Genome-wide Identication of the Aspergillus oryzae GATA Transcription Factor Gene Family and expression Analysis under Temperature or Salt Stresses

GATA transcription factors (TFs) are transcriptional regulatory proteins that contain a characteristic type-IV zinc nger and recognize the conserved GATA motif in the promoter region. Previous studies demonstrate that GATA TFs are involved in the regulation of diverse growth processes and various environmental stimuli stresses. Although the analysis of GATA TFs involved in abiotic stress have been performed in model plants and some fungi, information regarding GATA TFs in A. oryzae is extremely poor. A. oryzae GATA Six A. oryzae GATA

oryzae GATA TFs were classi ed into six subgroups, while the novel AoSnf5 also clustered into NSDD subgroups together with AoNsdD in the NJ_tree of all Aspergillus GATA TFs. Conserved motifs demonstrated that GATA TFs with similar motif compositions clustered into one subgroup, which suggests they might have similar genetic functions and further con rms the accuracy of the phylogenetic relationship of Aspergillus GATA TFs. The expression patterns of seven A. oryzae GATA TFs exhibited diversity under temperature and salt stresses. The expression analyses of AoLreA and AoLreB demonstrates AoLreA mainly played role in salt stress and AoLreB did under temperature stress. AoSreA was shown to positively regulate the expression of AoCreA and might act as a negative regulator in temperature and high salt stress response. In addition, the AoNsdD, AoSnf5, AoAreB, and AoAreA strongly responsed to salt stresses, while AoAreB and AoAreA showed opposite expression trends at high temperature. Overall, the expression patterns of these A. oryzae GATA TFs under distinct environmental conditions provided useful information for the further analysis of GATA TFs in regulation of various abiotic stress in A. oryzae.

Conclusion
In conclusion, the comprehensive analysis data of A. oryzae GATA TFs will provide insights into the critical role of A. oryzae GATA TFs in resistance to temperature and salt stresses in A. oryzae.
Background GATA TFs are widely distributed in fungi, plants, and animals [1]. They constitute a protein family that is characterized by the presence of one or two highly conserved type-IV zinc ngers (Cys-X 2 -Cys-X 17 − 20 -Cys- sequence of target genes [1]. Although most GATA domains harbor a class-IV zinc-nger motif of Cys-X 2 -Cys -X 17 − 20 -Cys-X 2 -Cys, this structure differs among kingdoms [1]. 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 [2,3]. 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 [4]. 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 [5]. The majority of fungal GATA TFs contain a single zinc-nger domain and they mostly fall into two different categories: animal-like with 17-residue loops Cys-X 2 -Cys-X 17 -Cys-X 2 -Cys (IVa), and plant-like with 18-residue loops Cys-X 2 -Cys-X 18 -Cys-X 2 -Cys (IVb) [4,5,6]. In addition, nineteen-and 20residue zinc-nger loops are also found, albeit rarely, in fungi [6]. For example, the Saccharomyces cerevisiae ASH1 of GATA TF contains 20-residue in the zinc-nger loop that binds to the promoter of the HO nuclease gene [7].
The functions of GATA TFs have been widely studied in fungi, animals, and plants. Apart from their active involvement in nitrogen metabolism, owering growth and development in plants, GATA TFs also play a key role in response to various environmental stimuli stress such as salinity, drought, and temperature stresses [8,9]. Diverse roles governed by GATA TFs in fungus mainly involved in nitrogen regulation and light responses, regulation of sexual and/or asexual reproduction, and secondary metabolism [10,11,12,13]. Research has demonstrated that the AreB and AreA GATA TFs are regulators that 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 in lamentous fungi [11,12]. The SreA involves in regulation of siderophore biosynthesis and the control of iron uptake [10,14], and NsdD is a global regulator that regulates sexual and/or asexual reproduction and the production of SMs in A. nidulans and A. fumigatus [15,16]. Fungal GATA TFs are a combination of both plant and animal GATA TFs in terms of amino acid residues present in the zinc-nger loop [5,6,8]. Thus, few fungal GATA TFs also play important role in response to the abiotic stresses like plant GATA TFs. For example, in Alternaria alternata, GATA TF SreA is related with the maintenance of cell wall integrity, and the ΔsreA increases resistance to calco uor white, Congo red and H 2 O 2 [10]. SreB strongly expresses and contributes to lamentous growth at 22 ℃ via lipid metabolism in Blastomyces dermatitidis [17]. Additionally, GLN3 and GAT1 of GATA TFs have been shown to be involved in salt tolerance in Saccharomyces cerevisiae [18]. 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, that is widely used in East Asian traditional fermented food products, such as soy sauce and sake fermentation [19,20]. A. oryzae secretes synthetic and hydrolytic enzymes, and accumulates avor compounds, which enhance the nutritional and avor pro le of fermented foods during fermentation [19,21]. Simultaneously, A. oryzae is exposed to environmental stress factors during fermentation process. For example, 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 [22,23]. In addition, high sodium chloride concentrations are added to soy sauce mash to inhibit the growth of spoilage bacteria during fermentation process, but high salt concentrations also inhibit the growth of A. oryzae [24,25]. 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 plants and few fungi [6,8,9,18]. Although the 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 secondary metabolism [11,12,26], there is lack of research on a comprehensive analysis of GATA TF structural characteristics, evolutionary features, conserved motifs and expression level under different environmental stress factors in A. oryzae. Therefore, the aim of this study was to identify the GATA TFs in the whole A. oryzae 3.042 genome, to analyze their domain structure, evolutionary features, and conserved motifs, and to provide a basis for the cloning of GATA TFs in A. oryzae. Furthermore, the results of protein-protein interaction prediction and the expression patterns of A. oryzae GATA TFs under different temperatures and high salt stresses can establish a good foundation for further study on the function and the mechanism of A. oryzae GATA TFs involved in abiotic stress responses in A. oryzae.

Results
Identi cation of A. oryzae GATA zinc nger 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, AoSreA corresponding to the names of fungal orthologs (Table 1). A. oryzae GATA 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 seven A. oryzae GATA proteins showed differences. Six A. oryzae GATA domains contained the Cys-X 2 -Cys-X 17/ 18 -Cys-X 2 -Cys motif as the reported in other fungi, while the zinc-nger loop of AoSnf5 had 20-residue between the Cys-X 2 -Cys motifs which has rarely been found in fungi [5,6] (Table 1 and Fig.1 A). 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.1 A) that two conserved type-IV zinc-nger 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, Fig.1 B). Previous studies have demonstrated that the PAS domain mainly functions in sensing environmental or physiological signals including oxidative and heat stress [27,28]. Therefore, extra domains presenting in A. oryzae GATA may also play the same role in diverse environmental stresses and could facilitate the functional analysis of A. oryzae GATA TFs.
In addition, chromosomal location of A. oryzae GATA TFs reveals their random distribution in the A. oryzae genome. 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.

Phylogenetic analysis of the Aspergillus GATA TFs
A neighbor-joining phylogenetic tree (NJ_tree) was constructed by using MEGA6.0 for the multiple sequence alignment of all Aspergillus GATA TFs with 1000 bootstrap replications to analyze phylogenetic relationships between the A. oryzae GATA TFs and other Aspergillus GATA TFs with the ZnF_GATA domains. All the Aspergillus GATA TFs divided into seven subgroups in the NJ_tree based on the number of ZnF_GATA domains and zinc nger motif of GATA domain sequences with other Aspergillus GATA TFs from FTFD, including six known subgroups and one unknown function subgroup (Fig. 2). A. oryzae GATA TFs were scattered in six subgroups with other Aspergillus GATA TFs which functions have been reported, while the novel AoSnf5 encoding GATA TF also clustered into NSDD subgroups together with AoNsdD. The different GATA subgroups perform different functions. For example, the GATA TFs of WC-1 and WC-2 subgroups mainly involve in the regulation of blue-and red-light responses [13,29]. Nitrogen regulation is regulated by the process of nitrogen catabolite repression which controls gene expression through GATA TFs of NIT2 and ASD4 subgroup family in yeasts and lamentous [12,30,31]. 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 [15,32], 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, all the Aspergillus GATA TFs 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 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 all GATA TFs also had different variable regions (motif 3, -4, -5). 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 GATA TFs. The distribution of motifs in different subgroups implied sources of functional differentiation in GATA TFs in the evolutionary processes.
Effects of different temperature and salinity treatments on the growth of A. oryzae The temperature and salt concentration are two of the most important environmental factors affecting the growth of A. oryzae during fermentation process [21,22,23,24,25]. 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 conditions of 22 and 40 ℃ (Fig. 4A, a-e; 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; 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 temperature and salt concentration (Fig. 5). Seven A. oryzae GATA TFs exhibited expression diversity under different temperatures and salt stresses. Except the AoLreA and AoSnf5, ve A. oryzae GATA TFs strongly responded to low-and high-temperatures (Fig. 5A). The expression levels of AoAreA, AoSreA and AoNsdD showed opposite trends, which were signi cantly induced at low-temperature (22 ℃) and inhibited at high-temperature (40 ℃) compared with CK (30 ℃). Besides, AoAreB and AoLreB expression levels were upregulated under low-and high-temperature stresses compared with 30 ℃(CK),especially under high-temperature (Fig. 5A). Furthermore, the expression level of AoAreA, AoSreA, and AoAreB was signi cantly downregulated under high-salt stress. In addition, AoLreA, AoNsdD, and AoSnf5 expression level exhibited upregulated under 5 and 10 mg/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 [33]. Therefore, the expression levels of AoAreA, AoSreA, and AoCreA were analyzed under temperature and salt stresses. Interestingly, three genes showed the same expression patterns under temperature and salt stresses ( Fig. 6C and D), which demonstrates that AoCreA may be positively coregulated by both AoAreA and AoSreA under temperature and salt stresses. Additionally, AoAreA protein interacted with CADAORAP00007152 (glutathione S-transferase) that is critical to abiotic stress [34]. 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 [35]. GATA TFs are transcriptional regulatory proteins that contain a characteristic type-IV zinc nger (Cys-X 2 -Cys-X 17 − 20 -Cys-X 2 -Cys) and a DNA-binding domain recognize the conserved GATA motif in the promoter sequence of target genes [1]. Fungal GATA TFs are mainly involved in the relation of nitrogen metabolism [12,31], light responses [13,29], siderophore biosynthesis and mating-type switching [10; 36]. Few GATA TFs in fungus also take part in response to the abiotic stresses, such as the SreA, SreB, LreA, LreB, GLN3 and GAT1 [10,13,17,18,29]. The number of the GATA TFs is conserved among A. clavatus, A. avus, A. fumigatus, A. nidulans, A. niger and A. oryaze that possess six GATA TFs, suggesting that lamentous fungi share an identical composition of GATA TFs with each other [26]. Here 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. [26], 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. Conserved motifs demonstrated that GATA TF menbers 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 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.
Therefore, the features of A. oryzae GATA TFs strongly demonstrate that A. oryzae GATA TFs might be the combination of both plant and animal GATA TFs, which is consistent with the report that fungal GATA TFs are combination of both plant and animal GATA TFs in terms of the numbers of ZnF-GATA domains and amino acid residues present in the zinc-nger loop [5,8].  [11,12,31], which indicated AoAreB and AoAreA might also act as positive and negative transcriptional regulators under temperature and salt stresses. The AoNsdD and AoSnf5, clustering into NSDD subgroup in the NJ_tree (Fig. 2), were strongly induced under high salt stresses. NsdD is a key repressor affecting the quantity of asexual spores in Aspergillus [32], but there is lack of research on NsdD in response to adversity stress in Aspergillus. 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.
Apart from the regulation of siderophore biosynthesis and iron metabolism, GATA TF 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 [10,14].The expression level of AoSreA was signi cantly downregulated under 40 ℃ 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 [17]. 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 [33]. 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 [13,29]. 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 and Fig. 1B. Previous studies have demonstrated that the PAS domain also functions in sensing environmental or physiological signals including oxidative and heat stress [27,28]. Therefore, except for the regulation of blue-and red-light responses, the PAS domains presenting in AoLreA and AoLreB may facilitate the environmental response analysis of A. oryzae GATA TFs. Additionally, LreA and LreB is a regulatory complex of the global regulator VeA, while VeA plays a critical role in environmental stress responses in A. cristatus, and the ΔveA mutants are more sensitive to high salt, osmotic pressure, and temperature stress [38,39]. AoLreA was signi cantly induced expression under 5 and 10 mg/mL NaCl stresses, while the expression level of AoLreB was increased under low-(22 ℃) and high-temperature (40 ℃) stresses compared with the CK (30℃). This result demonstrated that AoLreA and AoLreB might act as a regulatory complex of the global regulator VeA in response to different temperature and high salt stresses in A.oryzae.

Conclusion
We identi ed seven GATA TFs from A. oryzae 3.042 genome, including the novel AoSnf5 with 20-residue in the zinc-nger loops (Cys-X 2 -Cys-X 20 -Cys-X 2 -Cys), which was found in Aspergillus for the rst time. Six known A. oryzae GATA TFs were classi ed into six subgroups with other Aspergillus GATA TFs, while the novel AoSnf5 also clustered into NSDD subgroups together with AoNsdD. Conserved motifs demonstrated that GATA TFs with similar motif compositions clustered into one subgroup, which suggests they might have similar genetic functions and further con rms the accuracy of the phylogenetic relationship of Aspergillus GATA TFs. Seven A. oryzae GATA TFs exhibited expression diversity under different temperature and salt stresses. The AoNsdD and AoSnf5, clustering into NSDD subgroup in the NJ_tree, were strongly induced under high salt stresses. The expression level of AoAreB and AoAreA showed opposite trends at high temperature (40 ℃) compared with CK (30 ℃) in A. oryzae, while both them were inhibited under high salt stresses, which indicated AoAreB and AoAreA might act as negative or positive transcriptional regulators under temperature or salt stresses. AoLreA and AoLreB, with typical PAS dimerization domains that functions in sensing environmental and heat stress, exhibited different patterns under temperature and salt stresses, which demonstrates AoLreA mainly played role in salt stress and AoLreB did under temperature stress. AoSreA was shown to positively regulate the expression of AoCreA regulatory gene and participate in A. oryzae response to temperature and high salt stresses. In conclusion, the comprehensive analysis data of A. oryzae GATA TFs will be better to further study their functional characterization and evolution of A. oryzae GATA TFs and established a foundation for understanding the roles 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 [40].

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 [41]. 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 [42]. 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. 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 TM 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 [43].

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 [44].

Abbreviations
Not applicable.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
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Availability of data and materials
All the necessary data in this study has been provided in the manuscript and the Supplementary les. The software used to infer networks is open source/freely available and has been cited in this study.

Competing interests
The authors declare that they have no con ict of interests.

Additional Files
Additional le 1 File contains supplementary tables and gures referenced in this manuscript. Table S1. QRT-PCR primerws of A. oryzae GATA gene expression in response to abiotic stress. Table S2. The detailed information of the proteins in the PPI network. Figure S1. The ve structural motifs in A. oryzae GATA TF proteins. Figure S2. The predicted amino acid sequence of AoSreA aligned with SreB. AoSreA and SreB contained several conserved domains including two ZnF_GATA (N-terminal and C-terminal) separated by a cysteine-rich region (CRR) and a conserved C-terminus (CCT) with a predicted coiled-coil domain. Figure 1 The

Supplementary Files
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