Characterization of the Chitinase Gene Family in Mulberry (Morus notabilis) and MnChi18 Involved in Resistance to Botrytis cinerea

Chitinase is a hydrolase that uses chitin as a substrate. It plays an important role in plant resistance to fungal pathogens by degrading chitin. Here, we conducted bioinformatics analysis and transcriptome data analysis of the mulberry (Morus notabilis) chitinase gene family to determine its role in the resistance to Botrytis cinerea. A total of 26 chitinase genes were identified, belonging to the GH18 and GH19 families. Among them, six chitinase genes were differentially expressed under the infection of B. cinerea. MnChi18, which significantly responded to B. cinerea, was heterologously expressed in Arabidopsis (Arabidopsis thaliana). The resistance of MnChi18 transgenic Arabidopsis to B. cinerea was significantly enhanced, and after inoculation with B. cinerea, the activity of catalase (CAT) increased and the content of malondialdehyde (MDA) decreased. This shows that overexpression of MnChi18 can protect cells from damage. In addition, our study also indicated that MnChi18 may be involved in B. cinerea resistance through other resistance-related genes. This study provides an important basis for further understanding the function of mulberry chitinase.


Introduction
Plants have several defense mechanisms to resist the invasion of pathogens, including pathogenesis-related (PR) proteins. Chitin is an insoluble polymer, β-1,4-linked N-acetyl-Dglucosamine. It is an important component of the cell wall of pathogenic fungi, but it does not exist in plants. Chitinase (EC 3.2.1.14), a subgroup of PR proteins [1], exists in a variety of organisms and catalyzes the hydrolysis of the β-1-4-linkage in the N-acetyl-D-glucosamine polymer of chitin. The resulting chitin fragments act as powerful pathogen-associated molecular patterns (PAMPs) that induce PAMP-triggered immunity [2,3]. Therefore, chitinase is considered to be a defense-related gene against pathogens containing chitin. Plant chitinases are divided into PR-3, PR-4, PR-8 and PR-11 [4]. Some studies have shown that the increase in chitinase levels is a response to pathogen attack [5][6][7][8][9]. Chitinases are either directly induced by pathogen elicitors or are constitutively expressed in the attacked tissue [10,11]. Chitinases isolated from plants can limit the growth of chitin-containing fungi in vitro [12,13] and in vivo [14], and the overexpressed chitinases in plants can resist infection by different fungal pathogens [15][16][17][18][19].
According to the similarity of amino acid sequence in the catalytic domain, chitinases can be divided into glycosyl hydrolases families 18 and 19 (GH18 and GH19). According to their phylogeny, catalytic reaction mechanism, three-dimensional (3D) structure and sensitivity to inhibitors, these families are further divided into five different classes (Classes I-V). GH18 chitinases (Classes III and V) are widely distributed in various organisms, while GH19 chitinases (Classes I, II and IV) mainly exist in plants and are the main source of chitinolytic activity [20].
Mulberry (Morus notabilis) is a typical perennial woody plant with very important economic and medicinal value, because mulberry contains abundant secondary metabolites beneficial to human health [21][22][23][24]. B. cinerea is a necrotizing fungal pathogen that can infect more than 200 plant species in the world, including important economic horticultural crops [25][26][27]. B. cinerea is also one of the main pathogens affecting mulberry [28]. So far, there are only a few reports of mulberry genes that are effective against B. cinerea [28,29]. Plant chitinases resist fungal infection by producing hypersensitivity reactions and inducing defense reactions. Therefore, chitinase is a good target for studying the defense response to B. cinerea. However, so far, the role of mulberry chitinase genes in resistance has not been systematically studied.
The availability of the mulberry genome and transcriptome data in response to B. cinerea infection has facilitated the identification of genome-wide chitinase gene families and the study of their resistance to B. cinerea infection [29,30]. This study reports the genome-wide identification and analysis of the mulberry chitinase gene family. The resistance of the chitinase gene to B. cinerea infection was studied. In addition, MnChi18 was heterologous expressed in Arabidopsis to study its function. Diverse approaches were used to study the resistance of transgenic Arabidopsis to B. cinerea, confirming that the MnChi18 gene is involved in the defense mechanism of transgenic plants. These findings may provide effective genetic resources for improving mulberry resistance to B. cinerea.

Identification of Chitinase Genes in Mulberry
In order to identify the mulberry chitinase genes, genome sequence and annotation data were obtained from the Morus notabilis genome project [30]. The Hidden Markov Model (HMM) seed profiles of Glyco_hydro_18 (PF00704) and Glyco_hydro_19 (PF00182) from the Pfam database were downloaded [31]. HMMER3 (v.3.0) software was used to identify the mulberry chitinase gene [32]. Then, the presence of conserved domains of Glyco_hydro_18 or Glyco_hydro_19 was manually performed on all predicted chitinase genes.

Phylogenetic Tree of Chitinase Genes
To study the evolutionary relationship, ClustalW was used to align the full-length amino acid sequence of the chitinase protein under default settings, and we used MEGA6 to construct a neighbor-joining (NJ) phylogenetic tree [33]. Bootstrap analysis was performed with 1000 replicates.

Quantitative Real-Time PCR
Total RNA was isolated from the samples with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The QuantiNova™ SYBR Green PCR kit (Qiagen, Hilden, Germany) and StepOnePlus™ Real-time PCR system (Applied Biosystems, Waltham, MA, USA) were used for qRT-PCR detection. AtActin and MnActin genes were used as internal control genes in Arabidopsis and mulberry, respectively. The qRT-PCR test performed three biological replicates. Details of the qRT-PCR primers are shown in Table S1.

Plasmid Construction and Plant Transformation
Under the control of the CaMV35S promoter, full-length coding sequences of MnChi18 (GenBank accession number: EXB55192.1) were cloned into the KpnI (5 -GGGTACCATGGCC TCTCCCAATCCAA-3 ) and SalI (5 -GCGTCGACTTAGCAAGTGAGATTGGATCCA-3 ) restriction sites of the pLGNL vector. Afterwards, the CaMV35S::MnChi18 recombinant plasmid was obtained. The recombinant pLGNL expression vector was transformed into Agrobacterium tumefaciens strain GV3101. MnChi18 was finally transferred into Arabidopsis (Columbia-0) by the floral dip method [34]. The homozygous lines of the T 3 generation were studied.

Resistance Analysis of Transgenic Arabidopsis to B. cinerea
The resistance test was used to detect the ability of transgenic Arabidopsis plants to resist B. cinerea. Transgenic seeds were germinated on 1/2 Murashige and Skoog (MS) agar medium. Seven-day-old seedlings were transferred to pots of nutrient soil and grown at 24 • C/22 • C under a 16-h light/8-h dark photoperiod. The hyphal fragments were placed on the leaves of 21-day-old plants. The inoculated Arabidopsis plants were observed every 12 hours and photographed 36 hours later. The transgenic Arabidopsis plants with pLGNL were used as a control. The content of malondialdehyde (MDA) and the activity of catalase (CAT) were determined with a Malondialdehyde Assay Kit (Solarbio, Beijing, China) and Catalase Assay Kit (Solarbio, Beijing, China) according to the manufacturer's instructions. All treatments were repeated three times.
The contents of superoxide radical (O 2 − ) and hydrogen peroxide (H 2 O 2 ) in leaves were determined by histochemical staining. The leaves were inserted into 0.1% nitroblue tetrazole (NBT) containing a 50 mM potassium phosphate buffer (pH 7.8) for O 2 − detection [35]. For the detection of H 2 O 2 , a 3,3 -diaminobenzidine (DAB) solution was used in agro-infiltrated leaves [36]. The samples were placed in a 1.0 mg/mL DAB-HCl solution, darkly covered for 12 h at room temperature, and then placed in 95% ethanol for 5 minutes until brown spots of H 2 O 2 and blue O 2 − precipitate appeared on the leaves.

Statistical Analyses
All data were calculated using SPSS 26.0 statistical software (SPSS Inc., Chicago, IL, USA) and Excel 2013 (Microsoft, Redmond, CA, USA). The results are expressed as the mean ± standard error. Significant differences (P < 0.05) were measured by Student's t-test analysis.

Genome-Wide Identification and Phylogenetic Analysis of Chitinase Genes in Mulberry
A total of 26 chitinase genes were identified from the mulberry genome sequence, among which 15 belonged to the GH18 subfamily (7 Class III and 8 Class V) and 11 belonged to the GH19 subfamily (3 Class I, 4 Class II and 4 Class IV) ( Table 1). The 26 predicted chitinase proteins ranged in length from 104 (MnChi15) to 881 amino acids (aa) (MnChi23). The relative molecular mass ranged from 11.78 kDa (MnChi15) to 96.77 kDa (MnChi23). The theoretical isoelectric points (pI) ranged from 4.56 (MnChi16) to 8.77 (MnChi19). The phylogenetic analysis of the 31 chitinase sequences was carried out using the neighbor-joining method (Figure 1), and five types of chitinase proteins were identified, which was consistent with the previous Ammopiptanthus nanus chitinases [37]. The mulberry chitinase genes were divided into two large branches: one was composed of Classes I, II and IV, and the other was composed of Classes III and V. . Figure 1. Phylogenetic tree of the chitinase genes from M. notabilis and Arabidopsis. The neighborjoining (NJ) was used to construct a phylogenetic tree. The tree was generated by chitinase amino acid sequences using MEGA6. The numbers represent confidence percentages.

Expression Pattern of Mulberry Chitinases under the Infection of B. cinerea
To study the resistance of mulberry chitinase genes to B. cinerea, we analyzed the expression pattern of chitinases based on our previous transcriptome data of mock-treated (Mock) and B. cinerea-inoculated (Inoculated) M. notabilis leaves [29]. With FPKM > 1.0, a total of 14 MnChis were found to be expressed ( Figure 2 and Table S2). With |Fold Change (Inoculated/Mock)| > 2, the expression of four chitinases (MnChi3/14/17/18) in mulberry leaves was significantly upregulated after B. cinerea infection, and the expression of two chitinases (MnChi20/23) was significantly downregulated. These highly expressed chitinase genes suggested that they may be involved in mulberry resistance to B. cinerea. MnChi14 and MnChi18 were the two genes with the most increased expression after infec- The neighborjoining (NJ) was used to construct a phylogenetic tree. The tree was generated by chitinase amino acid sequences using MEGA6. The numbers represent confidence percentages.

Expression Pattern of Mulberry Chitinases under the Infection of B. cinerea
To study the resistance of mulberry chitinase genes to B. cinerea, we analyzed the expression pattern of chitinases based on our previous transcriptome data of mock-treated (Mock) and B. cinerea-inoculated (Inoculated) M. notabilis leaves [29]. With FPKM > 1.0, a total of 14 MnChis were found to be expressed ( Figure 2 and Table S2). With |Fold Change (Inoculated/Mock)| > 2, the expression of four chitinases (MnChi3/14/17/18) in mulberry leaves was significantly upregulated after B. cinerea infection, and the expression of two chitinases (MnChi20/23) was significantly downregulated. These highly expressed chitinase genes suggested that they may be involved in mulberry resistance to B. cinerea. MnChi14 and MnChi18 were the two genes with the most increased expression after infection of B. cinerea, and they may play an important role in resistance to B. cinerea infection. Then, the two genes were verified by qRT-PCR, and the results were consistent with the transcriptome data ( Figure 3).

Ectopic Expression of MnChi18 in Arabidopsis Enhances Resistance to B. cinerea
Under the control of Cauliflower mosaic virus (CaMV) 35S promoter, Arabidopsis plants were transformed with MnChi18 cDNA, and several T 3 transgenic lines were obtained. The MnChi18 gene expression was confirmed by analysis of transgenic Arabidopsis plants ( Figure 4A). Three lines with significantly higher expression than the empty vector control were selected for follow-up study. To investigate the resistance of transgenic Arabidopsis with the MnChi18 gene to B. cinerea, the leaves of the transgenic Arabidopsis with an empty vector and MnChi18 were inoculated with an agar block containing B. cinerea hyphae ( Figure 4B). Compared with the empty vector control leaves that showed severe disease symptoms 36 hours after inoculation, all the leaves of the MnChi18 overexpressed lines showed only slight lesions. Quantitative analysis showed that transgenic Arabidopsis with MnChi18 gene inhibited the growth of B. cinerea compared with transgenic Arabidopsis with the empty vector ( Figure 4C). In addition, the production of reactive oxygen species (ROS) is the response of plants to stress. The DAB and NBT staining methods were used to detect the hydrogen peroxide (H 2 O 2 ) and superoxide (O 2 − ) in leaves, respectively. In terms of phenotype, Arabidopsis transferred with empty vector showed large patches of dark brown after DAB staining, an indication of H 2 O 2 accumulation, and large patches of dark blue after NBT staining, a marker for O 2 − , compared with the MnChi18 transgenic Arabidopsis ( Figure 4D).

Ectopic Expression of MnChi18 in Arabidopsis Enhances Resistance to B. cinerea
Under the control of Cauliflower mosaic virus (CaMV) 35S promoter, Arabidopsis plants were transformed with MnChi18 cDNA, and several T3 transgenic lines were obtained. The MnChi18 gene expression was confirmed by analysis of transgenic Arabidopsis plants ( Figure 4A). Three lines with significantly higher expression than the empty vector control were selected for follow-up study. To investigate the resistance of transgenic Arabidopsis with the MnChi18 gene to B. cinerea, the leaves of the transgenic Arabidopsis with an empty vector and MnChi18 were inoculated with an agar block containing B. cinerea hyphae (Figure 4B). Compared with the empty vector control leaves that showed severe disease symptoms 36 hours after inoculation, all the leaves of the MnChi18 overexpressed lines showed only slight lesions. Quantitative analysis showed that transgenic Arabidopsis with MnChi18 gene inhibited the growth of B. cinerea compared with transgenic Arabidopsis with the empty vector ( Figure 4C). In addition, the production of reactive oxygen species (ROS) is the response of plants to stress. The DAB and NBT staining methods were used to detect the hydrogen peroxide (H2O2) and superoxide (O2 -) in leaves, respectively. In terms of phenotype, Arabidopsis transferred with empty vector showed large patches of dark brown after DAB staining, an indication of H2O2 accumulation, and large patches of dark blue after NBT staining, a marker for O2 -, compared with the MnChi18 transgenic Arabidopsis ( Figure 4D).

Detection of Biochemical Indices
In order to verify the physiological changes of transgenic Arabidopsis with MnChi18 and the empty vector, the MDA content and CAT activity were determined ( Figure 5). Before

Detection of Biochemical Indices
In order to verify the physiological changes of transgenic Arabidopsis with MnChi18 and the empty vector, the MDA content and CAT activity were determined ( Figure 5

The Enhanced Expressions of Resistance-Related Genes in MnChi18 Transgenic Plants
PR1, WRKY33, β-1,3-glucanase 2 (BG2) and hypersensitive induced reaction 1 (HIR1) are the defense-associated marker genes of a plant. The results showed that there was no significant difference between AtPR1 and AtWRKY33 in transgenic Arabidopsis with MnChi18 and the empty vector before and after B. cinerea infection ( Figure S1). AtBG2 and AtHIR1 had no significant difference in transgenic Arabidopsis with MnChi18 and the empty vector before the infection of B. cinerea. However, the expression levels of AtBG2 and AtHIR1 were upregulated in both MnChi18 and the empty vector transgenic plants after 36 h of B. cinerea infection, and the MnChi18 transgenic plants were significantly higher than the empty vector transgenic plants ( Figure 6). These results indicated that when the MnChi18 gene was introduced into Arabidopsis, it could resist the infection of B. cinerea by inducing the expression of resistance-related genes.

The Enhanced Expressions of Resistance-Related Genes in MnChi18 Transgenic Plants
PR1, WRKY33, β-1,3-glucanase 2 (BG2) and hypersensitive induced reaction 1 (HIR1) are the defense-associated marker genes of a plant. The results showed that there was no significant difference between AtPR1 and AtWRKY33 in transgenic Arabidopsis with MnChi18 and the empty vector before and after B. cinerea infection ( Figure S1). AtBG2 and AtHIR1 had no significant difference in transgenic Arabidopsis with MnChi18 and the empty vector before the infection of B. cinerea. However, the expression levels of AtBG2 and AtHIR1 were upregulated in both MnChi18 and the empty vector transgenic plants after 36 h of B. cinerea infection, and the MnChi18 transgenic plants were significantly higher than the empty vector transgenic plants ( Figure 6). These results indicated that when the MnChi18 gene was introduced into Arabidopsis, it could resist the infection of B. cinerea by inducing the expression of resistance-related genes.

Discussion
Chitinase genes are a large gene family, which play an important role in plant resistance. Clarifying the function of chitinase genes in plants is of great significance to plant-resistance breeding. So far, systematic genome-wide investigations of chitinase genes have been reported in many species. However, there is no systematic research report on mulberry chitinase genes. We identified a chitinase gene family in M. notabilis

Discussion
Chitinase genes are a large gene family, which play an important role in plant resistance. Clarifying the function of chitinase genes in plants is of great significance to plant-resistance breeding. So far, systematic genome-wide investigations of chitinase genes have been reported in many species. However, there is no systematic research report on mulberry chitinase genes. We identified a chitinase gene family in M. notabilis (Table 1 and Figure 1), for which a total of 26 mulberry chitinase genes were identified. Compared with the number of chitinase genes in other plants, mulberry is relatively small, but more than Arabidopsis [37]. This indicates that during the evolution process, the chitinase genes of mulberry have not been significantly amplified.
PR genes, including chitinase, are silenced or constitutively expressed at low levels in plants in the absence of pathogens, but are significantly induced in the presence of pathogens [38][39][40]. Consistent with previous reports, the expression of MnChis were both constitutive and inducible ( Figure 2 and Table S2). The results showed that at least six MnChi genes can be induced after inoculation with B. cinerea. Class I (MnChi3), III (MnChi14) and IV (MnChi17/18) were significantly upregulated, Class II had no significantly induced expression, and Class V (MnChi20/23) was significantly down regulated. These findings indicate that the mulberry chitinase genes may have a different mechanism of action. Interestingly, the expression pattern of Class V was opposite to that of other plants [8,41], suggesting that the Class V chitinases in mulberry may have evolved different functions, which needs to be further studied.
To verify the function of the MnChi18 gene, we overexpressed this gene in Arabidopsis. Overexpressed MnChi18 plants were inoculated with B. cinerea, and ROS activity was detected by DAB and NBT staining ( Figure 4). Compared with the empty vector plants, the plants overexpressing MnChi18 had less leaf damage and ROS accumulation, thus enhancing the resistance of Arabidopsis leaves to B. cinerea infection. Our results were consistent with previous studies that CaChiIV1 gene interference in peppers significantly reduces its resistance [42]. MnChi18 may indirectly participate in the defense mechanism of transgenic plants by changing the transcription of other PR genes ( Figure 6). Plant β-1,3-Glucanases (BG) are members of the PR2 family and one of the 17 PR protein families. It plays a key role in the response to biotic and abiotic stress. Overexpression of maize BG gene ZmGns in Arabidopsis can significantly increase the resistance to B. cinerea [43]. Arabidopsis hypersensitive-induced reaction (AtHIR) protein plays an important in plant innate immunity. Overexpression of AtHIR1 inhibited the growth of Pto DC3000 [44]. Overexpression of MnChi18 changes the expression of defense-related genes (BG2 and HIR1), which indicates that there is an interaction between them. Overexpression of the chitinase gene can usually increase the expression of the PR1 gene to enhance resistance [18,45], but overexpression of MnChi18 did not enhance the expression of AtPR1 ( Figure S1), indicating that the mulberry chitin gene may have different mechanisms in plant resistance.
MDA is an important lipid peroxidation product involved in defense signal transduction in plants under biotic and abiotic stress [46]. However, our results suggested that overexpression of MnChi18 resulted in decreased MDA accumulation ( Figure 5A). The MDA levels are usually associated with oxidative stress in plants. Therefore, overexpression of MnChi18 gene avoids cell membrane damage. When plants are invaded by pathogenic microorganisms, it will cause the accumulation of ROS and the activation of plant defense enzymes, which help maintain cell integrity and eliminate peroxides [47]. After 36 hours of infection by B. cinerea, the CAT activity of MnChi18 transgenic Arabidopsis was significantly higher than that of the empty vector transgenic Arabidopsis ( Figure 5B). This indicates that the overexpression of MnChi18 in Arabidopsis increases the ability to maintain cell integrity and thus resist B. cinerea infection.

Conclusions
This study identified three Class I, four Class II, seven Class III, four Class IV and eight Class V chitinase genes from the M. notabilis genome sequence. The ectopic expression of MnChi18 in Arabidopsis increased its resistance to B. cinerea, and the disease symptoms were lighter. Overexpression of MnChi18 protected plant cells from damage and enhanced the expression of plant resistance genes. This study will provide basic insights into the role of the MnChi18 gene in the resistance pathway.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3 390/genes13010098/s1: Figure S1: Relative expression of pathogen-related genes in empty vector transgenic (CK) and MnChi18 transgenic (OE) Arabidopsis leaves before and after of B. cinerea inoculation; Table S1: Primers for real-time PCR; Table S2: Differential expression analysis of chitinase gene in Mock and Inoculated.
Author Contributions: Y.X. and X.J. conceived and designed the experiments; D.W. and S.H. performed the experiments; S.L., N.G. and Y.F. analyzed the data; Y.X. and D.W. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.