Cy-1, a major QTL for tomato leaf curl New Delhi virus resistance, harbors a gene encoding a DFDGD-Class RNA-dependent RNA polymerase in cucumber (Cucumis sativus)

doi:10.21203/rs.3.rs-3784112/v1

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Research Article

Cy-1, a major QTL for tomato leaf curl New Delhi virus resistance, harbors a gene encoding a DFDGD-Class RNA-dependent RNA polymerase in cucumber (Cucumis sativus)

https://doi.org/10.21203/rs.3.rs-3784112/v1

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published 02 Oct, 2024

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Background

Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) is a significant threat to cucumber (Cucumis sativus) production in many regions. Previous studies have reported the genetic mapping of loci related to ToLCNDV resistance, but no resistance genes have been identified.

Results

We conducted map-based cloning of the ToLCNDV resistance gene in cucumber accession No.44. Agroinfiltration and graft-inoculation analyses confirmed the resistance of No.44 to ToLCNDV isolates from the Mediterranean and Asian countries. Initial mapping involving two rounds of phenotyping with two independent F₂ populations generated by crossing the begomovirus-susceptible cultivar SHF and No.44 consistently detected major quantitative trait loci (QTLs) on chromosomes 1 and 2 that confer resistance to ToLCNDV. Fine-mapping of Cy-1, the dominant QTL on chromosome 1, using F₃ populations narrowed the candidate region to a 209-kb genomic segment harboring 24 predicted genes. Among these genes, DFDGD-class RNA-dependent RNA polymerase (CsRDR3), an ortholog of Ty-1/Ty-3 of tomato and Pepy-2 of capsicum, was found to be a strong candidate conferring ToLCNDV resistance. The CsRDR3 sequence of No.44 contained multiple amino acid substitutions; the promoter region of CsRDR3 in No.44 had a large deletion; and the CsRDR3 transcript levels were greater in No.44 than in SHF. Virus-induced gene silencing (VIGS) of CsRDR3 using two chromosome segment substitution lines harboring chromosome 1 segments derived from No.44 compromised resistance to ToLCNDV.

Conclusions

Forward and reverse genetic approaches identified CsRDR3, which encodes a DFDGD-class RNA-dependent RNA polymerase, as the gene responsible for ToLCNDV resistance at the major QTL Cy-1 on chromosome 1 in cucumber. Marker-assisted breeding of ToLCNDV resistance in cucumber will be expedited by using No.44 and the DNA markers developed in this study.

begomovirus

cucurbit

geminivirus

genetic mapping

marker-assisted breeding

resistance gene

RDR

ToLCNDV

Virus-induced gene silencing (VIGS)

Cucumber (Cucumis sativus) cultivated worldwide is an economically important vegetable crop, and its fruit is consumed fresh or pickled [1]. Cucumber is understood to have originated in India and was domesticated in Asia approximately 3,000 years ago [2]. The global production of cucumber was 93.5 million tonnes in 2021, and the largest cucumber producers is China followed by Turkey, Russia, Ukraine, and Mexico [3].

The genus Begomovirus (family Geminiviridae) contains 445 virus species [4], each of which carries a circular, monopartite or bipartite, single-stranded DNA genome [5]. Most begomoviruses have bipartite genomes organized of two circular DNA components (DNAs A and B), each approximately 2,800 nucleotides (nt) in length [6]. A bipartite begomovirus tomato leaf curl New Delhi virus (ToLCNDV) that infects Solanaceae crops (tomato, pepper, eggplant, potato) and cucurbit crops (cucumber, melon, zucchini, luffa) [7, 8], is the second most important begomovirus after the monopartite tomato yellow leaf curl virus (TYLCV) and has a high economic impact on horticultural production. ToLCNDV was first detected in India in 1995, and its isolation has subsequently been reported across the Indian subcontinent (India, Pakistan, and Bangladesh) and Southeast Asia (Thailand, Laos, Indonesia, and Taiwan) [9–15]. ToLCNDV mainly threaten the production of cucurbit crops such as cucumber, melon (Cucumis melo), squash (Cucurbita spp.), bottle gourd (Lagenaria siceraria), Luffa spp., and Sechium edule in Asian countries [9, 11, 16–18]. Based on reports from Iran and Turkey in the Middle East and from Spain, Portugal, Italy, Greece, Morocco, Tunisia, and Algeria in the Mediterranean Basin, from 2012 onward, ToLCNDV has spread westward, affecting cucurbit crops such as zucchini squash (Cucurbita pepo), melon, and cucumber [19–28].

The insect vector, whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), has driven the remarkable emergence of begomoviruses [29]. In tropical and subtropical regions, the distribution and polyphagous feeding habits of B. tabaci critically affecting the prevalence and economic importance of begomoviral diseases. Generally, insecticides that target B. tabaci populations are used to control begomoviruses caused diseases; however, insecticide resistance in B. tabaci has emerged by intensive and unregulated use of insecticides [30, 31]. An integrated pest management approach which uses begomovirus-resistant cultivars can be an effective alternative to control begomoviruses. Several begomovirus resistance genes have been cloned from Solanaceae crops using elaborate forward and reverse genetic methods [32–36]. Among the identified resistance genes, Ty-1/Ty-3 in tomato and Pepy-2 in capsicum encode DFDGD-class RNA-dependent RNA polymerases (RDRs) and are dominant resistance genes to multiple begomoviruses [33, 35]. Furthermore, it was demonstrated that the replication of begomoviral DNA is restricted by RDR via a transcriptional gene silencing pathway [37]. Breeding for begomovirus-resistant tomato plants has advanced, and Ty-1 has been introgressed into most of the TYLCV-resistant hybrid cultivars bred worldwide [38, 39]. Several studies have reported ToLCNDV resistance in cucurbits, such as melon, C. moschata, and L. cylindrica accessions [40–43]. In cucumber, ToLCNDV-resistant materials and the detection of quantitative trait loci (QTLs) on chromosomes 1 and 2 have been described in international patents from Vilmorin & Cie, Rijk Zwaan, and Nunhems [44–46] More recently, several ToLCNDV-resistant cucumber accessions and detected a QTL for resistance on chromosome 2 [47]. However, no resistance genes have been identified.

In this study, ToLCNDV resistance of a cucumber accession No.44 were evaluated. Reliable QTLs were detected on chromosomes 1 and 2 by two rounds of phenotyping with two independent F₂ populations generated by crossing the begomovirus-susceptible cultivar SHF and No.44. Further fine-mapping and functional analysis of a candidate gene on chromosome 1 identified CsRDR3 as the gene responsible for ToLCNDV resistance at the QTL.

No.44 is resistant to ToLCNDV isolates from the Mediterranean Basin and Southeast Asia

The resistance of cucumber accession No.44 to two ToLCNDV isolates was evaluated by agroinfiltration. In the first experiment, agroinfiltration of ToLCNDV-Es resulted in 100% infection in the susceptible cultivar ‘Sagami Hanjiro Fushinari’ (SHF), and the plants consistently exhibited severe symptoms, with an average disease severity index (DSI) score of 4 (Fig. 1A, Table 1). Moreover, only 26% of the inoculated No.44 plants were virus-infected, and the infected plants showed no symptoms, with an average DSI score of 0 at 30 dpi. F₁ showed moderate resistance to ToLCNDV-Es, with an average DSI score of 2.8 at 30 dpi (Table 1). In the second experiment, ToLCNDV-Es was agroinfiltrated into No.44 plants and another susceptible cultivar ‘Natsu Suzumi’ (NS) (Table 1). Again, No.44 was resistant to ToLCNDV-Es, while NS was susceptible to virus infection. Moreover, the accumulation of ToLCNDV-Es viral DNA was significantly lower in No.44 plants than in NS plants (Fig. 2A). In the third experiment, agroinfiltration of ToLCNDV-Idn [15] with higher pathogenicity induced severe symptoms in susceptible SHF plants (Fig. 1B, Table 1). Meanwhile, No.44 was resistant to ToLCNDV-Idn with an average DSI score of 1.4 at 30 dpi. Moreover, the accumulation of ToLCNDV-Idn viral DNA was significantly lower in No.44 plants than in SHF plants (Fig. 2B). From these results, we concluded that No.44 is resistant to ToLCNDV isolates from multiple cucumber-producing regions, including the Mediterranean Basin and Southeast Asia.

ToLCNDV-Es was transmitted from a symptomatic NS scion to healthy No.44 or SHF rootstock and evaluated for resistance at 30 and 60 days after grafting. ToLCNDV-Es infection of the NS scion was confirmed by PCR before grafting. At 30 days after grafting, No.44 plants exhibited vigorous growth compared with the susceptible SHF plants (Fig. 1C). The average DSI values of the fifth and 10th true leaves of No.44 plants were significantly lower than those of their counterparts in SHF plants (Table 2). Moreover, at 60 days after grafting, intense yellowing was observed on the ninth true leaf of SHF plants, but only a few spots with mild yellowing were observed on the leaves of No.44, and the average DSI was significantly lower for No.44 than for SHF (Fig. 1D, Table 2). On the 20th true leaf, symptoms were relatively mild in both No.44 and SHF, but the average DSI was significantly lower in No.44 than in SHF (Table 2). Quantification of ToLCNDV-Es DNA in leaves revealed that significantly less ToLCNDV-Es DNA accumulated in the fifth and 10th true leaves of No.44 than in those of SHF at 30 days after grafting (Fig. 2C). At 60 days after grafting, there was no significant difference in the amount of viral DNA in the ninth true leaf between No.44 and SHF plants, but there was significantly less viral DNA in the 20th true leaf of No.44 than in its counterpart in SHF (Fig. 2D). Taken together, these results show that No.44 is resistant to ToLCNDV-Es not only at the young seedling stage but also at the fruit-setting stage.

Genetic mapping of ToLCNDV resistance in F₂ populations

For the genetic mapping of ToLCNDV-Es resistance, two rounds of phenotyping were conducted with two independent F₂ populations generated by crossing the begomovirus-susceptible cultivar SHF and No.44. In the first F₂ population, 187 out of the 203 individuals agroinfiltrated with ToLCNDV tested positive for virus infection at 30 dpi, as determined by PCR. These ToLCNDV-positive plants were analyzed further. At 30 dpi, number of resistant individuals (DSI 0–1) to susceptible individuals (DSI 2–4) segregated into 1:3 for the F₂ population, as shown via the χ² test, indicating that ToLCNDV resistance was controlled by a single recessive gene (Table S1). Linkage analysis of ToLCNDV resistance in the first round of the SHF × No.44 F₂ population (n = 187) was performed using 309 SNPs discovered by restriction site-associated DNA sequencing (RAD-seq). The number of linkage groups was the same as the chromosome number of cucumber (C. sativus), and the total size of the linkage map was 647.8 cM (average marker distance = 2.1 cM). Two significant QTLs, one on chromosome 1 and the other on chromosome 2, were detected by a composite interval mapping (CIM) analysis of the SHF × No.44 F₂ population; these QTLs were denoted as cucumber yellow leaf curl disease resistance-1 (Cy-1) and Cy-2, respectively (Fig. 3A, Table 3). The QTLs explaining less than 10.0% of the phenotypic variation were defined as minor QTLs, whereas the QTLs explaining more than 10% of the phenotypic variation were defined as major QTLs according to previous report [48]. The highest peaks and narrowest intervals of QTLs were detected for the DSI score at 30 dpi. A major QTL Cy-1 with logarithm of odds (LOD) score of 15.1 was detected at the physical position of 24,837,399 on chromosome 1 of the reference sequence (Chinese Long, ver. 3), and the other major QTL Cy-2 (LOD score = 22.2) was detected at the physical position of 17,866,822 on chromosome 2. Cy-1 and Cy-2 explained 20.0% and 30.7% of the total phenotypic variation, respectively.

In the second F₂ population, 143 out of 149 individuals tested positive for ToLCNDV-Es infection at 30 dpi, and the segregation ratio indicated that ToLCNDV resistance was conferred by a single recessive gene (Table S1). Linkage analysis of ToLCNDV resistance was performed using 455 SNPs discovered by RAD-seq. The number of linkage groups was the same as the chromosome number of cucumber, and the linkage map size was 623.3 cM (average marker distance = 2.0 cM). A CIM analysis of the SHF × No.44 F₂ population detected three significant QTLs on chromosomes 1, 2, and 6 (Fig. 3B, Table 3). The highest peaks and narrowest intervals of QTLs were detected for the DSI score at 30 dpi. Among the three QTLs detected, two major QTLs had genomic regions that overlapped with Cy-1 and Cy-2 identified in the first round of QTL analysis. The Cy-1 QTL (LOD score = 13.7) was detected at position 24,211,834 on chromosome 1, and the Cy-2 QTL (LOD score = 21.6) was detected at position 17,695,694 on chromosome 2. Cy-1 and Cy-2 explained 18.0% and 36.3% of the total phenotypic variation, respectively. Additionally, the third minor QTL Cy-6 (LOD score = 8.1) located at the physical position 11,725,560 on chromosome 6 explained 8.5% of the total phenotypic variation.

The major QTLs Cy-1 and Cy-2 that were consistently detected in the two rounds of genetic mapping were further analyzed. Individuals with different allelic combinations of Cy-1 and Cy-2 were identified from the first-round F₂ population on the basis of the genotypes of the closest markers at the LOD peaks. Plants homozygous for the No.44 allele at marker S2_17866822 on chromosome 2 were more resistant to ToLCNDV and had lower virus titers than plants heterozygous or homozygous for the SHF allele. These results indicated that Cy-2 acts recessively to increase resistance to ToLCNDV (Fig. S1A and B). In light of these results, this locus is hereinafter referred to as cy-2. In contrast, plants homozygous and heterozygous for the No.44 allele at marker S1_24837399 on chromosome 1 were more resistant to ToLCNDV and had lower virus titers than those of plants homozygous for the SHF allele, indicating that Cy-1 acts dominantly (Fig. S1A and B). Similar results were observed for the second-round F₂ population (Fig. S1C). The overlapping regions of Cy-1 and cy-2 identified in the two rounds of linkage analyses included 468 and 573 candidate genes, respectively (Table S2).

Fine mapping of the ToLCNDV resistance QTL Cy-1

We further finely mapped the ToLCNDV resistance QTL Cy-1 (Fig. 4). Two KASP markers (Cuc24327273-KASP and Cuc25380586-KASP) were developed and five recombinants (No.8, No.12, No.270, No.420, and No.437) were screened from the newly prepared F₂ population (n = 752) to narrow down the candidate region. The recombination points were verified using 13 additional markers, consisting of eight KASP and five high-resolution melting (HRM) markers. F₃ populations were obtained by self-pollinating these F₂ recombinants. After phenotyping and genotyping these F₃ populations infected with ToLCNDV-Es, the ToLCNDV resistance QTL Cy-1 was mapped to a 209-kb region between the Cuc25061147-HRM marker and the Cuc25270268-KASP marker. In the Chinese Long genome (ver.3), 24 putative genes were identified in this target region (Table 4). Whole-genome resequencing of the two parents revealed that, compared with those of SHF and Chinese Long, only two genes, CsaV3_1G039730 and CsaV3_1G039870, had nonsynonymous substitutions in their open reading frames in No.44. Furthermore, the transcript level of CsaV3_1G039730 was significantly greater in ToLCNDV-infected and mock-inoculated No.44 than in ToLCNDV-infected and mock-inoculated SHF plants, as determined by RNA-seq analysis (Table 4). On the basis of these results, CsaV3_1G039730, which encodes an RNA-dependent RNA polymerase (RDR), was identified as Cy-1 conferring resistance to ToLCNDV.

Analysis of CsRDR3 encoding an RNA-dependent RNA polymerase

The full-length sequences of CsaV3_1G039730, CsRDR3, from No.44 and SHF were isolated and analyzed. These analyses revealed that CsRDR3 consisted of 19 exons (Fig. 5A). CsRDR3 showed high sequence similarly with SlRDR3 (Ty-1/Ty-3) of tomato and CaRDR3a (Pepy-2) of capsicum (Fig. 5B). Seven nonsynonymous substitutions were detected in the amino acid sequence of CsRDR3 in No.44 compared with its counterpart in SHF (Fig. 5B). In addition, compared with those in SHF, an approximately 1.9-kb deletion and a 62-bp insertion were identified in the CsRDR3 promoter region in No.44 (Fig. 5A). A BLAST analysis of this 1.9-kb fragment in the genomic DNA of SHF and Chinese Long revealed no similarity to any annotated sequences, including transposable elements. Multiplex PCR successfully amplified a 454-bp band from plants homozygous for the No.44-allele, an 805-bp band from plants homozygous for the SHF-allele, and 454-bp and 805-bp bands from heterozygous plants (Fig. 5A). These primers can be used for genotyping the resistant and susceptible alleles of CsRDR3. According to the results of the phylogenetic analysis, the RDRs from different plant species formed two clusters, namely α and γ -clades of RDR (Fig. 6). Phylogenetic analysis showed that the CsRDR3s of No.44 and SHF exhibited high amino acid sequence similarity with RDR3, RDR4, and RDR5 of Arabidopsis, which are γ-clade RDRs. The RDRs of tomato (SlRDR3), pepper (SlRDR3a), potato (StRDR3), and tobacco (NtRDR3) constituted an independent clade from the CsRDR3s within the γ-clade RDRs.

Virus-induced gene silencing of CsRDR3

All the analyses so far support CsRDR3 as a strong candidate for the gene conferring ToLCNDV resistance at the QTL Cy-1. To further verify this hypothesis, we conducted virus induced gene silencing (VIGS) of CsRDR3 to confirm its involvement in ToLCNDV resistance. In an initial experiment in which CsRDR3 was silenced in No.44 by VIGS, changes in resistance were not detected because of the effect of another major ToLCNDV resistance QTL cy-2 on chromosome 2. To address this problem, we used two chromosome segment substitution lines, No.148 and No.489, which were homozygous for the No.44 genotype at the target region on chromosome 1 and homozygous for the SHF genotype at the target region on chromosome 2. Moreover, we used CsRDR3-specific gene fragments as inserts for the apple latent spherical virus (ALSV) vector to avoid the off-target effects of VIGS.

For VIGS of CsRDR3 in No.148, the ALSV construct pBICAL2::CsRDR3-189 was used. The characteristic photobleaching effect appeared in the topmost leaves after 28 dpi and was subsequently enhanced in the phytoene desaturase (PDS) gene-silenced No.148 plants (Fig. 7A). Plants with mixed ToLCNDV and ALSV infections, as confirmed by PCR, were selected for further analysis. The control No.148 plants with mixed infection of ToLCNDV and wild-type ALSV were resistant to ToLCNDV. In contrast, the CsRDR3-silenced No.148 plants exhibited mild leaf-yellowing symptoms and accumulated nine times more ToLCNDV viral DNA, which was significantly greater than that of the control plants. The transcript levels of CsRDR3 tended to be lower in CsRDR3-silenced No.148 plants than in control plants at 40 dpi, but with no statistical differences. Although the first experiment partially supported our hypothesis that CsRDR3 is involved in ToLCNDV resistance, further experiments are needed.

Since the efficiency of VIGS by ALSV is reportedly affected by the genotype of the host plant and the size of the fragment inserted into the virus vector [49, 50], we used another chromosome segment substitution line, No.489, and ALSV construct pBICAL2::CsRDR3-102, which has a shorter fragment from a different part of the CsRDR3 gene (Fig. 7B). The PDS gene-silenced No.489 plants started to show a photobleached phenotype in the developing leaves at 28 dpi. Since the growth of No.489 was more vigorous than that of No.148 under the same experimental conditions, we cut back the plants at 28 dpi and used the newly elongating lateral branches for analyses. At 40 dpi, the PDS-silenced No.489 plants exhibited a highly uniform photobleached phenotype. The control No.489 plants with mixed infection of ToLCNDV and wild-type ALSV were resistant to ToLCNDV. Moreover, the CsRDR3-silenced No.489 plants exhibited severe leaf curling and yellowing symptoms. In addition, there were 51-fold more ToLCNDV-accumulating viral DNA in the CsRDR3-silenced No.489 plants than in the control plants. Moreover, the CsRDR3 transcript level was significantly lower in the CsRDR3-silenced No.489 plants than in the control No.489 plants. We also conducted VIGS for several other genes located in the candidate genomic region, such as CsaV3_1G039720, CsaV3_1G039750, CsaV3_1G039790, CsaV3_1G039870, and CsaV3_1G039910, which had sequence variances in No.44 or higher gene expression in ToLCNDV-infected No.44 than in SHF (Table 4), but no loss-of-resistance phenotypes were observed in the gene-silenced plants (data not shown). In conclusion, that CsRDR3 is most likely the gene underlying ToLCNDV resistance at QTL Cy-1 in No.44.

ToLCNDV was first isolated in India from diseased tomato plants [14]. Since then, its distribution has been reported from the Indian subcontinent, Southeast Asia, the Middle East, and the Mediterranean Basin [8]. The ToLCNDV isolates from Spain, Italy, Morocco, Algeria, and Tunisia in the Mediterranean Basin are monophyletic and form a nested clade within a much more diverse group of virus isolates from the Indian subcontinent. This strongly indicates that the ToLCNDV population in the Mediterranean Basin was founded by a single virus that originated from the Indian subcontinent [7, 8, 16]. A similar phylogenetic pattern was displayed for Southeast Asian ToLCNDV isolates from Thailand, Laos, Indonesia, and Taiwan. In the present study, we demonstrated that No.44 is resistant to the ToLCNDV-Es isolate from Spain and to the highly pathogenic Indonesian isolate, which are the representative ToLCNDV isolates from the Mediterranean Basin and Southeast Asia, respectively.

High-throughput sequencing technology has enabled us to obtain DNA polymorphisms easily, even for nonmodel plant species. In contrast, successful genetic mapping of the target gene depends on accurate phenotyping of target traits which requires deliberate and laborious work. In our linkage analyses, ToLCNDV-agroinfiltrated cucumber plants were grown under constant climate conditions of laboratory where we can also avoid any unpredictable infection by other viruses or pathogens. Additionally, to exclude the use of inoculation escapes, which can significantly reduce mapping quality, plants that tested positive for ToLCNDV infection according to PCR were selected for analysis. Two rounds of phenotyping with two independent F₂ populations consistently mapped two major QTLs for ToLCNDV resistance on chromosomes 1 and 2. Further fine-mapping using F₃ populations narrowed the candidate region of chromosome 1 to a genomic fragment containing 24 genes. We cannot dismiss the possibility that the other 23 candidate genes, in addition to RDR in the target genomic region, also contributed to ToLCNDV resistance in No.44 according to our forward genetic analysis. However, the results of whole-genome sequencing and RNA-seq analyses strongly indicated that RDR is most likely the gene responsible for ToLCNDV resistance.

To strengthen the scientific basis of our hypothesis, we conducted reverse genetic analyses by using ALSV-based VIGS in cucumber. The efficiency of VIGS was greater in the second experiment in which chromosome segment substitution line No.489 and the ALSV construct pBICAL2::CsRDR3-102 were used than the first experiment, in which chromosome segment substitution line No.148 and the ALSV construct pBICAL2::CsRDR3-189 were used. There are several possible explanations for these results. Because it is reported that the effectiveness of ALSV-mediated VIGS is genotype dependent in soybean [49], the difference in genotype between cucumber No.489 and No.148 may have affected the VIGS efficiency of CsRDR3. The length and/or position of inserted sequences are also reported to affect the efficiency of VIGS [50]. Since we used CsRDR3 at different positions and fragment lengths for pBICAL2::CsRDR3-102 and pBICAL2::CsRDR3-189, this may have resulted in differences in VIGS efficiency between the two experiments. Notably, there was good correspondence between the VIGS efficiency of CsRDR3 and the symptomology observed or the accumulating ToLCNDV viral DNA. Moreover, because the VIGS of other genes located in the candidate genomic region induced no loss of resistance, as did the CsRDR3-silenced plants, we concluded that CsRDR3, encoding an RDR, is the gene responsible for ToLCNDV resistance at the major QTL Cy-1.

Several begomovirus resistance genes have been cloned from Solanaceae plants using elaborate forward and reverse genetic methods. In tomato, the TYLCV resistance genes Ty-1/Ty-3 encode RDRs, Ty-2 encodes a nucleotide-binding leucine-rich repeat (NB-LRR) protein, and ty-5 encodes Pelota [34–36]. More recently, we identified pepy-1 encoding Pelota and Pepy-2 encoding an RDR that confer resistance to bipartite begomoviruses in capsicum [32, 33]. The resistance gene Ty-1, which is widely used in the breeding of F₁ hybrid cultivars of tomato plants, also confers resistance to the curtovirus, the leafhopper-transmitted beet curly top virus, suggesting that RDR-based resistance represents broad resistance to geminiviruses [51]. Previously conducted genetic mapping of ToLCNDV resistance in cucumbers revealed QTLs in the overlapping genomic region of chromosome 1, as detected in our study [44–46]. These previous findings indicate that Cy-1, which harbors CsRDR3, confers resistance to ToLCNDV in multiple cucumber resources, not only in No.44.

In plants, the long double-stranded RNA molecules are cleaved to produce siRNAs by RNA-silencing pathways. The RDR protein participates in these pathways as a part of the antiviral defense mechanism. RDRs share a special conserved RDR catalytic domain, and eukaryotic RDRs can be grouped into RDRα, RDRβ, and RDRγ [52]. Members of the RDRβ clade are not found in plants but are conserved mainly among fungi [53]. In the genome of A. thaliana, there are six RDRs which are grouped into the RDRα and RDRγ clades [54]. Arabidopsis α-type RDRs have the DLDGD motif of eukaryotic RDRs but are not essential for the production of viral siRNAs in geminivirus-infected Arabidopsis [54, 55]. The functions of the three RDRγs in Arabidopsis with a DFDGD amino acid motif in the catalytic domain have not yet been described. In contrast, the function of γ-type RDRs related to geminivirus resistance in Solanaceae is better understood. According to our phylogenetic analysis, the cucumber CsRDR3 was clustered with γ-type RDRs, specifically SlRDR3 (Ty-1/Ty-3) of tomato and CaRDR3a (Pepy-2) of capsicum. Furthermore, it was demonstrated that the replication of begomoviral DNA is restricted by RDR via a transcriptional gene silencing pathway in tomato plants harboring Ty-1 [37]. We speculate that CsRDR3 confers ToLCNDV resistance via a similar mechanism in cucumber. Analyses of the transcript levels and sequence differences in CsRDR3 between ToLCNDV-resistant No.44 and susceptible SHF revealed higher gene transcript levels in No.44, as well as multiple amino acid substitutions in its CsRDR3a protein. Similar observations have been reported for Ty-1 and its encoded RDR [35, 56]. However, further research is needed to determine whether one or both of these differences in CsRDR3 is required for conferring resistance. Since γ-type RDR was demonstrated to confer resistance to begomovirus not only in Solanaceae but also in cucumber, other vegetable crops in cucurbit may also harbor a resistance allele of γ-type RDR. From a practical viewpoint, the multiplex PCR-based system developed in this study to distinguish between resistant and susceptible alleles of CsRDR3 will be practically beneficial for DNA marker-assisted breeding of ToLCNDV resistance in cucumber using No.44 as a resistant source.

In this study, our genetic mapping analyses revealed three QTLs for ToLCNDV resistance on chromosomes 1, 2, and 6 in the cucumber genome. However, the minor QTL on chromosome 6 was detected only in the second-round mapping population, and its effect on resistance was rather small. Moreover, cy-2 on chromosome 2 was identified as a major QTL conferring resistance to ToLCNDV. The ty-5 and pepy-1 genes, both of which encode Pelota, are well-known recessive genes involved in resistance to begomoviruses [32, 34]. No Pelota-encoding gene was present in the target region of cy-2, suggesting that this region contains a novel recessive resistance gene that is potentially useful as a plant disease susceptibility (S) gene. This gene could be the target of genome editing or artificial mutant screening for the application of resistance mechanisms to other crop species. Fine mapping and reverse genetic analysis of cy-2 are now in progress. These analyses will aid in DNA marker-assisted breeding for durable and wide-spectrum begomovirus resistance when combined with Cy-1 and will provide a theoretical basis for the practical application of this resistance mechanism in various crops.

We conducted fine-mapping of a major QTL Cy-1 on chromosome 1 for ToLCNDV resistance in cucumber accession No.44. Among 24 genes located on the candidate region, CsRDR3, an ortholog of Ty-1/Ty-3 of tomato and Pepy-2 of capsicum, was found to be a strong candidate conferring ToLCNDV resistance. Reverse genetic analysis of CsRDR3 by VIGS also supported that this gene is responsible for ToLCNDV resistance. DNA marker developed in this study to distinguish the resistant and susceptible alleles of CsRDR3 will be practically beneficial for marker-assisted breeding of ToLCNDV resistance in cucumber using No.44 as a resistant source.

Plant material

The cucumber (C. sativus) accession No.44, which is resistant to ToLCNDV, and the susceptible cultivars ‘Natsu Suzumi’ (NS) (Takii seeds, Kyoto, Kyoto, Japan) and ‘Sagami Hanjiro Fushinari’ (SHF) were used in this study. The F₂ and F₃ populations generated by crossing SHF and No.44 were used for genetic mapping. Plants were cultivated in a growth room controlled with temperatures between 23°C to 30°C and a photoperiod of 13-h light/11-h dark.

ToLCNDV inoculation and detection

Two ToLCNDV isolates were used in this study: the ToLCNDV-Es isolate ES-Alm-Cuc-16 (GenBank accession numbers for DNA-A: LC596380, DNA-B: LC596383) from Spain and the ToLCNDV-Idn isolate BACu-20 (DNA-A: LC511775, DNA-B: LC511780) from Indonesia [15, 57]. Full details about the infectious clones and the inoculation method have been reported elsewhere [15, 58]. In brief, agrobacteria bearing the plasmids pGreenII-p35S-ToLCNDV-[ES-Alm-Cuc-16]-DNA-A and pGreenII-p35S-ToLCNDV-[ES-Alm-Cuc-16]-DNA-B or pGreenII-p35S-ToLCNDV-[BACu-20]-DNA-A and pGreenII-p35S-ToLCNDV-[BACu-20]-DNA-B were cultured to an optical density of 0.3 and subsequently used to agroinfiltrate cotyledons of No.44, SHF, NS, and SHF × No.44 F₁, F₂, and F₃ plants before the first true leaf developed. The disease symptoms of each plant were scored at 15 and/or 30 days postinoculation (dpi) as the disease severity index (DSI). The DSI ranged from 0 to 4 and was scored as follows: 0, no symptoms; 1, a few yellow spots on the leaf; 2, yellow spots across the whole leaf; 3, yellowing of the leaf with mild curling; and 4, intense yellowing and curling of the leaf. For DNA and RNA extraction, the young upper leaves were collected and stored at − 80°C. Statistical analyses for DSI scores were conducted using the Bonferroni–Dunn test or Mann–Whitney U test with Excel Toukei ver. 7.0, and a p value less than 0.05 was considered statistically significant.

An inoculation experiment was conducted to evaluate the graft transmission of ToLCNDV-Es to No.44 and SHF plants. A scion of ToLCNDV-Es-infected NS plants was grafted onto uninoculated No.44 and SHF plants (as rootstocks), and the newly elongated lateral branches from the rootstock were evaluated for ToLCNDV resistance. Plants were grown in a greenhouse. Disease symptoms were surveyed at 30 and 60 days after grafting, and leaves were collected and stored at − 80°C until DNA extraction.

A Nucleon PhytoPure Kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) was used to extract DNA from cucumber leaves. Conventional PCR and qPCR were conducted to detect and quantify the viral DNA of ToLCNDV following the methods of [59]. The primer sequences and PCR conditions used in these analyses are listed in Supplementary Tables S3 and S4. Statistical analysis for accumulating viral DNA amount was conducted using the Tukey–Kramer test in Excel Toukei ver. 7.0 or Student’s t test, and a p value less than 0.05 was considered statistically significant.

QTL mapping of ToLCNDV resistance

In this study, two rounds of phenotyping were conducted for two independent F₂ populations consisting of 187 and 149 individuals infected with ToLCNDV-Es, and the extracted DNA was subjected to RAD-seq analysis. The RAD-seq libraries were created for F₂ individuals and their parents and sequenced with the NovaSeq 6000 platform (Illumina, Hercules, CA, USA) following the methods of [60]. The obtained reads were trimmed and mapped to the genome sequence of C. sativus (Chinese Long, ver.3) [61]. The precise methodology used for the data analysis is described elsewhere [32]. The genetic linkage maps were constructed from the single nucleotide polymorphism (SNP) RAD tags, and R/qtl was used to conduct QTL analyses by composite interval mapping (CIM) [62].

Fine mapping

The NovaSeq 6000 platform (Illumina) was used for whole-genome resequencing of No.44 and SHF, and the data analysis of the obtained reads was conducted following the methods reported in [32]. Additional SNP markers were developed for the KBiosciences KASPar assay (LGC Genomics GmbH, Berlin, Germany) to further narrow the region of the QTL, and recombinants were screened from F₂ individuals. Five F₂ recombinants, namely, No.8, No.12, No.270, No.420, and No.437, were screened, and the additional KASP markers and high-resolution melting (HRM) markers were used to identify the recombination points in each F₂ individual. HRM analysis was conducted following the methods reported in [32]. The primer sequences and PCR conditions used for fine mapping is listed in Supplementary Table S4 and Table S3. The recombinants were self-pollinated to obtain F₃ populations.

Analyses of the candidate gene

Sepasol-RNA I Super G extraction buffer (Nacalai Tesque, Kyoto, Japan) and High-Salt Solution for Precipitation (Plant) (Takara Bio, Shiga, Japan) were used to extract and purify the RNA from leaves. Transcriptome profiling was conducted via RNA-seq using the NovaSeq 6000 platform (Illumina) for mock-inoculated and ToLCNDV-Es-infected No.44 and SHF plants according to the previously reported methods [33]. Reverse-transcription PCR (RT‒PCR) was conducted following the previously reporte methods using the cDNA template, KOD-plus Neo (Toyobo, Osaka, Japan), and the CsRDR3-specific primer pairs to amplify the open reading frame of the candidate gene [32]. The amplified PCR products were cloned into the TOPO vector (Thermo Fisher Scientific, MA, USA) and used for subsequent sequencing. Supplementary Table S3 contains the list of the sequences of the primers used for PCR. Multiple sequence comparisons of the predicted RDR amino acid sequences of Arabidopsis thaliana, Oryza sativa (rice), Nicotiana tabacum, Solanum tuberosum, S. lycopersicum, and C. annuum were conducted using the MUSCLE program [63]. MEGA 7.0 was used to construct the phylogenetic tree via the neighbor‒joining method, with 1,000 bootstrap replicates [64].

Reverse genetic analysis by virus-induced gene silencing

Apple latent spherical virus (ALSV) vectors were used for VIGS [50, 65]. In brief, partial coding sequences of CsPDS (102 bp), CsRDR3 (189 bp) or CsRDR3 (102 bp) were amplified from No.44 with the CsPDS-102-Xho/Bam, CsRDR3-189-Xho/Bam or CsRDR3-102-Xho/Bam primers, respectively (Supplementary Table S3). The obtained amplicons digested with XhoI and BamHI were ligated with XhoI- and BamHI-digested pBICAL2 to construct pBICAL2::CsPDS, pBICAL2::CsRDR3-189, and pBICAL2::CsRDR3-102. To avoid the effects of off-target genes, a BLASTn analysis was conducted using PCR fragment sequences as queries to design the gene-specific primers for each gene.

For VIGS, we used two chromosome segment substitution lines, No.148 and No.489, which were homozygous for the No.44 genotype at the target region on chromosome 1 and homozygous for the SHF genotype at the target region on chromosome 2. Cultures of Agrobacterium containing pBICAL1 and pBICAL2, pBICAL2::CsPDS, pBICAL2::CsRDR3-189, or pBICAL2::CsRDR3-102 were mixed at a ratio of 1:1 for agroinfiltration. Before the first true leaf developed, cotyledons were agroinfiltrated with the bacterial suspension at an optical density of 0.3. One day after agroinfiltration of ALSV, ToLCNDV-Es was agroinoculated following the methods reported previously [15, 66]. For agroinoculation, a colony inoculation procedure was performed on the hypocotyl just below the apex of the seedlings using Agrobacterium transformed with pGreenII-p35S-ToLCNDV-[ES-Alm-Cuc-16]-DNA-A + B. No.489 plants were cut back at 28 dpi to allow for elongation of lateral branches, which were subsequently used for analysis. Symptom survey and collection of young upper leaves were conducted at 40 dpi.

The viral DNA-A of ToLCNDV-Es was detected by conventional PCR and quantified by qPCR. ALSV infection was confirmed by conventional PCR using the ALSV-F/R primer pair (Table S3). The transcript levels of CsRDR3 were analyzed via qRT‒PCR following the previously reported methods [32]. The primer sequences and PCR conditions used for real-time qRT‒PCR analyses are listed in Supplementary Tables S3 and S4. The CsActin reference gene was used to normalize the transcript level of the candidate gene, and 2^−ΔΔCt method was applied to calculate relative gene transcript levels. Statistical analyses for gene expression and viral DNA accumulation were conducted using Student’s t test, and a p value less than 0.05 was considered statistically significant.

Acknowledgment

We thank Shinya Kanzaki (Kindai University, Japan), Hayato Shiragane (Takii seeds, Japan), and Ryohei Arimoto (Takii seeds, Japan) for useful discussion. We thank Nobuyuki Yoshikawa (Iwate University) and Masanori Kaido (Setsunan University) for providing the ALSV vector and Takashi Kawai (Okayama University) for offering technical advice on VIGS. We thank Jennifer Smith, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

Authors' contributions

SK designed the experiments, performed the genetic mapping and VIGS, analyzed the data, interpreted the results and wrote the manuscript. CY and HY performed the virus inoculation, resistance evaluation, genetic mapping, gene expression analysis, and VIGS. KF performed the initial screening of the resistant material. RM and MO performed the VIGS. AJN performed the RAD-seq. T.M. prepared the crossing populations and conducted graft-inoculation and genetic mapping. All the authors read and approved the final manuscript.

Funding

This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant numbers 19H02950, 21KK0109, and 23H02207) awarded to S. Koeda.

Availability of data and material

Data is provided within the manuscript or supplementary information files. The accession numbers for each of the gene sequences referred to in this work are as follows: mRNA sequences of No.44 (LC778230) and SHF (LC778231). Further data is available from the authors upon reasonable request.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Tables 1 to 4 are available in the Supplementary Files section.

No competing interests reported.

Fig.S1.pdf
Figure S1. Effect of quantitative trait loci (QTLs) on ToLCNDV resistance in cucumber. a Interaction effect of chromosome 1 (S1_24837399 marker) and chromosome 2 (S2_17866822 marker) on DSI scores at 30 days postinoculation (dpi) and b the level of ToLCNDV-ES viral DNA accumulation at 30 dpi in the first-round ‘Sagami Hanjiro Fushinari’ (SHF) × No.44 F₂ population (n = 187). c Interaction effect of chromosome 1 (S1_24211834 marker) and chromosome 2 (S2_17695694 marker) on DSI scores at 30 dpi in the second-round SHF × No.44 F₂ population (n = 143). No.44 and SHF indicate plants homozygous for the No.44 and SHF alleles, respectively; h indicates heterozygous plants.
TableS1.pdf
Table S1. Segregation of resistant and susceptible plants in the F₁ and F₂ populations derived from ‘Sagami Hanjiro Fushinari’ (SHF) × No.44 crosses.
TableS2.xlsx
Table S2. Candidate genes located at quantitative trait loci (QTLs) on chromosomes 1, 2, and 6 in cucumber.
TableS3.pdf
Table S3. Primer list used in this study.
TableS4.pdf
Table S4. Conditions for PCR in this study.
TableS5.pdf
Table S5. DNA markers developed in this study.
Table1.pdf
Table2.pdf
Table3.pdf
Table4.pdf

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Cy-1, a major QTL for tomato leaf curl New Delhi virus resistance, harbors a gene encoding a DFDGD-Class RNA-dependent RNA polymerase in cucumber (Cucumis sativus)

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Abstract

Background

Results

Conclusions

Figures

Background

Results

No.44 is resistant to ToLCNDV isolates from the Mediterranean Basin and Southeast Asia

Genetic mapping of ToLCNDV resistance in F₂ populations

Discussion

Conclusions

Methods

Plant material

ToLCNDV inoculation and detection

QTL mapping of ToLCNDV resistance

Fine mapping

Analyses of the candidate gene

Reverse genetic analysis by virus-induced gene silencing

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Cy-1, a major QTL for tomato leaf curl New Delhi virus resistance, harbors a gene encoding a DFDGD-Class RNA-dependent RNA polymerase in cucumber (Cucumis sativus)

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Results

No.44 is resistant to ToLCNDV isolates from the Mediterranean Basin and Southeast Asia

Genetic mapping of ToLCNDV resistance in F2 populations

Discussion

Conclusions

Methods

Plant material

ToLCNDV inoculation and detection

QTL mapping of ToLCNDV resistance

Fine mapping

Analyses of the candidate gene

Reverse genetic analysis by virus-induced gene silencing

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Genetic mapping of ToLCNDV resistance in F₂ populations