bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

1 Overexpression of the RNA-binding protein NrdA affects global gene expression

2 and secondary metabolism in Aspergillus species

3

4 Chihiro Kadooka1,2*, Kosuke Izumitsu3, Teigo Asai4, Kazuki Mori5, Kayu Okutsu2,

5 Yumiko Yoshizaki1,2, Kazunori Takamine1,2, Masatoshi Goto1,6, Hisanori Tamaki1,2,

6 Taiki Futagami1,2#

7

8 1 United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24

9 Korimoto, Kagoshima, 890-0065, Japan

10 2 Education and Research Centre for Fermentation Studies, Faculty of Agriculture,

11 Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan.

12 3 Graduate School of Environmental Science, University of Shiga Prefecture, 2500

13 Hassaka-cho, Hikone, Shiga, 522-8533, Japan.

14 4 Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki,

15 Aoba-ku, Sendai, Miyagi, 980-8578, Japan

16 5 Cell Innovator Co., Ltd., 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.

17 6 Department of Applied Biochemistry and Food Science, Faculty of Agriculture, Saga

18 University, 1 Honjo-machi, Saga, 840-8502, Japan.

19

20 Running title: RNA-binding protein NrdA in Aspergillus

21

22 * Present address: Chihiro Kadooka, Faculty of Life and Environmental Sciences,

23 University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan.

24 # Address correspondence to Taiki Futagami, [email protected]

25

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26 ABSTRACT

27 RNA-binding protein Nrd1 plays a role in RNA polymerase II transcription termination.

28 In this study, we showed that the orthologous NrdA is important in global mRNA

29 expression and secondary metabolism in Aspergillus species. We constructed an nrdA

30 conditional expression strain using the Tet-On system in Aspergillus luchuenesis mut.

31 kawachii. Downregulation of nrdA caused a severe growth defect, indicating that NrdA

32 is essential for the proliferation of A. kawachii. Parallel RNA-sequencing and RNA

33 immunoprecipitation-sequencing analysis identified potential NrdA-interacting

34 transcripts, corresponding to 32% of the predicted protein coding genes of A. kawachii.

35 Subsequent gene ontology analysis suggested that overexpression of NrdA affects the

36 production of secondary metabolites. To clarify this, we constructed

37 NrdA-overexpressing strains of Aspergillus nidulans, Aspergillus fumigatus, and

38 Aspergillus oryzae. Overexpression of NrdA reduced the production of sterigmatocystin

39 and penicillin in A. nidulans, as well as that of helvolic acid and pyripyropene A in A.

40 fumigatus. Moreover, it increased the production of kojic acid and reduced production

41 of penicillin in A. oryzae. These effects were accompanied by almost consistent

42 transcriptional changes in the relevant genes. Collectively, these results suggest that

43 NrdA is the essential RNA-binding protein, which plays a significant role in global gene

44 expression and secondary metabolism in Aspergillus species.

45

46 IMPORTANCE

47 Nrd1, a component of the Nrd1–Nab3–Sen1 complex, is an essential RNA-binding

48 protein involved in transcriptional termination in yeast. However, its role in filamentous

49 fungi has not been studied. In this study, we characterized an orthologous NrdA in the

50 Aspergillus species, identified potential NrdA-interacting mRNA, and investigated the

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51 effect of overexpression of NrdA on mRNA expression in Aspergillus luchuensis mut.

52 kawachii. The results indicated that NrdA controls global gene expression involved in

53 versatile metabolic pathways, including the secondary metabolic process. We

54 demonstrated that NrdA overexpression significantly affected the production of

55 secondary metabolites in Aspergillus nidulans, Aspergillus oryzae, and Aspergillus

56 fumigatus. Our findings are of importance to the fungal research community because the

57 secondary metabolism is an industrially and clinically important aspect for the

58 Aspergillus species.

59

60 KEYWORDS

61 Aspergillus, RNA-binding protein, NrdA, gene expression, secondary metabolism

62

63

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64 INTRODUCTION

65 There are at least two pathways for transcription termination in the budding yeast

66 Saccharomyces cerevisiae, namely the RNA polymerase II cleavage/polyadenylation

67 factor-related pathway and the Nrd1–Nab3–Sen1 (NNS)-related pathway (1, 2). The

68 former cleavage/polyadenylation factor pathway is required for polyadenylation of the 3’

69 end of precursor mRNA. The latter pathway is required for the early termination of

70 transcripts to produce the shorter polyadenylated transcripts. Moreover, it is involved in

71 the subsequent production of non-coding transcripts, such as small nucleolar RNAs,

72 small nuclear RNAs, cryptic unstable transcripts, and stable uncharacterized transcripts

73 with largely unknown functions. The Nrd1 and Nab3 are essential RNA-binding

74 proteins and bind specific sequences in target RNA, whereas Sen1 is a helicase

75 responsible for the termination of transcription. The NNS complex has been extensively

76 studied from the view point of non-coding RNA production. However, it has been

77 recently suggested that the NNS-related pathway also plays a significant role in

78 protein-coding mRNA involved in response to nutrient starvation (3, 4). It was proposed

79 that binding of Nrd1 and Nab3 led to premature transcription termination, resulting in

80 the downregulation of mRNA levels. In S. cerevisiae, it was estimated that Nrd1 and

81 Nab3 directly regulate a variety of protein-coding genes, representing 20%–30% of

82 protein-coding transcripts (5). In addition, the Nrd1 ortholog Seb1 drives transcription

83 termination for both protein-coding and non-coding sequences in the fission yeast

84 Schizosaccharomyces pombe (6, 7).

85 We have investigated the mechanism of citric acid accumulation by the white koji

86 fungus Aspergillus luchuensis mut. kawachii, which is used for the production of

87 shochu, a Japanese traditional distilled spirit. A. kawachii is used as a producer of

88 starch-degrading enzymes, such as α-amylase and glucoamylase (8, 9). In addition, A.

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89 kawachii also produces a large amount of citric acid, which prevents the growth of

90 microbial contaminants during the fermentation process (10). During the course of the

91 study, we found that the Nrd1 ortholog encoding nrdA was located in a syntenic region

92 together with mitochondrial citrate synthase-encoding citA and mitochondrial citrate

93 transporter encoding yhmA in the subdivision Pezizomycotina (including genus

94 Aspergillus). However, it was not found in the subdivisions Saccharomycotina

95 (including genus Saccharomyces) and Taphrinomycotina (including genus

96 Schizosaccharomyces) (11). Considering the numerous examples of metabolic gene

97 clusters in fungi and plants (12, 13), this finding motivated us to study the function of

98 NrdA in Aspergillus species in terms of metabolic activity.

99 In this study, we showed that NrdA is an essential RNA-binding protein involved

100 in the production of citric acid in A. kawachii using the nrdA conditional expression

101 strain. Nevertheless, whether nrdA is functionally related to citA and yhmA remains

102 unclear. By combining RNA-sequencing (RNA-seq) and RNA

103 immunoprecipitation-sequencing (RIP-seq) analyses, we identified potential NrdA–

104 mRNA interactions, corresponding to approximately 32% of protein-coding genes of the

105 A. kawachii genome. In addition, we showed that overexpression of nrdA causes

106 significant changes in the expression levels of NrdA-interacting mRNA involved in

107 secondary metabolism. Furthermore, we demonstrated that overexpression of nrdA also

108 caused changes in the production of secondary metabolites in Aspergillus nidulans,

109 Aspergillus fumigatus, and Aspergillus oryzae. These results suggested that the

110 NrdA-associated transcription termination pathway potentially regulates the secondary

111 metabolic process in the Aspergillus species.

112

113 RESULTS AND DISCUSSION

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114 Sequence feature of NrdA. The A. kawachii nrdA gene encodes a protein

115 composed of 742 amino acid residues. BLASTP analysis

116 (https://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that the amino acid sequence percent

117 identities between A. kawachii NrdA and S. cerevisiae Nrd1, and between A. kawachii

118 NrdA and Schiz. pombe Seb1 were 48.57% and 34.52%, respectively. The RNA

119 polymerase II C-terminal domain-interacting domain (CID) (1–150 amino acid residues

120 of S. cerevisiae Nrd1) (14-16) and RNA recognition motif (RRM) (339–407 amino acid

121 residues of S. cerevisiae Nrd1) (16, 17) are well conserved in A. kawachii NrdA (Fig.

122 S1). The Pfam domain analysis (https://pfam.xfam.org/) also confirmed the presence of

123 CID and RRM in the relevant regions of A. kawachii NrdA (data not shown). On the

124 other hand, the amino acid residues of the Nab3-binding domain,

125 arginine-glutamate/arginine-serine-rich domain, and the C-terminal proline/glutamine

126 (P/Q)-rich low-complexity domain (LCD) (151–214, 245–265, and 513–575 amino acid

127 residues of S. cerevisiae Nrd1, respectively) (16, 18, 19) were less conserved in the A.

128 kawachii NrdA. The Nab3-binding domain, arginine-glutamate/arginine-serine-rich

129 domain, and P/Q rich LCD of A. kawachii NrdA were more similar to those of Schiz.

130 pombe Seb1 than those of S. cerevisiae Nrd1. In addition, amino acid restudies between

131 RRM and P/Q rich LCD were well conserved among S. cerevisiae Nrd1, Schiz. pombe

132 Seb1, and A. kawachii NrdA. Phylogenetic analysis supported that Nrd1 orthologs of

133 subdivision Pezizomycotina (including A. kawachii NrdA) are closer to those of

134 subdivision Taphrinomycotina (including Schiz. pombe Seb1) than those of subdivision

135 Saccharomycotina (including S. cerevisiae Nrd1) (Fig. S2).

136 Phenotype of the Tet-nrdA strain. To investigate the physiologic role of nrdA,

137 we attempted to construct A. kawachii nrdA disruptant. However, all of transformants

138 obtained by the nrdA disruption cassette were heterokaryotic gene disruptants (data not

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139 shown). Therefore, we constructed a Tet-nrdA strain that conditionally expressed nrdA

140 under the control of a Tet-On promoter. The Tet-nrdA strain exhibited significantly

141 deficient growth in yeast extract sucrose (YES) agar medium without doxycycline

142 (Dox) (nrdA depletion condition) (Fig. 1A), indicating that disruption of nrdA induces

143 lethality. This result was consistent with those of previous reports stating that NRD1 and

144 seb1 are essential genes in S. cerevisiae and Schiz. pombe, respectively (20, 21).

145 Next, we tested the effect of NrdA expression on citric acid production, because

146 nrdA was located in the syntenic region with mitochondrial citrate synthase encoding

147 citA and citrate transporter encoding yhmA (11). We compared the production of citric

148 acid by A. kawachii control and Tet-nrdA strains (Fig. 1B). The control strain was

149 precultivated in YES medium at 30°C for 16 h, transferred to citric acid production

150 (CAP) medium (an optimized medium for CAP) (11), and further cultured at 30°C for

151 48 h. Because the Tet-nrdA strain showed severe defect growth in the absence of Dox, it

152 was precultivated in YES medium with Dox and transferred to CAP medium with or

153 without Dox. Following cultivation, the concentration of citric acid in the culture

154 supernatant and mycelial biomass was measured to determine the extracellular CAP per

155 mycelial weight. Based on the level of citric acid in the culture supernatant and amount

156 of mycelial biomass produced, the Tet-nrdA strain cultivated without Dox showed

157 similar CAP compared with the control strain. In contrast, the Tet-nrdA strain cultivated

158 with Dox exhibited only approximately 9% of the CAP of the control strain.

159 Subcellular localization of NrdA. To analyze the subcellular localization of

160 NrdA in A. kawachii, NrdA tagged with green fluorescent protein (GFP-NrdA) and

161 histone H2B tagged with monomeric red fluorescent protein (mRFP) (H2B-mRFP, a

162 nuclear marker protein) were co-expressed in the Tet-nrdA strain. The GFP-NrdA was

163 expressed by the native nrdA promoter. Functional expression of GFP-NrdA was

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164 confirmed by the viable phenotype of the Tet-nrdA plus GFP-nrdA plus H2B-mRFP

165 strain in YES agar medium without Dox (Fig. 2A). The Tet-nrdA plus GFP-nrdA plus

166 H2B-mRFP strain was cultivated in minimal liquid medium for 16 h without shaking

167 and observed by fluorescence microscopy. Green fluorescence associated with

168 GFP-NrdA merged with the red fluorescence of H2B-mRFP (Fig. 2B), indicating that

169 the GFP-NrdA localizes in the nucleus. In addition, speckles of green fluorescence were

170 observed in the nucleus. It was reported that the Nrd1-dependent nuclear speckles

171 appear in S. cerevisiae under glucose starvation conditions (3). Thus, it might be

172 possible that NrdA also forms these speckles in A. kawachii in response to some

173 environmental stress.

174 Examination of experimental conditions for transcriptome analysis. We

175 performed transcriptome analysis to investigate the NrdA-associated transcripts. For

176 this purpose, we constructed a Tet-S-nrdA strain, which expresses S-tagged NrdA

177 (S-NrdA) under the control of the Tet-On promoter. The Tet-S-nrdA strain formed

178 colonies in the YES agar medium with Dox, whereas it showed severe growth defect in

179 the YES agar medium without Dox. These findings confirmed that the Tet-On system

180 regulates the functional S-NrdA (Fig. 3A). In addition, the expression of S-NrdA was

181 confirmed by the detection of a band of the predicted size of the S-NrdA protein through

182 S-protein affinity purification (Fig. 3B left panel) and detection by immunoblotting

183 using an anti-S-tag antibody (Fig. 3B right panel).

184 For the identification of NrdA–mRNA interaction and evaluation of the

185 destination of mRNA interacted with NrdA, we performed an experiment under the

186 following conditions. The Tet-S-nrdA strain was precultivated in YES medium with

187 Dox; next, mycelia were transferred to YES medium with or without Dox and further

188 cultivated (Fig. 3C). The resultant mycelial cells cultivated with Dox were separated

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189 into two portions and used for RNA-seq analysis and RIP-seq analysis to identify the

190 NrdA–mRNA interaction by comparing their RNA pools. In addition, the resultant

191 mycelial cells cultivated without Dox were used for RNA-seq analysis to investigate the

192 expression levels of NrdA-interacting mRNA in the presence or absence of Dox.

193 For the latter purpose, we evaluated the expression level of S-nrdA in the

194 Tet-S-nrdA strain cultivated in the presence or absence of Dox. The Tet-S-nrdA strain

195 was precultivated in YES medium with Dox for 16 h; next, mycelia were transferred to

196 YES medium with or without Dox and further cultivated for 4, 6, 12, or 24 h. In

197 addition, the A. kawachii control strain was precultivated in the YES medium for 16 h;

198 subsequently, the mycelia were transferred to YES medium and further cultivated for 24

199 h. After the cultivations, total RNA was extracted and used to compare the expression

200 levels of S-nrdA (Fig. 3D). The levels of S-nrdA in the Tet-S-nrdA strain cultivated in

201 the medium with Dox was significantly higher (17.3–37.2-fold higher) than those of

202 nrdA in the control strain, indicating that S-NrdA was overexpressed throughout the

203 cultivation period. On the other hand, the expression level of S-nrdA in the Tet-S-nrdA

204 strain cultivated in the medium without Dox was gradually decreased; however, it

205 remained comparable to the expression level of nrdA in the control strain after

206 cultivation for 12 h and 24 h. This result indicated that the S-nrdA transcript was not

207 depleted even after cultivation without Dox for 24 h. Thus, we considered cultivation of

208 the Tet-S-nrdA strain with and without Dox as S-nrdA overexpression and expression

209 condition, respectively. To confirm this consideration, we additionally performed

210 RNA-seq analysis of the control strain cultivated in YES medium without Dox (Fig. 3C,

211 an experimental condition was surrounded by the dotted line), followed by clustering

212 analysis of RNA-seq along with the RNA-seq and RIP-seq data obtained using the

213 Tet-S-nrdA strain (Fig. 3E). The results indicated that the transcriptomic profile of the

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214 control strain is more similar to that of the Tet-S-nrdA strain cultivated without Dox

215 than that of the Tet-S-nrdA strain cultivated with Dox. In addition, this is consistent with

216 the data showing that the Tet-nrdA strain cultivated without Dox showed similar CAP

217 compared with the control strain. In contrast, the Tet-nrdA strain cultivated with Dox

218 exhibited significantly reduced CAP compared with the control strain (Fig. 1B).

219 Analysis of NrdA-interacting transcripts and their destination. Based on the

220 examination of experimental conditions, we cultivated the Tet-S-nrdA strain in YES

221 medium with or without Dox as S-nrdA overexpression and expression condition,

222 respectively, and the mycelia were subjected to RNA-seq and RIP-seq (Fig. 3C). A

223 4.4-fold higher amount of RNA was obtained from the mycelia of the Tet-S-nrdA strain

224 cultivated with Dox by RIP using the anti-S-tag antibody compared with that obtained

225 using the normal rabbit IgG (negative control) (Fig. 4A). This result indicated that the

226 enrichment of NrdA-associated RNA was successful. To identify the NrdA–mRNA

227 interaction, we compared the mRNA profiles of the Tet-S-nrdA strain cultivated with

228 Dox obtained from the RNA-seq and RIP-seq analyses (Fig. 3C). We predicted NrdA–

229 mRNA interaction by identifying the mRNA significantly enriched by RIP based on the

230 following criteria: log2 fold change >0 and q-value <0.05 (see Data Set S1 in the

231 supplemental material). This analysis identified 3,676 mRNA transcripts that represent

232 32% of the total 11,474 predicted coding sequences of A. kawachii. This is consistent

233 with the estimation that Nrd1 and Nab3 directly regulate a variety of protein-coding

234 genes, representing 20%–30% of the protein-coding transcripts of S. cerevisiae (5).

235 This study was initiated due to an interest in the nrdA genes localized on the

236 syntenic region with citA and yhmA on the genome. However, the transcripts of citA

237 (AKAW_06279) and yhmA (AKAW_06280) were not predicted to interact with NrdA

238 (see Data Set S1 in the supplemental material). In addition, citA and yhmA are not

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239 essential genes in A. kawachii (11, data not shown), indicating that they are not

240 responsible for the lethality caused by nrdA disruption.

241 We performed Gene Ontology (GO) enrichment analysis of mRNA transcripts,

242 which were predicted to interact with NrdA. The results of this analysis identified

243 metabolic process-, transport process-, and transcriptional regulation-related terms

244 (Table 1). In addition, enriched terms included numerous secondary metabolic related

245 terms, such as the secondary metabolic process (GO:0019748) and mycotoxin

246 biosynthetic process (GO:0043386). It should be noted that A. kawachii does not

247 produce mycotoxins, such as ochratoxin A and fumonisin B, and is considered safe for

248 use in the food and beverage industry (22, 23).

249 Next, to examine the significance of NrdA–mRNA interaction, differential gene

250 expression between the S-NrdA expression and overexpression conditions was

251 compared using RNA-seq (see Data Set S1 in the supplemental material). A q-value

252 <0.05 denoted statistically significant changes in gene expression. Based on this

253 criterion, the predicted 3,676 NrdA-associated mRNAs included 1,768 downregulated

254 genes and 674 upregulated genes by the overexpression of S-NrdA. These findings

255 indicated that approximately half of NrdA-associated mRNAs were downregulated by

256 the overexpression of S-NrdA. In addition, RIP-seq and RNA-seq plotting analyses

257 showed that the proportion of downregulated genes was increased in the higher enriched

258 mRNA transcript by RIP (predicted strong interaction of mRNA with S-NrdA) (Fig. 4B).

259 For example, the higher enriched mRNA transcript (with log2 fold change >5) included

260 10 genes downregulated by the overexpression of S-NrdA (surrounded by dotted line in

261 Fig. 4B). Based on the information obtained from domain search using the Pfam

262 database, these may have beta lactamase (AKAW_10564), G-protein coupled receptor

263 (AKAW_10304), aldehyde reductase (AKAW_01947), or tannanase (AKAW_10686

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264 and AKAW_04699) functions; however, most of them are hypothetical proteins with

265 unknown function (Table S1). In addition, S. cerevisiae and Schiz. pombe have

266 homologs only for AKAW_01947 among these 10 genes. The physiological reason

267 responsible for the interaction of the transcripts of these genes with NrdA remains

268 unclear. Nevertheless, the overexpression of NrdA associated with transcriptional

269 downregulation may be attributed to the negative effect exerted by the NNS complex

270 pathway on the mRNA expression through the termination of transcription (3-5). Hence,

271 the overexpression of S-NrdA may cause the downregulation of interacted RNA

272 molecules by the transcription termination.

273 NrdA regulates the production of secondary metabolites in A. nidulans, A.

274 fumigatus, and A. oryzae. GO analysis indicated that the significant change observed in

275 the transcriptional levels of secondary metabolism was related to genes induced by the

276 overexpression of NrdA in A. kawachii (Table 1). This result was interesting because the

277 relationships between the NNS complex pathway and the production of secondary

278 metabolites have not been studied. However, the secondary metabolites of A. kawachii

279 are currently largely unknown. Therefore, we tested the effect of overexpression of

280 intrinsic NrdA on the production of secondary metabolites in A. nidulans, A. fumigatus,

281 and A. oryzae.

282 A. nidulans OE-nrdA strain showed a slightly fluffy phenotype (Fig. 5A) with

283 reduced number of conidia (Fig. 5B). To investigate the production of penicillin and

284 sterigmatocystin, A. nidulans strains were precultivated in YES medium at 30°C for

285 16 h, transferred to minimal medium, and further cultivated at 30°C for 24, 48, or 72 h.

286 The results of the halo assay indicated that the OE-nrdA strain showed significantly

287 reduced penicillin production compared with the control strain (Fig. 5C). The sizes of

288 the halos were significantly different using culture supernatant obtained after 24 and 48

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289 h, whereas they were similar at 72 h. This evidence indicates that overexpression of

290 NrdA suppressed the production of penicillin in the early cultivation period. In addition,

291 the production of sterigmatocystin by OE-nrdA was 26% of that produced by the control

292 strain after 24 h of cultivation (Fig. 5D, left side). This difference was reduced after 48 h

293 of cultivation (Fig. 5D, right side). We confirmed that transcriptional levels of nrdA in

294 OE-nrdA were 15-fold higher compared with those detected in the control strain at 12 h

295 (Fig. 5E). The differences in the transcriptional levels of nrdA in OE-nrdA were not

296 statistically significant at 24 and 48 h, indicating that nrdA is overexpressed in the early

297 cultivation period of the OE-nrdA strain. Next, we investigated whether the reduced

298 production of penicillin and sterigmatocystin was controlled at the transcriptional level.

299 The transcriptional levels of ipnA (an isopenicillin-N synthase with a role in penicillin

300 biosynthesis) (24), aflR (a transcriptional regulator involved in the production of

301 sterigmatocystin) (25), and stcU (a putative versicolorin reductase involved in the

302 biosynthesis of sterigmatocystin) (26, 27) were significantly reduced in the OE-nrdA

303 strain at 12 and 24 h compared with the control strain. However, these levels became

304 similar at 48 h. This is consistent with the data showing that the higher transcriptional

305 level of nrdA and reduced productions of penicillin and sterigmatocystin were

306 significant in the early cultivation period.

307 The A. fumigatus OE-nrdA strain showed similar colony sizes (Fig. 6A) with

308 reduced number of conidia (Fig. 6B). Next, we analyzed the levels of fumagillin,

309 helvolic acid, and pyripyropene A in culture supernatant (Fig. 6C). A. fumigatus strains

310 were cultivated in minimal liquid medium with fetal bovine serum (FBS) at 30°C for

311 24 h. The difference in the production of fumagillin between the control and OE-nrdA

312 strains were not statistically significant. In contrast, the production of helvolic acid and

313 pyripyropene by OE-nrdA was significantly reduced to 47% and 3% of that observed in

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314 the control strain, respectively. We confirmed that the OE-nrdA strain exhibited higher

315 expression of nrdA compared with the control strain (Fig. 6D). Subsequently, we

316 investigated the gene expression of fumR (a putative C6 type transcription

317 factor-encoding gene involved in fumagillin biosynthesis) (28), helA (an oxidosqualene

318 cyclase-encoding gene involved in the production of helvolic acid) (29), and pyr2 (a

319 polyketide synthase-encoding gene involved in the biosynthesis of pyripyropene A) (30).

320 Although the transcriptional levels of fumR in the control and OE-nrdA strains were

321 similar, those of helA and pyr2 were lower in the OE-nrdA strain (Fig. 6D). This was

322 consistent with the results demonstrating that the production of helvolic acid and

323 pyripyropene A was significantly suppressed by the overexpression of NrdA.

324 A. oryzae control and OE-nrdA strains were cultivated in medium containing

325 ferric ion (31), whose color turns red if it is chelated by kojic acid (Fig. 7A). The

326 OE-nrdA strain showed a reduced number of conidia (Fig. 7B). In addition, the number

327 of sclerotia increased in the OE-nrdA strain compared with the control strain (Fig. 7A).

328 The bottom side of the colony of the OE-nrdA strain exhibited a red color, indicating

329 that this strain produces higher amounts of kojic acid compared with the control strain.

330 To investigate the transcriptional levels of genes involved in the production of kojic acid,

331 the strains were cultivated in kojic acid production liquid medium at 30°C for 5 days.

332 Furthermore, we confirmed a higher transcriptional level of nrdA in the OE-nrdA strain

333 compared with that detected in the control strain (Fig. 7C). In addition, the gene

334 expression of kojR, kojA, and kojT, which are involved in the production of kojic acid

335 (31), also exhibited higher levels compared with those measured in the control strain. To

336 investigate the production of penicillin, strains were precultured in YES medium,

337 transferred to minimal medium, and further cultivated for 24 or 48 h. The results of the

338 halo assay using the culture supernatant at 48 h revealed significantly reduced

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339 production of penicillin by the OE-nrdA strain (Fig. 7D). In addition, the transcriptional

340 analysis confirmed that the OE-nrdA strain showed higher and similar expression levels

341 of nrdA compared with the control strain at 12 and 24 h, respectively (Fig. 7E). In

342 addition, the OE-nrdA strain showed higher and lower transcriptional levels of ipnA

343 compared with the control strain at 12 and 24 h, respectively. The significantly lower

344 transcriptional level of ipnA is consistent with the data indicating that penicillin

345 production was suppressed by the overexpression of nrdA at 48 h.

346 The overexpression of nrdA significantly affected the production of secondary

347 metabolites in A. nidulans, A. fumigatus, and A. oryzae. A hypothesis is that NrdA may

348 be directly involved in the silencing of secondary metabolite gene clusters via a

349 NNS-related transcription termination system because the overexpression of nrdA

350 suppressed the gene expression levels of ipnA, aflR, and stcU in A. nidulans (Fig. 5E),

351 helA and pyr2 in A. fumigatus (Fig. 6D), and ipnA in A. oryzae (Fig. 7E). Another

352 hypothesis is that these genes may be indirectly regulated via transcriptional factors

353 and/or histone modification and chromatin remodeling, which often regulate the

354 production of secondary metabolites in fungi (32). The transcriptional factor BrlA

355 regulates the production of fumagillin, helvolic acid, and pyripyropene A in A.

356 fumigatus (33). The production of sterigmatocystin and penicillin is regulated by the

357 putative methyltransferase LaeA in A. nidulans (34). The production of penicillin and

358 kojic acid is regulated by histone deacetylase Hst4 and LaeA (35, 36). In S. cerevisiae,

359 Nrd1 and exosome-dependent RNA degradation leads to gene silencing by histone

360 modification. The euchromatin mark, such as histone H3 trimethylated at lysine 4

361 (H3K4me3) and histone H3 acetylated at lysine 9 (H3K9ac) at non-transcribed spacer

362 locus of ribosomal DNA, a heterochromatin region is enriched by the depletion of Nrd1

363 (37). Recently, it was also reported that heterochromatin assembly occurs by the pausing

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364 of RNA polymerase II promoted by Seb1 in Schiz. pombe (38). Thus, overexpression of

365 NrdA might contribute to the formation of heterochromatin at the secondary metabolite

366 gene cluster regions in Aspergillus spp.

367 We could not determine the reason for the co-localization of nrdA with citA and

368 yhmA in subdivision Pezizomycotina; however, NrdA plays a significant role in global

369 gene regulation and is involved in the production of secondary metabolites in

370 Aspergillus species. CAP is regulated by the regulator of secondary metabolism LaeA in

371 Aspergillus niger, Aspergillus carbonarius, and A. kawachii (39-41). Therefore, NrdA

372 may be linked to CAP via the regulation of secondary metabolism. Although the

373 NNS-associated RNA surveillance system has been extensively investigated in the

374 yeasts S. cerevisiae and Schiz. pombe, the present findings may enhance the

375 understanding of the NNS-associated production of secondary metabolites in

376 Aspergillus spp., including industrially and clinically important fungi.

377

378 MATERIALS AND METHODS

379 Strains and culture conditions. In this study, A. kawachii SO2 (42), A. nidulans

380 A26 (Fungal Genetics Stock Center [FGSC; Manhattan, KS]), A. fumigatus A1151

381 (FGSC), and A. oryzae ΔligD plus pGNA (43, 44) were used as parental strains (Table

382 2). Control strains were defined to show an identical auxotrophic background for

383 comparison with the respective nrdA overexpression strains.

384 Strains were cultivated in minimal medium (45; FGSC

385 [http://www.fgsc.net/methods/anidmed.html]) with or without 0.211% (wt/vol) arginine

386 and/or 0.15% (wt/vol) methionine, and/or 0.1 µg/ml pyrimethamine. CAP medium (11),

387 kojic acid production medium with 1 mM ferric ion (31), or YES (15% sucrose [wt/vol],

388 2% yeast extract [wt/vol]) medium were used appropriately for the fungal growth

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389 experiments. Minimal and CAP media were adjusted to the required pH with NaOH and

390 HCl, respectively. For the cultivation of A. nidulans strains, minimal medium was

391 supplemented with 5 µg/ml biotin.

392 Construction of A. kawachii Tet-nrdA and Tet-S-nrdA strains. To achieve

393 Dox-inducible conditional expression of the nrdA gene, 2 kb of the 5’-end of nrdA, 2.2

394 kb of argB, 0.8 kb of cgrA terminator and Tet-On promoter, and 1.8 kb of a part of the

395 ORF region of nrdA were constructed by recombinant PCR using the primer pairs

396 AKtet-nrdA-FC/AKtet-nrdA-R1, AKtet-nrdA-F2/AKtet-nrdA-R2,

397 AKtet-nrdA-F3/AKtet-nrdA-R3, and AKtet-nrdA-F4/AKtet-nrdA-RC, respectively (see

398 Table S2 in the supplemental material). For amplification of the argB gene and Tet-On

399 promoter, A. nidulans A26 genomic DNA and plasmid pVG2.2ANsC were used as

400 template DNA, respectively (11, 46). The resultant DNA fragment was amplified with

401 the primers AKtet-nrdA-F1 and AKtet-nrdA-R4 and used to transform the A. kawachii

402 strain CK3, which carried pVG2.2ANsC, yielding the Tet-nrdA strain. Transformants

403 were selected on minimal agar medium without arginine. Introduction of this cassette

404 into the target locus was confirmed with PCR using the primer pairs AKtet-nrdA-FC

405 and AKtet-nrdA-RC (see Fig. S3 in the supplemental material).

406 To confirm the functional expression of S-tagged NrdA, we constructed a S-NrdA

407 expression strain (data not shown). The 2 kb of the 5’-end of nrdA, 1.8 kb of ptrA, and

408 1.8 kb of a part of the ORF region of S-nrdA were constructed by recombinant PCR

409 using the primer pairs Aktet-nrdA-FC/AkS-nrdA-ptrA-R1,

410 AkS-nrdA-ptrA-F2/AkS-nrdA-ptrA-R2, and AkS-nrdA-ptrA-F3/AKtet-nrdA-RC,

411 respectively (see Table S2 in the supplemental material). For amplification of the ptrA

412 gene, pPTR I (Takara bio, Shiga, Japan) (47) was used as template DNA. The resultant

413 DNA fragment was amplified with the primers AKtet-nrdA-F1 and AKtet-nrdA-R4 and

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414 used to transform the A. kawachii strain SO2, yielding the S-nrdA strain. Transformants

415 were selected on minimal agar medium with pyrithiamine.

416 To achieve Dox-inducible conditional expression of the S-nrdA gene, 2 kb of the

417 5’-end of nrdA, 2.2 kb of argB, 0.8 kb of cgrA terminator and Tet-On promoter, and 2

418 kb of a part of ORF region of nrdA were constructed by recombinant PCR using the

419 primer pairs AKtet-nrdA-FC/AKtet-S-nrdA-R3, and AKtet-S-nrdA-F4/AKtet-nrdA-RC,

420 respectively (see Table S2 in the supplemental material). For amplification of the 5’-end

421 nrdA-argB-Tet-On promoter and S-nrdA, the genomic DNA of strain Tet-nrdA and strain

422 S-nrdA were used as template DNA, respectively. The resultant DNA fragment was

423 amplified with the primers AKtet-nrdA-F1 and AKtet-nrdA-R4 and used to transform

424 the A. kawachii strain CK3, yielding the Tet-S-nrdA strain. Transformants were selected

425 on minimal agar medium without arginine. Introduction of this cassette into the target

426 locus was confirmed with PCR using the primer pairs AKtet-nrdA-FC and

427 AKtet-nrdA-RC (see Fig. S3 in the supplemental material). The expression of S-tagged

428 NrdA was confirmed by purification using S-protein agarose (Merck Millipore,

429 Darmstadt, Germany) as previously described (11, 48). The purified protein was

430 subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and

431 immunoblotting analysis using an anti-S-tag antibody (Medical and Biological

432 Laboratories, Nagoya, Japan) (Fig. 3B).

433 Construction of an A. kawachii strain expressing GFP-NrdA and H2B-mRFP.

434 The plasmid pPTR I (Takara Bio), which carries the ptrA gene, was used to construct

435 the expression vector for GFP-NrdA. The 5’-end of nrdA (until start codon) and gfp and

436 nrdA ORF (without start codon) were amplified by PCR using the primer pairs

437 pPTRI-gfp-nrdA-F1/pPTRI-gfp-nrdA-R1, pPTRI-gfp-nrdA-F2/pPTRI-gfp-nrdA-R2,

438 and pPTRI-gfp-nrdA-F3/pPTRI-gfp-nrdA-R3, respectively. For the amplification of gfp,

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439 plasmid pFNO3 (49) was used as template DNA. The resultant DNA fragment was

440 amplified with the primers pPTRI-gfp-nrdA-inf-F/pPTRI-gfp-nrdA-inf-R (see Table S2

441 in the supplemental material). The amplified fragment was cloned into the SmaI site of

442 pPTR I using an In-Fusion HD cloning kit (Takara Bio). The pPTR I-GFP-nrdA plasmid

443 was used to transform the A. kawachii strain Tet-nrdA, yielding the GFP-NrdA strain.

444 Transformants were selected on minimal agar medium with pyrithiamine. After

445 fluorescence microscopy testing, the pGbar-H2B-mRFP plasmid (50) was used to

446 transform the A. kawachii strain GFP-NrdA, yielding the GFP-NrdA H2B-mRFP strain.

447 Transformants were selected on minimal agar medium with glufosinate extracted from

448 the herbicide Basta (Bayer Crop Science, Bayer Japan, Tokyo, Japan).

449 Fluorescence microscopy. A strain expressing GFP-NrdA and H2B-mRFP was

450 cultured in minimal medium supplemented with arginine. After cultivation in minimal

451 medium supplemented with arginine for 12 h, the mycelia were observed under a

452 DMI6000B inverted-type fluorescent microscope (Leica Microsystems, Wetzlar,

453 Germany). Image contrast was adjusted using the LAS AF Lite software, version 2.3.0,

454 build 5131 (Leica Microsystems).

455 Measurement of citric acid. To measure the levels of extracellular citric acid,

456 conidia (2 × 107 cells) of the A. kawachii control strain were inoculated into 100 ml of

457 YES medium and precultivated with shaking (180 rpm) at 30°C for 16 h. Subsequently,

458 they were transferred to 50 ml of CAP medium with arginine and further cultivated with

459 shaking (163 rpm) at 30°C for 48 h. Prior to their transfer to CAP medium, the mycelia

460 were washed using fresh CAP medium. The Tet-nrdA strain was precultured in YES

461 medium with 1 µg/ml Dox and transferred to CAP medium with or without Dox. The

462 culture supernatant was filtered through a PTFE filter (pore size: 0.2 μm) (Toyo Roshi

463 Kaisha, Tokyo, Japan) and used as the extracellular fraction.

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464 The concentration of citric acid was determined using a Prominence HPLC

465 system (Shimadzu, Kyoto, Japan) equipped with a CDD-10AVP conductivity detector

466 (Shimadzu). The organic acids were separated with tandem Shimadzu Shim-pack

467 SCR-102H columns (300 × 8 mm I.D.; Shimadzu) at 50°C using 4 mM

468 p-toluenesulfonic acid monohydrate as the mobile phase at a flow rate of 0.8 ml/min.

469 The flow rate of the post-column reaction solution (4 mM p-toluenesulfonic acid

470 monohydrate, 16 mM bis-Tris, and 80 µM ethylenediaminetetraacetic acid [EDTA])

471 was 0.8 ml/min.

472 RNA preparation. To identify the transcriptome of A. kawachii control and

473 Tet-S-nrdA strains, conidia (2 × 107 cells) were inoculated into 100 ml of YES medium

474 with 1 µg/ml Dox (for Tet-S-nrdA strain) and precultured for 14 h at 30°C. Subsequently,

475 they were transferred to 50 ml of YES medium with or without Dox (for Tet-S-nrdA

476 strain) and further cultivated with shaking (163 rpm) at 30°C for 24 h. Prior to their

477 transfer to YES medium with or without Dox, the mycelia were washed with fresh YES

478 medium. Following incubation, the mycelia were collected and ground to a powder in

479 the presence of liquid nitrogen. Total RNA was extracted using RNAiso Plus (Takara

480 Bio) according to the instructions provided by the manufacturer and quantified using a

481 NanoDrop-8000 (Thermo Fisher Scientific).

482 RIP assay. To identify NrdA interacting RNA, A. kawachii Tet-S-nrdA strain was

483 cultivated using YES medium with Dox under the condition described for the RNA

484 preparation. The mycelia were collected and ground to a powder in the presence of

485 liquid nitrogen. The powdered mycelia (1 g wet weight) were dissolved in 13 ml of

486 ice-cold nuclear extraction buffer (25 mM N-2-hydroxyethylpiperazine-N’-2-ethane

487 sulfonic acid [HEPES; pH 6.8], 1 M sorbitol, 250 μg/ml phenylmethylsulfonyl fluoride

488 [PMSF], cOmplete [EDTA-free protease inhibitor cocktail; Roche, Basel, Switzerland])

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489 and vigorously mixed using a vortexer. Cell debris was removed by filtration using

490 Miracloth, and the solution was centrifuged at 10,000 × g at 4°C for 15 min. The

491 supernatant was removed and the pellet was dissolved in 500 µl of ice-cold nuclear

492 solubilization buffer (25 mM HEPES [pH 6.8], 1 M sorbitol, 0.5% NP-40, 250 μg/ml

493 PMSF, cOmplete [EDTA-free protease inhibitor cocktail; Roche]). Following

494 incubation for 30 min at 4°C, the debris was removed by centrifugation at 2,000 × g at

495 4°C for 15 min. Twenty-five µL of Protein A Sepharose beads (50% slurry contained

496 phosphate-buffered saline buffer) (GE healthcare, Chicago, IL) was added to the

497 resultant supernatant, and the resulting mixture was gently mixed for 1 h at 4°C using a

498 rotator. The mixture was separated by centrifugation at 2,000 × g at 4°C for 1 min, and

499 25 µl of anti-S-tag antibody (Medical and Biological Laboratories) or normal rabbit IgG

500 (Medical and Biological Laboratories) cross-linked protein A Sepharose was added to

501 the supernatant. Subsequently, these mixtures were gently mixed for 3 h at 4°C using a

502 rotator. The RNA interacted with NrdA was purified using RiboCluster Profiler

503 (Medical and Biological Laboratories) according to the instructions provided by the

504 manufacturer and quantified using a NanoDrop-8000 (Thermo Fisher Scientific).

505 Library preparation, sequencing, and data analysis for RNA-seq and RIP-seq

506 were performed by Kabushiki Kaisha DNAFORM (Yokohama, Japan). All RNA

507 samples were treated with RiboZero (Human/Mouse/Rat) (Illumina, San Diego, CA) for

508 the depletion of ribosomal RNA. All RNA-seq and RIP-seq experiments were

509 performed thrice with RNA samples obtained from independently prepared mycelia

510 using the NextSeq 500 system (Illumina) and mapped to the A. kawachii IFO 4308

511 genome (51) using a pipeline of trimming (trim_galore

512 [http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/], trimmomatic [52],

513 cutadapt [53]), mapping (STAR) (54), counting gene levels (featureCount) (55),

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514 differential expression analysis (DEseq2) (56), and clustering analysis (MBCluster.Seq)

515 (57). A gene set of A. kawachii were converted to a homologous gene set of A. niger to

516 perform the GO term enrichment analysis using the GO Term Finder of the Aspergillus

517 genome database (AspGD, http://www.aspgd.org/) (58). This species was used because

518 A. niger is closely related to A. kawachii (9, 59, 60).

519 Construction of the A. nidulans nrdA overexpression strain. The plasmid

520 pPTR I (Takara Bio) was used to construct the overexpression vector for the A. nidulans

521 nrdA gene (Locus tag, AN8276). The A. nidulans gpdA promoter and nrdA were

522 amplified by PCR using the primer pairs

523 pPTRI-PgpdA-nrdA-inf-F1/pPTRI-PgpdA-nrdA-inf-R1 and

524 pPTRI-PgpdA-nrdA-inf-F2/pPTRI-PgpdA-nrdA-inf-R2 (see Table S2 in the

525 supplemental material). The amplified fragments were cloned into the SmaI site of

526 pPTR I using an In-Fusion HD cloning kit (Takara Bio). Transformants were selected on

527 minimal agar medium with pyrithiamine.

528 Construction of the A. fumigatus nrdA overexpression strain. For the

529 overexpression analysis of nrdA in A. fumigatus (Locus tag, AFUB_052760), a gene

530 replacement cassette encompassing a homology arm at the 5’ end of nrdA, ptrA

531 selection marker, A. nidulans gpdA promoter, and homology arm at the nrdA locus was

532 constructed through recombinant PCR using the primer pairs

533 AfPgpdA-nrdA-FC/AfPgpdA-nrdA-R1, AfPgpdA-nrdA-F2/PgpdA-nrdA-R2,

534 AfPgpdA-nrdA-F3/PgpdA-nrdA-R3, and AfPgpdA-nrdA-F4/AfPgpdA-nrdA-RC,

535 respectively (see Table S2 in the supplemental material). For the amplification of DNA

536 fragments, A. fumigatus FGSC A1151 genomic DNA, A. nidulans A26 genomic DNA,

537 or plasmid pPTR I (Takara Bio) were used as template DNA. The resultant DNA

538 fragments amplified with primers AfPgpdA-nrdA-F1/AfPgpdA-nrdA-R4 were used to

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539 transform the A. fumigatus FGSC A1151, yielding the A. fumigatus OE-nrdA strain.

540 Transformants were selected on minimal agar medium supplemented with 0.1 µg/ml

541 pyrithiamine. Introduction of the ptrA and A. nidulans gpdA promoter into the target

542 locus was confirmed by PCR using primers AfPgpdA-nrdA-FC/AfPgpdA-nrdA-RC (see

543 Fig. S4 in the supplemental material).

544 Construction of the A. oryzae nrdA overexpression strain. For the overexpression

545 analysis of nrdA in A. oryzae (Locus tag, AO090102000629), a gene replacement

546 cassette encompassing a homology arm at the 5’ end of nrdA, ptrA selection marker, A.

547 nidulans gpdA promoter, and homology arm at the nrdA locus of A. oryzae was

548 constructed with recombinant PCR using the primer pairs

549 AoPgpdA-nrdA-FC/AoPgpdA-nrdA-R1, AoPgpdA-nrdA-F2/PgpdA-nrdA-R2,

550 AoPgpdA-nrdA-F3/PgpdA-nrdA-R3, and AoPgpdA-nrdA-F4/AoPgpdA-nrdA-RC,

551 respectively (see Table S2 in the supplemental material). For the amplification of DNA

552 fragments, A. oryzae RIB40 wild-type genomic DNA, A. nidulans A26 genomic DNA,

553 and plasmid pPTR I (Takara Bio) were used as template DNA. These resultant DNA

554 fragments amplified with primers AoPgpdA-nrdA-F1/AoPgpdA-nrdA-R4 were used to

555 transform the A. oryzae strain ΔligD plus pGNA (43, 44), yielding the A. oryzae strain

556 OE-nrdA. Transformants were selected on minimal agar medium supplemented with 0.1

557 µg/ml pyrithiamine. Introduction of the ptrA and A. nidulans gpdA promoter into the

558 target locus was confirmed by PCR using primers

559 AoPgpdA-nrdA-FC/AoPgpdA-nrdA-RC (see Fig. S5 in the supplemental material).

560 Analysis of secondary metabolites by liquid chromatography-mass

561 spectrometry. Conidia (2 × 107 cells) were inoculated into 100 ml of YES medium and

562 precultivated with shaking (180 rpm) at 30°C for 16 h. Subsequently, they were

563 transferred to 50 ml of minimal medium supplemented with biotin (for A. nidulans

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564 strains) or with 5% (vol/vol) FBS (for A. fumigatus strains) and further cultivated with

565 shaking (163 rpm) at 30°C for 24 or 48 h. The mycelia were collected and used to

566 measure the weight of freeze-dried mycelia. Culture supernatant (5 ml) was collected

567 and mixed with an equal volume of ethyl acetate for A. nidulans or chloroform for A.

568 fumigatus. The mixture was centrifuged at 10,000 × g at 4°C for 15 min. The ethyl

569 acetate fraction was collected, evaporated by N2 gas spraying, and resuspended in 100

570 µl of acetonitrile. The concentration of sterigmatocystin, fumagillin, helvolic acid, and

571 pyripyropene A was determined using a Prominence ultra-high-performance liquid

572 chromatograph system (Shimadzu) equipped with a 3200 QTRAP system (AB SCIEX,

573 Framingham, MA) in the multiple reaction monitoring mode. The secondary

574 metabolites were separated with an ACQUITY UPLC CSH C18 Column (1.7 µm, 2.1

575 mm × 50 mm) (Waters, Milford, MA) at 40°C using 0.05% formic acid in acetonitrile as

576 the organic phase and 0.05% formic acid in water as the aqueous phase at a flow rate of

577 0.2 ml/min. The solvent gradient started at 20% organic for 2 min, followed by a linear

578 increase to 60% organic over 10 min, a linear increase to 100% organic over 1 min, and

579 final maintenance at 100% organic for 20 min.

580 Penicillin bioassay. Conidia (2 × 107 cells) of the A. nidulans control and

581 OE-nrdA strains and A. oryzae control and OE-nrdA strains were inoculated into 100 ml

582 of YES medium and precultivated with shaking (180 rpm) at 30°C for 16 h.

583 Subsequently, they were transferred to 50 ml of minimal medium supplemented with

584 biotin and further cultivated with shaking (163 rpm) at 30°C for 24, 48, or 72 h. Culture

585 supernatant (5 ml) was evaporated by freeze drying and resuspended in 500 µl of water.

586 Penicillin bioassay was performed using Kocuria rhizophila (NBRC 12708) as

587 previously reported (34).

588 Kojic acid assay. Conidia (1 × 104 cells) of A. oryzae strains were inoculated

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589 onto kojic acid production agar medium with ferric ion (chelation by kojic acid changes

590 the color of the medium to red) (31). After cultivation at 30°C for 5 days, the color of

591 the medium was observed to evaluate the production of kojic acid.

592 Transcriptional analysis. To evaluate the expression level of S-nrdA in the A.

593 kawachii Tet-S-nrdA strain, the Tet-S-nrdA strain was precultivated in YES medium

594 with Dox for 16 h. Next, the mycelia were transferred to YES medium with or without

595 Dox and further cultivated for 4, 6, 12, or 24 h with shaking (163 rpm) at 30°C. In

596 addition, the A. kawachii control strain was precultivated in the YES medium for 16 h.

597 The mycelia were transferred to YES medium and further cultivated for 24 h.

598 To evaluate the gene expression level involved in the production of secondary

599 metabolites, A. nidulans, A. fumigatus, and A. oryzae strains were cultivated under the

600 condition described for the analysis of secondary metabolites. For the analysis of

601 transcripts related to the production of kojic acid in A. oryzae, conidia (2 × 107 cells)

602 were inoculated onto kojic acid production medium with ferric ion (31) and incubated

603 with shaking (163 rpm) at 30°C for 5 days. The mycelia were collected from the liquid

604 culture using a gauze and ground to a powder in the presence of liquid nitrogen.

605 Total RNA was extracted using RNAiso Plus (Takara Bio) according to the

606 instructions provided by the manufacturer and quantified using a NanoDrop-8000

607 (Thermo Fisher Scientific). cDNA was synthesized from total RNA using a PrimeScript

608 Perfect real-time reagent kit (Takara Bio) according to the instructions provided by the

609 manufacturer. Real-time reverse transcription-PCR was performed using a Thermal

610 Cycler Dice real-time system MRQ (Takara Bio) with TB Green Premix Ex Taq II (Tli

611 RNaseH Plus) (Takara Bio). The following primer sets were used: ANnrdA-RT-F and

612 ANnrdA-RT-R for A. nidulans nrdA, ANipnA-RT-F and ANipnA-RT-R for A. nidulans

613 ipnA, ANaflR-RT-F and ANaflR-RT-R for A. nidulans aflR, ANstcU-RT-F and

25 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

614 ANstcU-RT-R for A. nidulans stcU, ANacnA-RT-F and ANacnA-RT-R for A. nidulans

615 acnA, AFnrdA-RT-F and AFnrdA-RT-R for A. fumigatus nrdA, AFfumR-RT-F and

616 AFfumR-RT-R for A. fumigatus fumR, AFhelA-RT-F and AFhelA-RT-R for A. fumigatus

617 helA, AFpyr2-RT-F and AFpyr2-RT-R for A. fumigatus pyr2, AFact1-RT-F and

618 AFact1-RT-R for A. fumigatus act1, AOnrdA-RT-F and AOnrdA-RT-R for A. oryzae

619 nrdA, AOkojR-RT-F and AOkojR-RT-R for A. oryzae kojR, AOkojA-RT-F and

620 AOkojA-RT-R for A. oryzae kojA, AOkojT-RT-F and AOkojT-RT-R for A. oryzae kojT,

621 AOipnA-RT-F and AOipnA-RT-R for A. oryzae ipnA, and AOactA-RT-F and

622 AOactA-RT-R for A. oryzae actA (see Table S2 in the supplemental material). The gene

623 expression levels of acnA, act1, and actA were used to calibrate those of A. nidulans, A.

624 fumigatus, and A. oryzae, respectively.

625 Data availability. The data obtained from the RNA-seq and RIP-seq analyses in

626 this study were deposited in the Gene Expression Omnibus under accession number

627 GSE164392.

628

629 ACKNOWLEDGMENTS

630 We thank the Division of Instrumental Analysis Research Support Center of

631 Kagoshima University for technical support. This study was supported in part by

632 KAKENHI (grant numbers: 16K07672, 18K05394, and 19K05773) and a research grant

633 from Kagoshima University based on a scholarship donation from SUNUS Co., Ltd. C.

634 K. received a Grant-in-Aid for JSPS Research Fellows (grant number: 17J02753).

635

636

26 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

637 FIGURE LEGENDS

638 FIG 1 (A) Colony formation of A. kawachii control and Tet-nrdA strains. Conidia (104)

639 were inoculated onto YES medium. Strains were cultured at 30°C for 5 days on YES

640 medium with or without doxycycline (Dox). Conidia of each strain (1 × 104) were

641 inoculated onto agar medium. (B) Citric acid production of control and Tet-nrdA strains.

642 Strains were precultured in YES medium with 1 µg/ml Dox for 16 h, transferred to CAP

643 medium, and further cultivated for 48 h. The mean and standard deviation were

644 determined from the results of 3 independent cultivations. *, statistically significant

645 difference (p < 0.05, Welch’s t-test) relative to the data obtained for the control strain.

646

647 FIG 2 (A) Colony formation of A. kawachii Tet-nrdA strain and its complemented strain

648 with GFP-NrdA and H2B-mRFP. Conidia (1 × 104) were inoculated onto YES agar

649 medium with or without 1µg/ml Dox and incubated at 30°C for 5 days. (B)

650 Fluorescence microscopic observation of the GFP-NrdA- and H2B-mRFP-expressing

651 strain. Scale bars indicate 10 μm.

652

653 FIG 3 (A) Colony formation of A. kawachii control and Tet-S-nrdA strains. Strains were

654 cultured at 30°C for 5 days on YES medium with or without Dox. Conidia of each strain

655 (1 × 104) were inoculated onto agar medium. (B) Immunoblotting analysis of purified

656 S-NrdA protein from the A. kawachii Tet-S-nrdA strain. The apparent molecular mass of

657 S-NrdA was 80.7 kDa. (C) Scheme of experimental design for the RNA-seq and

658 RIP-seq analyses to identify the NrdA-interacting transcripts and investigate the effect

659 of NrdA overexpression. (D) Quantitative RT-PCR analysis to evaluate the expression

660 levels of nrdA and S-nrdA in A. kawachii control and Tet-S-nrdA strains. (E) Cluster

661 analysis to confirm the S-nrdA expression condition in the A. kawachii Tet-S-nrdA strain

27 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

662 in the presence or absence of Dox. The mean and standard deviation were determined

663 from the results of 3 independent cultivations. *, statistically significant difference (p <

664 0.05, Welch’s t-test) relative to the data obtained for the control strain.

665

666 FIG 4 (A) The RNA obtained by immunoprecipitation from the Tet-S-nrdA strain

667 cultivated in the presence of Dox. Anti-S-tag antibody and normal rabbit IgG (as a

668 negative control) were used for the RNA immunoprecipitation. The obtained RNA was

669 measured by NanoDrop-8000 (Thermo Fisher Scientific). The mean and standard

670 deviation were determined from the results of 3 independent cultivations. *, statistically

671 significant difference (p < 0.05, Welch’s t-test) relative to the negative control. (B)

672 Change in gene expression of putative NrdA-interacting transcripts by the

673 overexpression of NrdA. Predicted NrdA-interacting transcripts were plotted according

674 to the log2 fold enrichment detected by the RIP analysis on the X-axis and the log2 fold

675 change in gene expression observed after the overexpression of NrdA on the Y-axis. All

676 the plotted data showed statistical significance (q < 0.05).

677

678 FIG 5 (A) Colony formation of the A. nidulans control and OE-nrdA strains. Conidia (1

679 × 104) were inoculated onto minimal agar medium with biotin and cultured at 30°C for

680 7 days. (B) Conidiation of the control and OE-nrdA strains. (C) Penicillin bioassay of

681 the A. nidulans control and OE-nrdA strains. (D) Production of sterigmatocystin by the

682 control and OE-nrdA strains. (E) Transcriptional levels of nrdA, ipnA, aflR, and stcU.

683 The control and OE-nrdA strains were precultured in YES medium for 16 h, transferred

684 to minimal medium with biotin, and further cultured for 12, 24, and 48 h. The mean and

685 standard deviation were determined from the results of 3 independent cultivations.

686 Asterisks indicate significant differences (* p < 0.05, Welch’s t-test) versus the results

28 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

687 obtained for the control strain.

688

689 FIG 6 (A) Colony formation of the A. fumigatus control and OE-nrdA strains. Conidia

690 (1 × 104) were inoculated onto minimal agar medium and cultured at 30°C or 42°C for 5

691 days. (B) Conidiation of the control and OE-nrdA strains. (C) Production of fumagillin,

692 helvolic acid, and pyripyropene A by the control and OE-nrdA strains. Conidia (2 × 107

693 cells) were inoculated in minimal liquid medium with 5% FBS and cultivated with

694 shaking (163 rpm) at 30°C for 24 h. (D) Transcriptional levels of nrdA, fumR, helA, and

695 pyr2. The strains were cultivated in minimal liquid medium with 5% FBS for 12 h. The

696 mean and standard deviation were determined from the results of 3 independent

697 cultivations. Asterisks indicate significant differences (* p < 0.05, Welch’s t-test) versus

698 the results obtained for the control strain.

699

700 FIG 7 (A) Colony formation of the A. oryzae control and OE-nrdA strains. Conidia (1 ×

701 104) were inoculated onto kojic acid production agar medium and cultured at 30°C for 5

702 days. (B) Conidiation of the control and OE-nrdA strains. (C) Transcriptional levels of

703 nrdA, kojR, kojA, and kojT. Conidia (2 × 107 cells) were inoculated in kojic acid

704 production liquid medium and cultivated with shaking (163 rpm) at 30°C for 5 days. (D)

705 Penicillin bioassay of the A. oryzae control and OE-nrdA strains. (E) Transcriptional

706 levels of nrdA and ipnA. The mean and standard deviation were determined from the

707 results of 3 independent cultivations. Asterisks indicate significant differences (* p <

708 0.05, Welch’s t-test) versus the results obtained for the control strain.

709

29 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

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39 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A

Control

Tet-nrdA

Dox (µg/ml) 0 0.1 0.25 0.5 1

B 1000

800

600

400

/g dry mycelia weight) mycelia dry /g 200

mol * Citric acid concentration concentration acid Citric

(µ 0 Control1 Tet-2nrdA Tet3 -nrdA (+Dox) (-Dox)

FIG 1 (A) Colony formation of A. kawachii control and Tet-nrdA strains. Conidia (104) were inoculated onto YES medium. Strains were cultured at 30°C for 5 days on YES medium with or without doxycycline (Dox). Conidia of each strain (1 × 104) were inoculated onto agar medium. (B) Citric acid production of control and Tet-nrdA strains. Strains were precultured in YES medium with 1 µg/ml Dox for 16 h, transferred to CAP medium, and further cultivated for 48 h. The mean and standard deviation were determined from the results of 3 independent cultivations. *, statistically significant difference (p < 0.05, Welch’s t-test) relative to the data obtained for the control strain. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A

Tet-nrdA

Tet-nrdA plus GFP-nrdA plus H2B-mRFP Dox (µg/ml) 0 1 B

DIC

GFP-NrdA

H2B-mRFP

Merge

FIG 2 (A) Colony formation of A. kawachii Tet-nrdA strain and its complemented strain with GFP-NrdA and H2B-mRFP. Conidia (1 × 104) were inoculated onto YES agar medium with or without 1µg/ml Dox and incubated at 30°C for 5 days. (B) Fluorescence microscopic observation of the GFP-NrdA- and H2B-mRFP-expressing strain. Scale bars indicate 10 μm. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Immunoblot using A B Silver staining anti-S-tag antibody S-NrdA S-NrdA

250 150 100 Control 75

50

37

Tet-S-nrdA 25

25 MW Dox (µg/ml) 0 1 /kDa

C RIP-seq Identification of NrdA-mRNA Tet-S-nrdA interaction Evaluation of YES RNA-seq the destination of + Dox mRNA interacted with NrdA

YES RNA-seq + Dox E RIP #3 YES RIP #1 RIP #2 Control Dox Plus #3 Dox Plus #2 Dox Plus #1 RNA-seq Control #3 Control #2 YES YES Control #1 Dox Minus #3 Additional experiment Dox Minus #2 Dox Minus #1 D 0.00 0.05 0.10 0.15 50 40 * 30 * * 20 *

nrdA * - 10 S 5 *

or 4 3 2

nrdA 1

of 0

Relative copy numberRelative copy 24 4 6 12 24 4 6 12 24 Time (h) － ＋ ＋ ＋ ＋ － － － － Dox Control Tet-S-nrdA

FIG 3 (A) Colony formation of A. kawachii control and Tet-S-nrdA strains. Strains were cultured at 30°C for 5 days on YES medium with or without Dox. Conidia of each strain (1 × 104) were inoculated onto agar medium. (B) Immunoblotting analysis of purified S-NrdA protein from the A. kawachii Tet-S-nrdA strain. The apparent molecular mass of S-NrdA was 80.7 kDa. (C) Scheme of experimental design for the RNA-seq and RIP-seq analyses to identify the NrdA- interacting transcripts and investigate the effect of NrdA overexpression. (D) Quantitative RT-PCR analysis to evaluate the expression levels of nrdA and S-nrdA in A. kawachii control and Tet-S-nrdA strains. (E) Cluster analysis to confirm the S- nrdA expression condition in the A. kawachii Tet-S-nrdA strain in the presence or absence of Dox. The mean and standard deviation were determined from the results of 3 independent cultivations. *, statistically significant difference (p < 0.05, Welch’s t-test) relative to the data obtained for the control strain. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

rosA brlA A

400 300 * 200

RNA (ng) 100 0 Normal1 Anti2-S-tag Rabbit IgG antibody B

Predicted weaker Predicted stronger interaction to NrdA interaction to NrdA 8

6

4 overexpression Upregulation Upregulation by NrdA 2 condition) - 0

fold change 0 1 2 3 4 5 6 7 2 seq of Dox seq of Dox+ condition/ -2 Log - - RNA (RNA -4

(listed in Table S1)

-6 overexpression Downregulation by Downregulation NrdA -8 Log2 fold Enrichment (RIP-seq of Dox+ condition/ RNA-seq of Dox+ condition)

FIG 4 (A) The RNA obtained by immunoprecipitation from the Tet-S-nrdA strain cultivated in the presence of Dox. Anti-S-tag antibody and normal rabbit IgG (as a negative control) were used for the RNA immunoprecipitation. The obtained RNA was measured by NanoDrop- 8000 (Thermo Fisher Scientific). The mean and standard deviation were determined from the results of 3 independent cultivations. *, statistically significant difference (p < 0.05, Welch’s t-test) relative to the negative control. (B) Change in gene expression of putative NrdA-interacting transcripts by the overexpression of NrdA. Predicted NrdA-interacting

transcripts were plotted according to the log2 fold enrichment detected by the RIP analysis on the X-axis and the log2 fold change in gene expression observed after the overexpression of NrdA on the Y-axis. All the plotted data showed statistical significance (q < 0.05). bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B )

2 4 per ) 6 3 10 2 × * 1 0 onidia ( onidia colony area (cm area colony

C Control1 OE2-nrdA Control OE-nrdA C Control OE-nrdA D 24 h 50 24 h 1000 48 h 40 800 30 600 48 h 20 * 400 10 200 0 0

Sterigmatocystin Control1 OE2 -nrdA Control1 OE2-nrdA (mg/ g dry mycelia) dry g (mg/ 72 h

E nrdA ipnA aflR stcU 20 10 14 40 * 8 12 15 10 30 6 8 10 20 4 6 73% 4 70% 8% 5 2 1% 10 26% 2% * 2 0 0 * 0 * * 0 * * Relative expression Relative 121 242 483 121 242 483 121 242 483 121 242 483

Cultivation time (h) Control OE-nrdA

FIG 5 (A) Colony formation of the A. nidulans control and OE-nrdA strains. Conidia (1 × 104) were inoculated onto minimal agar medium with biotin and cultured at 30°C for 7 days. (B) Conidiation of the control and OE-nrdA strains. (C) Penicillin bioassay of the A. nidulans control and OE-nrdA strains. (D) Production of sterigmatocystin by the control and OE-nrdA strains. (E) Transcriptional levels of nrdA, ipnA, aflR, and stcU. The control and OE-nrdA strains were precultured in YES medium for 16 h, transferred to minimal medium with biotin, and further cultured for 12, 24, and 48 h. The mean and standard deviation were determined from the results of 3 independent cultivations. Asterisks indicate significant differences (* p < 0.05, Welch’s t-test) versus the results obtained for the control strain. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B

30℃ ℃ ) 30 42℃ 2 4 per 4 ) 6 3 3 10

× 2 2 1 42℃ 1 *

onidia ( onidia * colony area (cm area colony 0 0 C Control1 OE2-nrdA Control1 OE2-nrdA Control OE-nrdA C

2500 1000 250 A 2000 800 200 acid 1500 600 150 1000 400 * 100

Fumagillin 500 200 50 Helvolic * 0 Pyripyropene 0 (µg/g dry mycelia) dry (µg/g

0 mycelia) dry (µg/g (µg/g dry mycelia) dry (µg/g Control1 OE2-nrdA Control1 OE2-nrdA Control1 OE2-nrdA

D 3 * 2.5 Control

2 OE-nrdA

1.5

1

0.5 * * Relative expression Relative

0 nrdA1 fumR2 helA3 pyr24

FIG 6 (A) Colony formation of the A. fumigatus control and OE-nrdA strains. Conidia (1 × 104) were inoculated onto minimal agar medium and cultured at 30°C or 42°C for 5 days. (B) Conidiation of the control and OE-nrdA strains. (C) Production of fumagillin, helvolic acid, and pyripyropene A by the control and OE-nrdA strains. Conidia (2 × 107 cells) were inoculated in minimal liquid medium with 5% FBS and cultivated with shaking (163 rpm) at 30°C for 24 h. (D) Transcriptional levels of nrdA, fumR, helA, and pyr2. The strains were cultivated in minimal liquid medium with 5% FBS for 12 h. The mean and standard deviation were determined from the results of 3 independent cultivations. Asterisks indicate significant differences (* p < 0.05, Welch’s t-test) versus the results obtained for the control strain. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

A B )

2 8 per ) 6 6 10

× 4 Top 2 * onidia ( onidia

colony area (cm area colony 0 C Control1 OE2-nrdA C

12 * * Bottom 10 * 8 * 6 4 2 0 Relative expression Relative Control OE-nrdA nrdA1 kojR2 kojA3 kojT4

Control OE-nrdA

D E Control OE-nrdA 12 nrdA ipnA 10 24 h 8 6 4 * * 2 * Relative expression Relative 0 48 h 121 242 123 244 Cultivation time (h)

Control OE-nrdA

FIG 7 (A) Colony formation of the A. oryzae control and OE-nrdA strains. Conidia (1 × 104) were inoculated onto kojic acid production agar medium and cultured at 30°C for 5 days. (B) Conidiation of the control and OE-nrdA strains. (C) Transcriptional levels of nrdA, kojR, kojA, and kojT. Conidia (2 × 107 cells) were inoculated in kojic acid production liquid medium and cultivated with shaking (163 rpm) at 30°C for 5 days. (D) Penicillin bioassay of the A. oryzae control and OE-nrdA strains. (E) Transcriptional levels of nrdA and ipnA. The mean and standard deviation were determined from the results of 3 independent cultivations. Asterisks indicate significant differences (* p < 0.05, Welch’s t-test) versus the results obtained for the control strain. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Table 1. Biological process GO terms of potential NrdA-interacting mRNA identified by AsgGD GO Term Finder. GO ID GO term Number in gene set Number in background P-value

GO:0019748 secondary metabolic process 160 310 1.23E-27

GO:0055085 transmembrane transport 307 799 2.68E-24

GO:0055114 oxidation-reduction process 339 984 1.49E-17

GO:0044550 secondary metabolite biosynthetic process 115 243 2.21E-15

GO:0008152 metabolic process 1226 4844 7.69E-09

GO:0043386 mycotoxin biosynthetic process 56 113 1.80E-07

GO:0043385 mycotoxin metabolic process 57 116 1.88E-07

GO:0009404 toxin metabolic process 58 119 1.95E-07

GO:0018958 phenol-containing compound metabolic process 43 77 2.01E-07

GO:0009403 toxin biosynthetic process 56 115 4.28E-07

GO:0051234 establishment of localization 378 1315 1.68E-06

GO:0042537 benzene-containing compound metabolic process 31 50 2.45E-06

GO:0018130 heterocycle biosynthetic process 237 766 3.76E-06

GO:1901362 organic cyclic compound biosynthetic process 253 828 3.86E-06

GO:0046189 phenol-containing compound biosynthetic process 35 61 4.15E-06

GO:0006810 transport 369 1289 4.73E-06

GO:1901376 organic heteropentacyclic compound metabolic process 44 86 5.18E-06

GO:1901378 organic heteropentacyclic compound biosynthetic process 43 84 7.69E-06

GO:0051179 localization 394 1402 1.47E-05

GO:1903506 regulation of nucleic acid-templated transcription 216 697 1.76E-05

GO:0006355 regulation of transcription, DNA-templated 216 697 1.76E-05

GO:2001141 regulation of RNA biosynthetic process 216 698 2.01E-05

GO:1900813 monodictyphenone metabolic process 23 34 3.07E-05

GO:1900815 monodictyphenone biosynthetic process 23 34 3.07E-05

GO:0051252 regulation of RNA metabolic process 219 714 3.57E-05

GO:1900555 emericellamide metabolic process 22 32 4.03E-05

GO:1900557 emericellamide biosynthetic process 22 32 4.03E-05

GO:0050761 depsipeptide metabolic process 22 32 4.03E-05

GO:0050763 depsipeptide biosynthetic process 22 32 4.03E-05

GO:1901334 lactone metabolic process 24 37 5.21E-05

GO:1901336 lactone biosynthetic process 24 37 5.21E-05

GO:0042180 cellular ketone metabolic process 44 94 0.00016

GO:0072330 monocarboxylic acid biosynthetic process 50 113 0.0002

GO:1901503 ether biosynthetic process 22 34 0.00021

GO:0030638 polyketide metabolic process 22 34 0.00021

GO:0030639 polyketide biosynthetic process 21 32 0.00029

GO:0005975 carbohydrate metabolic process 138 422 0.00032

GO:0042181 ketone biosynthetic process 39 81 0.00034

GO:1900582 o-orsellinic acid metabolic process 16 21 0.00038

GO:1900584 o-orsellinic acid biosynthetic process 16 21 0.00038

GO:1900552 asperfuranone metabolic process 20 30 0.00039

GO:1900554 asperfuranone biosynthetic process 20 30 0.00039

GO:1902644 tertiary alcohol metabolic process 20 30 0.00039 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

GO:1902645 tertiary alcohol biosynthetic process 20 30 0.00039

GO:0045460 sterigmatocystin metabolic process 36 73 0.00047

GO:2000112 regulation of cellular macromolecule biosynthetic process 229 775 0.00048

GO:0010556 regulation of macromolecule biosynthetic process 230 779 0.00049

GO:0031326 regulation of cellular biosynthetic process 238 813 0.00063

GO:0019438 aromatic compound biosynthetic process 211 707 0.00066

GO:0045461 sterigmatocystin biosynthetic process 23 71 0.00071

GO:0009889 regulation of biosynthetic process 240 822 0.00071

GO:0034311 diol metabolic process 25 44 0.0011

GO:0034312 diol biosynthetic process 25 44 0.0011

GO:0019219 regulation of nucleobase-containing compound metabolic process 222 755 0.00112

GO:1901615 organic hydroxy compound metabolic process 91 258 0.00116

GO:1901617 organic hydroxy compound biosynthetic process 62 158 0.00127

GO:0010468 regulation of gene expression 232 797 0.00145

GO:0006351 transcription, DNA-templated 124 384 0.00284

GO:0097659 nucleic acid-templated transcription 124 384 0.00284

GO:0009820 alkaloid metabolic process 26 49 0.00387

GO:0009821 alkaloid biosynthetic process 26 49 0.00387

GO:0032774 RNA biosynthetic process 124 387 0.00443

GO:0080090 regulation of primary metabolic process 253 899 0.00828

GO:0018904 ether metabolic process 23 43 0.01271

GO:0060255 regulation of macromolecule metabolic process 256 919 0.01666

GO:0035834 indole alkaloid metabolic process 23 45 0.03425

GO:0035835 indole alkaloid biosynthetic process 23 45 0.03425

bioRxiv preprint doi: https://doi.org/10.1101/2021.03.15.435561; this version posted March 16, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.

Table 2. Aspergillus strains used in this study Strain name or Genotype References description Aspergillus luchuensis mut. kawachii IFO 4308a wild type IFO SO2 ligD- argB::hph sC- 42 CK3 ligD- argB::hph sC- pVG2.2ANsC This study Tet-nrdA ligD- argB::hph sC- pVG2.2ANsC nrdA::argB-Tet-nrdA This study Tet-nrdA plus ligD- argB::hph sC- pVG2.2ANsC nrdA::argB-Tet-nrdA This study GFP-nrdA plus pPTRI-GFP-nrdA pGbar-H2B-mRFP H2B-mRFP Tet-S-nrdA ligD- argB::hph sC- pVG2.2ANsC This study nrdA::argB-Tet-S-nrdA Aspergillus nidulans A26a veA1 biA1 FGSC OE-nrdA veA1 biA1 pPTRI-PgpdA-nrdA This study Aspergillus fumigatus A1151a akuB::pyrG FGSC OE-nrdA akuB::pyrG nrdA::ptrA-PgpdA-nrdA This study Aspergillus oryzae ΔligD plus pGNA sC- niaD- ligD::sC pGNA 44 OE-nrdA sC- niaD- ligD::sC pGNA nrdA::ptrA-PgpdA-nrdA This study aThese strains were used as controls. IFO, Institute for Fermentation, Osaka; FGSC, Fungal Genetics Stock Center.