Characteristics of the Aspergillus oryzae GATA Transcription Factor Family and Expression Analysis under Temperature or Salt Stresses

GATA transcription factors(cid:0)TFs(cid:0)are involved in the regulation of diverse growth processes and various environmental stimuli stresses. Although the analysis of GATA TFs involved in abiotic stress has been performed in plants and some fungi, information regarding GATA TFs in A. oryzae is extremely poor. Therefore, we identied seven GATA TFs from A. oryzae 3.042 genome and classied into six subgroups in NJ_tree, including a novel AoSnf5 with 20-residue between the Cys-X 2 -Cys motifs which was found in Aspergillus for the rst time. Conserved motifs demonstrated that Aspergillus GATA TFs with similar motif compositions clustered into one subgroup, which suggests they might have similar genetic functions and further conrms the accuracy of the phylogenetic relationship. Moreover, the expression patterns of seven A. oryzae GATA TFs under temperature and salt stresses indicated that A. oryzae GATA TFs were mainly responsive to high-temperature and high salt stress. The PPI network of A. oryzae GATA TFs proposed some potentially interacting proteins. The comprehensive analysis of A. oryzae GATA TFs will be benecial to understand their functional and evolutionary features and provide useful information for the further analyzing the role of GATA TFs in regulation of distinct environmental conditions in A. oryzae. 2008; Tan al. In our study, AoLreA and AoLreB was increased under high-temperature (42 ℃ ) stresses, and AoLreA was signicantly induced expression under 5.0 and 10.0 g/100 mL NaCl stresses. The results demonstrated that AoLreA and AoLreB might act as a regulatory complex of the global regulator VeA in response to temperature and high salt stresse in A.oryzae. Hence, the expression patterns of these A. oryzae GATA TFs under distinct environmental conditions provided useful information for the further analysis of A. oryzae GATA TFs in regulation of various abiotic stress responses in Aspergillus.


Introduction
GATA transcription factors (TFs) constitute a protein family that is characterized by the presence of one or two highly conserved type-IV zinc ngers and a DNA-binding domain that recognizes the (A/C/T)-G-A-T-A-(A/G) sequence in the promoter sequence of target genes (Scazzocchio 2000). In fungi, diverse roles governed by GATA TFs mainly involved in nitrogen regulation and light responses, regulation of sexual and/or asexual reproduction, and secondary metabolism. GATA TFs AreB and AreA are not only involved in the nitrogen and carbon metabolism, but also in the control of several complex cellular processes such as transport and secondary metabolism (SM) (Pfannmüller et al. 2017; Chudzicka-Ormaniec et al. 2019). The SreA involves in regulation of siderophore biosynthesis and iron uptake (Oberegger et al. 2010;Schrettl et al. 2008), and NsdD regulates sexual and/or asexual reproduction and the production of SMs (Lee et al. 2014;Niehaus et al. 2017). Furthermore, few fungal GATA TFs also play important role in response to the abiotic stresses. Alternaria alternata SreA is related with the maintenance of cell wall integrity (Chung et al. 2020), while Blastomyces dermatitidis SreB strongly expresses and contributes to lamentous growth at 22 ℃ via lipid metabolism (Marty et al. 2015). Additionally, GLN3 and GAT1 have been shown to be involved in salt tolerance in Saccharomyces cerevisiae (Crespo et al. 2001). However, there are still very few reports regarding the function of lamentous fungal GATA TFs in response to abiotic stress factors.
Aspergillus oryzae is an important lamentous fungus, which is widely used in East Asian traditional fermented food products (Kitamoto 2015). Simultaneously, A. oryzae is exposed to various environmental stress factors during fermentation process. Temperature is the most important environmental factor affecting the growth and activity of microorganisms and can directly affect the activity of enzymes involved in substrate digestion during fermentation process (Chen et al. 2011; Bechman et al. 2012). In addition, high sodium chloride concentration, which inhibits the growth of spoilage bacteria in soy sauce mash, also affects the growth of A. oryzae (Wang et al. 2013;Fernandes et al. 2018). Therefore, the ability of A. oryzae to adapt to different temperatures and high salt concentration have attracted the attention of researchers, but the molecular mechanisms underlying their response to these stress factors are still unclear. The previous studies have demonstrated that GATA TFs mainly involved in regulation of various temperature and salt stimuli stress signaling in few fungi (Scazzocchio 2000;Crespo 2001;Marty et al. 2015). Although the Fungal Transcription Factor Database (FTFD) and Tetsuo Kobayashi et al publicized six A. oryzae GATA TFs which may involve in nitrogen regulation and light responses, regulation of sexual and/or asexual reproduction, and SM Kobayashi et al. 2007), there is lack of research on a comprehensive analysis of A. oryzae 3.042 GATA TF. Therefore, the aim of this study was to analyze A. oryzae GATA TF structural characteristics, evolutionary features, and conserved motifs, and the expression patterns of GATA TFs under temperature and salt stresses. Furthermore, the expression patterns and the results of PPI prediction can establish a good foundation for further study on the function and the mechanism of A. oryzae GATA TFs involved in abiotic stress responses.

Identi cation of A. oryzae GATA transcription factors
The Aspergillus oryzae 3.042 genome was downloaded from NCBI database (https://www.ncbi.nlm.nih.gov/genome/?term=Aspergillus+oryzae). The BLASTP program with a threshold e-value of 1e-10 was used to predict GATA TFs from the A. oryzae genome, using gene sequences from Aspergillus as query sequences. All potential A. oryzae GATA TF proteins were identi ed by HMMER3.1 and were predicted if they contained ZnF-GATA domains (PF00320). The sequences that resulted in GATA-type zinc nger genes hits with the GATA zinc-nger domains (PF00320) were considered as GATA TFs. CDD and PFAM databases were used to validate all the potential A. oryzae GATA TFs.
To determine the chromosomal locations of the seven identi ed A. oryzae GATA TFs, locus coordinates were downloaded from the A. oryzae RIB40 genomics database. The distribution of seven A. oryzae TFs on the chromosomes was drawn by MG2C (mg2c.iask.in/mg2c_v2.0/) and visualized using MapChart 2.2 (Voorrips 2002).

The multi sequences alignment and phylogenetic analysis
ClustalW was used to align A. oryzae GATA TF proteins. The protein sequences of known GATA TFs in all other Aspergillus were downloaded from fungal transcription factor databases (FTFD, http://ftfd.snu.ac.kr/index.php?a=view). The sequences of GATA TFs between A. oryzae and other Aspergillus species were also aligned using ClustalW to analyze the phylogenetic relationships of all Aspergillus GATA TFs. A Neighbor-Joining (NJ) tree was constructed based on aligned results in MEGA6.0 with bootstrap replications of 1000.

Motif analysis of A. oryzae and other Aspergillus GATA transcription factors
MEME was used to predict and analyze motifs of A. oryzae GATA proteins, which were visualized using TBtools (Chen et al. 2018). The parameters were set to zero or one of a contributing motif site per sequence, and the numbers of motifs were chosen as ve; motif widths were set to 6 and 50 (Wu et al. 2016). The other parameters were set to default values. Each motif was individually checked so that only motifs with an e-value of < 1e-10 were retained for motif detection in A. oryzae GATA proteins.
2.4 Effects of temperature and salinity treatment on the growth of A. oryzae A. oryzae 3.042 (CICC 40092), the main fermentation strain used in industry, was selected to test the growth of A. oryzae under temperature and salt stress. A. oryzae conidia were inoculated in fresh potato dextrose agar (PDA) medium and cultured at 22, 25, 30, 35 and 42 °C for 72 h to investigate the effects of temperature; the optimum growth temperature of A. oryzae, 30 °C, was used as the control temperature. PDA media with a nal salt concentration of 5.0, 10.0, 12.5 and 15.0 g/100 mL NaCl were prepared to test the effects of salinity stress on A. oryzae. Medium without salt was used as the control medium. Two microliters of freshly prepared A. oryzae suspension containing 1 × 10 7 conidia were inoculated on the medium to analyze phenotypes. To test the effect of two abiotic stress on fungal viability, 100 µL 1 × 10 7 conidia suspension was inoculated on per plate covered with cellophane (Solarbio, Beijing, China); the fungal mycelia were collected at 72 h incubation. Fungal mycelia were then dried overnight, and the biomass was tested. Material for RNA extraction was also collected as the same experimental operation.
Three replicates were performed each time for experiments.
2.5 qRT-PCR analysis of A. oryzae GATA TFs expression in response to temperature and salinity stress Total RNA was extracted using an Omega plant RNA kit (Omega Bio-Tek, Georgia, USA) according to the instructions provided by the manufacturer. One microgram of RNA was reverse-transcribed into cDNA using PrimeScript™ RT reagent with the gDNA Eraser kit (TaKaRa, Dalian, China). A. oryzae GATA TF primers were designed using the Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast) ( Table S1). Gene expression levels were determined by perfoming quantitative real-time polymerase chain reaction (qRT-PCR) on a Bio-rad CFX96 Touch instrument (Bio-Rad, USA) using TB Premix Ex Taq II (TaKaRa) according to the manufacturer's instructions. Data were analyzed using Bio-rad CFX96 software and the 2 −△△CT method (Livak and Schmittgen 2001).

Construction of protein-protein interaction network
Protein-protein interaction (PPI) data were obtained from the online database of STRING (https://stringdb.org/), which is an open source software for predicting and visualizing complex networks. These interactions were derived from literature of experimental validation including physical interactions and enzymatic reactions found in signal transduction pathways. The PPI networks were visualized in biological graph-visualization tool Cytoscape with the nodes representing proteins/genes (Pathan et al. 2015).

Characteristics of A. oryzae GATA TFs
BLASTP analysis was used to check predicted GATA TFs from the A. oryzae 3.042 genome. All potential A. oryzae GATA proteins were used to identify ZnF_GATA domains (PF00320) by HMMER3.1. In total, seven A. oryzae GATA TFs were identi ed, and were named AoAreA, AoAreB, AoLreA, AoLreB, AoNsdD, AoSnf5 and AoSreA corresponding to the names of fungal orthologs ( Table 1). The A. oryzae GATA TF amino acid lengths ranged from 313 (AoAreB) to 867 aa (AoAreA). The details of these A. oryzae GATA TFs, such as ZnF_GATA motif type, number domains of ZnF_GATA, sizes of the deduced peptides, and their homologous gene IDs, are listed in Table 1.
The GATA DNA binding domain is a conserved type-IV zinc-nger motif with the form Cys-X 2 -Cys-X 17 − 20 -Cys-X 2 -Cys. The zinc-nger motifs of Cys-X 2 -Cys -X 17 − 20 -Cys -X 2 -Cys among the  Fig. 1A). Interestingly, AoSreA harbored two highly conserved type-IV zinc-nger motifs with Cys-X 2 -Cys-X 17 -Cys-X 2 -Cys (Table 1 and Fig. 1A) that two conserved type-IV zincnger motifs usually occur in animals. Apart from the ZnF_GATA domain, additional domains such as TFIIB zinc-binding, AreA-N, SNF5/INI1, and PAS were also characterized ( Table 1 In addition, chromosomal location of A. oryzae GATA TFs reveals their random distribution in the A. oryzae genome. Here, the seven of A. oryzae strain 3.042 was mapping to the rst complete genome of A.
oryzae strain RIB40 chromosomes. The chromosomal distribution of A. oryzae GATA TFs was visualized by the MapChart program. Seven A. oryzae GATA TFs were randomly distributed on chromosomes 1, 3, 4, and 6 ( Fig. 1C). Interestingly, AoAreB, AoSreA, and AoSnf5 clustered into the same subgroup in the neighbor-joining tree (Fig. 1B) and were distributed on the same chromosome, which indicates a close evolutionary relationship exists among them. The chromosomal location of A. oryzae GATA TFs could help to determine the exact sequence of events. . Therefore, the AoLreA, AoLreB, AoAreA, and AoAreB divided respectively into WC-1, WC-2, NIT2, and ASD4 subgroups might also involve in light responses or nitrogen regulation as the reported. In addition, NsdD had been shown not only to affect sexual and asexual reproduction but also secondary metabolism in Aspergillus (Lee et al. 2014;, which could help to determine the function of the AoNsdD and Aosnf5 assigned to the NSDD subgroup.

Analysis of conserved motifs in A. oryzae GATA TFs
In order to obtain insights into the diversity of motifs compositions in A. oryzae GATA TFs, the A. oryzae GATA TFs and other Aspergillus' were predicted the conserved motifs Using MEME4.11.4 online software. A total ve conerved motifs were identi ed. The relative location of these motifs within the protein is represented in Fig. 3. The identi ed consensus sequence of the ve motifs is shown in Figure S1. A typical zinc-nger structure which was composed of motif 1 and motif 2 was observed in all Aspergillus GATA TFs, but the compositions of GATA TF motifs also had different variable regions. As expected, GATA menbers that had similar motif compositions could be clustered into one subgroup, which suggests they may have similar genetic functions within the same subgroups. In addition, the motif distribution further con rms the accuracy of the phylogenetic relationship of Aspergillus GATA TFs. The distribution of motifs in different subgroups implied sources of functional differentiation in GATA TFs in the evolutionary processes. . Therefore, we investigated the growth of A. oryzae under different temperature and salt concentration stresses. The optimum temperature for A. oryzae growth usually ranges from 30-35 °C. Low-and high-temperatures signi cantly inhibited the mycelial growth, especially at the temperature of 22 and 42 ℃ (Fig. 4A, a-e and Fig. 4B). In addition, the high salt concentration also signi cantly inhibited the hyphal growth and differentiation of A. oryzae, and the inhibitory effect increased with the salt concentration (Fig. 4A, f-j and Fig. 4C). Furthermore, the formation and development of A. oryzae spores, which shows yellow-green color in the middle of the fungal colony, were also inhibited under low-and high-temperature and high salinity stresses (Fig. 4A).

Expression patterns of A. oryzae GATA TFs in response to temperature and salinity stresses
To determine on the possible roles of A. oryzae GATA TFs in response to abiotic stresses, we analyzed the expression level of seven A. oryzae GATA TFs by qRT-PCR in A. oryzae that grew under different temperatures and salt concentrations (Fig. 5). Seven A. oryzae GATA TFs exhibited expression diversity under different temperatures and salt stresses. Except the AoSnf5, six A. oryzae GATA TFs strongly responded to low-or high-temperatures (Fig. 5A). AoSreA and AoNsdD showed the same expression trends that they were signi cantly induced at low-temperature (22 ℃) and inhibited at high-temperature (42 ℃) compared with CK (30 ℃). Besides, AoAreB, AoLreA and AoLreB expression levels were remarkably upregulated under high-temperature stresses compared with 30 ℃(CK),especially AoAreB (Fig. 5A). Interestingly, only AoAreA was inhibited under both low-and high-temperature stresses. Furthermore, the expression level of AoAreA, AoSreA, and AoAreB was signi cantly downregulated under high-salt concentration stress, while AoLreA, AoNsdD, and AoSnf5 expression level exhibited upregulated under 5.0 and 10.0 g/100 mL NaCl stresses (Fig. 5B). Together, the results demonstrate the importance of A. oryzae GATA TFs in response to temperature and high salt stresses and provide a basis information for future studies into the function of A. oryzae GATA in abiotic stresses.

Protein-protein interaction network of A. oryzae GATA TFs
To analyze the functions of A. oryzae GATA TFs proteins, protein-protein interaction (PPI) network was constructed using the data from STRING database, and only two independent PPI network of AoAreA and AoSreA proteins was obtained ( Fig. 6A and B). Furthermore, we found both AoAreA and AoSreA proteins interacted with CreA that CreA deletion mutants show less conidiation than wild type and mutants are sensitive to salt stress (Hou et al. 2018). Therefore, the expression levels of AoAreA, AoSreA, and AoCreA were analyzed under temperature and salt stresses. AoSreA and AoCreA showed the same expression patterns under both low-and high-temperature stresses, while the AoAreA and AoCreA exhibited opposite expression level at the temperature of 22 ℃ (Fig. 6C). Interestingly, three genes showed the same expression patterns under high salt concentration stresses (Fig. 6D), which demonstrates that AoCreA may be positively coregulated by both AoAreA and AoSreA under salt stresses. Additionally, the protein glutathione S-transferase (CADAORAP00007152) that is critical to abiotic stress was also found in the network of AoAreA (Favaloro et al. 2000). These results in this study were bene cial to identify more important proteins and biological modules that interacted with A. oryzae GATA TFs and understand the roles of A. oryzae GATA TFs in response to abiotic stresses. The detailed information of the proteins in the PPI network is listed in Table S2.

Discussion
Transcription factors (TFs) regulate expression of genes that mediate growth processes and environmental response and are employed as a principal source of the diversity and change that underlie evolution (Riechmann and Ratcliffe 2000). GATA TFs are transcriptional regulatory proteins that contain a characteristic type-IV zinc nger (Cys-  (Kobayashi et al. 2007). However, in this study, we identi ed seven A. oryzae GATA TFs from the A. oryzae 3.042 genome using an HMM model. Six known A. oryzae GATA TFs, consistent with the report of Kobayashi et al. (2007), were classi ed into six functional subgroups based on the number of ZnF_GATA domains and zinc nger motif of GATA domain sequences with other Aspergillus GATA TFs from FTFD, while the novel AoSnf5 encoding GATA TF also clustered into NSDD subgroups together with AoNsdD (Fig. 2). Conserved motifs demonstrated that GATA TF menbers had similar motif compositions could be clustered into one subgroup (Fig. 3), which suggests they may have similar genetic functions within the same subgroups. In addition, the motif distribution further con rms the accuracy of the phylogenetic relationship of Aspergillus GATA TFs. The analyses of phylogenetic tree and conserved motifs demonstrated that the evolution of GATA TFs among different Aspergillus was very conservative which might have the same evolutionary events and perform similar function among the Aspergillus GATA proteins within the same subgroups.
Although most GATA domains harbor a class-IV zinc-nger motif, this structure differs among kingdoms (Lowry and Atchley 2000). In plants, most GATA domains have a single Cys-X 2 -Cys-X 18 -Cys-X 2 -Cys motif, but some harbor more than two zinc-nger motifs or 20-residue within zinc-nger loops (Reyes et a. 2004; Behringer and Schwechheimer 2015). In animals, the GATA domain harbors two zinc-nger motifs with Cys-X 2 -Cys-X 17 -Cys-X 2 -Cys, but only the C-terminal nger is associated with DNA binding (Patient and Mcghee 2002). Fungal GATA TFs are combination of both animal and plant GATA TFs in terms of the amino acid residues present in the zinc-nger loop (Teakle and Gilmartin 1998). The majority of fungal GATA TFs contain a single zinc-nger domain and fall into two different categories: animal-like with 17residue loops(Cys-X 2 -Cys-X 17 -Cys-X 2 -Cys), and plant-like with 18-residue loops (Cys-X 2 -Cys-X 18 -Cys-X 2 -Cys) (Teakle and Gilmartin 1998;Scazzocchio 2000;Patient and Mcghee 2002). Nineteen-and 20-residue zinc-nger loops (Cys-X 2 -Cys-X 19 − 20 -Cys-X 2 -Cys) are also found in fungi, albeit rarely (Scazzocchio 2000; Maxon and Herskowitz 2001). Except for the 17-and 18-residue zinc-nger loops in A. oryzae GATA TFs, the novel AoSnf5 contains 20-residue in the zinc-nger loops (Cys-X 2 -Cys-X 20 -Cys-X 2 -Cys), which are rarely found in fungi (Table 1 and Fig. 1). To our knowledge, GATA TF with 20-residue zinc-nger loops was found in Aspergillus for the rst time. In addition, AoSreA harbors two ZnF-GATA domains with the form of Cys-X 2 -Cys-X 17 -Cys-X 2 -Cys, which is the typical GATA characteristic in animals (. Lowry and  (Fig. 5A), which indicated AoAreA and AoAreB might also act respectively as negative and positive transcriptional regulators under high-temperature stresses. The AoNsdD and AoSnf5, clustering into NSDD subgroup in the NJ_tree (Fig. 2), were strongly induced under high salt stresses. NsdD has been reported as a key repressor affecting the quantity of asexual spores in Aspergillus (Lee et al. 2014;, but there is lack of research on NsdD in response to adversity stress in Aspergillus. Apart from the regulation of siderophore biosynthesis and iron metabolism, SreA is also related with the maintenance of cell wall integrity and negatively impacts resistance as ΔsreA increases resistance to H 2 O 2 , calco uor white, and Congo red (Chung et al. 2020).The expression level of AoSreA was signi cantly downregulated under 42 ℃ and high salt stresses, which indicates AoSreA might negatively impact high-temperature and high salt resistance. In contrast, AoSreA was signi cantly upregulated at 22 ℃, and there is a report that the SreB strongly expresses and contributes to lamentous growth at 22 ℃ via lipid metabolism in Blastomyces dermatitidis (Marty et al. 2015). AoSreA and SreB shared the same conserved ZnF_GATA domain ( Figure S2), which demonstrates that overexpression AoSreA in A. oryzae might also enhance the growth of mycelium at 22 ℃. Moreover, AoCreA, interacting with AoSreA protein within the PPI network, has the same expression patterns as AoSreA, which indicates AoSreA might positively regulate the AoCreA under temperature and high salt stresses. Curiously, the expression level of AoCreA was inhibited under high salt stresses in A. oryzae, which con icted with the previous study that ΔcreA mutants of Fusarium graminearum are sensitive to salt stress (Hou and Wang 2018). However, the results provide insights into the critical role of SreA in resistance to different temperatures and high salt stresses in A. oryzae.
LreA and LreB, is the GATA TFs of WC-1 and WC-2 subgroups involve in the regulation of blue-and redlight responses (Purschwitz et al. 2008;Fuller et al.2013). AoLreA and AoLreB, dividing respectively into WC-1 and WC-2 subgroups in NJ_tree (Fig. 2), acts as a dimer and contain typical PAS dimerization domains that display in Table 1    Phylogenetic analysis of A. oryzae and other Aspergillus TFs. GATA protein sequences were aligned using ClustalW in MEGA6.0 software using default parameters. The consensus NJ_tree represent 1, 000 bootstrap replications. Bootstrap values are displayed with nodes. The protein sequences of Aspergillus GATA TFs were downloaded from FTFD. The Aspergillus GATA TFs are classed into seven subgroups in NJ_tree, including one group with unknown function. Seven A. oryzae GATA TFs are scattered in six known subgroups, and the novel AoSnf5 also clustered into NSDD subgroups together with AoNsdD.

Figure 3
The conserved motif arrangement of A. oryzae and other Aspergillus GATA TF proteins based on their phylogenetic relationships. A NJ_tree was predicted from the amino acid sequences of GATA TFs using ClustalW and MEGA6.0 with 1, 000 bootstrap replications. The conserved motifs in the GATA TFs were identi ed by MEME. In total, ve conserved motifs were identi ed and shown in different colors.   Supplementary Files