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Lee, Kim, Suh, and Park: Analysis of Basic Helix-Loop-Helix Gene Related to Virus Resistance in Squash


Viral diseases in squash are damaging during the early stages of development and cause loss of crop yield and economic value. To reduce the damage caused by viral diseases, resistant cultivars should be bred. The objective of this study was to determine whether there was a relationship between virus resistance and a basic helix-loop-helix (bHLH) gene in squash (Cucurbita pepo L.). To do this, the bHLH transcription factor, known to be related to virus resistance, was isolated and analyzed using C. pepo individuals that were resistant or susceptible to zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus (WMV). The results showed that a three bp (GAC) deletion in the nucleotide sequences were found in the regulatory region of the CpbHLH gene in susceptible lines. It was confirmed that the deletion region was located near the binding site of the MYB transcription factor associated with the bHLH gene. It could be hypothesized that the susceptibility of susceptible lines may have been due to the lack of important sequences near the transcription factor binding region due to the deletion in the regulatory region, thus suppressing the expression of the gene. Quantitative real-time PCR (qRT-PCR) revealed that the expression level of the bHLH gene in resistant lines was 2.17 times higher than in susceptible lines.


It is important to analyze the genes, proteins, signaling molecules, and transcription factors involved in resistance mechanisms in various crops. Among germplasms, resistant individuals displaying high expression levels of resistance-related factors can be selected as breeding materials. The expression of resistance-related factors can then be increased using various breeding methods to enhance disease resistance (Mackintosh et al. 2007). In addition, if the genes known to be involved in disease resistance in other crops were identified in the target crops and overexpressed through molecular genetic methods, disease-resistant individuals could be used as breeding materials (Park et al. 2001; Li and Steffens 2002; Malnoy et al. 2007).
Squash, Cucurbita pepo L., is an economically valuable vegetable crop used for a wide variety of food and cultural purposes, such as Halloween and Thanksgiving celebrations around the world. The region that produces the most squashes is Asia, providing approximately 63% of total global production. The countries that produce the most squashes are China and India (Faostat 2009). There are three cultivated species in the genus Cucurbita: C. pepo L., C. moschata Duchesne, and C. maxima L.; and these cultivated species are widely distributed (Paris 2001). The factors that classify these different species as cultivated are shape, size, color, taste, aroma, sugar content, sex expression, parthenocarpy, ploidy, and growing season (Bates and Robinson 1990).
Some of the viruses that commonly infect squashes are the watermelon mosaic virus (WMV) and the zucchini yellow mosaic virus (ZYMV), which belong to the genus, Potyvirus (Zitter et al. 1996). The WMV and ZYMV are usually characterized by a combination of infections (Fuchs et al. 1998), especially when they infect plants in the early stages of growth, resulting in almost a 50% decrease in crop yields. Even when virus infections occur during the flowering period, the number of fruits is reduced. Even if the fruit forms, the virus leads to weight loss and fruit blistering, resulting in economic loss (Fletcher et al. 2000).
To prevent this loss, cultivated methods are used, such as covering with vinyl mulching, removing aphids, and eliminating the infectious sources of the viruses. However, these methods are only temporary and cannot fundamentally solve WMV and ZYMV damage (Lecoq et al. 1991). Therefore, to prevent such economic loss, it is necessary to breed virus-resistant squash varieties.
In general, the basic helix-loop-helix (bHLH) transcription factor, MYC2, is known to be a negative regulator of the Jasmonate (JA)-responsive pathogen defense genes plant defensin 1.2 (PDF1.2), basic chitinase B/pathogenesis-related 3 (CHIB/PR3), and hevein-like/pathogenesis-related 4 (HEL/PR4) (Anderson et al. 2004; Lorenzo et al. 2004), upon infection with bacterial (Nickstadt et al. 2004; Laurie-Berry et al. 2006) or fungal pathogens (Anderson et al. 2004). However, when the pathogen is a virus, it has been reported that bHLH act as positive regulators in the defense system. The bHLH in tomato plants have been reported to be more highly expressed in tomato yellow leaf curl virus (TYLCV)-resistant plants than in TYLCV-susceptible plants (Wang et al. 2015). The bHLH are also involved in the expression of JA, which plays a role in signal transduction in response to various stresses such as wounding, lack of water, and pathogen attack, and is involved in various developmental processes such as root growth.
Therefore, in this study, in order to confirm whether bHLH gene are related to the resistance of WMV and ZYMV in squash, a bHLH gene was isolated and identified from squashes that displayed resistance and susceptibility to WMV and ZYMV. Finally, we compared the expression of these genes and analyzed the differences in gene structure that caused the differences in resistance.


Plant materials

In this study, ‘SY87’ (the squash line resistant to WMV and ZYMV) was crossed with ‘SS40’ (high-yield squash line) and backcrossed to obtain ‘BC2F7’ lines (Hongik Bio, Pyeongtaek-si, Gyeonggi-do; Fig. 1A). These ‘BC2F7’ lines were planted and separated according to their phenotype that showed either resistance or susceptibility to WMV and ZYMV (Fig. 1B). Segregated lines showing resistance and susceptibility to the virus were used to isolate and identify the CpbHLH gene. Finally, the CpbHLH gene structure and level of expression were compared between lines with resistance and susceptibility.

Genomic DNA extraction

Young leaves were taken from the susceptible and the resistant squash plant. The leaves were pulverized and put into a 2 mL tube in powder form. Then, 800 mL of 2.5 × CTAB [5.0 M NaCl, 1.0 M Tris-HCl (pH 8.0), 0.5 M EDTA, and 2.5% CTAB] was added to each tube containing the leaf powder. The tubes were then incubated in a water bath at 65℃ for 30 minutes. After 30 minutes, PCI (phenol, chloroform, and isoamyl alcohol; vol 25: 24: 1) reagent was added to each mixture and centrifuged at 13,041 g for 10 minutes. After centrifugation, 400 mL of supernatant was dispensed into 2 mL tubes. Subsequently, 40 mL of 3 M sodium acetate and 880 mL of 100% ethanol were added to the supernatant, which was slowly inverted 10 times and stored at ‒20℃ for 1 hour. After 1 hour, the pellet that had formed in the tube was washed with 70% ethanol and dried in a new 1.5 mL tube. Then, 40 mL of 1 × TE buffer and 2 mL of RNase (10 mg/mL) were added to dissolve the dried pellet. The concentration of extracted genomic DNA was measured using a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and stored at ‒20℃.

CpbHLH gene isolation

The CpbHLH gene, was isolated from each of the resistant and susceptible individuals by PCR. The PCR primers were designed based on the CpbHLH gene sequence of Cucurbita pepo using the Vector NTI® program (Life Technologies, USA; Fig. 2A). A primer set was designed containing 683 bp in the upstream direction of the putative transcription start site (TSS) and 192 bp in the downstream direction of the stop codon, which is the region that transcription factors use to control the gene expression (Fig. 1B). The PCR program used for the isolation was pre-denaturation at 95℃ for 5 minutes, followed by denaturation at 95℃ for 30 seconds, annealing at a temperature corresponding to the melting temperature (Tm) of each primer for 30 seconds, and extension at 72℃ for 1 minute. After 35 cycles, the final extension was performed at 72℃ for 10 minutes. The PCR products were sequenced at Macrogen Co. (Seoul, Korea). The structure of the CpbHLH gene was analyzed to predict the TSS and exon regions using the CLC Sequence Viewer (QIAGEN Bioinformatics, Germany) and the Fgenesh program from Softberry (

RNA extraction and quantitative real-time PCR (qRT-PCR) analysis

To isolate the total RNA from the resistant and susceptible lines, leaf tissue was ground in liquid nitrogen and total RNA was extracted using an extraction kit (MiniBEST Plant RNA Extraction Kit, TaKaRa, Tokyo, Japan), according to the manufacturer’s instructions. cDNA was synthesized using a cDNA synthesis premix (HiSenScriptTM RH [‒] RT PreMix, iNtRON Biotechnology Inc., Seongnam, Korea). The primers for reverse transcription PCR (RT-PCR) and qRT-PCR analysis were designed to target the exon region of the isolated CpbHLH gene and actin was used as an internal control (Table 1). qRT-PCR analysis of the CpbHLH gene from each of the resistant and susceptible lines was performed using a Rotor-Gene 6000 (Corbett Robotics, Brisbane, Australia) and the TransStart Top Green qPCR SuperMix (#AQ131-02, Transgen Biotech, Beijing, China), with the following conditions: 95℃ for 2 minutes, followed by 40 cycles of 10 seconds at 95℃ for denaturation and 30 seconds at 60℃ for annealing and extension. After melting analysis was performed for each PCR product using 0.5℃/s increments from 45 to 95℃. Fluorescence intensity data were collected at the end of each cycle and analyzed using the instrument’s software (Rotor-Gene Q–windows Platforms ver. 2.2.3; Qiagen, Hilden, Germany). The delta-delta CT values were calculated relative to the reference PCR values (Livak and Schmittgen 2001). Each qRT-PCR reaction was conducted in triplicate and presented as the standard error of the mean to compare differences between resistant and susceptible lines. Also, P-values were obtained using delta CT values and T-test. Difference of relative expression was considered significant at P < 0.05.

PCR analysis to verify if deletion affect susceptibility

PCR was performed on twenty-one resistant and nine susceptible lines derived from BC2F7 line. For verification, forward primer (5ʹ-CAT CGG TTG GGG AGG AGG AG - 3ʹ) and reverse primer (5ʹ-TGC TAC AAT TCC AAC AGT ATA CAG AGC A - 3ʹ) were designed to contain sequences corresponding to deletion in the regulatory region in susceptible lines. Predicted size of amplified product was 136 bp. PCR analysis was performed in the same method as used for CpbHLH gene isolation. Amplified PCR products were loaded on 1.0% agarose gels for electrophoresis.


Identification of CpbHLH gene structure in resistant and susceptible lines

The 5ʹ region containing the putative TSS, and from the first exon to second exon of the CpbHLH gene was isolated from each resistant and susceptible squash. The sequence from the putative TSS to the second exon of the CpbHLH gene was predicted to be 2,558 bp in length. To separate the 5ʹ region containing the putative TSS, and the first and second exon of the squash CpbHLH gene, this region was divided into five parts, and primer sets for isolation were designed from five different regions (Fig. 2B). By aligning the nucleotide sequences of each PCR product, 2,558 bp of the 5ʹ region of the CpbHLH gene with the putative TSS and the first and second exon, were obtained from each resistant and susceptible individual. In addition, a 683 bp region upstream of the putative TSS was isolated for analysis as it acts as a promoter binding site for gene expression and is the region at which the transcription factors control the expression of the gene. In addition, the 3ʹ region containing from the stop codon to the 192 bp downstream region in the 3ʹ direction was obtained from resistant and susceptible lines. Thus, a total sequence length of 3,433 bp was obtained.
The differences in the sequence of the CpbHLH genes between resistant and susceptible lines were compared (Fig. 3B). A 3 bp deletion occurred in the regulatory region, exactly 11 bp upstream of the putative TSS in the susceptible lines that was not present in the resistant lines (Fig. 3B).

Polymorphism in the regulatory region

The 3 bp deletion in the nucleotide sequences in the susceptible individual were found in the regulatory region of the CpbHLH gene (Fig. 3A). The regulatory region combines various promoter complexes to regulate the transcription of associated genes. From the above results, it could be hypothesized that the susceptibility of susceptible lines may be due to the lack of promoter binding due to the deletion in the regulatory region, thus suppressing the expression of the gene. Analyzing the deletion region using New PLACE (, identified the deletion of regulatory region was located near the MYB binding site (Lüscher and Eisenman 1990; Urao et al. 1993; Sablowski et al. 1994; Solano et al. 1995; Tamagnone et al. 1998; Fig. 3A).

Verification of CpbHLH gene expression in resistant and susceptible individuals

RT-PCR and qRT-PCR analysis were conducted to confirm whether the presence of the deletion region of the transcription factor binding site in the regulatory region affected the expression of the CpbHLH gene. The same primers were used for RT-PCR and qRT-PCR analysis. To confirm the expression of the CpbHLH gene, a primer set was designed to target the exon region in the isolated CpbHLH gene. A control primer set was also designed to target actin obtained from the reference sequence of C. pepo (Table 1). Amplification was confirmed by RT-PCR using the actin and CpbHLH gene primers for each individual cDNA (Fig. 4A). Because the expression of CpbHLH gene was similar between resistant and susceptible lines based on band intensity of RT-PCR, qRT-PCR was performed to compare more accurate gene expression levels using the same cDNA and primers used in RT-PCR. Quantitative analysis of CpbHLH gene expression in resistant and susceptible individuals by qRT-PCR revealed that the expression level of the CpbHLH gene in the resistant individuals was 2.17 times higher than in the susceptible individuals (Fig. 4B). A P-value of 0.027 showed that the relative expression levels of qRT-PCR analysis between resistant and susceptible lines were statistically significant.

Verify if deletion affect susceptibility

To verify deletion in the regulatory region of the CpbHLH gene was the major cause of susceptibility to viral disease in squash, PCR was performed on 21 resistant and 9 susceptible squash lines derived from BC2F7 line using primers designed from the deletion region. As a result, it was confirmed that the 136 bp size of PCR products were amplified only for the resistant lines and was not amplified for the susceptible lines (Fig. 5).


In this study, experiments were conducted to clarify the genetic differences between the resistance and susceptibility to ZYMV and WMV in C. pepo, which adversely affected the yield of squash. In our previous experiment to develop SSR markers, there was an amplified fragment showing polymorphism between the resistant and susceptible squash lines, and the sequence of the polymorphic fragment showed homology with the bHLH gene. The bHLH is a superfamily of transcription factors that play important roles in developmental and essential physiological processes in eukaryotes (Ledent and Vervoort 2001). Based on this result, this experiment was conducted to find out if the bHLH gene was related to virus resistance. In tomatoes, ‘SlybHLH131’ has been reported to be directly involved in resistance to TYLCV, which causes economic damage to tomato production (Wang et al. 2015). In plants, bHLH transcription factors are known to be involved in several roles. First, bHLH is thought to be involved in the physiological development of plants. Genes related to bHLH transcription factors were expressed at different specific sites such as the roots, stems, flowers, leaves, seeds, and stomata during the development of the plant (Ohashi-Ito and Bergmann 2007; Kanaoka et al. 2008). In addition, bHLH have been reported to be involved in a variety of abiotic stress tolerance mechanisms. When plants are exposed to cold conditions, the transcription activator, Inducer of CBF expression 1 (ICE1), which contains the bHLH domain, regulates the expression of CBF genes involved in the chilling and freezing resistance of plants (Chinnusamy et al. 2003). Furthermore, the bHLH transcription factor AtNIG1, has been reported to be associated with salt stress in plants and induces salt stress tolerance by binding to the 45Ca2+ or E-box-DNA sequence (CANNTG) present in the promoter region of several salt stress-related genes (Kim and Kim 2006). The RERJ1 gene, which is involved in the tolerance of drought stress and wound stress, also encodes a bHLH transcription factor (Kiribuchi et al. 2005). Another study reported that co-expression of a bHLH gene and a WRKY transcription factor gene induced stress tolerance when plants were exposed to mannitol and oxidative stress (Babitha 2013). In Arabidopsis, bHLH is known to play role in stress response, hormone signaling, anther and epidermal cell development, and regulation of fruit dehiscence (Feller et al. 2011). Based on these previous studies, it was assumed that bHLH gene was related to the resistance to ZYMV and WMV in squash. To confirm this assumption, sequence information of the bHLH gene was obtained from the reference sequence of C. pepo, and the bHLH gene of squash line showing resistance or susceptibility to ZYMV and WMV was compared. As a result, it was confirmed that there was a 3 bp deletion in the regulatory region in the CpbHLH gene of susceptible squash to the viruses. Analysis of the region where the 3 bp deletion occurred found that it was located near the binding site for the MYB transcription factor. MYB transcription factors regulate the expression of genes involved in development, differentiation, and metabolism in plants. MYB transcription factors also regulate the expression of genes associated with responses to biotic and abiotic stresses, thus acting as part of the defense mechanism and potentially leading to resistance (Ambawat et al. 2013). MYB transcription factors are also able to rapidly respond to environmental stress by interacting with bHLH to lead to the expression of genes in response to abscisic acid (ABA). ABA is a hormone that is related to environmental stress such as drought. When working in combination, MYB and bHLH transcription factors respond to stresses faster than when MYB or bHLH exist alone (Abe et al. 2003).
Overall, the results of this analysis and those of previous studies indicate that the phenotype of susceptibility to WMV and ZYMV in squash might be caused by a deletion in the nucleotide sequence corresponding to the MYB binding site and miRNA-854a 3ʹ-end region in the regulatory region of the CpbHLH gene. The qRT-PCR experiment was conducted on lines of resistant or susceptible squash to confirm the effect of deletion on the expression of CpbHLH gene. In susceptible lines, the expression of the CpbHLH gene was thus suppressed, which was related to the lack of virus resistance. In addition, to verify the relationship between the deletion in the regulatory region of CpbHLH gene and the susceptibility phenotype, PCR analysis was performed on 21 resistant and 9 susceptible squash lines using primers containing deletion region. As a result, it was confirmed that the expected amplifications of 136 bp size were occurred in the resistant lines, but not in the susceptible lines. This result indicated that the 3 bp deletion located regulatory region in the CpbHLH gene of susceptible squash line was the major cause of the phenotype of viral disease resistance.
The regulatory region that upstream of the TSS is a “transcription control region” in which promoters and a variety of transcription regulatory elements form binding that allow the promotion and regulation of gene expression (Yadav et al. 2016). If a polymorphism such as an indel is present in a sequence corresponding to this transcription control region, the expression of the associated gene changes. This has been shown in a previous study on rice: the presence of an indel polymorphism in the promoter binding region led to a difference in gene expression (Zhang et al. 2008). In this study, the expression of the CpbHLH gene in the resistant line was higher than in the susceptible line. The reason for this was expected to be that the expression of the CpbHLH gene was regulated differently due to the deletion polymorphism near the MYB transcription factor binding site.
In conclusion, in this study, the bHLH transcription factor, known to be related to virus resistance in plants, was analyzed using C. pepo lines that were resistant and susceptible to ZYMV and WMV. First, each CpbHLH gene sequence from resistant and susceptible C. pepo lines was identified and compared. These comparisons revealed that a 3 bp deletion in the regulatory region occurred in susceptible lines. This deletion appeared to suppress the expression of the CpbHLH gene in susceptible lines. Analysis of the promoter binding site at the deletion region revealed that it was located near the MYB transcription factor binding site.
RT-PCR and qRT-PCR were performed to identify the expression level of the CpbHLH gene in resistant and susceptible lines. The expression level of the CpbHLH gene in resistant lines was 2.17 times higher than that of the susceptible lines. Based on these results, it was assumed that the deletion polymorphism in the regulatory region of the CpbHLH gene in susceptible lines reduced the expression of the CpbHLH gene and resulted in the susceptibility trait.


This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Golden Seed Project, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (213007-05-4-SBB20).


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Fig. 1
Information on plant materials. (A) The pedigree of ‘BC2F7’ resistant and susceptible lines for this study. SY87, WMV and ZYMV resistant C. pepo line; SS40, high-yield recurrent parent C. moschata line; F1 hybrid, WMV and ZYMV resistant progeny line from the cross between SY87 and SS40. (B) The phenotype of segregated lines from BC2F7 lines. Left, resistant lines to ZYMV and WMV; Right, susceptible lines to ZYMV and WMV.
Fig. 2
Information on the isolated CpbHLH gene in Cucurbita pepo. (A) Structure of CpbHLH gene in the C. pepo genome and the information of each region. (1) to (5), each part was divided to isolate the CpbHLH gene in resistant and susceptible lines, respectively. TSS: transcription start site, (B) Primer sequences used for PCR to isolate.
Fig. 3
The 5ʹ region of the isolated CpbHLH gene in Cucurbita pepo and polymorphism in the regulatory region. (A) The 5ʹ region of the CpbHLH gene in the C. pepo genome and comparative analysis of the deletion region in CpbHLH gene of resistant and susceptible lines. Deletion in the CpbHLH gene of susceptible lines was located 11-bp upstream of the putative TSS, but not in the resistant individual. The MYB binding site was located upstream of the deletion region in the regulatory region. (B) Sequence information of the polymorphism region and putative TSS. Light gray box, 3 bp deletion region in susceptible lines; dark gray box, putative TSS.
Fig. 4
Expression analysis of the CpbHLH gene in resistant and susceptible lines (A) RT-PCR confirmation of CpbHLH gene in resistant and susceptible lines. R, resistant lines; S, susceptible lines. (B) Analysis of CpbHLH gene expression by quantitative real-time RT-PCR analysis. The vertical T-bars indicate the standard deviation.
Fig. 5
PCR analysis of twenty-one resistant and nine susceptible squash lines derived from BC2F7 line. Primers were designed to contain sequences corresponding to deletion in the regulatory region in susceptible line. M: 100 bp DNA ladder, R: resistant lines, S: susceptible lines.
Table 1
Primer sequences used for reverse transcription (RT-PCR) and quantitative real-time PCR (qRT-PCR) analysis.
Primer name Nucleotide sequences (5ʹ-3ʹ) Product size
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