MnASI1 Mediates Resistance to Botrytis cinerea in Mulberry (Morus notabilis)

Six α-amylase/subtilisin inhibitor genes (MnASIs) were identified from mulberry (Morus notabilis). In this study, bioinformatics and expression pattern analysis of six MnASIs were performed to determine their roles in resistance to B. cinerea. The expression of all six MnASIs was significantly increased under Botrytis cinerea infection. MnASI1, which responded strongly to B. cinerea, was overexpressed in Arabidopsis and mulberry. The resistance of Arabidopsis and mulberry overexpressing MnASI1 gene to B. cinerea was significantly improved, the catalase (CAT) activity was increased, and the malondialdehyde (MDA) content was decreased after inoculation with B. cinerea. At the same time, H2O2 and O2− levels were reduced in MnASI1 transgenic Arabidopsis, reducing the damage of ROS accumulation to plants. In addition, MnASI1 transgenic Arabidopsis increased the expression of the salicylic acid (SA) pathway-related gene AtPR1. This study provides an important reference for further revealing the function of α-amylase/subtilisin inhibitors.


Introduction
Plant protease inhibitors (PIs) are a class of small proteins with various biological functions. They regulate endogenous protease activity and apoptosis while protecting plants from animals, insects, and microorganisms [1][2][3]. PIs are divided into different families based on their identical cysteine pattern, overall 3D structure, and mechanism of action. They can also be grouped based on amino acid sequence similarity [4,5]. There are more than 10 different families of PIs from many plant species depending on the type of protease they act on [6][7][8]. Kunitz-type protease inhibitors (PKPIs) are usually present in the storage tissues of plants and can resist pathogen infection [9]. PKPIs have a molecular weight of about 20 to 22 kDa and may consist of one to two polypeptide chains linked by one to two disulfide bonds [5]. Their structures may be missing cysteine residues [10,11]. As a member of PKPIs, α-amylase/subtilisin inhibitors (ASIs) inhibit α-amylase in mammals and lepidopteran pests [12]. Therefore, ASIs can protect plants from lepidopteran pests. In addition, ASIs have an inhibitory effect on subtilisin and are therefore also associated with plant resistance to microorganisms [2].
Mulberry (Morus L.) is widely distributed all over the world and has important economic value in the production of food and medicine due to its rich content of secondary metabolites that are beneficial to humans [13][14][15][16]. Botrytis cinerea is a necrotizing fungal pathogen that can infect more than 200 species of plants around the world [17][18][19]. At the same time, B. cinerea is one of the main pathogenic fungi of mulberry [20,21]. Previous studies on ASIs mainly focused on identification and classification. Although it was found to be related to plant disease resistance, the mechanism of disease resistance was not completely clear. At the same time, there is no systematic study on the role of ASIs in mulberry resistance. The aim of this study is to reveal the underlying mechanism of disease resistance in mulberry ASIs. 2 of 12 The availability of transcriptomic data from mulberry (Morus notabilis) in response to B. cinerea infection facilitates studies on resistance of ASIs to B. cinerea infection [22]. Based on the previous transcriptome data, we found that the expression levels of six ASIs in M. notabilis were significantly increased during B. cinerea infection. The resistance of ASIs to B. cinerea infection was investigated. Furthermore, to investigate their functions, we performed MnASI1 expression in Arabidopsis and mulberry. The resistance of transgenic Arabidopsis and mulberry was studied by various methods, and it was confirmed that MnASI1 was involved in the defense response of transgenic plants. These results initially revealed the mechanism of ASIs disease resistance, laid the foundation for further understanding the function of ASIs, provided a reference for other plant ASI research, and provide potential target genes for enhancing the resistance of mulberry to B. cinerea.

Bioinformatics Analyses of MnASIs
A total of six α-amylase/subtilisin inhibitor genes were identified from the mulberry genome sequence. The six α-amylase/subtilisin inhibitor proteins ranged in length from 191 (MnASI1 and MnASI5) to 207 amino acids (aa) (MnASI3) ( Table 1). The relative molecular mass ranged from 20.82 kDa (MnASI1) to 22.65 kDa (MnASI3). The theoretical isoelectric points (pI) ranged from 4.46 (MnASI1) to 8.53 (MnASI6). The multiple alignment results of MnASIs and other plant α-amylase/subtilisin inhibitors showed that there were two regions of plant α-amylase/subtilisin inhibitors, namely the protease inhibitory region and the α-amylase inhibitory region ( Figure 1).
The protein sequences of other plant α-amylase/subtilisin inhibitors obtained from NCBI were multiple aligned. Phylogenetic and molecular evolutionary analyses were performed using MEGA 6 to explore the evolutionary relationships among different species ( Figure 2). The results showed that the MnASIs proteins of mulberry clustered together and were more distantly related to the α-amylase/subtilin inhibitors in Aegilops tauschii, Hordeum vulgare, and Brachypodium distachyon, and were more closely related to the αamylase/subtilin inhibitors in Vitis vinifera and Citrus sinensis.  The protein sequences of other plant α-amylase/subtilisin inhibitors obtained from NCBI were multiple aligned. Phylogenetic and molecular evolutionary analyses were performed using MEGA 6 to explore the evolutionary relationships among different species ( Figure 2). The results showed that the MnASIs proteins of mulberry clustered together and were more distantly related to the α-amylase/subtilin inhibitors in Aegilops tauschii, Hordeum vulgare, and Brachypodium distachyon, and were more closely related to the αamylase/subtilin inhibitors in Vitis vinifera and Citrus sinensis.

B. cinerea-Induced MnASIs Expression
The expression levels of six MnASIs in mulberry seedlings infected with B. cinerea were determined by qRT-PCR ( Figure 3). The expression levels of all six MnASIs were significantly increased 3 days after inoculation, which was consistent with our previous

B. cinerea-Induced MnASIs Expression
The expression levels of six MnASIs in mulberry seedlings infected with B. cinerea were determined by qRT-PCR ( Figure 3). The expression levels of all six MnASIs were significantly increased 3 days after inoculation, which was consistent with our previous transcriptome data (Table S1) [22]. These highly expressed α-amylase/subtilisin inhibitor genes may be involved in the resistance of mulberry to B. cinerea. MnASI1 is the gene with the most increased expression after B. cinerea infection, which may play an important role in resistance to B. cinerea infection. In order to further verify the disease resistance function of MnASIs, MnASI1 was selected for follow-up research.

Subcellular Localization of MnASI1
Subcellular localization of transgenic Arabidopsis root tips was used in confocal microscopy ( Figure 4). The results showed that the MnASI1 protein was localized on the cell membrane, which indicated that MnASI1 might play a disease-resistant role on the cell membrane.

Subcellular Localization of MnASI1
Subcellular localization of transgenic Arabidopsis root tips was used in confocal microscopy ( Figure 4). The results showed that the MnASI1 protein was localized on the cell membrane, which indicated that MnASI1 might play a disease-resistant role on the cell membrane.

Subcellular Localization of MnASI1
Subcellular localization of transgenic Arabidopsis root tips was used in confocal microscopy ( Figure 4). The results showed that the MnASI1 protein was localized on the cell membrane, which indicated that MnASI1 might play a disease-resistant role on the cell membrane.

Positive Regulation of MnASI1 for Resistance to B. cinerea
Arabidopsis was transformed with MnASI1 cDNA to obtain three T3 transgenic lines ( Figure 5). Afterward, the expression of MnASI1 was confirmed by qRT-PCR in transgenic Arabidopsis (Figure 5a). To study the resistance of Arabidopsis transgenic with MnASI1 to B. cinerea, the leaves of transgenic Arabidopsis were inoculated with an agar block containing B. cinerea hyphae (Figure 5b). Compared with severe lesions on control leaves 36 h after inoculation, only mild lesions appeared on leaves of MnASI1 overexpression lines. Quantitative analysis revealed that Arabidopsis transformed with MnASI1 inhibited the infection of B. cinerea (Figure 5c). Furthermore, the production of reactive oxygen species is a response to stress in plants. The contents of hydrogen peroxide (H2O2) and superoxide (O2 − ) in leaves were detected by DAB staining and NBT staining, respectively (Figure 5d). Compared with Arabidopsis transfected with MnASI1, large dark brown patches appeared after DAB staining in Arabidopsis transfected with empty vector, indicating the accumulation of H2O2, and large dark blue patches appeared after NBT staining, which was the accumulation of O2 − .

Detection of Biochemical Indices
To determine the physiological changes of transgenic Arabidopsis, MDA content and CAT activity were measured ( Figure 6). There was no significant difference in MDA content between MnASI1 transgenic Arabidopsis and empty vector transgenic Arabidopsis before B. cinerea infection (Figure 6a). After 36 h infection with B. cinerea, the MDA content of transgenic Arabidopsis increased, while the MDA content of MnASI1 transgenic Arabidopsis was significantly lower than that of empty vector transgenic Arabidopsis. These results indicated that the plasma membrane damage of empty vector transgenic Arabidopsis was more severe than that of MnASI1 transgenic Arabidopsis. Similarly, there was no significant difference in CAT activity between MnASI1 and empty vector transgenic Arabidopsis before infection with B. cinerea (Figure 6b). After 36 h infection with B. cinerea, the CAT activity of transgenic Arabidopsis increased, while the CAT content of MnASI1 transgenic Arabidopsis was significantly higher than that of empty vector transgenic Arabidopsis. These results suggest that overexpression of MnASI1 enhanced plant resistance to oxidative damage.

Detection of Biochemical Indices
To determine the physiological changes of transgenic Arabidopsis, MDA content and CAT activity were measured ( Figure 6). There was no significant difference in MDA content between MnASI1 transgenic Arabidopsis and empty vector transgenic Arabidopsis before B. cinerea infection (Figure 6a). After 36 h infection with B. cinerea, the MDA content of transgenic Arabidopsis increased, while the MDA content of MnASI1 transgenic Arabidopsis was significantly lower than that of empty vector transgenic Arabidopsis. These results indicated that the plasma membrane damage of empty vector transgenic Arabidopsis was more severe than that of MnASI1 transgenic Arabidopsis. Similarly, there was no significant difference in CAT activity between MnASI1 and empty vector transgenic Arabidopsis before infection with B. cinerea (Figure 6b). After 36 h infection with B. cinerea, the CAT activity of transgenic Arabidopsis increased, while the CAT content of MnASI1 transgenic Arabidopsis was significantly higher than that of empty vector transgenic Arabidopsis. These results suggest that overexpression of MnASI1 enhanced plant resistance to oxidative damage.  To investigate the role of MnASI1 in mulberry, transient overexpression was performed in mulberry (Figure 7). The beta-glucuronidase (GUS) histochemical analysis showed strong GUS staining in the leaves of mulberry seedlings, indicating that this transient expression system is effective in mulberry (Figure 7a). The expression levels of MnASI1 were up-regulated in mulberry leaves overexpressing MnASI1 compared with those overex-pressing empty vectors (Figure 7b). The resistance of mulberry overexpressing MnASI1 to B. cinerea was enhanced compared with the overexpressing empty vector (Figure 7c). Under the condition of B. cinerea infection, the transient expression of MnASI1 significantly reduced the content of MDA in mulberry seedlings (Figure 7d) and increased the content of CAT (Figure 7e), which was consistent with the previous results of MnASI1 transgenic Arabidopsis.
To investigate the role of MnASI1 in mulberry, transient overexpression was performed in mulberry (Figure 7). The beta-glucuronidase (GUS) histochemical analysis showed strong GUS staining in the leaves of mulberry seedlings, indicating that this transient expression system is effective in mulberry (Figure 7a). The expression levels of MnASI1 were up-regulated in mulberry leaves overexpressing MnASI1 compared with those overexpressing empty vectors (Figure 7b). The resistance of mulberry overexpressing MnASI1 to B. cinerea was enhanced compared with the overexpressing empty vector (Figure 7c). Under the condition of B. cinerea infection, the transient expression of MnASI1 significantly reduced the content of MDA in mulberry seedlings (Figure 7d) and increased the content of CAT (Figure 7e), which was consistent with the previous results of MnASI1 transgenic Arabidopsis.

MnASI1 Transgenic Plants Enhance PR1 Expression
PR1 is a plant defense-related marker gene. The results showed that the expression of AtPR1 in empty vector and MnASI1 transgenic Arabidopsis was up-regulated 36 h after B. cinerea infection, and empty vector transgenic Arabidopsis was significantly lower than MnASI1 transgenic Arabidopsis (Figure 8). This indicated that MnASI1 overexpression in Arabidopsis could enhance the resistance to B. cinerea by inducing the expression of the resistance-related gene.

Discussions
Many resistant plants have evolved specific PIs that both regulate plant protease activity and promote plant defense against pests and pathogens [23]. As a class of PIs, there are few studies on plant α-amylase/subtilin inhibitors genes, and most reports have not performed functional analysis. Previous studies related to ASIs disease resistance mainly focused on the study of recombinant ASIs protein. For example, after infection with P. palmivora spores pretreated with recombinant HbASI, the growth, lesion number, and scopolamine content of the spores were low, and the effect was better with the increase of recombinant HbASI content [2]. Similarly, Kunitz inhibitory fusion protein had a certain inhibitory effect on the growth of F. moniliforme mycelium [24]. The study of MnASIs gene in mulberry will help to further understand the characteristics and function of this gene. Based on amino acid sequence features (Figure 1), MnASIs can be classified as α-amylase/subtilisin inhibitors, belonging to group C of the kunitz protease inhibitor family [25]. Furthermore, phylogenetic tree results indicated that MnASIs clustered with other plant α-amylase/subtilin inhibitors (Figure 2). High homology between MnASIs and V. vinifera a-amylase/subtilisin inhibitors. Analysis showed that the mulberry MnASIs were intronless (Table 1). These results are consistent with previous reports on intronless PI genes [26]. Intronless genes may be a structural feature that provides a selective advantage to rapidly encode and flip transcripts in response to a variety of exogenous signals without significant delays [27].
In plants, the expression level of pathogenicity-related (PR) genes is generally low, and only high induced expression during pathogen infection [28][29][30]. Similar to PR genes, except for MnASI5, the other five MnASIs were only induced to express during B. cinerea infection (Figure 3). Inoculation of mulberry leaves with B. cinerea increased the expression of MnASIs, indicating that MnASIs are induced by biological elicitors and participate in plant defense responses. These results are consistent with previous reports [2,9]. The MnASI5 gene was highly expressed in mulberry leaves mock-treated and B. cinerea inoculated. This suggests that the MnASI5 gene may have dual functions of development and self-protection. The transcriptome data were consistent with the qRT-PCR results as a whole (Figure 3 and Table S1), and the expression of MnASIs was significantly enhanced, but the multiple of MnASIs expression enhancement was different, which may be caused by mulberry individual differences.
To further study the role of MnASIs in disease resistance, the empty vector and MnASI1 gene transgenic Arabidopsis and mulberry transiently overexpressed were

Discussions
Many resistant plants have evolved specific PIs that both regulate plant protease activity and promote plant defense against pests and pathogens [23]. As a class of PIs, there are few studies on plant α-amylase/subtilin inhibitors genes, and most reports have not performed functional analysis. Previous studies related to ASIs disease resistance mainly focused on the study of recombinant ASIs protein. For example, after infection with P. palmivora spores pretreated with recombinant HbASI, the growth, lesion number, and scopolamine content of the spores were low, and the effect was better with the increase of recombinant HbASI content [2]. Similarly, Kunitz inhibitory fusion protein had a certain inhibitory effect on the growth of F. moniliforme mycelium [24]. The study of MnASIs gene in mulberry will help to further understand the characteristics and function of this gene. Based on amino acid sequence features (Figure 1), MnASIs can be classified as α-amylase/subtilisin inhibitors, belonging to group C of the kunitz protease inhibitor family [25]. Furthermore, phylogenetic tree results indicated that MnASIs clustered with other plant α-amylase/subtilin inhibitors (Figure 2). High homology between MnASIs and V. vinifera a-amylase/subtilisin inhibitors. Analysis showed that the mulberry MnASIs were intronless (Table 1). These results are consistent with previous reports on intronless PI genes [26]. Intronless genes may be a structural feature that provides a selective advantage to rapidly encode and flip transcripts in response to a variety of exogenous signals without significant delays [27]. In plants, the expression level of pathogenicity-related (PR) genes is generally low, and only high induced expression during pathogen infection [28][29][30]. Similar to PR genes, except for MnASI5, the other five MnASIs were only induced to express during B. cinerea infection (Figure 3). Inoculation of mulberry leaves with B. cinerea increased the expression of MnASIs, indicating that MnASIs are induced by biological elicitors and participate in plant defense responses. These results are consistent with previous reports [2,9]. The MnASI5 gene was highly expressed in mulberry leaves mock-treated and B. cinerea inoculated. This suggests that the MnASI5 gene may have dual functions of development and self-protection. The transcriptome data were consistent with the qRT-PCR results as a whole ( Figure 3 and Table S1), and the expression of MnASIs was significantly enhanced, but the multiple of MnASIs expression enhancement was different, which may be caused by mulberry individual differences.
To further study the role of MnASIs in disease resistance, the empty vector and MnASI1 gene transgenic Arabidopsis and mulberry transiently overexpressed were constructed ( Figures 5 and 7). Compared with plants transformed with empty vector, after inoculation with B. cinerea, plants transformed with MnASI1 gene had smaller necrotic spots. The results showed that molecular and physiological responses to B. cinerea included ROS production and transcriptional responses. A more significant response of the SA-mediated defense gene was detected in B. cinerea-infected transgenic MnASI1 plants (Figure 8), suggesting that overexpression of MnASI1 enhances the defense capacity of transgenic plants by activating hypersensitive responses. The SA-dependent signaling pathway leads to the expression of the PR protein AtPR1, which promotes resistance. Consistent with previous reports, the SA signaling pathway plays an important role in response to B. cinerea infection [31]. Arabidopsis had obvious disease at 36 h of B. cinerea infection, while mulberry had obvious disease at 72 h of B. cinerea infection, indicating that mulberry was more resistant to B. cinerea.
MDA is a key lipid peroxidation product in plant defense [32]. The results showed that transgenic MnASI1 plants resulted in a decrease in MDA content (Figures 6a and 7d). MDA content in plants is often associated with oxidative stress. Transfection of MnASI1 can reduce cell membrane damage. When plants are infected with pathogens, the activity of plant defense-related enzymes is induced, which helps scavenge peroxides [33]. The CAT activity of MnASI1 overexpressing plants was significantly enhanced (Figures 6b and  7e), thus resisting B. cinerea infection.
Previous studies have shown that ASI recombinant protein has a disease-resistance effect, and this study is the first to report the disease-resistance mechanism of the ASI gene in mulberry, indicating that ASI has a disease-resistance effect both in vitro and in vivo. However, the upstream regulatory genes and downstream interacting genes of ASI are still unclear and need to be further studied. In addition, alginate encapsulation of plant biocontrol bacteria has been studied to some extent [34], and ASI protein may also be coated with alginate for plant disease control in the future.

Phylogenetic Tree of ASIs
To investigate evolutionary relationships, the full-length amino acid sequences of ASIs proteins were aligned using ClustalW under default settings, and a neighborhood-joining phylogenetic tree of ASIs was subsequently constructed using MEGA 6 [35]. Bootstrap analysis of 1000 replicates was performed.

Quantitative Real-Time PCR
Total RNA was extracted using RNAiso Plus Kit (Takara Bio., Kusatsu, Shiga, Japan). cDNA synthesis using the PrimeScript™ RT Reagent Kit (Takara Bio., Kusatsu, Shiga, Japan). qRT-PCR detection was performed using SYBR ® Premix Ex Taq™ II (Takara Bio., Kusatsu, Shiga, Japan) and StepOnePlus™ Real-time PCR system (Applied Biosystems, Waltham, MA, USA). The actin gene was used as an internal reference gene. qRT-PCR was performed in three technical replicates. qRT-PCR primers were shown in Table S2.

Transient Expression Analysis of Mulberry Gene Function
The A. tumefaciens GV3101 containing pLGNL-MnASI1 or pLGNL vectors prepared with transformation solution (1/2 MS, 5% sucrose, 200 µM acetosyringone and 0.05% Tween-20, pH 5.6) to a final OD 600 to 0.5. The 15-day-old mulberry seedlings were placed in the transformation solution of the GV3101 containing pLGNL-MnASI1 or pLGNL, and vacuumed at room temperature for 20 min.

Resistance Analysis of Transgenic Arabidopsis and Mulberry to B. cinerea
The hyphal fragments were placed on plant leaves. Photographs were taken 36 h after inoculation. The plants transformed into pLGNL were used as controls. Malondialdehyde (MDA) content and catalase (CAT) activity were determined using a Malondialdehyde Assay Kit (Solarbio, Beijing, China) and a Catalase Assay Kit (Solarbio, Beijing, China) according to the manufacturer's instructions. All treatments were repeated three times. The content of superoxide radical (O 2 − ) and hydrogen peroxide (H 2 O 2 ) in leaves was determined by nitroblue tetrazole (NBT) and 3,3 -diaminobenzidine (DAB) staining as described previously [37,38].

Statistical Analysis
All data were subjected to Student's t-test or one-way ANOVA with SPSS 26.0 software. These values are expressed as mean ± standard deviation (SD). p < 0.05 was considered statistically significant.