Introduction
Alkaline protease (Alp) is one of most important enzymes required for the production of soy sauce and is used to hydrolyze  the  raw  materials—an important step that determines the flavor of soy sauce (1).
Alp is a serine protease with an alkaline pH optimum. Under alkaline conditions, a signal transferred via the pal ambient pH signal transduction pathway (comprising palA, palB, palC, palF, palH, and palI genes) induces the activation of the transcription factor PacC (2). The zinc finger transcription PacC is synthesized as a factor 674-residue    precursor.    At    alkaline ambient pH, conformational changes lead to an open conformation in which PacC is accessible to a processing protease, and this protease then removes 400 residues from the C terminus, including a negative-acting domain. The resulting product is fully competent in transcriptional regulation      through 5`-GCCARG-3'   sites in the promoters of both alkaline (activated by PacC) and acidic (repressed by PacC) genes (3). This activation of PacC is assumed to be related to the activation of the alp gene. The alkaline pH-sensing module in the plasma membrane is composed of a receptor 7-transmembrane-domain (PalH),      a      3-transmembrane-domain protein (PalI), and an arrestin-related protein (PalF) (4). PalH is an ambient pH sensor, and PalI promotes the plasma membrane      localization     of      PalH. Arrestin-like PalF, which contains arrestin  N-terminal  and  C-terminal domains, interacts strongly with PalH. PalF is ubiquitylated in a receptor (PalH)- (alkaline and signal pH)-dependent manner, and ubiquitylation of arrestin-like PalF leads to constitutive signaling (5). However, the direct relationship between PalF and PacC in A. oryzae remains unclear. In a previous study, we analyzed the functions of PalI (6) and PalH (7) in A. oryzae. Therefore, in this study, we reported the effects of palF disruption on pacC and alp expression in A. oryzae.
Materials and Methods
Strains, media, and culture conditions A. oryzae △LigD, △niaD, and △pryG were used as hosts for transformation. The control strain was transformed using only the pyrG gene. The palF disruption strain and control strains were grown on yeast-peptone-dextrose (YPD) medium at 30°C for 24 h. DNA was extracted using     a     Wizard     Genomic     DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's protocol.
Construction of transformant strains
The palF disruption strain was using    previously    described created procedures (8). The palF gene disruption cassette was generated by fusion polymerase chain reaction (PCR) using an  Expand  High  Fidelity  PCR  System Diagnostics,       Mannheim, (Roche, Germany). The 5'- and 3'-arms of the palF gene were amplified from genomic DNA with the primers LU/LL (5'-arm) and RU/RL (3'-arm). The pyrG gene was amplified with the primer pair PU/PL. Amplified fragments were purified using a Wizard Gel Extraction kit (Promega). The PCR products were used for a second round of PCR with the primer pair LU/RL to fuse the 5' and 3' regions of the target gene at each end of the pyrG gene. The primer sequences (5'→3') as were follows: ATCCGACTCGCCGTTTGTACAT (LU), CACAGGGTACGTCTGTTGTCAGTG GATACGCACACTGATGCTAC  (LL), GTGCGTATCCACTGACAACAGACG TACCCTGTGATGTTC (PU), GTTGCTCATCCGACTAACTGCACCT CAGAAGAAAGGATG (PL), TTCTTCTGAGGTGCAGTTAGTCGG ATGAGCAACAGCAGTCAAA (RU), and ACGCTTGTGGCACTGTATTCTC (RL). The amplified fragment was purified using a Wizard Gel Extraction kit and was subsequently used for transformation. Fungal transformation was carried out essentially as previously described (9). Expression analysis by real-time PCR To analyze the expression of pacC and alp using real-time PCR, we isolated total RNA and mRNA from the control and disrupted strains. These strains were precultured at 30°C for 24 h in minimum medium (MM; 2% glucose, 1.2% NaNO 3 , 0.3% KH 2 PO 4 , 0.1% KCl, and 0.1% MgSO 4 ) containing 0.025% yeast extract. The main culture was then established for 3 h at 30°C in MM 0.2%     casamino     acids, containing buffered to pH 4.0 using 0.1 M citrate-phosphate (pH 3.8) and to pH 8.0 using 0.1 M Tris-HCl (pH 9.5). Total RNA was extracted using RNAiso Plus (TaKaRa, Shiga, Japan), according to the manufacturer’s instructions. mRNA was purified using an Oligotex-dT30<super> mRNA Purification Kit (TaKaRa), following the manufacturer's protocol. Reverse transcription and real-time PCR were performed as described previously (10) using a SuperScript III Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen, Carlsbad, CA, USA). The sequences (5'→3') of the pacC primers were GACTGCGGCTGCTCCTATTG (forward primer) and TGTCGTGGTAGCAGACTTGAG (reverse primer). The alp primers were synthesized according to previously described sequences (7). Histone H1 was used as an endogenous control. a palF disruption strain of A. oryzae was constructed. The transformants constructed by homologous recombination with the palF-disruption gene cassette were confirmed by PCR (Fig. 1).
Results and Discussion
Gene disruption and phenotype analysis First, we identified the palF gene from A. oryzae (XM_003189575). A. oryzae palF was homologous to palF from other organisms. For example, A. oryzae palF showed 71% homology to palF from A. nidulans, 75% homology to palF from A. fumigatus, and 99% homology to palF from A. flavus. Therefore, these data demonstrated that the palF gene was highly conserved in Aspergillus. To analyze PalF function in A. oryzae, Physical maps of the palF locus and the gene disruption cassette. The primers used for PCR amplification are indicated. The white arrow indicates amplified products from disruption constructs, and the black arrow indicates amplified products from the palF gene. The band sizes of palF of the control strain and the palF disruption strain were 2.3 and 4.5 kbp, respectively. results  showed These successful homologous  recombination  at  the resident palF locus. The palF disruption strain showed reduced growth on alkaline pH plates (Fig. 2), but normal growth on acid pH plates. Under alkaline pH conditions, growth of the A. oryzae palH-disrupted strain was completely inhibited (7), whereas the palI-disrupted strain showed moderate growth (6). The control and palF disruption strains were grown at 30°C for 5 days on MM+0.6% casamino acid agar. The values are colony sizes (cm). These data suggested that PalF played an important role in the pH signal transduction pathway of A. oryzae , but was not essential for this process.
Analysis of pacC gene expression
Figure 3 shows the relative expression levels of pacC in the control and disrupted strains (n = 3). 3. Comparison of pacC gene expression by real-time PCR Gene expression in the control strain at pH 4.0 was set at 1.0. The error bars indicate standard deviations. At pH 4.0, the expression level of pacC differed slightly between the control and palF disruption strains. However, at pH 8.0, the expression level of pacC in the palF disruption strain was about 7.9-fold less than that of the control strain. The transcription factor PacC was strongly expressed in the control strains under alkaline conditions. In A. nidulans, pacC is expressed under alkaline conditions and is subjected to autogenous  transcriptional  activation (11). pacC transcript levels are low in the absence of pal signal transduction. Therefore, the pal signal transduction pathway could not function in the palF disruption strain, and the expression level of pacC decreased. This finding suggested that PalF was important for the expression of pacC under alkaline conditions.
Analysis of alp gene expression
Next, we investigated the influence of palF disruption on alp gene expression at pH 8.0. The relative expression levels of alp in the control strain were approximately 2.6-fold higher than those in the palF disruption strain (Fig. 4). Expression of the alp gene decreased in the palF disruption strain because the pal signal transduction pathway did not function in this strain. The Alp activity in the palF disruption strain decreased to 8.7% that of the control (data not shown). Gene expression in the control strain was set at 1.0. The error bars indicate standard deviations. Thus, our data demonstrated that palF disruption had significant effects on alp gene expression. We concluded that pH signal transduction was interrupted by the disruption of palF . On the other hand, Alp activity in the palH-disrupted strain decreased to 6.3% that of the control (7). These results indicated that PalH was more important than PalF in the pH signal transduction pathway. In conclusion, we analyzed the role of the  ambient  pH signal PalF in transduction pathway in A. oryzae. We found that palF disruption affected PacC expression. PalF had an important role in the ambient pH signal transduction pathway of A. oryzae. The palF disruption strain exhibited decreased expression of pacC and reduced growth on alkaline pH plates. Therefore, PalF did not play an essential role in the pH signal transduction pathway. We intend to analyze this ambient pH signal transduction pathway in future studies. This work was supported by MEXT (Ministry of Education, Culture, Sports, Science and Technology) -Supported Program for the Strategic Research Foundation at    Private    Universities, 2009-2013. We thank Harue Kitagawa for technical assistance. 1) Murakami  K,  Ishida  Y,  Masaki  A, Tatsumi H, Murakami S, Nakano E, Motai H, Kawabe H, and Arimura H, Agric Biol Chem, 55, 2807-2811 (1991). 2) Selvig K, and Alspaugh JA, Mycobiology, 39, 249-256 (2011). 3) Mingot JM, Espeso EA, Diez E, and Penalva MA, Mol Cell Biol, 21, 1688-1699 (2001). 4) Calcagno-Pizarelli AM, Negrete-Urtasun S, Denison SH, Rudnicka HJ, Bussink JD, Munera-Huertas T, Stanton L, Hervas-Aguilar A, Espeso EA, Tilburn J, Arst HN, Jr., and Penalva MA, Eukaryot Cell, 6, 2365-2375 (2007). 5) Hervas-Aguilar  A,  Galindo  A,  and Penalva MA, J Biol Chem, 285, 18095-18102 (2010). 6)   Sano M, Kobayashi A and Ohashi S, Food Sci. Technol. Res, 20, 155-159 (2014). 7) Dohmoto M, Inoue Y, Kobayashi A, Ohashi S, and Sano M, Biosci 10) Kobayashi  A,  Sano  M,  Oda  K, Biotechnol Biochem, 74, 188-190 (2010). Hisada H, Hata Y, and Ohashi S, K, Satoh Y, Biosci 71, Ishii Biochem, T, 8) Tamano Biotechnol 1797-1799 (2007). Terabayashi  Y,  Ohtaki  S,  Sano  M, Takahashi T, Koyama Y, Mizutani O, Abe K, and Machida M, Biosci Biotechnol   Biochem,   71,   926-934 (2007). 11) Tilburn  J,  Sarkar  S,  Widdick  DA, Espeso EA, Orejas M, Mungroo J, Penalva MA, and Arst HN, Jr., Embo J, 14, 779-790 (1995). 9) Takahashi T, Hatamoto O, Koyama Y, and  Abe  K,  Mol  Genet  Genomics, 272, 344-352 (2004). Communicated by Ishijima Sumio 132