The Sodium/Calcium Exchanger PcNCX1-Mediated Ca 2+ Efﬂux Is Involved in Cinnamaldehyde-Induced Cell-Wall Defects of Phytophthora capsici

: Phytophthora capsici is one of the devastating pathogens, causing foliar blight, root rot, and fruit rot in peppers. Cinnamaldehyde (CA) is a natural compound coming from Cinnamomum cassia . The medicinal properties of CA have been widely identiﬁed. Limited knowledge is known about the application of CA in agriculture. In this study, CA signiﬁcantly inhibited P. capsici , which further suppressed Phytophthora blights in both pepper seedlings and pepper fruits. Treatment with CA resulted in collapsed and fragmented hyphae, accompanying the increase in MDA (malondialdehyde) content and the decrease in intercellular glycerol content in hyphae. CA also inhibited the growth of wild type yeast. The yeast mutant ∆ Yvc1 with a deletion of Yvc1 (a Ca 2+ transporter) showed decreased sensitivity to CA. The transformation of PcNCX1 , a sodium/calcium exchanger from P. capsici , into ∆ Yvc1 restored its sensitivity to CA. The transformant carrying PcNCX1 also showed restored Ca 2+ efﬂux upon CA treatment. RNA-seq analysis showed that CA treatments resulted in the down-regulation of a set of genes encoding for calcium-related proteins. Collectively, our study demonstrates that the antifungal activity of CA against P. capsici may be associated with PcNCX1-mediated Ca 2+ efﬂux. Our results provide crucial insights into the antimicrobial action of CA. P. in fruit Similar results were obtained with pepper plants 1B). Inoculation with P. capsici at the base of stem resulted in the infection of stem and roots, further leading to seedling lodging and leaf withering. Treatment with CA was able to rescue seedling growth from the infection of P. capsici (Figure 1B). As a kind of soil borne disease fungus, P. capsici can infect plant roots to cause root cell death. We analyzed root cell death using trypan blue staining. CA remarkably attenuated root cell death of seedlings inoculated with P. capsici (Figure 1C). These results indicated that CA effectively controlled the infection of P. capsici on pepper.


Introduction
Phytophthora blights caused by Phytophthora capsici are an increasingly severe disease on solanaceous and cucurbitaceous hosts, such as pepper, cucumber, tomato, eggplant, and pumpkin [1]. Chemical fungicides are frequently used to control Phytophthora blight diseases. However, the long-term application of chemical fungicides not only leads to the resistance of P. capsici but also poses potential threats on human health and environment [2]. Consequently, environmentally friendly fungicides need to be developed in order to control Phytophthora blight diseases effectively. it was purchased from Euroscarf (accessed on 15 May 2022, http://www.euroscarf.de/ search.php?search=YOR087W, Germany). Yeast cells were incubated on a liquid YPD (yeast extract peptone dextrose) medium containing CA at various concentration.

Inoculation of P. capsici on Pepper Seedlings and Plants
Zoospores of P. capsici were induced from 5-day-old sporangia by washing with sterile distilled water for 24 h at 25 • C and were adjusted to a concentration of 1 × 10 5 spores/mL for inoculations. Pepper (Capsicum annuum) fruits (Sujiao 5) were surface-sterilized with 1% NaClO for 10 min followed by washing with double-distilled water (ddH 2 O) for three times. A total of 50 µL of zoospores suspension (1 × 10 5 spores/mL) with 1 mM CA [21] were inoculated on the surface of each pepper fruit at three different sites. The inoculated pepper fruits were placed in a plastic box with a water-saturated filter paper and were incubated at 25 • C. The disease symptoms were observed at 4 days post inoculation (dpi). For pepper plants inoculation, a total of 50 µL of zoospores suspension (1 × 10 5 spores/mL) with 1 mM CA was inoculated at the stem base of 35-day-old pepper plants ( Sujiao 5). The inoculated plants were kept in a chamber with photosynthetic active radiation of 200 µmol/m 2 /s, a photoperiod of 12 h and a temperature at 25 • C for 4 days followed by the observation of disease symptoms.

Histochemical Staining
Trypan blue staining was used to evaluated cell death in the root pepper plant. The pepper roots at 4 dpi were carefully washed with ddH 2 O. Then, the roots were stained with Trypan blue (10 mg/L) for 1 h and detained in lactophenol for 1 h followed by photographing with a digital camera [22,23].
The intracellular Ca 2+ in yeast cells were labeled in situ with specific fluorescent probe Fluo-3-AM (Beyotime Biotechnology Institute, Haimen, China). Fluo-3-AM was added to yeast suspension at a final concentration of 150 µM. Then, the suspension was incubated at 37 • C for 1 h. Confocal microscopy was performed using a Zeiss Axiovert 200 M microscope equipped with a Zeiss LSM 710 META system using 940/1.2 NA and 963/1.2 NA C-Apochromat water immersion objectives. Images were acquired and processed using LSM 710 AIM version 4.2 SP1 software (Zeiss, Oberkochen, Germany).

Determination of Glycerol Content
The P. capsici strain grown in liquid V8 media were treated with 0, 0.8, 1.6, and 2 mM of CA [21] for 24 h. Then, the mycelia were harvested for the determination of glycerol content. Glycerol content was determined using cupric glycerinate colorimetric assay under the instructions of a glycerol detection kit (E1012; Beijing Applygen Technologies Inc., Beijing, China).

Determination of Malondialdehyde (MDA) Content
The content of MDA was determined as an indicator of the lipid peroxidation in CA-treated mycelia of P. capsici. An MDA detection kit (A003; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was selected to determine the MDA's level based on the spectrophotometric determination of the reaction between MDA and 1,3-diethyl-2thiobarbituric acid (TBA) assisted by trichloroacetic acid (TCA).

qRT-PCR Analysis
Quantitative RT-PCR was performed using the ABI 7500 Fast Real-Time PCR System (ABI Co., Carlsbad, CA, USA) and transcripts were analyzed using the 7500 System SDS software (ABI). To compare the relative abundances of target gene transcripts, the average threshold cycle (Ct) was normalized to that of Actin (Actin-F: ACTGCACGTTCCA-GACGATC; Actin-R: CCACCACCTTGATCTTCATG) for each of the treated samples as 2 −∆Ct , where −∆Ct = (C t, target gene − C t, actin ). Fold change was calculated as 2 −∆∆Ct , where −∆∆Ct = (C t, target gene − C t, actin ) treated with CA −(C t, control − C t, actin ) control . The primers were 131120-RT-F: ACTCGCTCTGGTATACGTGG and 131120-RT-R: AACAACAGTTTAC-CACCCGC.

Total RNA Isolation and Library Preparation for Transcriptome Sequencing
Total RNA was extracted from P. capsici mycelia treated with 1 mM CA for 24 h using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacture's protocol. P. capsici mycelia without CA were taken as control. The quality and integrity of RNA was evaluated using Bioanalyzer 2100 (Agilent Technologies, CA, USA) and agarose gel electrophoresis. A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparation. Sequencing libraries were generated using NEBNext Ultra TM RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following the manufacturer's recommendations. The mRNA was purified from total RNA using oligo (dT)-attached magnetic beads. The first strand cDNA was synthesized using random hexamer primer and M-MuLV reverse transcriptase. Second stand cDNA syntheses were subsequently performed using DNA polymerase I and RNase H. After second-strand cDNA synthesis and dual-index adaptor ligation, the paired-end library fragments with 150-200 bp in length were purified with AMPure XP system (Beckman Coulter, Beverly, CA, USA). The library quality was assessed using Agilent Bioanalyzer 2100 system. Then, four libraries (Control-1, -2 and CA-treatment-1, -2) preparations were sequenced on an Illumina Hiseq 2000 platform to generate 100 bp paired-end reads (BGI, Shenzhen, Guangzhou, China).

Quality Control, Transcriptome Assembly and Differential Expression Analysis of Unigenes
For quality control, raw sequencing data were filtered by Fast QC software, and lowquality reads were filtered out. Both clean reads were de novo assembled with Trinity with the default settings, with the exception that min_kmer_cov was set to 2 by default.
The analysis of DEGs (differential expression genes) between two samples were performed with the DEGseq R package. The p-value was adjusted using the q value. The q value < 0.005 and the absolute value of log 2 > 1 were set as the threshold for significant difference in gene expression. Gene ontology (GO) analyses of DEGs were implemented by BGI WEGO (Web Gene Ontology Annotation Plotting, accessed on 15 August 2015, http://wego.genomics.org.cn/cgi-bin/wego/index.pl) and agriGO (GO Analysis Toolkit and Database for Agricultural Community, accessed on 15 August 2015, http://bioinfo.cau.edu.cn/agriGO/index.php). Pathways were significantly enriched with KEGG (Q-value < 0.05).

Statistical Analysis
For the data of the measurement of physiological parameters, each result was presented as the mean and standard deviation (SD) of three replicates. The least significant difference (LSD) test was performed on all data following a one-way analysis of variance (ANOVA) to test for significant differences (p < 0.05) among different treatments.

CA Controlled the Pepper Phytophthora Blight
In our previous study, CA exerted efficient inhibitory effects on both mycelial growth (EC 50 = 0.75 mM) and zoospore germination (MIC = 0.4 mM) of P. capsici [6]. To investigate the role of CA in the pathogenicity of P. capsici, CA (1 mM) was applied on pepper fruit surface inoculated with P. capsici zoospore suspensions. Pepper fruit inoculated with P. capsicia showed severe disease symptoms while treatment with CA resulted in much fewer fruit rots at 4 dpi ( Figure 1A). Similar results were obtained with pepper plants ( Figure 1B). Inoculation with P. capsici at the base of stem resulted in the infection of stem and roots, further leading to seedling lodging and leaf withering. Treatment with CA was able to rescue seedling growth from the infection of P. capsici ( Figure 1B). As a kind of soil borne disease fungus, P. capsici can infect plant roots to cause root cell death. We analyzed root cell death using trypan blue staining. CA remarkably attenuated root cell death of seedlings inoculated with P. capsici ( Figure 1C). These results indicated that CA effectively controlled the infection of P. capsici on pepper.

CA Controlled the Pepper Phytophthora Blight
In our previous study, CA exerted efficient inhibitory effects on both mycelial growth (EC50 = 0.75 mM) and zoospore germination (MIC = 0.4 mM) of P. capsici [6]. To investigate the role of CA in the pathogenicity of P. capsici, CA (1 mM) was applied on pepper fruit surface inoculated with P. capsici zoospore suspensions. Pepper fruit inoculated with P. capsicia showed severe disease symptoms while treatment with CA resulted in much fewer fruit rots at 4 dpi ( Figure 1A). Similar results were obtained with pepper plants ( Figure 1B). Inoculation with P. capsici at the base of stem resulted in the infection of stem and roots, further leading to seedling lodging and leaf withering. Treatment with CA was able to rescue seedling growth from the infection of P. capsici ( Figure 1B). As a kind of soil borne disease fungus, P. capsici can infect plant roots to cause root cell death. We analyzed root cell death using trypan blue staining. CA remarkably attenuated root cell death of seedlings inoculated with P. capsici ( Figure 1C). These results indicated that CA effectively controlled the infection of P. capsici on pepper.

CA Impacted Cell Integrity of P. capsici
Results from the SEM revealed clear morphological alterations in the hyphae of P. capsici under CA treatments. Compared to the control group, collapsed and fragmented hyphae were observed in mycelia treated with CA ( Figure 2A). The accumulation of MDA is a typical indicator of oxidative injury and plasma membrane damage [24]. Treatment with CA led to significant increase in MDA content in the mycelia of P. capsici in a dose-dependent manner ( Figure 2B). The intercellular glycerol plays a critical role in the protection of fungal cells from osmotic stress [25]. We found that treatment with CA resulted in a significant decrease in intercellular glycerol content in the mycelia of P. capsici. The changes of MDA and glycerol content also confirmed the loss of cell integrity in the hyphae of P. capsici under CA treatment.

CA Impacted Cell Integrity of P. capsici
Results from the SEM revealed clear morphological alterations in the hyphae of P. capsici under CA treatments. Compared to the control group, collapsed and fragmented hyphae were observed in mycelia treated with CA ( Figure 2A). The accumulation of MDA is a typical indicator of oxidative injury and plasma membrane damage [24]. Treatment with CA led to significant increase in MDA content in the mycelia of P. capsici in a dosedependent manner ( Figure 2B). The intercellular glycerol plays a critical role in the protection of fungal cells from osmotic stress [25]. We found that treatment with CA resulted in a significant decrease in intercellular glycerol content in the mycelia of P. capsici. The changes of MDA and glycerol content also confirmed the loss of cell integrity in the hyphae of P. capsici under CA treatment.

Identification of CA-Regulated Ca 2+ Channels in P. capsici
We previously found that CA induced immediate Ca 2+ efflux from zoospores of P. capsici [9]. In this study, the yeast mutant ΔYvc1 was used to investigate CA-regulated Ca 2+ channel in P. capsici. ΔYvc1 was more tolerant to CA compared to the wild type (WT) yeast, indicating that Yvc1 played a role in response to CA ( Figure 3A). Then, we complemented the yeast mutant ΔYvc1 with PcNCX1 (the P. capsici sodium/calcium exchanger). The transformants (ΔYvc1/NCX1 #1 and ΔYvc1/NCX1 #2) expressing PcNCX1 showed recovered sensitivity to different concentration of CA ( Figure 3A), indicating that PcNCX1 maybe the potential target of CA. Then we tested the intracellular Ca 2+ level using specific fluorescent probe Fluo-3 AM. In CA-free medium, the intracellular Ca 2+ was successfully labeled with green fluorescence in WT yeast, the deletion mutant ΔYvc1, and the complemented strain ΔYvc1/PcNCX1 ( Figure 3B). Compared to WT and the ΔYvc1/PcNCX1 strains, the deletion mutant ΔYvc1 stilled showed strong fluorescent signal upon CA treatment ( Figure 3C). These results showed that the PcNCX1 can partly restore CA-induced Ca 2+ efflux in the yeast deletion mutant ΔYvc1.

Identification of CA-Regulated Ca 2+ Channels in P. capsici
We previously found that CA induced immediate Ca 2+ efflux from zoospores of P. capsici [9]. In this study, the yeast mutant ∆Yvc1 was used to investigate CA-regulated Ca 2+ channel in P. capsici. ∆Yvc1 was more tolerant to CA compared to the wild type (WT) yeast, indicating that Yvc1 played a role in response to CA ( Figure 3A). Then, we complemented the yeast mutant ∆Yvc1 with PcNCX1 (the P. capsici sodium/calcium exchanger). The transformants (∆Yvc1/NCX1 #1 and ∆Yvc1/NCX1 #2) expressing PcNCX1 showed recovered sensitivity to different concentration of CA ( Figure 3A), indicating that PcNCX1 maybe the potential target of CA. Then we tested the intracellular Ca 2+ level using specific fluorescent probe Fluo-3 AM. In CA-free medium, the intracellular Ca 2+ was successfully labeled with green fluorescence in WT yeast, the deletion mutant ∆Yvc1, and the complemented strain ∆Yvc1/PcNCX1 ( Figure 3B). Compared to WT and the ∆Yvc1/PcNCX1 strains, the deletion mutant ∆Yvc1 stilled showed strong fluorescent signal upon CA treatment ( Figure 3C). These results showed that the PcNCX1 can partly restore CA-induced Ca 2+ efflux in the yeast deletion mutant ∆Yvc1.

Transcriptome Analysis of P. capsici Treated with CA
To explore the mechanisms underlying the inhibition of P. capsici by CA, we performed RNA-sequencing of P. capsici hyphae under CA treatments and found that the hyphae are thin compared to control ( Figure 4A). Approximately 46 million (96.81%) and 44 million (98.33%) clean reads were obtained from CA-treatment and control, respectively (Table 1). Among all the clean reads, approximately 91.46% and 91.32% of the CA-treatment and control were mapped to the reference genome, respectively (Table 1). We identified 956 upregulated unigenes and 2575 down-regulated unigenes upon CA treatment ( Figure 4B). Enrichment analysis was performed to illustrate the biological functions of the identified DEGs. In total, 607, 1184, and 1198 DGEs of cellular components, molecular function, and biological process were enriched in 37 (gene ontology) GO terms. Five GO terms including "metabolic process" (805), "Cellular process" (578), and "Single-organism process" (457) in the category of biological process; "Catalytic activity" (1229) and "Transporter activity" (215) in the category of molecular function were significantly enriched in the CA-treatment sample ( Figure 5A). In addition, 1591 DEGs were enriched in KEGG pathways. The most enriched pathways were "Metabolic pathways" (388) and "Biosynthesis of secondary metabolites" (169) ( Figure 5B). Ca 2+ acts as a second messenger to modulate numerous intrinsic metabolic processes by regulating a set of Ca 2+ -binding proteins [26]. CA affected Ca 2+ homeostasis by regulating PcNCX1. We screened the expression level of genes coding for Ca 2+ -regulated proteins from RNA-sequencing data. As expected, in the CA-treatment samples, 25 and 5 calcium-related proteins were down-and up-regulated, respectively ( Table 2). This may due to decreased intracellular Ca 2+ levels in CA-treated P. capsici. In addition, we found that the expressions of four Ca 2+ pump genes (111,992, 109,877, 126,140, and 549,419) were also down-regulated by CA, indicating that the down-regulation of these genes may also contributed CA-suppressed intracellular Ca 2+ levels in P. capsici.

Transcriptome Analysis of P. capsici Treated with CA
To explore the mechanisms underlying the inhibition of P. capsici by CA, we performed RNA-sequencing of P. capsici hyphae under CA treatments and found that the hyphae are thin compared to control ( Figure 4A). Approximately 46 million (96.81%) and 44 million (98.33%) clean reads were obtained from CA-treatment and control, respectively (Table 1). Among all the clean reads, approximately 91.46% and 91.32% of the CAtreatment and control were mapped to the reference genome, respectively (Table 1). We identified 956 up-regulated unigenes and 2575 down-regulated unigenes upon CA treatment ( Figure 4B).  Enrichment analysis was performed to illustrate the biological functions of the identified DEGs. In total, 607, 1184, and 1198 DGEs of cellular components, molecular function, and biological process were enriched in 37 (gene ontology) GO terms. Five GO terms including "metabolic process" (805), "Cellular process" (578), and "Single-organism process" (457) in the category of biological process; "Catalytic activity" (1229) and "Transporter activity" (215) in the category of molecular function were significantly enriched in the CA-treatment sample ( Figure 5A). In addition, 1591 DEGs were enriched in KEGG pathways. The most enriched pathways were "Metabolic pathways" (388) and "Biosynthesis of secondary metabolites" (169) ( Figure 5B).   ABC (ATP-binding cassette) transporters mainly use the hydrolysis of ATP to fuel the transmembrane's transport and participates in the physiological process of drug resistance by facilitating drug efflux [27]. In this study, we found that 45 DEGs (including 34 downregulated and 11 up-regulated) encoded for ABC transporters and drug-resistance proteins in the CA-treated samples (Table S2). These results suggested that ABC transporters in P. capsici may be differentially regulated by CA.

Discussion
P. capsici is a highly destructive plant pathogen worldwide. CA is a widely used flavoring agent that has been used to improve food quality. In this study, we showed that CA was able to destroy the cell integrity of P. capsici hyphae and to control pepper phytophthora effectively. Yvc1 in S. cerevisiae is a vacuolar membrane localized calcium channel. Yvc1 mediates Ca 2+ release from the vacuole in response to hypertonic shock [28][29][30]. We found that PcNCX1 can partly restore the immediate Ca 2+ efflux of the yeast deletion mutant ∆Yvc1 under CA treatment. Thus, CA could target PcNCX1 to cause cell defects of P. capsici.
CA could be a safe antibacterial and antifungal alternative due to its food additive properties. Several studies have shown that CA can inhibit the growth, morphology, and mycelial formation of C. albicans. Our previous study indicated that CA can control the pepper blight by inhibiting the zoospore germination and vegetable growth of P. capsici [9]. In this study, we found that CA significantly controls the pepper Phytophthora blight by destroying the cell wall integrity of P. capsici (Figures 1 and 2). Cell-wall integrity plays a vital role in fungal growth and development as well as the ability to survive under stress conditions [31]. We found collapsed and fragmented hyphae in P. capsici mycelia treated with CA ( Figure 2A). In addition, MDA concentration and intercellular glycerol measured tests showed that the hyphae of P. capsici treated with CA was more sensitive to oxidative stress and osmotic stress ( Figure 2B). The pathogenicity of M. oryzae is dependent on several key genes (e.g., MoSWI6, MoRGS1, and MoPDEH) modulating cell wall integrity [32][33][34]. It is reported that CA can affect the synthesis of 1,3-β-D-glucans of Aspergillus fumigatus and can inhibit chitin synthase 1 in Saccharomyces cerevisiae [35]. RNA-seq analysis showed that the expressions of twelve glucan 1,3-β-glucosidase of P. capsici treated with CA were significantly different from control (Table S3), which suggested that CA affected cell-wall integrity by regulating the expression of glucan 1,3-β-glucosidase.
Ca 2+ is a second messenger involved in the response to different stress signals (e.g., hypotonic, hypertonic shock, and nutrients) in yeast [36]. Ca 2+ -binding proteins interact with specific transcription factors to modulate the cell-wall's integrity and pathogenicity of fungi [37,38]. Ca 2+ concentration is regulated precisely by the endoplasmic reticulum (ER), Golgi, and vacuole [39]. Yvc1 is involved in the regulation of cytoplasmic Ca 2+ homeostasis [40]. In this study, we transformed PcNCX1 (a sodium/calcium exchanger) to the yeast mutant ∆Yvc1. The transformants expressing PcNCX1 showed restored sensitivity to CA ( Figure 3A), coinciding with the decrease in intracellular Ca 2+ level in complemented strain ∆Yvc1/PcNCX1 upon CA treatment ( Figure 3B,C). In Candida albicans, CA introduced apoptosis through accumulating the intracellular reactive oxygen species and calcium in the cytoplasm [41]. In Brassica rapa, CA can be able to prime plant defense by regulating endogenous Ca 2+ in order to facilitate cadmium tolerance [42]. In our pervious study, we have found that CA inhibited the growth of P. capsici by stimulating Ca 2+ efflux [9]. Combining with the data in this study, it can be suggested that CA may regulate PcNCX1 to induce the decrease in intracellular Ca 2+ levels, further leading to the growth inhibition of P. capsici.
The Yeast complementation experiment suggested that PcNCX1 is a potential target of CA. Thus, we suspect that CA may directly interact with PcNCX1 to modulate its activity. CA has been identified as an agonist of hTRPA1 (human transient receptor potential ankyrin 1). CA directly binds to a set of cysteine residues in the N-terminus of hTRPA1, leading to covalent protein modification. These modifications may activate the channel's activity by altering subunit-subunit interactions or by modulating the association of hTRPA1 with other cellular proteins [43]. Whether CA can modulate the channel activity of PcNCX1 through similar mechanisms needs further studies. In addition, CA can modulate mammalian TRPs at a transcriptional level [44]. In this study, we found that CA down-regulated the expression of two Ca 2+ pump genes in P. capsici as well. Therefore, CA may modulate Ca 2+ flux by regulating Ca 2+ channels at transcriptional level.
The RNA-seq of P. capsici treated with CA was analyzed, and we found that 956 upregulated and 2575 down-regulated genes in the CA-treated sample and the functions of these DEGs varied ( Figure 4B). However, we could not detect PcNCX1 in the RNA-seq data. Moreover, we investigated the expression of PcNCX1 in the sample treated with CA with qRT-PCR and found that the expression level was significantly down-regulated ( Figure S1). In addition, most of the calcium-related genes (25/30) were down-regulated in CA-treated sample ( Table 2), suggesting that CA could introduced decreased intracellular Ca 2+ level in P. capsici. The chemical insensitivity may include non-specific efflux pumps and detoxification, which can be performed by proteins such as the ABC transporter and the cytochrome P450 protein, respectively [45]. CA treatments resulted in the downregulation of most ABC transporter genes (34/45) in P. capsici, suggesting that CA shows strong antifungal activities by possibly suppressing the detoxification system of P. capsici. In our next research, we will study the function of these Ca 2+ transporter associated genes using the CRISPER-CAS9 method, which further enrich the action mechanism of CA to P. capsici.

Conclusions
CA effectively control the pepper Phytophthora blight caused by P. capsici. The loss of cell-wall integrity is closely related to the antifungal effect of CA against P. capsici.
Yeast complementation experiments showed that the sodium/calcium exchanger PcNCX1 restored the immediate Ca 2+ efflux of ∆Yvc1. Our study demonstrates that PcNCX1mediated Ca 2+ efflux may be one of the important reasons to explain the antifungal effect of CA against P. capsici, which provides crucial insights into the antimicrobial action of CA.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/xxx/s1, Figure S1: The expression of PcNCX1 in the sample treated with CA by qRT-PCR. Error bars represent standard deviation; Table S1: The CDS sequence of PcNCX1; Table S2: Summary of DEGs encoding ABC transporter genes; Table S3: Summary of DEGs encoding glucan 1,3-beta-glucosidase genes.