The histone acetyltransferase GcnE (GCN5) plays a central role in the 1

regulation of Aspergillus asexual development 2

3

Running title: GcnE is essential for conidiation in Aspergillus 4

5

David Cánovas1,2¶*, Ana T. Marcos1¶, Agnieszka Gacek2¶, María S. Ramos1, Gabriel Gutiérrez1, 6

Yazmid Reyes-Domínguez2§, Joseph Strauss2,3 7

8

Affiliations: 9

1 Departmento de Genética, Facultad de Biología, Universidad de Sevilla, Spain 10

2 Fungal Genetics and Genomics Unit, Department of Applied Genetics and Cell Biology, 11

University of Natural Resources and Life Sciences (BOKU) Vienna, 3Department of Health and 12

Environment, Bioresources, Austrian Institute of Technology (AIT), Austria 13

14

¶ These authors have equally contributed to the work. 15

§ Current affiliation: Research Centre for Agriculture and Forestry Laimburg, Laimburg 6, 16

Auer/Ora, BZ, 39040, Italy. 17

18

Author for correspondence: Departamento de Genética, Facultad de Biología, Universidad de 19

Sevilla, Reina Mercedes 6, 41012 Sevilla, Spain, e-mail: davidc@us.es 20

21

Keywords: Gcn5; GcnE; histone acetylation; SAGA; Aspergillus; conidiation; asexual 22

development; brlA; fluffy genes 23

24

Genetics: Early Online, published on June 6, 2014 as 10.1534/genetics.114.165688

Copyright 2014.

2

Summary 1

2

Acetylation of histones is a key regulatory mechanism of gene expression in eukaryotes. GcnE is an 3

acetyltransferase of Aspergillus nidulans involved in the acetylation of histone H3 at lysine 9 and 4

lysine 14. Previous works have demonstrated that deletion of gcnE results in defects in primary and 5

secondary metabolism. Here we unveil the role of GcnE in development and show that a ∆gcnE 6

mutant strain has minor growth defects but is impaired in normal conidiophore development. No 7

signs of conidiation were found after 3 days of incubation, and immature and aberrant 8

conidiophores were found after one week of incubation. Centroid linkage clustering and principal 9

component analysis of transcriptomic data suggest that GcnE occupies a central position in 10

Aspergillus developmental regulation, and that it is essential for inducing conidiation genes. GcnE 11

function was found to be required for the acetylation of histone H3K9/K14 at the promoter of the 12

master regulator of conidiation, brlA, as well as at the promoters of the upstream developmental 13

regulators of conidiation flbA, flbB, flbC and flbD (fluffy genes). However, analysis of the gene 14

expression of brlA and the fluffy genes revealed that the lack of conidiation originated in a complete 15

absence of brlA expression in the ∆gcnE strain. Ectopic induction of brlA from a heterologous alcA 16

promoter did not remediate the conidiation defects in the ∆gcnE strain, suggesting that additional 17

GcnE-mediated mechanisms must operate. Therefore, we conclude that GcnE is the only 18

nonessential histone modifier with a strong role in fungal development found so far. 19

20

3

Introduction 1

2

Chromatin rearrangements are associated with the transcriptional regulation of gene expression in 3

eukaryotes. For example, facultative heterochromatin can be associated with the transcriptionally 4

active or silent states of developmentally regulated loci (GREWAL and JIA 2007). This is achieved in 5

part through histone Post Translational Modifications (PTM), which play a very important role in 6

the control of these active or silent chromatin states. Histone modifications include acetylation, 7

methylation, phosporylation and ubiquitination at different positions of the histone proteins. In 8

particular, acetylation of lysine 9 or lysine 14 in histone H3 has been associated with activation of 9

transcription. Acetylation of histones plays two roles in the regulation of transcription: it alters the 10

physical properties of the histone-DNA interaction, and it also provides a frame for the binding of 11

bromodomain proteins that remodel the chromatin and regulate gene expression (SPEDALE et al. 12

2012). These modifications regulate the nucleosome positioning at the gene promoters and the 13

recruitment of the regulatory proteins. One of these modifiers, the SAGA complex, is responsible 14

for the acetylation of several lysine residues in the N terminal region of histones, particularly 15

histone H3 lysine 9 (H3K9) and lysine 14 (H3K14) (KUO et al. 1996). The SAGA complex is a 16

multimeric protein association with several subunits including Ada2p, Ada3p, Spt3p and Tra1p 17

(GRANT et al. 1997; SPEDALE et al. 2012), where Gcn5p is the subunit with the histone 18

acetyltransferase (HAT) catalytic activity (GRANT et al. 1997). The SAGA complex is implicated 19

in several functions related to transcription, such as transcription initiation and elongation, histone 20

ubiquitination and interactions of TATA-binding proteins. In addition, SAGA has also been 21

implicated in mRNA export in yeasts and Drosophila (KURSHAKOVA et al. 2007; RODRIGUEZ-22

NAVARRO et al. 2004). In Saccharomyces cerevisiae the SAGA complex is involved in the 23

transcriptional regulation of 12% of the yeast genome. Approximately, a third of that 12% of the 24

yeast genome is downregulated and two thirds are upregulated in ΔGCN5 cells (LEE et al. 2000) 25

implying a direct or indirect negative role of Gcn5p. Interestingly, a high proportion of genes 26

4

regulated by SAGA are upregulated during the responses to environmental stresses (such as heat, 1

oxidation and starvation) (HUISINGA and PUGH 2004). The SAGA complex is also present in 2

metazoans, where it has diverged and evolved into four different complexes (two SAGA and two 3

ATAC complexes), while lower eukaryotes, like yeasts and other fungi, contain one single SAGA 4

complex. It was hypothesized that this evolution into a diverse set of complexes is involved in 5

cellular specialization during development and homeostasis in metazoans (SPEDALE et al. 2012). 6

The SAGA and ATAC complexes participate in the regulation of genes in response to intracellular 7

and extracellular signals: protein kinase C signalling, response to osmotic stress, UV-induced DNA 8

damage, arsenite-induced signalling, endoplasmic reticulum stress and nuclear receptor signalling 9

(SPEDALE et al. 2012). Likewise, plants also have multiple HATs. In Arabidopsis, AtGCN5 is 10

involved in many developmental processes (SERVET et al. 2010). 11

Although elegant experimental approaches using Neurospora crassa as a model system have 12

significantly contributed to general concepts of DNA methylation, genome defense and 13

heterochromatin formation (FREITAG et al. 2004; FREITAG et al. 2002; HONDA et al. 2010; 14

ROUNTREE and SELKER 2010; TAMARU and SELKER 2001), studies on transcriptionally related 15

chromatin rearrangements and histone modifications are still scarce in filamentous fungi, a broad 16

group of ecologically, industrially and clinically important organisms. In N. crassa the 17

transcriptional activation of the light-inducible gene al-3 requires the acetylation of histone H3K14 18

by a homologue of Gcn5p, NGF-1 (GRIMALDI et al. 2006), and in Aspergillus nidulans the 19

SAGA/ADA complex is involved in the acetylation of H3K9/K14 at the prnD-prnB bidirectional 20

promoter during inducing conditions, but increased levels of H3K9ac/K14ac are not required for 21

transcription (REYES-DOMINGUEZ et al. 2008). Georgakopoulos and colleagues reported the 22

delineation of the A. nidulans SAGA complex with a combined proteomics and bioinformatics 23

approach revealing a high conservation with the yeast SAGA complex except for the 24

deubiquitination-H2B-Ub complex (GEORGAKOPOULOS et al. 2013). Only recently, the relevance of 25

chromatin-based silencing of secondary metabolite gene clusters was recognized in several 26

5

Aspergillus and Fusarium species (BOK et al. 2009; LEE et al. 2009; REYES-DOMINGUEZ et al. 1

2010; SHWAB et al. 2007; STRAUSS and REYES-DOMINGUEZ 2010). For example, it has been 2

demonstrated that acetylation of histone H3 is required for the synthesis of secondary metabolites in 3

A. nidulans (BOK et al. 2013; NÜTZMANN et al. 2013; NÜTZMANN et al. 2011; REYES-DOMINGUEZ 4

et al. 2010). Reduction of heterochromatin marks leads to higher secondary metabolite production 5

in Aspergillus and Fusarium species (REYES-DOMINGUEZ et al. 2012; REYES-DOMINGUEZ et al. 6

2010), and it has also been found that it de-represses silent clusters leading to the production of 7

novel metabolites (BOK et al. 2009). In addition, adverse metabolic and morphologic effects are 8

also observed in histone modifier mutants, e.g. deletion of the histone H3K9 methyltransferase clrD 9

in A. fumigatus resulted in reduced radial growth, and also delayed transcriptional activation of brlA 10

and conidiation (PALMER et al. 2008). 11

Asexual reproduction, also called conidiation, results in the formation of mitotic propagules 12

(conidia) which are the infectious particles for pathogenic filamentous fungi. Conidiation is the 13

most common and proliferative reproductive mode in filamentous fungi. For this reason, 14

conidiation has been extensively studied in A. nidulans for several decades (for recent reviews see 15

(ETXEBESTE et al. 2010; KRIJGSHELD et al. 2013; PARK and YU 2012)). Conidiation is controlled by 16

a central regulatory pathway (Figure 1), encompassing three transcriptional activators BrlA, AbaA 17

and WetA (see reviews by (ADAMS et al. 1998; YU et al. 2006)). The first component in this 18

regulatory cascade, BrlA, is essential to drive conidiation (ADAMS et al. 1988). brlA expression is 19

silent during vegetative growth, and its expression during conidiation is controlled by a number of 20

genes, including the fluffy genes. Deletion of any of the fluffy genes gives a typical fluffy phenotype 21

with cotton-like colonies, lack of normal conidia and reduced levels of brlA expression (ADAMS et 22

al. 1998; YU et al. 2006). There are 6 fluffy genes: fluG and flbA-E. fluG encodes a protein similar 23

to bacterial glutamine synthetases (LEE and ADAMS 1994) and the FluG protein is responsible for 24

the synthesis of the extracellular factor dehydroaustinol that in conjunction with the orsellinic acid 25

derivative diorcinol induces conidiation (RODRIGUEZ-URRA et al. 2012). FluG works upstream of 26

6

the flbA-E genes (YU et al. 2006). Flb-genes operate in three parallel routes in A. nidulans to 1

regulate the expression of brlA upon induction of conidiation. FlbA is a repressor of the G-protein 2

signalling, which participates in a protein kinase A-dependent pathway to promote filamentous 3

growth and to inhibit conidiation (YU et al. 1996). FlbE interacts with FlbB at the fungal tip and is 4

required for proper activation of FlbB (GARZIA et al. 2009). FlbB is a bZip transcription factor that 5

activates the transcription of flbD, a cMyb-type regulator. Then, both FlbB and FlbD jointly activate 6

the transcription of brlA (GARZIA et al. 2010). FlbC is a putative C2H2 Zn finger protein that 7

constitutes a third route for the regulation of brlA expression (KWON et al. 2010). These fluffy genes 8

are expressed in vegetative mycelium, and are able to respond to intracellular stimuli to induce a 9

coordinated activation of the master regulator brlA (ETXEBESTE et al. 2010). 10

Previous work noticed that deletion of the SAGA subunits gcnE or adaB in A. nidulans resulted 11

in strongly reduced conidiation, while not affecting the activation (but repressibility) of the proline 12

utilization genes prnD-prnB, which are transcribed divergently from a bidirectional promoter 13

(REYES-DOMINGUEZ et al. 2008). Interestingly, also Georgakopoulos et al found a lack of acetate 14

repressibility in a SAGA-defective mutant (GEORGAKOPOULOS et al. 2012). Here, we clarify the 15

role of GcnE in the control of fungal development. 16

17

18

Materials and Methods 19

20

Strains, media and culture conditions 21

The A. nidulans strains used in this study are listed in Table S1. Strains were grown in complete or 22

minimal media containing the appropriate supplements (COVE 1966). Glucose was used as carbon 23

source and ammonium nitrate was used as nitrogen source. In the brlA overexpressing experiments 24

threonine and fructose were used as carbon sources to overexpress the brlA gene, and glucose was 25

used to repress the brlA gene under the control of the promoter of the alcA gene. Ammonium 26

7

tartrate was used as nitrogen source. Strains were obtained following standard procedures 1

(PONTECORVO et al. 1953). Trichostatin A, butyric acid and valproic acid were purchased from 2

Sigma and used as histone deacetylases (HDAC) inhibitors at a concentration of 5 µM. 3

4

RNA isolation and real time RT-PCR 5

Isolation of RNA and quantification of mRNA were performed as previously described (RUGER-6

HERREROS et al. 2011). Briefly, mycelia (100-200 mg) were disrupted in 1ml of TRI reagent 7

(Sigma) with 1.5 g of zirconium beads by using a cell homogeneizer (FastPrep-24, MP 8

Biomedicals). Cell debris was removed by centrifugation and RNA samples were further purified 9

using the NucleoSpin RNA II Kit (Macherey-Nagel). 10

The primers employed for real time RT-PCR are detailed in Table S2. Real time RT-PCR 11

experiments were performed in triplicates (technical replicates) in a LightCycler 480 II (Roche) by 12

using the One Step SYBR® PrimeScript™ RT-PCR Kit (Takara Bio Inc.). The fluorescent signal 13

obtained for each gene was normalized to the corresponding fluorescent signal obtained with the ß-14

tubulin gene benA to correct for sampling errors. Expression data are the average of at least three 15

independent biological replicates. 16

17

Microarray experiment 18

Strains were grown in complete liquid medium for 18 h at 37 ºC and then conidiation was induced 19

by transferring the vegetative cultures to complete solid media. Strains were further grown for 10 h 20

at 37 ºC. Samples were immediately frozen in liquid nitrogen upon harvesting and stored at – 80ºC 21

until processing. RNA was isolated from strains grown in liquid or solid media as previously 22

reported (SCHINKO et al. 2010). RNA samples were quality controlled with the Agilent 2100 23

Bioanalyzer using the RNA 6000 Nano Kit. For each array 1 µg of total RNA was labelled with 24

MessageAmpTMII-Biotin Enhanced RNA Kit (Ambion) according to the manufacturer's 25

instructions. Hybridizations were done automatically for 16h at 45 ºC using the GeniomRT® 26

8

Analyzer. The array underwent a stringent wash. Following the labeling procedure, a microfluidic-1

based primer extension assay was performed. This assay utilized the bound mRNAs as a primer for 2

an enzymatic elongation with labeled nucleotides. The elongation was done with Klenow Fragment 3

and biotinylated nucleotides at 37 ºC for 15 minutes. Finally, the array was washed automatically 4

and detection was achieved with streptavidin-phycoerythrin using Cy3 filter set in a GeniomRT® 5

Analyzer. Three independent biological replicates were obtained for each sample. 6

7

Analysis of microarray data 8

The experimental dataset is deposited in the GEO database (accession number GSE48426). For the 9

four conditions, further data analysis was performed in the Bioconductor R (http://www. 10

bioconductor.org/). Raw intensity values were imported into R for statistical analysis using the 11

Limma package (SMYTH 2005). First, we carried out a global background subtraction, i.e. for each 12

array the global background was computed and subtracted from the measured intensity. To account 13

for variations between the hybridized arrays the VSN (variance stabilizing normalization) was used. 14

The normalized data were thereby transformed to a so-called generalized log-scale. Thus, the fold 15

quotients were also calculated on a log scale (qmedian). To provide estimates of the fold quotients 16

we utilized the exponential function. This was roughly equivalent to using the natural logarithm 17

instead of log2 (log-qmedian). 18

For the detection of differentially regulated genes between vegetative growth and conidiation in 19

the wild type or the mutant strain, the Empirical Bayes test statistics (SMYTH 2005) was used. The 20

raw p-values were adjusted for multiple testing to control the False Discovery Rate by using the 21

Benjamini-Hochberg (BH) method (BENJAMINI and HOCHBERG 1995) with a cutoff of adjusted p-22

value < 0.05. Under this criterion all selected genes showed a minimum log-qmedian lower than -23

0.7 or higher than 0.7 (where a log-qmedian of 0.7 was roughly equivalent to a 2-fold change). 24

In order to study the effect of the different factors (mutant, wild type, vegetative growth and 25

conidiation) on gene expression we performed the ANOVA test of the normalized intensities using 26

9

the Babelomics 4 suite (MEDINA et al. 2010). Differentially expressed genes were selected using a 1

cut-off of adjusted p-values (BH method) of 0.05. The normalized intensities of the genes selected 2

by the ANOVA test were used for PC analysis and clustering of the 12 samples (4 different 3

conditions with 3 replicate samples each). The PC analysis was performed with PAST (HAMMER et 4

al. 2001). Cluster analysis was performed by centroid linkage clustering of the euclidean distances 5

in Eisen’s modified Cluster 3.0 (DE HOON et al. 2004). Gene Ontology (GO) analysis was 6

performed with the GO Term Finder tool at the Aspergillus Genome Database (AspGD) site, 7

http://www.aspergillusgenome.org (ARNAUD et al. 2010). 8

9

Chromatin immunoprecipitation (ChIP) 10

ChIP assays were carried out as previously described (REYES-DOMINGUEZ et al. 2008) with primers 11

listed in Table S2. DNA was immunoprecipitated with antibodies recognizing acetylated K9 and 12

K14 of histone H3 (Millipore ab 06-599) or the C-terminus of histone H3 (Abcam ab1791). For 13

each sample the absolute amount of the specific DNA fragment in the immunoprecipitated sample 14

was divided by the amount of this fragment in the sample before precipitation (normalizing to input 15

DNA). The values shown are the averages of at least three biological repetitions. Standard errors are 16

indicated. 17

18

Scanning electron microscopy 19

Strains were grown on complete solid medium for 7 days at 37ºC. Samples were prepared for 20

electron microscopy as previously reported (CANOVAS et al. 2011) with some modifications. 21

Briefly, excised cubes of agar containing fungal mats were fixed with 2.5% glutaraldehyde in 22

cacodylate buffer for 2 hours at 4ºC and then treated with 1% OsO4 in cacodylate buffer for 2 hours 23

at 4ºC. Samples were slowly dehydrated firstly using increasing concentrations of ethanol from 24

10% to 70% and then using increasing concentrations of acetone from 70% to 100%. Samples were 25

10

dried in a Balzers CPD 030 Critical Point Dryer and gold coated. Samples were examined with a 1

JEOL 6460LV Scanning Electron Microscope. 2

3

4

Results 5

6

∆gcnE strain does not undergo asexual development 7

Reyes-Domínguez et al. noticed that deletion of the SAGA/ADA components gcnE or adaB 8

resulted in strongly reduced conidiation (REYES-DOMINGUEZ et al. 2008). Here, we followed the 9

developmental process in complete medium in a time course experiment. As shown in Figure 2A, 10

conidiophore heads were already evident after 10 h of induction in a wild type strain. After 72 h of 11

induction, conidiophores were completely mature with heads displaying the regular cylindric 12

morphology. However, the ∆gcnE mutant strain did not show any evidence of conidiophore 13

formation even after 72 h of induction. Complementation of the ∆gcnE deletion restored the 14

conidiation defects (Figure S1). 15

The phenotype of the deletion strain was compared to a set of strains harbouring deletions in 16

genes of the central regulatory pathway (brlA, abaA or wetA) to search at which step of conidiation 17

was blocked. As shown in Figure 2B, the brlA mutant produced the stalk cells than continued 18

growing rather than developing the conidiophore vesicles, metulae, phialides and conidia, which 19

gave it a bristly appearance under the stereo microscope. Mutations in abaA and wetA interfered in 20

later stages of the conidiophore development, and white structures corresponding to the vesicles, 21

metulae and phialides could be observed under the stereo microscope. The phenotypic differences 22

between abaA and wetA mutants with regards to the formation of conidia could not be observed at 23

this magnification. Nevertheless, the phenotype of ∆gcnE did not resemble ∆abaA or ∆wetA strains, 24

suggesting that developmental defects probably originate in genes upstream of abaA. Indeed, the 25

∆gcnE mutant looked more alike a ∆brlA strain. When the ∆gcnE strain was allowed to grow for 26

11

one week, some conidiophores could be observed (Figure 2C). The colony showed a very low 1

density of conidiphores as compared to a wild type strain. In addition, higher SEM magnifications 2

revealed that the conidiophores were not completely developed, harbouring rows of four conidia at 3

most, even after one week of growth. Most remarkably, these conidiophores displayed aberrant 4

morphologies, e.g. a conidium arising from a hyphal tip, rama growing out of stalk cells, or 5

sterimagta cells budding off what could be stalk or hyphal cells (Figure 2D). 6

We reasoned that the conidiation defects could be due to the growth reduction previously 7

reported (REYES-DOMINGUEZ et al. 2008). In order to test this, we quantified the linear growth rate 8

of both the wild type and an isogenic ∆gcnE mutant strain on complete and minimal solid media 9

(Figure 3A). Consistent with the previous report, the wild type strain grew faster than the ∆gcnE 10

strain on both complete and minimal media. However, the growth reduction observed in the mutant 11

strain is not strong enough to explain the conidiation defects. When both the wild type and the 12

∆gcnE strains were point-inoculated on plates and allowed to grow until they reach the same colony 13

size (diameter) the wild type showed strong conidia developement whereas the mutant strain did not 14

show any signs of conidiation (Figure 3B). This suggests that the growth reduction in the mutant 15

strain is not responsible for the conidiation defects. 16

17

Transcriptome analysis of conidiation 18

The fact that the gcnE-deletion mutant phenotype was most similar to the ∆brlA mutant suggested 19

that conidiation is blocked at an early stage of development, and no or very few conidiophore heads 20

are produced. Thus, the mutant cells are most likely defective in the expression of the upstream 21

developmental regulators or genes in the central regulatory pathway. As chromatin modifyers 22

impact on the expression of a large set of genes, we globally compared gene expression in the wild 23

type and in the ∆gcnE during vegetative growth and development. In a first approach, the 24

transcriptomes of wild type cells grown vegetatively in liquid medium (time 0, non-induced) and at 25

10 h after induction of conidiation on solid medium (time 10 h, inducing conditions) were 26

12

compared using two colors expression microarray. At this time, genes in the central regulatory 1

pathway are already activated. The analysis of the data by Empirical Bayes Test statistics revealed 2

that 1,225 genes were differentially regulated (i.e. 13.6% of the total number of genes in the chip) 3

between these two conditions. Out of these 1,225 differentially regulated genes, 600 were 4

downregulated and 625 were upregulated (Figure S2; Tables S3-4 list the Top 25 up- and down-5

regulated genes). This suggests that after 10 h of induction a major re-programming of the gene 6

expression profile occurred in the conidiating cultures. Some of the upregulated genes are relevant 7

for the regulation of conidiation (see Table 1 for a list of genes), e.g. the fluffy gene flbC, and the 8

central regulatory cascade of transcriptional activators (brlA, abaA and wetA) involved in the 9

temporal and spatial regulation of the conidiation genes (MIRABITO et al. 1989). Other genes 10

involved in conidiation were also found to be upregulated, such as vosA, medA, ivoB, yA, rodA and 11

dewA. Gene ontology (GO) term analysis of the significant genes upregulated during conidiation 12

revealed that some terms were enriched (see Figure S3), e.g carbohydrate metabolism, in which 60 13

genes out of the 584 upregulated genes having GO descriptions were induced, including 14

polysaccharide (25), pectin (7), alcohol (31), xylan (6) and pentose (9) metabolism genes. Another 15

GO term was found to be involved in secondary metabolism and toxin synthesis corresponding to 16

20 genes out the 584 upregulated genes having GO descriptions (e.g. 8 genes of the 17

sterigmatocystin biosynthesis cluster and the regulator aflR). Another GO group that was enriched 18

is related to cell wall biogenesis (18 out 584), which includes conidiation genes involved in the 19

synthesis of the spores layers (dewA, rodA, wetA). sdeA and sdeB, which are required for 20

development in A. nidulans (WILSON et al. 2004) belong to another enriched GO. 21

GO term analysis of the significant genes downregulated during conidiation (i.e. vegetative 22

genes) identified small molecule metabolism (89 genes out of the 579 downregulated genes having 23

GO descriptions), which included metabolism of ketone (52), carboxilic acids (51) and cellular 24

nitrogen (39), and biosynthesis of heterocycle (cofactor and coenzyme) (25) and nucleotides (17) 25

(see Figure S3). This suggests that genes involved in primary metabolism were expressed stronger 26

13

during vegetative growth, while genes related to development or secondary metabolite production 1

were expressed to a higher level during the conidiation program. 2

3

Transcriptome analysis of gcnE-dependent and independent control of gene expression during 4

development 5

In contrast to the 1,225 genes differentially regulated in the wild type strain, only 319 genes were 6

differentially regulated in the ∆gcnE strain when vegetative and conidiation conditions were 7

compared (Figure S2). This corresponds to only 26% of the number of genes (319 in the ∆gcnE 8

strain vs 1,225 in the wild type) differentially regulated in the wild type strain. Therefore, deletion 9

of gcnE appeared to affect the expression of a large number of genes regulated during development. 10

Out of these differentially expressed genes, 181 genes were upregulated and 138 genes were 11

downregulated in the ∆gcnE mutant (Figure S2; Tables S5-6 list the Top 25 up- and down-regulated 12

genes). In agreement with the phenotype of the mutant some regulators of conidiation were not 13

upregulated during conidiation in the ∆gcnE strain, for example brlA, abaA, wetA, vosA and medA 14

(Table 1). In addition, genes required for the synthesis of secondary metabolites (such as laeA or the 15

sterigmatocystin gene cluster) were not upregulated either. 16

Several GO groups mainly related to primary metabolism showed a higher expression level 17

during vegetative growth in the mutant but only one GO group, namely xylan metabolism, was 18

more strongly expressed in the gcnE mutant under conidiation conditions (Figure S4). Therefore, 19

wild type and ∆gcnE strains shared most of the GO terms of genes up-regulated during vegetative 20

growth but not during conidiation, suggesting that deletion of gcnE mainly affected the regulation 21

of genes during development. Out of the 625 genes upregulated during development in the wild 22

type, 41 were also upregulated in the ∆gcnE, which suggests that these genes are gcnE-independent. 23

74 genes appeared to be gcnE-independent in the down-regulated genes (Figure S2). 24

25

14

Transcriptome analysis reveals that GcnE is involved in the regulation of conidiation and 1

secondary metabolism genes 2

The transcriptome data of the wild type and ∆gcnE cells was further analyzed by ANOVA to allow 3

the comparision of all four conditions at the same time, i.e. wild type vegetative, wild type 4

conidiation, ∆gcnE vegetative and ∆gcnE conidiation. Using this type of analysis 1,162 genes were 5

found to be differentially expressed in at least one of the conditions. This corresponds to 10.9% of 6

the total number of predicted A. nidulans genes. The expression pattern of these 1,162 differentially 7

expressed genes was grouped by using centroid linkage clustering of the Euclidean distances. The 8

resulting dendrogram shows that the ∆gcnE strain grown under conidiation conditions clustered 9

together with vegetative cultures of both the wild type and the mutant strain (Figure 4A). We 10

further analyzed the differentially expressed gene set by Principal Component (PC) analysis to 11

assess the contribution of the genetic background or the growth mode to the gene expression 12

pattern. PC analysis assigns coordinates (components) to the variation in gene expression 13

representing the largest, second largest, third largest and so on variance in the corresponding axis. 14

As shown in Figure 4B, the major variation between vegetative growth and conidiation in the wild 15

type strain was depicted in the first PC (the X-axis), while the second PC (the Y-axis) showed small 16

differences between these 2 conditions in comparison (ca. 50 and 11 units, respectively). On the 17

other hand, differences between the wild type strain and the ∆gcnE mutant appeared mainly in the 18

second PC (Y-axis), and not in the X-axis, when the strains were grown vegetatively (ca. 4 units in 19

the X-axis vs 16 units in the Y-axis). This difference between the wild type and mutant strains in 20

the Y-axis strongly increased when conidation was induced (ca. 31 units). PC analysis assigned 21

49% of the variation to the mode of growth (vegetative vs conidiation) and 26% to the genetic 22

variation (wild type vs ∆gcnE). These results mean that the gene expression profile of the 23

vegetative wild type strain was similar to the ∆gcnE strain under both vegetative and conidiation 24

conditions in the X-axis/first PC (ca. 4 and 13 units, respectively; Figure 4B). In other words, the 25

gcnE mutant under conidiation conditions was more similar to the wild type strain growing 26

15

vegetatively than conidiating (the gene expression profile of the gcnE mutant was similar to the 1

vegetatively growing wild type regardless of the growth mode of the mutant). The results of this 2

statistical analysis enforces the view that GcnE plays a more important role in regulation of 3

development and seems to be less involved in the regulation of transcription under the conditions of 4

vegetative growth used in this set of experiments. Among the top 20 genes differentially expressed 5

during conidiation in the wild type strain (positive side of the first PC), 9 genes are known to have a 6

role in conidiation or secondary metabolism (Table S7). The negative side of the first PC includes 7

genes involved in oxidoreduction or other metabolic activities, such as hydrolases and peptidases 8

(Table S8). Using this approach we thus identified a group of genes that were specifically expressed 9

in the wild type strain during conidiation but were not expressed in the gcnE mutant under any 10

condition. 11

Two interesting genes appearing in this list were nkuA and nkuB. Knock-out strains of nkuA have 12

become widely used in Aspergillus and Neurospora laboratories after the discovery that deletion of 13

the KU80 or KU70 homologs results in a high rate of homologous integration but does not affect 14

development (DA SILVA FERREIRA et al. 2006; KRAPPMANN et al. 2006; NAYAK et al. 2006; 15

NINOMIYA et al. 2004). However, both genes were found to be upregulated during conidiation and 16

their induction seems to be GcnE-independent (upregulated also in the ∆gcnE mutant). A more 17

complete analysis of the GO terms of genes found to be differentially regulated by ANOVA is 18

shown in supplementary material (Figure S5-6 and Tables S7-10). 19

20

brlA is a major target of GcnE 21

Analysis of the transcriptomes revealed that conidiation genes and their regulators were induced in 22

the wild type strain after 10 h of induction but most of these genes remained unchanged in the 23

∆gcnE strain (Table 1). As expected, we found brlA as an induced gene in the wild type, but this 24

central regulator was not upregulated in the ∆gcnE strain. To get further details and to confirm 25

transcriptome data, we studied the expression of brlA by RT-qPCR in the wild type and the gcnE 26

16

mutant strain grown under the same conditions as employed for the microarray experiment and, in 1

addition, in cultures harvested 72 h after induction of conidiation (Figure 5A). In the wild type 2

strain brlA expression was high at both time points whereas the ∆gcnE strain did not show any 3

accumulation of brlA mRNA after 10 h. Interestingly, brlA mRNA can be detected in this strain at 4

72 h post-induction (still around 30-fold lower than in the wild type strain), which is in agreement 5

with microarray data and with the ΔgcnE phenotype, in which some conidia are produced upon 6

prolongued incubation on solid media . 7

To find out whether the effect of gcnE deletion on brlA is direct or indirect, we determined the 8

acetylation pattern of histone H3K9 and H3K14 at the brlA promoter by ChIP. Because the 9

promoter of brlA covers more than 2 kb from the ATG of brlAα (GARZIA et al. 2010; KWON et al. 10

2010), we employed two different primer pairs. Primers brlAp1 were located at a distal position 11

from the ATG of brlAα (–2303 to -2483 bps), spanning the FlbB binding site, while primers brlAp3 12

were located at a proximal position to the ATG (-60 to -245 bps). The acetylation levels of H3K9 13

and H3K14 increased after induction of conidiation in both regions of the promoter in the wild type 14

strain (Figure 5B). The levels of acetylation were higher at 10 h than at 72 h after induction of 15

conidiation. The overall acetylation pattern was similar in both regions of the promoter although the 16

levels were higher in the proximal region to the ATG than in the distal region. This can be 17

explained by the fact that highly acetylated nucleosomes +1 in the open reading frames are not 18

evicted during transcriptional activation (in contrast to promoter nucleosomes) (WORKMAN 2006), 19

and the DNA fragments encompassing nucleosome + 1 are captured by the proximal PCR primers. 20

On the contrary, in the ∆gcnE strain the acetylation levels at the promoter of brlA were lower than 21

in the wild type strain, and did not increase over the basal levels of the wild type strain growing 22

vegetatively. This was consistent with the lack of brlA expression and consequently, with the 23

absence of conidiophore formation in the mutant. Analysis of the total amount of histone H3 at the 24

brlA promoter by ChIP revealed that the total amount of histone H3 decreased in the wild type after 25

induction of conidiation and reached a minimum at 72 h (Figure S7). Opposite to the wild type 26

17

strain, the total levels of histone H3 did not decrease, but even increased in ∆gcnE strain upon 1

induction of conidiation consistent with an inactive promoter (Figure S7). 2

3

Inhibitors of histone deacetylation do not recover conidiation in ΔgcnE 4

Surprisingly, although the histone H3K9 and H3K14 acetylation levels were below the wild type 5

basal levels in the ∆gcnE strain, there was a slight increase in acetylation after the induction of 6

conidiation. We reasoned that alternative histone H3 acetyl-transferases may be operating at these 7

genes under induction conditions as around 40 putative acetyl transferases are present in the 8

genome of A. nidulans (NÜTZMANN et al. 2011). To test this possibility, we employed HDAC 9

inhibitors to block deacetylation. This presumably would lead to increased acetylation levels of 10

histone H3 and may recover conidiation in the ∆gcnE mutant. Trichostatin A was already shown to 11

be an effective HDAC inhibitor in A. nidulans in previous studies (SHWAB et al. 2007). Addition of 12

trichostatin A or a cocktail of inhibitors (trichostatin A + butyric acid + valproate) did not result in 13

restoration of conidiation, not even partially (Figure S8). Therefore, the most plausible explanation 14

is that the histone H3 acetylation levels in the ∆gcnE strain corresponded to background levels and 15

that a functional SAGA complex is necessary for brlA expression. 16

17

Expression of the upstream regulatory genes controlling conidiation in the ∆gcnE strain is 18

deregulated 19

There are three parallel routes for the activation of conidiation, consisting of FlbA, FlbB/D and 20

FlbC (ADAMS et al. 1998; ETXEBESTE et al. 2010). The transcriptome studies revealed a slight up-21

regulation of flbC, one of the upstream factors for conidiation genes and that this pattern was 22

affected by the gcnE deletion (Table 1). To get further details and to confirm transcriptome data, we 23

tested the expression of flbC and four of the other upstream regulators in the wild type and gcnE 24

mutant strain grown under the conditions described above in Figure 5. As shown in Figure 6, none 25

of these genes were upregulated in the wild type strain after 10 h of induction (the conditions used 26

18

for the microarray experiment). However, after a longer period of induction (72 h) four of these 1

genes (flbA, flbB, flbC and flbD) showed higher steady-state mRNA levels compared to vegetative 2

mycelia or after 10h induction in the wild type strain. When mRNA levels of these 5 genes were 3

compared between both strains in vegetative mycelia, flbA showed a three-fold higher expression in 4

the ∆gcnE strain than in the wild type, whereas the other genes were basically identical. There was 5

no significant difference between wild type and ∆gcnE in expression of the upstream regulators at 6

time point 10 h induction with the exception of flbC, but interestingly, at 72 h, flbA expression is 7

reduced whereas flbB expression is around two-fold higher in the gcnE mutant compared to the wild 8

type strain. In the case of fluG, there was a big variation from sample to sample at the 72h time 9

point, so no conclusion could be drawn from these results. These slight differences in mRNA levels 10

suggest that the lack of conidiation in the ∆gcnE did not probably originate from defects on the 11

fluffy genes expression. This is also consistent with the fact that expression of the fluffy genes was 12

already detected in vegetative mycelia, and their transcriptional upregulation is not the critical 13

regulatory point for their function in the transcriptional activation of brlA. 14

Consistent with the moderate transcriptional activation of flbA, flbB, flbC and flbD genes, their 15

promoters showed higher levels of H3 acetylation during induction of conidiation (data not shown). 16

This increase was associated with a decrease in the amount of total histone H3 present at these 17

promoters probably due to partial eviction of nucleosomes from these regions. This difference was 18

not observed in the mutant strain which showed very low H3 occupancy at all fluffy gene promoters 19

before and after conidial induction (data not shown). 20

21

Are there additional targets mediating the GcnE effects on development? 22

During the analysis of the microarray data, we observed that the orcinol / orsellinic acid cluster 23

was not expressed in the ∆gcnE strain. This is in agreement with a previous report by Nützmann et 24

al. (NÜTZMANN et al. 2011). Diorcinol is a derivative of orsellinic acid and functions together with 25

dehydroaustinol one of the signals required for the induction of conidiation (RODRIGUEZ-URRA et 26

19

al. 2012). One possibility is that the absence of the expression of this cluster contributes to the 1

conidiation defects. However, addition of different concentrations of orcinol ranging from 50 µg to 2

50 mg did not recover the conidiation defects of the ∆gcnE strain (Figure 7A). Furthermore, the 3

deletion of the polyketide synthase orsA responsible for the biosynthesis of orsellinic acid 4

(SCHROECKH et al. 2009), a precurssor of diorcinol, did not show any conidiation defects (data not 5

shown). 6

Therefore, this result further implied that the conidiation defects of ∆gcnE strain could be 7

directly related to SAGA function at the brlA promoter. We tested this by expressing the brlA gene 8

under the control of the heterologous inducible promoter of the alcA gene at an ectopic location 9

(ADAMS et al. 1988). ∆gcnE alcA(p)::brlA strains were constructed by crossing. The parentals and 10

two strains from the progeny were grown in liquid medium for 24 h and then transferred to inducing 11

(threonine) or repressing (glucose) liquid medium and grown for further 24 h. Inspection of the 12

fungal pellets by light microscopy (Figure 7B) revealed that the wild type strain harbouring the 13

alcA(p)::brlA construct produced primitive conidiophores and conidia under inducing conditions as 14

previously reported (ADAMS et al. 1988). However, none of the ∆gcnE alcA(p)::brlA strains 15

produced any of these primitive conidiophores or conidia. The experiment was repeated twice with 16

two independent strains from the progeny. Next, we transferred the strains grown for 24 h in 17

glucose liquid media (repressing conditions) to solid media containing threonine (inducing) or 18

glucose (repressing) conditions. Not even under these conditions, the ∆gcnE alcA(p)::brlA could 19

conidiate (Figure 7C). However it is interesting that growth restriction upon induction of 20

alcA(p)::brlA on threonine was observed in both the wild type and the ∆gcnE strains 21

overexpressing brlA (Figure 7D). This effect was already observed by Adams and coworkers 22

(ADAMS et al. 1988) and may be suggestive for a BrlA role in halting vegetative growth, maybe 23

through a crosstalk with the FlbA/G-protein signalling pathway regulating vegetative growth. 24

25

Discussion 26

27

20

The data obtained during this work revealed that GcnE is the only nonessential histone modifier 1

found so far with an essential function in fungal development. In previous work we and others 2

established that histone acetylation only plays a minor role in the regulation of some selected 3

primary metabolic systems (GEORGAKOPOULOS et al. 2012; REYES-DOMINGUEZ et al. 2008) but 4

significantly regulates secondary metabolism (BOK et al. 2013; NÜTZMANN et al. 2013; NÜTZMANN 5

et al. 2011; SHWAB et al. 2007). Data presented here enlarge our picture of GcnE function to a 6

genome-wide scale and from these experiments it is becoming clear that GcnE is a minor regulator 7

of primary metabolism but an essential component to drive A. nidulans developmental processes. 8

Among the diverse set of reversible histone modifications, acetylation and methylation have been 9

the most extensively studied ones in filamentous fungi (GACEK and STRAUSS 2012). It has been 10

shown that the histone H3K9 methyltransferase ClrD and the heterochromatin-protein 1 (HepA) are 11

regulators of secondary metabolite gene clusters (GACEK and STRAUSS 2012; REYES-DOMINGUEZ et 12

al. 2010). Deletion of the histone H3K9 methyltransferase clrD or the histone H3K4 13

methyltransferase cclA in A. nidulans has no growth or conidiation phenotype (BOK et al. 2009; 14

REYES-DOMINGUEZ et al. 2010). However, a decrease in radial growth and delayed conidiation due 15

to later brlA expression is observed in the equivalent A. fumigatus clrD deletion mutant (PALMER et 16

al. 2008). The histone deacetylase RpdA is essential for growth in A. nidulans and consequently a 17

direct and unequivocal effect on development has not been tested yet (TRIBUS et al. 2010). The 18

histone H4K12 acetyltransferase EsaA is also essential for growth and cooperates with the general 19

secondary metabolite regulator LaeA to mediate histone H4 acetylation and transcriptional 20

activation of selected secondary metabolite clusters (SOUKUP et al. 2012), but due to its essential 21

nature the involvement in conidiation remains elusive. Similarly, deletion of the histone deacetylase 22

hdaA was reported to have effects on secondary metabolism but not on development (BOK et al. 23

2009; SHWAB et al. 2007). Therefore, other histone modifiers have either shown a minor role during 24

development or they are essential for growth, while GcnE is required for conidiation but not 25

essential for growth. The SAGA complex is also involved in the regulation of development in 26

21

higher eukaryotes, such as plants (SERVET et al. 2010), and metazoans (SPEDALE et al. 2012). In 1

Arabidopsis, AtGCN5 plays an essential role in the development of root and shoot and flower 2

meristems, in leaf cell differentiation and responses to light. AtGCN5 also appears to regulate the 3

expression of a large number of genes, likely mediated by direct or indirect interactions with DNA-4

binding transcription factors (SERVET et al. 2010). In metazoans it was suggested that Gcn5 may be 5

required to maintain pluripotent states and is important for the differentiation of rat mesenchymal 6

stem cells into cardiomyocytes. Indeed, loss of Gcn5 resulted in a hard-pack chromatin structure at 7

the cardiomyocyte-specific genes GATA4 and NKx2.5 and elevated levels of apoptosis during 8

embrionic development (LI et al. 2010; LIN et al. 2007). It is intriguing that, while metazoans have 9

evolved 4 HAT complexes acetylating histone H3 specialized in different cellular processes 10

(SPEDALE et al. 2012), A. nidulans only has one. 11

The SAGA complex plays a general role in transcriptional activation in yeasts. TAFII145 and 12

Gcn5 are apparently functionally redudant in yeast (LEE et al. 2000), although there is some 13

specialization of the SAGA complex in stress related genes (HUISINGA and PUGH 2004). Although 14

the SAGA complex is very similar in A. nidulans to the yeast counterpart, the role of GcnE seems 15

to be significantly different to the role of its orthologs in yeasts. Thus, according to genome-wide 16

expression analysis and the observation of mutant phenotypes, it appears that the main role of GcnE 17

is to regulate development and some specific secondary metabolism gene clusters in A. nidulans. 18

Some of the secondary metabolites can be considered as “weapons” utilized only under stressing 19

conditions in nature as a defense mechanism. For example, it was found that GcnE played a major 20

role during the induction of biosynthetic gene clusters of sterigmatocystin, terrequinone and 21

penicillin (NÜTZMANN et al. 2011). An interesting case is the polyketide orsellinic acid, which is 22

produced by A. nidulans in response to the interaction with a Streptomycete species in a GcnE-23

dependent manner (NÜTZMANN et al. 2011). Synthesis of orsellinic acid derivatives F9775A and B, 24

which is induced in a ∆veA strain, is lost in the double mutant ∆gcnE ∆veA (BOK et al. 2013). Thus, 25

the reported role of GcnE in the regulation of secondary metabolism are consistent with our 26

22

microarray expression analysis. Orsellinic acid is also the precursor of diorcinol, which makes an 1

adduct with the bioactive compound dehydroaustinol, produced by FluG, to induce conidiation 2

(RODRIGUEZ-URRA et al. 2012). Although the absence of GcnE activity could lead to a lack of this 3

conidiation inducer adduct, addition of external orcinol did not restore conidiation. In support of 4

this, the deletion of the polyketide synthase orsA responsible for the biosynthesis of orsellinic acid 5

(SCHROECKH et al. 2009) did not show any conidiation defects either. Therefore, a scenario in 6

which brlA expression is not turned on in the gcnE mutant after induction due to the lack of the 7

inducer adduct seems unlikely. Instead, GcnE appears to be responsible for histone H3K9/K14 8

acetylation at the brlA promoter, which in turn is a prerequisite for brlA expression. Consequently, 9

deletion of gcnE blunts brlA expression under inducing conditions. However this is only a part of 10

the whole picture because forced expression of brlA from the inducible alcA promoter could not 11

restore conidiation in a ∆gcnE background. As expression of upstream-regulators of the fluffy-12

family of genes (flbA, flbB, flbC, flbD and fluG) were also not significantly affected by the gcnE 13

deletion and the phenotype does not conform to the mutants of the central regulatory pathway 14

abaA-wetA either, we have to assume that other regulators of conidiation may be “hidden” targets 15

of this SAGA complex component. One such target may be the velvet complex members veA, velB 16

or velC (BAYRAM et al. 2008b) or the light receptors fphA, lreA/B or cryA (BAYRAM et al. 2008a; 17

PURSCHWITZ et al. 2008). However, we did not observe any significant change in expression of 18

these regulators and photoreceptors comparing the wild type and the gcnE mutant and these genes 19

are not even responsive to induction of conidiation in the wild type. Thus it is unlikely, that some of 20

these known genes involved in developmental regulation are targets of the SAGA complex. At the 21

moment it remains elusive which of the differentially regulated genes (apart form brlA) may be 22

responsible for the strong conidiation-deficient phenotype of the gcnE deletion strain. In addition, 23

the SAGA complex participates in more cellular functions as it is not only necessary for the 24

activation of gene expression but also for transcriptional elongation, splicing, nuclear mRNA export 25

and as a general platform for the recruitment of regulatory factors (BAKER and GRANT 2007; 26

GUNDERSON and JOHNSON 2009; GUNDERSON et al. 2011; MILLAR and GRUNSTEIN 2006). 27

23

Therefore, the conidiation defects in the ∆gcnE strain could be mediated through a combination of 1

several targets and diverse molecular activities, which deserve further investigations. 2

In this study we compared vegetative cells grown in liquid cultures immediately before and after 3

10 hours of shift to conidiation conditions and found 1,225 of genes differentially regulated, of 4

which 625 were up-regulated. Garzia et al. analysed the conidiation-specific transcriptome after 5 5

hours of induction and found 2,222 genes differentially regulated (corresponding to 20,3% of the 6

genes present in the genome), out of which only 187 were up-regulated (GARZIA et al. 2013). These 7

numbers are much higher than the 533 genes found to be differentially regulated in response to light 8

in A. nidulans (RUGER-HERREROS et al. 2011). It suggests that induction of asexual development 9

actually results in a major cellular re-programming over time. For example, master regulators of 10

carbon (creA) and nitrogen (areA) metabolism did not appear to be differentially regulated at 10 h 11

after the induction of conidiation in our analysis, but they were down-regulated after 5 h. The 12

comparison of both transcriptomic experiments at different time points suggests that in a first stage 13

there is a major down-regulation of genes expressed in the vegetative phase to produce a growth 14

arrest. In a next stage many genes are up-regulated to accommodate all morphogenetic requirements 15

for asexual reproduction. Ten hours after the transition from vegetative growth to conidiation, the 16

number of up-regulated genes aproximates to the prediction of 1,200 unique mRNAs postulated by 17

Timberlake (TIMBERLAKE 1980). However, Martinelli and Clutterbuck estimated that only between 18

45 and 150 genes are specifically required for conidiation (MARTINELLI and CLUTTERBUCK 1971). 19

This difference could be explained with genes that are not specifically required for conidiation, but 20

rather play additional roles or simply indirectly respond to the changing environmental conditions 21

(exposure to oxygen, light, solid interphase, different nutrient signalling, etc.). For example, the 22

osmotic stress MAPK hogA is still up-regulated after 10h (so it is after 5h) of induction of 23

conidiation, but not some of its targets or downstream regulators (atfA, srrA, tcsA). None of the 24

chromatin regulators known in A. nidulans appear to be regulated at the transcriptional level upon 25

induction of conidiation (gcnE, adaB, clrD, hepA, cclA, rpdA, dmtA and laeA), and they do not 26

24

require GcnE for their constitutive expression (they are not affected by the ∆gcnE deletion). 1

Noteworthily, one of the most heavily affected GO categories found in the list of genes upregulated 2

in the ∆gcnE mutant during conidiation was related to responses to stress and in particular to DNA 3

damage (nkuA, nkuB, uvsC, and other putative genes). It can be argued that GcnE is required for 4

genome stability maintenance and/or DNA repair during conidiation and consequently the absence 5

of GcnE may generate DNA damage stress. The function of the spores is not only dispersion in the 6

environment but also protection of the genome (PARK and YU 2012). Indeed, isolation of yeast 7

mutants affected in components of the SAGA/ADA complex showed a phenotype of increased 8

Rad52 foci and sister chromatid recombination (MUNOZ-GALVAN et al. 2013). Whether GcnE and 9

the SAGA complex plays similar roles in filamentous fungi is not known yet but the importance of 10

GcnE in conidiospore production may justify speculations on a similar role in these organisms. 11

In conclusion, GcnE plays an essential role in asexual development and is required for the 12

expression of the master regulator of conidiation brlA and some yet unidentified conidiation-13

specific genes. It is, to the best of our knowledge, so far the only nonessential histone modifier with 14

such a role. One of the questions to be followed up is to identify the other GcnE-dependent 15

mechanisms required for initiation of development and to elucidate the factors that recruit the 16

SAGA complex to the promoter of brlA. 17

18

19

Acknowledgements 20

21

We would like to thank Jae-Hyuk Yu, Nancy Keller, Axel Brakhage and the FGSC for sharing 22

strains. We would like to thank Juan Luis Ribas and Cristina Vaquero (Servicio de Microscopía, 23

Centro de Investigación Tecnología e Innovación, Universidad de Sevilla) for help with SEM. D.C. 24

would like to thank the University of Sevilla for supporting his stay at BOKU-University of Natural 25

Resources and Life Sciences, Vienna. At this place, work was funded by grant SFB- F37-3 from the 26

25

Austrian Science Fund FWF and grant LS12-009 (EpiMed) of the NÖ-Forschung und Bildung 1

Fund to JS. 2

3

4

References 5

6

ADAMS, T. H., M. T. BOYLAN and W. E. TIMBERLAKE, 1988 brlA is necessary and sufficient to 7

direct conidiophore development in Aspergillus nidulans. Cell 54: 353-362. 8

ADAMS, T. H., J. K. WIESER and J. H. YU, 1998 Asexual sporulation in Aspergillus nidulans. 9

Microbiol Mol Biol Rev 62: 35-54. 10

ARNAUD, M. B., M. C. CHIBUCOS, M. C. COSTANZO, J. CRABTREE, D. O. INGLIS et al., 2010 The 11

Aspergillus Genome Database, a curated comparative genomics resource for gene, protein and 12

sequence information for the Aspergillus research community. Nucleic Acids Res 38: D420-427. 13

BAKER, S. P., and P. A. GRANT, 2007 The SAGA continues: expanding the cellular role of a 14

transcriptional co-activator complex. Oncogene 26: 5329-5340. 15

BAYRAM, O., C. BIESEMANN, S. KRAPPMANN, P. GALLAND and G. H. BRAUS, 2008a More than a 16

repair enzyme: Aspergillus nidulans photolyase-like CryA is a regulator of sexual development. 17

Mol Biol Cell 19: 3254-3262. 18

BAYRAM, O., S. KRAPPMANN, M. NI, J. W. BOK, K. HELMSTAEDT et al., 2008b VelB/VeA/LaeA 19

complex coordinates light signal with fungal development and secondary metabolism. Science 20

320: 1504-1506. 21

BENJAMINI, Y., and Y. HOCHBERG, 1995 Controlling the false discovery rate: A practical and 22

powerful approach to multiple testing. J R Statist Soc B 57: 289-300. 23

BOK, J. W., Y. M. CHIANG, E. SZEWCZYK, Y. REYES-DOMINGUEZ, A. D. DAVIDSON et al., 2009 24

Chromatin-level regulation of biosynthetic gene clusters. Nat Chem Biol 5: 462-464. 25

26

BOK, J. W., A. A. SOUKUP, E. CHADWICK, Y. M. CHIANG, C. C. WANG et al., 2013 VeA and MvlA 1

repression of the cryptic orsellinic acid gene cluster in Aspergillus nidulans involves histone 3 2

acetylation. Mol Microbiol 89: 963-974. 3

CANOVAS, D., K. J. BOYCE and A. ANDRIANOPOULOS, 2011 The fungal type II myosin in 4

Penicillium marneffei, MyoB, is essential for chitin deposition at nascent septation sites but not 5

actin localization. Eukaryot Cell 10: 302-312. 6

COVE, D. J., 1966 The induction and repression of nitrate reductase in the fungus Aspergillus 7

nidulans. Biochim Biophys Acta 113: 51-56. 8

DA SILVA FERREIRA, M. E., M. R. KRESS, M. SAVOLDI, M. H. GOLDMAN, A. HARTL et al., 2006 9

The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for 10

analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell 5: 207-211. 11

DE HOON, M. J., S. IMOTO, J. NOLAN and S. MIYANO, 2004 Open source clustering software. 12

Bioinformatics 20: 1453-1454. 13

ETXEBESTE, O., A. GARZIA, E. A. ESPESO and U. UGALDE, 2010 Aspergillus nidulans asexual 14

development: making the most of cellular modules. Trends Microbiol 18: 569-576. 15

FREITAG, M., P. C. HICKEY, T. K. KHLAFALLAH, N. D. READ and E. U. SELKER, 2004 HP1 is 16

essential for DNA methylation in Neurospora. Mol Cell 13: 427-434. 17

FREITAG, M., R. L. WILLIAMS, G. O. KOTHE and E. U. SELKER, 2002 A cytosine methyltransferase 18

homologue is essential for repeat-induced point mutation in Neurospora crassa. Proc Natl Acad 19

Sci U S A 99: 8802-8807. 20

GACEK, A., and J. STRAUSS, 2012 The chromatin code of fungal secondary metabolite gene clusters. 21

Appl Microbiol Biotechnol 95: 1389-1404. 22

GARZIA, A., O. ETXEBESTE, E. HERRERO-GARCIA, R. FISCHER, E. A. ESPESO et al., 2009 23

Aspergillus nidulans FlbE is an upstream developmental activator of conidiation functionally 24

associated with the putative transcription factor FlbB. Mol Microbiol 71: 172-184. 25

27

GARZIA, A., O. ETXEBESTE, E. HERRERO-GARCIA, U. UGALDE and E. A. ESPESO, 2010 The 1

concerted action of bZip and cMyb transcription factors FlbB and FlbD induces brlA expression 2

and asexual development in Aspergillus nidulans. Mol Microbiol 75: 1314-1324. 3

GARZIA, A., O. ETXEBESTE, J. RODRIGUEZ-ROMERO, R. FISCHER, E. A. ESPESO et al., 2013 4

Transcriptional changes in the transition from vegetative cells to asexual development in the 5

model fungus Aspergillus nidulans. Eukaryot Cell 12: 311-321. 6

GEORGAKOPOULOS, P., R. A. LOCKINGTON and J. M. KELLY, 2012 SAGA complex components and 7

acetate repression in Aspergillus nidulans. G3 2: 1357-1367. 8

GEORGAKOPOULOS, P., R. A. LOCKINGTON and J. M. KELLY, 2013 The Spt-Ada-Gcn5 9

Acetyltransferase (SAGA) complex in Aspergillus nidulans. PLoS One 8: e65221. 10

GRANT, P. A., L. DUGGAN, J. COTE, S. M. ROBERTS, J. E. BROWNELL et al., 1997 Yeast Gcn5 11

functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of 12

an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev 11: 1640-1650. 13

GREWAL, S. I., and S. JIA, 2007 Heterochromatin revisited. Nat Rev Genet 8: 35-46. 14

GRIMALDI, B., P. COIRO, P. FILETICI, E. BERGE, J. R. DOBOSY et al., 2006 The Neurospora crassa 15

White Collar-1 dependent blue light response requires acetylation of histone H3 lysine 14 by 16

NGF-1. Mol Biol Cell 17: 4576-4583. 17

GUNDERSON, F. Q., and T. L. JOHNSON, 2009 Acetylation by the transcriptional coactivator Gcn5 18

plays a novel role in co-transcriptional spliceosome assembly. PLoS Genet 5: e1000682. 19

GUNDERSON, F. Q., E. C. MERKHOFER and T. L. JOHNSON, 2011 Dynamic histone acetylation is 20

critical for cotranscriptional spliceosome assembly and spliceosomal rearrangements. Proc Natl 21

Acad Sci U S A 108: 2004-2009. 22

HAMMER, Ø., D. HARPER and P. D. RYAN, 2001 PAST:Paleontological Statistics Software Package 23

for Education and Data Analysis. Palaeontologia Electronica 4: 9pp. 24

28

HONDA, S., Z. A. LEWIS, M. HUARTE, L. Y. CHO, L. L. DAVID et al., 2010 The DMM complex 1

prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa. 2

Genes Dev 24: 443-454. 3

HUISINGA, K. L., and B. F. PUGH, 2004 A genome-wide housekeeping role for TFIID and a highly 4

regulated stress-related role for SAGA in Saccharomyces cerevisiae. Mol Cell 13: 573-585. 5

KRAPPMANN, S., C. SASSE and G. H. BRAUS, 2006 Gene targeting in Aspergillus fumigatus by 6

homologous recombination is facilitated in a nonhomologous end- joining-deficient genetic 7

background. Eukaryot Cell 5: 212-215. 8

KRIJGSHELD, P., R. BLEICHRODT, G. J. VAN VELUW, F. WANG, W. H. MULLER et al., 2013 9

Development in Aspergillus. Stud Mycol 74: 1-29. 10

KUO, M. H., J. E. BROWNELL, R. E. SOBEL, T. A. RANALLI, R. G. COOK et al., 1996 Transcription-11

linked acetylation by Gcn5p of histones H3 and H4 at specific lysines. Nature 383: 269-272. 12

KURSHAKOVA, M. M., A. N. KRASNOV, D. V. KOPYTOVA, Y. V. SHIDLOVSKII, J. V. NIKOLENKO et 13

al., 2007 SAGA and a novel Drosophila export complex anchor efficient transcription and 14

mRNA export to NPC. EMBO J 26: 4956-4965. 15

KWON, N. J., A. GARZIA, E. A. ESPESO, U. UGALDE and J. H. YU, 2010 FlbC is a putative nuclear 16

C(2)H(2) transcription factor regulating development in Aspergillus nidulans. Mol Microbiol 77: 17

1203-1219. 18

LEE, B. N., and T. H. ADAMS, 1994 The Aspergillus nidulans fluG gene is required for production 19

of an extracellular developmental signal and is related to prokaryotic glutamine synthetase I. 20

Genes Dev 8: 641-651. 21

LEE, I., J. H. OH, E. K. SHWAB, T. R. DAGENAIS, D. ANDES et al., 2009 HdaA, a class 2 histone 22

deacetylase of Aspergillus fumigatus, affects germination and secondary metabolite production. 23

Fungal Genet Biol 46: 782-790. 24

LEE, T. I., H. C. CAUSTON, F. C. HOLSTEGE, W. C. SHEN, N. HANNETT et al., 2000 Redundant roles 25

for the TFIID and SAGA complexes in global transcription. Nature 405: 701-704. 26

29

LI, L., J. ZHU, J. TIAN, X. LIU and C. FENG, 2010 A role for Gcn5 in cardiomyocyte differentiation 1

of rat mesenchymal stem cells. Mol Cell Biochem 345: 309-316. 2

LIN, W., G. SRAJER, Y. A. EVRARD, H. M. PHAN, Y. FURUTA et al., 2007 Developmental potential 3

of Gcn5(-/-) embryonic stem cells in vivo and in vitro. Dev Dyn 236: 1547-1557. 4

MARTINELLI, S. D., and A. J. CLUTTERBUCK, 1971 A quantitative survey of conidiation mutants in 5

Aspergillus nidulans. J Gen Microbiol 69: 261-268. 6

MEDINA, I., J. CARBONELL, L. PULIDO, S. C. MADEIRA, S. GOETZ et al., 2010 Babelomics: an 7

integrative platform for the analysis of transcriptomics, proteomics and genomic data with 8

advanced functional profiling. Nucleic Acids Res 38: W210-213. 9

MELIN, P., J. SCHNURER and E. G. WAGNER, 2003 Characterization of phiA, a gene essential for 10

phialide development in Aspergillus nidulans. Fungal Genet Biol 40: 234-241. 11

MILLAR, C. B., and M. GRUNSTEIN, 2006 Genome-wide patterns of histone modifications in yeast. 12

Nat Rev Mol Cell Biol 7: 657-666. 13

MIRABITO, P. M., T. H. ADAMS and W. E. TIMBERLAKE, 1989 Interactions of three sequentially 14

expressed genes control temporal and spatial specificity in Aspergillus development. Cell 57: 15

859-868. 16

MUNOZ-GALVAN, S., S. JIMENO, R. ROTHSTEIN and A. AGUILERA, 2013 Histone H3K56 17

acetylation, Rad52, and non-DNA repair factors control double-strand break repair choice with 18

the sister chromatid. PLoS Genet 9: e1003237. 19

NAYAK, T., E. SZEWCZYK, C. E. OAKLEY, A. OSMANI, L. UKIL et al., 2006 A versatile and efficient 20

gene targeting system for Aspergillus nidulans. Genetics 172: 1557-1566. 21

NINOMIYA, Y., K. SUZUKI, C. ISHII and H. INOUE, 2004 Highly efficient gene replacements in 22

Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci U S A 101: 23

12248-12253. 24

30

NÜTZMANN, H. W., J. FISCHER, K. SCHERLACH, C. HERTWECK and A. A. BRAKHAGE, 2013 Distinct 1

Amino Acids of Histone H3 Control Secondary Metabolism in Aspergillus nidulans. Appl 2

Environ Microbiol 79: 6102-6109. 3

NÜTZMANN, H. W., Y. REYES-DOMINGUEZ, K. SCHERLACH, V. SCHROECKH, F. HORN et al., 2011 4

Bacteria-induced natural product formation in the fungus Aspergillus nidulans requires 5

Saga/Ada-mediated histone acetylation. Proc Natl Acad Sci U S A 108: 14282-14287. 6

PALMER, J. M., R. M. PERRIN, T. R. DAGENAIS and N. P. KELLER, 2008 H3K9 methylation 7

regulates growth and development in Aspergillus fumigatus. Eukaryot Cell 7: 2052-2060. 8

PARK, H. S., and J. H. YU, 2012 Genetic control of asexual sporulation in filamentous fungi. Curr 9

Opin Microbiol 15: 669-677. 10

PONTECORVO, G., J. A. ROPER, L. M. HEMMONS, K. D. MACDONALD and A. W. BUFTON, 1953 The 11

genetics of Aspergillus nidulans. Adv Genet 5: 141-238. 12

PURSCHWITZ, J., S. MULLER, C. KASTNER, M. SCHOSER, H. HAAS et al., 2008 Functional and 13

physical interaction of blue- and red-light sensors in Aspergillus nidulans. Curr Biol 18: 255-14

259. 15

REYES-DOMINGUEZ, Y., S. BOEDI, M. SULYOK, G. WIESENBERGER, N. STOPPACHER et al., 2012 16

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium 17

graminearum. Fungal Genet Biol 49: 39-47. 18

REYES-DOMINGUEZ, Y., J. W. BOK, H. BERGER, E. K. SHWAB, A. BASHEER et al., 2010 19

Heterochromatic marks are associated with the repression of secondary metabolism clusters in 20

Aspergillus nidulans. Mol Microbiol 76: 1376-1386. 21

REYES-DOMINGUEZ, Y., F. NARENDJA, H. BERGER, A. GALLMETZER, R. FERNANDEZ-MARTIN et al., 22

2008 Nucleosome positioning and histone H3 acetylation are independent processes in the 23

Aspergillus nidulans prnD-prnB bidirectional promoter. Eukaryot Cell 7: 656-663. 24

31

RODRIGUEZ-NAVARRO, S., T. FISCHER, M. J. LUO, O. ANTUNEZ, S. BRETTSCHNEIDER et al., 2004 1

Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-2

associated mRNA export machinery. Cell 116: 75-86. 3

RODRIGUEZ-URRA, A. B., C. JIMENEZ, M. I. NIETO, J. RODRIGUEZ, H. HAYASHI et al., 2012 4

Signaling the induction of sporulation involves the interaction of two secondary metabolites in 5

Aspergillus nidulans. ACS Chem Biol 7: 599-606. 6

ROUNTREE, M. R., and E. U. SELKER, 2010 DNA methylation and the formation of heterochromatin 7

in Neurospora crassa. Heredity (Edinb) 105: 38-44. 8

RUGER-HERREROS, C., J. RODRIGUEZ-ROMERO, R. FERNANDEZ-BARRANCO, M. OLMEDO, R. 9

FISCHER et al., 2011 Regulation of conidiation by light in Aspergillus nidulans. Genetics 188: 10

809-822. 11

SCHINKO, T., H. BERGER, W. LEE, A. GALLMETZER, K. PIRKER et al., 2010 Transcriptome analysis 12

of nitrate assimilation in Aspergillus nidulans reveals connections to nitric oxide metabolism. 13

Mol Microbiol 78: 720-738. 14

SCHROECKH, V., K. SCHERLACH, H. W. NUTZMANN, E. SHELEST, W. SCHMIDT-HECK et al., 2009 15

Intimate bacterial-fungal interaction triggers biosynthesis of archetypal polyketides in 16

Aspergillus nidulans. Proc Natl Acad Sci U S A 106: 14558-14563. 17

SERVET, C., N. CONDE E SILVA and D. X. ZHOU, 2010 Histone acetyltransferase AtGCN5/HAG1 is 18

a versatile regulator of developmental and inducible gene expression in Arabidopsis. Mol Plant 19

3: 670-677. 20

SHWAB, E. K., J. W. BOK, M. TRIBUS, J. GALEHR, S. GRAESSLE et al., 2007 Histone deacetylase 21

activity regulates chemical diversity in Aspergillus. Eukaryot Cell 6: 1656-1664. 22

SMYTH, G. K., 2005 Limma: linear models for microarray data., pp. 397-420 in Computational 23

Biology Solutions using R and Bioconductor, edited by V. C. R. GENTLEMAN, S. DUDOIT, R. 24

IRIZARRY, W. HUBER. Springer, New York. 25

32

SOUKUP, A. A., Y. M. CHIANG, J. W. BOK, Y. REYES-DOMINGUEZ, B. R. OAKLEY et al., 2012 1

Overexpression of the Aspergillus nidulans histone 4 acetyltransferase EsaA increases activation 2

of secondary metabolite production. Mol Microbiol 86: 314-330. 3

SPEDALE, G., H. T. TIMMERS and W. W. PIJNAPPEL, 2012 ATAC-king the complexity of SAGA 4

during evolution. Genes Dev 26: 527-541. 5

STRAUSS, J., and Y. REYES-DOMINGUEZ, 2010 Regulation of secondary metabolism by chromatin 6

structure and epigenetic codes. Fungal Genet Biol in press. 7

TAMARU, H., and E. U. SELKER, 2001 A histone H3 methyltransferase controls DNA methylation in 8

Neurospora crassa. Nature 414: 277-283. 9

TIMBERLAKE, W. E., 1980 Developmental gene regulation in Aspergillus nidulans. Dev Biol 78: 10

497-510w. 11

TRIBUS, M., I. BAUER, J. GALEHR, G. RIESER, P. TROJER et al., 2010 A novel motif in fungal class 1 12

histone deacetylases is essential for growth and development of Aspergillus. Mol Biol Cell 21: 13

345-353. 14

TSITSIGIANNIS, D. I., R. ZARNOWSKI and N. P. KELLER, 2004 The lipid body protein, PpoA, 15

coordinates sexual and asexual sporulation in Aspergillus nidulans. J Biol Chem 279: 11344-16

11353. 17

WILSON, R. A., P. K. CHANG, A. DOBRZYN, J. M. NTAMBI, R. ZARNOWSKI et al., 2004 Two Delta9-18

stearic acid desaturases are required for Aspergillus nidulans growth and development. Fungal 19

Genet Biol 41: 501-509. 20

WORKMAN, J. L., 2006 Nucleosome displacement in transcription. Genes Dev 20: 2009-2017. 21

YU, J. H., J. H. MAH and J. A. SEO, 2006 Growth and developmental control in the model and 22

pathogenic aspergilli. Eukaryot Cell 5: 1577-1584. 23

YU, J. H., J. WIESER and T. H. ADAMS, 1996 The Aspergillus FlbA RGS domain protein 24

antagonizes G protein signaling to block proliferation and allow development. Embo J. 15: 25

5184-5190. 26

33

1

Table 1 Differentially regulated genes that are involved in development (sexual or asexual) 2

and secondary metabolism in A. nidulans. Absent values indicate that the gene is not 3

differentially regulated under those conditions in that strain in particular. 4

5

WT

veg vs con

∆gcnE

veg vs con

gene

name

gene

code

qmedian1 Log

qmedian

qmedian Log

qmedian

Description of gene function

dewA AN8006 160.8 5.1 - - Hydrophobin, protein of the conidium

wall responsible for hydrophobicity of

conidium surface.

yA AN6635 28.2 3.3 - - Conidial laccase (p-diphenol oxidase)

involved in dark green pigment

production of conidium wall.

pclA AN0453 11.6 2.4 5.4 1.7 G1/S cyclin; mutants produce abnormal

conidiophores with extra layers of

phialides

ivoB AN0231 11.1 2.4 3.7 1.3 Conidiophore-specific phenol oxidase.

wetA AN1937 11.0 2.4 - - Regulatory protein involved in conidial

development.

aflR AN7820 10.0 2.3 - - Transcriptional activator of the

sterigmatocystin biosynthesis gene

cluster.

abaA AN0422 8.9 2.2 - - TEA/ATTS domain transcriptional

34

activator involved in regulation of

conidiation; required for phialide

differentiation.

brlA AN0973 5.9 1.8 - - C2H2 zinc finger transcription factor,

master regulator of conidiophore

development.

rodA AN8803 5.5 1.7 - - Hydrophobin

medA AN6230 5.3 1.7 - - Protein involved in regulation of

conidiophore development; required for

normal temporal expression of brlA.

hogA AN1017 4.2 1.4 - - Mitogen-activated protein kinase

(MAPK) involved in osmotic stress

response; required for sexual

development and conidiation.

flbC AN2421 3.7 1.3 - - C2H2 zinc finger transcription factor;

involved in regulation of conidiophore

development.

vosA AN1959 3.6 1.3 - - Nuclear protein involved in spore

formation and trehalose accumulation.

imeB AN6243 0.4 -0.9 - - Serine/threonine protein kinase involved

in light-mediated regulation of sexual

development and sterigmatocystin

production

nsdC AN4263 0.3 -1.2 - - C2H2 zinc finger transcription factor;

required for sexual development.

35

laeA AN0807 0.2 -1.9 - - Methyltransferase-domain protein;

velvet complex component composed of

VelB, VeA and LaeA; coordinates

asexual development in response to

light; regulates secondary metabolism

and is required for Hülle cell formation

pipA AN2513 0.1 -1.9 - - Serine/threonine protein kinase involved

in hyphal growth and asexual

development

sskA AN7697 - - 0.2 -1.6 Response regulator, part of a two-

component signal transducer involved in

the HOG signaling pathway that

regulates osmotic stress response; null

spores are heat labile and lose viability

at 4 degrees

rosA AN5170 - - 13.6 2.6 Zn(II)2Cys6 transcription factor;

negative regulator of sexual

development

1

1 qmedian values higher than 1 (or positive log qmedian values) indicate that the gene is expressed 2

during conidiation, while qmedian values lower than 1 (or negative log qmedian values) indicate 3

that the gene is expressed during vegetative growth. An absence of value indicates that gene is not 4

differentially expressed in that strain. 5

- Not differentially regulated in the strain. 6

7

36

Figure legends 1

2

Figure 1. Simplified model of the genetic regulation of conidiation. 3

Only some of the regulators studied in this work are shown for clarity. FluG is responsible for the 4

synthesis of an extracellular factor that induces the rest of the fluffy genes in the three parallel 5

routes. FlbE (not shown) interacts and activates FlbB. FlbB and FlbD are transcription factors that 6

jointly bind to the promoter of brlA activating its transcription. FlbC is another transcription factor 7

activating the expression of brlA. FlbA is a regulator of G protein activity that positively regulates 8

the transcription of brlA. Activation of brlA is necessary and sufficient to induce conidiation. Ovals 9

indicate the promoter regions and in front of brlA correspond for the two sites analysed by ChIP. 10

11

12

Figure 2. The ∆gcnE mutant is impaired in conidiation. 13

(A) Wild type (WT) and ∆gcnE strains were grown vegetatively for 18 h and then conidiation was 14

induced in complete medium. Progression of the developmental program was followed under the 15

stereo microscope at the indicated time points. Conidiophore heads were evident after 10 h of 16

induction in the wild type strain. Yellow conidia were evident 24 h after induction. No such 17

structures were seen in the ∆gcnE strain even after 72 h of induction. 18

(B) Comparision of the conidiation phenotype of wild type and ∆gcnE strain with the phenotypes of 19

the mutants in the central regulatory pathway (∆brlA, ∆abaA or ∆wetA) after four days of growth. 20

The brlA mutant produced the stalk cells than continued growing rather than developing the 21

conidiophore vesicles, metulae, phialides and conidia. Mutations in abaA and wetA interfered in 22

later stages of the conidiophore development and were capable of producing white structures 23

corresponding to the vesicles, metulae and phialides. The ∆gcnE strain resembles a ∆brlA 24

phenotype. 25

37

(C) SEM images of the wild type and ∆gcnE strains grown for one week. A very low density of 1

immature conidiophores can be observed in the ∆gcnE strain, compared to the complete 2

development of the wild type conidiophores. Bar = 50 µm. 3

(D) Details of SEM images comparing the wild type conidiophores with the aberrant ∆gcnE 4

conidiophore morphologies (indicated by arrows). Arrows indicate details of aberrant 5

conidiophores. The double lined arrow points to a severe example, where sterigmata cells seem to 6

bud off from a hyphal or stalk cell. A higher magnification of this example is shown as a separate 7

image on the top right panel. Bar = 10 µm, except for the top right image where the bar corresponds 8

to 5 µm. 9

10

Figure 3. Differences in growth rate do not explain the conidiation defects in ∆gcnE. 11

(A) Growth of wild type and ∆gcnE strains was followed on complete and minimal solid media 12

over a period of 5 days. The linear growth rate of the mutant was only slightly lower in comparison 13

with the wild type on both media. The growth rate is shown as the increment in the colony diameter 14

on solid media per day. Error bars show the standard error of at least 3 independent experiments 15

performed in duplicates. 16

(B) Wild type and ∆gcnE strains were point inoculated on complete media plates and allowed to 17

grow at 37 ºC. Plates were photographed after the colonies reached the same size. 18

19

Figure 4. Global expression analysis of wild type and ∆gcnE strain growing under vegetative or 20

conidiation conditions. 21

Both strains were grown vegetatively for 18 h and then, conidiation was induced for 10 h. The 22

global expression of genes in the 4 conditions (wild type vegetative, WT-VEG; wild type-23

conidiation, WT-CON; ∆gcnE vegetative, GCN-VEG; ∆gcnE conidiation, GCN-CON) was 24

compared by using microarray hibridization. 1,162 differentially expressed genes were identified by 25

ANOVA. (A) A dendogram was obtained by centroid linkage clustering using euclidean distances 26

38

of the 1,162 differentially regulated genes in the 12 samples (4 conditions with 3 biological 1

replicates each). The ∆gcnE strain grown under conidiation conditions was more similar to 2

vegetative growth than to the conidiating wild type. (B) Principal Component (PC) analysis of the 3

genes differentially regulated in at least one of the 4 different conditions. The X-axis shows the first 4

PC with a variation of 49% due to the growth mode (vegetative vs conidiation). The Y-axis shows 5

the second PC with a variation of 26% due to the genetic background (wild type vs ∆gcnE). The 6

results obtained by clustering (A) and PC (B) analysis are in agreement. 7

8

Figure 5. brlA is not expressed and acetylation of histone H3K9/K14 at the brlA promoter is 9

reduced in the ∆gcnE strain. 10

(A) Both wild type and ∆gcnE strains were grown vegetatively for 18h and then conidiation was 11

induced for 10 h or 72 h. RNA was isolated and gene expression was quantified by RT-qPCR. Data 12

is shown normalized to the tubulin gene (benA) as an internal standard. 13

(B) ChIP was carried out by immunoprecipitation of crosslinked DNA with an antibody recognizing 14

acetylated histone H3K9ac and H3K14ac, followed by qPCR analysis of the promoter regions. brlA 15

showed an increase in the immunoprecipitated DNA in both distal (brlAp1) and proximal (brlAp3) 16

regions of the promoter in the wild type. In the ∆gcnE strain acetylation levels were grossly reduced 17

and conidiation-specific increases were not observed. Values were normalized to input DNA 18

(before IP) and are shown as the mean with standard errors of the mean of at least 3 biologically 19

independent experiments. 20

21

Figure 6. Expression of the fluffy genes during conidiation is de-regulated in the ∆gcnE strain. 22

Both wild type and ∆gcnE strains were grown vegetatively for 18h and then conidiation was 23

induced for 10 h or 72 h. RNA was isolated and gene expression was quantified by RT-qPCR. Data 24

are shown normalized to the tubulin gene (benA) as an internal standard. Values are the mean and 25

standard error of the mean of at least 3 independent experiments. 26

39

1

Figure 7. GcnE has additional yet-to-know targets mediating the developmental effects. 2

(A) Wild type and ∆gcnE strains were pre-grown for 24h before addition of different concentrations 3

of orcinol (50 µg to 50 mg) on top of the colony. Plates were incubated for 3 additional days and 4

photographed. The highest concentration of orcinol had some slightly negative effects on colony 5

development in both strains. 6

(B) Strains indicated at the left of the panels were pre-grown for 24 h in liquid media under 7

repressing conditions (glucose) and then transferred to fresh liquid medium containing inducing 8

threonine or repressing glucose and incubation continued for additional 24 hours. Fungal pellets 9

were photographed under the light microscope. The parental strains harboured either a construct 10

overexpressing brlA from the alcA promoter (OE::brlA) or the gcnE deletion (∆gcnE). Two 11

independent strains of the cross progeny (DKA234, DKA235) were used in this experiment. Black 12

arrows indicate conidiophore-like structures, black arrowheads point to individual conidia produced 13

in liquid cultures, white dotted arrows point to vegetative hyphal tips. 14

(C) Strains were pre-grown as in panel B for 24 h under repressing condintions but then transferred 15

to solid medium containing threonine or glucose, and incubation continued for one day. Fungal 16

colonies were photographed under a stereo microscope at the same magnification. The OEbrlA 17

strain (brlA+; alcA(p)::brlA) conidiated on glucose plates due to brlA expression from its native 18

promoter. Two independent strains of the progeny were also used in this experiment. 19

(D) Strains pre-grown for 24 h under repressing condintions (as in panel B) were transferred to 20

solid medium containing threonine or glucose and incubation continued. Plates were photographed 21

after 3 days of growth. Growth inhibition could be observed in the strains overexpressing brlA in 22

both wild type and ∆gcnE background only under brlA-inducing conditions (threonine). 23

24

25

Figure 1

Conidiation brlA

FlbA FlbB

FluG FluG factor fluG

FlbD FlbC

BrlA

flbD flbB flbC flbA

Figure 1. Simplified model of the genetic regulation of conidiation. Only some of the regulators studied in this work are shown for clarity. FluG is responsible for the synthesis of an extracellular factor that induces the rest of the fluffy genes in the three parallel routes. FlbE (not shown) interacts and activates FlbB. FlbB and FlbD are transcription factors that jointly bind to the promoter of brlA activating its transcription. FlbC is another transcription factor activating the expression of brlA. FlbA is a regulator of G protein activity that positively regulates the transcription of brlA. Activation of brlA is necessary and sufficient to induce conidiation. Ovals indicate the promoter regions and in front of brlA correspond for the two sites analysed by ChIP.

10 h 24h 48h 72hW

T∆gcnE

Figure 2A

B

∆brlA

∆abaA ∆wetA

WT

∆gcnE

∆brlA

∆abaA ∆wetA

WT

∆gcnE

Figure 2. The ∆gcnE mutant is impaired in conidiation.(A) Wild type (WT) and ∆gcnE strains were grown vegetatively for 18 h and then conidiation was induced in complete medium.Progression of the developmental program was followed under the stereo microscope at the indicated time points. Conidiophoreheads were evident after 10 h of induction in the wild type strain. Yellow conidia were evident 24 h after induction. No suchstructures were seen in the ∆gcnE strain even after 72 h of induction.(B) Comparision of the conidiation phenotype of wild type and ∆gcnE strain with the phenotypes of the mutants in the centralregulatory pathway (∆brlA, ∆abaA or ∆wetA) after four days of growth. The brlA mutant produced the stalk cells than continuedgrowing rather than developing the conidiophore vesicles, metulae, phialides and conidia. Mutations in abaA and wetA interfered inlater stages of the conidiophore development and were capable of producing white structures corresponding to the vesicles, metulaeand phialides. The ∆gcnE strain resembles a ∆brlA phenotype.

∆gcnE

WT

WT

∆gcnE ∆gcnE

∆gcnE

Figure 2

C

D

(C) SEM images of the wild type and ∆gcnE strains grown for one week. A very low density of immature conidiophores can beobserved in the ∆gcnE strain, compared to the complete development of the wild type conidiophores. Bar = 50 µm.(D) Details of SEM images comparing the wild type conidiophores with the aberrant ∆gcnE conidiophore morphologies (indicated byarrows). Arrows indicate details of aberrant conidiophores. The double lined arrow points to a severe example, where sterigmata cellsseem to bud off from a hyphal or stalk cell. A higher magnification of this example is shown as a separate image on the top right panel.Bar = 10 µm, except for the top right image where the bar corresponds to 5 µm.

Figure 3

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

WT ∆gcnE WT ∆gcnE

Col

ony

grow

th ra

te

(cm

/day

)

Complete medium

Minimal medium

A

B WT ∆gcnE

Figure 3. Differences in growth rate do not explain the conidiation defects in ∆gcnE. (A) Growth of wild type and ∆gcnE strains was followed on complete and minimal solid media over a period of 5 days. The linear growth rate of the mutant was only slightly lower in comparison with the wild type on both media. The growth rate is shown as the increment in the colony diameter on solid media per day. Error bars show the standard error of at least 3 independent experiments performed in duplicates. (B) Wild type and ∆gcnE strains were point inoculated on complete media plates and allowed to grow at 37 ºC. Plates were photographed after the colonies reached the same size.

A Figure 4

B

-30 -25 -20 -15 -10 -5 0 5

10 15 20

-20 -15 -10 -5 0 5 10 15 20 25 30 35 40 1st PC 49%

2nd

PC 2

6%

WT-CON

GCN-CON

WT-VEG

GCN-VEG

WT-CON

GCN-CON

WT-VEG

GCN-VEG

0.1

Figure 4. Global expression analysis of wild type and ∆gcnE strain growing under vegetative or conidiation conditions. Both strains were grown vegetatively for 18 h and then, conidiation was induced for 10 h. The global expression of genes in the 4 conditions (wild type vegetative, WT-VEG; wild type-conidiation, WT-CON; ∆gcnE vegetative, GCN-VEG; ∆gcnE conidiation, GCN-CON) was compared by using microarray hibridization. 1,162 differentially expressed genes were identified by ANOVA. (A) A dendogram was obtained by centroid linkage clustering using euclidean distances of the 1,162 differentially regulated genes in the 12 samples (4 conditions with 3 biological replicates each). The ∆gcnE strain grown under conidiation conditions was more similar to vegetative growth than to the conidiating wild type. (B) Principal Component (PC) analysis of the genes differentially regulated in at least one of the 4 different conditions. The X-axis shows the first PC with a variation of 49% due to the growth mode (vegetative vs conidiation). The Y-axis shows the second PC with a variation of 26% due to the genetic background (wild type vs ∆gcnE). The results obtained by clustering (A) and PC (B) analysis are in agreement.

A

B

brlA expression

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

rel.

exp.

(A.U

.)

veg con 10h con 72h veg con 10h con 72h

WT ∆gcnE

Figure 5

0  

1  

2  

3  

4  

5  

WT 0h   WT 10h   WT 72h   ∆  g  c  n  E  0  h   ∆  g  c  n  E  1  0  h   ∆  g  c  n  E  7  2  h  veg con 10h con 72h veg con 10h con 72h

WT ∆gcnE

H3K9ac/K14ac / input

Rel

. am

ount

o

f IP

DN

A

brlAp1 brlAp3

Figure 5. brlA is not expressed and acetylation of histone H3K9/K14 at the brlA promoter is reduced in the ∆gcnE strain. (A) Both wild type and ∆gcnE strains were grown vegetatively for 18h and then conidiation was induced for 10 h or 72 h. RNA was isolated and gene expression was quantified by RT-qPCR. Data is shown normalized to the tubulin gene (benA) as an internal standard. (B) ChIP was carried out by immunoprecipitation of crosslinked DNA with an antibody recognizing acetylated histone H3K9ac and H3K14ac, followed by qPCR analysis of the promoter regions. brlA showed an increase in the immunoprecipitated DNA in both distal (brlAp1) and proximal (brlAp3) regions of the promoter in the wild type. In the ∆gcnE strain acetylation levels were grossly reduced and conidiation-specific increases were not observed. Values were normalized to input DNA (before IP) and are shown as the mean with standard errors of the mean of at least 3 biologically independent experiments.

flbA

0 0.1 0.2 0.3 0.4 0.5

Rel

. Exp

. (A

.U.)

flbB

0 0.05 0.10 0.15 0.20 0.25

Rel

. Exp

. (A

.U.)

flbC

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07

Rel

. Exp

. (A

.U.)

flbD

0 0.2 0.4 0.6 0.8 1.0 1.2

Rel

. Exp

. (A

.U.)

fluG

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Rel

. Exp

. (A

.U.)

veg con 10h

con 72h

veg con 10h

con 72h

WT ∆gcnE

Figure 6 Gene expression

Figure 6. Expression of the fluffy genes during conidiation is de-regulated in the ∆gcnE strain. Both wild type and ∆gcnE strains were grown vegetatively for 18h and then conidiation was induced for 10 h or 72 h. RNA was isolated and gene expression was quantified by RT-qPCR. Data are shown normalized to the tubulin gene (benA) as an internal standard. Values are the mean and standard error of the mean of at least 3 independent experiments.

WT

∆gcnE

Orcinol - Figure 7

B

A

Glucose Threonine

OE:

:brlA

∆g

cnE

DK

A23

4 ∆g

cnE

OE:

:brlA

DK

A23

5 ∆g

cnE

OE:

:brlA

Figure 7. GcnE has additional yet-to-know targets mediating the developmental effects. (A) Wild type and ∆gcnE strains were pre-grown for 24h before addition of different concentrations of orcinol (50 µg to 50 mg) on top of the colony. Plates were incubated for 3 additional days and photographed. The highest concentration of orcinol had some slightly negative effects on colony development in both strains. (B) Strains indicated at the left of the panels were pre-grown for 24 h in liquid media under repressing conditions (glucose) and then transferred to fresh liquid medium containing inducing threonine or repressing glucose and incubation continued for additional 24 hours. Fungal pellets were photographed under the light microscope. The parental strains harboured either a construct overexpressing brlA from the alcA promoter (OE::brlA) or the gcnE deletion (∆gcnE). Two independent strains of the cross progeny (DKA234, DKA235) were used in this experiment. Black arrows indicate conidiophore-like structures, black arrowheads point to individual conidia produced in liquid cultures, white dotted arrows point to vegetative hyphal tips. (C) Strains were pre-grown as in panel B for 24 h under repressing condintions but then transferred to solid medium containing threonine or glucose, and incubation continued for one day. Fungal colonies were photographed under a stereo microscope at the same magnification. The OEbrlA strain (brlA+; alcA(p)::brlA) conidiated on glucose plates due to brlA expression from its native promoter. Two independent strains of the progeny were also used in this experiment. (D) Strains pre-grown for 24 h under repressing condintions (as in panel B) were transferred to solid medium containing threonine or glucose and incubation continued. Plates were photographed after 3 days of growth. Growth inhibition could be observed in the strains overexpressing brlA in both wild type and ∆gcnE background only under brlA-inducing conditions (threonine).

Figure 7

D

OE:

:brlA

DK

A23

4 ∆g

cnE

OE:

:brlA

DK

A23

5 ∆g

cnE

OE:

:brlA

Glucose Threonine

∆gcn

E

C

OE:

:brlA

DK

A23

5 ∆g

cnE

OE:

:brlA

Glucose Threonine

∆gcn

E