Transcriptome analysis reveals ClWRKY75 enhanced the resistance to Colletotrichum Fructicola via activating the Phenylalanine Ammonia-Lyase Pathway in Watermelon

Transcriptome analysis reveals ClWRKY75 enhanced the resistance to Colletotrichum Fructicola via activating the Phenylalanine Ammonia-Lyase Pathway in Watermelon | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 4 November 2025 V1 Latest version Share on Transcriptome analysis reveals ClWRKY75 enhanced the resistance to Colletotrichum Fructicola via activating the Phenylalanine Ammonia-Lyase Pathway in Watermelon Authors : Shiqi Gong , Dengke Wang , Shan Yuan , Zihao Yang , Guang Liu , Changzheng He , Fengjiao Bu , … Show All … , Longjun Sun , Zhongquan Chen , Shan Li , Xiaowu Sun , Sihui Dai , and Bingqian Tang [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.176224947.72313670/v1 191 views 157 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Watermelon, a significant horticultural crop, faces threats from various fungal diseases throughout its growth cycle. Colletotrichum fructicola , a member of the Colletotrichum spp ., is particularly notorious for causing anthracnose, resulting in severe damage to both seedlings and fruits of watermelon. Chemical control is the primary method for managing anthracnose, which significantly hinders the green and sustainable development of the watermelon industry. However, reports on relevant disease resistance genes and their molecular mechanisms against C olletotrichum fructicola in watermelon are scarce. In this study, we identified a WRKY transcription factor ( ClWRKY75 ) via transcriptome analysis; it was highly expressed in the resistant line (M10). Moreover, integrated transcriptomic and metabolomic analyses indicated that ClPAL20 is involved in lignin biosynthesis post-Colletotrichum fructicola infection. Individual VIGS-mediated downregulation of ClWRKY75 or ClPAL20 in M10 rendered positive plants sensitive to C. fructicola . Additionally, Y1H, EMSA, and Dual-Luc assays verified ClWRKY75 binds to and activates ClPAL20 expression. These findings of this study provide essential theoretical references and genetic resources for the targeted breeding of anthracnose-resistant watermelon varieties. These results have substantial practical implications for expediting the progress of watermelon disease resistance breeding and promoting the development of new resistant cultivars. Transcriptome analysis reveals ClWRKY75 enhanced the resistance to Colletotrichum Fructicola via activating the Phenylalanine Ammonia-Lyase Pathway in Watermelon Shiqi Gong 1, 2, * , Dengke Wang 1, 2, * , Shan Yuan 1, 2 , Zihao Yang 1, 2 , Guang Liu 3 , Changzheng He 1, 2 , Fengjiao Bu 1, 2 , Longjun Sun 4 , Zhongquan Chen 4 , Shan Li 1, 2 , Xiaowu Sun 1, 2, † , Sihui Dai 1, 2, † , Bingqian Tang 1, 2, † 1 College of Horticulture, Hunan Agricultural University, Changsha, 410128, China∣2 Yuelushan Laboratory, Changsha, 410128, China∣3 Institute of Vegetable Research, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China∣4 Hunan Xuefeng Seeds Co., Ltd., Shaoyang, 422001, China * These authers contributed equally to this work. † Corresponding auther: Bingqian Tang (Email: [email protected] ), Sihui Dai (Email: [email protected] ) and Xiaowu Sun (Email: [email protected] ) Abstract Watermelon, a significant horticultural crop, faces threats from various fungal diseases throughout its growth cycle. Colletotrichum fructicola , a member of the Colletotrichum spp ., is particularly notorious for causing anthracnose, resulting in severe damage to both seedlings and fruits of watermelon. Chemical control is the primary method for managing anthracnose, which significantly hinders the green and sustainable development of the watermelon industry. However, reports on relevant disease resistance genes and their molecular mechanisms against C olletotrichum fructicola in watermelon are scarce. In this study, we identified a WRKY transcription factor ( ClWRKY75 ) via transcriptome analysis; it was highly expressed in the resistant line (M10). Moreover, integrated transcriptomic and metabolomic analyses indicated that ClPAL20 is involved in lignin biosynthesis post-Colletotrichum fructicola infection. Individual VIGS-mediated downregulation of ClWRKY75 or ClPAL20 in M10 rendered positive plants sensitive to C. fructicola . Additionally, Y1H, EMSA, and Dual-Luc assays verified ClWRKY75 binds to and activates ClPAL20 expression. These findings of this study provide essential theoretical references and genetic resources for the targeted breeding of anthracnose-resistant watermelon varieties. These results have substantial practical implications for expediting the progress of watermelon disease resistance breeding and promoting the development of new resistant cultivars. Keywords: watermelon, Colletotrichum fructicola , ClWRKY75 , phenylalanine ammonia-lyase pathway, lignin synthesis Instruction Watermelon ( Citrullus lanatus ) belonging to Cucurbitaceae family, is an important horticultural crop. China is the world’s largest producer and consumer of watermelon; Colletotrichum spp. greatly constrain the development of the watermelon industry in China. Over the past decade, C. karstii , C. magnum , C. scovillei , C. orbiculare and C. incanum have also been identified as the major pathogens causing watermelon anthracnose, based on the strategy of multi-locus combined analysis combined with morphological characteristic observation (Damm et al., 2013; Damm et al., 2012; Damm et al., 2019; Goh et al., 2022). In a recent study, a novel pathogen Colletotrichum fructicola , which belongs to Colletotrichum gloeosporioides species complex ( CGSO ), has also been proven to cause watermelon anthracnose, expanding the known range of causal agents of this disease (Guo, 2023). Phenylpropanoid metabolism is one of the most important metabolic pathways in plants, playing crucial roles in plant development and plant-environment interactions, while phenylalanine ammonia lyase (PAL) serves as a key enzyme in the general phenylpropanoid pathway. Lignin, in turn, represents the major downstream product of this pathway; its synthesis and accumulation can influence fruit quality, postharvest physiological properties, and lodging resistance in horticultural crops (Li et al., 2024; Wang et al., 2021a; Wang et al., 2025b). Additionally, lignin biosynthesis plays a vital role in plant disease resistance. Upon pathogenic infection, the expression of genes encoding lignin synthases and lignin content are both increased, which enhances the mechanical strength of cell walls and improves their resistance to cell wall-degrading enzymes secreted by pathogens. In recent years, numerous studies have revealed that expression of lignin biosynthesis genes is regulated by transcription factors (TFs), including MYB, MYC and NAC. PpMYC2 is upregulated after infection by Monilinia fructicola (brown rot pathogen), and five lignin biosynthesis genes ( PpPAL1 , PpC4H , Pp4CL1 , PpCSE , PpCCoAOMT1 ) were identified as the targets of PpMYC2 by using DNA Affinity Purification Sequencing (DAP-seq) and RNA-seq (Li et al., 2025b). The MYB transcription factors AtMYB20 , AtMYB42 , AtMYB43 , and AtMYB85 are transcriptional regulators that directly activate lignin biosynthesis genes (Geng et al., 2020). However, some R2R3-MYB TFs are the repressors that are crucial regulators of lignin biosynthesis, including AtMYB4 , AtMYB7 and AtMYB32 (Miyamoto et al., 2020). FER is specifically upregulated in the root xylem in response to Ralstonia solanacearum infection. It phosphorylates the NAC transcription factor RESPONSIVE TO DESSICATION 26 (RD26), leading to the degradation of RD26, which in turn inhibits lignin biosynthesis and plant resistance to this pathogen (Wang et al., 2025a). The WRKY transcription factor gene family is one of the largest gene families in higher plants, and many of its members are involved in regulation of plant stress resistance under diverse stress environments, including salt stress, drought stress and temperature stress(Yang et al., 2025). Additionally, some of its members have been reported to be involved in responding to biotic stress. OsWRKY70 is a hub gene participating in resistance to Chilo. Suppressalis , and its function depends on the damage-associated molecular patterns that can trigger the Mitogen-activated protein kinase (MAPK) signaling pathway, OsMAPK3 / 6 then directly interact and phosphorylate OsWRKY70 , thereby increasing its transcriptional activation activity (Li et al., 2015). In horticultural species, recent studies have revealed that many WRKY TFs have been identified as being involved in regulation of resistance to Ralstonia solanacearum , Botrytis cinerea , Pseudoperonospora spp ., Colletotrichum spp . and Botryosphaeria dothidea (Cheng et al., 2024; Li et al., 2023; Liu et al., 2020; Luan et al., 2019; Zhao et al., 2020). For example, silencing CaWRKY50 enhances pepper resistance to Colletotrichum musae , indicating that this gene acts as a negative regulator of anthracnose resistance in pepper (Li et al., 2023). In contrast, MdWRKY100 serves as a positive regulator of anthracnose resistance in apple, and overexpressing MdWRKY100 strengthens apple resistance to Colletotrichum spp . (Zhang et al., 2019). In recent years, significant progress has been made in research on genome and pan-genome assembly and annotation, plant architecture improvement, fruit quality optimization, seed development and high-throughput breeding tool development in watermelon (Duan et al., 2025; Ren et al., 2021; Sun et al., 2024; Wang et al., 2021b; Zhang et al., 2024). However, there are few reports on watermelon’s responses to biotic stresses, particularly to anthracnose. Here, transcriptome data analysis revealed that a WRKY transcription factor, ClWRKY75 , was more highly expressed in the resistant line. Silencing ClWRKY75 resulted in reduced lignin content in the plant and a significant decrease in resistance to C. fructicola . Similarly, we also found that silencing ClPAL20 or ClCCoAOMT also resulted in reduced lignin content and significantly decreased resistance to C. fructicola in the plants. Additionally, ClWRKY75 could directly bind to and activate the expression of ClPAL20 to promote lignin biosynthesis. These findings highlight the crucial role of ClWRKY75 in watermelon resistance to C. fructicola and provide valuable insights for developing anthracnose-resistant watermelon accessions. Materials and methods Plant materials, C. Fructicola infection and growth conditions The Citrullus lanatus “M10” and “HX6” were used in this study and cultivated in a greenhouse under controlled conditions at 25 ℃ with a 16/8 h light/dark cycle. Conidia of C. Fructicola were collected after incubation at 30 °C in the dark for 7 days, and the concentration of conidia was adjusted to 1×10⁶ conidia/mL using sterile ddH₂O. The plants were inoculated by spraying with the anthracnose conidial suspension at the two-leaf stage. After inoculation, they were first treated in the dark for 48 hours, followed by cultivation under a normal light-dark cycle (16 h light / 8 h dark) at a temperature regime of 28 °C / 20 °C. Determination biochemical indicators and Histological analysis Leaves were sampled at 0 day post-inoculation (dpi) and 5 dpi, further were placed into grinding tubes, quickly immersed in liquid nitrogen for flash freezing, and stored for subsequent analysis. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) were determined using enzyme activity assay kits (Solarbio) respectively. Detailed experimental procedures refer to the kit instructions. After drying at 75 °C to a constant weight, the lignin content of leaves was determined using the Lignin Content Assay Kit (Solarbio). Leave samples of M10 and HX6 collected at 0 dpi and 5 dpi were subjected to paraffin embedding and sectioning. Subsequently, the sections were subjected to dewaxing, mordanting, and staining, followed by photography. Three biological replicates were conducted for the entire experiment. RNA-seq analysis and sequence analysis Leaves of ‘M10’ and ‘HX6’ at three time points—pre-inoculation, 3 dpi, and 4 dpi—were collected for total RNA extraction. RNA sequencing (RNA-seq) was performed on 18 libraries using the Illumina HiSeq™ 2500 platform. After removing the low quality, the clean reads were aligned to “97103” watermelon reference genome version 2 (Guo et al., 2019). The aligned reads of each sample are assembled by StringTie (v1.3.3b), using featureCounts v1.5.0-p3 to calculate the number of gene reads, and calculate FPKM based on gene length. The differential expression of genes (DEGs) was proceeded by DESeq2 ( P -value1.0). The Mfuzz R package was used to conduct a temporal trend analysis of gene expression and divide the clustering groups (Kumar and Matthias, 2007). The hisat2, Samtools and Vcftools were used to identified sequence difference between M10 and HX6 (Danecek et al., 2011; Kim et al., 2019; Li et al., 2009). Untargeted metabolomics analysis 30 mg of sample powder and add 1500 μL of -20 °C pre-cooled 70% methanolic aqueous internal standard extract. All samples were acquired by the LC-MS system followed machine orders. The analytical conditions were as follows, UPLC: column, Waters ACQUITY UPLC HSS T3 1.8 µm, 2.1 mm*100 mm; column temperature, 40 °C; flow rate, 0.40 mL/min; injection volume, 4 μL; solvent system, water (0.1% formic acid): acetonitrile (0.1% formic acid). The data acquisition was operated using the information-dependent acquisition (IDA) mode using Analyst TF 1.7.1 Software (Sciex, Concord, ON, Canada). Subcellular localization and qRT-PCR The coding sequence of ClWRKY75 without the stop codon was inserted into the Bam HI and Xba I sites of the p2300. The recombinant plasmids transferred into the Agrobacterium tumefaciens strain GV3101 (pSoup). The p2300-ClWRKY75-GFP and the p2300-NLS-mcherry were transiently co-transformed into N.benthamiana leaves. After 48 hours, the fluorescence was visualized and photographed with a confocal laser scanning microscope (LSM800, Zeiss, Germany). Total RNA was extracted from different watermelon tissues of M10 and HX6 using the FastPure Complex Tissue-Cell Total RNA isolation Kit (Vazyme, China). The RNA was then used to synthesized cDNA using HiScript II Reverse Transcriptase (Vazyme, China). For qRT-PCR, specific primers for ClWRKY75, ClPAL10, ClPAL20, ClPAL30, ClPAL50, ClCCR, ClCCoAOMT and ClNPR1 were designed by using primer3 and primer check of Tbtools (Chen et al., 2023). qRT-PCR was carried out with the ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) on a LightCycler PCR system (Roche, Switzerland). The 2 −△△Ct method was used for the calculation of relative expression of genes. The primer sequences are shown in Supplementary Table 1. Virus-induced gene silencing The 300-bp specific sequence of ClWRKY75 , ClPAL20 and ClCCoAOMT were selected by using the SGN VIGS tool (https://vigs.solgenomics.net/). These sequences were cloned into the Eco RI and Bam HI sites of the pTRV2 vector, respectively. The recombinant plasmids transferred into the Agrobacterium tumefaciens strain GV3101 (pSoup). The pTRV1 bacterial solution was mixed with pTRV2-empty, pTRV2- ClWRKY75 , pTRV2- ClPAL20 and pTRV2- ClCCoAOMT bacterial solution for inoculation M10. After inoculation, the plants were cultivated in the dark at 18 °C for 3 days in an artificial climate box. Then all plants were cultivated in a greenhouse under controlled conditions at 25 ℃ with a 16/8 h light/dark cycle. The primer sequences are shown in Supplementary Table 1. Yeast one-hybrid assay A Y1H assay was conducted by using the Matchmaker Gold Yeast One-Hybrid System (Coolaber, China), following the manufacturer’s recommendations. The 2-kb promoter sequence of ClPAL20 was cloned into the Hin dIII and Xho I sites of the pAbAi vector. The minimum concentrations of Aureobasidin A (AbA) required to repress pAbAi- ClPAL20 -Pro self-activation. The CDS of ClWRKY75 was cloned into the EcoR I and BamH I sites of the pGADT7 vector. Then the recombinant plasmids were transformed into the Y1H gold strain (Coolaber, China). By observing the growth of co-transformants on synthetic dextrose minimal medium lacking leucine with 75 ng/mL AbA. The growth condition of yeast cell at 29 ℃. The primer sequences are shown in Supplementary Table 1. Dual-luciferase transient expression assay The CDS of ClWRKY75 was cloned into the Eco RI and Hin dIII sites of the pGreenII 62-SK, and the 2-kb promoter sequence of ClPAL10, ClPAL20, ClPAL30, ClPAL50, ClCCOAOMT were inserted into the Hin dIII and Bam HI of the vector pGreenII 0800-LUC, respectively. These recombinant plasmids were transferred into the Agrobacterium tumefaciens strain GV3101 (pSoup) and then transformed into N. benthamiana leaves. After 48 h injection, the N. benthamiana leaves were collected for fluorescence signal observation and LUC activity detection by using D-Luciferin (Beyotime,China) in LucRD plant vivo imaging system (YPH-Bio, China). Then the activity of renilla luciferase and firefly luciferase was examined using dual-luciferase assay reagents (TransGen, China). The primer sequences are shown in Supplementary Table 1. Electrophoretic mobility shift assay The full-length CDS sequence of ClWRKY75 was inserted into the Sac I and Bam HI of the pCold-His vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3) PlysS cells (WEIDI). The E. coli cultures at OD 600nm = 0.6 were treated with isopropyl-β-D-thiogalactoside to induce expression of fusion protein at 37 °C overnight. The recombinant protein ClWRKY75-His was purified by using His Label Protein Purification Kit (Beyotime,China). An electrophoretic mobility shift assay was performed using the Light Shift Chemiluminescent EMSA kit (Beyotime, China). Biotin-labeled DNA fragments containing the W-box cis -element were used as probes. The primer sequences are shown in Supplementary Table 1. Statistical analysis Statistical significance was determined using the Student’s t test or one-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test. Differences were considered statistically significant at P < 0.05. Post C. Fructicola infection symptomatic comparison between ‘M10’ and ‘HX6’ M10 is an inbred line resistant to C. fructicola , while HX6 is an inbred line sensitive to this pathogen (Fig.1, A). To explore variations in susceptibility to C. fructicola between two inbred lines, seedlings of both lines were inoculated with this pathogen. At the 3dpi, leaves were stained with trypan blue: a large number of blue spots appeared on HX6 leaves, while only a large amount of brown substances were observed on M10 leaves (Fig.S1). Additionally, prior to inoculation, no significant differences were detected in the activities of SOD, POD, and CAT, nor in the malondialdehyde (MDA) content, between the leaves of M10 and HX6 (Fig.1, B). In contrast, at 5 dpi, the activities of SOD, POD, and CAT in the leaves of M10 were significantly higher than those in HX6, while the MDA content in M10 was significantly lower than that in HX6 (Fig.1, B). Subsequently, we analyzed metabolite variations in leaves of the two inbred lines pre- and post-inoculation. Among the detected compounds, 2,654 differential metabolites were identified, and many differential metabolites were involved in PAL pathway. Of particular interest, p-coumaryl alcohol 4-O-glucoside, 2-Hydroxycinnamic acid, and 4-Methoxycinnamic acid are the intermediate products in lignin biosynthesis (Fig.1, C). Histological analysis showed that more lignin deposition could be observed in M10 after inoculation (Fig.1, D). To further investigate whether lignin content affects disease resistance, we determined the lignin content of the two materials pre- and post- inoculation. The results showed that no significant difference in leaf lignin content was detected between M10 and HX6 pre-inoculation; however, 5 dpi, the lignin content in M10 leaves was significantly higher than that in HX6 (Fig.1 E). These results suggested that the difference in lignin content post-inoculation plays a critical role in determining the C. fructicola resistance of the two materials. Identification of key transcripts involved in C. fructicola infection response via transcriptome sequencing To screen for genes whose expression is induced by C. fructicola , the Illumina HiSeq platform was used to sequence a total of 18 samples. The samples included leaves of two materials were collected pre-inoculation, and at 3 dpi and 4 dpi. After quality control, the 18 libraries yielded 119.09 Gb of clean reads in total, where the number of high-quality reads produced by each library fell between 19.04 million and 27.94 million (Tab.S2). Prior to inoculation, there were only 1,013 differentially expressed genes (DEGs) between M10 and HX6 (Fig.2, A-B). However, we found that the infection of the pathogen caused transcriptome changes in the two materials at 3 dpi (Fig.2, A). A total of 11,311 and 7,751 DEGs were identified, respectively, through the following comparisons: M10_0dpi vs. M10_3dpi, and HX6_0dpi vs. HX6_3dpi (Fig.2, C-D). Among these, 6,341 DEGs were detected in both groups, and 338 DEGs of these genes exhibited an up-regulated expression trend in M10 post-inoculation (Fig.2, E-F and Fig.S2). Further KEGG analysis of these 338 DEGs revealed that a large number of genes were enriched in the ‘Metabolism’, ‘Biosynthesis of other secondary metabolites’, ‘Phenylpropanoids biosynthesis’, and ‘Transcription factors’ (Fig.2, G). We identified a WRKY transcription factor, designated Cla97C05g095150 , named ClWRKY75 , which exhibited a high expression level in M10 post-inoculation. Subsequently, we analyzed the expression of all members of the WRKY family in the transcriptome, and only this gene was upregulated by nearly 100-fold after inoculation (Fig.S3). Which indicated that the expression of ClWRKY75 may induced by C. fructicola in M10. After re-inoculating the two inbred lines with anthracnose pathogen, we found that the expression level of ClWRKY75 in M10 was consistently higher than that in HX6, and showed a continuous upward trend with the progression of post-inoculation time (Fig.2, H). These results indicated that ClWRKY75 is the key transcript involved in the response of M10 to C. fructicola infection. ClWRKY75 positively regulates the resistance of watermelon to C. fructicola Being a transcription factor, ClWRKY75 showed a strong presence within the nucleus, and was highly expressed in root, stem and leaf (Fig.3, A-B). In addition, we analyzed the sequence differences of ClWRKY75 between the two inbred lines by using re-sequencing data. No high-confidence SNPs or Indels were detected between M10 and HX6 (Fig.S4). Then, we used Virus-Induced Gene Silencing (VIGS) technology to silence ClWRKY75 in M10, the positive plants exhibited sensitivity to C. fructicola (Fig.3, C and E). Compared to the control, the activities of three antioxidant enzymes SOD, POD, and CAT in ClWRKY75 silenced plants were significantly lower, while the MDA content was significantly increased (Fig.3, F-I). Furthermore, the lignin content in ClWRKY75 -silenced plants was significantly lower than that in control plants (Fig.3, D). These results suggested that C lWRKY75 is a positive factor contributing to the resistance of watermelon to C. fructicola . Lignin biosynthesis genes involved in resistance of watermelon to C. fructicola The cluster of genes consisting of the 338 DEGs that exhibited an up-regulated expression trend in M10 post-inoculation contains many genes associated with lignin biosynthesis, including ClPALs , Cl4CL , ClCCR and ClCCoAOMT (Fig.4, A). Among these genes, qRT-PCR results demonstrated that the expression levels of ClPAL10 , ClPAL20 , ClPAL30 and ClCCR in M10 leaves post-inoculation were higher than those in HX6 leaves (Fig.S5, A). Furthermore, we found that the expression of ClPAL10 , ClPAL20 , ClPAL30 and ClCCR was notably downregulated in ClWRKY75 -silenced plants (Fig.4, B-E), while the ClCCoAOMT exhibited a different expression level in different ClWRKY75- silenced plants (Fig.4, F). To verify whether these genes are involved in regulating watermelon resistance against C. fructicola , we further employed VIGS technology to individually silence ClPAL20 and ClCCoAOMT in M10. Consistent with our previous observations, both ClPAL20 -silenced and ClCCoAOMT -silenced plants displayed significantly increased sensitivity to C. fructicola , compared to control plants (Fig.4, G-H and Fig.S5, B-C). And the lignin content in both ClPAL20 -silenced and ClCCoAOMT -silenced plants decreased significantly (Fig.4, I and Fig.S5, D). These results suggested that the genes of lignin biosynthesis are involved in resistance to C. fructicola in watermelon. ClWRKY75 physically binds to and activates ClPAL20 Based on these findings, we hypothesized that ClWRKY75 may regulate the expression of lignin biosynthesis genes, thereby contributing to resistance against C. fructicola . To explore the underlying molecular mechanism, we analyzed the promoter of ClPAL20 (Fig.S6, A). Notably, the cis -acting element W-box, which is specifically recognized and bound by WRKY family, and were detected in ClPAL20 . Therefore, we performed a Y1H assay and found that ClWRKY75 could interact with the ClPAL20 promoter (Fig.5, A and Fig.S6, B). To further confirm the interaction between ClWRKY75 and ClPAL20 , an electrophoretic mobility shift assay (EMSA) was performed. The results showed that the ClWRKY75-His fusion protein can interact with specific fragments (-147 to -118 bp) containing W-box cis- acting element, while increasing the cold probe levels reduced the binding of ClWRKY75-His fusion protein to the ClPAL20 promoter (Fig.5, B). We also conducted a dual-luciferase reporter assay to clarify the effect of ClWRKY75 on ClPAL20 promoter activity. Compared to the control N. benthamiana leaves (pGreenII 62-SK + pGreenII 0800: ClPAL20- Pro::LUC), the leaves co-transformed with pGreenII 62-SK:ClWRKY75 and pGreenII 0800: ClPAL20- Pro::LUC exhibited stronger luciferase signals, indicating that ClWRKY75 activates the transcription of ClPAL20 . Taken together, these results demonstrate that ClWRKY75 acts as an upstream activator of ClPAL20 (Fig.5, C and D). In addition, we also observed W-box elements in the promoter of ClPAL10 , ClPAL30 , ClCCR and ClCCoAOMT (Fig.S6, A). Thus, we further performed dual-luciferase reporter assays to clarify the effect of ClWRKY75 on promoter activity of these genes. In the co-transformed groups with the 62SK-ClWRKY75 construct and each of the reporter constructs (LUC- ClPAL10 , LUC- ClPAL30 , LUC- ClPAL50 , LUC- ClCCR , and LUC- ClCCoAOMT ), significantly elevated dual-luciferase activity was observed, relative to the control group (Fig.5, E). These results suggested that ClPAL20 is involved in regulating watermelon resistance against C. fructicola , and may function downstream of ClWRKY75 in the disease resistance regulatory pathway. Discussion Colletotrichum fructicola , which belongs to CGSC , a newly identified anthracnose pathogen in Hubei, Hunan and Jiangxi provinces of China, that can infect the leaves and stems of watermelon. Chemical control is the main method for anthrax prevention and control, but it gives rise to concerns about food safety. It is crucial to investigating key resistance-related genes, unraveling the underlying disease resistance mechanisms, and employing molecular breeding approaches are the effective ways to protect and improve watermelon industry sustainable development. Plant resistance mechanisms are sophisticated, which are usually controlled by multiple quantitative trait locis (QTLs) of minor to moderate effects. With the improvement of the watermelon genome, researchers have identified multiple QTL loci or genes related to powdery mildew and bacterial fruit blotch (Deng et al., 2024; Yeo et al., 2022). In recent years, mining relevant disease resistance genes using transcriptomics has become an important approach in horticultural crops (Hao et al., 2025; Jeyaraj et al., 2019; Jiang et al., 2025) . Cslnc170 , a novel long noncoding RNA, activates CsLOX14 expression via loop 4 of its secondary structure and the CsLOX4 promoter region, thereby enhance resistance to Colletotrichum camelliae in tea (Jiang et al., 2025). RNA-seq analysis revealed a high expression level gene, CsNAC17 ，the interaction between CsbHLH62 promotes CsRPM1 expression following Colletotrichum gloeosporioides infection in the resistant accessions.(Han et al., 2025). In the present research, we identified 338 genes highly expressed in the M10 post-inoculation C. fructicola leaves by RNA-seq analysis. The KEGG results showed that many genes were enriched in the “biosynthesis of secondary metabolites”, “biosynthesis of phenylpropanoids”, and “transcription factors” (Fig.2 E, Fig.S2 and Fig.S3). Lignin is one of the components that make up plant cell walls and plays a vital role in helping plants cope with both biotic and abiotic stress. Recent studies have shown that the biosynthesis of lignin enables plants to resist infection by a variety of pathogens, including Colletotrichum , Ralstonia solanacearum , Botryosphaeria dothidea , Rhizoctonia solani and Xoo (Cheng et al., 2024; Li et al., 2023; Lin et al., 2022; Liu et al., 2020; Yang et al., 2024) . The biosynthesis of lignin relies on relevant genes in the phenylalanine ammonia-lyase (PAL) metabolic pathway. In our study, multiple DEGs and differential metabolites involved were identified in the PAL pathway in metabolomics and transcriptomics analyses (Fig.1 C and Fig.4 A). We further found lignin could enhance the resistance to C. fructicola in watermelon, through histological observation and content determination (Fig.1 D and E). Silencing the expression of ClPAL20 and ClCCoAOMT in M10 significantly decreased both its lignin content and resistance to C. fructicola (Fig.4, G-I and Fig.S5, B-D). In addition, Liu found that PAL genes mediated a substantial increase in Salicylic Acid levels around 8 h after inoculation as treat-ment of Pst DC3000 triggers in N. benthamiana (Liu et al., 2025) . In the ClWRKY75- silenced plants, the expression of ClPAL10 , ClPAL20 , ClPAL30 , ClPAL50 and ClNPR1 were decreased compared by control plants. This finding suggests that SA may be involved in the resistance of watermelon to C. fructicola . Generally, the expression of PAL genes is often directly activated or repressed by transcription factors (Fig.4, B-F and Fig.S5, E)(Geng et al., 2020; Miyamoto et al., 2020). However, recent studies showed that DNA demethylation could influence the expression of PAL genes. Li found that Strigolactone-mediated DNA demethylation could increase the demethylation of SlPAL5 at CHG site in gene body, SlPAL at CHG site in promoter and SlHDC at CHG site in promoter may result in the transcription of the genes, activating its expression. (Li et al., 2025a). In present study, we identified W-box elements in the promoters of several PAL pathway genes. Several Dua-LUC assays showed these genes were activated by ClWRKY75 (Fig.5, E). Y1H and EMAS assays indicated that ClWRKY75 could bind the promoter of ClPAL20 (Fig.5, A-D and Fig.S6, B). The WRKY gene family is one of the transcription factor families in plants, regulating plant stress resistance. WRKY TFs were distinguished by the presence of one or two WRKY domains (the conserved motif WRKYGQK) at their N-termini and zinc-finger-like motifs at their C-termini, which could specifically bind to W-box (TTGAC[T/C]) within the target genes promoter(Wang et al., 2023a). The expression of WRKY TFs is induced by many environment factors, for example, salt, drought, and pathogenic bacteria(He et al., 2016; Huangfu et al., 2016; Jiang and Deyholos, 2008). After pathogen invasion, some transcription activator-like effectors (TALEs) can directly bind to WRKY transcription factors, thereby increasing the plant’s susceptibility to the pathogen. Specifically, Xoo1219 and Xoo2145 activate the expression of OsWRKY104 and OsWRKY55 , respectively, through direct interactions(Im et al., 2025). However, some WRKY TFs can be activated by Mitogen-Activated Protein Kinase signaling pathway. For instance, OsWRKY31 and OsWRKY70 could interact with some OsMKKs or OsMPKs , and the phosphorylated form of OsWRKY31 and OsWRKY70 has increased DNA binding activity(Li et al., 2015; Wang et al., 2023b). In the present study, we found the expression level of ClWRKY75 differs between M10 and HX6 after C. fructicola (Fig.2, F). Interestingly, there were no difference in the coding region sequences and promoter sequences of ClRKY75 between M10 and HX6 (Fig.S4). This finding indicated that differences in the upstream genes of ClWRKY75 , such as members of mitogen-activated protein kinases lead to variations in its expression level among different materials. Conclusion The watermelon anthracnose resistance-related gene ClWRKY75 was identified through screening based on transcriptomic technology. Using gene silencing, we clearly elucidated the core role of ClWRKY75 in regulating the resistance response of watermelon to anthracnose. Specifically, ClWRKY75 specifically binds to and activates the expression of ClPAL20 , a key gene in the PAL metabolic pathway. This activation promotes the biosynthesis of lignin and its accumulation in leaves, which in turn enhances the physical structural stability of leaf cell walls. In summary, our study revealed the molecular mechanism underlying watermelon’s response to Colletotrichum fructicola mediated by ClWRKY75 (Fig. 6). Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Declaration of interests All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research was supported by the Yuelushan Laboratory Breeding Program (Grant No. YLS-2025-ZY02015), the China Agriculture Research System-25 (Grant No. CARS 25) and the Graduate Student Scientific Research and Innovation Program of Hunan Agricultural University (Grant No. CX20251126). Author contributions B.T., S.D. and X.S. designed the research; S.G., and D.W. conducted the research; S.Y., Z.Y., G.L., C.H. contributed to experimental design, data interpretation; F.B., L.S., Z.C. edited the manuscript; S.G. and D.W. participated in data analysis and manuscript writing. Reference Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, Liu Y, Feng J, Chen H, He Y, Xia R , 2023. 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(E) The lignin content of M10 and HX6 pre- and post- inoculation. Figure 2. ClWRKY75 is associate with the resistance to C. fructicola. (A) PCA analysis of transcriptome samples. (B) Number of DEGs in HX6_0 dpi_VS_M10_0dpi. (C) Number of DEGs in HX6_0dpi_VS_ HX6_3dpi. (D) Number of DEGs in M10_0dpi_VS_M10_3dpi. (E) Venn Diagram Analysis of Differentially Expressed Genes Between 0 dpi and 3 dpi in HX6 and M10. (F) A cluster of genes up-regulated in M10 after pathogen inoculation in time-series clustering analysis. (G) KEGG enrichment analysis of 338 annotated genes in the 6th cluster of time-series clustering. (H) Expression levels of ClWRKY75 in M10 and HX6 at 2-5 days after re-inoculation with pathogens. Figure 3. Function analysis of ClWRKY75 . (A) Subcellular localization of ClWRKY75 in N. benthamiana leaves. Mcherry, mCherry signal; Bright, Bright field; GFP, GFP signal; Merged, combined Bright, GFP and mCherry signals. (B) Expression pattern of ClWRKY75 in different tissues between M10 and HX6. R, Root; S, Stem; L, Leaf; Ff, Female flower; Fm, Male flower. (C) The relative expression level of ClWRKY75 between positive plants and control. (D) The lignin content difference between ClWRKY75 -silenced plants and control plants. *** means significantly difference. (E) The phenotype difference between ClWRKY75 -silenced plants and control plants after inoculation with C. fructicola . (F) (G) (H) (I) The activates difference of SOD, POD, CAT and MDA between ClWRKY75 -silenced plants and control plants. ** , *** means significantly difference. Figure 4. Lignin biosynthesis genes involved in the resistance to C. fructicola. (A) Expression trends of genes related to the lignin biosynthesis pathway in the transcriptome. (B) (C) (D) (E) (F) Expression level of ClPAL10 , ClPAL20 , ClPAL30 , ClCCR and ClCCoAOMT in ClWRKY75 -silenced plants. (G) Phenotypes of control plants and ClPAL20 -silenced plants after inoculation with C. fructicola . (H) Identification of ClPAL20 -silenced plants. (I) Lignin content in leaves of ClPAL20 -silenced plants after inoculation with C. fructicola . Figure 5. Lignin biosynthesis genes may downstream of ClWRKY75. (A) Yeast one-hybrid assay verified that ClWRK75 can bind to the promoter element of ClPAL20 . (B) Electrophoretic Mobility Shift Assay (EMSA) verified the specific binding of ClWRKY75 to the promoter element of ClPAL20 . (C) Schematic diagram of Dual-LUC assay vector construction. (D) LUC/REN Ratio of ClWRKY75 and ClPAL20 . ** means significantly difference. (E) Dua-LUC analysis of ClWRKY75 with ClPAL10 , ClPAL30 , ClPALCCR and ClCCoAOMT. ** , *** means significantly difference. Figure 6. A regulatory model of the resistance of watermelon to C. fructicola mediated by ClWRKY75 . Supplemental Data Supplemental Figure 1. Trypan blue staining of leaves from M10 and HX6 after inoculation with pathogens at 0 d, 3 d, and 5 d Supplemental Figure 2. Temporal clustering of 6,341 differential expression genes Supplemental Figure 3. The heatmap of WRKY gene family Supplemental Figure4. The sequence analysis of ClWRKY75 between M10 and HX6 Supplemental Figure 5. The expression of PAL genes and function analysis of ClCCoAOMT. (A) The expression of PAL genes in pre- and post-inoculation between M10 and HX6. (B) The phenotype of ClCCoAOMT -silenced plants and control plant. (C) The expression check of positive plants. (D) The lignin content of ClCCoAOMT -silenced plants and control plant. (E) The expression of ClNPR1 in ClWRKY75 -silenced plants. Supplemental Figure 6. (A) The W-box element location in PAL genes promoters. (B) The Self-activation analysis of the ClPAL20 promoter. Supplemental Table 1. Primer sequences Supplemental Table 2. Summary of transcriptomic data Information & Authors Information Version history V1 Version 1 04 November 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords clwrky75 colletotrichum fructicola lignin synthesis metabalome watermelon Authors Affiliations Shiqi Gong Hunan Agricultural University View all articles by this author Dengke Wang Hunan Agricultural University View all articles by this author Shan Yuan Hunan Agricultural University View all articles by this author Zihao Yang Hunan Agricultural University View all articles by this author Guang Liu Jiangsu Academy of Agricultural Sciences View all articles by this author Changzheng He Hunan Agricultural University View all articles by this author Fengjiao Bu Hunan Agricultural University View all articles by this author Longjun Sun Hunan Xuefeng Seeds Co Ltd View all articles by this author Zhongquan Chen Hunan Xuefeng Seeds Co Ltd View all articles by this author Shan Li Hunan Agricultural University View all articles by this author Xiaowu Sun Hunan Agricultural University View all articles by this author Sihui Dai Hunan Agricultural University View all articles by this author Bingqian Tang [email protected] Hunan Agricultural University View all articles by this author Metrics & Citations Metrics Article Usage 191 views 157 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shiqi Gong, Dengke Wang, Shan Yuan, et al. Transcriptome analysis reveals ClWRKY75 enhanced the resistance to Colletotrichum Fructicola via activating the Phenylalanine Ammonia-Lyase Pathway in Watermelon. Authorea . 04 November 2025. DOI: https://doi.org/10.22541/au.176224947.72313670/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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