Identification of A. oryzae GATA zinc finger 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 identified, 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.
Table 1 GATA TFs identified from A. oryzae.
|
Name
|
Protein ID
|
Peptide (aa)
|
ZnF_GATA Motif type
|
Number domain of ZnF_GATA
|
Homologous ID
|
Extra domain
|
AoSreA
|
EIT82081.1
|
567
|
Cys-X2-Cys-X17-Cys- X2-Cys
|
2
|
KOC08900.1
|
TFIIB zinc-binding
|
AoAreB
|
EIT79032.1
|
313
|
Cys-X2-Cys-X17-Cys- X2-Cys
|
1
|
XP_002379623.1
|
TFIIB zinc-binding
|
AoAreA
|
EIT72728.1
|
867
|
Cys-X2-Cys-X17-Cys- X2-Cys
|
1
|
RAQ50831.1
|
AreA_N
|
AoLreB
|
EIT79273.1
|
496
|
Cys-X2-Cys-X18-Cys- X2-Cys
|
1
|
RAQ50386.1
|
PAS
|
AoNsdD
|
EIT79449.1
|
504
|
Cys-X2-Cys-X18-Cys- X2-Cys
|
1
|
KOC07076.1
|
-
|
AoLreA
|
EIT77832.1
|
283
|
Cys-X2-Cys-X18-Cys- X2-Cys
|
1
|
XP_002384232.1
|
PAS
|
AoSnf5
|
EIT78280.1
|
570
|
Cys-X2-Cys-X20-Cys- X2-Cys
|
1
|
XP_022385751.1
|
SNF5/INI1
|
The GATA DNA binding domain is a conserved type-IV zinc-finger motif with the form Cys-X2-Cys-X17-20-Cys-X2-Cys. The zinc-finger motifs of Cys-X2-Cys -X17-20-Cys -X2-Cys among the seven A. oryzae GATA proteins showed differences. Six A. oryzae GATA domains contained the Cys-X2-Cys-X17/ 18-Cys-X2-Cys motif as the reported in other fungi, while the zinc-finger loop of AoSnf5 had 20-residue between the Cys-X2-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-finger motifs with Cys-X2-Cys-X17-Cys-X2-Cys (Table 1 and Fig.1 A) that two conserved type-IV zinc-finger 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 finger 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 filamentous [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 five conerved motifs were identified. The relative location of these motifs within the protein is represented in Fig. 3. The identified consensus sequence of the motifs is shown in Figure S1. A typical zinc-finger 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 confirms 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 significantly 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 significantly 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, five 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 significantly 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 significantly 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 beneficial 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.