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Phytoalexin deficient 4 is associated with the lesion mimic trait in watermelon clalm mutant (Citrullus lanatus)

BMC Plant Biology volume 25, Article number: 92 (2025) Cite this article

Abstract

In watermelon (Citrullus lanatus), lesion mimic is a rare, valuable trait that can be used by breeders for selection at early growth stages. In this study, we tested a seven-generation family to determine the inheritance and genetic basis of this trait. As revealed by analysis of the lesion mimic mutant clalm, this trait is controlled by a single dominant gene. Whole genome resequencing–bulked segregant analysis demonstrated that this gene is located on chromosome 4 from 3,760,000 bp to 7,440,000 bp, a region corresponding to a physical distance of 3.68 Mb encompassing approximately 72 annotated genes. There are 6 genes with non-synonymous mutation SNP sites. The predicted target gene, ClCG04G001930, encodes a Phytoalexin deficient 4 (PAD4), a protein that plays an important regulatory role in leaf senescence in many plant species. According to quantitative real-time PCR analysis, the expression level of ClCG04G001930 was significantly higher in the clalm mutant than in normal watermelon. Twenty-five SNPs were identified in the ClCG04G001930 gene of F2 individuals of the clalm mutant. Overexpression the ClCG04G001930 gene, designated as ClPAD4, yielded transgenic lines whose leaves gradually developed chlorotic lesions over 3 weeks. RNA interference of the ClPAD4 yielded transgenic lines whose cotyledon prone to diseased over 2 weeks. Our results suggest that ClPAD4 might be the candidate gene responsible for lesion mimic in the clalm mutant. Our findings may serve as a foundation for elucidating the mechanism underlying the molecular metabolism of programmed cell death and should be useful for marker-assisted selection breeding in watermelon.

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Introduction

Plant lesion mimic mutants (LMMs) exhibit spontaneous cell death in the absence of pathogen attack [1]. The LMMs of rice, such as spl7, spl11, spl28, spl30 and lmr, exhibit small, reddish-brown lesions or spots that develop at leaf tips or bases and gradually spread on fully expanded leaves [2,3,4,5]. In Arabidopsis, overexpression of UGT76D1, which plays an important role in SA homeostasis, results in a hypersensitive response (HR)-like lesion mimic phenotype [6]. In birch, BpGH3.5, an early auxin-response factor regulating root elongation, gives rise to typical LMM characteristics and accelerates leaf senescence [7]. In switchgrass, co-silencing of methylenetetrahydrofolate reductase and caffeic acid O-methyltransferase results in a novel lesion-mimic leaf phenotype [8]. In contrast to these findings, the genetic mechanism underlying the lesion-mimic leaf phenotype of watermelon (Citrullus lanatus) remains unknown.

The genome of watermelon (2n = 2x = 22) comprises 11 chromosomes and has an approximate size of 425 Mb. The annotated watermelon genome sequence, which was released in 2013, includes 23,440 predicted protein-coding genes and is thus suitable for next-generation sequencing to accelerate the identification of candidate genes controlling important agronomic traits in this species. Watermelon fruit shape, skin color, and rind stripe patterning have been previously investigated by bulked segregant analysis and next-generation sequencing technology (BSA-seq) [9,10,11]. Many dwarfing genes have been identified through BSA-seq. Two single-nucleotide polymorphism (SNP) mutation of GA20ox, one SNP mutation of GA3ox and a SNP deletion in an ABC transporter gene lead to dwarfism phenotypes in different watermelon [12,13,14,15]. The SNP-based genetic linkage map was constructed using a recombinant inbred lines derived from a cross between the 97103 (Citrullus lanatus var lanatus) and the 296341-FR (Citrullus lanatus var citroides) [16,17,18]. The objective of the present study was the identification of the LMM gene in the watermelon clalm mutant. We re-sequenced the whole genome of two DNA bulks (i.e., spotted leaf and normal pools) developed from plants in an F2 population. Our study results provide preliminary evidence that ClCG04G001930 encoding phytoalexin deficient 4 (PAD4) might be the candidate gene responsible for. To our knowledge, our study is the first reported gene mapping and elucidation of the inheritance mechanism controlling lesion mimic in watermelon. To improve the quality of watermelon cultivars, breeders have focused on the introduction of novel traits into existing germplasm. The application of the spotted leaf trait as a selection tool in breeding programs will thus help improve the ability of breeders to make selections at early growth stages, thus accelerating the watermelon breeding program. In practical production and application, the seedlings with spots should be retained, and the seedlings without spots should be removed. The purity of watermelon can be guaranteed by observation, which is simple and fast.

Methods

Plant materials

The clalm mutant (lesion mimic mutants of Citrullus lanatus var lanatus) was detected in an inbred watermelon line derived from ‘V603’ (Citrullus lanatus var lanatus) in July 2012, after years of screening and cultivating. The clalm mutant were analyzed by conducting testcross experiments. The clalm mutant were hand-pollinated with pollen from four inbred lines ‘Kexi’, ‘Hei2’, ‘Cai10’, and ‘K1’ (Citrullus lanatus var lanatus) to generate the F1 generation in the spring of 2013. The wild type watermelon cultivars ‘Zd’ (Citrullus lanatus var lanatus) is male parent with no spotting phenotypes. The clalm mutant is female parent with yellow spots spreading over leaves and fruits. Watermelon cultivars ‘Zd’ and clalm mutant were self-pollinated for seven generations to obtain stable phenotypes. F2 population obtained by clalm and ‘Zd’ crosses. For use as BSA-seq research materials, 60 F2 segregation populations (30 + 30 mixed pools with extreme characteristics), 10 parent plants clalm and ‘Zd’ were grown and evaluated at the Henan University Genetics and Breeding Base in the spring of 2018.

Trypan blue staining

To analyze the state of lesion mimic leaf cells, leaves were incubated in fixative solution, immersed and rinsed five times in ultrapure water, and placed on filter paper. After absorption of excess water, the leaves were stained at room temperature for 2 to 6 h in darkness. Next, the leaves were immersed and rinsed five time in ultrapure water and then immersed and stored in plant trypan blue dye solution B for 3 to 16 h. After incubation for 30 min at room temperature in 20 ml of plant trypan blue dye solution, the leaves were photographed.

Microscopic observations

For light microscopic examination, leaves were fixed in fixative solution (4% paraformaldehyde) for 24 h and then dehydrated, embedded in paraffin, and sliced. The slices were stained with toluidine blue and sealed in neutral gum heated to 38 °C. The prepared samples were observed with an Eclipse E100 microscope (Nikon, Tokyo, Japan) at 100 × magnification. For staining prior to transmission electron microscopy (TEM) examination, targeted fresh tissues were selected to minimize mechanical damage. Fresh tissue blocks (≤ 1 mm2) were quickly cut with a sharp blade and harvested within 1 to 3 min. The washed tissue blocks were immediately fixed with electron microscopy fixative for 2 h at room temperature and then transferred to 4 °C for preservation and transportation.

Samples were fixed in a solution of 1% OsO4 in PB (0.1 M, pH 7.4) for 7 h at room temperature. After removal of OsO4, the tissues were rinsed three times in 0.1 M PB (pH 7.4), dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%) for 1 h at each concentration, resin penetrated, and embedded at 37 °C in a graded reagent series (3:1 acetone:EMBed 812 for 2–4 h; 1:1 acetone:EMBed 812 overnight; 1:3 acetone:EMBed 812 for 2–4 h; and pure EMBed 812 for 5–8 h). Tissues were inserted into embedding molds filled with pure EMBed 812 resin and then incubated overnight in a 37 °C oven. The embedding molds containing resin and samples were moved to a 65 °C oven and allowed to polymerize for more than 48 h. The resin blocks were cut into 60- to 80-nm sections using an ultramicrotome, and the tissues were fished out onto 150-mesh cuprum grids with formvar film. The grid-attached tissues were stained with 2% uranium acetate-saturated alcohol solution in darkness for 8 min, rinsed three times each with 70% ethanol and ultrapure water, stained with 2.6% lead citrate in the absence of CO2 for 8 min, and rinsed three times with ultrapure water. After blotting with filter paper, the cuprum grids were placed on a grid board and dried overnight at room temperature. The samples were examined under an HT7700 transmission electron microscope (Hitachi, Japan).

Sample collection and BSA library preparation

Young leaves of the parents and F2 plants were collected and immediately placed in liquid nitrogen, and total genomic DNA was isolated using the CTAB method. Genomic DNA samples of 60 F2 segregation populations and 10 parent plants were subjected to whole-genome resequencing performed by the Beijing Biomarker Technologies Corporation (Beijing, China). To generate bulked samples, equal amounts of DNA from each plant per group were mixed to form yellow-spot (Y-pool) and normal (N-pool) sets at a final concentration of 40 ng/μL. Prior to high-throughput sequencing, DNA samples were sonicated to produce 350-bp fragments. After trimming of barcodes, clean, high-quality reads from identical samples were mapped onto the C. lanatus subsp. vulgaris ‘Charleston Gray’ genome sequence (http://cucurbitgenomics.org/organism/4). The reason for selecting C. lanatus subsp. vulgaris ‘Charleston Gray’ genome sequence as the reference genome is that the character of clalm mutant is similar to that. All identified SNPs shared across the bulk were considered polymorphic in association studies. To represent the difference between the SNP index of the two pools, we calculated ΔSNP and ΔInDel indexes, which are association analysis metrics used to find significant differences in genotype frequency between two pools [19, 20]. Candidate regions over the threshold (99th percentile) were extracted from each linkage group.

Expression analysis of candidate genes

We investigated the expression patterns of ClCG04G000300, ClCG04G000420, ClCG04G001450, ClCG04G001740, ClCG04G001900, and ClCG04G001930 using qRT-PCR. 'Zd' and clalm mutant seedlings were grown for 10, 30, and 60 days. Spotted leaves and normal leaves were collected from three plants each, and each sample collection was repeated three times. After addition of SYBR Green I to each reaction mixture, qRT-PCR amplifications were performed on a Roche LightCycler 480 II instrument using the following thermal profile: a 30-s hot start at 95 °C followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The ClYLS8 gene was included as a control for normalizing gene expression data [12]. The primers used to amplify genes are listed in Table 1.

Table 1 Sequences of primers used for quantitative real-time PCR
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Bioinformatic analysis

The homology of nucleotide sequences of candidate gene was analyzed online (http://www.ncbi.nlm.nih.gov). A phylogenetic tree comprising the target gene and its homologous sequences was constructed using the MEGA 7.0 software [21]. A bootstrap analysis (1,000 replicates) was completed to assess the reliability of the tree [22]. The high quality tree figure was created online (https://itol.embl.de/). The protein tertiary structure of ClPAD4 was predicted online ( https://swissmodel.expasy.org/) [23].

Watermelon transformation

The binary vectors pCambia 1300 were constructed. The candidate gene was inserted between SalI and SpeI sites in the vector. The expression vector pCAM1300 were constructed. The candidate gene was inserted between SalI and SpeI sites in the vector. The binary RNAi interference vectors DHpart27RNAi-FADP1P4 were constructed as described by Dong et al. (2021) [24]. In brief, 300-bp target fragments of the candidate gene were inserted between HindIII and XbalI sites in the vector, and the reverse sequence was inserted between XhoI and KpnI sites. The 300-bp target fragments contain the non-synonymous coding SNP site. The watermelon explants were transformed according to a modified method [17, 25, 26]. In particular, surface-sterilized watermelon seeds of watermelon cultivars Kexi (the wild type) were sown on basic Murashige-Skoog solid medium supplemented with 3% Suc and maintained for 2 days in darkness and for 2 to 3 days under a 16-h/8-h light/dark photoperiod. The cotyledons were excised, and hypocotyls and the apical portion were discarded. Agrobacterium tumefaciens strain GV3101 harboring the binary vector was used for the transformation. The cotyledon explants were co-cultivated in darkness for 4 days. After transfer and maintenance on selective induction medium for 4 weeks, the explants were transferred onto selective elongation medium containing 0.2 mg/L KT, 60 mg/L Kan, and 400 mg/L Cef and incubated for 2 weeks. Plantlets with well-developed roots were taken from the rooting medium and placed in plastic cups containing vermiculite. The traits of T0 plants were detected and the transgenic plants following self-pollination. The transgenic plants were self-pollinated for five generations to obtain stable phenotypes.

Results

Morphology of clalm mutant

To evaluate the genetic stability, clalm mutant were hand-pollinated using pollen from four inbred lines (‘Kexi’, ‘Hei2’, ‘Cai10’, and ‘K1’) to produce F1 and F2 generations. All plants of the F1 generation developed spotted leaves (Fig. 1 a–d), whereas the ratio of spotted- to non-spotted-leaf plants in the F2 population conformed to a Mendelian segregation ratio of 3:1 (χ2 ≤ 0.5, P < 0.05) (Table 2). The parents, F1 and F2 phenotypes of the constructed population were shown in fig. S3.

Fig. 1
figure 1

The Characteristics of F1 hybrids’generation. a F1 hybrid between Kexi and clalm mutant. b F1 hybrid between Hei2 and clalm mutant. c F1 hybrid between Cai10 and clalm mutant. d F1 hybrid between K1 and clalm mutant

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Table 2 The segregation analysis on the vine and fruit type of F2 population
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Necrotic tissue of clalm mutant spotted-leaf

In contrast to living tissue, necrotic tissue can be stained with trypan blue. Plants of clalm mutant were self-pollinated for seven generations to yield stable lesion mimic on leaves and fruits. Yellow spots appeared on leaves 10 days after sowing and then gradually spread over entire leaves and fruits during the life cycle of clalm mutant, the female parent (Fig. 2 a-d). Dead leaf-membrane cells will appear dark blue, whereas other parts should be almost colorless. We therefore stained leaves of the wild type and clalm mutant with trypan blue, which revealed that leaves of clalm mutant, but not those of the wild type, harbored small, spontaneous lesions (Fig. 2 e, f).

Fig. 2
figure 2

Comparative morphology of clalm mutantand wild-type ‘Zd’ watermelon. a-b Plants of lesion mimic mutant clalm (a) and ‘Zd’ (b) growing at the experimental breeding site. (c) Spotted leaf of lesion mimic mutant clalm. d Non-spotted leaf of ‘Zd’. e-f Leaves of lesion mimic mutant clalm (e) and ‘Zd’. f stained with trypan blue

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Microscopic observations

No significant differences between speckled leaf areas and normal leaves were observed under a light microscope (Fig. 3a, b). Obvious differences were detected, however, using TEM. First, mesophyll cells of normal leaves were rich in chloroplasts, whereas those of clalm mutant spotted leaves contained very little chlorophyll (Fig. 3c, e). Second, mesophyll cell chloroplasts of normal leaves were rich in starch grains, but starch grains were absent from those of spotted leaves (Fig. 3d, f). Finally, mesophyll cell chloroplasts of normal leaves were rich in grana; in contrast, those of spotted leaves had no grana and only a few thylakoids (Fig. 3d, f).

Fig. 3
figure 3

Optical microscope imaging of wild-type ‘Zd’ (a) and lesion mimic mutant clalm (b) leaf cross sections. Electron microscope imaging of wild-type ‘Zd’ (c, d) lesion mimic mutant clalm (e, f) leaf cross sections. E, epidermis; PM, palisade mesophyll cells; SM, spongy mesophyll cells; S, stoma; V, vein; C, chloroplasts; A, amyloplasts; P, peroxisome; SG, starch grain

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Whole-genome resequencing analysis

Filtering of the raw data yielded 43.89 G of clean data for further analysis. The Q30 ratio of the clean data was 85%, and the GC content was 41.67%, with an average genome resequencing depth of 22.75 × and a genome coverage of 98.81%. The data have been submitted to the NCBI database. Compared with the reference genome, 165,590 SNPs and 71,589 insertions–deletions (InDels) were identified between spotted- and non-spotted-leaf parents (Fig. S1). A Circos plot of the chromosomal distribution of candidate regions among samples is shown in Fig. 4. In addition, 12,784 SNPs and 19,212 InDels were found in the two bulk segregant populations. Association analyses between the two bulks based on SNP and InDel indexes narrowed the candidate region to approximately 4.04 Mb (Fig. 5 a) and 4.00 Mb (Fig. 5 b), respectively. Collectively, these two indexes indicated the candidate gene was located in an approximately 3.68 Mb region on chromosome 4 containing roughly 72 annotated genes. There are 6 genes with non-synonymous mutation SNP sites, ClCG04G000300, ClCG04G000420, ClCG04G001450, ClCG04G001740, ClCG04G001900, and ClCG04G001930, which were likely directly associated with leaf spotting.

Fig. 4
figure 4

Circos plot of the chromosomal distribution of candidate regions among watermelon samples. Proceeding from the outside to the inside, the circles indicate (1) chromosomal coordinates and the distributions of (2) genes, (3) SNP densities, (4) ED values, and (5) ΔSNP index values

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Fig. 5
figure 5

Chromosomal distributions of SNP, ΔSNP, InDel, and ΔInDel index values. a Chromosomal distributions of SNP index values in the non-spotted-leaf plant bulk from the F2 population (top), SNP index values in the spotted-leaf bulk from the F2 population (middle), and the difference in SNP index values (ΔSNP) between the two bulks (bottom). b Distributions of InDel index values in the non-spotted-leaf plant bulk from the F2 population (top),InDel index values of the spotted-leaf bulk from the F2 population (middle), andΔIndel index values between the two bulks (bottom). The 99% threshold in each figure is indicated by a red dashed line, and Citrullus lanatus chromosome numbers are shown at the bottom

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Identification of the LMM gene

We examined expression levels of six candidate genes (ClCG04G000300, ClCG04G000420, ClCG04G001450, ClCG04G001740, ClCG04G001900, and ClCG04G001930) in the two parental watermelon lines by qRT-PCR to assess whether their expression levels were correlated with the spotted leaf phenotype (Fig. 6). The expression level of ClCG04G001930 was significantly higher in the clalm mutant than in normal watermelon (P < 0.05), thus suggesting that this gene might be the candidate gene responsible for leaf spotting in clalm mutant. The SNP of ClCG04G001930 gene in the F2 population was examined by sequencing. Twenty-five SNPs were identified in ClCG04G001930, including a non-synonymous coding SNP, five SNPs in the ClCG04G001930 promoter and seventeen SNPs in the intron (Table 3). The SNP analysis resulted in one non-synonymous coding SNP locating in the ClCG04G001930 gene exon. Mutation of amino acid codon from GTA to ATA (Fig. S2). Amino acid changes from Ile to Val.

Fig. 6
figure 6

Relative expression levels of six candidate genes in clalm mutant and wild-type ‘Zd’ watermelon. Error bars represent the standard deviation of mean values. Significant differences (P < 0.05) between parents are indicated by different letters

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Table 3 Chromosomal positions and codons of 20 identified SNPs in ClPAD4
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ClCG04G001930 was predicted to encode a PAD4, a protein that plays an important regulatory role in leaf senescence in many plant species. We therefore hypothesized that ClCG04G001930 is a PAD4 homolog in watermelon and accordingly named this gene ClPAD4. According to an amino acid sequence multiple alignment (Fig. 7 a), the ClPAD4 protein is 100% similar to PAD4 proteins of Cucumis sativus (accession number XP_011653897.1), Cucumis melo (XP_008442139.1), Cucurbita maxima (XP_022966244.1), and Momordica charantia (XP_022149409.1). The constructed phylogenetic tree clarified the molecular evolutionary relationship between ClPAD4 and its homologs (Fig. 7 b).

Fig. 7
figure 7

Bioinformatics characteristics of ClPAD4. a Results of Blast comparison of ClPAD4 proteins from various angiosperms. Query_10001 = Citrullus lanatus PAD4 (Gene ID:ClCG04G001930); Query_10002 = Benincasa hispida PAD4 (gene ID:LOC120072712); Query_10003 = Cucumis sativus PAD4 (gene ID:LOC101222098); Query_10004 = Cucumis melo PAD4 (gene ID:LOC103486086); Query_10005 = Cucurbita maxima PAD4 (gene ID:LOC111465963); Query_10006 = Momordica charantia PAD4 (gene ID:LOC111017842); Query_10007 = Quercus suber PAD4 (gene ID:LOC112032821); Query_10008 = Ziziphus jujuba PAD4 (gene ID:LOC107404877); Query_10009 = Tripterygium wilfordii PAD4 (gene ID:LOC119990818); Query_10010 = Arabidopsis thaliana PAD4 (gene ID:At3g52430). b Phylogenetic tree based on ClPAD4 in different organisms. The GenBank accession numbers are provided. The sequences are in Supplementary Material

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Over-expression of ClPAD4 in watermelon

To characterize the function of ClPAD4, we generated transgenic watermelon plants for this gene. We obtained 35 transgenic plants and screened them for positive clones (Fig. 8a, b, c). The yellow spots gradually appeared on leaves of T0 ClPAD4 over-expression after Agrobacterium infection (Fig. 8c). The rooted shoots were transferred to plastic cups containing vermiculite (Fig. 8d). The chlorotic lesions on the transgenic leaves are marked with red boxes.

Fig. 8
figure 8

Growth stages of transgenic watermelon. a Precultured watermelon cotyledons. b Adventitious shoots regenerated from cotyledons after 1 weeks. c Adventitious shoots regenerated from cotyledons after 3 weeks. d Transgenic plants after transplantation

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RNAi of ClPAD4 in watermelon

To characterize the function of ClPAD4, we generated transgenic knockdown watermelon plants for this gene. We obtained 56 transgenic plants and screened them for positive clones (Fig. 9a). The rooted shoots were transferred to plastic cups containing vermiculite (Fig. 9b). And the transgenic plants do not exhibit specific traits. The homozygous fruits were produced by the transgenic plants following self-pollination (Fig. 9c). It's very interesting that the cotyledons of T5 ClPAD4 RNAi watermelon seedlings susceptible to mosaic virus infection in a normal production environment which was not sterile environment (Fig. 9d). After transplantation, the leaves of the transgenic seedlings grew normally (Fig. 9e).

Fig. 9
figure 9

Growth stages of transgenic watermelon. a Adventitious shoots regenerated from cotyledons after 3 weeks. b Enlarged adventitious shoots. c T0 generation watermelon fruit. d-e 1-week-old (d) and 1-month-old (e) T5 generation seedlings

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Discussion

The strategy used by watermelon breeders to improve the quality of watermelon cultivars is the introduction of novel traits, especially ones useful for seedling screening, into existing germplasm. The underlying gene in LMM of watermelon and other cucurbitaceous crops has not yet been well studied.

The timing of spot appearance can be used to circumscribe vegetative, reproductive, and complete growth periods [1, 27]. In our study, lesion mimic emerged on watermelon cotyledons 10 days after sowing, which makes their presence an obvious marker for identifying impure and aberrant varieties at the seedling stage. The seedlings grow very slowly and flowered late, which was consistent with previous reports. In rice LMMs, leaf spots appear during the seedling stage and are relatively weakly correlated with the duration of the entire growth period [28]. We found that watermelon lesion mimicry is controlled by a single dominant gene. LMMs are relatively rare in cucurbitaceous species, with the spotting trait mainly manifested in fruits and seeds. In addition, a single dominant gene is responsible for fruit spotting in non-spotted varieties of Cucumis melo, Cucurbita pepo, and watermelon [6, 29,30,31,32]. In rice, a single dominant gene controls the LMM phenotype of NH1, spl12, spl13, spl15, and spl24 mutants, whose leaves display small, reddish-brown lesions or spots [33,34,35]. Many LMMs exhibit altered disease resistance and are thus considered ideal for studying signaling pathways in species such as Arabidopsis, maize, and rice [36,37,38,39,40].

Watermelon LMM genes are valuable experimental materials for studying molecular mechanisms and creating new germplasm resources. The advent of BSA-seq has accelerated the identification of candidate genes controlling important agronomic traits [12, 41]. This method has been used to map major QTLs for powdery mildew resistance to chromosome 12 in melon [42]. In addition, the candidate genes Csa2M435460.1 and Csa5M579560.1 conferring resistance to cucumber powdery mildew have been identified using BSA-seq [43]. Furthermore, a genome-wide analysis of SNPs resulted in the detection of a genomic region harboring the candidate dwarfism gene Cla010726 [12]. In contrast, few researchers have investigated the gene responsible for spontaneous lesion mimicry in watermelon and other cucurbitaceous crops. In this study, we used BSA-seq to map ClCG04G001930 of watermelon for the first time, which might be the candidate gene responsible for lesion mimic. We localized this gene to chromosome 4 in a region between 3,760,000 bp to 7,440,000 bp, corresponding to a physical distance of 3.68 Mb. ClCG04G001930 is a PAD4 homolog in watermelon. ClPAD4 and AtPAD4 have a protein sequence similarity of 62.72%, which suggests that they have similar functions (Fig. 8). The PAD4 orthologs are present in many plant species and essential for systemic resistance against biotic stress in angiosperms [44,45,46,47,48]. AtPAD4 is a member of a small family of sequence-related immunity regulator [49, 50]. Heterologous expression of the AtPAD4 gene in soybean roots inhibits the development of plant parasitic nematodes. GbPAD4 is up-regulated in cotton during pathogen infection [51] and LePAD4 expression is elevated in response to green peach aphid infestation in tomato [52]. The PAD4 protein in grape supports the response of the SA defense pathway to biotic stress [53]. ICS1, NPR1-3, PRs, EDS1, PAD4, and FMO1 signaling is strongly elicited during rust disease infection in rice [54].

The PAD4 is involved in the regulation of programmed cell death and acclimation to biotic and abiotic stresses [47, 48]. We detected an obvious difference between LMM and normal watermelon leaves under an electron microscope: the cells of watermelon LMM leaves contained very little chlorophyll, whereas those of normal leaves were rich in chloroplasts and grana (Fig. 3). Chloroplasts play an important role in regulating PAD4-modulated stress responses [55,56,57]. Our experimental result is thus very interesting and requires detailed study. The ClPAD4 over-expression transgenic watermelon plants exhibited spontaneous lesions (Fig. 8b). Overexpression of TaPAD4 produce necrotic spots to prevent the spread of powdery mildew, which validate the function of TaPAD4 in wheat powdery mildew resistance [58]. The ClPAD4 RNAi transgenic watermelon seedlings susceptible to mosaic virus infection (Fig. 9d). Silencing of OsPAD4 increases sensitivity to biotrophic pathogens in rice [46], and silencing of GmPAD4 reduces SA accumulation and enhances soybean susceptibility to virulent pathogens [59]. Previous studies have shown that the LMM phenotype of the mutant cpr5 is controlled by PAD4-dependent SA accumulation [37, 60]. Our experimental results, which are consistent with previous studies, indicate that the mutation of ClPAD4 in watermelon most likely led to the spontaneous lesions.

Genes used as screening markers for resistance breeding, biomass production, and productivity enhancement are important from an agricultural point of view. Watermelon breeders have always focused on the introduction of novel traits into existing germplasm as a means of improving cultivar quality. The clalm mutant exhibited obvious chlorotic lesions, which makes this application useful for seedling screening. Future elucidation of the mechanism of the PAD4 gene with respect to the spotted leaf trait should be useful in molecular marker-assisted selection breeding of watermelon.

Data availability

The datasets generated during the current study are available in the NCBI (BioSample accessions: SAMN45962846, SAMN45962847, SAMN45962848, SAMN45962849, SAMN45962850, SAMN45962851, SAMN45962852, SAMN45962853).

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Acknowledgements

We gratefully acknowledge the support provided by the National Natural Science Foundation of China (31801882) and Henan Province Key Science and Technology Projects (242102111085).

Funding

This work was supported by funding from the National Natural Science Foundation of China (31801882) and Henan Province Key Science and Technology Projects (242102111085).

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Authors and Affiliations

  1. School of Life Science, Henan University, Kaifeng, Henan, 475004, People’s Republic of China

    Yuanyuan Qin, Qingqing Liu, Shengqi Hua, Defeng Wu & Wei Dong

  2. State Key Laboratory of Cotton Biology, School of Life Sciences, Henan University, Kaifeng, 475001, China

    Jiale Shi & Congji Yang

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  1. Jiale Shi
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Contributions

W.D. wrote the main manuscript text. W.D. and D.W. prepared figures 1-3 and figure S3. J.S., C.Y., Y.Q., Q.L. and S.H. prepared figure 4-9 and figure S1-S2. J.S. and C.Y. contributed equally. All authors reviewed the manuscript.

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Correspondence to Wei Dong.

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Supplementary Information

12870_2025_6071_MOESM1_ESM.pdf

Supplementary Material 1. Fig. S1 Venn diagrams of small InDels (a) and SNPs (b) in spotted-leaf and non-spotted-leaf watermelon. R01, clalm mutant; R02, ‘Zd’; R03, spotted-leaf plants in the F2 population; R04, non-spotted-leaf plants in the F2 population.

12870_2025_6071_MOESM2_ESM.pdf

Supplementary Material 2. Fig. S2 Predicted protein tertiary structure of ClPAD4. Mutation of amino acid codon from Ile to Val. Marked with red boxes and tables.

12870_2025_6071_MOESM3_ESM.pdf

Supplementary Material 3. Fig. S3 The photos of F1 and F2 phenotypes of the constructed population. (a) The experimental materials before collection. (b) All plants of the F1 generation developed spotted leaves. (c) The non-spotted-leaf plants in the F2. (d) The spotted-leaf plants in the F2.

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Shi, J., Yang, C., Qin, Y. et al. Phytoalexin deficient 4 is associated with the lesion mimic trait in watermelon clalm mutant (Citrullus lanatus). BMC Plant Biol 25, 92 (2025). https://doi.org/10.1186/s12870-025-06071-2

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BMC Plant Biology

ISSN: 1471-2229

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