CaWRKY50 Acts as a Negative Regulator in Response to Colletotrichum scovillei Infection in Pepper

Chili anthracnose is one of the most common and destructive fungal pathogens that affects the yield and quality of pepper. Although WRKY proteins play crucial roles in pepper resistance to a variety of pathogens, the mechanism of their resistance to anthracnose is still unknown. In this study, we found that CaWRKY50 expression was obviously induced by Colletotrichum scovillei infection and salicylic acid (SA) treatments. CaWRKY50-silencing enhanced pepper resistance to C. scovillei, while transient overexpression of CaWRKY50 in pepper increased susceptibility to C. scovillei. We further found that overexpression of CaWRKY50 in tomatoes significantly decreased resistance to C. scovillei by SA and reactive oxygen species (ROS) signaling pathways. Moreover, CaWRKY50 suppressed the expression of two SA-related genes, CaEDS1 (enhanced disease susceptibility 1) and CaSAMT1 (salicylate carboxymethyltransferase 1), by directly binding to the W-box motif in their promoters. Additionally, we demonstrated that CaWRKY50 interacts with CaWRKY42 and CaMIEL1 in the nucleus. Thus, our findings revealed that CaWRKY50 plays a negative role in pepper resistance to C. scovillei through the SA-mediated signaling pathway and the antioxidant defense system. These results provide a theoretical foundation for molecular breeding of pepper varieties resistant to anthracnose.


Introduction
Pepper (Capsicum annuum L.) is one of the most commercially significant vegetables in the world [1]. However, pepper cultivation is often damaged by pathogenic fungi, including chili anthracnose caused by Colletotrichum spp. [2]. Among these fungi, C. scovillei is one of the most devastating fungal diseases in pepper [3]. Currently, traditional chemical treatments are widely used to control chili anthracnose, but they are harmful to the environment and can negatively impact food safety. The breeding of resistant pepper varieties is the most effective and economical approach to controlling plant pathogens [4]. Therefore, it is important to study the mechanisms of pepper resistance to anthracnose.
Salicylic acid (SA) serves as a vital hormone for plant defense and plays a crucial role in many aspects of plant immunity. SA is involved not only in systemic acquired resistance (SAR) but also in PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) [5]. The PTI and ETI immune systems cooperate to promote downstream responses, leading to the production of ROS and plant hormones, as well as the activation of a hypersensitive response (HR) to trigger associated defense mechanisms [6]. When plants are attacked by pathogens, SA stimulates the synthesis of pathogenesis-related (PR) proteins involved in plant defense [7]. Under stress combinations, plants can enhance their levels of antioxidant enzymes to decrease ROS (particularly H 2 O 2 and O 2 − ) accumulation and reduce their damage by maintaining a dynamic balance [8].

Characterization of CaWRKY50
To explore the potential function of CaWRKY50 in response to anthracnose, we used qRT-PCR to investigate any change in CaWRKY50 expression in the susceptible cv. R25 under C. scovillei infection. We found that CaWRKY50 expression was significantly induced from 1 to 7 dpi compared to before infection, indicating that CaWRKY50 was involved in disease resistance following C. scovillei infection in pepper fruits (Figure 1a). The coding sequence of CaWRKY50 (Capana10g001548) was cloned from cv. R25 and encodes a protein consisting of 232 amino acids. Phylogenetic analysis revealed that CaWRKY50 was highly identical to NtWRKY70 (XP_016492893.1) and homologous to AtWRKY54 (AT2G40750.1), SlWRKY81 (NP_001266272.1), StWRKY6 (NP_001275414.1), VvWRKY70 (XP_002275401.1), CsWRKY70 (KAH9673391.1), OsWRKY41 (XP_015638413.1), and TaWRKY19 (XP_04438106) based on BLAST analysis in the NCBI database ( Figure 1b). The CaWRKY50 protein contained one conserved WRKYGQK domain, a C2HC zinc finger, and was clustered in subgroup III members (Figure 1c). The CaWRKY50 protein contained one conserved WRKYGQK domain, a C2HC zinc finger, and was clustered in subgroup III members (Figure 1c).  (AY486137) was used as the internal control. Data are shown as means (±SD) from three biological replicates. Significant differences are shown by asterisks (* p < 0.05, ** p < 0.01, Student's t test). (f) Localization of the CaWRKY50 protein in N. benthamiana epidermal cells (Scale bar = 50 µm). The 35S:GFP protein is used as a control. (g) Transcriptional activation of CaWRKY50 in the yeast. The positive control is pGBKT7-53 and pGADT7-T, and the negative control is pGBKT7-Lam and pGADT7-T.
To understand the effect of SA and jasmonic acid (JA) on pepper fruit resistance against C. scovillei, we analyzed the expression patterns of the CaWRKY50 gene after treatment with 5 mM SA and 100 µM JA. The results show that CaWRKY50 was strongly induced at 3-12 hpt with 5 mM SA but did not respond to 100 µM JA (Figure 1d). We also analyzed the transcript levels of CaWRKY50 following C. scovillei infection after exogenous 5 mM SA treatment using qRT-PCR. The results indicate that CaWRKY50 expression was significantly decreased compared to the control at 2 and 7 dpi (Figure 1e). These findings imply that the response of CaWRKY50 to C. scovillei may be related to SA signaling pathways.
Sequence analysis using the PSORT online program revealed that the predicted CaWRKY50 protein contains two putative nuclear localization signals (Figure 1c). To confirm this speculation, the recombinant CaWRKY50-GFP protein was transiently expressed in tobacco epidermal cells. The results show that the CaWRKY50-GFP protein only appeared in the nucleus, while the empty GFP protein was present throughout the cell (Figure 1f). To analyze the transcriptional activity of CaWRKY50, we used the yeast two-hybrid (Y2H) system. The positive control grew well and turned blue in the SD/-Leu/-Trp/-Ade/-His medium with X-α-gal (20 µg mL −1 ), while pGBKT7-CaWRKY50 and the negative control did not express X-α-gal activity (Figure 1g). These results suggest that CaWRKY50 has no transcriptional activity in yeast cells.

CaWRKY50 Silencing Improves Pepper Resistance to C. scovillei
To investigate the function of CaWRKY50 in anthracnose resistance, we downregulated its expression on detached pepper fruits (cv. R25) using virus-induced gene silencing (VIGS) methods. CaWRKY50 expression was significantly lower in silenced pepper fruits compared to control fruits at 15 days post-infiltration, which indicates that CaWRKY50 were successfully silenced ( Figure 2a). The phenotypic symptoms and lesion diameters of CaWRKY50-silenced fruits were considerably smaller in comparison to control fruits at 7 dpi (Figure 2b,c). Furthermore, the contents of malondialdehyde (MDA) and H 2 O 2 in CaWRKY50-silenced fruits were remarkably lower than those in control fruits at 7 dpi (Figure 2d,e). Conversely, the catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities of CaWRKY50-silenced fruits were significantly higher at 7 dpi (Figure 2f-h).
Furthermore, the expression of CaWRKY50 was markedly lower in CaWRKY50-silenced fruits compared to control fruits at 4 and 7 dpi (Figure 3a). To determine whether CaWRKY50 affects the expression of SA and ROS defense-related genes in pepper, qRT-PCR was used to evaluate the expression levels of these genes at various days after C. scovillei infection. The transcript levels of CaPR1, CaPR10, CaSAR8.2, and CaPO1 (SA-related genes) were significantly increased in CaWRKY50-silenced fruits relative to control fruits at 7 dpi (Figure 3b-e). Similarly, the transcript patterns of CaCAT, CaPOD, and CaSOD (ROS-related genes) were consistent with the SA-related genes (Figure 3f-h). Overall, these results indicate that silencing CaWRKY50 improves the resistance of pepper fruits to C. scovillei.
CaWRKY50-silenced fruits were considerably smaller in comparison to control fruits at 7 dpi (Figure 2b,c). Furthermore, the contents of malondialdehyde (MDA) and H2O2 in CaWRKY50-silenced fruits were remarkably lower than those in control fruits at 7 dpi (Figure 2d,e). Conversely, the catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities of CaWRKY50-silenced fruits were significantly higher at 7 dpi (Figure 2f-h). Furthermore, the expression of CaWRKY50 was markedly lower in CaWRKY50-silenced fruits compared to control fruits at 4 and 7 dpi (Figure 3a). To determine whether CaWRKY50 affects the expression of SA and ROS defense-related genes in pepper, qRT-PCR was used to evaluate the expression levels of these genes at various days after C. scovillei infection. The transcript levels of CaPR1, CaPR10, CaSAR8.2, and CaPO1 (SA-related genes) were significantly increased in CaWRKY50-silenced fruits relative to control  Overall, these results indicate that silencing CaWRKY50 improves the resistance of pepper fruits to C. scovillei.

Transient CaWRKY50 Overexpression Enhances Susceptibility to C. scovillei in Pepper
To further explore the role of CaWRKY50 in disease resistance to anthracnose, we performed transient overexpression of CaWRKY50 in detached pepper fruits (cv. R25) and

Transient CaWRKY50 Overexpression Enhances Susceptibility to C. scovillei in Pepper
To further explore the role of CaWRKY50 in disease resistance to anthracnose, we performed transient overexpression of CaWRKY50 in detached pepper fruits (cv. R25) and infected them with C. scovillei. CaWRKY50 was successfully overexpressed in the pepper fruits, as confirmed by qRT-PCR analysis at 3 days post-infiltration (Figure 4a). The disease symptoms and lesion diameters of CaWRKY50-overexpressing fruits were markedly larger than those of control fruits at 7 dpi (Figure 4b,c). Additionally, MDA and H 2 O 2 levels in CaWRKY50-overexpressing fruits were dramatically increased compared to the control fruits at 7 dpi (Figure 4d,e). Furthermore, the CAT, POD, and SOD activities of CaWRKY50-overexpressing fruits were significantly lower than those of control fruits at 7 dpi (Figure 4f  Additionally, CaWRKY50 expression was obviously increased in CaWRKY50-overexpressing fruits compared to control fruits at 4 and 7 dpi (Figure 5a). To understand whether CaWRKY50 regulates the expression of SA and ROS-related defense genes, we analyzed the transcript levels of these genes in CaWRKY50-overexpressing fruits as well as control fruits at various days after C. scovillei infection. The transcript levels of CaPR1, CaPR10, CaSAR8.2, and CaPO1 (SA-related genes) were downregulated in the CaWRKY50-overexpressing fruits relative to the control at 7 dpi (Figure 5b-e). Similarly, the expression patterns of CaCAT, CaPOD and CaSOD (ROS-related genes) were consistent with the SA-related genes under C. scovillei infection at 7 dpi (Figure 5f-h). Taken together, our data show that CaWRKY50 negatively regulates pepper fruit resistance to C. scovillei. Additionally, CaWRKY50 expression was obviously increased in CaWRKY50-overexpressing fruits compared to control fruits at 4 and 7 dpi (Figure 5a). To understand whether CaWRKY50 regulates the expression of SA and ROS-related defense genes, we analyzed the transcript levels of these genes in CaWRKY50-overexpressing fruits as well as control fruits at various days after C. scovillei infection. The transcript levels of CaPR1, CaPR10, CaSAR8.2, and CaPO1 (SA-related genes) were downregulated in the CaWRKY50overexpressing fruits relative to the control at 7 dpi (Figure 5b-e). Similarly, the expression patterns of CaCAT, CaPOD and CaSOD (ROS-related genes) were consistent with the SArelated genes under C. scovillei infection at 7 dpi (Figure 5f-h). Taken together, our data show that CaWRKY50 negatively regulates pepper fruit resistance to C. scovillei. CaPR10, CaSAR8.2, and CaPO1 (SA-related genes) were downregulated in the CaWRKY50-overexpressing fruits relative to the control at 7 dpi (Figure 5b-e). Similarly, the expression patterns of CaCAT, CaPOD and CaSOD (ROS-related genes) were consistent with the SA-related genes under C. scovillei infection at 7 dpi (Figure 5f-h). Taken together, our data show that CaWRKY50 negatively regulates pepper fruit resistance to C. scovillei.

Overexpression of CaWRKY50 in Tomato Decreases Resistance to C. scovillei
To better understand the function of CaWRKY50 in anthracnose resistance, CaWRKY50 was stably expressed in tomato by Agrobacterium-mediated genetic transformation. Three transgenic T 2 lines (OE-97, OE-110, and OE-113) with high levels of CaWRKY50 transcript ( Figure 6a) were infected with C. scovillei to assess the role of CaWRKY50 in anthracnose resistance. At 3 dpi, the transgenic fruits exhibited greater lesion diameters than WT fruits (Figure 6b,c). MDA and H 2 O 2 content were evaluated in OE and WT fruits, and compared to WT fruits, MDA and H 2 O 2 levels were greatly increased in OE fruits at 3 dpi (Figure 6d,e). Moreover, the activities of CAT, POD, and SOD were obviously lower in OE fruits than WT fruits at 3 dpi (Figure 6f-h).
Additionally, we analyzed the expression levels of SA and ROS related defense genes in both CaWRKY50-overexpressing and control fruits. The transcript patterns of SA-related genes SlPR1, SlNPR1, and SlSABP2 were markedly downregulated in CaWKRY50 OE fruits compared to WT fruits at 3 dpi (Figure 6i-k). Consistently, the transcript patterns of the ROS-related genes SlPOD, SlAPX2, and SlCAT were also significantly downregulated in OE fruits relative to WT fruits at 3 dpi (Figure 6l-n). In conclusion, our studies demonstrate that CaWRKY50 has a negative regulatory effect on tomato fruit resistance to C. scovillei.

CaWRKY50 Specifically Binds the Promoter of CaEDS1 and CaSAMT1
To identify which genes are directly regulated by CaWRKY50 for anthracnose resistance in pepper, we looked for two SA-related defense genes (CaEDS1 and CaSAMT1), whose promoters contain W-box elements (TTGACC/T). Subsequently, we performed yeast one-hybrid (Y1H) assays to confirm whether CaWRKY50 binds to the promoter of CaEDS1 and CaSAMT1. We transformed CaWRKY50-pGADT7 into the Y1H strain by integrating the proCaEDS1/proCaSAMT1-pAbAi vector, which produced yeast cells that grew well on SD/-Leu medium with 150 and 250 ng ml −1 AbA. In contrast, the negative control failed to grow (Figure 7a). The Y1H assay confirmed that CaWRKY50 specifically binds to the promoters of CaEDS1 and CaSAMT1.
To investigate the effect of CaWRKY50 on the promoters of CaEDS1 and CaSAMT1, we performed GUS activity and dual-luciferase assays in tobacco leaves. The staining

CaWRKY50 Specifically Binds the Promoter of CaEDS1 and CaSAMT1
To identify which genes are directly regulated by CaWRKY50 for anthracnose resistance in pepper, we looked for two SA-related defense genes (CaEDS1 and CaSAMT1), whose promoters contain W-box elements (TTGACC/T). Subsequently, we performed yeast one-hybrid (Y1H) assays to confirm whether CaWRKY50 binds to the promoter of CaEDS1 and CaSAMT1. We transformed CaWRKY50-pGADT7 into the Y1H strain by integrating the proCaEDS1/proCaSAMT1-pAbAi vector, which produced yeast cells that grew well on SD/-Leu medium with 150 and 250 ng ml −1 AbA. In contrast, the negative control failed to grow (Figure 7a). The Y1H assay confirmed that CaWRKY50 specifically binds to the promoters of CaEDS1 and CaSAMT1.
To investigate the effect of CaWRKY50 on the promoters of CaEDS1 and CaSAMT1, we performed GUS activity and dual-luciferase assays in tobacco leaves. The staining intensity and activity of GUS were significantly decreased compared to the control when CaWRKY50-pGADT7 was co-expressed with the proCaEDS1/proCaSAMT1-GUS vector in tobacco leaves (Figure 7b,c). Furthermore, we observed that the luciferase activity was significantly inhibited compared to control when CaWRKY50:62-SK was co-expressed with the proCaEDS1/proCaSAMT1-LUC vector in tobacco leaves (Figure 7d,e). Taken together, these results indicate that CaWRKY50 specifically binds to the promoters of CaEDS1 and CaSAMT1, suppressing their expression in response to C. scovillei.
x FOR PEER REVIEW 9 of 18 intensity and activity of GUS were significantly decreased compared to the control when CaWRKY50-pGADT7 was co-expressed with the proCaEDS1/proCaSAMT1-GUS vector in tobacco leaves (Figure 7b,c). Furthermore, we observed that the luciferase activity was significantly inhibited compared to control when CaWRKY50:62-SK was co-expressed with the proCaEDS1/proCaSAMT1-LUC vector in tobacco leaves (Figure 7d,e). Taken together, these results indicate that CaWRKY50 specifically binds to the promoters of CaEDS1 and CaSAMT1, suppressing their expression in response to C. scovillei.

CaWRKY50 Interacts with CaWRKY42 and CaMIEL1
In order to identify the interacting partner of CaWRKY50 in the regulation of pepper defense responses, we screened a pepper cDNA library by Y2H assay and isolated CaWRKY42 (Capana08g001044) and CaMIEL1 (XM_016719768) as CaWRKY50-interacting proteins. CaMIEL1 encodes a RING E3 ubiquitin-protein ligase. Subsequently, we carried out a Y2H assay to confirm whether CaWRKY50 interacted with CaWRKY42/CaMIEL1. The yeast cells co-transformed with CaWRKY50-pGBKT7 and CaWRKY42/CaMIEL1-pGADT7 plasmid combinations were viable on SD/-Leu/-Trp/-Ade/-His medium and turned blue in X-α-gal (20 µg mL −1 ) (Figure 8a). This confirmed The LUC to REN ratio was assessed as transcriptional activity. The empty vector 62-SK was used as a control. Data are shown as means (±SD) from three biological replicates. Significant differences are shown by asterisks (* p < 0.05, ** p < 0.01, Student's t test).

CaWRKY50 Interacts with CaWRKY42 and CaMIEL1
In order to identify the interacting partner of CaWRKY50 in the regulation of pepper defense responses, we screened a pepper cDNA library by Y2H assay and isolated CaWRKY42 (Capana08g001044) and CaMIEL1 (XM_016719768) as CaWRKY50-interacting proteins. CaMIEL1 encodes a RING E3 ubiquitin-protein ligase. Subsequently, we carried out a Y2H assay to confirm whether CaWRKY50 interacted with CaWRKY42/CaMIEL1. The yeast cells co-transformed with CaWRKY50-pGBKT7 and CaWRKY42/CaMIEL1-pGADT7 plasmid combinations were viable on SD/-Leu/-Trp/-Ade/-His medium and turned blue in X-α-gal (20 µg mL −1 ) (Figure 8a). This confirmed that CaWRKY50 strongly interacted with CaWRKY42 and CaMIEL1.
Moreover, we performed luciferase complementation imaging (LCI) and bimolecular fluorescence complementation (BiFC) assays to confirm the interactions in living plant cells. In the LCl assay, we transiently co-expressed CaWRKY50-nLUC and CaWRKY42/CaMIEL1-cLUC in tobacco leaves and found luminescence signals in the co-expressed CaWRKY50-nLUC and CaWRKY42/CaMIEL1-cLUC regions but not in controls (Figure 8b). In the BiFC assay, we transiently co-expressed CaWRKY50-nYFP and CaWRKY42/CaMIEL1-cYFP in tobacco leaves and observed the interaction between CaWRKY50 and CaWRKY42/CaMIEL1 generating YFP fluorescence in the nucleus (Figure 8c). In summary, our data demonstrate that CaWRKY50 interacts with CaWRKY42/CaMIEL1 in the nucleus. Moreover, we performed luciferase complementation imaging (LCI) and bimolecular fluorescence complementation (BiFC) assays to confirm the interactions in living plant cells. In the LCl assay, we transiently co-expressed CaWRKY50-nLUC and CaWRKY42/CaMIEL1-cLUC in tobacco leaves and found luminescence signals in the coexpressed CaWRKY50-nLUC and CaWRKY42/CaMIEL1-cLUC regions but not in controls (Figure 8b). In the BiFC assay, we transiently co-expressed CaWRKY50-nYFP and CaWRKY42/CaMIEL1-cYFP in tobacco leaves and observed the interaction between CaWRKY50 and CaWRKY42/CaMIEL1 generating YFP fluorescence in the nucleus (Figure 8c). In summary, our data demonstrate that CaWRKY50 interacts with CaWRKY42/CaMIEL1 in the nucleus.

Discussion
WRKY proteins play a critical role in plant resistance to various pathogens. According to the Zunla-1 genome sequence, a total of 62 WRKY genes have been identified in pepper [30]. In our study, we selected CaWRKY50 as a candidate gene based on previous RNA-seq and VIGS analysis. Sequence analysis indicated that CaWRKY50 is highly homologous with NtWRKY70 in tobacco, AtWRKY54 in Arabidopsis, and CsWRKY70 in citrus. These proteins belong to subgroup III members (Figure 1b,c), for which AtWRKY54 plays a positive regulatory role in pathogen infection and a negative regulatory role in leaf senescence [31,32]. CsWRKY70 actively participates in SA-mediated disease resistance by regulating the transcription of the CsSAMT genes [11]. Thus, it was speculated that CaWRKY50 might also be involved in plant disease resistance. However, the underlying molecular mechanism of CaWRKY50 in pepper anthracnose resistance remains unknown, and further investigation is needed to understand its function.
In this study, a transactivation assay was used to investigate CaWRKY50's role in transcriptional activity, and it was found that it did not possess transcriptional activity in yeast cells (Figure 1g). Similar findings have been reported for grapevine VvWRKY8 [33],

Discussion
WRKY proteins play a critical role in plant resistance to various pathogens. According to the Zunla-1 genome sequence, a total of 62 WRKY genes have been identified in pepper [30]. In our study, we selected CaWRKY50 as a candidate gene based on previous RNA-seq and VIGS analysis. Sequence analysis indicated that CaWRKY50 is highly homologous with NtWRKY70 in tobacco, AtWRKY54 in Arabidopsis, and CsWRKY70 in citrus. These proteins belong to subgroup III members (Figure 1b,c), for which AtWRKY54 plays a positive regulatory role in pathogen infection and a negative regulatory role in leaf senescence [31,32]. CsWRKY70 actively participates in SA-mediated disease resistance by regulating the transcription of the CsSAMT genes [11]. Thus, it was speculated that CaWRKY50 might also be involved in plant disease resistance. However, the underlying molecular mechanism of CaWRKY50 in pepper anthracnose resistance remains unknown, and further investigation is needed to understand its function.
In this study, a transactivation assay was used to investigate CaWRKY50's role in transcriptional activity, and it was found that it did not possess transcriptional activity in yeast cells (Figure 1g). Similar findings have been reported for grapevine VvWRKY8 [33], maize ZmWRKY17 [34], soybean GmWRKY13, GmWRKY27, GmWRKY40, GmWRKY54 [35], and Brachypodium distachyon BdWRKY36 [36]. This phenomenon could be explained by the fact that the activation of these proteins may be dependent on posttranslational modifications or require activation by unknown upstream proteins [37].
In this study, the transcript levels of CaWRKY50 were markedly induced by C. scovillei and SA treatments, as shown in Figure 1a,d. By performing VIGS, we found that CaWRKY50-silencing enhanced pepper resistance to C. scovillei inoculation (Figures 2 and 3). Also, CaWRKY50 overexpression resulted in enhanced susceptibility to infection with C. scovillei in pepper and tomato fruits (Figures 4-6). These results suggest that CaWRKY50 acts as a negative regulator of pepper resistance to C. scovillei.
Recently, studies have revealed that plants can actively generate ROS, which can affect a variety of physiological processes, including biotic and abiotic stress as well as pathogen defense [8]. To counteract ROS damage, plants possess a comprehensive antioxidative system that reduces ROS damage and maintains cell homeostasis [38]. Malondialdehyde (MDA) is the main product of membrane lipid peroxidation and is commonly used as a marker for oxidative stress in plants [39]. For instance, overexpression of CaCIPK13 in pepper enhanced cold tolerance by regulating the antioxidant defense system, which increased CAT, POD, and SOD activities while decreasing MDA and H 2 O 2 content [40]. Similarly, CaCIPK3-overexpression improved drought stress resistance in pepper by increasing CAT, POD, and SOD activities, while decreasing MDA and H 2 O 2 content [41]. Hence, we measured the contents of MDA and H 2 O 2 as well as the levels of ROS scavenging enzymes (CAT, POD, and SOD) in CaWRKY50-overexpressing and CaWRKY50silenced fruits under C. scovillei infection. Our results revealed that the contents of MDA and H 2 O 2 in CaWRKY50-overexpressing fruits were noticeably higher than control fruits under C. scovillei infection (Figures 4 and 6). In contrast, the results were reversed in CaWRKY50-silenced fruits, as shown in Figure 2. Furthermore, the activities of CAT, POD, and SOD in CaWRKY50-overexpressing fruits were significantly lower than control fruits under C. scovillei infection (Figures 4 and 6), which was contrary to CaWRKY50-silenced fruits (Figure 2). To corroborate these findings, we measured the expression of ROSscavenging genes (CaCAT/SlCAT, CaPOD/SlPOD, CaSOD, and SlAPX2) using qRT-PCR and found that the results were consistent with ROS-scavenging enzyme activity results (Figures 3, 5, and 6). These findings suggest that CaWRKY50 plays a crucial role in regulating oxidative stress caused by C. scovillei infection.
SA signaling is known to play a key role in regulating the defense response of plants to pathogenic infections. Previous studies have shown that SA-related defense genes can be induced by SA to modulate plant defense responses to pathogen infection. [42]. Previous results indicated that CaSBP12, CaSBP08, and CaSBP11 negatively regulated pepper resistance to phytophthora capsici by inhibiting the transcript of SA-related defense genes [39,43,44]. In another study, VaERF16-overexpressing improved disease resistance to B. cinerea in Arabidopsis thaliana by increasing the expression of AtPR1 and AtNPR1 [45]. Our research revealed that the transcript levels of CaPR1, CaPR10, CaSAR8.2, and CaPO1 were significantly downregulated in transient CaWRKY50-overexpression pepper fruits in comparison to control fruits at 7 dpi. (Figure 5). In contrast, these genes were dramatically upregulated in CaWRKY50-silenced pepper fruits at 7 dpi ( Figure 3). Furthermore, the expression of SlPR1, SlNPR1, and SlSABP2 in CaWRKY50-overexpressing tomato fruits was markedly lower than in control fruits at 7 dpi (Figure 6i-k). In summary, these results indicate that CaWRKY50 acts as a negative regulator in response to C. scovillei through the SA signaling pathway.
WRKY TFs can specifically bind to W-box cis-elements (TTGACC/T) to regulate the expression of defense-associated genes. The expression of SA signaling-related genes (TGA2 and TGA6) was increased by WRKY70, which directly bound their promoter elements to regulate Verticillium dahliae toxin infection [46]. In the absence of a pathogen, WRKY70 is directly bound to the promoter of SARD1 to repress its expression and regulate the expression of SA-related genes [47]. In another study, MdWRKY17 specifically bound to the MdDMR6 promoter to enhance its expression, which negatively regulated resistance to C. fructicola in apples [17]. In the present study, we found that CaWRKY50 specifically binds to the promoter of CaEDS1 and CaSAMT1 by Y1H (Figure 7a). To elucidate how CaWRKY50 regulates the expression of CaEDS1 and CaSAMT1, we conducted GUS activity and dual-luciferase assays, which demonstrate that CaWRKY50 could inhibit CaEDS1 and CaSAMT1 transcription (Figure 7b-e). In plants, SAMT catalyzes SA to produce MeSA. CsWRKY70 specifically binds to the promoter of CsSAMT and activates its expression, thereby positively regulating resistance to Penicillium digitatum in citrus fruit [11]. EDS1 plays an important function in plant basal defense and the SA signaling pathway [12]. PcAvh103 interacts with EDS1 to inhibit plant immunity by the EDS1-PAD4 signaling pathway [48]. These findings suggest that CaWRKY50 can suppress the expression of CaEDS1 and CaSAMT1 to reduce disease resistance.
WRKY proteins can interact with other proteins to regulate plant immunity [14]. For example, WRKY33 interacts with WRKY12 to increase hypoxia tolerance in Arabidopsis [49]. JrWRKY21 was shown to improve walnut resistance to C. gloeosporioides by interacting with JrPTI5L (a PR protein) [50]. SlWRKY31 interacts with SlVQ15 to regulate the resistance to B. cinerea in tomatoes [51]. It is plausible that the CaWRKY50 protein also interacts with other defense proteins in response to various pathogen infections. In this study, we determined the interaction between CaWRKY50 with CaWRKY42/CaMIEL1 through Y2H, LCI, and BiFC assays (Figure 8). A previous study showed that CaWRKY41 (CA08g08240), which is the same gene as CaWRKY42 (Capana08g001044) in our study, could positively regulate pepper resistance to R. solanacearum inoculation [25]. CaMIEL1, an E3 ubiquitin ligase, inhibited tolerance to bacterial inoculation in Arabidopsis by degrading MYB30 [52]. These results suggest that CaWRKY50 may regulate resistance to C. scovillei by interacting with CaWRKY42 and CaMIEL1.
In conclusion, using overexpression and silenced techniques, we demonstrated that CaWRKY50 serves as a negative regulator in response to C. scovillei through the SA and ROS signaling pathways. CaWRKY50 directly binds to the promoters of CaEDS1 and CaSAMT1 to suppress their expression. Furthermore, CaWRKY50 participates in disease resistance by interacting with CaWRKY42 and CaMIEL1 (Figure 9). Our findings shed light on the mechanisms by which WRKY50 functions in response to anthracnose resistance. bound to the MdDMR6 promoter to enhance its expression, which negatively regulated resistance to C. fructicola in apples [17]. In the present study, we found that CaWRKY50 specifically binds to the promoter of CaEDS1 and CaSAMT1 by Y1H (Figure 7a). To elucidate how CaWRKY50 regulates the expression of CaEDS1 and CaSAMT1, we conducted GUS activity and dual-luciferase assays, which demonstrate that CaWRKY50 could inhibit CaEDS1 and CaSAMT1 transcription (Figure 7b-e). In plants, SAMT catalyzes SA to produce MeSA. CsWRKY70 specifically binds to the promoter of CsSAMT and activates its expression, thereby positively regulating resistance to Penicillium digitatum in citrus fruit [11]. EDS1 plays an important function in plant basal defense and the SA signaling pathway [12]. PcAvh103 interacts with EDS1 to inhibit plant immunity by the EDS1-PAD4 signaling pathway [47]. These findings suggest that CaWRKY50 can suppress the expression of CaEDS1 and CaSAMT1 to reduce disease resistance. WRKY proteins can interact with other proteins to regulate plant immunity [14]. For example, WRKY33 interacts with WRKY12 to increase hypoxia tolerance in Arabidopsis [48]. JrWRKY21 was shown to improve walnut resistance to C. gloeosporioides by interacting with JrPTI5L (a PR protein) [49]. SlWRKY31 interacts with SlVQ15 to regulate the resistance to B. cinerea in tomatoes [50]. It is plausible that the CaWRKY50 protein also interacts with other defense proteins in response to various pathogen infections. In this study, we determined the interaction between CaWRKY50 with CaWRKY42/CaMIEL1 through Y2H, LCI, and BiFC assays ( Figure 8). A previous study showed that CaWRKY41 (CA08g08240), which is the same gene as CaWRKY42 (Capana08g001044) in our study, could positively regulate pepper resistance to R. solanacearum inoculation [25]. CaMIEL1, an E3 ubiquitin ligase, inhibited tolerance to bacterial inoculation in Arabidopsis by degrading MYB30 [51]. These results suggest that CaWRKY50 may regulate resistance to C. scovillei by interacting with CaWRKY42 and CaMIEL1.
In conclusion, using overexpression and silenced techniques, we demonstrated that CaWRKY50 serves as a negative regulator in response to C. scovillei through the SA and ROS signaling pathways. CaWRKY50 directly binds to the promoters of CaEDS1 and CaSAMT1 to suppress their expression. Furthermore, CaWRKY50 participates in disease resistance by interacting with CaWRKY42 and CaMIEL1 (Figure 9). Our findings shed light on the mechanisms by which WRKY50 functions in response to anthracnose resistance. C. scovillei (SXBJ23) were cultured on potato dextrose agar (PDA) medium at 28 • C for 7 days. For pathogen inoculation assays, mature fruits were inoculated with C. scovillei using the previously described method [53]. In brief, the surfaces of mature fruits were injected with 2 µL of suspension (5 × 10 5 spores mL −1 ) using the microinjection method [54]. The inoculated fruits were stored at 28 • C for 7 days. Measurements of lesion diameter (LD) were performed by the crossing method as previously described [55]. Pepper fruit samples were collected at 0, 1, 2, 4, and 7 days post incubation (dpi). Tomato fruit samples were collected at 0 and 3 dpi. Three biological replicates were performed for each treatment.
For the study of CaWRKY50 transcript levels in response to SA and JA, cv. R25 mature fruits were soaked with 5 mM SA and 100 µM MeJA for 1 h [22]. Control fruits were soaked in sterile water. The pepper pericarps were collected after 0, 3, 6, 12, and 24 h post treatment (hpt) and then inoculated with 2 µL of C. scovillei after 24 hpt. Pepper fruits were stored at 28 • C for 7 days.

RNA Extraction and qRT-PCR Analysis
The total RNA was extracted by the RNAprep pure plant kit (TSP411, Tsingke, Beijing, China) according to the manufacturer's protocol. The first-strand cDNA was synthesized using ToloScript RT EasyMix (22106, Tolobio, Shanghai, China). qRT-PCR analysis was performed using the SYBR Green Master qPCR Mix (TSE201, Tsingke, Beijing, China). CaUBI3 (AY486137) and SlACTIN (NM_001308447.1) were used as reference genes for pepper and tomato to normalize transcript levels according to the 2 −∆∆Ct method. Each qRT-PCR experiment consisted of three biological replicates. The qRT-PCR primers listed in Supplemental Table S1.

Phylogenetic Analysis and Subcellular Localization
To analyze the relationship between CaWRKY50 and its homologs, the WRKY TFs, the protein sequence alignment was carried out by the DNAMAN software. The construction of the phylogenetic tree was performed using MEGA X software according to neighborjoining (NJ).
The PSORT online program (https://www.genscript.com/psort.html (accessed on 26 April 2023)) was used to predict the subcellular localization of the CaWRKY50 protein.
To confirm the subcellular localization, the coding sequence of CaWRKY50 without the stop codon was inserted into pVBG2307-GFP vectors. The recombinant construct (pVBG2307-CaWRKY50-GFP) was transformed into Agrobacterium tumefaciens strain GV3101 and then transiently infiltrated into tobacco (Nicotiana benthamiana) leaves. The empty vector serves as a negative control. The GFP fluorescence signals were detected using a BX63 Olympus (Tokyo, Japan) after 2-3 days. Fluorescence was evaluated using at least three independent replicates.

Transcriptional Activity Assays in Yeast
The coding sequence of CaWRKY50 was amplified and cloned into the pGBKT7 vector to investigate the transcriptional activity. Co-transformation of the CaWRKY50-pGBKT7 recombinant vector and the pGADT7-T empty vector into Y2H yeast cells according to the manufacturer's procedures (PT1183, Shaanxi Pyeast Bio.CO.LTD, Shaanxi, China). Transformants were cultured on SD/-Leu/-Trp/-Ade/-His medium containing X-α-gal (20 µg/mL) at 30 • C for 3 days.

Virus-Induced Gene Silencing (VIGS)
For the VIGS assays, CaWRKY50 silencing was performed following a previously reported procedure [56]. To generate the CaWRKY50-silenced vector, a 300 bp fragment of CaWRKY50 was obtained using the VIGS Tool (https://vigs.solgenomics.net/ (accessed on 4 December 2019)). The Agrobacterium tumefaciens strain GV3101 cells containing TRV2:00 and TRV2:CaWRKY50 were combined in a 1:1 ratio with TRV1 and then infiltrated into detached mature pepper fruits of cv. R25. The treated pepper fruits were placed in growth chambers at 24/18 • C under a 16/8 h photoperiod day/night cycle. After 15 days of infiltration, the transcript levels of CaWRKY50 were measured by qRT-PCR, and then the fruits were injected with 2 µL C. scovillei. Pepper fruit samples were collected at 0, 1, 2, 4, and 7 dpi, and the lesion diameters were measured at 7 dpi. Three biological replicates were performed for each treatment. The experiment was repeated three times with similar results.

Transient and Transgenic Overexpression
We carried out transient overexpression assays by A. tumefaciens-mediated infiltration [57]. The fusion protein pVBG2307-CaWRKY50-GFP was infiltrated into detached mature pepper fruits of cv. R25. The expression level of CaWRKY50 was detected at 3 days post-infiltration by qRT-PCR, and the fruits were subsequently inoculated with 2 µL C. scovillei. Pepper fruit samples were collected at 0, 1, 4, and 7 dpi, and the lesion diameters were measured at 7 dpi. Three biological replicates were performed for each treatment. The experiment was repeated three times with similar results.
The transgenic overexpression assays were performed as previously reported methods, with slight modifications [58]. Transcript levels in transgenic plants were evaluated by qRT-PCR. T 2 transgenic lines (OE-97, OE-110, and OE-113) were employed for further investigation. The mature red fruits were inoculated with 2 µL of C. scovillei at 28 • C for 3 days. Fruit samples were collected at 0 and 3 dpi, and the lesion diameters were measured at 3 dpi. Three biological replicates were performed for each treatment.

Measurement of Physiological Indicators
The content of malondialdehyde (MDA) was detected according to the previously described method [40]. The catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) activities as well as H 2 O 2 contents were quantified using the corresponding assay kits (Sangon Biotech, Shanghai, China) in accordance with the manufacturer's procedures. Three biological replicates were performed for each determination. The experiment was repeated three times with similar results.

Protein Interaction Assays
The coding sequence of CaWRKY50 was cloned into the pGBKT7 vector as the bait, and the coding sequences of CaWRKY42 and CaMIEL1 were cloned into the pGADT7 vector as the prey. Co-transformation of the bait and prey vectors into Y2H yeast cells according to the manufacturer's procedures (PT1183, Shaanxi Pyeast Bio.CO.LTD, Shaanxi, China). The protein interactions were screened using SD/-Leu/-Trp/-Ade/-His medium containing X-α-gal (20 µg/mL).
For the bimolecular fluorescence complementation (BiFC) assays, the recombinant constructs CaWRKY50-pSPYNE and CaWRKY42/CaMIEL1-pSPYCE were transformed into A. tumefaciens strain GV3101 and co-infiltrated in tobacco leaves. An automated fluorescence microscope (BX63, Olympus, Japan) was used to image YFP signals 2 days after infiltration. The BiFC assays were carried out as previously reported [60].

Yeast One-Hybrid (Y1H) Assay
The coding sequence of CaWRKY50 was cloned into the pGADT7 vector, and the CaEDS1 (799 bp) and CaSAMT1 (521 bp) promoter regions containing W-box elements (TTGACT/C) were cloned into the pAbAi vector as a bait vector. The restriction enzyme BstB1 was used to linearize the bait vectors before inserting them into Y1H Gold yeast cells. Then, these yeast cells were grown at 30 • C for 3 days on SD/-Leu medium with 150 and 250 ng mL −1 of Aureobasidin A (AbA).

GUS Activity Analysis and Dual Luciferase Reporter Assay
For the GUS activity analysis, the CaEDS1 and CaSAMT1 promoter regions were amplified from the genomic DNA of cv. R25 and inserted into the pCAMBIA1381-GUS vector as reporters. The effector plasmid, pVBG2307-CaWRKY50-GFP, was identical to that used in the previous subcellular localization assay. The recombinant constructs were transformed into A. tumefaciens strain GV3101 and co-infiltrated in tobacco leaves for 48 h. The GUS staining and enzyme activity detection methods were carried out as previously described [61].
For the dual luciferase reporter assays, the CaEDS1 and CaSAMT1 promoter regions were amplified from the genomic DNA of cv. R25 and inserted into the pGreenII-0800-LUC vector to generate a reporter construct. The coding sequence of CaWRKY50 was cloned into the pGreenII 62-SK vector to create an effector construct. The recombinant constructs were transformed into A. tumefaciens strain GV3101 (pSoup) and co-infiltrated in tobacco leaves for 48 h. The luciferase activity was measured according to the method in the Dual Luciferase Reporter Gene Assay Kit (11402ES60; Yeasen, Shanghai, China).

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12101962/s1, Table S1: Primers used in this study.  Data Availability Statement: All data supporting the findings of this study are available within the paper and its Supplementary Data. The GenBank accession numbers of ITS, ACT, and GAPDH for SXBJ23 are OQ119154, OQ127267, and OQ127268, respectively.