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Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production
Fungal Genetics and Biology 85 (2015) 1–6
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Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production Mizuki Tanaka, Takahiro Shintani, Katsuya Gomi ⇑ Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
a r t i c l e
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Article history: Received 26 September 2015 Revised 19 October 2015 Accepted 20 October 2015 Available online 21 October 2015 Keywords: Aspergillus oryzae Unfolded protein response IreA HacA Endoplasmic reticulum stress Hydrolytic enzyme production
a b s t r a c t Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireArepressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction In eukaryotic cells, nascent polypeptides of secretory and membrane proteins are appropriately folded and assembled in the endoplasmic reticulum (ER). However, these proteins are often misfolded, and the accumulation of such unfolded proteins elicits stress to the ER. To decrease ER stress, eukaryotic cells possess an intracellular signaling pathway called the unfolded protein response (UPR) (Mori, 2009; Ron and Walter, 2011). In UPR, unfolded proteins trigger oligomerization and phosphorylation of the ER transmembrane protein IRE1, resulting in the activation of its cytosolic RNase domain (Gardner et al., 2013). In the budding yeast Saccharomyces cerevisiae, activated Ire1 cleaves out a 252nucleotide (nt) intron of HAC1 mRNA, and the functional basic leucine zipper-type transcription factor Hac1 is translated from this unconventionally spliced form of HAC1 mRNA (Kaufman, 1999). Hac1 induces the expression of ER chaperone genes and ERassociated degradation (ERAD) genes for refolding or degradation of unfolded proteins. ⇑ Corresponding author. E-mail address: [email protected] (K. Gomi). http://dx.doi.org/10.1016/j.fgb.2015.10.003 1087-1845/Ó 2015 Elsevier Inc. All rights reserved.
Aspergillus oryzae is a filamentous fungus used for the production of sake, soy sauce, and soybean paste (miso) in Japan (Machida et al., 2008). A. oryzae secretes a copious amount of extracellular enzyme proteins, such as amylolytic enzymes, with enzyme production reaching >50 g/L culture (Oda et al., 2006). Thus, strong ER stress might be induced in A. oryzae under conditions inducing secretory protein production. The UPR mechanism has been well studied in various filamentous fungi (Heimel, 2015), and ER stress has been found to promote unconventional splicing of 20- or 23-nt intron within the yeast HAC1 ortholog genes. This unconventional splicing of HAC1 ortholog mRNAs results in the inducible expression of ERAD and ER chaperone genes, such as bipA and pdiA (Benz et al., 2014; Joubert et al., 2011; Mulder et al., 2004; Ohno et al., 2011; Saloheimo et al., 2003). However, most of the previous studies were conducted under artificial conditions where ER stress was triggered by chemical compounds or heterologous protein expression. Although recent studies have reported that UPR is induced in Neurospora crassa grown under conditions inducing cellulolytic enzyme production (Benz et al., 2014; Fan et al., 2015), the contribution of UPR to ER stress adaptation under physiological conditions in filamentous fungi remains unclear.
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To examine the role of UPR under conditions inducing secretory protein production in A. oryzae, we attempted to construct a disruption mutant of IRE1 ortholog (ireA). However, no homokaryotic ireA disruption mutant could be obtained. Moreover, because no homokaryotic ireA disruption mutant could be obtained in Aspergillus niger (Carvalho et al., 2010; Mulder and Nikolaev, 2009), we hypothesized that ireA is essential for A. oryzae growth. Hence, we generated an ireA conditionally expressing strain and examined the impact of ireA knockdown on the growth of A. oryzae under conditions inducing enzymatic protein production. 2. Materials and methods 2.1. A. oryzae strains and media For the construction of the ireA conditionally expressing strain, the A. oryzae NS4 strain (niaD, sC) (Yamada et al., 1997) was used as the recipient. The ireA conditionally expressing strain was also constructed in the A. oryzae DligD::loxP pyrG strain (DligD::loxP, niaD, sC, pyrG), derived from the DligD::loxP strain (Mizutani et al., 2012), for the disruption of amyR. In addition, the DligD::loxP pyrG strain was also used as the recipient strain for ireA disruption after the introduction of synthetic intronless hacA (hacA-i). The DligD::loxP pyrG::niaD strain (Ichinose et al., 2014) was used as the control for DligD::loxP pyrG-derived strains. The A. oryzae strains used in this study are summarized in Table 1. For A. oryzae cultivation, Czapek Dox medium (0.6% NaNO3, 0.05% KCl, 0.2% KH2PO4, 0.05% MgSO4, trace amounts of FeSO4, ZnSO4, CuSO4, MnSO4, Na2B4O7, and (NH4)6Mo7O24, and 1% sugar supplemented with nutrient requirements) and YPD medium (0.5% yeast extract, 0.5% polypeptone, and 1% glucose) were used as the minimal and complete medium, respectively. 2.2. Construction of plasmid DNAs The DNA fragment for the replacement of the ireA upstream region plus the 50 -untranslated region (50 -UTR) with the nmtA promoter plus its 50 -UTR was constructed as follows: a DNA fragment approximately 1 kb upstream of the ireA coding region was amplified by PCR using ireAupsenKpnI and ireAupantiBamHI primers. The KpnI/BamHI-digested fragment was inserted into the plasmid pUSC–PnmtA (Hiramoto et al., 2015), which contains the nmtA promoter plus the 50 -UTR and A. nidulans ATP sulfurylase gene sC yielding pireAupsCPnmtA. A DNA fragment comprising the partial ireA coding region was obtained by NotI/SphI digestion of the fragment amplified by PCR using the primers ireAsenNotI and ireAdownantiSphI. The acquired DNA fragment was inserted into NotI/SphI-digested pireAupsCPnmtA, yielding psCPnmtAireA. The DNA fragment obtained from SpeI/SphI-digested psCPnmtAireA was used for A. oryzae transformation.
The DNA fragment for the deletion of amyR using orotidine-50 decarboxylase gene (pyrG) as the selectable marker was constructed as follows: a DNA fragment containing Aspergillus nidulans pyrG was amplified by PCR from A. nidulans genomic DNA using the primers AnpyrGsen and AnpyrGantiPstI. A modified pUC119, containing the amyR fragment obtained by KpnI/HindIII digestion of the plasmid pGL20 (Gomi et al., 2000), was digested with PstI and NcoI to remove the internal coding region of the amyR. The PstI/NcoI-digested pyrG fragment was then inserted into the resulting plasmid, yielding pDamyR::pyrG. The DNA fragment obtained from ClaI/EcoRI-digested pDamyR::pyrG was used for A. oryzae transformation. Expression plasmids containing the intact hacA and hacA-i lacking 20-nt intron were constructed as follows: a DNA fragment of hacA-i was constructed by overlap extension PCR. In brief, two hacA fragments, each amplified by PCR using primer sets hacAsenNotI + hacADintronanti or hacADintronsen + hacAantiEcoRV with A. oryzae genomic DNA as the template, were mixed, and a second round of PCR with the primers hacAsenBamHI and hacAantiEcoRV was performed. This amplified fragment lacking 20-nt intron was digested with NotI and EcoRV and inserted into pBluescriptKS(+), which yielded pBS/hacA-i. The niaD fragment encoding nitrate reductase was obtained from pNPthiA vector (Suzuki et al., 2015) by digestion with NruI and insertion into EcoRV-digested pBS/ hacA-i, which generated pBShacA-i /niaD. Similarly, intact hacA fragment was amplified by PCR using the primers hacAsenNotI and hacAantiEcoRV with A. oryzae genomic DNA, and the NotI/ EcoRV-digested fragment was inserted into pBluescriptKS(+), followed by insertion of the niaD fragment, which resulted in the plasmid pBShacA/niaD. The DNA fragments for ireA disruption were constructed as follows: a DNA fragment approximately 1 kb upstream of the ireA coding region was amplified by PCR using ireAupsenKpnI and ireAupantiBamHI primers, and the KpnI/BamHI-digested fragment was inserted into the plasmid pUSC (Yamada et al., 1997) containing the A. nidulans sC, which yielded pUSCireAup. In addition, a DNA fragment of approximately 1 kb of the ireA 30 -region was also amplified by PCR using ireAdownsenPstI and ireAdownantiSphI primers, and the PstI/SphI-digested fragment was inserted into the plasmid pUSCireAup, yielding pDireA::sC. To generate a plasmid for ireA disruption using pyrG as the selection marker, the BglII/PstI-digested A. nidulans pyrG fragment was ligated to BamHI/PstI-digested pDireA::sC, which resulted in the plasmid pDireA::pyrG. The DNA fragment obtained by KpnI/SphI digestion of pDireA::sC and the fragment amplified by PCR using the ireAupsenKpnI and ireAdownantiSphI primers with pDireA::pyrG as the template were used for A. oryzae transformation. The nt sequences of all primers used in this study are shown in Supplementary Table S1.
Table 1 The Aspergillus oryzae strains used in this study. Strain
Strain origin
Genotype
Reference
NS4 nmtAp-ireA nmtAp-ireA pyrG nmtAp-ireA DamyR DamyR nmtAp-ireA + hacA nmtAp-ireA + hacA-i DligD::loxP pyrG + hacA-i DireA + hacA-i DligD::loxP + hacA-i DligD::loxP pyrG::niaD
RIB40 NS4 DligD::loxP nmtAp-ireA NS4 nmtAp-ireA nmtAp-ireA DligD::loxP DligD::loxP DligD::loxP DligD::loxP
niaD; sC niaD; nmtAp-ireA::sC niaD; pyrG; nmtAp-ireA::sC niaD; nmtAp-ireA::sC; DamyR::pyrG niaD; DamyR::sC nmtAp-ireA::sC; niaD::hacA nmtAp-ireA::sC; niaD::hacA-i sC; pyrG; niaD::hacA-i sC; niaD::hacA-i; DireA::pyrG sC; niaD::hacA-i; niaD::pyrG sC; niaD::pyrG
Yamada et al. (1997) This study This study This study Hasegawa et al. (2010) This study This study This study This study This study Ichinose et al. (2014)
pyrG pyrG
pyrG pyrG + hacA-i pyrG + hacA-i pyrG
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2.3. A. oryzae transformation Transformation of A. oryzae was performed according to the method of Gomi et al. (1987). 2.4. Nucleic acid blotting
A
sC Genomic DNA (Host strain)
ireA BglII
BglII
Southern and northern blot analyses were performed as previously described (Tanaka et al., 2012). 2.5. RT-PCR analysis
Genomic DNA (Transformant)
sC BglII
First-strand cDNAs were synthesized as previously described (Ichinose et al., 2014). PCR using cDNAs as template was performed for 20 cycles. Quantitative RT-PCR was performed as previously described (Ichinose et al., 2014). The nt sequences of primers used for RT-PCR are shown in Supplementary Table S1.
ireA BglII
probe
1 kb
B
kb
WT
nmtAp -ireA
9.4 3. Results
6.6
3.1. Growth of the ireA conditionally expressing strain Because deletion of ireA seemed lethal in A. oryzae, an ireA conditionally expression strain was constructed by replacing the ireA promoter plus the 50 -UTR with the nmtA promoter plus its 50 -UTR which contains a thiamine riboswitch. The nmtA promoter plus the 50 -UTR (nmtAp) is sufficient for gene silencing in A. oryzae (Hiramoto et al., 2015). The upstream region of ireA (AO090005000934) was replaced with nmtAp to generate the ireA conditionally expressing strain (Fig. 1A), and the resultant transformant was labeled as the nmtAp-ireA strain. The replacement of the ireA upstream region with nmtAp was confirmed by Southern blot analysis (Fig. 1B), and the growth of the nmtAp-ireA strain was observed on minimal agar media containing various carbon sources (Fig. 1C). When thiamine was added to the medium containing glucose or fructose as the sole carbon source for ireA silencing, the nmtAp-ireA strain exhibited poor growth. In contrast, ireA silencing by thiamine addition to the medium containing maltose and starch, which induce amylolytic gene expression, resulted in the complete inhibition of A. oryzae growth. In addition to amylolytic enzymes, A. oryzae secretes xylanolytic and cellulolytic enzymes when xylan is used as the sole carbon source (Noguchi et al., 2009). The ireA-repressing strain showed no growth on medium containing xylan as the sole carbon source. Even under thiamine-deficient conditions in which ireA was expressed by nmtAp at a moderate level, the nmtAp-ireA strain exhibited almost no growth on media with starch and xylan as the carbon source. Moreover, the nmtAp-ireA strain could not grow on medium containing skim milk as the sole nitrogen and carbon source, which is an inducer of proteolytic gene expression. These results indicated that ireA was required for A. oryzae growth under conditions inducing secretory hydrolytic enzyme production.
4.4
Without thiamine
C
WT
nmtAp -ireA
+ 10 µM thiamine WT
nmtAp -ireA
Glucose
Fructose
Maltose
Starch
Xylan
Skim milk
3.2. UPR is induced by amylolytic gene expression The expression of amylolytic genes in A. oryzae is regulated by the Zn(II)2Cys6-type transcription activator AmyR (Gomi et al., 2000; Petersen et al., 1999). To examine whether the growth defect by ireA silencing on maltose medium was caused by inducible amylolytic enzyme production, amyR was disrupted in the nmtAp-ireA strain. As shown in Fig. 2, amyR disruption partially restored the growth of the nmtAp-ireA strain on maltose medium supplemented with thiamine, which repressed ireA expression. This result suggested that amylolytic enzyme production correlated with growth defect under the condition of ireA silencing. To
Fig. 1. Growth of the A. oryzae ireA conditionally expressing strain. (A) Construction of the ireA conditionally expressing strain by replacement of the ireA upstream region with 50 -untranslated region (50 -UTR) attached to the nmtA promoter. The nmtA promoter and 50 -UTR are represented by a gray box. (B) Southern blot analysis of the ireA conditionally expressing strain. The BglII-digested genomic DNAs from the wild-type (WT) and ireA conditionally expressing strains were electrophoresed on a 0.8% agarose gel and transferred on to a nylon membrane. A labeled DNA fragment of the ireA upstream region was used as a probe. (C) Growth of the ireA conditionally expressing strain. Approximately 104 conidiospores of the WT (NS4) and nmtAp-ireA strains were grown on minimal agar plates containing 1% carbon sources indicated, with or without 10 lM thiamine, at 30 °C for 3 days. Skim milk was also used as the sole nitrogen source.
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WT
nmtAp -ireA
nmtAp -ireA ΔamyR
WT
nmtAp -ireA
nmtAp -ireA +hacA
nmtAp -ireA +hacA-i
Without thiamine
+ 10 µM thiamine
Glucose
Without thiamine
+10 µM thiamine
Fig. 2. Effect of amyR deletion on the growth of the ireA conditionally expressing strain. Approximately 104 conidiospores of each strain were grown on minimal agar plates containing 1% maltose, with or without 10 lM thiamine, at 30 °C for 3 days. The DligD::loxP pyrG::niaD strain was used as the wild-type strain.
A
Wild-type
Maltose
ΔamyR
0 30 40 50 60 70 80 90 0 30 40 50 60 70 80 90 (min)
bipA pdiA
Starch
amyA/B/C
18S rRNA
B
Wild-type
ΔamyR
G 0 30 40 50 60 70 80 0 30 40 50 60 70 80 (min)
hacA actA Fig. 3. Expression analysis of the UPR genes. (A) Northern blot analysis of bipA and pdiA. The wild-type (NS4) and DamyR mutant strains were grown in liquid Czapek Dox + 0.1% polypeptone medium containing 1% fructose as the carbon source for 24 h, followed by transfer to liquid minimal medium containing 1% maltose. The mycelia were harvested at the time points indicated, and the total RNA was extracted from the harvested mycelia. Approximately 20 lg of the total RNA was subjected to northern blot analysis, and the digoxigenin-labeled fragment of each gene was used as a probe. The loading control used was 18S rRNA. (B) RT-PCR analysis of hacA. The cDNAs were synthesized from total RNA used for northern blot analysis. PCR using the resulting cDNAs as template was performed for 20 cycles. The PCR products of hacA and actin gene (actA) were electrophoresed on 4% agarose gel with TBE buffer and 2% agarose gel with TAE buffer, respectively. The PCR products derived from genomic DNA were also electrophoresed (lane G).
confirm that amylolytic enzyme production essentially induced UPR, the expression levels of bipA and pdiA in the wild-type and DamyR strains were examined by northern blot analysis after incubation in the liquid maltose medium (Fig. 3A). After incubating the wild-type strain in maltose medium for 60 min, the expression of
Fig. 4. Effect of hacA-i introduction on the growth of the ireA conditionally expressing strain. Approximately 104 conidiospores of each strain were grown on minimal agar plates containing 1% carbon sources indicated, with or without 10 lM thiamine, at 30 °C for 3 days.
both bipA and pdiA was strongly induced. In contrast, the expression levels of these genes in the DamyR strain were significantly low. Furthermore, RT-PCR analysis showed that splicing of 20-nt intron within hacA mRNA was actively promoted in the wild-type strain following 50 min of incubation, whereas such a splicing of unconventional intron of hacA was not evident in the DamyR strain (Fig. 3B). Moreover, both hacA intron splicing and ER chaperone gene (bipA and pdiA) expression in the wild-type strain were preceded by a-amylase gene expression (Fig. 3). These results clearly indicated that UPR was induced by amylolytic gene expression in A. oryzae. 3.3. Involvement of unconventional splicing of hacA intron during A. oryzae growth To examine whether the growth defect of the ireA-repressing strain is caused by the suppression of unconventional hacA intron splicing, synthetic hacA DNA fragment lacking 20-nt intron was introduced into the chromosome of the nmtAp-ireA strain and was referred to as hacA-i. The introduction of hacA-i resulted in
M. Tanaka et al. / Fungal Genetics and Biology 85 (2015) 1–6
the growth restoration of the nmtAp-ireA strain on media containing glucose, maltose, and starch as the sole carbon source (Fig. 4). This growth restoration was also observed on media containing fructose, xylan, and skim milk (Supplementary Fig. S1). In contrast, introduction of the intact hacA had no apparent effect on the growth of the ireA-repressing strain. These results suggested that the growth defect of the nmtAp-ireA strain mainly resulted from the suppression of unconventional hacA intron splicing. Based on these findings, we hypothesized that ireA could be disrupted
A
pyrG
Genomic DNA (+hacA-i strain)
ireA
BglII
BglII
Genomic DNA (Transformant)
pyrG
BglII
B
BglII
probe
1 kb
WT ΔireA
kb 9.4 6.6 4.4
C
WT
+hacA-i
Glucose
+hacA-i ΔireA
Maltose
Fig. 5. Construction of an ireA disruption mutant using the hacA-i harboring strain. (A) Construction of an ireA disruption mutant using the hacA-i harboring strain as the host. (B) Southern blot analysis of the ireA disruption mutant. Genomic DNAs were examined as described in Fig. 1B. (C) Growth of the ireA disruption mutant. Approximately 104 conidiospores of each strain were grown on minimal agar plates containing 1% glucose or maltose at 30 °C for 3 days. The DligD::loxP pyrG::niaD strain was used as the wild-type strain.
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together with the concomitant introduction of hacA-i. Predictably, we successfully obtained the homokaryotic ireA disruption mutant when the strain harboring hacA-i was used as the host for ireA disruption (Fig. 5A and B). The resulting ireA disruption mutant exhibited significant growth defect on all media (Fig. 5C). This result indicated that the function of IreA was not restricted to unconventional splicing of the hacA intron. 4. Discussion UPR is a key cellular response for homeostatic adaptation to ER stress. In this study, we examined the contribution of UPR to A. oryzae growth under conditions inducing secretory hydrolytic enzyme production by constructing the ireA conditionally expressing strain. The failure to obtain a homokaryotic ireA disruption mutant led us to speculate that ireA disruption is lethal to A. oryzae. However, poor growth of the nmtAp-ireA strain harboring a conditionally expressed ireA construct was observed in the presence of thiamine even under the secretion repressing condition of glucose or fructose as the sole carbon source (Fig. 1C). We assumed that this weak growth could be due to the leaky expression of ireA because nmtAp may not be completely repressed in the presence of thiamine. In contrast to A. oryzae and A. niger, a homokaryotic ireA disruption mutant was successfully obtained in the fungal pathogen Aspergillus fumigatus (Feng et al., 2011). The growth of A. fumigatus ireA disruption mutant at 30 °C was nearly normal; however, its growth was inhibited at 42 °C (Feng et al., 2011). It would be possible that the function of IreA probably differs between A. fumigatus and its related Aspergillus spp., such as A. oryzae and A. niger, which possess greater ability to produce large amount of secretory hydrolytic enzymes. The essentiality of ireA for A. oryzae growth under conditions not inducing hydrolytic enzyme production remains controversial. In the present study, ireA repression completely inhibited A. oryzae growth under conditions inducing hydrolytic enzyme production, whereas the introduction of hacA-i restored its viability (Figs. 1C and 4). In addition, UPR was induced by amylolytic enzyme production (Fig. 3), and severe growth defect of the ireA-repressing strain was partially restored by amyR deletion (Fig. 2). These results clearly indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by the production of secretory hydrolytic enzymes. It has been previously reported that the A. fumigatus DireA strain exhibited a striking growth defect on maltose medium, and this defect was decreased by the expression of hacA cDNA using the gpdA promoter (Feng et al., 2011). However, there is no evidence that UPR was essentially induced in A. fumigatus cultured in maltose medium. The results obtained in the present study provide the first experimental validation for the essential role of ireA in filamentous fungal growth under UPR-inducing condition. Introduction of hacA-i into the ireA-repressing strain was not sufficient for complete growth recovery under the UPR-inducing condition (Fig. 4). In addition, the ireA disruption mutant obtained using the hacA-i harboring strain as the host exhibited poor growth under all growth conditions tested. These results suggest that the role of IreA is not restricted to the unconventional splicing of the hacA intron and IreA has other important function(s) required for A. oryzae growth, as also suggested in A. fumigatus (Feng et al., 2011). In addition to the unconventional intron of XBP1, a homolog of yeast HAC1, metazoan IRE1a also cleaves mRNAs encoding membrane and secretory proteins, and this mRNA turnover pathway is called regulated Ire1-dependent decay (RIDD) (Hollien and Weissman, 2006; Hollien et al., 2009; Maurel et al., 2014). Recently, RIDD was also found in fission and budding yeasts (Kimmig et al., 2012; Tam et al., 2014). However, there is still no information on RIDD in filamentous fungi, and we are now trying to detect the presence of RIDD pathway in Aspergillus spp., including A. oryzae, for the further understanding of IreA function.
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The nmtAp-ireA strain showed nearly normal growth on media containing glucose and fructose as the carbon source and without thiamine. In contrast, severe growth defect of the strain was observed on maltose and xylan media without thiamine (Fig. 1C). Furthermore, the nmtAp-ireA strain exhibited complete growth inhibition on media containing starch and skim milk, regardless of the presence or absence of thiamine (Fig. 1C). These results suggested that the expression level of ireA driven by nmtAp was not sufficient for the normal growth of A. oryzae and that a higher ireA expression level was required for A. oryzae growth under UPR-inducing conditions. This notion was supported by the induced ireA expression in the wild-type strain after incubation in maltose and starch media; the observation that induction of the ireA expression by starch was higher than that by maltose (Supplementary Fig. S2) also suggested that a higher ireA expression level was required for growth on starch medium without thiamine comparable to the growth on maltose medium. However, in the skim milk medium, the influence of thiamine originally included in skim milk should be considered. The requirement of unconventional splicing of the hacA intron for A. oryzae growth suggests that hacA is also essential for the growth of this fungus. In fact, we obtained A. oryzae hacA disruption mutant only as a heterokaryon (data not shown). However, homokaryotic hacA disruption mutant has been successfully generated in A. niger and A. fumigatus, with hacA disruption resulting in drastic growth defect and nearly normal growth in A. niger and A. fumigatus, respectively (Carvalho et al., 2010; Feng et al., 2011; Mulder and Nikolaev, 2009). A recent report showed that the disruption of N. crassa hac-1 (hacA ortholog) resulted in growth inhibition on cellulose medium, whereas growth on other media was normal (Montenegro-Montero et al., 2015). An examination of A. oryzae hacA essentiality will support our further understanding of UPR contribution to ER stress adaptation in filamentous fungi. However, the importance of hacA 50 -UTR in UPR activation suggests that promoter replacement is not appropriate to examine hacA essentiality (Mulder and Nikolaev, 2009), and another approach for hacA silencing is required. Acknowledgments We thank Osamu Mizutani for kindly providing the DligD::loxP pyrG mutant strain and Tomoko Shintani for technical assistance. This work was supported in part by JSPS KAKENHI (Grant no. 17019001), Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2015.10.003. References Benz, J.P., Chau, B.H., Zheng, D., Bauer, S., Glass, N.L., Somerville, C.R., 2014. A comparative systems analysis of polysaccharide-elicited responses in Neurospora crassa reveals carbon source-specific cellular adaptations. Mol. Microbiol. 91, 275–299. Carvalho, N.D.S.P., Arentshorst, M., Kwon, M.J., Meyer, V., Ram, A.F.J., 2010. Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses. Appl. Microbiol. Biotechnol. 87, 1463–1473. Fan, F., Ma, G., Li, J., Liu, Q., Benz, J.P., Tian, C., Ma, Y., 2015. Genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase production in Neurospora crassa. Biotechnol. Biofuels 8, 66. Feng, X., Krishnan, K., Richie, D.L., Aimanianda, V., Hartl, L., Grahl, N., PowersFletcher, M.V., Zhang, M., Fuller, K.K., Nierman, W.C., Lu, L.J., Latgé, J.P., Woollett, L., Newman, S.L., Cramer Jr., R.A., Rhodes, J.C., Askew, D.S., 2011. HacAindependent functions of the ER stress sensor IreA synergize with the canonical
UPR to influence virulence traits in Aspergillus fumigatus. PLoS Pathog. 7, e1002330. Gardner, B.M., Pincus, D., Gotthardt, K., Gallagher, C.M., Walter, P., 2013. Endoplasmic reticulum stress sensing in the unfolded protein response. Cold Spring Harb. Perspect. Biol. 5, a013169. Gomi, K., Akeno, T., Minetoki, T., Ozeki, K., Kumagai, C., Okazaki, N., Iimura, Y., 2000. Molecular cloning and characterization of a transcriptional activator gene, amyR, involved in the amylolytic gene expression in Aspergillus oryzae. Biosci. Biotechnol. Biochem. 64, 816–827. Gomi, K., Iimura, Y., Hara, S., 1987. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem. 51, 2549–2555. Hasegawa, S., Takizawa, M., Suyama, H., Shintani, T., Gomi, K., 2010. Characterization and expression analysis of a maltose-utilizing (MAL) cluster in Aspergillus oryzae. Fungal Genet. Biol. 47, 1–9. Heimel, K., 2015. Unfolded protein response in filamentous fungi-implications in biotechnology. Appl. Microbiol. Biotechnol. 99, 121–132. Hiramoto, T., Tanaka, M., Ichikawa, T., Matsuura, Y., Hasegawa-Shiro, S., Shintani, T., Gomi, K., 2015. Endocytosis of a maltose permease is induced when amylolytic enzyme production is repressed in Aspergillus oryzae. Fungal Genet. Biol. 82, 136–144. Hollien, J., Lin, J.H., Li, H., Stevens, N., Walter, P., Weissman, J.S., 2009. Regulated Ire1dependent decay of messenger RNAs in mammalian cells. J. Cell Biol. 186, 323–331. Hollien, J., Weissman, J.S., 2006. Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response. Science 313, 104–107. Ichinose, S., Tanaka, M., Shintani, T., Gomi, K., 2014. Improved a-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression. Appl. Microbiol. Biotechnol. 98, 335–343. Joubert, A., Simoneau, P., Campion, C., Bataillé-Simoneau, N., Iacomi-Vasilescu, B., Poupard, P., François, J.M., Georgeault, S., Sellier, E., Guillemette, T., 2011. Impact of the unfolded protein response on the pathogenicity of the necrotrophic fungus Alternaria brassicicola. Mol. Microbiol. 79, 1305–1324. Kaufman, R.J., 1999. Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev. 13, 1211–1233. Kimmig, P., Diaz, M., Zheng, J., Williams, C.C., Lang, A., Aragón, T., Li, H., Walter, P., 2012. The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis. Elife 1, e00048. Machida, M., Yamada, O., Gomi, K., 2008. Genomics of Aspergillus oryzae: learning from the history of koji mold and exploration of its future. DNA Res. 15, 173– 183. Maurel, M., Chevet, E., Tavernier, J., Gerlo, S., 2014. Getting RIDD of RNA: IRE1 in cell fate regulation. Trends Biochem. Sci. 39, 245–254. Mizutani, O., Masaki, K., Gomi, K., Iefuji, H., 2012. Modified Cre-loxP recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue. Appl. Environ. Microbiol. 78, 4126–4133. Montenegro-Montero, A., Goity, A., Larrondo, L.F., 2015. The bZIP transcription factor HAC-1 is involved in the unfolded protein response and is necessary for growth on cellulose in Neurospora crassa. PLoS ONE 10, e0131415. Mori, K., 2009. Signalling pathways in the unfolded protein response: development from yeast to mammals. J. Biochem. 146, 743–750. Mulder, H.J., Nikolaev, I., 2009. HacA-dependent transcriptional switch releases hacA mRNA from a translational block upon endoplasmic reticulum stress. Eukaryot. Cell 8, 665–675. Mulder, H.J., Saloheimo, M., Penttilä, M., Madrid, S.M., 2004. The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and upregulates its own transcription. Mol. Genet. Genomics 271, 130–140. Noguchi, Y., Sano, M., Kanamaru, K., Ko, T., Takeuchi, M., Kato, M., Kobayashi, T., 2009. Genes regulated by AoXlnR, the xylanolytic and cellulolytic transcriptional regulator, in Aspergillus oryzae. Appl. Microbiol. Biotechnol. 85, 2236–2239. Oda, K., Kakizono, D., Yamada, O., Iefuji, H., Akita, O., Iwashita, K., 2006. Proteomic analysis of extracellular proteins from Aspergillus oryzae grown under submerged and solid-state culture conditions. Appl. Environ. Microbiol. 72, 3448–3457. Ohno, A., Maruyama, J., Nemoto, T., Arioka, M., Kitamoto, K., 2011. A carrier fusion significantly induces unfolded protein response in heterologous protein production by Aspergillus oryzae. Appl. Microbiol. Biotechnol. 92, 1197–1206. Petersen, K.L., Lehmbeck, J., Christensen, T., 1999. A new transcriptional activator for amylase genes in Aspergillus. Mol. Gen. Genet. 262, 668–676. Ron, D., Walter, P., 2011. 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Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production Mizuki Tanaka, Takahiro Shintani, Katsuya Gomi ⇑ Laboratory of Bioindustrial Genomics, Department of Bioindustrial Informatics and Genomics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
a r t i c l e
i n f o
Article history: Received 26 September 2015 Revised 19 October 2015 Accepted 20 October 2015 Available online 21 October 2015 Keywords: Aspergillus oryzae Unfolded protein response IreA HacA Endoplasmic reticulum stress Hydrolytic enzyme production
a b s t r a c t Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireArepressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction In eukaryotic cells, nascent polypeptides of secretory and membrane proteins are appropriately folded and assembled in the endoplasmic reticulum (ER). However, these proteins are often misfolded, and the accumulation of such unfolded proteins elicits stress to the ER. To decrease ER stress, eukaryotic cells possess an intracellular signaling pathway called the unfolded protein response (UPR) (Mori, 2009; Ron and Walter, 2011). In UPR, unfolded proteins trigger oligomerization and phosphorylation of the ER transmembrane protein IRE1, resulting in the activation of its cytosolic RNase domain (Gardner et al., 2013). In the budding yeast Saccharomyces cerevisiae, activated Ire1 cleaves out a 252nucleotide (nt) intron of HAC1 mRNA, and the functional basic leucine zipper-type transcription factor Hac1 is translated from this unconventionally spliced form of HAC1 mRNA (Kaufman, 1999). Hac1 induces the expression of ER chaperone genes and ERassociated degradation (ERAD) genes for refolding or degradation of unfolded proteins. ⇑ Corresponding author. E-mail address: [email protected] (K. Gomi). http://dx.doi.org/10.1016/j.fgb.2015.10.003 1087-1845/Ó 2015 Elsevier Inc. All rights reserved.
Aspergillus oryzae is a filamentous fungus used for the production of sake, soy sauce, and soybean paste (miso) in Japan (Machida et al., 2008). A. oryzae secretes a copious amount of extracellular enzyme proteins, such as amylolytic enzymes, with enzyme production reaching >50 g/L culture (Oda et al., 2006). Thus, strong ER stress might be induced in A. oryzae under conditions inducing secretory protein production. The UPR mechanism has been well studied in various filamentous fungi (Heimel, 2015), and ER stress has been found to promote unconventional splicing of 20- or 23-nt intron within the yeast HAC1 ortholog genes. This unconventional splicing of HAC1 ortholog mRNAs results in the inducible expression of ERAD and ER chaperone genes, such as bipA and pdiA (Benz et al., 2014; Joubert et al., 2011; Mulder et al., 2004; Ohno et al., 2011; Saloheimo et al., 2003). However, most of the previous studies were conducted under artificial conditions where ER stress was triggered by chemical compounds or heterologous protein expression. Although recent studies have reported that UPR is induced in Neurospora crassa grown under conditions inducing cellulolytic enzyme production (Benz et al., 2014; Fan et al., 2015), the contribution of UPR to ER stress adaptation under physiological conditions in filamentous fungi remains unclear.
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To examine the role of UPR under conditions inducing secretory protein production in A. oryzae, we attempted to construct a disruption mutant of IRE1 ortholog (ireA). However, no homokaryotic ireA disruption mutant could be obtained. Moreover, because no homokaryotic ireA disruption mutant could be obtained in Aspergillus niger (Carvalho et al., 2010; Mulder and Nikolaev, 2009), we hypothesized that ireA is essential for A. oryzae growth. Hence, we generated an ireA conditionally expressing strain and examined the impact of ireA knockdown on the growth of A. oryzae under conditions inducing enzymatic protein production. 2. Materials and methods 2.1. A. oryzae strains and media For the construction of the ireA conditionally expressing strain, the A. oryzae NS4 strain (niaD, sC) (Yamada et al., 1997) was used as the recipient. The ireA conditionally expressing strain was also constructed in the A. oryzae DligD::loxP pyrG strain (DligD::loxP, niaD, sC, pyrG), derived from the DligD::loxP strain (Mizutani et al., 2012), for the disruption of amyR. In addition, the DligD::loxP pyrG strain was also used as the recipient strain for ireA disruption after the introduction of synthetic intronless hacA (hacA-i). The DligD::loxP pyrG::niaD strain (Ichinose et al., 2014) was used as the control for DligD::loxP pyrG-derived strains. The A. oryzae strains used in this study are summarized in Table 1. For A. oryzae cultivation, Czapek Dox medium (0.6% NaNO3, 0.05% KCl, 0.2% KH2PO4, 0.05% MgSO4, trace amounts of FeSO4, ZnSO4, CuSO4, MnSO4, Na2B4O7, and (NH4)6Mo7O24, and 1% sugar supplemented with nutrient requirements) and YPD medium (0.5% yeast extract, 0.5% polypeptone, and 1% glucose) were used as the minimal and complete medium, respectively. 2.2. Construction of plasmid DNAs The DNA fragment for the replacement of the ireA upstream region plus the 50 -untranslated region (50 -UTR) with the nmtA promoter plus its 50 -UTR was constructed as follows: a DNA fragment approximately 1 kb upstream of the ireA coding region was amplified by PCR using ireAupsenKpnI and ireAupantiBamHI primers. The KpnI/BamHI-digested fragment was inserted into the plasmid pUSC–PnmtA (Hiramoto et al., 2015), which contains the nmtA promoter plus the 50 -UTR and A. nidulans ATP sulfurylase gene sC yielding pireAupsCPnmtA. A DNA fragment comprising the partial ireA coding region was obtained by NotI/SphI digestion of the fragment amplified by PCR using the primers ireAsenNotI and ireAdownantiSphI. The acquired DNA fragment was inserted into NotI/SphI-digested pireAupsCPnmtA, yielding psCPnmtAireA. The DNA fragment obtained from SpeI/SphI-digested psCPnmtAireA was used for A. oryzae transformation.
The DNA fragment for the deletion of amyR using orotidine-50 decarboxylase gene (pyrG) as the selectable marker was constructed as follows: a DNA fragment containing Aspergillus nidulans pyrG was amplified by PCR from A. nidulans genomic DNA using the primers AnpyrGsen and AnpyrGantiPstI. A modified pUC119, containing the amyR fragment obtained by KpnI/HindIII digestion of the plasmid pGL20 (Gomi et al., 2000), was digested with PstI and NcoI to remove the internal coding region of the amyR. The PstI/NcoI-digested pyrG fragment was then inserted into the resulting plasmid, yielding pDamyR::pyrG. The DNA fragment obtained from ClaI/EcoRI-digested pDamyR::pyrG was used for A. oryzae transformation. Expression plasmids containing the intact hacA and hacA-i lacking 20-nt intron were constructed as follows: a DNA fragment of hacA-i was constructed by overlap extension PCR. In brief, two hacA fragments, each amplified by PCR using primer sets hacAsenNotI + hacADintronanti or hacADintronsen + hacAantiEcoRV with A. oryzae genomic DNA as the template, were mixed, and a second round of PCR with the primers hacAsenBamHI and hacAantiEcoRV was performed. This amplified fragment lacking 20-nt intron was digested with NotI and EcoRV and inserted into pBluescriptKS(+), which yielded pBS/hacA-i. The niaD fragment encoding nitrate reductase was obtained from pNPthiA vector (Suzuki et al., 2015) by digestion with NruI and insertion into EcoRV-digested pBS/ hacA-i, which generated pBShacA-i /niaD. Similarly, intact hacA fragment was amplified by PCR using the primers hacAsenNotI and hacAantiEcoRV with A. oryzae genomic DNA, and the NotI/ EcoRV-digested fragment was inserted into pBluescriptKS(+), followed by insertion of the niaD fragment, which resulted in the plasmid pBShacA/niaD. The DNA fragments for ireA disruption were constructed as follows: a DNA fragment approximately 1 kb upstream of the ireA coding region was amplified by PCR using ireAupsenKpnI and ireAupantiBamHI primers, and the KpnI/BamHI-digested fragment was inserted into the plasmid pUSC (Yamada et al., 1997) containing the A. nidulans sC, which yielded pUSCireAup. In addition, a DNA fragment of approximately 1 kb of the ireA 30 -region was also amplified by PCR using ireAdownsenPstI and ireAdownantiSphI primers, and the PstI/SphI-digested fragment was inserted into the plasmid pUSCireAup, yielding pDireA::sC. To generate a plasmid for ireA disruption using pyrG as the selection marker, the BglII/PstI-digested A. nidulans pyrG fragment was ligated to BamHI/PstI-digested pDireA::sC, which resulted in the plasmid pDireA::pyrG. The DNA fragment obtained by KpnI/SphI digestion of pDireA::sC and the fragment amplified by PCR using the ireAupsenKpnI and ireAdownantiSphI primers with pDireA::pyrG as the template were used for A. oryzae transformation. The nt sequences of all primers used in this study are shown in Supplementary Table S1.
Table 1 The Aspergillus oryzae strains used in this study. Strain
Strain origin
Genotype
Reference
NS4 nmtAp-ireA nmtAp-ireA pyrG nmtAp-ireA DamyR DamyR nmtAp-ireA + hacA nmtAp-ireA + hacA-i DligD::loxP pyrG + hacA-i DireA + hacA-i DligD::loxP + hacA-i DligD::loxP pyrG::niaD
RIB40 NS4 DligD::loxP nmtAp-ireA NS4 nmtAp-ireA nmtAp-ireA DligD::loxP DligD::loxP DligD::loxP DligD::loxP
niaD; sC niaD; nmtAp-ireA::sC niaD; pyrG; nmtAp-ireA::sC niaD; nmtAp-ireA::sC; DamyR::pyrG niaD; DamyR::sC nmtAp-ireA::sC; niaD::hacA nmtAp-ireA::sC; niaD::hacA-i sC; pyrG; niaD::hacA-i sC; niaD::hacA-i; DireA::pyrG sC; niaD::hacA-i; niaD::pyrG sC; niaD::pyrG
Yamada et al. (1997) This study This study This study Hasegawa et al. (2010) This study This study This study This study This study Ichinose et al. (2014)
pyrG pyrG
pyrG pyrG + hacA-i pyrG + hacA-i pyrG
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2.3. A. oryzae transformation Transformation of A. oryzae was performed according to the method of Gomi et al. (1987). 2.4. Nucleic acid blotting
A
sC Genomic DNA (Host strain)
ireA BglII
BglII
Southern and northern blot analyses were performed as previously described (Tanaka et al., 2012). 2.5. RT-PCR analysis
Genomic DNA (Transformant)
sC BglII
First-strand cDNAs were synthesized as previously described (Ichinose et al., 2014). PCR using cDNAs as template was performed for 20 cycles. Quantitative RT-PCR was performed as previously described (Ichinose et al., 2014). The nt sequences of primers used for RT-PCR are shown in Supplementary Table S1.
ireA BglII
probe
1 kb
B
kb
WT
nmtAp -ireA
9.4 3. Results
6.6
3.1. Growth of the ireA conditionally expressing strain Because deletion of ireA seemed lethal in A. oryzae, an ireA conditionally expression strain was constructed by replacing the ireA promoter plus the 50 -UTR with the nmtA promoter plus its 50 -UTR which contains a thiamine riboswitch. The nmtA promoter plus the 50 -UTR (nmtAp) is sufficient for gene silencing in A. oryzae (Hiramoto et al., 2015). The upstream region of ireA (AO090005000934) was replaced with nmtAp to generate the ireA conditionally expressing strain (Fig. 1A), and the resultant transformant was labeled as the nmtAp-ireA strain. The replacement of the ireA upstream region with nmtAp was confirmed by Southern blot analysis (Fig. 1B), and the growth of the nmtAp-ireA strain was observed on minimal agar media containing various carbon sources (Fig. 1C). When thiamine was added to the medium containing glucose or fructose as the sole carbon source for ireA silencing, the nmtAp-ireA strain exhibited poor growth. In contrast, ireA silencing by thiamine addition to the medium containing maltose and starch, which induce amylolytic gene expression, resulted in the complete inhibition of A. oryzae growth. In addition to amylolytic enzymes, A. oryzae secretes xylanolytic and cellulolytic enzymes when xylan is used as the sole carbon source (Noguchi et al., 2009). The ireA-repressing strain showed no growth on medium containing xylan as the sole carbon source. Even under thiamine-deficient conditions in which ireA was expressed by nmtAp at a moderate level, the nmtAp-ireA strain exhibited almost no growth on media with starch and xylan as the carbon source. Moreover, the nmtAp-ireA strain could not grow on medium containing skim milk as the sole nitrogen and carbon source, which is an inducer of proteolytic gene expression. These results indicated that ireA was required for A. oryzae growth under conditions inducing secretory hydrolytic enzyme production.
4.4
Without thiamine
C
WT
nmtAp -ireA
+ 10 µM thiamine WT
nmtAp -ireA
Glucose
Fructose
Maltose
Starch
Xylan
Skim milk
3.2. UPR is induced by amylolytic gene expression The expression of amylolytic genes in A. oryzae is regulated by the Zn(II)2Cys6-type transcription activator AmyR (Gomi et al., 2000; Petersen et al., 1999). To examine whether the growth defect by ireA silencing on maltose medium was caused by inducible amylolytic enzyme production, amyR was disrupted in the nmtAp-ireA strain. As shown in Fig. 2, amyR disruption partially restored the growth of the nmtAp-ireA strain on maltose medium supplemented with thiamine, which repressed ireA expression. This result suggested that amylolytic enzyme production correlated with growth defect under the condition of ireA silencing. To
Fig. 1. Growth of the A. oryzae ireA conditionally expressing strain. (A) Construction of the ireA conditionally expressing strain by replacement of the ireA upstream region with 50 -untranslated region (50 -UTR) attached to the nmtA promoter. The nmtA promoter and 50 -UTR are represented by a gray box. (B) Southern blot analysis of the ireA conditionally expressing strain. The BglII-digested genomic DNAs from the wild-type (WT) and ireA conditionally expressing strains were electrophoresed on a 0.8% agarose gel and transferred on to a nylon membrane. A labeled DNA fragment of the ireA upstream region was used as a probe. (C) Growth of the ireA conditionally expressing strain. Approximately 104 conidiospores of the WT (NS4) and nmtAp-ireA strains were grown on minimal agar plates containing 1% carbon sources indicated, with or without 10 lM thiamine, at 30 °C for 3 days. Skim milk was also used as the sole nitrogen source.
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WT
nmtAp -ireA
nmtAp -ireA ΔamyR
WT
nmtAp -ireA
nmtAp -ireA +hacA
nmtAp -ireA +hacA-i
Without thiamine
+ 10 µM thiamine
Glucose
Without thiamine
+10 µM thiamine
Fig. 2. Effect of amyR deletion on the growth of the ireA conditionally expressing strain. Approximately 104 conidiospores of each strain were grown on minimal agar plates containing 1% maltose, with or without 10 lM thiamine, at 30 °C for 3 days. The DligD::loxP pyrG::niaD strain was used as the wild-type strain.
A
Wild-type
Maltose
ΔamyR
0 30 40 50 60 70 80 90 0 30 40 50 60 70 80 90 (min)
bipA pdiA
Starch
amyA/B/C
18S rRNA
B
Wild-type
ΔamyR
G 0 30 40 50 60 70 80 0 30 40 50 60 70 80 (min)
hacA actA Fig. 3. Expression analysis of the UPR genes. (A) Northern blot analysis of bipA and pdiA. The wild-type (NS4) and DamyR mutant strains were grown in liquid Czapek Dox + 0.1% polypeptone medium containing 1% fructose as the carbon source for 24 h, followed by transfer to liquid minimal medium containing 1% maltose. The mycelia were harvested at the time points indicated, and the total RNA was extracted from the harvested mycelia. Approximately 20 lg of the total RNA was subjected to northern blot analysis, and the digoxigenin-labeled fragment of each gene was used as a probe. The loading control used was 18S rRNA. (B) RT-PCR analysis of hacA. The cDNAs were synthesized from total RNA used for northern blot analysis. PCR using the resulting cDNAs as template was performed for 20 cycles. The PCR products of hacA and actin gene (actA) were electrophoresed on 4% agarose gel with TBE buffer and 2% agarose gel with TAE buffer, respectively. The PCR products derived from genomic DNA were also electrophoresed (lane G).
confirm that amylolytic enzyme production essentially induced UPR, the expression levels of bipA and pdiA in the wild-type and DamyR strains were examined by northern blot analysis after incubation in the liquid maltose medium (Fig. 3A). After incubating the wild-type strain in maltose medium for 60 min, the expression of
Fig. 4. Effect of hacA-i introduction on the growth of the ireA conditionally expressing strain. Approximately 104 conidiospores of each strain were grown on minimal agar plates containing 1% carbon sources indicated, with or without 10 lM thiamine, at 30 °C for 3 days.
both bipA and pdiA was strongly induced. In contrast, the expression levels of these genes in the DamyR strain were significantly low. Furthermore, RT-PCR analysis showed that splicing of 20-nt intron within hacA mRNA was actively promoted in the wild-type strain following 50 min of incubation, whereas such a splicing of unconventional intron of hacA was not evident in the DamyR strain (Fig. 3B). Moreover, both hacA intron splicing and ER chaperone gene (bipA and pdiA) expression in the wild-type strain were preceded by a-amylase gene expression (Fig. 3). These results clearly indicated that UPR was induced by amylolytic gene expression in A. oryzae. 3.3. Involvement of unconventional splicing of hacA intron during A. oryzae growth To examine whether the growth defect of the ireA-repressing strain is caused by the suppression of unconventional hacA intron splicing, synthetic hacA DNA fragment lacking 20-nt intron was introduced into the chromosome of the nmtAp-ireA strain and was referred to as hacA-i. The introduction of hacA-i resulted in
M. Tanaka et al. / Fungal Genetics and Biology 85 (2015) 1–6
the growth restoration of the nmtAp-ireA strain on media containing glucose, maltose, and starch as the sole carbon source (Fig. 4). This growth restoration was also observed on media containing fructose, xylan, and skim milk (Supplementary Fig. S1). In contrast, introduction of the intact hacA had no apparent effect on the growth of the ireA-repressing strain. These results suggested that the growth defect of the nmtAp-ireA strain mainly resulted from the suppression of unconventional hacA intron splicing. Based on these findings, we hypothesized that ireA could be disrupted
A
pyrG
Genomic DNA (+hacA-i strain)
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kb 9.4 6.6 4.4
C
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+hacA-i ΔireA
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Fig. 5. Construction of an ireA disruption mutant using the hacA-i harboring strain. (A) Construction of an ireA disruption mutant using the hacA-i harboring strain as the host. (B) Southern blot analysis of the ireA disruption mutant. Genomic DNAs were examined as described in Fig. 1B. (C) Growth of the ireA disruption mutant. Approximately 104 conidiospores of each strain were grown on minimal agar plates containing 1% glucose or maltose at 30 °C for 3 days. The DligD::loxP pyrG::niaD strain was used as the wild-type strain.
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together with the concomitant introduction of hacA-i. Predictably, we successfully obtained the homokaryotic ireA disruption mutant when the strain harboring hacA-i was used as the host for ireA disruption (Fig. 5A and B). The resulting ireA disruption mutant exhibited significant growth defect on all media (Fig. 5C). This result indicated that the function of IreA was not restricted to unconventional splicing of the hacA intron. 4. Discussion UPR is a key cellular response for homeostatic adaptation to ER stress. In this study, we examined the contribution of UPR to A. oryzae growth under conditions inducing secretory hydrolytic enzyme production by constructing the ireA conditionally expressing strain. The failure to obtain a homokaryotic ireA disruption mutant led us to speculate that ireA disruption is lethal to A. oryzae. However, poor growth of the nmtAp-ireA strain harboring a conditionally expressed ireA construct was observed in the presence of thiamine even under the secretion repressing condition of glucose or fructose as the sole carbon source (Fig. 1C). We assumed that this weak growth could be due to the leaky expression of ireA because nmtAp may not be completely repressed in the presence of thiamine. In contrast to A. oryzae and A. niger, a homokaryotic ireA disruption mutant was successfully obtained in the fungal pathogen Aspergillus fumigatus (Feng et al., 2011). The growth of A. fumigatus ireA disruption mutant at 30 °C was nearly normal; however, its growth was inhibited at 42 °C (Feng et al., 2011). It would be possible that the function of IreA probably differs between A. fumigatus and its related Aspergillus spp., such as A. oryzae and A. niger, which possess greater ability to produce large amount of secretory hydrolytic enzymes. The essentiality of ireA for A. oryzae growth under conditions not inducing hydrolytic enzyme production remains controversial. In the present study, ireA repression completely inhibited A. oryzae growth under conditions inducing hydrolytic enzyme production, whereas the introduction of hacA-i restored its viability (Figs. 1C and 4). In addition, UPR was induced by amylolytic enzyme production (Fig. 3), and severe growth defect of the ireA-repressing strain was partially restored by amyR deletion (Fig. 2). These results clearly indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by the production of secretory hydrolytic enzymes. It has been previously reported that the A. fumigatus DireA strain exhibited a striking growth defect on maltose medium, and this defect was decreased by the expression of hacA cDNA using the gpdA promoter (Feng et al., 2011). However, there is no evidence that UPR was essentially induced in A. fumigatus cultured in maltose medium. The results obtained in the present study provide the first experimental validation for the essential role of ireA in filamentous fungal growth under UPR-inducing condition. Introduction of hacA-i into the ireA-repressing strain was not sufficient for complete growth recovery under the UPR-inducing condition (Fig. 4). In addition, the ireA disruption mutant obtained using the hacA-i harboring strain as the host exhibited poor growth under all growth conditions tested. These results suggest that the role of IreA is not restricted to the unconventional splicing of the hacA intron and IreA has other important function(s) required for A. oryzae growth, as also suggested in A. fumigatus (Feng et al., 2011). In addition to the unconventional intron of XBP1, a homolog of yeast HAC1, metazoan IRE1a also cleaves mRNAs encoding membrane and secretory proteins, and this mRNA turnover pathway is called regulated Ire1-dependent decay (RIDD) (Hollien and Weissman, 2006; Hollien et al., 2009; Maurel et al., 2014). Recently, RIDD was also found in fission and budding yeasts (Kimmig et al., 2012; Tam et al., 2014). However, there is still no information on RIDD in filamentous fungi, and we are now trying to detect the presence of RIDD pathway in Aspergillus spp., including A. oryzae, for the further understanding of IreA function.
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The nmtAp-ireA strain showed nearly normal growth on media containing glucose and fructose as the carbon source and without thiamine. In contrast, severe growth defect of the strain was observed on maltose and xylan media without thiamine (Fig. 1C). Furthermore, the nmtAp-ireA strain exhibited complete growth inhibition on media containing starch and skim milk, regardless of the presence or absence of thiamine (Fig. 1C). These results suggested that the expression level of ireA driven by nmtAp was not sufficient for the normal growth of A. oryzae and that a higher ireA expression level was required for A. oryzae growth under UPR-inducing conditions. This notion was supported by the induced ireA expression in the wild-type strain after incubation in maltose and starch media; the observation that induction of the ireA expression by starch was higher than that by maltose (Supplementary Fig. S2) also suggested that a higher ireA expression level was required for growth on starch medium without thiamine comparable to the growth on maltose medium. However, in the skim milk medium, the influence of thiamine originally included in skim milk should be considered. The requirement of unconventional splicing of the hacA intron for A. oryzae growth suggests that hacA is also essential for the growth of this fungus. In fact, we obtained A. oryzae hacA disruption mutant only as a heterokaryon (data not shown). However, homokaryotic hacA disruption mutant has been successfully generated in A. niger and A. fumigatus, with hacA disruption resulting in drastic growth defect and nearly normal growth in A. niger and A. fumigatus, respectively (Carvalho et al., 2010; Feng et al., 2011; Mulder and Nikolaev, 2009). A recent report showed that the disruption of N. crassa hac-1 (hacA ortholog) resulted in growth inhibition on cellulose medium, whereas growth on other media was normal (Montenegro-Montero et al., 2015). An examination of A. oryzae hacA essentiality will support our further understanding of UPR contribution to ER stress adaptation in filamentous fungi. However, the importance of hacA 50 -UTR in UPR activation suggests that promoter replacement is not appropriate to examine hacA essentiality (Mulder and Nikolaev, 2009), and another approach for hacA silencing is required. Acknowledgments We thank Osamu Mizutani for kindly providing the DligD::loxP pyrG mutant strain and Tomoko Shintani for technical assistance. This work was supported in part by JSPS KAKENHI (Grant no. 17019001), Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2015.10.003. References Benz, J.P., Chau, B.H., Zheng, D., Bauer, S., Glass, N.L., Somerville, C.R., 2014. A comparative systems analysis of polysaccharide-elicited responses in Neurospora crassa reveals carbon source-specific cellular adaptations. Mol. Microbiol. 91, 275–299. Carvalho, N.D.S.P., Arentshorst, M., Kwon, M.J., Meyer, V., Ram, A.F.J., 2010. Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses. Appl. Microbiol. Biotechnol. 87, 1463–1473. Fan, F., Ma, G., Li, J., Liu, Q., Benz, J.P., Tian, C., Ma, Y., 2015. Genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase production in Neurospora crassa. Biotechnol. Biofuels 8, 66. Feng, X., Krishnan, K., Richie, D.L., Aimanianda, V., Hartl, L., Grahl, N., PowersFletcher, M.V., Zhang, M., Fuller, K.K., Nierman, W.C., Lu, L.J., Latgé, J.P., Woollett, L., Newman, S.L., Cramer Jr., R.A., Rhodes, J.C., Askew, D.S., 2011. HacAindependent functions of the ER stress sensor IreA synergize with the canonical
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