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. 2019 Feb 19;20(4):902. doi: 10.3390/ijms20040902

Genome-Wide Characterization of NBS-Encoding Genes in Watermelon and Their Potential Association with Gummy Stem Blight Resistance

Md Zahid Hassan 1, Md Abdur Rahim 1, Hee-Jeong Jung 1, Jong-In Park 1, Hoy-Taek Kim 1, Ill-Sup Nou 1,*
PMCID: PMC6412240  PMID: 30791419

Abstract

Watermelon (Citrullus lanatus) is a nutritionally rich and economically important horticultural crop of the Cucurbitaceae family. Gummy stem blight (GSB) is a major disease of watermelon, which is caused by the fungus Didymella bryoniae, and results in substantial economic losses in terms of yield and quality. However, only a few molecular studies have focused on GSB resistance in watermelon. Nucleotide binding site (NBS)-encoding resistance (R) genes play important roles in plant defense responses to several pathogens, but little is known about the role of NBS-encoding genes in disease resistance in watermelon. The analyzed NBS-encoding R genes comprises several domains, including Toll/interleukin-1 receptor(TIR), NBS, leucine-rich repeat (LRR), resistance to powdery mildew8(RPW8) and coiled coil (CC), which are known to be involved in disease resistance. We determined the expression patterns of these R genes in resistant and susceptible watermelon lines at different time points after D. bryoniae infection by quantitative RT-PCR. The R genes exhibited various expression patterns in the resistant watermelon compared to the susceptible watermelon. Only six R genes exhibited consistent expression patterns (Cla001821, Cla019863, Cla020705, Cla012430, Cla012433 and Cla012439), which were higher in the resistant line compared to the susceptible line. Our study provides fundamental insights into the NBS-LRR gene family in watermelon in response to D. bryoniae infection. Further functional studies of these six candidate resistance genes should help to advance breeding programs aimed at improving disease resistance in watermelons.

Keywords: gummy stem blight, watermelon, resistance, NBS genes, expression

1. Introduction

Watermelon (Citrullus lanatus) is one of the most important cucurbit crops worldwide. This crop is well known in all tropical and subtropical regions of the world, where it is primarily grown for the fresh consumption of the juicy, sweet flesh of its mature fruit [1]. Watermelon plays a significant role in human health by providing important nutritional compounds, such as sugars and lycopene, and cardiovascular health-promoting amino acids, including arginine, glutathione and citrulline [2].

However, watermelon is frequently affected by several pathogens and insects, including fungi, bacteria, viruses and aphids. Gummy stem blight (GSB) is one of the most destructive diseases of watermelons. GSB is caused by the soil, seed and airborne fungus Didymella bryoniae [3,4]. This disease also affects other cucurbit crops, including melon, cucumber and squash [5,6]. GSB in watermelon causes crown blight, stem cankers and extensive defoliation, with symptoms detected in cotyledons, hypocotyls, leaves and fruits. This results in severe economic losses in the field and fruit losses during storage [4,7]. It is difficult to properly control GSB using chemical methods and the frequent use of fungicides is not desirable due to their negative impact on the environment [4,8]. Consequently, developing GSB-resistant watermelon cultivars containing major resistance genes through gene pyramiding represents the most environmentally sustainable and economically viable means of GSB management [4,9].

Due to the agricultural importance of this disease, early studies have focused on identifying the sources of genetic resistance to GSB [10]. However, few such sources have been reported in watermelon [4,7]. The identification of linked markers is essential for exploring GSB resistance in watermelon genotypes from diverse germplasm. However, no molecular studies of GSB resistance in watermelon have thus far been reported and current efforts are still focused on developing a GSB-resistant watermelon cultivar.

Resistance (R) genes play important roles in plant immune systems in response to various pathogens and insects, including viruses, bacteria, fungi, aphids and nematodes [11]. Disease resistance in plants involves the interaction between the avirulence (avr) genes of the pathogen and specific disease resistance (R) genes of plants, with such interactions described by the gene-for-gene model [12,13]. This form of plant resistance can be lost due to the development of new races of pathogens via evolution or as a consequence of the evolutionary loss of R genes [14].

Most R genes in plants encode proteins that are comprised of a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs), which play vital roles in plant–pathogen recognition [15]. The NBS domains, which bind to and hydrolyze adenosine triphosphate (ATP) or guanosine triphosphate (GTP), are involved in signaling, whereas LRRs are highly adaptable structural domains that are responsible for protein–protein interactions [15]. Plant NBS-LRR proteins can be classified into two subgroups based on the identity of the sequences that precede the NBS domain: TIR-NBS-LRR (TNL) proteins that have Toll-like domains and CC-NBS-LRR (CNL) proteins that are characterized by their coiled-coil domains. Watermelon contains 44 NBS-LRR genes, as revealed by genomic analysis [1]. R genes have been identified in a number of plant species, including Arabidopsis thaliana [16], rice [17,18], melon [19], cucumber [20] and apple [21]. Initially, we identified GSB-resistant watermelon lines through an extensive bioassay. Since NBS-encoding R genes have been reported to confer resistance against various pathogens and insects in different plant species, the R genes were explored in watermelon. Further, the availability of the complete genome sequences of C. lanatus allowed us to systematically analyze the NBS-encoding R genes in watermelon.

However, little is known about NBS-encoding R genes in resistant and susceptible watermelon lines/cultivars. Therefore, in this study, we analyzed the expression patterns of NBS-encoding genes in D. bryoniae-resistant and -susceptible watermelon lines to identify candidate R genes conferring resistance to GSB, which could be highly useful for breeding programs.

2. Results

2.1. Distribution of 44 NBS-Encoding Genes in Watermelon Chromosomes

A total of 44 NBS-encoding R genes were previously identified in the watermelon genome [1]. In this study, we found that the genes are differentially expressed in susceptible compared to resistant C. lanatus lines. Forty-four R genes are distributed across nine C. lanatus chromosomes, ranging from 1 to 10 genes per chromosome (Figure 1, Table 1). The highest number of NBS-encoding genes was found on Chr2 (Cla006803, Cla006813, Cla006820, Cla019844, Cla019831, Cla019854, Cla019855, Cla019856, Cla019857 and Cla019863) and Chr8 (Cla001017, Cla012424, Cla012425, Cla012427, Cla012428, Cla012430, Cla012431, Cla012434 and Cla012439), whereas the lowest number was found on Chr0 (Cla000024). Chr1, Chr5, Chr9 and Chr11 each contain three NBS-encoding genes, while Chr7 contains five and Chr10 contains six genes (Figure 1).

Figure 1.

Figure 1

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Distribution of the 44 NBS-encoding R genes on the watermelon chromosomes. The genes shown in blue are the candidate NBS-encoding genes for gummy stem blight resistance in watermelon. The positions of genes on the watermelon chromosomes were drawn using MapChart software [22].

Table 1.

Location of 44 NBS-encoding genes on the watermelon chromosomes.

Sl. No. Gene Name a Chr Start End CDS (bp) Protein (aa)
1 Cla000024 Chr0 573,701 575,732 1725 574
2 Cla001821 Chr1 26,529,719 26,532,585 2448 815
3 Cla003651 Chr1 5,737,988 5,740,048 2061 686
4 Cla003652 Chr1 5,741,726 5,742,592 867 288
5 Cla006803 Chr2 9,631,689 9,634,916 3228 1075
6 Cla006813 Chr2 9,860,817 9,862,469 1653 550
7 Cla006820 Chr2 9,989,292 9,992,234 2943 980
8 Cla019831 Chr2 26,750,001 26,753,327 2445 814
9 Cla019844 Chr2 26,582,380 26,589,679 4227 1408
10 Cla019854 Chr2 26,456,943 26,459,976 2667 888
11 Cla019855 Chr2 26,449,200 26,453,033 2826 941
12 Cla019856 Chr2 26,439,873 26,444,126 2832 943
13 Cla019857 Chr2 26,432,098 26,437,657 3036 1011
14 Cla019863 Chr2 26,383,499 26,388,744 3588 1195
15 Cla020705 Chr5 27,501,959 27,505,144 2466 821
16 Cla021846 Chr5 6,824,170 6,829,020 1239 412
17 Cla021858 Chr5 6,953,920 6,957,366 3447 1148
18 Cla002280 Chr7 1,370,278 1,374,401 3930 1309
19 Cla002282 Chr7 1,381,169 1,385,954 2979 992
20 Cla010826 Chr7 30,211,275 30,212,050 606 201
21 Cla010833 Chr7 30,251,502 30,254,738 3237 1078
22 Cla010834 Chr7 30,256,839 30,260,099 3261 1086
23 Cla001017 Chr8 11,759,681 11,762,959 3279 1092
24 Cla012424 Chr8 1,647,030 1,648,988 1377 458
25 Cla012425 Chr8 1,641,639 1,643,644 1299 432
26 Cla012427 Chr8 1,609,802 1,613,337 3042 1013
27 Cla012428 Chr8 1,595,322 1,601,397 4353 1450
28 Cla012430 Chr8 1,580,252 1,584,048 1278 425
29 Cla012431 Chr8 1,547,018 1,555,932 4578 1525
30 Cla012433 Chr8 1,530,729 1,534,838 2454 817
31 Cla012434 Chr8 1,420,444 1,427,949 3423 1140
32 Cla012439 Chr8 1,331,566 1,335,184 2469 822
33 Cla015218 Chr9 3,429,200 3,430,883 1404 467
34 Cla015257 Chr9 3,116,792 3,119,902 3111 1036
35 Cla015258 Chr9 3,111,030 3,115,364 3444 3444
36 Cla001168 Chr10 4,050,530 4,052,711 1050 349
37 Cla002913 Chr10 21,882,174 21,883,565 1392 463
38 Cla002924 Chr10 21,745,526 21,748,738 3213 1070
39 Cla016986 Chr10 21,332,030 21,336,749 3402 1133
40 Cla017475 Chr10 22,939,927 22,942,518 2592 863
41 Cla017478 Chr10 22,955,431 22,957,062 1632 543
42 Cla007904 Chr11 9,507,150 9,514,341 4062 1353
43 Cla007937 Chr11 8,764,524 8,779,874 3391 1155
44 Cla011937 Chr11 3,833,117 3,836,951 1725 574
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a Genomic information retrieved from the Cucurbit Genomics Database (http://cucurbitgenomics.org) using 97103 as the reference genome for watermelon.

2.2. Exon–Intron Structure

To better understand the genomic structures of the 44 NBS-encoding R genes, we generated exon–intron diagrams of the genes by comparing their coding sequences with the corresponding genomic sequences using the online tool GSDS2.0 (http://gsds.cbi.pku.edu.cn/). The number of exons per gene was 1–8 (Figure 2). The highest number of exons was found in Cla019855 and Cla021846, while the lowest was found in Cla001017, Cla006813, Cla012424, Cla003651, Cla003652, Cla006803, Cla002913, Cla006820, Cla010833, Cla010834, Cla015257, Cla017475, Cla017478 and Cla021858. Three genes (Cla007937, Cla011937 and Cla012428) contain six exons while three other genes (Cla012431, Cla019857 and Cla019863) contain seven exons. The number of introns per gene was 0–7. Cla019855 and Cla021846 contain the most introns, whereas 14 genes lack introns (Figure 2).

Figure 2.

Figure 2

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Exon–intron structures of the 44 NBS-encoding R genes from watermelon. Rectangles and gray lines indicate exons and introns, respectively. Left and right arrows indicate position of forward and reverse primers, respectively.

2.3. Conserved Domain and Motif Analysis

We analyzed the conserved domains of the 44 NBS-encoding R genes using the Conserved Domain Database (CDD) of NCBI and the Pfam protein database v30.0 (https://www.ncbi.nlm.nih.gov/structure/cdd/wrpsb.cgi). These results are shown in Figure 3 and Table 2. All 44 proteins have a highly conserved NBS (NB-ARC) domain. The R genes were grouped in different classes based on the presence of the following conserved domains: (i) NBS, (ii) NBS-LRR, (iii) LRR, (iv) RPW8-NBS-LRR, (v) TIR, (vi) TIR-LRR, (vii) CC-NBS and (viii) CC-NBS-LRR (Table 3). Three genes (Cla001821, Cla019831 and Cla020705) encode proteins with both RPW8 and NBS-LRR domains. We subjected the 44 NBS proteins to motif analysis using the MEME Suite (http://meme-suite.org/tools/meme). These results are shown in Figure 4 and Table 4. We detected 20 conserved motifs, each being comprised of over 13 amino acids. The greatest number of motifs was identified in the CC-NBS-LRR domain-containing gene Cla021858, whereas the fewest were detected in Cla001168 and Cla021846.

Figure 3.

Figure 3

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Domains present in NBS proteins in watermelon. The conserved domains were identified using the Conserved Domain Database (CDD) of NCBI against the Pfam v30.0 database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Detailed descriptions of these domains are provided in Table 2. Specific domains in each protein are shown in the diagram.

Table 2.

Key domains in the 44 watermelon NBS proteins.

Sl. No. Domain Name Description Function Reference
1 RPW8 Resistance to Powdery Mildew8 Involved in powdery mildew resistance [23]
2 LRR Leucine-rich repeats Disease resistance [24]
3 NB-ARC Nucleotide-binding adaptor shared by APAF-1, R proteins and CED-4 Disease resistance [25,26]
4 TIR Toll-interleukin 1-receptor Plant defense [25]
5 CC Coiled-coiled Disease resistance [27,28]
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Table 3.

Classification of the 44 Citrullus lanatus NBS-encoding genes.

Sl. No. Type Gene Name
1 NBS Cla003652, Cla002280, Cla002282 and Cla001168
2 NBS-LRR Cla001821, Cla003651, Cla006820, Cla019831, Cla012433 and Cla017478
3 LRR Cla002924, Cla011937 and Cla000024
4 RPW8-NBS-LRR Cla001821, Cla019831 and Cla020705
5 TIR Cla012424, Cla012425 and Cla012430
6 TIR-LRR Cla019854, Cla019855, Cla019856, Cla019857, Cla019863, Cla012427, Cla012428, Cla012431, Cla012439, Cla016986 and Cla007937
7 CC-NBS Cla006813, Cla010826, Cla015218 and Cla002913
8 CC-NBS-LRR Cla006803, Cla021858, Cla010833, Cla010834, Cla001017, Cla015257 and Cla017475
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Figure 4.

Figure 4

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Conserved motifs of NBS-encoding R genes in the watermelon genome. Motifs are indicated by different colored rectangles. Detailed information is provided in Table 4.

Table 4.

Putative conserved motifs in the 44 watermelon NBS proteins.

Motif Name E-Value Sites Width Motif Sequence
Motif 1 1.2 × 10−386 38 29 DDVRFVGIVGMGGIGKTTLAKAVYNHILI
Motif 2 2.30 × 10−290 40 29 LLDLSKEIVKYCGGLPLAJKVLGSSLRGK
Motif 3 1.70 × 10−232 17 37 IIKNRLSSKKVLJVLDDVDELEQLZALAGGRDWFGPG
Motif 4 1.50 × 10−220 33 29 JKELPESIGNLTSLKTLNLKNCSNLKELP
Motif 5 1.30 × 10−204 15 29 YDVFLSFRGEDTRRNFTSHLYEALRQKGI
Motif 6 4.90 × 10−189 15 29 VLPVFYKVDPSDVRKQTGSFGZAFAKHEA
Motif 7 1.70 × 10−187 34 21 KVKMHDLIQDMARTIVRKZSV
Motif 8 1.80 × 10−167 37 21 YNVEKLSDEEALELFSKHAFG
Motif 9 3.40 × 10−119 35 13 SKIIVTTRNEHLL
Motif 10 1.00 × 10−117 21 21 PSSSLKQCFLYCSLFPKDYKF
Motif 11 1.10 × 10−171 28 41 PDFSSLPNLEKLDJEGCTNLVKLHESIGSLKKLIKLDJKDC
Motif 12 1.50 × 10−157 8 50 MAEFLWTFAVZEVLKKTLKLAAEQIGLAWGFKKELSKLRKSLLKVEAILR
Motif 13 7.60 × 10−122 13 21 FSENYASSKWCLEELVKIIEC
Motif 14 2.10 × 10−126 22 29 KHFDKVIWVCVSZPFDVKKILEEIJESLN
Motif 15 1.10 × 10−109 15 27 KEIFLDIACFFKGEDVELVKEILEACG
Motif 16 6.60 × 10−92 11 29 EHHSVKJWVEKLZDIVYEADDLLDELSYE
Motif 17 8.20 × 10−90 10 34 NSSGGLDSKEALLRELQKELHGKRYFLVLDDVWN
Motif 18 5.70 × 10−92 11 29 TMEDIGDKYFNELLSRSLFQDVVKDKRGR
Motif 19 4.90 × 10−78 13 29 SWHGFPFKSLPSDFHPENLVELDLRYSCI
Motif 20 7.60 × 10−97 22 29 SLRVLDLSNTNITKLPNSIGQLKHLRYLD
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2.4. Synteny Analysis of 44 NBS-Encoding R Genes of C. lanatus Compared with Cucumis melo, Cucumis sativus, and A. thaliana

We performed comparative analysis to identify the homologous NBS-encoding R genes among C. lanatus, Cucumis melo, Cucumis sativus and A. thaliana, with the results shown in Figure 5. Most R genes from C. lanatus share homologous relationships with those of Cucumis melo, Cucumis sativus and A. thaliana. However, five genes (Cla000024, Cla019844, Cla002280, Cla002282 and Cla011937) lack homologues in Cucumis melo and three (Cla000024, Cla019844 and Cla002280) lack homologues in Cucumis sativus. On the other hand, all 44 R genes of C. lanatus have homologues in A. thaliana.

Figure 5.

Figure 5

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Microsynteny analysis of the 44 watermelon NBS-encoding R genes compared to those of Cucumis melo, Cucumis sativus and A. thaliana. Brown orange, blue and green indicate C. lanatus, Cucumis melo, Cucumis sativus and A. thaliana chromosomes, respectively.

2.5. Expression Patterns of the NBS-Encoding R Genes in Resistant and Susceptible Watermelon Lines

GSB, which is one of the most devastating diseases of cucurbits, significantly reduces the yield and quality of watermelon. To gain insight into the roles of NBS-encoding genes in the response to GSB in watermelon, we designed specific primers for the 44 NBS-encoding R genes and analyzed their expression patterns following inoculation with D. bryoniae at various time points. Several genes were differentially expressed in the leaf tissue of the resistant compared to susceptible watermelon lines (Figure 6). Among these, six genes (Cla001821, Cla019863, Cla020705, Cla012430, Cla012433 and Cla012439) were expressed at higher levels in the resistant line compared to the susceptible line. These genes belong to the same cluster in the heat map (Figure 6 and Figure 7). The transcript levels of five of these six genes reached a peak at 12 h postinoculation with D. bryoniae in the resistant line, whereas Cla001821 transcript levels reached a peak at 72 h postinoculation. Finally, the transcript levels of Cla020705 peaked at both 12 and 72 h postinoculation in the resistant line.

Figure 6.

Figure 6

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Heat map of the expression patterns of the 44 NBS-encoding R genes determined by qRT-PCR in gummy stem blight resistant and susceptible watermelon lines subjected to Didymella bryoniae infection at various time points. The expression levels were normalized with the Actin gene. The values were obtained from the means of three technical replicates. Red and blue represent the maximum and minimum values, respectively. The heat map was generated with an online tool ‘Heatmapper’ (http://www.heatmapper.ca/expression/).

Figure 7.

Figure 7

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Relative expression levels of candidate NBS-encoding R genes in Didymella bryoniae-resistant and susceptible watermelon lines. Error bars represent ± SE of the means of three technical replicates. Different letters above the bars indicate significant differences.

3. Discussion

Watermelon is an economically important fruit crop that is widely cultivated throughout the tropical and subtropical regions of the world. Watermelon is frequently affected by fungal, bacterial, viral and insect pests. GSB is a severe disease of watermelon caused by the fungus D. bryoniae, which significantly reduces fruit yields and quality. To reduce crop losses due to GSB, it is important to investigate the mechanism underlying the resistance to this disease. The ultimate target of both breeders and researchers is to develop GSB-resistant cultivars, since chemical treatment is not an environmentally suitable approach for controlling GSB [29]. Gene pyramiding is an effective way to increase the chances of conferring stable resistance to plant diseases, but disease resistance can break down due to increasing mutation rates in the pathogen population [9,30].

NBS-encoding R genes play important roles in plant protection against a diverse range of pathogens, including fungi, bacteria, viruses, aphids and nematodes [15,31]. For example, the R genes Fom1, Prv and Vat are responsible for resistance to Fusarium, Papaya ringspot virus and aphid resistance in melon, respectively [32,33]. The RPS6 gene plays a role in resistance against the bacterium Pseudomonas syringae in A. thaliana [14]. In addition, the R gene Bo1037156 (FOC1) confers resistance to the fungal pathogen Fusarium in Brassica oleracea [34]. NBS-LRR disease resistance genes have been extensively studied in various plant species, such as Cucumis melo [35], Arabidopsis [36], Oryza sativa [17], Zea mays [37], Solanum tuberosum [38] and Glycine max [39]. However, the roles of NBS-LRR genes in response to D. bryoniae infection in watermelon have not been reported. The analysis of 44 NBS-encoding genes performed in this study revealed that these genes encode TIR-LRR, CC-NBS and CC-NBS-LRR proteins (Table 3). These genes are distributed throughout all watermelon chromosomes except Chr3, Chr4 and Chr6, with at least one gene per chromosome (Figure 1).

The analysis of qRT-PCR expression revealed that various genes were differentially expressed in resistant compared to susceptible watermelon. Consistent expression patterns were detected for six genes, with higher levels of expression in the resistant line compared to the susceptible line (Figure 6 and Figure 7). These candidate genes belong to different categories, including NBS-LRR (Cla012433), RPW8-NBS-LRR (Cla001821), RPW8-LRR (Cla020705), TIR (Cla012430) and TIR-LRR (Cla012439 and Cla019863) (Table 3). NBS-encoding genes contribute to disease resistance in various plant species. For example, TIR-NBS-LRR-type R genes confer resistance to tobacco mosaic virus in Nicotiana benthamiana and GSB in Cucumis melo [5,40]. In indica rice, the R gene Os11g0704100 (Pia), which encodes an NBS-LRR domain-containing protein, functions in rice blast resistance, with significantly higher expression detected in resistant compared to susceptible land races both before and after pathogen inoculation [41]. In Arabidopsis, the expression levels of four TIR-NBS (TN) genes increased upon treatment with different pathogens [42]. Finally, three NBS-encoding genes (Bo1010559, Bo129866 and Bo1042121) were recently shown to function in resistance to black rot in cabbage (Brassica oleracea var. capitata), with higher expression levels detected in the resistant line compared to the susceptible lines [43].

In this study, we identified six candidate genes for GSB resistance, including Cla012430, Cla012433 and Cla012439 on Chr8 and Cla001821, Cla019863 and Cla020705 on Chr1, Chr2 and Chr5, respectively. The existence of homologues of these candidate genes in melon, cucumber and Arabidopsis (Figure 5) indicates that these genes likely play similar roles in these plants. Although GSB is a major yield-limiting factor for watermelon, few genetic studies have focused on this disease. Moreover, to date, no quantitative trait loci associated with GSB resistance in watermelon have been identified. To our knowledge, this is the first report of candidate genes for GSB resistance in watermelon.

4. Materials and Methods

4.1. Experimental Materials

The GSB-resistant ‘PI189225’ and -susceptible ‘PI438676’ (Charleston Gray) watermelon inbred lines used in this study were obtained from National Plant Germplasm System (NPGS), U.S. Department of Agriculture (USDA). The resistance and susceptibility of these lines were previously assessed via bioassay screening using the fungus D. bryoniae [4,44]. In this study, these lines were reexamined using an intensive bioassay, which confirmed the GSB resistance of ‘PI189225’ and the susceptibility of ‘PI438676’ (Figure 8). The seeds were sown in a commercial soil mixture in a 32-hole plastic tray and transferred to a plant growth chamber at a constant temperature of 25 ± 1 °C, relative humidity of 60% and light intensity of 80–120 µmol.m−2·s−1. Two weeks after germination, the plants were transferred to plastic pots and grown in a glasshouse where plants were inoculated with D. bryoniae, which was maintained at 24 ± 2 °C temperature with 90% relative humidity.

Figure 8.

Figure 8

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Phenotypes of watermelon lines PI189225 (resistant) and PI438676 (susceptible) after inoculation with Didymella bryoniae.

4.2. Pathogen Inoculation and Sampling

A D. bryoniae fungal isolate (13-020) was collected from National Institute of Horticulture and Herbal Sciences (NIHHS), Republic of Korea. The fungus was cultured in potato dextrose agar (PDA) medium at 24 ± 2 °C under alternating periods of 12-h fluorescent light (40–90 µmol·m−2.sec−1 PPFD) and 12-h darkness for 2–3 weeks until pycnidia formed. The final concentration of the fungal spores was adjusted to 5 × 105 spores mL−1 with deionized water. For fungal inoculation, 28-day-old resistant and susceptible plants were inoculated with D. bryoniae by hand using a spray bottle, whereas control resistant and susceptible plants were sprayed with plain water. The inoculated plants were incubated in a growth chamber with a relative humidity of 90–95% and a temperature of 24 °C. Samples were collected from the third and fourth true leaves of the plants at the time points 12, 24 and 72 h after inoculation and from control plants at the same time points. The samples were immediately frozen in liquid nitrogen and stored at −80 °C.

4.3. Total RNA Isolation and cDNA Synthesis

Infected and control watermelon leaves were ground to a powder in liquid nitrogen and 100 mg of each sample was subjected to total RNA extraction using an RNeasy Mini kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. First-strand cDNA was synthesized from total RNA with a SuperScript III First-Strand Synthesis System kit (Invitrogen, Gaithersburg, MD).

4.4. Exploring NBS-Encoding Genes in C. lanatus

Few genetic studies of GSB resistance in watermelon have been performed. However, 44 NBS-LRR genes have been identified by genomic analysis [1] and these genes were subjected to expression analysis in this study (Table 5).

Table 5.

Primers for the 44 NBS-encoding genes in watermelon.

Sl. No. Gene Name Forward Primer Reverse Primer Product Size (bp)
1 Cla001821 ACTGTCTAACGAGTCTTGAACG TTCTCCAGATTTATCAGCGGT 94
2 Cla003651 ATCTCTCGATTATGTGGGCGG TTGGGTGGCACGGTTACTCTG 118
3 Cla003652 TCCCAAAATCGCTCTTCTGC AGATACCGTTTGCCTCTCAGT 126
4 Cla006803 GGAAACGATCCAAACCATAGG CTTCCCTTTGCTCCAAGTTGA 85
5 Cla006813 AACTCATGGAAACACGCCCTA CCAATGGTACGCCACCAACT 165
6 Cla006820 AGGTTGTATCAGAGATGAGTTC CATATTCATTAGAAGCCGTGGGT 108
7 Cla019831 GGAGTGTTTCATGGACTTGG TTCATGTGCTTCGTTTCTCA 178
8 Cla019844 AGTGTTGAAGGAAGAGCAGCC TAAATCAAGTCCCTCCCACA 96
9 Cla019854 ACAAATGGGTCGCACAATCGC TGGCTTTAACTGCTCTTGCT 131
10 Cla019855 GTGAGGTACGAAAACAAACTGG ACAACCCAGCAGCAGTAGTC 122
11 Cla019856 GTCAACAAACATCCGAGGATTA TCAAGGTTTAATGTCGCCGAG 116
12 Cla019857 CCAGTCTTCCTTGTTGGATGG CCATCCAACAAGGAAGACTGG 171
13 Cla019863 GTATAGGAAGCTGTGGCGTCC TCCCCAGTGTCCGGATTTTGC 127
14 Cla020705 GTTGTCACGAGGCGAGCCTAT TTTGTCTTCAGGAAAGCATCCC 133
15 Cla021846 CCTGACCCATCTTCTCCTATT ATCTTGCACTTTCGCATGAACA 135
16 Cla021858 GAAACCATGGAAGACATAGGAG TGAGACAGAATAAGCAAGATCG 144
17 Cla002280 AGATGGGGAGCGGAAGTATCA CCCATTTCTATACTCAACTCAG 146
18 Cla002282 GGTTAGAAGATGATAGACGTGG GGAAACTCCAACTCCAGGGGA 91
19 Cla010826 TGTGGCATGGGGCTTGGACA GATCCTTCACCCACAATCTCAC 128
20 Cla010833 AGTGTGGATCGCCTACCGTCA CATTCCTTTCCTCAGATGGTCG 145
21 Cla010834 CTCCGACCCTTGTGATGCTA ACATCCATCATCAAACCCAACT 100
22 Cla001017 GCAAACATTGTGGAGACGCAT CAAGCACTTCTCGAACTACA 145
23 Cla012424 GTGGGGATGTGTAGTATTGGTA ATCAATGCGAAGGAAGCAACTA 99
24 Cla012425 AGCGGGAGGTGATTCAAAGC GAACCTTTCTACCACTCAGTCG 143
25 Cla012427 CAGCCGAGTGAACTATTGGAAC GCACCATTGACAGATCAGGG 145
26 Cla012428 TCCAGCATTCGATGTTTACTCG ACCATTCCAATAGGTCAACATC 99
27 Cla012430 TGGTTGGAATTAGCCACAGATT CCAATTCCACCCATTCCCCATA 100
28 Cla012431 GCTTGGATCCTCAATCACAGTC GAGAGTTTTGTAGCACTTGGAAG 116
29 Cla012433 GGATTTGGATGAGGAAGGAGAA GTATCCATGCCAATTGAGAAAC 151
30 Cla012434 CTCTCATCTTCCAACAAGCATA TCCTTGGAGGTAGCTCTGGGA 101
31 Cla012439 GCGGCATTGGCAAGACGACA AATAAGGAAGATTGTGGGTCTC 120
32 Cla015218 GTTGAAAGGGTCTCCTCTTGC GTGCAAAGTTCACTGTCCTTGA 102
33 Cla015257 GGGAAGTTGAGTTGTCTACAGA CTAAACTGACTTCAGATGGACT 157
34 Cla015258 GTGGAGTCAAATTTCCCAACTG CCAAATATAGAGAATGGAGAGC 132
35 Cla001168 GTGTACCAAGCGTTTGGAGTT TACTTAACCTGCCCCTCAACT 163
36 Cla002913 AGAATGGCTTATGGGACGAGC TGGTTGCCACTTCTAGGTTCC 102
37 Cla002924 CTCAATTCCCTTCAAACACTGA GCTCACACCCCCATATTGCC 112
38 Cla017475 GGTCGTTACCGGAAGATACTCA CATCTAGCTCATTCAGAGGGC 123
39 Cla017478 CCCAGTCACAGAACCTAAATCT CAGCATTTGGTATTTCCCGTAG 149
40 Cla016986 CTTTTGGAAGGAGTGTAAGTTG TTCTCTAGATTTGGGAGTTTGG 108
41 Cla007904 GCAGCATCCACACAGTGCCTT GGAAAGACATTTGTGGAAGCC 104
42 Cla007937 AAGATCGACCGCCTCCACGA CCGATCTTCTGCAGTTACAAC 154
43 Cla011937 GAGAGGATGTTGAACTTGCCAG GCTTTCCACATCTCTTGAATCA 154
44 Cla000024 GACATTGAAGGCATAGTGATGG CCATGCCAATTGAGAAACCTC 167
Actin Cla007792 CCATGTATGTTGCCATCCAG GGATAGCATGGGGTAGAGCA 140
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4.5. Quantitative RT-PCR Analysis

The expression patterns of the 44 NBS-encoding genes were analyzed by quantitative RT-PCR (qRT-PCR) in a LightCycler® instrument (Roche, Mannheim, Germany) following the manufacturer’s instructions. The gene sequences used in this study were retrieved from the Cucurbit Genomics Database (http://cucurbitgenomics.org/) considering ‘97103’ as the reference genome for watermelon. Gene-specific primers for qRT-PCR were designed using Primer3Plus (https://primer3plus.com/cgi-bin/dev/primer3plus.cgi) (Table 5 and Table S1). The reactions were performed in a 10-µL volume consisting of 5 µL of 2× qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems, London, UK), 5 pmol of primers and cDNA templates diluted to the appropriate concentrations. The PCR conditions were as follows: 5 min at 95 °C, followed by 3-step amplifications at 95 °C for 15 s, 58 °C for 15 s and 72 °C for 20 s for 45 cycles. The relative expression level of each gene was evaluated using the comparative 2−ΔΔCT method, with Actin used as an internal control [45].

4.6. Statistical Analysis

Analysis of variance (ANOVA) and significance tests were carried out using the normalized gene expression values with MINITAB17 software (Minitab Inc., State College, PA, USA). The means were separated by Tukey’s pairwise comparisons.

5. Conclusions

We identified six candidate genes that might be involved in the response of watermelon to D. bryoniae infection based on their expression profiles in resistant and susceptible watermelon lines. This study provides the basis for further functional studies to confirm the association of NBS-LRR genes with GSB resistance in watermelon. In addition, our results should facilitate marker-assisted breeding for developing GSB-resistant watermelon cultivars through gene pyramiding.

Supplementary Materials

Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/4/902/s1.

Click here for additional data file. (64.8KB, pdf)

Author Contributions

I.-S.N., H.-T.K. and J.-I.P. conceived the study. M.Z.H. conducted the experiments and wrote the manuscript. H.-J.J. helped carry out the qRT-PCR assay. M.A.R. analyzed the qRT-PCR data, created the figures, performed the bioinformatics analysis and edited the manuscript. All authors read and approved the final version of the manuscript.

Funding

This study was supported by the Golden Seed Project (Center for Horticultural Seed Development, grant number 213007-05-3-CG100) of the Ministry of Agriculture, Food and Rural Affairs (MAFRA) of the Republic of Korea.

Conflicts of Interest

The authors declare no conflict of interest.

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Supplementary Materials

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