Open Access

Comparative transcriptome analysis revealing dormant conidia and germination associated genes in Aspergillus species: an essential role for AtfA in conidial dormancy

  • Daisuke Hagiwara1Email author,
  • Hiroki Takahashi1, 2,
  • Yoko Kusuya1,
  • Susumu Kawamoto1,
  • Katsuhiko Kamei1 and
  • Tohru Gonoi1
BMC Genomics201617:358

https://doi.org/10.1186/s12864-016-2689-z

Received: 9 January 2016

Accepted: 4 May 2016

Published: 17 May 2016

Abstract

Background

Fungal conidia are usually dormant unless the extracellular conditions are right for germination. Despite the importance of dormancy, little is known about the molecular mechanism underlying entry to, maintenance of, and exit from dormancy. To gain comprehensive and inter-species insights, transcriptome analyses were conducted across Aspergillus fumigatus, Aspergillus niger, and Aspergillus oryzae.

Results

We found transcripts of 687, 694, and 812 genes were enriched in the resting conidia compared with hyphae in A. fumigatus, A. niger, and A. oryzae, respectively (conidia-associated genes). Similarly, transcripts of 766, 1,241, and 749 genes were increased in the 1 h-cultured conidia compared with the resting conidia (germination-associated genes). Among the three Aspergillus species, we identified orthologous 6,172 genes, 91 and 391 of which are common conidia- and germination-associated genes, respectively. A variety of stress-related genes, including the catalase genes, were found in the common conidia-associated gene set, and ribosome-related genes were significantly enriched among the germination-associated genes. Among the germination-associated genes, we found that calA-family genes encoding a thaumatin-like protein were extraordinary expressed in early germination stage in all Aspergillus species tested here. In A. fumigatus 63 % of the common conidia-associated genes were expressed in a bZIP-type transcriptional regulator AtfA-dependent manner, indicating that AtfA plays a pivotal role in the maintenance of resting conidial physiology. Unexpectedly, the precocious expression of the germination-associated calA and an abnormal metabolic activity were detected in the resting conidia of the atfA mutant, suggesting that AtfA was involved in the retention of conidial dormancy.

Conclusions

A comparison among transcriptomes of hyphae, resting conidia, and 1 h-grown conidia in the three Aspergillus species revealed likely common factors involved in conidial dormancy. AtfA positively regulates conidial stress-related genes and negatively mediates the gene expressions related to germination, suggesting a major role for AtfA in Aspergillus conidial dormancy.

Keywords

Conidia Dormancy Germination Aspergillus AtfA Transcriptome

Background

Conidia are, in general, stress-tolerant reproductive structures, and filamentous fungi vigorously produce conidia under the appropriate conditions [1]. In the presence of water and appropriate nutrients, conidia germinate, whereas conidia enter dormancy when the environment is not appropriate. Dormant conidia are metabolically inactive and are viable for a long time (more than a year) [2]. Therefore, dormant conidia do not consume energy prior to encountering the appropriate conditions for germination. This mechanism allows conidia to find an environment where the fungi can prosper, which consequently contributes to their ubiquity and prosperity in nature. Despite its significance to conidial physiology, however, the molecular mechanisms underlying entering and exiting dormancy remain largely unknown.

To gain insights into dormancy mechanisms, genes and proteins that are specifically highly expressed in conidia, or during germination, have been investigated by transcriptomic or proteomic approaches in this decade. Van Leeuwen et al. [3] found that 4,628 out of 14,253 genes were expressed in Aspergillus niger dormant conidia using Affymetrix microarray chips. Additionally, more than half of the genes changed their expression levels at the beginning of the early germination stage (~2 h). Novodvorska et al. [4] showed that 6,519 genes (42.3 %) were differentially expressed [> twofold fragments per kilobase of transcript per million mapped reads (FPKM)] during the first hour of germination in A. niger, as detected by an RNA-sequencing analysis. Of these, 2,626 genes had increased expression levels, and functional categories related to RNA processing, protein synthesis, and nitrogen metabolism were enriched in the gene set. Proteome analyses regarding Aspergillus conidia were reported by two groups in 2010. Teutschbein et al. [5] presented a proteome of Aspergillus fumigatus’ resting conidia. They detected 449 proteins [4.7 % of predicted open reading frames (ORFs)] in the conidia and 57 were overrepresented compared with in hyphae. Interestingly, pyruvate decarboxylase and alcohol dehydrogenase were found in dormant conidia, suggesting that alcoholic fermentation might occur during dormancy. They also revealed the presence of several A. fumigatus allergens, including Asp f3, Asp f13, Asp f22, Asp f27, and CatA in the conidia. However, Oh et al. [6] investigated the Aspergillus nidulans proteome 1 h after germination and 144 proteins were found to be differentially expressed. These two proteomic data partly overlapped the data from the transcriptomic analysis, even though they were performed using different Aspergillus species under different culture conditions.

Genome sequence data with annotations for four representatives of the Aspergillus species (A. nidulans, A. fumigatus, A. niger, and Aspergillus oryzae) are now available from Aspergillus genome database, AspGD (http://www.aspgd.org/), which is advantageous to the investigation of the universality and diversity of the intra-genus genomes. Indeed, a comparative genome analysis was conducted and provided a vast amount of information on the biology and physiology of filamentous fungi [79]. However, studies comparing different species at the transcriptional level have not been undertaken. Because cost-effective RNA-sequencing technology is available, we are now able to determine the transcriptomes of different Aspergillus species and make inter-species comparisons, which could provide new findings.

In the present study, we mainly addressed the issue of dormancy mechanisms in Aspergillus conidia by performing a comparative transcriptome analysis within the genus. Indeed, we focused on the conidia and 1 h-grown conidia of A. fumigatus, A. niger, and A. oryzae for our comparative transcriptome analysis. First, we defined the genes that were dominantly expressed in conidia or germinating conidia as conidia-associated genes (CAGs) or germination-associated genes (GeAGs), respectively. Subsequent comparisons of the CAGs or GeAGs among three species exposed the common conidial features of these filamentous fungi.

Results

Transcriptome determination using RNA-sequencing

To compare the conidial transcriptomes of different Aspergillus species, the conidia of each strain should be harvested from physiologically similar cultivation conditions. We chose potato dextrose agar (PDA) and potato dextrose broth (PDB) for the culture media because all of the strains (A. fumigatus Af293, A. niger IFM 58835, and A. oryzae RIB40) produced a large amount of conidia on PDA. Based on the colony expansion rates, conidia production rates, and germination rates (Additional file 1), we grew them at preferred temperature condition, namely A. fumigatus were grown at 37 °C, A. niger at 30 °C, and A. oryzae at 30 °C on PDA.

We first made comparisons among hyphae, dormant conidia, and 1 h-grown conidia in each species. The conidia were harvested from a 7-day-old culture at temperature specified above, and RNA was extracted from the conidia. Similarly, RNA in hyphae was prepared from mycelia cultivated in PDB at the sub-stationary phase according to dry weight (Additional file 1). To get insight into early response of conidia to the condition preferable to germinate, RNA was extracted from the conidia incubated in PDB at the appropriate temperature for 1 h. At the time-point, no germ tubes were appeared at all and the conidia do not start swelling in all species.

Transcriptomes from three different growth phases (hyphae, conidia, and 1 h-grown conidia) of A. fumigatus, A. niger, and A. oryzae were determined by RNA-sequencing analyses. In A. fumigatus, 97.8, 96.5, and 95.5 % of the ORFs were expressed in hyphae, conidia, and 1 h-grown conidia, respectively (Table 1). In A. niger, 86.7, 83.3, and 72.0 % of ORFs were expressed in hyphae, conidia, and 1 h-grown conidia, respectively, while in A. oryzae, 87.0, 85.8, and 77.1 % of ORFs were expressed in hyphae, conidia, and 1 h-grown conidia, respectively. There were 93, 1,251, and 815 genes whose expression levels were not detected in any of the three phases in A. fumigatus, A. niger, and A. oryzae, respectively (Table 1). The mean FPKM for each transcriptome was calculated and 16.3–19.4 %, 12.0–15.3 %, and 11.0–18.8 % of genes showed FPKM values higher than the mean FPKM in A. fumigatus, A. niger, and A. oryzae, respectively (Table 1).
Table 1

The numbers of ORFs expressed in each phase of the Aspergillus fungi

  

Hyphae

Conidia

1 h-grown conidia

 

# of ORFs

# of expressed ORFs

%

mean FPKM

# > mean

%

# of expressed ORFs

%

mean FPKM

# > mean

%

# of expressed ORFs

%

mean FPKM

# > mean

%

A. fumigatus

9783

9569

97.8

69.00

1900

19.4

9445

96.5

87.67

1599

16.3

9341

95.5

91.33

1906

19.5

A. niger

14056

12183

86.7

40.83

2146

15.3

11711

83.3

41.82

2083

14.8

10123

72.0

108.12

1682

12.0

A. oryzae

11902

10353

87.0

31.79

2234

18.8

10217

85.8

49.97

1948

16.4

9173

77.1

108.5

1313

11.0

Determination of CAGs and GeAGs

To determine the genes whose expression is enriched in conidia or 1 h-grown conidia, we compared transcriptomes between hyphae and conidia, or between conidia and 1 h-grown conidia, respectively. The CAGs and GeAGs were defined as follows: 1) The expression levels in conidia or 1 h-grown conidia increased more than fourfold compared with that in hyphae or conidia, respectively; and 2) The expression levels in conidia or 1 h-grown conidia conidia were higher than the mean FPKM value. Using these criteria, we identified 687, 694, and 812 CAGs in A. fumigatus, A. niger, and A. oryzae, respectively. Similarly, 766, 1,241, and 749 GeAGs were identified in A. fumigatus, A. niger, and A. oryzae, respectively (Fig. 1).
Fig. 1

Experimental settings and a summary of the comparative transcriptomic analysis. The conidia-associated genes (CAGs) were determined by comparisons between FPKMs of conidia and hyphae (C/H). The germination-associated genes (GeAGs) were determined by comparisons between FPKMs of 1 h-grown conidia and conidia (Ge/C)

We used freshly harvested conidia to investigate the conidial transcriptome. Thus, whether the transcriptome could change during preservation (7 d) after drying, when the resting conidia should be metabolically inactive, was of interest. To address this, we compared A. fumigatus transcriptomes between the freshly harvested conidia and the dried resting conidia, focusing on 687 CAGs (data not shown). In the resting conidia, only 4 and 6 % of the CAGs showed a more than fourfold increase and a less than ¼ decrease, respectively, compared with the freshly harvested conidia. Whereas 58 % of the CAGs showed an unchanged expression level, less than twofold, during preservation, and 69 % of all of the genes were unchanged in their expression levels. These results suggested that the mRNA profiles of the conidia were largely unaffected during the 7-day incubation, and specific mRNA degradation did not occur during drying.

Functional classifications of CAGs and GeAGs using gene ontology (GO) terms

To gain a comprehensive insight into the specific molecular functions of the CAGs and GeAGs, a functional classification analysis was conducted. The total numbers of GO terms for A. fumigatus, A. niger, and A. oryzae were 233, 244, and 235, respectively. Among them, 8, 2, and 14 terms were significantly overrepresented or underrepresented in the CAGs, respectively (Additional file 2). The three Aspergillus species shared only one CAG-enriched GO term that is associated with glyoxysome (Table 2). In addition, functional classes related to adaptation to the intracellular oxidative state, such as oxidation-reduction process, oxidoreductase activity, and response to oxidative stress, were found in each fungus (Additional file 2). In the same way, 50, 70, and 59 terms were found in the GeAGs, respectively (Additional file 3). The larger number of terms found in GeAGs suggested that more molecular functions were triggered during the beginning of germination in the all three fungi. Indeed, 30 GO terms were commonly overrepresented in the GeAG of the three species (Table 2). Most were associated with ribosome function and primary cellular activities, such as translation, respiration, and metabolism, which supported the previous view that the construction of translational machinery starts at a very early stage in germination [24].
Table 2

Common CAG- and GeAG-enriched GO terms

   

Count (changed: not changed)

 

GO

terms

A. fumigatus

A. niger

A. oryzae

Over (+)/under (−)

CAG-enriched

# C — Cellular component

 

GO:0009514

glyoxysome

13:24

10:28

15:21

+

GeAG-enriched

# C — Cellular component

 

GO:0005730

nucleolus

113:56

119:40

106:50

+

 

GO:0030686

90S preribosome

37:0

34:1

33:1

+

 

GO:0005829

cytosol

243:1105

333:986

225:1050

+

 

GO:0032040

small-subunit processome

32:2

35:0

33:2

+

 

GO:0005840

ribosome

44:22

57:16

44:21

+

 

GO:0005762

mitochondrial large ribosomal subunit

26:4

30:0

21:4

+

 

GO:0030687

preribosome, large subunit precursor

24:3

20:1

17:4

+

 

GO:0005739

mitochondrion

71:304

95:297

57:315

+

 

GO:0009986

cell surface

21:51

29:46

25:52

+

 

GO:0031966

mitochondrial membrane

12:13

14:13

11:11

+

 

GO:0005886

plasma membrane

42:224

73:191

43:214

+

 

GO:0005634

nucleus

154:1289

238:1331

140:1322

+

 

GO:0005819

spindle

8:13

10:12

7:13

+

 

GO:0016021

integral component of membrane

15:555

36:695

17:780

-

# F — Molecular function

 

GO:0003735

structural constituent of ribosome

110:18

126:3

95:9

+

 

GO:0003723

RNA binding

44:83

57:55

42:64

+

 

GO:0008026

ATP-dependent helicase activity

14:25

19:22

12:27

+

 

GO:0003743

translation initiation factor activity

12:16

23:6

15:12

+

 

GO:0051082

unfolded protein binding

14:30

21:25

13:28

+

 

GO:0005524

ATP binding

69:439

102:475

64:506

+

 

GO:0003676

nucleic acid binding

51:286

76:236

49:227

+

 

GO:0005525

GTP binding

18:75

24:68

21:81

+

 

GO:0000166

nucleotide binding

35:208

53:208

36:244

+

 

GO:0016491

oxidoreductase activity

8:391

20:634

12:635

-

 

GO:0000981

sequence-specific DNA binding RNA polymerase II transcription factor activity

2:238

12:326

2:248

-

# P — Biological process

 

GO:0006412

translation

86:14

97:3

72:11

+

 

GO:0006364

rRNA processing

26:7

24:4

23:9

+

 

GO:0000447

endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA)

20:2

21:1

19:2

+

 

GO:0000027

ribosomal large subunit assembly

18:4

19:1

17:3

+

 

GO:0035690

cellular response to drug

34:151

49:135

37:157

+

 

GO:0006696

ergosterol biosynthetic process

10:16

11:15

8:19

+

 

GO:0006413

translational initiation

9:12

17:5

11:9

+

 

GO:0009060

aerobic respiration

9:18

12:11

9:16

+

 

GO:0008152

metabolic process

9:324

16:468

6:513

-

 

GO:0055085

transmembrane transport

16:405

21:552

16:651

-

Expression profile of genes encoding allergenic proteins

A. fumigatus was reported to possess 22 known allergenic proteins, which is the highest number in fungi found to date [10]. A comprehensive view of the allergenic gene expression would be useful for understanding which cellular forms of the fungus were potential allergens. Our transcriptome data revealed that 14, 9, and 9 allergen-related genes showed FPKM values higher than the corresponding mean FPKM in hyphae, conidia, and 1 h-grown conidia, respectively (Additional file 4). Among the highly expressed genes, 8 genes were commonly found in all cellular forms, 5 transcripts (aspf3, aspf8, cyp4, hsp90, and rpL3) of which were most abundant in 1 h-grown conidia. Notably, previous proteome research revealed that AspF3 and AspF8 were presented in the A. fumigatus germinating conidia [11]. In addition, the other A. fumigatus allergenic genes (Asp f1, Asp f4, sod3, and Asp f7) were highly expressed in hyphae but relatively silent in conidia. These transcriptional data suggest that higher amount of allergenic proteins may be present in hyphae and germinating conidia of A. fumigatus.

Expression profiles of common orthologous genes

In addition to species-specific allergenic genes, the Aspergillus species should share a large number of genes that are involved in common biological functions, such as primary metabolism and biosynthesis for cellular components. To gain more general insights into Aspergillus biology, we focused on the sets of orthologous genes that were found in each genome of A. fumigatus, A. niger, and A. oryzae and that had the highest homology levels. Using the three homology searches, A. fumigatus vs. A. niger, A. fumigatus vs. A. oryzae, and A. niger vs. A. oryzae, we found 6,172 sets of common genes whose counterparts represented 63.1, 43.9, and 51.9 % of all of the genes in A. fumigatus, A. niger, and A. oryzae, respectively (Additional file 5). The RNA-sequencing data showed that 5,972 (96.3 %), 5,758 (93.3 %), and 5,636 (91.3 %) of the common genes were expressed in A. fumigatus, A. niger, and A. oryzae, respectively.

We next sought to identify the common genes in the CAGs and GeAGs. Among the CAGs of the three species, 91 genes were found to be common, which was only 11.2–13.2 % of the CAGs in each species (Fig. 2a). These ratios were lower than those for whole gene sets in each species (43.9–63.1 %), suggesting that the CAGs are more diverse and strain specific compared with the non-CAGs. Of the 91 genes, 43 were greater than 10-fold the FPKM ratio of conidia to hyphae in all of the species (Table 3). While there was no information on 17 of the genes’ encoded protein functions, the rest included several genes that were previously reported to be conidia-specific or expressed during the asexual developmental stage. CatA is a conidia-specific catalase and is responsible for the stress tolerance of conidia, which was well documented in A. fumigatus and A. oryzae, as well as A. nidulans [1214]. ConJ is a conidia-specific protein that was studied in A. nidulans [15], and was reported to be expressed during asexual development in A. fumigatus [16]. VosA plays an important role in conidia formation and trehalose accumulation in conidia, as shown in A. fumigatus and A. nidulans [17, 18]. PilB is enriched in the conidia of A. fumigatus [5], and fhk1, encoding a hybrid histidine kinase, was reported to be up-regulated during asexual development in A. fumigatus [16]. These results suggested that most of the common CAGs were involved in conidia-specific functions and played an essential role in the conidial biology of Aspergillus species.
Fig. 2

Venn diagrams comparing the CAGs and GeAGs from the three Aspergillus species. The common CAGs (a) and common GeAGs (b) were identified

Table 3

A list of common CAGs (Ratio > 10)

   

FPKM ratio of conidia to hyphae*1

   

A. fumigatus

A. niger

A. oryzae

A. fumigatus

A. niger

A. oryzae

atfA-dependency *2

Gene name in Af

Annotation in A. fumigatus

Afu2g00200

An12g10720

AO090113000153

-

458.5

-

full

cat3

Catalase, putative

Afu8g01530

An12g10710

AO090113000154

1751.2

1334.9

2307.7

n.d.

-

HHE domain protein

Afu1g01490

An15g07300

AO090102000259

1291.8

1040.6

467.4

n.d.

-

Hypothetical protein

Afu2g14330

An17g01885

AO090009000665

1014.6

238.2

780.3

n.d.

-

Hypothetical protein

Afu6g03210

An12g10240

AO090011000656

798.1

439.3

1367.5

full

conJ

Conidiation-specific protein 10

Afu8g05810

An15g04670

AO090005000570

584.1

1287.1

28.3

n.d.

-

DUF1295 domain protein

Afu5g10160

An14g04530

AO090010000533

560.4

133.2

22.4

n.d.

-

ActVA 4 protein

Afu8g00600

An03g00920

AO090010000696

525.8

-

-

n.d.

-

Conserved hypothetical protein

Afu3g01210

An01g10950

AO090026000081

454.0

867.9

583.9

n.d.

-

ThiJ/PfpI family protein

Afu5g01160

An03g02190

AO090020000259

338.5

21.7

12.7

full

-

Monosaccharide transporter

Afu8g06020

An15g04770

AO090005000539

275.9

74.8

297.8

full

-

Glutamate decarboxylase

Afu6g03890

An09g03130

AO090701000158

249.3

350.6

194.9

full

catA

Catalase A

Afu1g03580

An18g04120

AO090009000418

196.6

288.5

629.2

n.d.

-

Hypothetical protein

Afu3g00640

An11g08160

AO090009000116

165.1

189.5

25.2

n.d.

-

Peptidoglycan binding domain protein

Afu2g01590

An03g04860

AO090701000790

125.5

22.0

74.4

full

nce102

Non-classical export protein (Nce2), putative

Afu2g15740

An15g05990

AO090012000251

103.7

261.2

-

full

-

Oxidoreductase, short chaindehydrogenase/reductase family

Afu5g03930

An09g06270

AO090102000598

100.6

220.2

70.0

partial

-

Alcohol dehydrogenase, putative

Afu1g13530

An08g06600

AO090012000522

100.5

74.2

35.6

n.d.

-

Hypothetical protein

Afu3g03940

An01g00280

AO090124000046

91.1

24.5

47.6

full

-

2,3-diketo-5-methylthio-1-phosphopentanephosphatase, putative

Afu3g11550

An02g07350

AO090003000710

88.6

42.0

360.8

n.d.

-

LEA domain protein

Afu5g03269

An09g05520

AO090102000529

88.6

54.5

72.9

n.d.

-

Unknown

Afu6g08320

An11g01750

AO090003000094

87.1

81.9

134.4

n.d.

pilB

Putative conserved eisosome component

Afu3g12760

An02g08740

AO090012000846

81.3

85.1

268.0

n.d.

-

Hypothetical protein

Afu4g03390

An14g02450

AO090011000203

79.2

15.6

21.0

full

-

Aquaporin

Afu5g13100

An14g06050

AO090120000435

77.1

16.0

78.7

n.d.

-

Hypothetical protein

Afu6g13470

An08g03030

AO090020000658

60.5

-

496.2

n.d.

-

Conserved hypothetical protein

Afu1g13550

An08g06620

AO090012000521

45.4

97.8

113.2

n.d.

-

Hypothetical protein

Afu5g14310

An09g01150

AO090020000287

43.8

137.2

13.0

not CAG

-

Short chain dehydrogenase/reductase familyprotein

Afu3g10480

An16g04420

AO090003000594

31.5

76.5

134.2

n.d.

-

Conserved hypothetical protein

Afu6g08730

An11g06120

AO090001000547

30.2

49.1

699.1

not CAG

-

6-phosphogluconate dehydrogenase,decarboxylating

Afu4g09250

An04g04280

AO090023000611

29.7

11.6

18.2

n.d.

-

Hypothetical protein

Afu4g05900

An04g00100

AO090023001009

25.1

35.1

24.7

n.d.

-

Conserved hypothetical protein

Afu4g01020

An18g01860

AO090003001579

24.1

28.6

99.1

n.d.

fhk1

Sensor histidine kinase/response regulator, putative

Afu6g13860

An08g08500

AO090103000362

23.9

370.9

429.6

n.d.

-

Conserved hypothetical protein

Afu5g09180

An07g03930

AO090020000514

23.7

671.7

448.4

n.d.

-

Hypothetical protein

Afu4g10860

An04g05790

AO090003001124

23.2

22.7

24.9

n.d.

vosA

Velvet family protein

Afu5g01290

An14g07380

AO090010000635

22.4

680.6

42.7

partial

-

Oxidoreductase, zinc-binding dehydrogenasefamily, putative

Afu1g03090

An01g05320

AO090005000810

21.7

17.9

16.1

n.d.

-

Conserved hypothetical protein

Afu2g04200

An11g02200

AO090003000208

20.8

16.6

37.7

not CAG

hppD

4-hydroxyphenylpyruvate dioxygenase

Afu7g01430

An12g01460

AO090010000221

16.5

109.6

37.5

full

-

Opsin 1

Afu2g16930

An04g09030

AO090102000125

14.8

33.2

103.7

not CAG

-

Succinate:fumarate antiporter (Acr1), putative

Afu2g10020

An16g05030

AO090011000634

10.1

1755.9

87.7

n.d.

-

Hypothetical protein

Afu2g02310

An07g06530

AO090011000512

10.0

19.7

71.1

not CAG

sur7

sur7 protein, putative

*1: “-”, not expressed in hyphae

*2: AtfA-dependency is clarified by real-time RT PCR. n.d., not determined

Likewise, we found 391 common genes in the GeAGs of the three species (Fig. 2b). This corresponded to 31.5–52.2 % of the GeAGs, and 46 of them showed more than 10-fold the FPKM ratio in 1 h-grown conidia compared with conidia in all of the species (Table 4). There were 13 genes that encoded a putative ribosome protein or a ribosome-related protein, which supported the view that de novo ribosome-complex biosynthesis begins at an early stage of germination. Notably, A. fumigatus calA (Afu3g09690) and the corresponding genes of A. niger (An16g03330) and A. oryzae (AO090005001280) showed quite high expression levels in 1 h-grown conidia (Table 4). The verification of the expression profiles during 2 h of germination using real-time PCR revealed that the A. fumigatus calA expression level was strongly induced at an early stage of germination (Fig. 3), suggesting that the CalA protein functions during the A. fumigatus germination process. Although the detailed function of CalA in germination remains unclear, CalA was detected on the swollen conidial surface by the binding of anti-CalA serum, and recombinant CalA protein was demonstrated to bind with laminin and murine lung cells [19]. CalA is a thaumatin-like protein, and A. fumigatus has the paralogous proteins encoded by Afu8g01710 and Afu3g00510 (calB and calC, respectively) (Additional file 6). Interestingly, the expression levels of calB and calC were induced during germination as well, while the expression level of calB was markedly high in hyphae (Fig. 3). In A. niger and A. oryzae, only a single protein, AoCalA and AnCalA, respectively, with high homology to the thaumatin-like protein was found, and the expression levels were also up-regulated in 1 h-grown conidia (Table 4, Additional file 6). Notably, A. nidulans has two thaumatin-like proteins, CetA and CalA, and the deletion of both genes was reported to result in synthetic lethality [20], suggesting an important role of the thaumatin-like proteins in Aspergillus conidial germination. The genetic distribution among the Aspergillus species (Additional file 6) suggested that the germination-related thaumatin-like protein is duplicated in some species and that their roles might be indispensable for filamentous fungi.
Table 4

A list of common GeAGs (Ratio > 10)

   

FPKM ratio of 1 h-grown conidia to conidia

  

A. fumigatus

A. niger

A. oryzae

A. fumigatus

A. niger

A. oryzae

Gene name in Af

Annotation in A. fumigatus

Afu3g09690

An16g03330

AO090005001280

2140.3

7365.7

1063.5

-

Extracellular thaumatin domain protein, putative

Afu4g08110

An04g02550

AO090023000758

75.3

163.0

12.4

-

Translation elongation factor G1, putative

Afu4g07630

An04g02000

AO090023000812

53.9

74.1

11.6

-

Microtubule associated protein (Ytm1), putative

Afu6g02690

An12g08230

AO090120000155

48.5

87.3

16.2

mtfA

C2H2 finger domain protein, putative

Afu1g10990

An08g03290

AO090038000305

43.2

729.2

13.4

-

Ribosomal RNA processing protein, putative

Afu1g04370

An01g03230

AO090003000912

39.7

2627.6

18.7

-

Hypothetical protein

Afu1g05310

An01g04590

AO090003000828

39.1

194.0

13.0

-

DUF699 ATPase, putative

Afu2g10070

An16g04970

AO090011000630

35.6

11.6

26.2

-

Carbamoyl-phosphate synthase, large subunit

Afu4g07540

An04g01900

AO090023000821

35.1

42.8

11.6

-

Small nucleolar ribonucleoprotein complexsubunit, putative

Afu2g16040

An15g06390

AO090012000197

30.2

73.6

10.6

-

rRNA biogenesis protein, putative

Afu2g09860

An16g05290

AO090011000649

29.0

71.4

33.7

-

Purine-cytosine permease

Afu1g10440

An08g02390

AO090038000383

25.7

100.8

10.2

-

Conserved hypothetical protein

Afu7g04700

An13g01230

AO090005000164

25.5

11.8

28.7

-

Conserved hypothetical portein

Afu5g03870

An09g06310

AO090102000594

23.7

42.9

10.7

-

Conserved hypothetical protein

Afu4g06350

An04g00680

AO090023000955

23.6

130.8

10.3

-

rna binding protein

Afu3g06320

An11g10000

AO090020000118

21.3

29.8

14.0

-

Conserved hypothetical protein

Afu2g02190

An07g06640

AO090011000497

21.0

52.7

28.0

-

Hypothetical nuclear protein

Afu6g08720

An11g06110

AO090001000546

20.3

38.3

23.7

-

5’-methylthioadenosine phosphorylase

Afu4g03650

An14g01560

AO090011000292

20.1

68.9

12.4

-

Ribosome associated DnaJ chaperone Zuotin, putative

Afu6g13370

An08g09160

AO090003000462

17.9

10.0

11.3

utp10

SSU processome component (Utp10), putative

Afu2g05950

An02g14340

AO090001000712

17.2

28.2

14.7

-

snRNP and snoRNP protein (Snu13), putative

Afu3g13320

An02g09200

AO090012000801

17.0

14.3

10.7

rps0

40S ribosomal protein S0, putative

Afu1g15730

An01g14080

AO090005001132

16.9

31.5

10.4

-

Ribosomal protein S8

Afu2g17200

An04g09270

AO090102000099

16.8

26.2

11.1

-

Hypothetical protein

Afu2g11810

An02g03860

AO090026000462

16.7

77.0

16.8

-

Conserved hypothetical protein

Afu5g11470

An18g04470

AO090026000687

16.0

83.7

24.6

-

MYB DNA binding protein (Tbf1), putative

Afu5g06430

An17g01270

AO090009000609

15.7

76.2

10.5

-

Mitochondrial large ribosomal subunit L7, putative

Afu2g02150

An07g06760

AO090011000490

15.5

20.0

12.7

S10a

Ribosomal protein S10

Afu6g04330

An15g01160

AO090701000118

15.1

35.8

12.0

-

DEAH-box RNA helicase (Dhr1), putative

Afu1g05390

An18g04220

AO090009000405

14.8

28.8

10.6

-

Mitochondrial ADP,ATP carrier protein (Ant), putative

Afu1g11710

An08g03910

AO090038000249

13.9

16.2

13.2

-

60S ribosomal protein L1

Afu8g05330

An16g07400

AO090005000616

13.9

16.1

10.8

-

Methylenetetrahydrofolate dehydrogenase

Afu3g10800

An02g06530

AO090003000629

13.7

10.4

10.9

-

Eukaryotic translation initiation factor 3subunit CLU1/TIF31, putative

Afu3g11260

An02g07010

AO090003000679

13.6

37.3

16.5

ubiC

Ubiquitin (UbiC), putative

Afu3g12300

An02g08080

AO090005000737

13.4

20.5

11.2

-

Ribosomal L22e protein family

Afu5g12360

An14g06860

AO090120000354

13.3

15.2

12.8

-

Mitochondrial oxaloacetate transporter (Oac),putative

Afu3g06580

An11g09740

AO090020000342

13.1

38.1

14.1

-

WD repeat protein

Afu3g05490

An11g11150

AO090020000020

13.0

72.7

13.3

-

Nrap protein superfamily

Afu6g10460

An11g00990

AO090023000242

12.8

10.8

15.5

lag1

Ceramide synthase membrane component (Lag1),putative

Afu6g09990

An11g04985

AO090038000466

12.1

29.5

10.7

-

Importin beta-4 subunit, putative

Afu6g08580

An11g05510

AO090001000519

11.9

16.0

10.4

fkbp4

FKBP-type peptidyl-prolyl isomerase, putative

Afu3g10660

An02g06320

AO090003000611

11.5

23.0

47.8

erg13

Hydroxymethylglutaryl-CoA synthase

Afu4g13170

An01g08850

AO090009000264

11.2

23.6

16.9

cpcB

Guanine nucleotide-binding protein subunit, putative

Afu3g00880

An09g03120

AO090003001496

11.1

486.0

48.1

-

Conserved hypothetical protein

Afu6g09060

An11g06810

AO090001000583

10.7

50.8

11.3

-

Mitochondrial 60S ribosomal protein L6precursor

Afu2g03380

An07g07840

AO090120000249

10.6

15.7

11.2

-

Alkaline serine protease

Fig. 3

Quantitative real-time RT-PCR analyses of calA-family genes in A. fumgiatus. Expression profiles of calA, calB, and calC during the germination process (~2 h) and hyphae (hyph) were analyzed. Each value represents the expression ratio relative to that of the actin gene. Data presented are the averages of three replicates, and the bar indicates standard deviation

Role of the AtfA transcription factor in regulating CAGs

In our previous study, we demonstrated that A. fumigatus catA, conJ, and fhk1 were up-regulated during the asexual stage in an AtfA-dependent manner [16]. AtfA is a bZip-type transcription factor that is involved in conidial stress tolerance, and the molecular function of AtfA is largely conserved in some Aspergillus species [16, 21, 22]. As described above, the known AtfA-dependent genes were commonly enriched in the conidia of A. oryzae, A. niger, and A. fumigatus (Table 3). Thus, it was of interest to investigate whether conidia-accumulated transcripts of common CAGs were dependent on the AtfA transcription factor. We cultivated the A. fumigatus control strain (AfS35) and atfA deletion mutant, and the expression levels of the selected genes in hyphae and 4- and 8-day-old conidia were analyzed using quantitative real-time RT-PCR. Most of the CAGs tested showed AtfA-dependent expression in resting conidia (Fig. 4). This clearly indicated that AtfA played a major role in conidial biology.
Fig. 4

Quantitative real-time RT-PCR analyses of the common CAGs in A. fumgiatus. Expression levels of the common CAGs in 4- and 8-day-old conidia of Afs35 (WT) and ΔatfA were analyzed. Each value represents a fold increase in the expression ratios compared with those in hyphae. Data presented are averages of three replicates, and the bar indicates standard deviation

In our previous study, the deletion of AtfA resulted in stress-labile conidia and delayed germ-tube formation [16]. To gain further insight into role of AtfA in conidial longevity, conidial transcriptomes were compared between the wildtype and the atfA mutant (Fig. 5). Among the whole genes, 10 and 13 % showed increased (>4 times) and decreased (< ¼ times) expression levels, respectively, in conidia of the atfA deletion mutant. For the CAGs, 54 % showed a decreased level (more than fourfold) of expression in the atfA mutant conidia compared with the wildtype. For the common CAGs, 63 % had lower expression levels in the A. fumigatus atfA mutant conidia (data not shown), which possibly led to deleterious defects in stress-homeostasis. Unexpectedly, a portion of GeAGs showed higher expression levels in the conidia of the atfA mutant. Particularly, calA and calB were highly up-regulated in the atfA conidia (Fig. 6). This derepression of the germination-associated genes in the resting conidia suggested that uncontrolled exit from dormancy might occur in the conidia.
Fig. 5

Distribution of genes with differential expression level in the atfA mutant conidia. Comparison between conidia of A. fumgiatus Afs35 (WT) and ΔatfA. From the FPKM values, the relative expression ratios of all of the genes (left), the CAGs (middle), and the GeAGs (right) were calculated as ΔatfA/WT conidia

Fig. 6

Expression levels of calA, calB, and calC in 4- and 8-day-old conidia of Afs35 (WT) and ΔatfA were analyzed. Each value represents the expression ratios compared with those in WT hyphae. Data presented are averages of three replicates, and the bar indicates standard deviation

Conidia of the null atfA mutant revealed germination-related traits

The first step toward germination, after sensing available carbon sources and water, is isotropic swelling, which is followed by polarized growth with germ tube formation [23]. During the isotropic growth, de novo ergosterol biosynthesis begins and a sterol-rich domain is observed in a polar position of the swollen conidia [24]. To investigate if conidia defective in the atfA gene showed precocious swelling, the freshly harvested conidia of the atfA mutant were microscopically observed by staining with filipin, which binds to ergosterol. Most of the conidia of the atfA mutant, but not of wildtype, were stained with filipin (Fig. 7a). Notably, some atfA mutant conidia showed sterol-rich domain formation and filipin staining at putative organelle membranes (Fig. 7a). These results supported the idea that resting conidia lacking AtfA began germination process without any available nutrients, such as carbon sources.
Fig. 7

Metabolic activities in ΔatfA resting conidia. a Ergosterols were stained by filipin in the resting conidia of A. fumigatus Afs35 (WT), ΔatfA, and the complemented strain (Co-atfA). The test was repeated twice, and more than three sections were observed. The representative photos were shown. b The resazurin assay indicates metabolic activity in the ΔatfA resting conidia, which are without nutrients. c The XTT assay shows metabolic activity in the ΔatfA resting conidia, which are without nutrients

We then investigated the metabolic activity of the resting conidia using respiration indicators, resazurin, and XTT reagents (Fig. 7b and c). When incubated in water without any carbon sources, the wildtype conidia showed no change in color, indicating a metabolically inactive state. On the contrary, the water containing atfA mutant conidia changed color based on the respiration markers, suggesting that the conidia defective in AtfA were somehow metabolically active even in the absence of nutrients. Collectively, these germination-related traits suggested that AtfA has a role in maintaining conidial dormancy, and the deletion of AtfA led to the derepression of dormancy.

Discussion

Dormant conidia of filamentous fungi are easy-to-disperse, stress-tolerant structures [1]. These properties enable the fungi to survive in a variety of harsh environments. Thus, there is a keen interest in the molecular mechanisms of conidial dormancy and germination. We used Aspergillus conidia as a model to gain a transcriptomic view of dormancy and the exit from dormancy. We used three Aspergillus species for transcriptome comparisons, which enabled us to achieve more universal findings.

The comparative analysis identified the common CAGs and GeAGs, which revealed that transcripts associated with stress tolerance and ribosome biogenesis are abundant in the conidia and 1 h-grown conidia, respectively. In fact, CatA, Cu/Zn superoxide dismutase (sod1), and trehalose synthase (tpsA) genes were found to be highly expressed in the conidia, which was consistent with the previous studies [13, 25, 26]. The conidia devoid of CatA showed an increased sensitivity to hydrogen peroxide in A. fumigatus and A. nidulans, indicating that oxidative stress tolerance in the Aspergillus conidia requires CatA [12, 13]. Our transcriptome data also showed that unstudied putative catalase genes (Afu2g00200, An12g10720, and AO090113000153) were commonly up-regulated in the conidia (Table 3). The amino acid sequences of the proteins are well conserved among the Aspergillus species, and they have a catalase core domain (IPRO: 11614), suggesting that it may play an important role in oxidative stress adaptation in the conidia. Notably, this putative catalase (here designated as Cat3), as well as CatA, was expressed in an AtfA-dependent manner (Fig. 4). Another catalase gene, cat1/catB, showed mycelia-specific expression in A. niger and A. oryzae, but not in A. fumigatus in which the expression level of cat1 (Afu3g02270) was quite low in all of the cell forms tested (Additional file 5). The bifunctional catalase-peroxidase cat2/cpeA/catD was highly expressed in the conidia of A. fumigatus (cat2), whereas the expression levels were quite low in A. oryzae (Additional file 5). Collectively, A. fumigatus conidia contained high transcript levels of the three catalase genes, catA, cat2, and cat3, which might contribute to the protection of resting conidia from oxidative stresses in the environments.

In addition to catalases, the CAGs included an array of genes associated with conidial biology. VosA, a velvet-family protein, is involved in trehalose accumulation in the conidia and UV-tolerance [18]. The opsin 1 gene encodes a protein homologous to Neurospora crassa rhodopsin NOP-1 that is expressed in a conidiation-related manner [27]. The most recent report demonstrated that Fusarium fujikuroi rhodopsin was highly accumulated in the conidia produced under light conditions and the F. fujikuroi rhodopsin functioned as a light-dependent proton pump [28]. One could hypothesize that light-dependent biological roles are conserved across most fungal spores/conidia. We also found that aquaporin genes were specifically expressed in the conidia. Although the function of aquaporin remains to be investigated in filamentous fungi, it was demonstrated that S. cerevisiae aquaporin was required for normal sporulation and freeze tolerance [29, 30], from which one could hypothesize that aquaporin might play an important role in conidial maturation by allowing water outflow. Taking these results into account, our comparative transcriptome analysis provided a novel insight into the molecular mechanisms underlying conidial dormancy and maturation. However, the GeAGs mainly included genes involved in fundamental cellular processes, such as ribosome biogenesis, nucleotide biogenesis, ubiquitin, and translation factors (Table 4). This corroborated previous transcriptome and proteome studies for Aspergillus species [2, 4, 6, 11].

In general, comparisons between transcriptome and proteome data provide results that may aid in understanding cellular functions and mechanisms. Teutschbein et al. [5] previously presented 449 proteins from the A. fumigatus resting conidia. We compared the conidial proteins against the transcriptome data from the present study, revealing that 448 had transcripts in the conidia and 284 (63.4 %) exhibited no less than the mean FPKM value. The genes corresponding to the proteins from the 40 most abundant spots in the conidial proteome analysis all showed greater than average FPKM values, except rodA, and 21 (52.5 %) were more highly expressed in the conidia than in the hyphae (> fourfold). This suggested a good accordance between the proteins expressed and the genes present in the resting conidia. However, when we compared the 100 most highly expressed CAGs against the 448 conidial proteins, only 26 corresponding proteins were found. When we compared the 687 CAGs against the proteins, 104 of the CAGs (15.1 %) were found in the conidial proteome. This comparison suggested that the transcripts with high FPKM levels were not necessarily translated in the resting conidia and that they might exist in an mRNA form. If this hypothesis is true, the conidial dormancy (metabolic inactive state) could be resulted from the absence of proteins necessary for metabolic and cellular activities in the resting conidia. Furthermore, the pre-packed mRNAs in resting conidia could contribute to an immediate response to exit from dormancy when the conidia sense appropriate nutrients in the extracellular environment. However, there is an open question regarding the mechanism underlying the preservation of mRNA in the resting conidia during dormancy.

In the present study, more than half of the A. fumigatus CAGs were regulated for their expression in an AtfA-dependent manner (Fig. 5). Compared with the A. fumigatus CAGs, the common CAGs contained a higher percentage (64 %) of AtfA-dependent genes, indicating that AtfA plays a more crucial role in the biology of Aspergillus resting conidia than was previously thought. The function of AtfA has been intensely studied in A. nidulans, A. oryzae, and A. fumigatus, in which the deletion of the atfA gene commonly resulted in extremely stress-labile conidia and the down-regulation of catA expression in the conidia [16, 21, 22]. As stated above, our data showed that not only catA, but also other catalase genes were highly expressed in the conidia of A. fumigatus in an AtfA-dependent manner. The dehydrin-like proteins DprA, DprB, and DprC, which play roles in the stress adaptation of conidia, were also enriched in the A. fumigatus conidia, and the transcriptional regulation of DprA and DprC was largely dependent on AtfA (Additional file 7). Notably, the atfA deletion mutants of A. fumigatus and A. nidulans are able to produce as many conidia as the wildtype [16, 21]. This clearly indicated that AtfA has no role in conidiation, and suggested that AtfA is likely to be essential for the conidial maturation process, in which the conidia enter dormancy and become more tolerant to environmental stresses.

In the germination process, calA was highly up-regulated in all of the species tested. This suggested that CalA protein commonly play an important role in initiating the germination of Aspergillus conidia. In fact, it was previously reported in A. nidulans that CalA and the paralog protein CetA were required for normal germination and that the GFP-fused proteins were localized to the hyphal periphery [20]. Intriguingly, plant thaumatin-like proteins are able to bind to polysaccharides like beta-glucan and showed beta-1,3-glucanase activity [3133]. Some were identified as antifungal proteins induced by pathogen exposure. From these characteristics, one could hypothesize that fungal thaumatin-like proteins are involved particularly in conidial isotropic growth (swelling) by loosening cell walls with hydrolyzing activity. This hypothesis was partly supported by the findings that the abnormal expression of calA and calB (Fig. 6) and an abnormal progression of swelling (Fig. 7a) were found in the atfA mutant’s resting conidia. The fungal thaumatin-like proteins were widely conserved in ascomycetes to basidiomycetes, including plant pathogens, which suggested an important role in the biology of filamentous fungi [34]. Therefore, a further functional analysis of the CalA-family protein may increase our understanding of the germination mechanism, as well as the interactions, between plants and pathogens.

One of the notable findings is the derepression of dormancy in the conidia of the A. fumigatus atfA deletion mutant, in which filipin-positive staining and metabolic activity, that were absent in the wildtype conidia, were found. These findings clearly indicated an abnormal exit from dormancy and the partial progression of the germination process. In general, the germination process of Aspergillus conidia starts when the cells perceive carbohydrates, like glucose. The nutrient cues are sensed and signaled via G-proteins and the cAMP-dependent protein kinase A signaling pathway [35, 36]. In the atfA mutant conidia, the signaling pathway might be constitutively activated by an unknown mechanism and subsequently the germination process starts without any extracellular nutrients. The source of the energy for boosting the metabolism in the resting conidia is unknown. One possibility is the glucose derived from the degradation of conidia-accumulated trehalose, which is thought to be used during germination in wildtype conidia. In fact, a lowered level of trehalose was found in the atfA deleted conidia compared with the wildtype conidia [16]. It may be possible that trehalose is abnormally degraded in the resting conidia of the atfA mutant, which in turn results in the exit from dormancy. Besides extracellular glucose, precocious consumption of the immobilized intracellular glucose may account for delayed germination phenotype of the atfA mutant. Although the detailed mechanisms are still obscure, AtfA is essential for the maintenance of metabolically inactive dormancy in resting conidia.

Conclusions

Comparisons among transcriptomes of hyphae, conidia, and 1 h-grown conidia of Aspergillus species provided a wide array of genes differentially expressed in each form. These findings further highlighted the essential role of the AtfA transcription factor in conidial dormancy. In addition, the thaumatin-like proteins are exclusively expressed at the germination stage, suggesting an important role in the germination of Aspergillus species. These results will facilitate the further investigation of molecular mechanisms underlying conidial dormancy and germination.

Methods

Strains and growth conditions

A. fumigatus strain Af293, A. niger strain IFM 58835, and A. oryzae strain RIB40 were used for the transcriptome analysis. To investigate AtfA roles, ΔatfA and the corresponding parental strain Afs35 of A. fumigatus were used [16]. All strains were cultivated on PDA plates. To collect hyphae, the strains were cultured in PDB for 18 h (A. fumigatus) or 26 h (A. niger and A. oryzae).

Conidia preparation

Conidia of each strain were stored in 20 % glycerol in a −80 °C freezer. To prepare fresh conidia, the stored conidia were inoculated on a PDA slant and incubated at 37 °C (A. fumigatus) or 30 °C (A. niger and A. oryzae) for 1 week. The conidia were harvested with phosphate-buffered saline (PBS) containing 0.1 % Tween 20, and the concentration was calculated by counting the conidia with a hemocytometer (Watoson, Kobe, Japan). To collect conidia for RNA purification, conidia were mixed with 15 mL of PDA (final concentration, approximately 104 conidia/mL) before the medium solidified in a 100-mL flask, and they were incubated at 30 or 37 °C for 7 d in the dark. After cultivation, conidia were harvested with PBS-Tween 20, filtered through a Miracloth, counted with a hemocytometer, and washed once with PBS-Tween 20. Microscopic observations revealed that there were no hyphal fragment contaminants in the conidial suspension, which contained exclusively conidia.

When 1 h-grown conidia were collected, approximately 109 conidia were shaken in 10 mL PDB for 1 h and then centrifuged. To prepare the dried conidia, freshly harvested conidia were centrifuged and the supernatant was discarded. The conidia were dried in a vacuum for 2 h, and then incubated at room temperature for 7 d.

RNA and cDNA preparation

Mycelia, conidia, and 1 h-grown conidia from duplicate independent culture were pooled and frozen in liquid nitrogen, and total RNA was isolated using the FastRNA Pro Red Kit (MP Biomedicals, Santa Ana, CA, USA). To obtain cDNA pools from the total RNA, possible contaminating genomic DNA was removed, and reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo).

RNA-sequencing analysis

An indexed cDNA library was prepared using the SureSelect Strand-Specific RNA Seq (Agilent Technologies) according to standard protocols. Briefly, each total RNA sample (1 μg) was enriched for mRNA using oligo(dT)-tagged beads. RNA samples were fragmented into smaller pieces and used to synthesize cDNAs. The library construction involved end repair, A-tailing, adapter ligation, and amplification. The mean length of each library was approximately 290 bp. Sequencing was performed in a single-end 60-base pair mode on a Hiseq system (Illumina). The RNA-sequencing read data was deposited to the DDBJ Sequence Read Archive under accession No. PRJDB4747.

Expression analysis

Illumina data sets were trimmed using fastq-mcf in ea-utils (v1.1.2-484) [37], where sequencing adapters and sequences with low-quality scores (Phred score Q < 20) were removed. Cleaned reads were mapped to the genome sequence of A. fumigatus Af293 (29,420,142 bp; genome version: s03-m04-r31), A. niger CBS (33,975,768 bp; genome version: s01-m06-r19), or A. oryzae RIB40 (37,912,014 bp; genome version: s01-m08-r26) from AspGD (http://www.aspgd.org/) using TopHat (v2.0.4) with the default parameters [38]. FPKMs were calculated using cuffdiff in Cufflinks (v2.1.1) with default parameters [39]. Data analyses were conducted using the R programming language (https://www.r-project.org/), and cummeRbund software [40].

GO analysis

Genes were functionally categorized using their GO information [41] obtained from AspGD, and overrepresented GO terms were identified using Fisher’s exact test. The one-tailed Fisher’s exact p-value corresponding to the overrepresentation of GO categories with equal to, or greater than, 20 genes was calculated based on counts in 2 × 2 contingency tables [42]. p-values were corrected by the false discovery rate method [43], and the threshold was set as 0.01.

Identification of orthologous genes

BLASTP (v2.2.28+) analyses [44] querying all of the protein sequences from one Aspergillus species against those of the other Aspergillus species were conducted using BLOSUM 80. The reciprocal best-hit pairs between two species—7,302 genes between A. fumigatus and A. niger; 7,216 between A. fumigatus and A. oryzae, and 7,636 between A. niger and A. oryzae—were extracted. Finally, 6,172 orthologous gene sets were identified among the three species and used in this study.

Quantitative real-time RT-PCR

Real-time RT-PCR was performed using SYBR Green detection as described previously [45]. The primer sets used in this study are listed in Additional file 8. The relative expression ratios were calculated by the comparative cycle threshold (Ct) (ΔΔCt) method. The actin gene was used as a normalization reference (internal control). Each sample was tested in triplicate.

Filipin staining

The resting conidia of interest were harvested as described above. 108 conidia were centrifuged and then washed once with PBS + Tween 20. They were dissolved in 200 μL of PBS + Tween20 (final concentration, 5 × 108/mL). A stock solution (1 mM) of filipin (Sigma) was prepared in DMSO, and 5 μL of the solution was added to 50 μL of the conidial suspensions. After incubation at room temperature for 3 min, they were centrifuged, immediately dissolved by an equivalent volume of PBS + Tween20, and observed under a fluorescent microscope.

Metabolic activity assay

To compare the respiration activity in conidia, a resazurin assay was conducted. The conidia (2 × 107–2 × 106) of each strain were incubated in 1 mL of distilled water with resazurin (final concentration, 0.1 mM) at 37 °C for 20 h and then photographed. The tetrazolium salt 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay was conducted. The conidia (2 × 107–2 × 106) of each strain were incubated in 0.4 mL of distilled water with 0.1 mL of XTT solution, containing 1 mg/mL XTT and 50 μM menadione, at 37 °C for 4 h and then photographed.

Ethics approval

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

The datasets, supporting the conclusions of this article, is available in a BioProject PRJDB4747 deposited to DDBJ. All fungal strains are available from National BioResource Project of Mycology Research Center of Chiba University (https://daphne.pf.chiba-u.jp/distribution/catalog).

Abbreviations

PDA: 

potato dextrose agar

BLOSUM: 

Blocks substitution matrix

bZIP: 

basic leucine zipper

CAG: 

conidia-associated gene

DMSO: 

dimethyl sulfoxide

FPKM: 

fragments per kilobase of transcript per million mapped reads

GeAG: 

germination-associated gene

GFP: 

green fluorescent protein

GO: 

gene ontology

PBS: 

phosphate-buffered saline

PDB: 

potato dextrose broth

RT-PCR: 

reverse transcription polymerase chain reaction

Declarations

Acknowledgments

We would like to thank Dr. Atsushi Iwama, Dr. Motohiko Ohshima, and Dr. Atsunori Saraya (Chiba University) for technical support with the Illumina HiSeq 1500.

Funding

This work was supported by a grant from the Noda Institute for Science Research to D.H. This work was also supported in part by the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) Special Budget for Research Project: The Project on Controlling Aspergillosis and the Related Emerging Mycoses, a Cooperative Research Grant of NEKKEN (2014–2015), and the National BioResource Project for Pathogenic Microbes funded by the MEXT, Japan (http://www.nbrp.jp/). MEXT provided funding to H.T. and Y.K. under grant number 221S0002.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Medical Mycology Research Center (MMRC), Chiba University
(2)
Molecular Chirality Research Center, Chiba University

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© Hagiwara et al. 2016

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