Potential Involvement of MnCYP710A11 in Botrytis cinerea Resistance in Arabidopsis thaliana and Morus notabilis

Cytochrome P450 (CYP) is a crucial oxidoreductase enzyme that plays a significant role in plant defense mechanisms. In this study, a specific cytochrome P450 gene (MnCYP710A11) was discovered in mulberry (Morus notabilis). Bioinformatic analysis and expression pattern analysis were conducted to elucidate the involvement of MnCYP710A11 in combating Botrytis cinerea infection. After the infection of B. cinerea, there was a notable increase in the expression of MnCYP710A11. MnCYP710A11 is overexpressed in Arabidopsis and mulberry and strongly reacts to B. cinerea. The overexpression of the MnCYP710A11 gene in Arabidopsis and mulberry led to a substantial enhancement in resistance against B. cinerea, elevated catalase (CAT) activity, increased proline content, and reduced malondialdehyde (MDA) levels. At the same time, H2O2 and O2− levels in MnCYP710A11 transgenic Arabidopsis were decreased, which reduced the damage of ROS accumulation to plants. Furthermore, our research indicates the potential involvement of MnCYP710A11 in B. cinerea resistance through the modulation of other resistance-related genes. These findings establish a crucial foundation for gaining deeper insights into the role of cytochrome P450 in mulberry plants.


Introduction
Cytochrome P450 (CYP) is one of the most important enzymes in the oxidoreductase family, which uses heme-thiolate as a cofactor [1].CYP functions as a protective agent for plants, defending them against various pathogenic microorganisms and pests by facilitating the synthesis and metabolism of numerous physiologically essential compounds [2][3][4][5][6].CYP operates within an intricate network of plant defense mechanisms [7].These defense mechanisms encompass hypersensitivity and the suppression of specific plant pathogen growth [8,9].The infection of Pseudomonas syringae triggered a hypersensitive response (HR), leading to an upregulation of the CYP76C2 gene in Arabidopsis thaliana.The activation of the CYP76C2 gene is associated with injury, senescent cell culture, and leaf senescence [10,11].CYP51H10 is responsible for the synthesis of antimicrobial oleanane-triterpene saponins, which confer resistance to root-infecting fungi in oats [12].
CYP710 is a member of the CYP superfamily and is believed to have originated from the CYP51 family through evolution [13].The transformation of β-sitosterol into stigmasterol occurs through a singular enzymatic process catalyzed by sterol C-22 desaturase, facilitated by the cytochrome P450 710A11 (CYP710A11) enzyme [14].As of now, there is limited knowledge regarding the function of CYP710A11 in the context of B. cinerea infection.
Mulberry is an important economic tree.Its leaves are the main feed of silkworm, and the fruit is rich in nutrition, active substances, good taste, and has high edible and medicinal value [15][16][17][18].In addition, mulberry is also used for ecological control because of its strong resistance to stress [19].B. cinerea is a necrotrophic fungal pathogen capable of infecting a broad spectrum of plant species, including significant agricultural crops [20,21].Its primary targets are tender tissues like fruits, vegetables, and flowers.This fungus is prevalent globally and is recognized as a highly economically impactful plant pathogen, leading to substantial crop losses pre-and post-harvest [22].Simultaneously, B. cinerea stands as a primary pathogenic fungus affecting mulberry plants [23].
The transcriptomic data detailing the resistance of mulberry (M.notabilis) to B. cinerea infection provide valuable insights for investigating the resistance of MnCYP710A11 to B. cinerea infection [24].According to the preceding transcriptome data, we observed a significant increase in MnCYP710A11 expression levels in M. notabilis during B. cinerea infection.The resistance conferred by MnCYP710A11 against B. cinerea infection was investigated.In addition, in order to explore the role of MnCYP710A11, we induced the overexpression of MnCYP710A11 in both Arabidopsis and mulberry trees.We examined the resistance of transgenic Arabidopsis and transiently overexpressed mulberry using diverse methods, confirming the involvement of MnCYP710A11 in the defense response of these transgenic plants.These results offer preliminary insights into the resistance mechanism of CYP710A11 and have established a basis for further elucidating the function of CYP710A11.At the same time, this serves as a reference for investigating CYP710A11 in other plant species and presents a potential target gene for enhancing mulberry's resistance to B. cinerea.

Plant Materials and Growth Conditions
The genetic background of A. thaliana was Columbia-0 (Col-0), which was cultured in a growth chamber at 22 • C, with 60-80% relative humidity and a 12 h day cycle.Mulberry trees were cultured in a growth chamber at 25 • C, with a 12 h day cycle and 75% humidity.

RNA Isolation and Quantitative Real-Time PCR
Plant tissues were processed for total RNA extraction utilizing CTAB-pBIOZOL (Bioer, Hangzhou, China) following the manufacturer's guidelines.The initial complementary DNA (cDNA) strand was generated using gDNA Eraser (Takara, Kusatsu, Shiga, Japan) in conjunction with the PrimeScript RT reagent Kit (Takara, Kusatsu, Shiga, Japan), subsequently followed by the synthesis of the second cDNA strand.The qRT-PCR analysis was conducted utilizing the SYBR Green PCR Master Mix (Takara, Kusatsu, Shiga, Japan) on both the Step One and Step OnePlus real-time PCR platforms (Applied Biosystems, Waltham, MA, USA).The Actin gene was employed as the internal reference gene.The qRT-PCR analysis was repeated using three different techniques, with the specific qRT-PCR primer sequences detailed in Table S1.The expression levels were analyzed by using the 2 −∆CT method [25].

Bioinformatic Analysis
The MnCYP710A11 (L484_021687) amino acid sequence was downloaded from a mulberry genome database (https://morus.biodb.org/morusdb/,accessed on 17 May 2023).Different species of CYP710A11 amino acid sequences were obtained from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/,accessed on 17 May 2023).ClustalX software (v.1.83) was used to align amino acid sequences [26].The phylogenetic tree was built utilizing the adjacency method as implemented in Molecular Evolutionary Genetic Analysis (MEGA 7) software with a bootstrap value of 1000 [27].

Transformation of A. thaliana
The complete coding sequence of the mulberry MnCYP710A11 gene was inserted into the pLGNL expression vector using KpnI and EcoRI restriction enzymes [28].Subsequently, the modified plasmid was introduced into the Agrobacterium tumefaciens LBA4404 strain.The positive A. tumefaciens containing MnCYP710A11 was transformed into Arabidopsis by using the flower dip method [29].The T3 homozygous lines were studied.

Histochemical GUS Staining
Histochemical analysis of GUS activity is a modification of the procedure previously described [30].First, the obtained homozygous Arabidopsis seeds overexpressing the MnCYP710A11 gene were sterilized, and then, the sterilized seeds were spread in an MS medium for culturing.After 2-3 days of cultivation, the plants underwent a water rinse followed by immersion in a GUS staining solution comprising 1 mM 5-bromo-4-chloro-3indolyl-β-d-glucuronic acid (X-Gluc), 50 mM sodium phosphate (pH = 7.0), 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% Triton X-100, 50 mM potassium ferricyanide, and 50 mM potassium ferrocyanide.The samples were then incubated at 37 • C for 8-16 h.To improve the visibility of GUS staining, chlorophyll was eliminated using 70% (v/v) ethanol.For GUS staining of mulberry leaves, first, the plant leaves were invaded by Agrobacterium tumefaciae containing the plant expression vector of the GUS fusion gene by using the vacuum immersion method.After co-culturing, the mulberry leaves were immersed in the GUS staining solution for staining, and finally decolorized with 70% (v/v) ethanol.

Transgenic Plants Inoculated with B. cinerea
To assess the resistance of transgenic Arabidopsis to B. cinerea (MM1 strain), a resistance test was conducted.Transgenic seeds were germinated on 1/2 Murashige and Skoog (MS) agar medium.After 7 days, the seedlings were transplanted into nutrient soil pots and cultivated under conditions of 24 • C/22 • C with a 16 h light and 8 h dark cycle.Mycelium fragments were then applied to the leaves of 21-day-old plants for the experiment.B. cinerea was initially cultured on a PDA plate and cultured in an incubator at 25 • C for 2~3 days.The fungus was then harvested from the edge of the B. cinerea fungus colony using a sterilized hole punch, and the fungus was inoculated on fresh and healthy Arabidopsis leaves.Before inoculation, we rinsed the leaf surface with distilled water and wiped the leaf surface dry.The diameter of the lesion was measured.The Arabidopsis plants were monitored at 12 h intervals post-inoculation, and photographs were taken 36 h later.

Evaluation of Resistance of Transgenic Plants to B. cinerea
Following the manufacturer's guidelines, the levels of malondialdehyde (MDA), proline, peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities were assessed using a plant detection kit (Solarbio, Beijing, China).Briefly, 0.1 g of plant sample was added to 1 mL of extraction liquid, homogenized, and then centrifuged; the supernatant was collected and finally determined by using a spectrophotometer.The levels of superoxide radicals (O 2 − ) and hydrogen peroxide (H 2 O 2 ) in the leaves were assessed through nitro blue tetrazolium (NBT) [32] and 3, 3 ′ -diaminobenzidine (DAB) staining [33].The leaf samples were vacuum-infiltrated with NBT solution, which reacts with superoxide anion radicals to form a dark blue formazan compound that is insoluble.After dyeing, the leaves were soaked in 70% ethanol (v/v) until all chlorophyll was completely removed.Another portion of the sample was immersed in DAB solution and stained for 12 h in the dark.Following this, these samples were also soaked in 70% ethanol (v/v) until all chlorophyll was entirely removed.

Statistical Analyses
The experiments in this study were replicated thrice.Data analysis was carried out using Excel 2021 (Microsoft, Redmond, WA, USA).The outcomes are presented as the mean ± standard deviation (SD).Statistical analysis was performed utilizing SPSS Statistics 26.0 software (SPSS Inc., Chicago, IL, USA), and graphical representations were generated using GraphPad Prism 10 software (GraphPad software Inc., La Jolla, CA, USA).

Phylogenetic Analysis of MnCYP710A11 Gene
Multiple alignment was performed with other plant CYP710A11 protein sequences obtained from the NCBI database.Subsequently, phylogenetic and molecular evolutionary analyses were conducted utilizing MEGA 7 to investigate the evolutionary connections among various species (Figure 1).The results indicated that CYP710A11 in mulberry was closely related to CYP710A11 in Malus domestica and more distantly related to CYP710A11 in Vitis vinifera and Helianthus annuus.levels of superoxide radicals (O2 − ) and hydrogen peroxide (H2O2) in the leaves were assessed through nitro blue tetrazolium (NBT) [32] and 3, 3′-diaminobenzidine (DAB) staining [33].The leaf samples were vacuum-infiltrated with NBT solution, which reacts with superoxide anion radicals to form a dark blue formazan compound that is insoluble.After dyeing, the leaves were soaked in 70% ethanol (v/v) until all chlorophyll was completely removed.Another portion of the sample was immersed in DAB solution and stained for 12 h in the dark.Following this, these samples were also soaked in 70% ethanol (v/v) until all chlorophyll was entirely removed.

Statistical Analyses
The experiments in this study were replicated thrice.Data analysis was carried out using Excel 2021 (Microsoft, Redmond, WA, USA).The outcomes are presented as the mean ± standard deviation (SD).Statistical analysis was performed utilizing SPSS Statistics 26.0 software (SPSS Inc., Chicago, IL, USA), and graphical representations were generated using GraphPad Prism 10 software (GraphPad software Inc., La Jolla, CA, USA).

Phylogenetic Analysis of MnCYP710A11 Gene
Multiple alignment was performed with other plant CYP710A11 protein sequences obtained from the NCBI database.Subsequently, phylogenetic and molecular evolutionary analyses were conducted utilizing MEGA 7 to investigate the evolutionary connections among various species (Figure 1).The results indicated that CYP710A11 in mulberry was closely related to CYP710A11 in Malus domestica and more distantly related to CYP710A11 in Vitis vinifera and Helianthus annuus.

B. cinerea-Induced MnCYP710A11 Expression
The expression level of MnCYP710A11 in mulberry seedlings infected with B. cinerea was examined using qRT-PCR analysis (Figure 2).Compared with 3 days of mock treatment, the expression level of MnCYP710A11 was significantly increased after 3 days of inoculation, consistent with our previous transcriptome findings [24].This suggests that the MnCYP710A11 gene could play a role in enhancing mulberry's resistance to B. cinerea.
was examined using qRT-PCR analysis (Figure 2).Compared with 3 days of mock treatment, the expression level of MnCYP710A11 was significantly increased after 3 days of inoculation, consistent with our previous transcriptome findings [24].This suggests that the MnCYP710A11 gene could play a role in enhancing mulberry's resistance to B. cinerea.

Ectopic Expression of MnCYP710A11
To further validate the role of the MnCYP710A11 gene in resistance, we introduced the MnCYP710A11 gene into Arabidopsis for heterologous expression.The cDNA of MnCYP710A11 was incorporated into Arabidopsis under the regulation of the Cauliflower mosaic virus 35S promoter, leading to the acquisition of T3 transgenic Arabidopsis plants following screening.First, the positive transgenic plants were verified by GUS staining.Following GUS staining, the transgenic plants exhibited a blue coloration (Figure 3a).Subsequent validation through qRT-PCR confirmed the overexpression of the MnCYP710A11 gene (Figure 3b).These outcomes affirm the successful overexpression of the MnCYP710A11 gene in Arabidopsis, resulting in the generation of positive transgenic plants.

Ectopic Expression of MnCYP710A11
To further validate the role of the MnCYP710A11 gene in resistance, we introduced the MnCYP710A11 gene into Arabidopsis for heterologous expression.The cDNA of Mn-CYP710A11 was incorporated into Arabidopsis under the regulation of the Cauliflower mosaic virus 35S promoter, leading to the acquisition of T3 transgenic Arabidopsis plants following screening.First, the positive transgenic plants were verified by GUS staining.Following GUS staining, the transgenic plants exhibited a blue coloration (Figure 3a).Subsequent validation through qRT-PCR confirmed the overexpression of the MnCYP710A11 gene (Figure 3b).These outcomes affirm the successful overexpression of the MnCYP710A11 gene in Arabidopsis, resulting in the generation of positive transgenic plants.

MnCYP710A11 Transgenic Plant Enhances Resistance to B. cinerea
To evaluate the resistance of Arabidopsis transgenic plants overexpressing MnCYP710A11 against B. cinerea, we conducted an experiment where transgenic Arabidopsis leaves were inoculated with agar blocks containing B. cinerea hyphae (Figure 4a).After 36 h of inoculation, while control leaves exhibited severe lesions, the leaves of MnCYP710A11 overexpression lines only showed mild lesions.Quantitative analysis

MnCYP710A11 Transgenic Plant Enhances Resistance to B. cinerea
To evaluate the resistance of Arabidopsis transgenic plants overexpressing MnCYP710A11 against B. cinerea, we conducted an experiment where transgenic Arabidopsis leaves were inoculated with agar blocks containing B. cinerea hyphae (Figure 4a).After 36 h of inoculation, while control leaves exhibited severe lesions, the leaves of MnCYP710A11 overexpression lines only showed mild lesions.Quantitative analysis clearly demonstrated that the overexpression of MnCYP710A11 in Arabidopsis effectively suppressed the infection induced by B. cinerea (Figure 4b).In addition, the production of reactive oxygen species represents a plant's reaction to stress.To determine this, we conducted DAB staining and NBT staining to detect the levels of hydrogen peroxide (H 2 O 2 ) and superoxide (O 2 − ) in the leaves, respectively (Figure 4c,d

Detection of Biochemical Indices
We assessed the variations in malondialdehyde (MDA) content under both normal conditions and during B. cinerea infection to assess cell membrane damage (Figure 5a).The findings revealed no notable disparity in MDA content between wild-type (WT) and transgenic Arabidopsis plants during normal growth conditions.However, the MDA content of WT and transgenic Arabidopsis increased 36 h after infection by B. cinerea compared with 0 h.Meanwhile, the MDA content of transgenic plants was significantly lower than that of WT plants after 36 h of infection with B. cinerea.These results indicate that plasma membrane damage is more pronounced in WT plants than in transgenic Arabidopsis.
During normal conditions, there was no substantial variance in proline content between wild-type (WT) Arabidopsis and those overexpressing MnCYP710A11 (Figure 5b).The proline content of wild-type and transgenic plants increased 36 h after B. cinerea infection compared with 0 h.At the same time, the proline content of transgenic Arabidopsis was significantly higher than that of wild-type plants 36 h after B. cinerea

Detection of Biochemical Indices
We assessed the variations in malondialdehyde (MDA) content under both normal conditions and during B. cinerea infection to assess cell membrane damage (Figure 5a).The findings revealed no notable disparity in MDA content between wild-type (WT) and transgenic Arabidopsis plants during normal growth conditions.However, the MDA content of WT and transgenic Arabidopsis increased 36 h after infection by B. cinerea compared with 0 h.Meanwhile, the MDA content of transgenic plants was significantly lower than that of WT plants after 36 h of infection with B. cinerea.These results indicate that plasma membrane damage is more pronounced in WT plants than in transgenic Arabidopsis.

Disease Resistance Analysis of Mulberry Seedlings Overexpressing MnCYP710A11
For a more in-depth exploration of MnCYP710A11's role in mulberry trees, a transient overexpression of MnCYP710A11 was induced in mulberry trees (Figure 6).Histochemical examination of β-glucuronidase (GUS) displayed intense GUS staining in the leaves of mulberry seedlings, confirming the successful overexpression of MnCYP710A11 in mulberry trees (Figure 6a).Compared with WT plants, mulberry trees overexpressing MnCYP710A11 showed increased resistance to B. cinerea (Figure 6b).Upon B. cinerea infection, the transient expression of MnCYP710A11 led to a notable reduction in malondialdehyde (MDA) content in mulberry seedlings (Figure 6c), while simultaneously enhancing proline content and catalase (CAT) activity (Figure 6d,e).These findings align with the earlier results observed in MnCYP710A11 transgenic Arabidopsis.During normal conditions, there was no substantial variance in proline content between wild-type (WT) Arabidopsis and those overexpressing MnCYP710A11 (Figure 5b).The proline content of wild-type and transgenic plants increased 36 h after B. cinerea infection compared with 0 h.At the same time, the proline content of transgenic Arabidopsis was significantly higher than that of wild-type plants 36 h after B. cinerea infection.
Catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) are crucial enzymes in plants that are pivotal in eliminating reactive oxygen species (ROS).During normal conditions, there were no notable variances in the activities of CAT, SOD, and POD between wild-type (WT) and transgenic Arabidopsis leaves (Figure 5c-e).However, the CAT, SOD, and POD activities of transgenic Arabidopsis were significantly increased compared with those of WT plants 36 h after infection with B. cinerea.

Disease Resistance Analysis of Mulberry Seedlings Overexpressing MnCYP710A11
For a more in-depth exploration of MnCYP710A11's role in mulberry trees, a transient overexpression of MnCYP710A11 was induced in mulberry trees (Figure 6).Histochemical examination of β-glucuronidase (GUS) displayed intense GUS staining in the leaves of mulberry seedlings, confirming the successful overexpression of MnCYP710A11 in mulberry trees (Figure 6a).Compared with WT plants, mulberry trees overexpressing MnCYP710A11 showed increased resistance to B. cinerea (Figure 6b).Upon B. cinerea infection, the transient expression of MnCYP710A11 led to a notable reduction in malondialdehyde (MDA) content in mulberry seedlings (Figure 6c), while simultaneously enhancing proline content and catalase (CAT) activity (Figure 6d,e).These findings align with the earlier results observed in MnCYP710A11 transgenic Arabidopsis.

Enhancement of AtBG2 Expression in MnCYP710A11 Transgenic Plants
β-1,3-glucanase 2 (BG2) serves as a marker gene associated with plant defense mechanisms.The findings revealed no notable variance in AtBG2 gene expression between transgenic Arabidopsis plants transfected with MnCYP710A11 and wild-type (WT) plants prior to B. cinerea infection (see Figure 7).However, compared with 0 h, AtBG2 expression levels in transgenic Arabidopsis plants transfected with MnCYP710A11 and WT plants were upregulated 36 h after infection with B. cinerea.At the same time, the expression level of transgenic plants was significantly higher than that of WT plants 36 h after infection with B. cinerea.These findings indicate that the introduction of the MnCYP710A11 gene into Arabidopsis can induce the expression of resistance-related genes to combat B. cinerea infection.

Enhancement of AtBG2 Expression in MnCYP710A11 Transgenic Plants
β-1,3-glucanase 2 (BG2) serves as a marker gene associated with plant defense mechanisms.The findings revealed no notable variance in AtBG2 gene expression between transgenic Arabidopsis plants transfected with MnCYP710A11 and wild-type (WT) plants prior to B. cinerea infection (see Figure 7).However, compared with 0 h, AtBG2 expression levels in transgenic Arabidopsis plants transfected with MnCYP710A11 and WT plants were upregulated 36 h after infection with B. cinerea.At the same time, the expression level of transgenic plants was significantly higher than that of WT plants 36 h after infection with B. cinerea.These findings indicate that the introduction of the MnCYP710A11 gene into Arabidopsis can induce the expression of resistance-related genes to combat B. cinerea infection.

Discussion
The identification of plant resistance genes is the basis of breeding resistant varieties.Transcriptomic analysis has been utilized to identify genes that govern plant resistance against pathogen infections [34,35].In our prior research, we conducted comparative transcriptomic analyses of mulberry trees after B. cinerea infection and obtained candidate genes that may regulate B. cinerea resistance.As a reactive enzyme, cytochrome P450 is extensively distributed among animals, plants, and microorganisms.Certain P450 enzymes have been documented to catalyze the production of diverse primary and secondary metabolites in plants.However, the roles of most cytochrome P450 genes in mulberry remain unknown.In this study, we chose the CYP450 protein-coding gene CYP710A11 to validate its role in disease resistance, thereby enhancing the precision of our findings.
Within plants, the CYP450 protein is categorized into 10 distinct clans spanning 61 families [36].In this study, we isolated and characterized a CYP450 gene, CYP710A11, from mulberry, classified under the CYP710 subfamily.The CYP710 family is widely distributed in plants, exhibiting diverse structures and functions, with its members playing a role in sterol biosynthesis [14,37,38].For example, overexpression of the WsCYP710A11 gene in transgenic hairy roots of Withania resulted in a significant elevation of withanolides and phytosterol levels [38].Although the role of CYP710 in sterol biosynthesis is well established, there is limited information on the disease resistance mechanisms of the CYP710 subfamily in plants.This study demonstrated that the overexpression of CYP710A11 improved the resistance of Arabidopsis and mulberry against B. cinerea.These findings offer fresh perspectives on the role of the P450 710 subfamily.
Reactive oxygen species (ROS) and ROS enzymes are crucial factors in enhancing plant resistance against B. cinerea.For example, ABA can reduce resistance to B. cinerea in tomatoes by reducing NO production, which also inhibits ROS and ethylene production [39].In addition, the disruption of cuticle integrity mediated by peroxide-dependent ROS accumulation plays an important role in the strong resistance of plants with altered homogalacturonan integrity to B. cinerea [40].ROS can harm cellular constituents such as lipids, proteins, and nucleic acids, leading to elevated levels of MDA, which serves as a marker for lipid peroxidation.[41].When faced with oxidative stress, plants trigger the activation of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) to eliminate reactive oxygen species (ROS) [42].Plants additionally

Discussion
The identification of plant resistance genes is the basis of breeding resistant varieties.Transcriptomic analysis has been utilized to identify genes that govern plant resistance against pathogen infections [34,35].In our prior research, we conducted comparative transcriptomic analyses of mulberry trees after B. cinerea infection and obtained candidate genes that may regulate B. cinerea resistance.As a reactive enzyme, cytochrome P450 is extensively distributed among animals, plants, and microorganisms.Certain P450 enzymes have been documented to catalyze the production of diverse primary and secondary metabolites in plants.However, the roles of most cytochrome P450 genes in mulberry remain unknown.In this study, we chose the CYP450 protein-coding gene CYP710A11 to validate its role in disease resistance, thereby enhancing the precision of our findings.
Within plants, the CYP450 protein is categorized into 10 distinct clans spanning 61 families [36].In this study, we isolated and characterized a CYP450 gene, CYP710A11, from mulberry, classified under the CYP710 subfamily.The CYP710 family is widely distributed in plants, exhibiting diverse structures and functions, with its members playing a role in sterol biosynthesis [14,37,38].For example, overexpression of the WsCYP710A11 gene in transgenic hairy roots of Withania resulted in a significant elevation of withanolides and phytosterol levels [38].Although the role of CYP710 in sterol biosynthesis is well established, there is limited information on the disease resistance mechanisms of the CYP710 subfamily in plants.This study demonstrated that the overexpression of CYP710A11 improved the resistance of Arabidopsis and mulberry against B. cinerea.These findings offer fresh perspectives on the role of the P450 710 subfamily.
Reactive oxygen species (ROS) and ROS enzymes are crucial factors in enhancing plant resistance against B. cinerea.For example, ABA can reduce resistance to B. cinerea in tomatoes by reducing NO production, which also inhibits ROS and ethylene production [39].In addition, the disruption of cuticle integrity mediated by peroxide-dependent ROS accumulation plays an important role in the strong resistance of plants with altered homogalacturonan integrity to B. cinerea [40].ROS can harm cellular constituents such as lipids, proteins, and nucleic acids, leading to elevated levels of MDA, which serves as a marker for lipid peroxidation.[41].When faced with oxidative stress, plants trigger the activation of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) to eliminate reactive oxygen species (ROS) [42].Plants additionally amass osmolytes like proline to uphold osmotic equilibrium and safeguard cellular structures against dehydration [43].In this study, in comparison to the WT, the parameters of Pro, SOD, CAT, and POD were significantly increased after infection with the gray mold, while the parameter of MDA was significantly decreased after the overexpression of CYP710A11.This indicates that the overexpression of MnCYP710A11 helps prevent cell membrane damage and enhances the capacity to preserve cell integrity in response to B. cinerea infection.Proline accumulation leads to hypersensitivity in incompatible interactions between plants and pathogens.At the same time, proline catabolism can act as a regulatory center of defense-related metabolism in many eukaryotes, connecting metabolic activities of different subcellular compartments and promoting and integrating ROS signals to regulate pathogen response [44].The overexpression of MnCYP710A11 may increase plant hypersensitivity by increasing proline content and may combine with ROS signaling pathways to enhance resistance against B. cinerea.
The level of callose (β-1,3-glucan) is controlled through the coordinated action of callose synthetase (CalSs) and β-1,3-glucanase [45,46].The deposition of callose between the plasma membrane (PM) and the cell wall serves as a complex defense mechanism exhibited by the plant host when encountering pathogen infection [47], for instance, stimulated by MAMP activation (such as bacterial flagellate epitopes like flg22 and chitosan), as well as by filamentous fungal attacks and physical injury [48][49][50].This callose accumulation helps fight diseases, such as fungal infections [51].The presence of β-1,3-glucanase in V. vinifera had an inhibitory effect on Plasmopara viticola [52].In our study, the BG2 gene associated with callose biosynthesis was upregulated in CYP710A11-OE plants, suggesting that this pathway is activated in CYP710A11-OE plants.

Conclusions
Our findings indicate that the CYP450 protein-coding gene CYP710A11 actively enhances the immune response in mulberry, thereby broadening our comprehension of the potential role of P450 proteins and offering valuable insights into the molecular mechanisms of plant immunity.Additionally, following the overexpression of MnCYP710A11, the resistance of mulberry to B. cinerea was augmented, thereby presenting significant genetic resources for the breeding of disease-resistant mulberry varieties.

Figure 2 .
Figure 2. The expression levels of MnCYP710A11 in mulberry leaves following mock treatment (Mock) and after inoculation with B. cinerea (Inoculated) was compared.The values represent averages, and the standard error (SE) is depicted as an error bar, based on three independent biological samples, each with three technical replicates (*** p < 0.001; two-tailed t-test).Mock, inoculated B. cinerea-free agar blocks; Inoculated, inoculated agar blocks containing B. cinerea.

Figure 2 .
Figure 2. The expression levels of MnCYP710A11 in mulberry leaves following mock treatment (Mock) and after inoculation with B. cinerea (Inoculated) was compared.The values represent averages, and the standard error (SE) is depicted as an error bar, based on three independent biological samples, each with three technical replicates (*** p < 0.001; two-tailed t-test).Mock, inoculated B. cinerea-free agar blocks; Inoculated, inoculated agar blocks containing B. cinerea.

Genes 2024 , 14 Figure 3 .
Figure 3.The identification of transgenic Arabidopsis.(a) Transgenic Arabidopsis GUS staining.(b) An assessment of the relative expression levels of MnCYP710A11 in transgenic Arabidopsis.WT, wild Arabidopsis; OE, MnCYP710A11 transgene Arabidopsis.The values represent averages, and the standard error (SE) is depicted as an error bar, based on three independent biological samples, each with three technical replicates (*** p < 0.001; two-tailed t-test).

Figure 3 .
Figure 3.The identification of transgenic Arabidopsis.(a) Transgenic Arabidopsis GUS staining.(b) An assessment of the relative expression levels of MnCYP710A11 in transgenic Arabidopsis.WT, wild Arabidopsis; OE, MnCYP710A11 transgene Arabidopsis.The values represent averages, and the standard error (SE) is depicted as an error bar, based on three independent biological samples, each with three technical replicates (*** p < 0.001; two-tailed t-test).
).The results showed that Arabidopsis plants transfected with MnCYP710A11 displayed minimal dark-brown patches after DAB staining, indicating lower levels of H 2 O 2 accumulation.Conversely, the WT Arabidopsis plants exhibited large dark-brown patches, suggesting higher H 2 O 2 accumulation.Similarly, NBT staining revealed minimal dark-blue patches in Arabidopsis plants transfected with MnCYP710A11, indicating lower levels of O 2 − accumulation, whereas large dark-blue patches were observed in the WT Arabidopsis plants, indicating higher O 2 − accumulation.Genes 2024, 15, x FOR PEER REVIEW 7 of 14

Figure 4 .
Figure 4. Evaluation of transgenic Arabidopsis resistance to B. cinerea.(a) Observation of leaf morphology in Arabidopsis 36 h post-infection with B. cinerea.(b) Quantitative assessment of resistance in transgenic Arabidopsis.(c) DAB staining showed H2O2 levels.(d) NBT staining showed O2 − levels.Values represent averages, and standard error (SE) is depicted as error bar, based on three independent biological samples, each with three technical replicates (*** p < 0.001; two-tailed t-test).

Figure 4 .
Figure 4. Evaluation of transgenic Arabidopsis resistance to B. cinerea.(a) Observation of leaf morphology in Arabidopsis 36 h post-infection with B. cinerea.(b) Quantitative assessment of resistance in transgenic Arabidopsis.(c) DAB staining showed H 2 O 2 levels.(d) NBT staining showed O 2 − levels.Values represent averages, and standard error (SE) is depicted as error bar, based on three independent biological samples, each with three technical replicates (*** p < 0.001; two-tailed t-test).

Figure 5 .
Figure 5.The determined physicochemical indexes before and after B. cinerea inoculation.(a) MDA content, (b) proline content, (c) CAT activity, (d) SOD activity, and (e) POD activity.The values represent averages, and the standard error (SE) is depicted as an error bar, based on three independent biological samples, each with three technical replicates (* p < 0.05, ** p < 0.01, and *** p < 0.001; two-tailed t-test).

Figure 5 .
Figure 5.The determined physicochemical indexes before and after B. cinerea inoculation.(a) MDA content, (b) proline content, (c) CAT activity, (d) SOD activity, and (e) POD activity.The values represent averages, and the standard error (SE) is depicted as an error bar, based on three independent biological samples, each with three technical replicates (* p < 0.05, ** p < 0.01, and *** p < 0.001; two-tailed t-test).

Figure 6 .
Figure 6.The resistance of MnCYP710A11 to B. cinerea was analyzed through transient expression in mulberry.(a) GUS staining of WT plants and transient overexpression after 72 h in mulberry leaves.WT, wild-type; Control, empty vector.(b) The photos were taken 72 h after infection with B. cinerea of mulberry leaves.(c) MDA content, (d) proline content, and (e) CAT activity.The values are averages and the standard error (SE) is represented by a bar representing three independent biological samples with three technical replicates per sample (** p < 0.01 and *** p < 0.001; two-tailed t-test).ns, no significant difference.

Figure 6 .
Figure 6.The resistance of MnCYP710A11 to B. cinerea was analyzed through transient expression in mulberry.(a) GUS staining of WT plants and transient overexpression after 72 h in mulberry leaves.WT, wild-type; Control, empty vector.(b) The photos were taken 72 h after infection with B. cinerea of mulberry leaves.(c) MDA content, (d) proline content, and (e) CAT activity.The values are averages and the standard error (SE) is represented by a bar representing three independent biological samples with three technical replicates per sample (** p < 0.01 and *** p < 0.001; two-tailed t-test).ns, no significant difference.

Figure 7 .
Figure 7.The relative expression of AtBG2 in leaves of Arabidopsis WT and MnCYP710A11 transgenic (OE) before and after inoculation with B. cinereal.The values are averages and the standard error (SE) is represented by a bar representing three independent biological samples with three technical replicates per sample (* p < 0.05; two-tailed t-test).

Figure 7 .
Figure 7.The relative expression of AtBG2 in leaves of Arabidopsis WT and MnCYP710A11 transgenic (OE) before and after inoculation with B. cinereal.The values are averages and the standard error (SE) is represented by a bar representing three independent biological samples with three technical replicates per sample (* p < 0.05; two-tailed t-test).