Analysis of the Properties of 44 ABC Transporter Genes from Biocontrol Agent Trichoderma asperellum ACCC30536 and Their Responses to Pathogenic Alternaria alternata Toxin Stress

ATP-binding cassette (ABC) transporters are involved in transporting multiple substrates, such as toxins, and may be important for the survival of Trichoderma when encountering biotic toxins. In this study, genome searching revealed that there are 44 ABC transporters encoded in the genome of Trichoderma asperellum. These ABC transporters were divided into six types based on three-dimensional (3D) structure prediction, of which four, represented by 39 ABCs, are involved in transport and the remaining two, represented by 5 ABCs, are involved in regulating translation. The characteristics of nucleotide-binding domain (NBD) are important in the identification of ABC proteins. Even though the 3D structures of the 79 NBDs in the 44 ABCs are similar, multiple sequence alignment showed they can be divided into three classes. In total, 794 motifs were found in the promoter regions of the 44 ABC genes, of which 541 were cis-regulators related to stress responses. To characterize how their ABCs respond when T. asperellum interact with fungi or plants, T. asperellum was cultivated in either minimal media (MM) control, C-hungry, N-hungry, or poplar medium (PdPap) to simulate normal conditions, competition with pathogens, interaction with pathogens, and interaction with plants, respectively. The results show that 17 of 39 transport ABCs are highly expressed in at least one condition, whereas four of the five translation-regulating ABCs are highly expressed in at least one condition. Of these 21 highly expressed ABCs, 6 were chosen for RT-qPCR expression under the toxin stress of phytopathogen Alternaria alternata, and the results show ABC01, ABC04, ABC05, and ABC31 were highly expressed and may be involved in pathogen interaction and detoxifying toxins from A. alternata.


Introduction
Many Trichoderma species, such as T. harzianum, T. asperellum, and T. virens, are biocontrol agents which are widely distributed in nature and are used to protect crop plants in the form of commercial fungicides [1]. The resistance mechanisms of Trichoderma against phytopathogens include mycoparasitism, induction of resistance genes, niche exclusion competition, and plant growth promotion [2,3]. Trichoderma spp. can also protect stored fruits and flowers against pathogens [4]; hence, they are considered promising and environmentally friendly agents for controlling plant diseases affecting cereals as well as vegetable crops [5]. However, to perform their biocontrol role, Trichoderma spp. must first survive in hostile environments in which they are exposed to pesticides, pathogen toxins, and phytoalexin. ATP-binding cassette (ABC) proteins play an important role in this process.
In this study, in order to determine the properties of ABC proteins in T. asperellum and further identify potential robust ABC proteins that can help T. asperellum survive under biotic stress, 44 ABC transporter genes were identified in the T. asperellum genome and characterized based on sequence analysis, structure analysis, functional prediction, and promoter analysis. Further, transcriptome-and transcription-level analysis of six genes under toxin conditions revealed which genes potentially respond to biotic stress. The findings expand our understanding of Trichoderma tolerance to pathogenic toxins or phytotoxins, and the characteristics of these potential genes can serve as a reference for identifying ABC genes or finding robust ABC genes in other Trichoderma species or organisms, which could ultimately assist with the development of improved environmentally friendly biocontrol agents.

Analysis of 44 ABC Transporter Genes in the Trichoderma asperellum CBS433.97 Genome
All ABC transporter gene sequences from the genome of T. asperellum CBS433.97 were obtained via keyword searching (http://genome.jgi.doe.gov/pages/tree-of-life.jsf, accessed on 3 March 2021). Protein molecular weight was predicted using the Sequence Manipulation Suite (http://www.bioinformatics.org/sms/, accessed on 9 March 2021), and proteins were ranked according to size from large to small. Conserved domains were predicted using the BLAST program and analyzed using Pfam (http://pfam.sanger.ac.uk/, accessed on 9 March 2021). All non-ABC transporter genes were omitted. The isoelectric point (pI) was predicted using the ProtParam tool (http://web.expasy.org/protparam/, accessed on 10 March 2021).

Multiple Sequence Alignment of 79 NBDs from the 44 ABCs of T. asperellum CBS433.97 and Phylogenetic Analysis of 44 ABCs
Highly conserved motifs were observed in most NBDs of ABCs and are important characteristics of NBDs, so a total of 79 NBDs were identified among the 44 ABC transporter genes and analyzed using Clustal Omega (http://www.ebi.ac.uk/services/proteins, accessed on 10 March 2021). Sequence logos were drawn using WebLogo (http://weblogo. berkeley.edu/logo.cgi, accessed on 10 March 2021).
To investigate the 44 ABCs, a phylogenetic tree was constructed for the amino acid sequences of 44 ABCs using the maximum-likelihood method (model: LG + G + I + F) with 1000 bootstrap replicates in the MEGA 7.0 program.

The Expression Analysis of ABC Transporter Genes under Different Media Based on RNA-Seq
Trichoderma asperellum ACCC30536 (obtained from the Agricultural Cultural Collection of China, inoculum amount: 1 × 10 7 spores) was cultured with shaking (180 r/min) at 25 • C for 2 d in potato dextrose (PD) broth medium, and mycelia were harvest using a sterile gauze and washed with sterile purified water under aseptic conditions, and the mycelia were then subjected to four different culture media: mineral medium (MM) as used in a previous study [19], C-hungry medium (MM with 0.5% w/v ammonium sulfate), N-hungry medium (MM with 0.5% w/v glucose), and PdPap (Populus davidiana × P. alba var. pyramidalis) medium (MM containing 1% w/v root powder, 1% w/v stem powder, or 1% w/v leaf powder from PdPap). After culturing with shaking (180 r/min) at 25 • C for 72 h, the mycelia were washed with sterile purified water and transferred into centrifuge tubes, which were stored at −70 • C until they were processed for total RNA extraction using TRIzol reagent (Invitrogen, Carlsbad, CA, USA, Kit) [20]. The total amount of 3 µg mRNA was obtained and fragmented into short fragments, followed by cDNA synthesis. The resulting library was sequenced using Illumina HiSeqTM 2000. The raw data were filtered, with the remaining reads referred to as "clean reads", which were aligned to reference genes and a genome file with SOAPaligner/SOAP2. The alignment data were utilized to calculate the distribution of reads on reference genes and perform coverage analysis. Quality control of alignment was then required to determine whether the samples needed resequencing. Qualified data were then subjected to several analyses, including gene expression, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation, alternative splicing, novel transcript prediction and annotation, and SNP analysis. Finally, four transcriptome libraries were constructed [21]. The gene expression level was calculated using the reads per kilobase of transcript per million mapped reads (RPKM) method. The dist method was used to determine the Euclidean distance, and the complete method includes the method for hierarchical clustering of ABC genes according to their expression.

Transcription Analysis of Six ABCs in Response to Toxins of Alternaria alternata CFCC82114
The poplar leaf blight strain A. alternata CFCC82114 (obtained from the China Forestry Culture Collection Center) was cultured with shaking (180 r/min) at 25 • C for 12 d in PD broth medium, the fermentation was filtered using a 2.5 µm filter, and the filtrate was stored at −80 • C. Trichoderma asperellum ACCC30536 (1 × 10 7 spores/mL) was cultured with shaking (180 r/min) at 25 • C for 2 d in PD broth medium, the mycelia were washed and transferred to MM and toxin-induced medium (MM with 10% fermentation filtrate) under aseptic conditions with harvesting at 0, 6, 24, and 48 h for RNA extraction. The filamentous total RNA was extracted using the CTAB method, digested with DNaseI (Promega, Madison, WI, USA), and reverse-transcribed into cDNA using the PrimeScript RT Kit (Takara, Japan) according to the manufacturer's instructions. The transcription level was determined using RT-qPCR and was calculated according to the 2 −∆∆Ct method, using cDNA as template, and actin, α-tubulin, and β-tubulin as reference genes [20]. Three RT-qPCR replicates were analyzed per cDNA sample, and the primers were designed using Primer premier 6.0 software ( Table 1). The experiment was set up with three replicates. The statistical significance of differences was determined using an independent-samples t-test (p < 0.05). The properties of the 44 ABC transporter genes identified in the T. asperellum CBS433.97 genome were analyzed (Table S1). ABC transporter genes were distributed on 17 different scaffolds, with the number of genes on each scaffold ranked from 1 to 7, and scaffold six includes the largest number (7). These genes contain different numbers of introns, ranging from 0 to 15; 17 genes (39%) have more than 4 introns, while 4 (9%), 13 (30%), 5 (11%), and 5 (11%) genes have 3, 2, 1, and 0 introns, respectively. The isoelectric point (pI) ranges from 5.48 to 9.87; 17 ABCs are basic (pI > 7) and 27 are acidic (pI < 7). Their molecular weight ranges from 55.33 to 187.56 kDa. Based on NCBI multidomain prediction, the 44 ABC transporter genes were classified according to nine different multidomains: ATM1 (4), CFTR (6), MdlB (5), MDR (8), MRP (9), Protein White (4), Rim protein (2), Uup (5), and 3a0123 (1). The 44 ABCs were divided into 11 different types based on the order of conserved domains. Furthermore, while most ABCs included both NBDs and TMDs, two proteins contain only NBDs, indicating that they are involved not in transport, but rather in translational regulation.  Figure 1.
The multiple sequence alignment shows the conserved amino acids (marked by shade) were different; they were mainly concentrated in the eighth and ninth amino acids in "Walker A" and the sixth, eighth, twelfth, and thirteenth amino acids in the "ATP-binding motif". The sequences were divided into three main classes according to these differences. Based on the functional prediction based on 3D structure and the annotation of the conserved domain, sequences in the Class 1 (top) include those classified with "Protein White" and "Rim protein" multidomain. Sequences in Class 2 (middle) are mostly aligned with the "Uup" multidomain classification, the proteins of which are reported to be involved in translational regulation. Meanwhile, most of the sequences in Class 3 (bottom) are proteins predicted to be involved in efflux (MRP, CFTR, MDR MdlB, and ATM1 multidomain). Class 3 can be further divided into three subclasses based on the different characteristics of conserved residues (marked by shadow). Furthermore, we found that the NBDs were clustered at the same position: the first NBD clustered together and the second NBD were clustered together, which is marked by shadowing of the name.

Phylogenetic Analysis of the 44 ABC Proteins
The phylogenetic relationships of 44 ABCs were investigated in further detail ( Figure 2). Their distribution in the phylogenetic tree is closely correlated with the multidomain. The MRPs and CFTRs clustered in Clade I, the ATM1s and MDRs clustered in Clade II, and the Uups clustered in Clade VI. Except for ABC06, which was distributed in Clade I, other MdlBs were distributed in Clade II. However, the ABCs in "Protein White" do not have close and stable phylogenetic relationships with each other, and they were distributed in different clades. ABC30, ABC40, and ABC41 have closer evolutionary relationships with the proteins in Clades I and II, while ABC19 has a closer evolutionary relation with Uups (Clade VI). The ABC30 was the only protein in Clade III, and it does not have close phylogenetic relationships with the proteins in Clades I, II, and IV. In Clade IV, we found that two "Rim protein" and two "Protein White" members had close evolutionary relationships. The phylogenetic relationships of 44 ABCs were investigated in further detail ( Figure  2). Their distribution in the phylogenetic tree is closely correlated with the multidomain. The MRPs and CFTRs clustered in Clade I, the ATM1s and MDRs clustered in Clade II, and the Uups clustered in Clade VI. Except for ABC06, which was distributed in Clade I, other MdlBs were distributed in Clade II. However, the ABCs in "Protein White" do not have close and stable phylogenetic relationships with each other, and they were distributed in different clades. ABC30, ABC40, and ABC41 have closer evolutionary relationships with the proteins in Clades I and II, while ABC19 has a closer evolutionary relation with Uups (Clade VI). The ABC30 was the only protein in Clade III, and it does not have close phylogenetic relationships with the proteins in Clades I, II, and IV. In Clade IV, we found that two "Rim protein" and two "Protein White" members had close evolutionary relationships.

Prediction of the 3D Structures of the 44 ABC Transporters in T. asperellum CBS433.97
The 44 ABC transporters in T. asperellum were divided into six types based on the predicted 3D structures, shown in different colors ( Figure 3). The first four types (ABC-3D-1 to ABC-3D-4) are involved in transmembrane transport, and the other two (ABC-3D-5 and ABC-3D-6) are involved in translational regulation.

Prediction of the 3D Structures of the 44 ABC Transporters in T. asperellum CBS433.97
The 44 ABC transporters in T. asperellum were divided into six types based on the predicted 3D structures, shown in different colors ( Figure 3). The first four types (ABC-3D-1 to ABC-3D-4) are involved in transmembrane transport, and the other two (ABC-3D-5 and ABC-3D-6) are involved in translational regulation.
protein to form a functional transporter. The two proteins in the ABC-3D-3 class belong to the multidomain category "Rim protein". The structure of ABC-3D-3 consists of two NBD regulatory domains (RDs) and one TMD. The four protein ABC-3D-4 class (pink in Figure 3) belong to the multidomain "Protein White", in both TMDs and NBDs, and the TMDs all belong to the ABC2-membrane superfam ABC-3D-1 and ABC-3D-2 members account for 75% of all ABC transporters, in their importance in T. asperellum. Members of ABC-3D-5 (red in Figure 3) and AB (dark in Figure 3) belong to the multidomain "Uup", the proteins in ABC-3D-5 on NBDs while the proteins in ABC-3D-6 have TMDs, and the TMDs in ABC-3D-6 al to "ABC-tran-2" superfamily. to the ABC-membrane, ABC-ATPase, ABC2-membrane, ABC-tran-2, and acyl-transfer-3 s ilies, respectively). The second line below the structure shows the multidomain type. The below the structure shows the protein molecular weight (kDa). . The number at the top right corner shows the protein number. The first line below the structure shows the order of conserved domains (A-E represents domains belonging to the ABC-membrane, ABC-ATPase, ABC2-membrane, ABC-tran-2, and acyl-transfer-3 superfamilies, respectively). The second line below the structure shows the multidomain type. The third line below the structure shows the protein molecular weight (kDa).
Based on Table 2 and Figure 3, we found that the ABC-3D-1 transport protein class (green in Figure 3) includes 27 of the 44 ABCs, all of which share a similar structure based on two pseudo-symmetric halves formed from a single polypeptide, with each half formed from several transmembrane helices (TMD), the NBD, and the various interconnecting loops and short helices. Proteins in the ABC-3D-2 class (blue in Figure 3) correspond with the half structures in the ABC-3D-1 class and must dimerize with another protein to form a functional transporter. The two proteins in the ABC-3D-3 class (yellow) belong to the multidomain category "Rim protein". The structure of ABC-3D-3 proteins consists of two NBD regulatory domains (RDs) and one TMD. The four proteins in the ABC-3D-4 class (pink in Figure 3) belong to the multidomain "Protein White", including both TMDs and NBDs, and the TMDs all belong to the ABC2-membrane superfamily. The ABC-3D-1 and ABC-3D-2 members account for 75% of all ABC transporters, indicating their importance in T. asperellum. Members of ABC-3D-5 (red in Figure 3) and ABC-3D-6 (dark in Figure 3) belong to the multidomain "Uup", the proteins in ABC-3D-5 only have NBDs while the proteins in ABC-3D-6 have TMDs, and the TMDs in ABC-3D-6 all belong to "ABC-tran-2" superfamily. Table 2. Relation among structure, multidomain, and conserved domain arrangement order. The 1000 bp upstream sequence of each of the 44 ABC transporter genes was cloned. The motifs in the promoter regions were predicted and located using the SCPD program ( Figure 4). A total of 794 motifs of 39 different types were identified across the 44 promoters. In each promoter, motif numbers ranged from 9 to 32 and the type of motif ranged from 5 to 14. Of these, 541 motifs, including GCR1, GCN4, ADR1, STRE, HSTF, PHO4, GC/FAR, and ABF1, are closely related to stress responses. The three most abundant motifs are GCN4, GCR1, and ADR1, which are involved in metabolism and cell defenses, and 165, 93, and 67 motifs, respectively, were found for these. Furthermore, "ATM1" ABCs have an average of 21 motifs, which is the most, and "Rim protein" ABCs have an average of 13 motifs, which is the fewest. The motifs in "Uup" ABCs are the most diverse, with each "Uup" ABC having an average of 11 kind of motifs, while the "Rim protein" ABCs only have an average of 7 kinds of motifs (the fewest). Genes with the greatest number of motifs belonged to the "MRP" protein category, and the gene with the most motif types belonged to that of "Uup" protein ( Figure 4).

The Expression of 44 ABC Transporter Genes in T. asperellum ACCC30536 under Four Different Conditions
The four transcriptomes were constructed, and the data were deposited in the SRA database (https://www.ncbi.nlm.nih.gov/sra, accessed on 26 August 2021) under the accession number PRJNA758014. Next, we examined the expression of ABC transporter genes in these four conditions: MM, C-hungry, N-hungry, and PdPap media ( Figure 5). We found that ABC31 and ABC42 were the most highly expressed in all four conditions. ABC31 expression was highest (59214 RPKM) in PdPap and lowest (8537 RPKM) in MM, whereas ABC42 expression was highest (11790 RPKM) in N-hungry and lowest (3957 RPKM) in MM.

The Expression of 44 ABC Transporter Genes in T. asperellum ACCC30536 under Four Different Conditions
The four transcriptomes were constructed, and the data were deposited in the SRA database (https://www.ncbi.nlm.nih.gov/sra, accessed on 26 August 2021) under the accession number PRJNA758014. Next, we examined the expression of ABC transporter genes in these four conditions: MM, C-hungry, N-hungry, and PdPap media ( Figure 5). We found that ABC31 and ABC42 were the most highly expressed in all four conditions. ABC31 expression was highest (59214 RPKM) in PdPap and lowest (8537 RPKM) in MM, whereas ABC42 expression was highest (11790 RPKM) in N-hungry and lowest (3957 RPKM) in MM.  ABCs highly expressed in all four conditions are mainly involved in three functions; eleven in multiple compound efflux, belonging to 3a0123 (ABC37), ATM1 (ABC32 and ABC38), CFTR (ABC04 and ABC11), MdlB (ABC34), MDR (ABC21 and ABC23), and MRP (ABC01, ABC05, and ABC 15) multidomain classes; two in transport of sterols belonging to the "Protein White" multidomain class (ABC19 and ABC30); and four in regulating transla-tion belonging to the Uup multidomain class (ABC29, ABC31, ABC35, and ABC 42). These genes were thought to be involved in the stress response and should be further investigated.

Differential Expression of Six ABCs in Response to A. alternata CFCC82114 Toxins
To further investigate the function of ABCs under fungal phytopathogen A. alternata CFCC82114 toxin stress, RT-qPCR analysis was performed at six ABCs using total RNA extracted from T. asperellum ACCC30536 (Figure 6). The genes were chosen in combination with the comprehensive consideration of expression in transcriptome, functional prediction, and structure. In multidomain "Rim protein", "Protein White", and "Uup", we chose one gene per multidomain based on the relatively high transcription level in four transcriptomes; the proteins in other multidomains were identified and shared similar structures and predicted functions, so three encoded genes highly expressed in four transcriptomes were chosen. The six genes analyzed by RT-qPCR were ABC01 (MRP), ABC02 (Rim protein), ABC04 (CFTR), ABC05 (MRP), ABC30 (Protein White), and ABC31 (Uup). The results show that ABC01, ABC04, ABC05, and ABC31 were upregulated after induction by the toxin, with peak 2 3.13 -, 2 2.54 -, 2 4.92 -, and 2 5.09 -fold increases at 48, 6, 6, and 6 h, respectively ( Figure 6A,C,D,F). Even though ABC02 was obviously upregulated in the treatment group at 24 and 48 h, it was also obviously upregulated in the control group ( Figure 6B), so the induction of ABC02 by toxin is not evident. A peak 2 0.80 -fold decrease in the transcription level at 6 h and 2 0.29 -fold increased transcription level at 48 h. ABC30 was upregulated in the control group at 48 h but mostly downregulated in the treatment group ( Figure 6E), with peak 2 0.59 -fold increased and 2 1.83 -fold decreased transcription levels at 6 and 48 h, respectively. The difference between the control and treatment groups is significant (p < 0.05), so ABC30 was also obviously changed by the exposure to toxin. These results show that ABC01, ABC04, ABC05, ABC030, and ABC31 are genes potentially involved in responding to pathogens. (ABC01, ABC05, and ABC 15) multidomain classes; two in transport of sterols belonging to the "Protein White" multidomain class (ABC19 and ABC30); and four in regulating translation belonging to the Uup multidomain class (ABC29, ABC31, ABC35, and ABC 42). These genes were thought to be involved in the stress response and should be further investigated.

Differential Expression of Six ABCs in Response to A. alternata CFCC82114 Toxins
To further investigate the function of ABCs under fungal phytopathogen A. alternata CFCC82114 toxin stress, RT-qPCR analysis was performed at six ABCs using total RNA extracted from T. asperellum ACCC30536 (Figure 6). The genes were chosen in combination with the comprehensive consideration of expression in transcriptome, functional prediction, and structure. In multidomain "Rim protein", "Protein White", and "Uup", we chose one gene per multidomain based on the relatively high transcription level in four transcriptomes; the proteins in other multidomains were identified and shared similar structures and predicted functions, so three encoded genes highly expressed in four transcriptomes were chosen. The six genes analyzed by RT-qPCR were ABC01 (MRP), ABC02 (Rim protein), ABC04 (CFTR), ABC05 (MRP), ABC30 (Protein White), and ABC31 (Uup). The results show that ABC01, ABC04, ABC05, and ABC31 were upregulated after induction by the toxin, with peak 2 3.13 -, 2 2.54 -, 2 4.92 -, and 2 5.09 -fold increases at 48, 6, 6, and 6 h, respectively ( Figure 6A,C,D,F). Even though ABC02 was obviously upregulated in the treatment group at 24 and 48 h, it was also obviously upregulated in the control group ( Figure 6B), so the induction of ABC02 by toxin is not evident. A peak 2 0.80 -fold decrease in the transcription level at 6 h and 2 0.29 -fold increased transcription level at 48 h. ABC30 was upregulated in the control group at 48 h but mostly downregulated in the treatment group ( Figure 6E), with peak 2 0.59 -fold increased and 2 1.83 -fold decreased transcription levels at 6 and 48 h, respectively. The difference between the control and treatment groups is significant (p < 0.05), so ABC30 was also obviously changed by the exposure to toxin. These results show that ABC01, ABC04, ABC05, ABC030, and ABC31 are genes potentially involved in responding to pathogens.

Discussion
We found that the order of conserved domains in the ABC sequences is related to the multidomain, structure, and function (Table 2, Figure 3, Supplementary Table S1). Using this observation, we were able to rapidly predict other characteristics of the ABCs.
Multiple sequence alignment (Figure 1) revealed the sequence characteristics of 79 NBDs within the 44 ABC transporters in T. asperellum, which corresponds to the results for previous work on bacteria and yeast ABCs [22] or plant ABCs [22]. However, we identified mutations in some positions of all three motifs. In previous studies [9,22,23], the Walker A, ATP-binding, and Walker B motifs were "G-X(4)-G-K- " (Φ = any hydrophobic amino acid), respectively. These findings can help us better understand NBDs and achieve better characterization of these domains in the future. Interestingly, we found that the characteristics of sequences correspond with the protein multidomain and where the NBD is located in the protein chain. This suggests that the characteristics of ABC NBDs from different protein families or that their involvement in different functions varies. Meanwhile, there was clustering when the NBD was in the same location, suggesting the NBDs in different positions have different sequence characteristics. This caused us to hypothesize that NBDs in different positions have different functions, but this theory needs further investigation.
In the phylogenetic analysis (Figure 2), evolutionary relationships between 44 ABC proteins were investigated. These 44 ABC proteins were divided into six clades, and their distribution in the phylogenetic tree is closely related to their multidomain. ABCs in the same clade belong to similar multidomain types and are likely involved in similar functions. In contrast, proteins in different clades might perform different functions or play roles in different pathways. Furthermore, proteins belonging to the "CFTR" multidomain may be more closely related to those of the "MRP" multidomain, and they may share similar mechanisms and properties, while those in "MDR" multidomain class proteins appear to be more distinct. The proteins in "Protein White" were not strongly clustered in the same clade, suggesting that even though they are similar, they may have evolved from different ancestral protein types and may take part in different functions.
The 3D structures prediction showed that the 33 transporters in ABC-3D-1 and ABC-3D-2 represent 75% of all 44 ABCs. Proteins in ABC-3D-1 and ABC-3D-2 classes belong to different multidomains but share a similar structure. The similar structure also indicates a similar mechanism in transporting compounds, which has previously been investigated: the two half structures enclose a central pocket/cavity containing multiple discrete binding sites for ligands, drugs, and transporting substrates [24,25]. Interestingly, the structure of ABC25 is clearly different from that of ABC-3D-1, with both an inward-facing and outwardfacing TMD. Even though the multidomain, order of conserved domains, and predicted function are not different from other proteins in ABC-3D-1, it may have unique functions, and this requires further investigation. Two "Rim protein" members were found to have a structure (ABC-3D-3) similar to those reported to be involved in transporting sugars [26]. Four "Protein White" proteins were found to have a structure (ABC-3D-4) similar to those reported involved in transporting sterols [27]. The five ABCs in ABC-3D-5 and ABC-3D-6 all belong to Uup multidomain, each of which has more than one NBD, and these NBDs are separated by a 1-80 residue linker. Their other structural characteristics are similar to those described previously for "Uup" proteins [28]. The "Uup" proteins are reportedly involved in protein synthesis and regulating translation [29]. One mechanism to regulate translation is controlling the tRNA binding site or tRNA exit site of ribosomes [30]. This suggests that "Uup" ABC proteins may be involved in similar functions.
The promoter reflects what can activate the expression of a gene, which in turn helps us understand the function in which the gene may be involved. In this study, many of the motifs are involved in stress responses upstream of ABC transporter genes ( Figure 4). These motifs are closely related to metabolism, stress responses, and cellular defense, suggesting ABC transporter genes in T. asperellum may play a role in resistance and enhance survival in harsh environments. "ATM1", "Protein White", and "Uup" ABCs have relatively more motifs and may be more sensitive to environmental stimuli. In addition, numerous HSTF type motifs are located in the "Protein White" multidomain, and numerous ADR1 and GCR1 type motifs were found in genes belonging to the "MdlB" multidomain, further indicating that these genes are highly sensitive to changes in environmental conditions. In particular, GCR1, ADR1, and HSTF are closely related to G-proteins and their receptors, and since G-protein receptors act as sensors for oligopeptides, this suggests ABCs in "ATM1" and "Protein White" may be more sensitive when it comes to detecting phytopathogens based on the oligopeptides that they secrete [31]. This sensing likely stimulates the synthesis and secretion of secondary metabolites and the expression of disease-resistant genes in Trichoderma. Therefore, promoter analysis provides guidance on what role ABC transporter genes may play in the defense responses of T. asperellum.
The differential expression of 44 ABC transporter genes was measured through RT-qPCR in four different culture conditions: MM (control), C-hungry (simulating competing with other fungi), N-hungry (simulating interacting with other fungi), and PdPap (simulating interacting with plants) medium [31], showing that 13 of 39 predicted transport ABCs and 4 of 5 predicted translational regulation ABCs may be related to these stress responses ( Figure 5). A previous study showed that proteins belonging to the multidomain "Uup" regulate translation upon nutrient starvation and depletion [32]. Our results are consistent with this observation; 4 of 5 Uup proteins were significantly upregulated under starvation conditions. Interestingly, of the highly expressed proteins, eleven are considered to be involved in multiple compound transport and belong to ABC-3D-1 and ABC-3D-2 subclasses, suggesting ABC genes in these two subclasses may be particularly important when dealing with pathogens and plants. We preliminarily and roughly filtered potential ABCs related to biocontrol through three simulated conditions, and these potential candidates should be further investigated in detail.
Leaf blight caused by A. alternata is a serious leaf disease in many plant species and needs to be well controlled. Trichoderma species are necrotrophic or destructive mycoparasites that can kill A. alternata and have been already used as useful biocontrol agents [32]. For further investigation, six genes were subjected to RT-qPCR analysis under phytopathogenic toxin stress of A. alternata ( Figure 6). Genes ABC01 (encoded a MRP protein, MRP), ABC05 (MRP), and ABC31 (Uup) were upregulated in the treatment group, while they were all downregulated in the control group, which suggests that they participate in detoxification and can help T. asperellum ACCC30536 survive in an environment full of other pathogens. ABC04 (CFTR) was upregulated in the treatment group, but also in the control group, which suggests that it may be involved in functions other than detoxification. During structure prediction, we found that ABC31 only has NBDs without transmembrane, and in a previous study, it was shown that the protein only has NBDs and can increase the resistance of Streptomyces antibioticus to oleandomycin [33]. Thus, we hypothesize that the highly expressed gene ABC31 may be very important in toxin resistance. In Escherichia coli, Uup protein can bind DNA [34] and may be involved in the excision of transposons [35]. The expression of ABC31 (Uup) was downregulated in the control, suggesting it is suppressed in general. When Trichoderma encountered toxins, however, ABC31 was rapidly induced, with the highest expression value at 6 h, so we hypothesize that it is involved in responding to stress as an early key regulator. The expression of ABC02 (Rim protein) is not obviously changed under toxin stress, but it was also highly expressed in the control group, as seen in the combined transcriptome data, which may indicate that in Rim protein genes are involved not in fungal phytopathogen toxin stress responses but are involved in some regular functions. The mechanism of ABCs in exporting substrates was due to ABCs functioning independently or with the help of a cofactor (such as membrane fused protein, outer-membrane factor, and inner-membrane factor, etc.) in exporting substrates. When the multiple discrete binding sites in the central pocket/cavity formed by two TMDs recognize their substrate, NBDs hydrolyze ATP, produce energy, and change conformation, leading to a change in the conformation of TMDs, which ultimately leads to the substrate being transferred to the extracellular milieu [24,25,36,37]. In this study, how the upregulated genes participate in detoxification, what exact components in the fermentation liquid of A. alternata are exported by the ABCs, and how these components bind to the ABCs should be further investigated. The findings indicate reference genes for genetic engineering, with which we can construct transgenic strains for further analysis and expect to obtain a strain with good biocontrol ability that cannot easily suppressed by toxins.
In conclusion, conserved domain prediction showed that ABC proteins in T. asperellum can be divided into nine groups based on their multidomains: MRP, CFTR, MdlB, ATM1, MDR, Protein White, Rim protein, 3a0123, and Uup. The NBD sequence characteristics, phylogenetic relationship, and structures are correlated with the multidomain. Numerous motifs related to stress responses were also found in their promoter regions. In addition, transcriptome analysis showed that when T. asperellum ACCC30536 was under pathogenic and botanic stress, 17 of 39 predicted transport ABCs and 4 of 5 predicted translational regulation ABCs had higher expression. In further RT-qPCR analysis, ABC01, ABC04, ABC05, ABC30, and ABC31 were found to be significantly altered. They are involved in the response to pathogenic A. alternata toxin stresses and, hence, are worthy of further study. In this study, the properties of ABC proteins in T. asperellum were characterized, which lays the foundation for understanding how they help T. asperellum survive under stress, and we identified potential ABC proteins that may be involved in toxin stress responses, which is also important for revealing the biocontrol mechanism of Trichoderma.

Disclosure
All experiments undertaken in this study comply with the current laws of the country in which they were performed.

Conflicts of Interest:
The authors declare no conflict of interest.