotenoid content was, however, notably reduced in the transformant in the absence of Dox, probably due to basal carS expression in the Tet-on system (Figure CII.6.B) and to a local effect of the 91 Chapter II genomic integration. Although the decrease in carB expression did not significantly decrease in SG262 in comparison to SG39 in control media, carRA transcripts were reduced to half. The CarRA enzyme contains the phytoene synthase domain required for the synthesis of coloured carotenoids. A lower amount in this enzyme could also explain a reduction in the final NX product. Figure CII.7. Repression of the carotenoid biosynthesis pathway by carS overexpression in the carS mutant: A. Transcripts levels for the carB and carRA genes in the TETcarS transformant (SG262) and the carS mutant (SG39). Cultures of SG39 and SG262 grown for 18 h were induced with 20 μg/ml of Dox and then incubated for up to three days in darkness. The noninduced controls were grown for three days without Dox. Quantitative RT-PCR values represent average and standard deviations of two independent experiments. B. Carotenoid content from the same strains grown on DG agar medium with 20 μg/ml of Dox, and without Dox as control, for seven days in darkness. Mycelial samples were taken from three independent experiments. ** p < 0.01; *** p < 0.001; **** p < 0.0001. In contrast to the wild strain and the transformants SG260 and SG262, the carS mutant SG39 exhibited slower growth in the presence of 20 μg/ml Dox in the light, indicating that this strain is more stressed than the others under this condition. To check the sensitivity of the carS mutant, we investigated the effect of different Dox concentrations on SG39 growth in comparison to the other strains (Figure CII.8). The results confirmed the reduced growth of SG39 with 20 μg/ml Dox, also detected to a minor extent for SG262 at the same concentration, but hardly exhibited by the wild strain 92 Chapter II and its derived SG260 TETcarS-expressing transformant. However, no differences in growth were detected between the four strains at lower Dox concentrations. Figure CII.8. Growth and pigmentation of strains induced with a range of Dox concentrations. TETcarS transformants (SG260 and SG262), the wild strain (WT) and carS mutant (SG39) were grown on minimal DG agar medium with different Dox concentrations (0, 2.5, 5, 10, and 20 μg/ml) at 30 °C, for five days under light. The growth inhibition of SG39 and SG262 observed in Figure CII.8, in the presence of 20 μg/ml, suggests that these conditions are too stressful for these strains. For this reason, to use physiological conditions more similar to those of the control, new experiments were conducted, halving the amount of Dox, and adding it to 48 hours old cultures, which allowed induction to be carried out for 24 hours. When the TETcarS transformant SG262, the carS mutant SG39, and the wild strain were induced with 10 μg/ml Dox under these experimental conditions, there was no variation of carS transcript levels in the wild strain and SG39 in comparison to the control without Dox, while in SG262, there was an eightfold increase (Figure CII.8.A). However, the increase in carS mRNA in SG262 was only twofold when induced with 20 μg/ml Dox (Figure CII.6.B). As stated above, the structural genes carRA and carB were chosen as representatives of carS-regulated genes of carotenogenesis. Data were normalized to those of the wild strain in order to detect a 93 Chapter II presumptive downregulation caused by CarS. The results confirmed that the levels of mRNA of the carRA and carB genes in the carS mutant SG39 were not affected by 10 μg/ml of Dox in the medium. Figure CII.9. Effect of optimized Dox concentration on gene expression. A. Transcripts levels for the carS gene in the wild strain (WT), carS mutant SG39 and TETcarS transformant (SG262). B. Transcript levels for the mluc gene in TETluc transformants SG253 and SG255. Values refer to those of SG253 in the absence of Dox. C. Transcript levels for the carRA gene in WT, SG39, and SG262. D. Transcript levels for the carB gene in WT, SG39 and SG262. The strains were grown for two days in DG medium in darkness, then induced with 10 μg/ml of Dox (10 Dox) for 24 hours; noninduced cultures (control) were incubated for the same time. Data of qRT- PCR are the average and standard deviation of three independent experiments. Values were 94 Chapter II normalized to those of the noninduced wild strain, except for the mluc gene. * p < 0.05; ** p < 0.01; *** p < 0.001. However, they showed a strong downregulation of their mRNA levels in the SG262 strain with this Dox concentration, approximately 7-fold for carRA and 12-fold for carB (Figure CII.9.C and D), even more drastic that the 3-fold and 2-fold decrease found for the same genes with 20 μg/ml of the inducer in the same transformant (Figure CII.7.A). Experiments were also conducted with the TETluc transformants SG253 and SG255 with 10 μg/ml of Dox in the dark to study mluc expression. As expected, no mluc mRNA was detected in the wild strain under the same conditions, and the transformants showed a correlation between their mluc transcript levels (Figure CII.9.B) and their luminescence emission pattern (Figure CII.3.A), which explains the higher levels of luminescence emission detected in SG255. 2.4 Effect of constitutive carS overexpression on the carotenoid biosynthesis pathway Due to the culture limitations and the possible influence of Dox on the strains as a stressing agent, we used an alternative strategy to investigate the effect of carS overexpression. Plasmid pJM2 was constructed, containing the coding carS sequence under control of the A. nidulans PgpdA promoter and the amdS gene as selection marker. The wild strain was transformed with plasmid pJM2, and three transformants were analyzed. Two of them contained the PgpdA-carS construct, as confirmed by the results obtained from PCR amplification with appropriate primers and the correct bands observed in a Southern blot hybridization (Figure CII.10). 95 Chapter II Figure CII.10. Molecular analysis of OEcarS transformants of wild F. fujikuroi. A. PCR amplification from wild strain (WT) and transformants SG263, SG264 and SG271 using primers S1carS-1F and amiE-1R of gDNA. Plasmid pJM2 was used as positive control. Below, map showing pJM2 plasmid integrated in OEcarS transformants. Blue continuous line represents amplicon after PCR with primers S1carS-1F and amiE-1R. Primers are represented by arrowheads. B. Southern blot of WT and OEcarS transformants genomic DNA digested with ScaI or SacI and hybridized with a probe for carS gene. C. Genomic region of WT and transformants OEcarS with integration of plasmid pJM2. Enzyme restriction sites are indicated with triangles. Discontinuous lines represent DNA fragments after digestion with enzymes ScaI and SacI. Sizes are indicated below the lines. Code of colours: Blue for ScaI and red for SacI. Transformants SG263 and SG264 were studied in more detail in this work. Transformants SG263 and SG264 are albino either in darkness or under light (Figure CII.11.A), and no carotenoids could be detected in their mycelia (Figure CII.11.B). In accordance with this result, carB and carRA mRNA levels were practically indetectable in any of the transformants, and no induction by light could be observed (Figure CII.11.D). The amount of carS mRNA detected by qRT-PCR was higher in SG263 and SG264 (Figure 102 103 Chapter III Chapter III: Influence of light and CarS regulation in Fusarium fujikuroi metabolome 1 Introduction The filamentous fungus F. fujikuroi, a settled biotechnological organism for its great capacity to produce gibberellins (Rodríguez et al., 2012; Tudzynski, 2005), is the bestknown natural source of neurosporaxanthin, a carboxylic xanthophyll with a high antioxidant potential and promising applications for industry (Parra-Rivero et al., 2020). Recently, feeding experiments in mice have shown that neurosporaxanthin is readily incorporated in this mammal model, and it serves as a source of vitamin A (Miller et al., 2023). These observations have increased the interest in the study of neurosporaxanthin synthesis and its regulatory mechanisms in Fusarium. Light is a key environmental signal in the activation of carotenoid biosynthesis in Fusarium, a process tightly controlled by the negative regulator CarS (Avalos et al., 2017), encoded by the gene carS (FFUJ_08714). Non-functional CarS, resulting from carS mutant alleles holding mutations in conserved residues of the predicted protein (Rodríguez-Ortiz et al., 2013), leads to the overproduction of carotenoids under all culture conditions (Avalos and Cerdá-Olmedo, 1987). Neurosporaxanthin is also produced by Neurospora crassa, the fungus where it was initially discovered, but such mutants are unknown in this species, whose NX-producing capacity in submerged cultures is not as high as the production levels reached by the F. fujikuroi carS mutants (Luque et al., 2012; Youssar et al., 2005). CarS expression is subjected to a finely tuned regulation, in which is involved a long non-coding RNA (lncRNA) called carP, upstream to the carS gene and transcribed from the same strand. Deletion of carP (ΔcarP) results in albino strains, showing a deficiency in carotenoid production due to an increase in carS transcripts (Parra-Rivero et al., 2020). A similar phenotype was obtained by overexpressing carS (OEcarS strains) (Marente et al., 2021), highlighting the role of CarS in keeping carotenoid biosynthetic homeostasis. Former RNA-seq data revealed that the CarS protein affects directly or indirectly the expression of hundreds of genes, involved in a large diversity of cellular processes, many of them involved in primary metabolism (Ruger-Herreros et al., 2019). This work was done 104 Chapter III in parallel to the study of the effect of light, which led to find an important overlap on the genes affected by light and those affected by the carS mutation. So, many genes activated in the dark in the absence of a functional CarS protein were also activated by light. This suggested that at least a major function of CarS is to keep at low levels in the dark the expression of many genes that are stimulated by light (Ruger-Herreros et al., 2019). Carotenoids are a visually striking case of such dual regulation because they are visually tractable pigments, but genes for other metabolic processes are also controlled by this interconnected regulatory network, including genes for basal metabolism. Notably, a significant number of these genes, up-regulated by the carS mutation, were downregulated by carP deletion (Pardo-Medina et al., 2021b). A large variety of genes are affected by CarS, comprises primary metabolism and stress response. These fact highlights that the influence of CarS extends beyond its regulatory role in carotenoids biosynthesis, potentially engaging in a variety of biological processes. To delve deeper into understanding the impact of CarS on diverse cellular functions and identify regulatory connections with the light response, we used GC-MS and HPLC-PDA as metabolomic tools to analyze the metabolic profile of the wild-type strain and selected mutants involved in carotenoid regulation in F. fujikuroi on the basis of different CarS activities: carS mutants, carS overexpressing strains (OEcarS) and ΔcarP mutant. To understand its connection with the effect of light, as formerly done for the RNA-seq study, the effect of carS mutation was investigated in cultures of wild type and carS mutants grown in the dark or under illumination. Metabolomic tools has been previously employed to analyze the impact of mutations in different car genes in the fungus Phycomyces blakesleeanus. The identification of distinct metabolic profiles for each mutant type contributed to a more comprehensive understanding of the complex metabolic networks linking intermediary and secondary metabolism (Alcalde and Fraser, 2016). In this chapter, the analysis of metabolic changes in strains affected in carotenoid production allowed us to identify specific metabolic patterns associated with CarS availability and with illumination. The observed metabolic profiles revealed disparities among strains, some of them consistent with transcriptomic data, providing clues on the influence of CarS function on F. fujikuroi metabolism. 105 Chapter III 2 Results 2.1 Multivariate analysis of metabolic profiles of F. fujikuroi strains affected in carotenoid production To further investigate the effects of the carS mutation or its resulting carotenoid overaccumulation on other aspects of F. fujikuroi metabolism, cultures of the wild-type strain, the carS mutants SG22 and SG39, and the complemented strain SG256, obtained by reintroduction of the wild-type carS allele into the SG39 mutant, were grown in submerged conditions in the dark or under illumination and their metabolomic profiles were analysed. In addition, as non-carotenoid-producing controls, the strain with overexpression of the carS gene SG263 (Marente et al., 2021) and the mutant lacking the carP lncRNA SG268 (Parra-Rivero, Pardo-Medina, et al., 2020b) were included in the analysis under illumination. These latter two strains have a very low carotenoid content because they have a higher expression of the carS gene for different reasons. A total of 314 metabolites were detected by GC-MS analysis in the investigated strains, of which the chemical identity of 259 were characterized and 55 compounds could not be determined (marked as unknown), and they were discarded for the multivariate analysis (Annex, Table S3.1). Out of the 259 metabolites identified, specific identities were not determined for 10 disaccharides, 9 hexoses, 20 hydrocarbons, 18 sugars, 3 sterols, and 2 steroids. Principal components analysis (PCA) was performed to reduce the dimensionality of data and find significant patterns among strains. The first two principal components (PC1 and PC2) account for 45.08 % of the total variance in the dataset and revealed a clear tendency to correlate the metabolomic profiles of strains with their distinct carotenoid phenotypes or illumination conditions (Figure CIII.1). Notably, PCA values exhibited significant disparities between strains exposed to dark conditions and those exposed to light, particularly in the PC1. Strains in darkness clustered close together, implying a higher degree of metabolic similarity between them, whereas strains in light were more separated, suggesting that light plays a significant role in modulating their metabolic profiles. 106 Chapter III Figure CIII.4. Principal component analysis (PCA) score plot from GC-MS data for strains wild-type, carS mutants SG22 and SG39, complemented carS+ mutant SG256, ΔcarP SG268 and OEcarS SG263 strains, in darkness or exposed to light. Coloured ellipses comprise closed groups that correlate with similar phenotype in light (carotenoid overproducer, wild-type or/and albino phenotype) or indicates the group of strains grown in dark. The PCA was performed with the mean of data from three independent biological replicates. It is worth to mention that PCA values from both carS mutants in light were in the same quadrant, whose proximity along PC2 indicates a degree of metabolic similarity between them in that condition. This similarity may be attributed to the loss of carS functions, and the divergences could be due either to specific allelic effects, since each strain has different mutations (Rodríguez-Ortiz et al., 2013), or to mutations on other locations in the genome. However, the values in the principal components observed in SG263 in light differ from those of the other strains under the same condition, particularly that observed in the two carS mutants. SG263 clusters together with the strains in darkness, despite being in a different quadrant, suggesting that SG263 exhibits a unique metabolic profile in response to light compared to the other strains, and an opposite metabolic profile to the carS mutants, suggesting a role or effect of CarS in the regulation of metabolism. These observations are based on the PCA analysis, but a nonparametric Kruskal-Wallis ANOVA test performed on the GC-MS data from all strains did not yield 107 Chapter III significant results. However, the reliability of the PCA results is supported by correlations with genotypes and carotenoid phenotypes, because SG263 overexpresses carS and cannot accumulate carotenoids under any environmental conditions, whereas SG22 and SG39 mutant strains express a non-functional CarS protein with respect to the regulation of carotenogenesis, that result in an overproduction of carotenoids in all culture conditions. The distribution of strains observed in the PCA has provided a first insight into the results. Possible correlations between strains and illumination conditions are studied in more detail in the following sections, with special attention to statistical significance. 2.2 Identification of metabolic changes in response to carS expression and light in F. fujikuroi The complete GC-MS datasets were represented in a hierarchical heatmap to get an overview of the data structure, and to get insight into the clustering patterns between strains and their interconnections under light or dark conditions. Figure CIII.5. Hierarchical clustering heatmap showing metabolic changes determined by GC-MS analysis among the strains in light and in the dark conditions. The GC-MS data were normalized using unit variance scaling to highlight similarities or differences between strains. 108 Chapter III An increase in numerous metabolites was observed in light relative to dark conditions among the strains, except for OEcarS strain, which shared the similar metabolic profile of the strains in the dark (Figure CIII.2). Although OEcarS and ΔcarP have a similar albino phenotype, their metabolic profiles did not closely align in this hierarchical clustering heatmap, and ΔcarP clustered with the wild type and SG256. Both carS mutants, SG22 and SG39, clustered together in light conditions, although these similarities were not as pronounced in the dark. The prominence of metabolic differences among strains under light conditions underlines the importance of light as a regulatory factor, corroborating transcriptomic results related to the regulatory role of light in F. fujikuroi (Ruger-Herreros et al., 2019; Pardo-Medina et al., 2021a). It also highlights the negative impact of CarS in modulating this signal, supported by the association of the OEcarS strain with the metabolic profile of strains grown in the dark. Although the Kruskal-Wallis ANOVA test resulted insignificant, we performed a multiple t-test to compare strains in dark and light conditions, at the sensitivity of this test to even minor differences (Table III.1). Table CIII.1. Significantly different metabolites in at least one of the strains in light conditions in comparison to the dark. Data of wild-type (WT), carS mutants SG22 and SG39, and complemented carS SG256 strains were obtained from GC-MS analysis. Each value represents the mean of three biological experiments. Values for each strain for which the difference between light and dark was statistically significant are indicated in bold (multiple comparisons test, with Benjamini-Hochberg-Yekutieli correction test): A q < 0.05; B q < 0.005, C q < 0.0005, D q < 0.00005). Dark Light C-carS- carS- carS- C-carS- carS- carS- Metabolites WT SG256 SG39 SG22 WT SG256 SG39 SG22 Aminoacids L-Threonine 0.084 0.14 0.0001 0.0001 B 0.23 0.21 0.0001 0.0001 L-Phenylalanine 0.0001 0.072 0.0001 0.0001 0.0001 A 0.0001 0.0001 0.0001 L-aspartic acid/asparagine 0.00009 0.2 0.0001 0.0001 A 0.19 0.0001 0.0001 0.3 Serine 0.57 0.17 0.14 0.0001 0.0001 0.19 0.23 D 0.17 109 Chapter III Note: -P: phosphate group The low number of differences observed explains why the result of the Kruskal- Wallis ANOVA test was not significant, and the few significant metabolites were visualized in a heatmap (Figure CIII.3). Certain metabolites accumulated in higher amounts consistently in all strains in the light, suggesting a light-induced response. These included two diterpenoids, the fatty acid palmitic acid, and the amino acid phosphoethanolamine. However, the accumulation of certain sugars varied among strains, and both carS mutants exhibited higher levels of Lyxose iso2 and hexose-phosphate-23.96 minutes in light, whereas Hexose 1_iso1 and Hexose-phosphate-23.96 minutes were increased only in SG22 and SG39, respectively, under dark conditions. Sugars Hexose -P - 26.96 min 0.0001 0.376 0.52 0.0001 0.0001 B 0.0001 0.347 0.32 Hexose iso1 0.34 0.099 0.42 6.43 0.18 A 0.26 0.41 0.43 Xylose iso2 0.0001 0.0001 0.0001 0.0001 0.0001 C 1.067 0.0001 0.0001 Lyxose iso1 0.00009 0.0001 5.6 6.3 A 1.3 0.0001 5.9 4.7 Lyxose iso2 0.6 0.8 0.0001 0.0001 C 0.0001 1.02 0.0001 0.00009 Melibiose 0.0001 0.0001 0.14 0.0001 0.0001 A 0.15 0.0001 0.0001 Terpenoids Diterpenoid 1 0.000004 0.000004 0.000004 0.000004 B 0.005 0.000004 0.006 0.006 Diterpenoid 2 0.000004 0.000004 0.000004 0.000004 A 0.0032 0.000004 0.004873 0.004 Steroid 24.23 min 0.000004 0.000004 0.000004 0.000004 0.01 0.01 0.02 0.03 Hydrocarbons Hydrocarbon 20.14 min 0.006 0.000004 0.007 0.000004 A 0.000004 0.006 0.000004 0.000004 Eicosene 0.006 0.008 0.007 0.006 A 0.009 0.007 0.008 0.006 Fatty acids Palmitic acid 0.000004 0.000004 0.000004 0.000004 D 1.22 1 1.32 D 1.48 Lipids Phosphoethanolamine 0.0001 0.0001 0.0001 0.0001 D 0.037 0.041 0.032 0.062 110 Chapter III Figure CIII.6. Hierarchical cluster analysis and heatmap showing significant differences among GC-MS metabolites of strains in the dark or exposed to light. Statistically significant metabolites determined by GC-MS analysis in the wild type (WT), carS mutants SG22 and SG39, and complemented carS+ SG256 strain under dark and light conditions. Data were normalised by rows by dividing each value by the average of the row. Each value comprises the mean of three biological experiments. Multiple t-tests with the Benjamini-Hochberg- Yekutieli correction test were performed to identify significant metabolites. 2.3 Effect of CarS in the F. fujikuroi metabolic profiling To compare the pairs of strains that were distinguished in the PCA under light conditions and delve in detail into the correlation of carotenoids phenotypes with the metabolic profiles of the strains in light, the carotenoid contents of the samples grown in the light were determined. Since the strains investigated differ in the availability of CarS, the carotenoid content can be taken as an indication of the activity of this regulatory protein. As expected, carotenoid levels were high in the carS mutants SG22 and SG39, low in the wild-type and in the carS+ complemented strain, and undetectable in the strains with higher levels of CarS activity, such as the carS overexpressing strain SG263 or the mutant lacking its carP regulatory lncRNA, SG268 (Figure CIII.4). 111 Chapter III Figure CIII.4. Carotenoids accumulated in the light in the investigated strains under the conditions used for the metabolome study. A. Total amount of carotenoids determined by HPLC from the wild type (WT), carS mutants SG22 and SG39, complemented carS+ SG256, ΔcarP SG268, and OEcarS SG263 strains, cultured for 3 days at 30 ºC and exposed to continuous light. B. Amounts of individual carotenoids in each strain in the samples described in A. The extracted metabolites were analysed by HPLC and quantified related to a β-carotene 118 Chapter III 3 Discussion Metabolomic analysis is an additional powerful tool to understand the regulatory roles of CarS, mainly in intermediary metabolism, and constitutes a complement to the previously published transcriptomic results aimed at studying the influence of light and carS mutation in F. fujikuroi (Ruger-Herreros et al., 2019; Pardo-Medina et al., 2021a). In this chapter, differences in the metabolome were observed that can be attributed to the absence of CarS activity. However, most of these differences were observed under light conditions, reinforcing previous conclusions associating CarS functions to regulation by light. The metabolome is the result of multiple biochemical and physiological interactions mediated by many hundreds of enzymes and governed by a large diversity of regulatory proteins and, therefore, the effects caused by a single regulatory mutation are not expected to be very noticeable. On the other hand, light is not expected to be an environmental agent that substantially alters the function of primary metabolism. It is not surprising, therefore, the number of metabolites showing statistically significant changes between the different strains and conditions investigated is rather small. Consequently, the metabolome appears to be less affected by light and the carS mutation compared to the transcriptome. In the case of the carS mutation, although the metabolites for which differences have been found reveal effects on intermediary metabolism by CarS, the reasons for these changes are uncertain. Thus, metabolomic effects might be a consequence on the regulatory functions of CarS on other proteins or might be a secondary outcome of variations in energy requirements associated with the notable change in carotenoid production. However, metabolic networks and interactions between metabolites can lead to compensatory responses, in which changes in one metabolite may be balanced by changes in others, although in many cases the changes are minor and therefore not detected as significantly different. It should be kept in mind that the margins of change in metabolism may often be small, and that biological significance is not always reflected in statistical significance. In other words, the high requirements for statistical tests to 119 Chapter III highlight significant differences may not always be within the ranges between which the concentrations of many metabolites may oscillate as a function of regulatory alterations. In the case of the data for the carS mutants, the study's robustness is enhanced by the comparison of two independent carS strains. The correlation between specific metabolic patterns and their phenotypic characteristics, determined by hierarchical clustering heatmap analysis of whole GC-MS data, must have a biological sense based in the shared features of the carS strains. Both mutants come from mutagenic treatments and therefore have different random mutations, but they have in common critical mutations in the carS gene resulting in carotenoid overproduction, and the coincidence of the metabolomic effects support the biological validity of the conclusions. Interestingly, the metabolic differences detected in light depend on CarS function. Furthermore, the influence of light on the metabolome is emphasised by the clustering in the principal components analysis of samples from strains grown in darkness, irrespective of carS expression, while the samples spread out from this cluster when the strains are illuminated, with differences depending on the presence of CarS function. It is noteworthy that some metabolites, as phosphoethanolamine, palmitic acid, and steroid 24.23 min, are produced exclusively under light conditions. Since these compounds were not detected in the dark even in the carS mutants, their accumulation is not triggered by carotenoid overaccumulation or lack of CarS activity. However, albino strains do not produce them in the light. So, both lack of CarS function and illumination are required for their synthesis, or for them not to be degraded, suggesting the participation of another regulatory protein controlled by CarS and light. Interestingly, these three metabolites are lipids. In filamentous fungi, plasma membranes interact with cell wall components, a connection that is particularly important in the coupling of both cellular structures during hyphal apical growth (Takeshita et al., 2014). In this interplay membrane lipids may play specific roles. Thus, specific components of cell plasma membrane lipids, such as sphingolipids, ergosterol, and phospholipids, participate in signalling pathways that regulate the integrity of fungal cell walls (Fabri et al., 2020). In S. cerevisiae the metabolite phosphoethanolamine is an intermediary of the Kennedy pathway to produce the phospholipid phosphatidylethanolamine (PE). Phospholipids are major structural components of cell membranes, whose structure is 120 Chapter III based on a glycerol-3-phosphate backbone in which fatty acyl groups are esterified, including palmitic acid as one of the most common esterified fatty acids (Carman and Han, 2011). The increase of both lipids specifically under light conditions in the wild type, carS+ complemented strain, and both carS mutants suggest that light plays a role in the CarS- independent regulation of lipid metabolism. Carotenoids are hydrophobic molecules that are assumed to be integrated into cell membranes, where they may protect against oxidation of cellular components. The increased presence of carotenoids in the cell membranes in the carS mutants could influence this regulation, thus explaining the absence of these metabolites in albino strains under light conditions. The presence of carotenoids is not sufficient, however, to explain these changes, as in darkness, carS mutants also accumulate carotenoids, but not these metabolites. A tentative hypothesis is that the quenching capacity of carotenoids could result in a change in the formation of a regulatory signal, formed in the membranes only in the light. A suggestive example is provided by the PPIX-1O2 signalling pathway in the bacteria Myxococcus xanthus, in which excitation of PPIX (Protoporphyrin IX) by light results in energy transfer to molecular O2 to generates the highly reactive 1O2, which interacts with a sensor molecule to trigger a response (Padmanabhan et al., 2021). Interestingly, neurosporaxanthin proved to be highly efficient in the protection of cell membranes against oxidation (Parra-Rivero, Paes de Barros, et al., 2020). A possible regulatory scenario in which a light-triggered oxidative radical is used as a signal, and whose formation is altered in the presence of an unusually high carotenoid content, including the particularly active neurosporaxanthin, cannot be ruled out, and could be worth of future investigation. Carotenoid overproduction may have other indirect effects on the metabolome. Fatty acid degradation contributes to the acetyl-CoA production, a precursor of the mevalonate pathway, essential to produce terpenoids. Thus, the presumptive decrease in the acetyl-CoA pool resulting from the higher demand for carotenoid production might result in a higher fatty acid degradation, motivated by the need of this precursor for other more essential metabolic pathways, e.g., the formation of sterols. A non-identified fatty acid, called in this study fatty acid – 19.33 min, increased only in albino strains. Because carotenoids and other terpenoids share common precursors, it is also consistent with the observed accumulation of two terpenoids only in the strains able to produce carotenoids, being higher in carS mutants, which overproduce carotenoids. 121 Chapter III The effect of carotenoid overproduction on the metabolome was previously investigated in the zygomycete P. blakesleeanus (Alcalde and Fraser, 2016). As in F. fujikuroi, alterations in carotenoid biosynthesis also resulted in alterations in intermediary metabolism. Thus, the carotene overproducers of P. blakesleeanus exhibited changes in other terpenoids, as well as in some sugars, amino acids, fatty acids, and TCA intermediates. However, the specific changes were different in both fungi. Some fatty acids and membrane precursors were also altered among the different P. blakesleeanus carotene mutants, but there was no direct correlation between the qualitative and quantitative levels of carotenes and the lipid/fatty acid content. Furthermore, the carotenoid overproducers in F. fujikuroi (carS mutants) accumulated higher levels of citric acid and higher number of sugars, and differences are also observed on the effects on some amino acids, as leucine, valine, isoleucine, and alanine that did not change in P. blakesleeanus mutants. The discrepancies in metabolomic effects between the two fungi are not unexpected. In addition to the considerable taxonomic distance between ascomycetes and zygomycetes, the biochemical reasons for carotenoid overproduction are very different in both fungi. P. blakesleeanus has a simpler carotene pathway, leading to the synthesis of β-carotene (Avalos, Pardo-Medina, et al., 2017b), but this does not accumulate as an end-product, β-carotene is the substrate for the sequential action of two carotene oxygenases that give rise to apocarotenoids, which are used as the source for trisporoids, a known family of derivatives (Medina et al., 2011). For this reason, structural gene mutants of the carotene pathway of P. blakesleeanus, such as those affected in the carB, carR (R domain of gene carRA), or carS genes accumulate large amounts of the corresponding enzyme substrate, phytoene, lycopene, or β-carotene, respectively (Salgado et al., 1991). So, these are not actually regulatory mutations and, unlike the carS gene in Fusarium, the responsible P. blakesleeanus genes, including carS (Tagua et al., 2012), are not involved in the regulation of other cellular processes. In addition, the P. blakesleeanus mutants lack apocarotenoids and trisporoids, with unknown consequences in the metabolome. The increases found in some sugars in strains lacking carS function, and the opposite effect observed in albino strains, are consistent with previous transcriptome data, which revealed roles of CarS on genes related to carbohydrate metabolism. Several 122 Chapter III carbohydrate metabolism-related genes were up-regulated by the carS mutation and down-regulated by the carP mutation, presumably because of carS overexpression. This included genes FFUJ_07515, FFUJ_10506 and FFUJ_06543, related to arabinose 5- phosphate isomerase (involved in the pentose phosphate pathway), ketoreductase and pseudomonas L-fucose dehydrogenase respectively; last one, with functions prediction of D-arabinose 1-dehydrogenase (NAD(+)), D-threo-aldose 1-dehydrogenase, and L- galactose 1-dehydrogenase. Under limited carbohydrate/sugar supply, fungi can use alternative carbon sources, such as amino acids, lipids, or organic acids, through the tricarboxylic acid (TCA) cycle to produce sugars (Suzuki et al., 2012). Citric acid is also increased in carS mutants, and the accumulation of this metabolite not only provides energy, but also regulates glycolysis (Nakamura et al., 2021). The increased levels of sugars and citric acid in carS mutants, compared to those in albino strains, could support that these metabolic changes are the result of different energy demands, increased in both carS mutants to keep the higher carotenoid production. The most interesting case is L-proline, as it specifically increases in albino strains and is hardly detected in the other strains. This amino acid can be used to produce pyruvate (Lacerda et al., 2021), and could also be converted to glutamate, increasing the activity of the TCA cycle, which contributes to energy demand. However, L-proline has also been widely studied for its involvement in stress responses. In yeast, it acts as osmoregulatory and inhibitor of cellular dehydration, as reacting oxygen species (ROS) scavenger, or by stabilising proteins and membranes (Takagi, 2008; Szabados and Savouré, 2010; Ben Rejeb et al., 2014). Moreover, in F. graminearum, increased L-proline levels were associated with protection against different stressors (Sevastos et al., 2018). In yeast, L- proline is synthesised from glutamate by γ-glutamyl kinase (encoded by the PRO1 gene), γ-glutamyl phosphate reductase (PRO2), and pyrroline-5-carboxylate reductase (PRO3), or alternatively by arginase (CAR1) and ornithine transaminase (CAR2), and converted to glutamate by proline oxidase (PUT1) and P5C dehydrogenase (PUT2) in yeast mitochondria (Takagi, 2008). In F. fujikuroi, FFUJ_02900 and FFUJ_07330 genes, homologous to yeast genes PRO1 and PRO2, are not affected either by the carS mutation or the carP deletion, and genes homologous to CAR1, CAR2, PUT1, and PUT2 were also unaffected. However, the FFUJ_11995 gene homologous to the yeast PRO3 gene is down- 123 Chapter III regulated by the carS mutation and up-regulated in ΔcarP under light conditions (Figure CIII.8). In plants, stress-induced L-proline accumulation correlates with transcriptional activation of the gene encoding P5C synthetase (orthologous of the S. cerevisiae P5CR enzyme, encode by the PRO3 gene), which is the key regulatory enzyme in this biosynthetic pathway (Takagi, 2008). According to GC-MS data, this amino acid is specifically increased in both albino strains under light, which correlates with the upregulation of the FFUJ_11995 gene in ΔcarP in the light, determined by RNA-seq. These results suggest an activation of proline-dependent mechanisms of stress by carS overexpression, providing further support for regulatory roles of CarS in stress responses. In relation to stress mechanisms and CarS, it was observed that the FFUJ_07916 gene, encoding a predicted glutathione S-transferase (GST) enzyme involved in the conversion of glutathione (GSH) to glutamate, is also up-regulated by the carS mutation (Figure CIII.8), and down-regulated by the carP deletion. Glutathione is crucial in eukaryotes as it plays a protective role against oxidative damage (Penninckx, 2002; Carlos et al., 2004), and in A. nidulans, the GST enzyme also contributes to resistance to heavy metal and xenobiotic by catalysing the conjugation of GSH to xenobiotics and endogenous products of oxidative stress (Fraser et al., 2002). Higher protection against oxidative damage would be expected in the carS mutant due to up-regulation of the gene encoding the GST enzyme. Contrary to what might be expected, the carS mutant SG39 exhibited a slight reduction of growth in Petri dishes with H2O2 compared to the wild type (Ruger-Herreros, 2016). The experimental approach to assess protection against oxidative damage was the addition of H2O2 to the medium; moreover, the carS mutants were more sensitive than the wild type to certain antibiotics (Ismael Borrego, personal communication), and a possible explanation could be a greater incorporation of these compounds into the cytoplasm across the cell membrane. In F. graminearum aquaporin 1 (FgAQP1), encoded by the FGSG_00811 gene, was found to play a role in regulating stress responses, development, and secondary metabolism, and the ΔAQP1 mutant also showed increased resistance to osmotic and oxidative stress, as well as cell wall perturbing agents (Ding et al., 2018). According to RNA-seq data, the carS mutant SG39 of F. fujikuroi has higher transcript levels of the FGSG_00811 orthologous gene FFUJ_00417 than the wild type (Figure CIII.8), and the higher levels of the FfAQP1 aquaporin could be related to the 124 Chapter III observed lower resistance against oxidative damage. In addition, the down-regulated effect on the pyrroline-5-carboxylate reductase gene by the carS mutation, and the consequent lower L-proline production, could be another reason of a worse protection of carS mutants under stressors such as oxidative damage. Figure CIII.8. Comparative mRNA levels of FFUJ_11995 gene and genes related to aquaporin 1 and to glutathione-S-transferase (GST) in carS-, carS+ complemented mutant and ΔcarP strains. Transcriptomic data were extracted from data sets already available from carS- mutant SG39 and carS+ complemented strain SG256 (Ruger-Herreros et al., 2019) and ΔcarP mutant SG268 (Pardo-Medina et al., 2021b). WT: Wild type. 125 Chapter III The large variety of genes influenced by CarS is an impediment to establishing direct targets and metabolic connections. However, our data are consistent with regulatory roles of CarS in oxidative stress response and intermediary metabolism. The potential effects on metabolic networks and their correlation with the observed changes in metabolomic data complement the transcriptomic data and has contributed to the identification of possible regulatory key steps whose modulation by light or CarS could provide us with a deeper understanding of some regulatory pathways in F. fujikuroi. Furthermore, it highlights the influence that variations in carotenoid production may have on the biosynthesis of other metabolites, although we still lack a complete understanding of their biological impact, which requires further investigation. 126 Chapter III 127 Chapter IV: Functional analysis of nutZ, a regulatory gene adjacent to the car cluster 134 Chapter IV Figure CIV.2. Confirmation of the deletion of nutZ gene in candidate transformants by PCR and Southern blot analysis. A. Genomic map of the nutZ region showing the target sites for the SalI restriction enzyme and the expected sizes for the corresponding bands, as well as the position of the primer set used for PCRs (a) and (b). B. PCR products in agarose gel electrophoresis of several transformants. Amplifications with genomic DNA from the wildtype strain (WT) or without DNA (0) were used as controls. (a) PCR was performed with the PC4.13-PC4.14 primer set, resulting in the amplification of a single 4.7 kb product as expected in the deletion mutants at T22 and T37. (b) PCRs were performed with the set of PC4.15- PC4.16 primers that amplified a product of 2.5 kb in T1 and T22, expected in nutZ deletion strains. C. Southern blot results. T1 and T22 transformants exhibited the expected band of 10.1 kb, which confirmed the accurate deletion of the nutZ gene, while the wild-type strain displayed a band of 3.4 kb. 135 Chapter IV 2.3 Complementation of nutZ deletion mutants and generation of nutZ overexpressing strains To complement the nutZ mutation, the plasmid pJM7 was constructed with the nutZ gene under the control of its own promoter (PnutZ). To overexpress nutZ, plasmid pJM6 was constructed with the nutZ gene under the control of the promoter of glyceraldehyde- 3-phosphate dehydrogenase A (PgpdA) of A. nidulans. Both plasmids were used to transform protoplasts obtained from SG295 and SG296 ΔnutZ strains. After selection on acetamide medium, potential transformants were genetically purified, and some of them were analysed by PCR. In the SG295 background, three potential positive transformants were obtained with plasmid pJM6 (OET13, OET13.1 and OET13.3) and two with plasmid pJM7 (C3-T1 and C1-T1). In the case of SG296, two possible positive transformants were obtained with plasmid pJM6 (OET22-3.1 and OE-T22-3.3) and three with plasmid pJM7 (C1- T22, C2-T22 and C3-T22). Strains generated with pJM6 were called OEgpdAnutZ. The complemented candidates obtained from SG296 with plasmid pJM7 were analysed by PCR with the PC4.19-PC4.24 primer set. The result showed the expected 3.7 kb positive band only in C3-T22, so the other two candidates were discarded (Figure CIV.3.Ba). Using primer set PC4.23-PC4.24, the five candidates obtained from SG295 exhibited the expected corresponding bands of ~1.6 kb (candidates OET13, OET13.1 and OET13.3), and ~1.8 kb (candidates C3-T1 and C1-T1) (Figure CIV.3.Bb), as well as the complemented candidates in the SG296 genetic background, which also exhibited the corresponding 1.6 kb. Genomic DNA samples of positive candidates were also subjected to Southern blot analysis, after digestion with SacI enzyme. For the analysis, wild-type strain, negative transformant C1-T22 and ΔnutZ strains SG295 and SG296, were also included as negative controls. As both vector sequences contain two ScaI restriction sites, bands of approximately 6.6 kb (for pJM6) or 6.8 kb (for pJM7) are expected in transformants that have integrated the corresponding plasmid into the genome as illustrated in Figure CIV.3.A. The presence of these bands in all the candidate transformants confirmed the successful vector integration. 136 Chapter IV Figure CIV.3. Molecular analysis of OEgpdAnutZ and OEnutZnutZ strains by PCR and Southern blot analysis. A. Left: Maps of the constructions to overexpress nutZ, pJM6 and pJM7, showing the target sites for ScaI restriction enzyme and the expected sizes for the corresponding bands, as well as the position of the primers used for PCRs (a) and (b). Right: genomic map of the nutZ region showing the target sites for ScaI restriction enzyme and the expected sizes for the corresponding bands. B. PCR products in agarose gel electrophoresis of several candidates. Amplifications with genomic DNA from the wild-type (WT) strain or without DNA 137 Chapter IV (0) were used as controls. (a) PCR performed with primer set PC4.19-PC4.24. Amplification of a product of 3.7 kb, expected in strains transformed with plasmid pJM7, was observed in the candidate transformant C3-T22. (b) PCRs performed with primer set PC4.23-PC4.24. Amplification of a product of 1.6 kb, expected in strains transformed with pJM6, or 1.8 kb, expected in strains transformed with pJM7, was observed in all candidates. C. Southern blot results. Aside from C1-T22, the rest of the candidates exhibited the expected band of 2.9 kb, corresponding to integration of plasmids pJM6 or pJM7, while the wild-type strain showed the expected band of 3.7 kb. Furthermore, as both nutZ gene and HygR cassette contain a ScaI restriction site, wild-type and ΔnutZ strains revealed their corresponding ~3.7 kb and ~2.9 kb bands, respectively (Figure CIV.3.C). Although in the mutant, the band signal was faint because the probe hybridised primarily with nutZ (Figure CIV.3.A), it was observed in both types of transformants, OEgpdAnutZ and nutZ complemented strains, confirming that the vector insertion was ectopic. Positive transformants were assigned names that corresponded to the strain collection numbers of the group. In the case of the OEgpdAnutZ transformants, strains OET13, OET13.1 and OET13.3 (obtained from SG295), and OET22-3.1 and OE-T22-3.3 (from SG296), were assigned collection numbers SG308, SG310, SG311, and SG312, respectively. The expression of nutZ was analysed by RT-qPCR from samples wild type and three candidate OEgpdAnutZ strains obtained from SG295: SG308, SG309, and SG310, grown in the dark or after under 1 hour of illumination. The results confirmed that SG308 and SG310 overexpressed nutZ under control of the gpdA promoter (Figure CIV.4), while SG309 did not express it, and it was discarded for future analysis. Complemented strains C3-T1 (from SG295) and C3-T22 (from SG296) were assigned collection numbers SG314 and SG315. Unexpectedly, as later observed in RNA-seq analysis, the complemented strain SG314 exhibited a high expression of the nutZ gene, and we will refer to it as OEnutZnutZ. 138 Chapter IV Figure CIV.4. Overexpression of the nutZ gene in OEgpdAnutZ strains. Transcripts levels of nutZ were determined by RT-qPCR in the wild type and OEgpdAnutZ transformant strains SG308, SG309 and SG310, grown in darkness or after 1 hour light exposure. The graphs show mean and standard error of the mean from three independent experiments. A two-way ANOVA statistical analysis was performed on data from the three biological replicates, and asterisks indicate the level of significance: *, p<0.05, **, p<0.01, ***, p< 0.001, ****, p<0.0001. 2.4 Effect of nutZ deletion or overexpression on carotenoids biosynthesis To check the possible role of NutZ in the regulation of carotenoids biosynthesis, expression of car genes was analysed by RT-qPCR in nutZ deletion mutants and nutZ overexpressing strains. More specifically, transcript levels of carRA, as a gene of the car cluster, and carT, as a car gene located outside the cluster, were determined in wild type, ΔnutZ strains, SG295 and SG296, and OEgpdAnutZ strains, SG308 and SG310, in the dark or after 1 hour of illumination. The results showed that neither nutZ deletion (Figure CIV.5.A) nor nutZ overexpression significantly affected the transcript levels of the investigated genes (Figure CIV.5.B). In addition, carotenoids were extracted from samples of the same strains grown for 7 days on solid DG medium at 30 °C and quantified. Data revealed that carotenoids 139 Chapter IV production was barely affected by nutZ deletion or overexpression, except for the OEgpdAnutZ SG310 strain, which showed a noticeable increase in carotenoid content in light (Figure CIV.5.C). This change was not correlated with increased transcript levels in carRA or carT genes or with higher levels of nutZ transcript since this strain showed the lowest levels of expression of all nutZ-overexpressing strains. On the other hand, both OEgpdAnutZ strains exhibited lower carotenoid levels in darkness, while slight increases were apparent in nutZ deletion strains. However, these changes were not remarkable, and the lack of effect of nutZ alterations on car genes transcript levels suggests that they cannot be directly attributed to NutZ. Figure CIV.5. Effect of nutZ deletion or overexpression on carotenoid production and on transcript levels of a selection of carotenoid biosynthesis genes. A. Transcript levels of carRA and carT genes in wild-type (WT) and nutZ deletion strains SG295 and SG296. B. Transcripts levels of carRA and carT genes in OEgpdAnutZ overexpressing strains SG308 and SG310. The strains were cultured for 3 days in the dark and exposed to light or dark for 1 hour. C. 140 Chapter IV Carotenoid content in mycelia from the wild type, nutZ deletion mutants SG295 and SG296, and OEgpdAnutZ transformant strains SG308 and SG310, cultured on solid DG medium for 7 days in the dark or under continuous illumination. The graphs show the mean and standard error of the mean of three independent experiments, analysed by quantitative RT-PCR. A twoway analysis of variance (ANOVA) statistical analysis was performed on data from the three biological replicates, and no significant differences were detected. 2.5 In vitro NutZ binding assays on car gene promoters To verify whether the NutZ protein can bind to car gene promoters, electrophoretic mobility shift assays (EMSA) were performed with NutZ produced in E. coli cells. The nutZ gene was cloned into the vectors pGEX-4T-3 and pQE-80L to express NutZ protein tagged with glutathione S-transferase (GST) or with 6xHis in E. coli, resulting in plasmids pJM8 and pJM10, respectively. After checking different strains of E. coli and culture conditions, the best NutZ production was obtained in Rosetta E. coli cultures grown at 22 °C overnight and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Bands of 106 kDa and 80 kDa, corresponding to expected sizes of GST-NutZ and 6His-NutZ, respectively, were detected in the acrylamide electrophoresis gel of total protein extracts from these cultures of E. coli (Figure CIV.6.A). Finally, GST-NutZ protein was purified from the corresponding total extracts with glutathione-agarose resin (Figure CIV.6.B) and used for EMSA experiments. Figure CIV. 6. Production of the NutZ protein in E. coli Rosetta strain. A. Visualisation on polyacrylamide gel electrophoresis of total protein extracts of E. coli Rosetta strain 141 Chapter IV transformed with plasmid pJM8 for GST-NutZ expression and plasmid pJM10 for 6His-NutZ expression. Lanes 0 h were loaded with total protein extracts from the cultures before protein induction, while lanes 16 h were loaded with total protein extracts after induction with 1 mM IPTG for 16 hours at 22 °C. Expected bands of 106 kDa for GST-NutZ and 80 kDa for 6HisNutZ were observed in lanes 16 h. B. Visualisation on polyacrylamide gel of GST-NutZ protein purified with glutathione agarose resin. Lanes E1, E2, and E3 correspond to the three consecutive elutions of the protein. For the EMSA assay, car gene promoters were divided into 2-3 overlapping segments, amplified by PCR, and subsequently labelled with biotin primers by PCR. This subdivision was necessary due to the size of the promoter sequences, which could potentially complicate complex migration. Since carRA and carX are transcribed in opposite directions from the same regulatory region, three promoter regions were analysed: carRA/carX, carB, and carO. Figure CIV.7. In vitro binding assay of GST-NutZ and promoter segments of carRA/carX, carB and carO genes. A. Map of the car cluster. The promoter segments used in the assay are indicated. B. EMSA results with GST-NutZ protein expressed in E. coli Rosetta strain. The assay was performed with 20 fmol of biotin-labelled promoter segments and 0.5 μg of GST-NutZ purified protein. Negative controls of free DNA (FD) of each promoter segment without protein were also included. C- and C+: control samples provided by the commercial kit. C-: 142 Chapter IV biotin-labelled DNA as negative control. C+: biotin-labelled DNA with control extract protein as positive control. The EMSA was performed using the Thermofisher LightShiftTM chemiluminescent EMSA kit, adapting the manufacturer's instructions to fit the characteristics of the NutZ protein. For the assay, 20 fmol of DNA biotin-labelled fragments were incubated with 0.5 μg of GST-NutZ purified protein or without protein (free DNA). In the EMSA assays, no mobility shifts could be appreciated in any of the DNA bands of the car promoters in the presence of the NutZ protein while a clear shift in the mobility of the controls was observed (Figure CIV.7). An additional EMSA was performed incubating 20 fmol of biotinlabelled DNA segments of carRA-carX and carB promoters with increasing amounts of GST-NutZ purified protein (0.5, 1, and 2 μg). Figure CIV.8. EMSA with increasing NutZ amounts on carRA/carX and carB promoter segments. Purified GST-NutZ protein was expressed in the E. coli Rosetta strain. The assay was performed with 20 fmol of biotin-labelled promoter segments and 0.5, 1 and 2 μg of purified GST-NutZ protein. Negative controls of free DNA (FD) from promoter segments 143 Chapter IV without protein were also included. C- and C+: control samples provided by the commercial kit, biotin-DNA as negative control (C-) and biotin-DNA with control protein as positive control (C+). The same negative result was obtained (Figure CIV.8), indicating that NutZ does not bind to the regulatory sequences of car cluster genes. This, together with the lack of effect of nutZ alterations on carRA and carT gene expression, indicated that NutZ does not play a direct role in the regulation of car genes. 2.6 Genes differentially expressed in nutZ overexpressing strains compared to wild-type and ΔnutZ strains Considering the lack of influence of NutZ on the regulation of car genes, a transcriptomic analysis was performed to determine the function of this TF. Samples from wild type, ΔnutZ (SG295), and the nutZ overexpressing strains OEgpdAnutZ (SG308) and OEnutZnutZ (SG314) were used in this analysis. In view of the lack of knowledge about the true function of NutZ, the strains were cultured in the standard minimal medium in the dark. The inclusion of nutZ overexpressing strains in these experiments was especially convenient, considering that under such conditions the nutZ gene shows a very low expression level in the wild type. Genes whose transcript levels changed at least two-fold (log2 = 1), with a p-value of 0.05, in the OEnutZ strains compared to wild-type or ΔnutZ strains were considered differentially expressed (Table CIV.1; complete lists of genes in Annex, Table S4.1. Overexpression of nutZ had a notable effect on the F. fujikuroi transcriptome, with at least 2.000 genes deregulated in both OEnutZ strains. However, the transcriptome was barely affected by nutZ deletion, with only five genes deregulated in the ΔnutZ strain. The numbers of up-regulated or down-regulated genes were alike in both OEnutZ strains: 1.094 up-regulated genes and 1.081 down-regulated genes in OEgpdAnutZ (SG308), 971 and 1.236 in the case of OEnutZnutZ (SG314). Due to the transcriptional similarity between wild type and ΔnutZ, the number of genes affected by nutZ overexpression in both types of OEnutZ strains were similar when compared with ΔnutZ than when compared to wild type. 246 247 Annex Table S3.1. Compounds identified through GC-MS analysis. Table S3.2 Genes ID and corresponding protein name of genes cluster by FunCat in the category “C-compounds and carbohydrates metabolism”, affected by carS and carP mutation. From Pardo-Medina et al., 2021b supplementary data. FunCat description: C-compounds and carbohydrates metabolism Gene ID Protein name FFUJ_07954 related to 6-hydroxy-D-nicotine oxidase FFUJ_07515 related to arabinose 5-phosphate isomerase FFUJ_08014 related to formaldehyde dehydrogenase FFUJ_05834 probable oxidoreductase CipA-like FFUJ_10649 related to toxD gene FFUJ_08504 related to formaldehyde dehydrogenase FFUJ_10506 related to ketoreductases FFUJ_07503 related to aldehyde dehydrogenase FFUJ_00322 related to ketoreductases FFUJ_10896 related to 2%2C5-diketo-D-gluconic acid reductase FFUJ_12693 related to reductases FFUJ_10702 related to isoamyl alcohol oxidase FFUJ_14760 related to acid phosphatase FFUJ_06988 Related to aryl-alcohol dehydrogenases FFUJ_12437 related to cytosine deaminase and related metal-dependent hydrolases FFUJ_14317 probable aldehyde dehydrogenase FFUJ_13902 related to dihydroxyacetone kinase FFUJ_14547 related to chitinase FFUJ_11537 uncharacterized protein FFUJ_10993 related to reductases FFUJ_00756 related to UDP-glucuronosyltransferase 2C1 microsomal FFUJ_11042 related to oxidoreductase FFUJ_14043 related to isoamyl alcohol oxidase FFUJ_06546 related to quinone oxidoreductase FFUJ_06628 related to NAD(P)H-dependent oxidoreductase FFUJ_06543 Related to Pseudomonas L-fucose dehydrogenase FFUJ_10321 probable alcohol dehydrogenase homolog Bli-4 Table S4.1. Differentially expressed genes after DESeq2 analysis (Log2(FC)>1, p < 0.05) in the comparisons: (A) OEgpdAnutZ vs. WT; (B) OEnutZnutZ vs. Wild type; (C) ΔnutZ vs. WT; (D) OEgpdAnutZ vs. ΔnutZ; (E) OEnutZnutZ vs. ΔnutZ; (F) OEgpdAnutZ vs. OEnutZnutZ. 248 Table S4.2. Genes differentially expressed in both OEnutZ (Log2(FC)2) Table S4.3. Cluster of genes affected by OEnutZ strains used to represent heatmaps. Table S4.4. Genes most affected in each OEnutZ strains. 249 References References Aasen, A.J., Jensen, S.L., Theander, O. (1965). Fungal Carotenoids. II. The structure of the carotenoid acid neurosporaxanthin. Acta Chem. Scand. 19: 1843–53. https://doi.org/10.3891/acta.chem.scand.19-1843. Adam, A., Deimel, S., Pardo-Medina, J., García-Martínez, J., Konte, T., Limón, M.C., Avalos, J., Terpitz, U. (2018). Protein activity of the Fusarium fujikuroi rhodopsins CarO and OpsA and their relation to fungus–plant interaction. 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Proline as a stress protectant in yeast: Physiological functions, metabolic regulations, and biotechnological applications. Applied Microbiology and Biotechnology, 81(2), 211–223. https://doi.org/10.1007/s00253-008-1698-5 278 A Carlos, mi compañero de vida. Para ti no existen palabras suficientes. Tú has sido quien estaba ahí para sostenerme cuando el laboratorio pesaba. Y también a quien invadía cuando volvía feliz. Gracias por haber sabido lo que necesitaba incluso mejor que yo misma, por gestionar(me) el huracán de emociones que ha sido esta tesis para mí. Simplemente somos el mejor equipo. A Toshi, mi querida bolita de pelo. Mi animal espiritual. Llegaste para sacarme de quicio a todas horas y maravillarme con tu profunda personalidad. Me has hecho más amena esta escritura, mientras me pisabas el teclado para hacerme recordar que había otras cosas importantes que atender. A Freddie. Aunque tengas cuatro patas y mucho pelo, has sido y serás mi mejor amigo. Fuiste mi compañero paciente en los días de estudio desde hace años. Y volviste a regalarme tu compañía estas navidades mientras escribía esta tesis. Gracias por todo el aprendizaje sobre la vida y el amor que me has dado. 279