Identification of CmbHLH Transcription Factor Family and Excavation of CmbHLHs Resistant to Necrotrophic Fungus Alternaria in Chrysanthemum

Chrysanthemum morifolium Ramat. ‘Huaihuang’ is a traditional Chinese medicinal plant. However, a black spot disease caused by Alternaria sp., a typical necrotrophic fungus, has a serious damaging influence on the field growth, yield, and quality of the plant. ‘Huaiju 2#’ being bred from ‘Huaihuang’, shows resistance to Alternaria sp. bHLH transcription factor has been widely studied because of their functions in growth development, signal transduction, and abiotic stress. However, the function of bHLH in biotic stress has rarely been studied. To characterize the resistance genes, the CmbHLH family was surveyed in ‘Huaiju 2#’. On the basis of the transcriptome database of ‘Huaiju 2#’ after Alternaria sp. inoculation, with the aid of the Chrysanthemum genome database, 71 CmbHLH genes were identified and divided into 17 subfamilies. Most (64.8%) of the CmbHLH proteins were rich in negatively charged amino acids. CmbHLH proteins are generally hydrophilic proteins with a high aliphatic amino acid content. Among the 71 CmbHLH proteins, five CmbHLHs were significantly upregulated by Alternaria sp. infection, and the expression of CmbHLH18 was the most significant. Furthermore, heterologous overexpression of CmbHLH18 could improve the resistance of Arabidopsis thaliana to necrotrophic fungus Alternaria brassicicola by enhancing callose deposition, preventing spores from entering leaves, reducing ROS accumulation, increasing the activities of antioxidant enzymes and defense enzymes, and promoting their gene expression levels. These results indicate that the five CmbHLHs, especially CmbHLH18, may be considered candidate genes for resistance to necrotrophic fungus. These findings not only increase our understanding of the role CmbHLHs play in biotic stress but also provide a basis by using CmbHLHs to breed a new variety of Chrysanthemum with high resistance to necrotrophic fungus.


Introduction
Chrysanthemum morifolium Ramat., a perennial herb with a wide variety of flower types, has been widely used in cut flowers, potting, and garden greening [1]. C. morifolium 'Huaijuhua' produced mainly in Jiaozuo city of Henan province, China, is one of the Four Famous Huai Herbals; it has been cultivated for more than three thousand years in China. Its annual planting area is approximately 800 hm 2 with an output value of one hundred and ninety million Yuan [2]. 'Huaijuhua' is rich in three kinds of medicinal active ingredients, namely luteoloside, chlorogenic acids, and 3, 5-dicaffeoyl-quinic acid [3]. As a traditional medicinal Chinese Chrysanthemum, besides its medicinal value, it can also be as pathogenic strains to inoculate the plants. The plants and fungi are maintained for research purposes at the Engineering Technology Research Center of Nursing and Utilization of Genuine Chinese Crude Drugs in Henan Province, Henan Normal University, Xinxiang, China.

Screening and Physicochemical Properties Analysis of the CmbHLH TF Family
Sequences annotated as the bHLH in the 'Huaiju 2 # ' transcriptome database (Accession number: PRJNA448499) were aligned to the Chrysanthemum genome database using BLAST on the http://www.amwayabrc.com/zh-cn/index.html (accessed on 22 January 2022). The retrieved candidate genes were checked through the CD-HIT website server (http://weizhongli-lab.org/cd-hit/, accessed on 9 November 2021) to delete redundant sequences [29]. NCBI CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 10 November 2021) and SMART (http://smart.embl-heidelberg, accessed on 11 November 2021) were used to remove sequences without conserved bHLH domains, incomplete CDS sequences were also removed, and sequences with typical bHLH domains were selected as putative bHLH TF family members [30,31].
Based on the identified amino acid sequence of the CmbHLH protein, the ExPASY online program (https://www.expasy.org/, accessed on 15 November 2021) was used to predict the physicochemical properties of the CmbHLH TFs [32].

Multiple Sequence Alignment, Phylogenetic, and Conserved Motifs Analysis of CmbHLH Family Proteins
To analyze the phylogenetic relationship of CmbHLH proteins, the conserved motifs of the CmbHLH proteins were searched and analyzed using the MEME online tool (http: //meme.nbcr.net/meme/tools/meme, accessed on 15 March 2022) (search criteria: motif length was set to 10-100, the maximum number of retrieved motifs was set to 40, and the default parameters were used for the rest) [33].
Using the NJ method, a neighbor-joining phylogenetic tree was constructed based on the alignment of the identified CmbHLHs with AtbHLHs from Arabidopsis using MEGA 7.0 with 1000 bootstraps (the others with default parameters) [34]. Protein sequences of 156 AtbHLH family members were downloaded from TAIR (https://www.Arabidopsis.org/ index.jsp, accessed on 12 November 2021) [35]. A phylogenetic tree was drawn using iTOL (http://itol.embl.de/, accessed on 9 November 2021) to group the CmbHLH proteins [36].

Excavation of Candidate CmbHLHs Resistant to Necrotrophic Fungi and Their Cis-Element Analysis
Based on the selection criteria for differential genes [37], the upregulated expression of CmbHLHs in response to Alternaria sp. innoculation were taken as potential candidate CmbHLHs resistant to necrotrophic fungi. Putative promoter sequences (2000 bp upstream of ATG) of the candidate genes were retrieved from the Chrysanthemum genome database (http://www.amwayabrc.com/zh-cn/index.html, accessed on 9 May 2021) (Table S1). cis-acting elements in the promoter sequences were predicted using the online software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 10 May 2021) [38]. The results were visualized and mapped using TBtools software v0.6673 [39].

Isolation and Arabidopsis Transformation of CmbHLH18
The total RNAs of 'Huaiju 2 # ' leaves were extracted using an RNAiso Plus kit (TaKaRa, Beijing, China) and purified using an ND-ONE-W spectrophotometer (Thermo, Waltham, MA, USA) according to the manufacturer's protocol. Reverse transcription into cDNA was performed using a HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) in a 20 µL reaction. cDNA was synthesized using specific primers (Table S6) with 500 ng of total RNA as the template.
The cDNA was used as a template and primer pair (CmbHLH18-F and CmbHLH18-R) (Table S6) were designed to amplify the coding area of CmbHLH18. The PCR program was The coding DNA sequence of CmbHLH18 (GenBank accession number: OP 313866) was constructed into the Super1300-35S vector [3]. The recombinant vector was introduced into Agrobacterium strains and transformed into Arabidopsis plants to produce plants overexpressing CmbHLH18 using the floral dip method [40]. After three generations of Hygromycin B resistance screening, a small amount of Arabidopsis leaf genomic DNA was extracted for PCR amplification using the cetyltrimethylammonium bromide (CTAB) method, and a PCR reaction system (50 µL) was prepared consisting of 100 ng of template DNA, 2 µL of primer super1300-F, 2 µL of primer CmbHLH18-V-R, 25 µL of 2× Pfu Mas-terMix (Dye) (Vazyme Biotech, Nanjing, China), and 19 µL of ddH 2 O. The PCR reaction program consisted of the following steps: 94 • C for 5 min, 35 cycles of 94 • C for 30 s, 56 • C for 30 s, 72 • C for 60 s, and finally, 72 • C for 10 min. Electrophoresis using 1% agarose was performed to separate the PCR products, and a gel imaging system was used to detect and identify transgenic Arabidopsis plants. This was also identified by RT-qPCR with the primer pair of CmbHLH18-At-F and CmbHLH18-At-R (Table S6), following Section 2.9 in this part.

Inoculation of Necrotrophic Fungi and Sampling
According to the method of Zhao et al. [3], Alternaria sp. and A. brassicicola were activated. When plants of 'Huaiju 2 # ' grew to 8-10 leaves, the middle and upper leaves were punctured with a needle (approximately 0.41 mm diameter) and inoculated with spores of Alternaria sp. [3]. When Arabidopsis plants grew to 4 weeks, the spore solution of A. brassicicola was sprayed on leaves until droplet runoff occurred [41]. The two spores were suspended and diluted to a concentration of 10 7 spores per milliliter in sterile distilled water (SDW) using the hemocytometer technique. After inoculation, plants were kept in a dark incubation chamber for 48 h at 25 • C with 100 % relative humidity (RH). Then, the plants were grown in the greenhouse with 40-60% RH, 20-25 • C, 60 mE·s −1 m −2 light intensity, and 14 h light/10 h dark light cycle for 'Huaiju 2 # ' [3], 10 h light/14 h dark light cycle for Arabidopsis.
Leaves of 'Huaiju 2 # ' were collected as samples at 0 d (before inoculation) and 1, 3, and 5 days post inoculation (dpi) with Alternaria sp. for expression profile analysis of candidate CmbHLHs. Leaves of the transgenic and WT Arabidopsis lines were collected at 0 d (before inoculation) and 1 dpi, 3 dpi, and 5 dpi with A. brassicicola for gene expression analysis and activity determination of antioxidant enzymes and defense enzymes.

Histochemical Staining and Microscopic Analysis after Inoculation with A. brassicicola in Arabidopsis and CmbHLH18 Transgenic Arabidopsis Lines
Evans Blue [43] was used to observe necrotic cells at 0, 5 dpi and fungal growth at 3, 5 dpi. Aniline blue staining [44] was used to observe callose deposition at 0 and 0.5 dpi. At 0 and 0.5 dpi, nitro blue tetrazolium (NBT) and diaminobenzidine (DAB) staining were used to detect the superoxide anion (O 2 − ) and hydrogen peroxide (H 2 O 2 ) of the leaves, respectively [45].

Determination of Enzyme Activity and Gene Expression Analysis of Arabidopsis Lines
Following the literature, we quantified the activities of antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), and defense enzymes, including phenylalanine ammonia lyase (PAL), peroxidase (POD), chitinase (CHT) and β-1,3-glucanase (GLU) in leaves of transgenic and WT Arabidopsis plants were measured following Liu et al. [46].
The method of obtaining cDNA is the same as 2.5; cDNA was used as a sample for the RT-qPCR of genes. RT-qPCR was done using an AceQ RT-qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). CiUBI (Cse2.0_LG8) was used as an internal reference for the expression level of CmbHLH18 in various tissues of Chrysanthemum. AtActin (823805) was selected as an internal reference for the expression level of genes post-inoculation in Arabidopsis. Specific primers were designed using primer 5.0 software [3] (Table S6). The expression levels in RT-qPCR were calculated using the 2 − CT method [47].

Statistical Analysis
A one-way ANOVA and Student's t-test were performed using SPSS 18.0 software (IBM, Armonk, NY, USA). One-way ANOVA tests were performed to test the DSI of the Arabidopsis lines after inoculation of A. brassicicola. Student's t-tests were used to test the gene expression level analysis and activities of antioxidant and defense enzymes after Alternaria sp. inoculation.

Hydrophilicity and High Aliphatic Amino Acid Content in 71 CmbHLH TFs Identified in Chrysanthemum
Our sequence analysis identified 71 proteins with a typical bHLH domain ( Figure S1) and designated as CmbHLH1, 2, 3 . . . 71, and their physical and chemical properties are summarized in Table 1. For these 71 CmbHLH proteins, the predicted polypeptide sequence lengths ranged from 158 to 937; the predicted molecular weights ranged from 17.67-103.49 kDa, and their theoretical isoelectric points (pIs) ranged between 4.75-9.2. Among them, the pIs of 15 proteins were greater than 7.5, while the pIs of 46 proteins were lower than 6.5. Most (64.8%) of these CmbHLH proteins were rich in negatively charged amino acids. The results of the protein hydropathicity analysis showed that the grand average of hydropathicity (GRAVY) of all 71 sequences was lower than 0, which indicated that the bHLH proteins in Chrysanthemum are generally hydrophilic proteins. The greater II (the instability index) value, the more unstable the protein. Therefore, most of the CmbHLH proteins were apparently unstable; the least stable of them was CmbHLH3 (CHR00034167-RA). Furthermore, these CmbHLHs had relatively high aliphatic amino acid (AI) contents, with CmbHLH22 (CHR00078227-RA) containing the highest number of AIs. Taken together, these analyses showed that Chrysanthemum harbors a wide array of bHLH TFs with hydrophilicity and high aliphatic amino acid content.

Phylogenetic Differences between Groups of bHLHs from Chrysanthemum and Arabidopsis
Phylogenetic analysis of the DNA-binding domain of the 71 CmbHLHs as well as 156 bHLHs from Arabidopsis suggested that there were 17 groups, none of which belonged to clade Ib, Va, VIIa (1), VII(a+b), VIIIc, XIV, and XV in Chrysanthemum ( Figure 1, Table S2). Five bHLHs were unique to Chrysanthemum, resulting in their classification as "Orphans". These results showed that the CmbHLH TF family probably expanded and contracted during the evolutionary process in Chrysanthemum. Motif analysis of the CmbHLH proteins showed that 40 putative motifs among the 71 CmbHLH proteins were identified ( Figure S2). Members of the same subfamily of CmbHLH had similar conservative motifs and numbers, and their location distribution was relatively conservative. This may help to analyze the phylogenetic relationship between bHLH TFs.

Domains of CmbHLHs Identified by Variation-Conserved Residues
To better understand the function of the 71 CmbHLH TFs identified in Chrysanthemum, their domains and DNA-binding capacities were analyzed. There were 24 amino acid residues that were highly conserved in >50% of the candidate proteins (Figure 2), and the R13, L23, L42, and L52 residues were conserved in >80% of the proteins (Table S3). The results of the domain analysis suggested that these conserved sites were likely involved in bHLH function. Further analysis showed that the basic regions of CmbHLHs were composed of 10-13 amino acids and contained a typical H5-E9-R13 sequence motif (His5-Glu9-Arg13) for binding DNA, which is necessary for bHLH to function as a transcription factor [48]. In particular, E9 and R13 residues have a function in the recognition and binding of E-box motifs in the promoter regions of target genes.

Excavation of Candidate CmbHLHs' Resistance to Necrotrophic Fungus Alternaria
Five CmbHLHs were highly upregulated in the transcriptome (Accession number: PRJNA448499) when plants were inoculated with Alternaria sp. and were potentially involved in the resistance to necrotrophic fungi. The five candidate genes were named CmbHLH16, CmbHLH18, CmbHLH28, CmbHLH30, and CmbHLH60 (Table S4). CmbHLH16, CmbHLH28, and CmbHLH30 belong to the IVd subfamily, CmbHLH18 belongs to the IVa subfamily, and CmbHLH60 belongs to the IX subfamily. They contain a total of 15 motifs ( Figure S2).

Analysis of Promoters of the Five Candidate CmbHLHs
To comprehend the transcriptional regulating function of the 71 CmbHLH TFs identified in Chrysanthemum, taken the five UP CmbHLHs as representative, sequence analysis identified 2000 bp fragments upstream of these five genes with their respective sequences in the Chrysanthemum genome database (http://www.amwayabrc.com/index.html, accessed on 9 May 2021). Then, a promoter analysis was conducted to identify the potential function of cis-acting elements within these regions ( Figure S3; Table 2) and found that three major types of cis-elements were present in the promoters of the UP CmbHLHs. The first type is responsive to plant hormones and includes ABRE, AuxRR, CGTCA/TGACG, P-box, and TCA elements, which are sensitive to ABA, IAA, JA, GA, and SA, respectively. The second type is responsive to defense and stress, including TC-rich repeats (defense and stress responsiveness, which are only in the promoter of CmbHLH18), MBS (drought stress), LTR (low-temperature stress), and ARE (anaerobic stress) elements. The third type participates in plant growth and development, such as that of the GCN4_Motif in endosperm expression. Our finding of hormone-responsive and defense-responsive promoter elements suggested that the CmbHLHs may have been upregulated specifically through these elements during Alternaria sp. infection and prompted us to investigate whether these elements could be related to an enhanced disease resistance phenotype.

Orphan
VIIa (2) Orphan VIIIa VII(a+b) Va XV III(a+c)   In order to better understand whether and how these promoter elements may contribute to transcriptional regulation of these five UP CmbHLHs to mediate a disease-resistant phenotype, we next inoculated Chrysanthemum with Alternaria sp. Then, their expression levels were examined (Figure 3). The results showed that these CmbHLHs were significantly induced by inoculation with Alternaria sp. compared with Mock. More specifically, CmbHLH18 expression peaked at 1 dpi and was 9.12 times higher than that of the Mock, whereas the expression of CmbHLH16, CmbHLH30, CmbHLH28, and CmbHLH60 reached their highest levels at 3 dpi (reaching 6.72-, 6.58-, 5.61-, and 3.93-fold, respectively, that of the Mock).  These results indicated that these five UP CmbHLHs might play a positive regulatory role in response to Alternaria sp. In particular, the expression of CmbHLH18 was strongly upregulated by Alternaria sp. (the necrotrophic fungus) infection. Therefore, CmbHLH18 is mostly expected to become a candidate gene resistance against the necrotrophic fungus Alternaria in plants.

CmbHLH18 Enhances Resistance against Necrotrophic Fungus Alternaria in Arabidopsis
In order to further verify the function of CmbHLH18 resistance against the necrotrophic fungus in plants, we isolated CmbHLH18 (GenBank accession number: OP 313866) and transformed it into Arabidopsis (Col). Transgenic plants were identified using PCR and RT-qPCR (Figure 4a,b). Transgenic plants show PCR products of 516 bp, while normal plants contain the amplified region with 0 bp in size (Figure 4a). There were no significant differences in morphology between the WT and transgenic plants before A. brassicicola inoculation. However, at 5 dpi, DSI was significantly higher in the WT than in transgenic plants (Figure 4c,d). This is reflected in cell activities being higher in transgenic plants than the WT (Figure 5a, Table S5). These results indicated that these five UP CmbHLHs might play a positive regulatory role in response to Alternaria sp. In particular, the expression of CmbHLH18 was strongly upregulated by Alternaria sp. (the necrotrophic fungus) infection. Therefore, CmbHLH18 is mostly expected to become a candidate gene resistance against the necrotrophic fungus Alternaria in plants.

CmbHLH18 Enhances Resistance against Necrotrophic Fungus Alternaria in Arabidopsis
In order to further verify the function of CmbHLH18 resistance against the necrotrophic fungus in plants, we isolated CmbHLH18 (GenBank accession number: OP 313866) and transformed it into Arabidopsis (Col). Transgenic plants were identified using PCR and RT-qPCR (Figure 4a,b). Transgenic plants show PCR products of 516 bp, while normal plants contain the amplified region with 0 bp in size (Figure 4a). There were no significant differences in morphology between the WT and transgenic plants before A. brassicicola inoculation. However, at 5 dpi, DSI was significantly higher in the WT than in transgenic plants (Figure 4c,d). This is reflected in cell activities being higher in transgenic plants than the WT (Figure 5a, Table S5).  The speed of spore growth of inoculated leaves at 3 dpi and 5 dpi was significantly slower in transgenic plants than in WT plants (Figure 5b). The leaves of transgenic plants showed more callose deposition compared with WT plants at 0.5 dpi with A. brassicicola (Figure 5c).
To further analyze the effect of CmbHLH18 on ROS accumulation in transformed Arabidopsis leaves after A. brassicicola inoculation, NBT staining and DAB staining were conducted. Transgenic plants of Arabidopsis after A. brassicicola inoculation exhibited a lower accumulation of ROS than the wild type plants at 0.5 dpi (Figure 6), as indicated by the NBT staining and DAB staining. The speed of spore growth of inoculated leaves at 3 dpi and 5 dpi was significantly slower in transgenic plants than in WT plants (Figure 5b). The leaves of transgenic plants  To further analyze the effect of CmbHLH18 on ROS accumulation in transformed Arabidopsis leaves after A. brassicicola inoculation, NBT staining and DAB staining were conducted. Transgenic plants of Arabidopsis after A. brassicicola inoculation exhibited a lower accumulation of ROS than the wild type plants at 0.5 dpi (Figure 6), as indicated by the NBT staining and DAB staining. The profiling of gene expression and activities of enzymes involved in ROS scavenging and plant defense suggested that after inoculation, there was a significant increase in ROS scavenging and defense abilities in transgenic plants (Figures 7 and 8). This indicated that CmbHLH18 enhanced resistance to A. brassicicola by increasing the ability of ROS scavenging and the activity of defense enzymes in Arabidopsis. The profiling of gene expression and activities of enzymes involved in ROS scavenging and plant defense suggested that after inoculation, there was a significant increase in ROS scavenging and defense abilities in transgenic plants (Figures 7 and 8). This indicated that CmbHLH18 enhanced resistance to A. brassicicola by increasing the ability of ROS scavenging and the activity of defense enzymes in Arabidopsis. (c-f) Expression levels of defense enzyme genes. Asterisks represent statistically significant differences between different lines by t-test (n = 3; *, p < 0.05; **, p < 0.01).

Discussion
The bHLH TF family exists widely in plants and animals and is one of the largest transcription factor families in plants [26,27]. This study reports the gene family for the first findings in Chrysanthemum. Our sequence analysis of genes identified 71 bHLHs of 17 subfamilies in Chrysanthemum, which is significantly smaller than that reported in Arabidopsis (162 bHLH TFs into 21 subfamilies, [7]), white poplar (202 bHLH TFs into 25 subfamilies, [17]), and rice (167 bHLH TFs into 22 subfamilies, [49]). Therefore, gene duplications occur more in some species than in others, such as Chrysanthemum, sacred lotus [50], and watermelon [51].
In general, genes with the same or similar structures are clustered in the same subfamily and may have a similar function [52]. For example, TT8 of the bHLH IIIf subfamily in Arabidopsis and TaPpb1 of the bHLH IIIf subfamilies in Triticum aestivum L. can both (c-f) Activities of defense enzymes. Asterisks represent statistically significant differences between different lines by t-test (n = 3; *, p < 0.05; **, p < 0.01).

Discussion
The bHLH TF family exists widely in plants and animals and is one of the largest transcription factor families in plants [26,27]. This study reports the gene family for the first findings in Chrysanthemum. Our sequence analysis of genes identified 71 bHLHs of 17 subfamilies in Chrysanthemum, which is significantly smaller than that reported in Arabidopsis (162 bHLH TFs into 21 subfamilies, [7]), white poplar (202 bHLH TFs into 25 subfamilies, [17]), and rice (167 bHLH TFs into 22 subfamilies, [49]). Therefore, gene duplications occur more in some species than in others, such as Chrysanthemum, sacred lotus [50], and watermelon [51].
In general, genes with the same or similar structures are clustered in the same subfamily and may have a similar function [52]. For example, TT8 of the bHLH IIIf subfamily in Arabidopsis and TaPpb1 of the bHLH IIIf subfamilies in Triticum aestivum L. can both regulate anthocyanin synthesis [19,53]. AtbHLH3 of the bHLH IIId subfamily in Arabidopsis and GmbHLH3 of the bHLH IIId subfamily in Glycine max (Linn.) Merr. can both regulate jasmonate-induced leaf senescence [54,55]. Therefore, cluster and comparative analysis of the phylogenetic tree classification results between CmbHLH proteins and the bHLH of other species with known functions can be helpful to predict the functions of some genes in Chrysanthemum (Figure 1). In other cases, there are some differences in function, even if these genes are clustered into the same subfamily. For example, NFL of the bHLH IIIb subfamily in Arabidopsis can promote flowering, but OsbHLH61 of the bHLH IIIb subfamily in Oryza sativa L. can increase resistance to the brown planthopper [56,57]. The GL3 of the bHLH IIIf subfamily in Arabidopsis can promote root epidermal development, which differs from the function of TaPpb1 in the same subfamily [19,58]. AtbHLH18 (belonging to IVa) achieved iron homeostasis by promoting JA-induced degradation of the Ferritin (FIT) protein and inhibiting iron absorption in Arabidopsis [59]. There may be a great difference in function between CmbHLH18 and AtbHLH18 in function.
The Weblogo diagram obtained by multi-sequence alignment of the CmbHLH domain shows that the bHLH domain is conserved in all CmbHLHs, which is similar to the Arabidopsis bHLH domain (Figure 2). In the CmbHLH domain, two amino acid residues, Leu-23 and Leu-52, are relatively conservative, accounting for 83% and 92%, respectively. The two residues are necessary for the formation of a homologous or heterodimer [9]. This indicates that, on the one hand, CmbHLH proteins probably form homodimers by itself, and on the other hand, CmbHLH proteins probably also interact with other TFs, such as R2R3-MYB, to form heterodimers [9]. Understanding its domains will deepen our understanding of the function.
bHLH TF family has a variety of biological functions [26][27][28]. However, there are still many unknowns about the function of bHLH TFs in biotic stress. The comparison of the cis-acting elements on the promoters of the five CmbHLHs showed that there are cis-elements involving plant hormone responsiveness, stress responsiveness, and plant growth and development. SA plays a crucial role in plant defense and is generally involved in the activation of defense responses against biotrophic and hemi-biotrophic pathogens, as well as the establishment of systemic acquired resistance [60]. By contrast, JA is usually associated with a defense against necrotrophic pathogens and herbivorous insects [61]. Interestingly, the TC-rich repeats were unique to the promoter of CmbHLH18, which was located at 515 bp upstream of ATG and was involved in defense and stress responsiveness ( Figure S3; Table 2). This may be related to the fact that CmbHLH18 can significantly respond to the necrotrophic fungus Alternaria sp. infection in Chrysanthemum. Surprisingly, CmbHLH18 may also enhance the resistance of Arabidopsis to A. brassicicola (Figure 4c), which is another typical necrotrophic fungus of Alternaria.
Callose is generally believed to positively regulate the stress response of plants [62][63][64][65]. Under abiotic and biotic stress, plants often trigger the accumulation of callose [64]. Callose deposition in the cell wall, plasmodesmata, and sieve pores controls the cell wall permeability, prevents further penetration of the pathogen into the tissue, and reduces the loss of cellular water and solute to maintain the internal stabilities of cells [66]. In this study, leaves of CmbHLH18-transformed Arabidopsis had more callose than WT after A. brassicicola inoculation, indicating that CmbHLH18 could enhance the defense of Arabidopsis against A. brassicicola by inducing callose deposition.
When plants are subjected to stress (including biotic and abiotic stress), a large amount of ROS (O 2 − , H 2 O 2 , etc.) is generated in the cells. If ROS is not cleared in time, it damages plant tissues. CmbHLH18-transformed Arabidopsis plants reduce the content of O 2 − and H 2 O 2 in response to fungal attacks. This is consistent with Yao et al. [67], who showed that overexpression of FtbHLH2 reduced the amount of ROS and increased the endurance of cold stress in Arabidopsis. In this study, the heterologous overexpression of CmbHLH18 enhanced resistance to A. brassicicola in Arabidopsis by decreasing the content of O 2 − and H 2 O 2 ( Figure 6).
Plants can scavenge ROS and be protected from oxidative stress damage through an antioxidant protecting system involving SOD and CAT [68]. Heterologous overexpression of VvbHLH1 in Arabidopsis [69] and NtbHLH123 in tobacco [16] both improved the antioxidant enzyme system and increased the antioxidant capacity of transgenic plants. Our results showed a similar pattern, suggesting that CmbHLH18 could decrease the amount of ROS, reduce plant damage, and enhance resistance to A. brassicicola by activating the expression of antioxidant genes and increasing the activities of antioxidant enzymes.
Plants can resist pathogen invasion through lignification. PAL and POD are key enzymes in the lignin synthesis pathway [70]. CHT and GLU are pathogenesis-related (PR) enzymes that can destroy fungal cell walls and prevent the invasion of pathogenic fungus [71]. Studies have shown that the expression levels of PAL, POD, CHT, and GLU are significantly increased by Magnaporthe grisea in rice [72]. In this study, after inoculation with A. brassicicola, the activities of PAL, POD, CHT, and GLU in transgenic leaves were significantly higher than those in WT leaves (Figure 8), and the corresponding gene expression levels in transgenic leaves were also higher than those in WT leaves (Figure 7).

Conclusions
We first identified 71 CmbHLHs genes of 17 subfamilies, and the genes in 'Huaiju 2 # ' are generally hydrophilic proteins and high aliphatic amino acid content. Additionally, combining the results of RT-qPCR analysis after Alternaria sp. inoculation, we found that CmbHLH16, 18, 28, 30, and 60 may be involved in resistance to biotic necrotrophic fungus. They all have hormone-responsive elements that are associated with enhanced disease resistance in their cis-acting elements, and CmbHLH18 may be a gene resistant to necrotrophic fungus in Chrysanthemum.
Heterologous overexpression of CmbHLH18 can enhance the resistance to necrotrophic fungus in Arabidopsis by a series of physiological and genetic activities, inducing callose deposition and increasing the activities and gene expression levels of antioxidant and defense enzymes.
The identification of CmbHLHs in this study will help to further identify candidate genes for resistance to necrotrophic fungus, elucidate the molecular mechanism of resistance to necrotrophic fungus in Chrysanthemum, and use CmbHLHs to breed a new variety of Chrysanthemum with high resistance to necrotrophic fungus.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/genes14020275/s1, Figure S1: Multiple sequence alignment of CmbHLH family proteins. The four-color modules at the top of the figure are represented, respectively: red represents the N-terminal basic region of bHLH, yellow, and green represent two amphiphilic regions, and blue represents a variable length loop. Process the data into pictures using Clustal W. Figure S2: Motif composition of the CmbHLH TF family. (a) Phylogenetic tree of CmbHLHs; (b) conserved motifs of the CmbHLHs; (c) sequence composition of each motif in CmbHLHs. Figure S3: Visualization of cis-elements in the promoter sequences of CmbHLHs. Different colors represent different cis-acting elements in the promoter sequences of CmbHLHs. Table S1: Promoter sequences of candidate resistant genes to necrotrophic fungus. Table S2: Number of bHLH TF in each subfamily of Chrysanthemum and Arabidopsis. Table S3: Proportion of conserved amino acids at each point of CmbHLH protein. Table S4: Selection criteria for differential genes. Table S5: Relative activity of leaf cells of WT and transgenic Chrysanthemum. Table S6: Primers used in this study.