Characterization of genetic resistance to cucumber mosaic virus (CMV) in spinach (Spinacia oleracea L.)

Abstract

Objective

Cucumber mosaic virus (CMV) is a significant pathogen causing quality loss in spinach. Although host genetic resistance is the primary method of managing CMV infection in this crop, CMV resistance genes are not widely utilized in spinach breeding programs as the genetic and molecular mechanisms underlying resistance are not yet fully understood. CMV infections were therefore studied in different lines of spinach plants, and their progeny, to develop a model of the genetic basis of CMV resistance.

Results

Visual observations and RT-PCR assays revealed that three monoecious lines (03-258, 03-263, and 03-336) were susceptible to CMV, while three traditional resistant cultivars and a near-isogenic line (NIL-M) exhibited resistance. A dioecious line (03–009) consisted of susceptible and resistant plants. Notably, resistant plants did not exhibit the lesions typical of the hypersensitive response. Genetic analysis of progeny from the cross NIL-M × 03-336 indicated that a single dominant allele (designated SRCm1, standing for Spinach Resistance to CMV 1) controlled CMV resistance; analysis of sib-cross progeny populations derived from line 03–009 supported this conclusion. These results offer a valuable model for CMV resistance in spinach and will enhance future breeding programs.

Introduction

Cucumber mosaic virus (CMV) has a wide range of hosts, infecting over 1000 plant species and causing significant economic losses in various crop [1]. CMV is a positive-sense RNA plant virus with a genome consisting of three single-stranded RNA molecules, designated RNA1, RNA2 and RNA3 [2]. Spinach is susceptible to 14 naturally occurring viral diseases, with CMV, historically known as ‘Spinach Blight,’ being particularly significant [3]. Early work by Pound and Cheo [4] characterized blight-resistant varieties such as ‘Virginia Savoy,’ emphasizing environmental factors in resistance. Early breeding efforts laid the foundation for spinach breeding programs, and host genetic resistance remains the primary tool for managing CMV infection [3,4,5]. As a result, the genetic control mechanisms responsible for the CMV resistance of spinach have not yet been fully understood and require further investigation.

Effector-triggered immunity (ETI) is a component of the plant immune system. It is activated by R genes, which encode nucleotide-binding and leucine-rich repeat domain proteins (NLRs), in response to pathogen attacks [6]. ETI can lead to a strong defense reaction known as the hypersensitive response (HR), which is characterized by localized cell death around the infection site [7, 8]. The HR is effective in suppressing pathogen infection and provides long-lasting protection. Extreme resistance (ER) is a type of antiviral resistance mediated by R genes that suppresses virus accumulation effectively in initially infected cells without triggering programmed cell death [9]. Arabidopsis thaliana ecotype C24 is resistant to the yellow strain of CMV (CMV-Y) and displays the HR following inoculation. Resistance is conferred by a single dominant allele of the RCY1 gene, which encodes a CC-type NLR protein [10, 11]. Similar R genes that confer CMV resistance have not yet been identified or isolated from other plants.

In this study, the resistance of various spinach cultivars and lines to CMV-Y was determined to establish a model system for understanding the genetic and molecular basis of CMV resistance. In addition to characterizing resistance phenotypically, we conducted genetic analyses of progeny from the resistant, susceptible, and segregating lines identified during the study.

Materials and methods

Plant material

This study assessed CMV resistance in four spinach lines (03-336, 03-258, 03-263, and 03–009) from Tohoku Seed Co., LTD (Utsunomiya, Japan), and a near-isogenic line, NIL-M, developed by introducing the monoecious gene from line 03-336 into the genetic background of dioecious line 03–009 through five generations of backcrossing (Table 1) [12, 13]. CMV resistance was also tested in five sib-crossed progeny populations (#10, #11, #13, #14, and #16) from dioecious line 03–009 (Table 2). These populations were produced by randomly crossing male and female plants without prior CMV resistance evaluation, as susceptible plants die before flowering after inoculation. F₁ and F₂ populations from the NIL-M × 03-336 cross were also studied (Table 2). Additionally, traditional blight-resistant cultivars (Tables 1 and 2) were included for comparison to compare their resistance with the spinach lines. All plants were grown in BioTRON growth chambers (Nippon Medical & Chemical Instruments, Osaka, Japan) at 20 °C with an 8-h photoperiod.

Table 1 Spinach lines and cultivars used in this study

Table 2 Responses of spinach cultivars and lines to inoculation with CMV-Y

CMV inoculation

The yellow strain of CMV (CMV-Y) was used in this study [14,15,16]. Nicotiana benthamiana leaves harboring CMV-Y were ground in 0.1 M phosphate buffer to produce leaf sap. Leaves of 4-week-old spinach plants at the 5–6 leaf stage were dusted with carborundum powder (600 mesh) (Nacalai Tesque, INC., Japan) and rub-inoculated with leaf sap. Plants mock-inoculated with phosphate buffer were used as negative controls. The viral titre was standardized across all inoculation tests to ensure consistent infection conditions.

RT-PCR analysis of CMV infections

Total cellular RNA was extracted from leaf tissues using the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). cDNA was synthesized by reverse transcription using GoScript™ Reverse Transcription Mix and random primers (Promega, Madison, USA). To confirm CMV infection in plants, a 285-bp cDNA fragment, reverse transcribed from CMV RNA3 [16], was amplified using GoTaq^® DNA Polymerase (Promega, Madison, USA) and the primers 5ʹ-TCCTCCCTCCGATCTCTGTGG-3ʹ and 5ʹ- TGGTCTCCTTTTGGAGGCCC-3ʹ, followed by 2% agarose gel electrophoresis. The spinach GAPDH (GenBank ID, NM_001426410) cDNA was amplified using the primers 5ʹ-TGTGTGCGACATCCCCCTCGTGTCAG-3ʹ and 5ʹ-CCACCCGTTGGCTGTAACCCCACTCAT-3ʹ as a positive control in the RT-PCR analysis.

Results & discussion

CMV-Y inoculation of spinach lines and cultivars

The response to CMV-Y was tested in 5–20 individual spinach plants from three cultivars and five lines (Tables 1 and 2). Plants from lines 03-336, 03-258, and 03-263 exhibited symptoms of infection. Inoculated leaves developed chlorotic spots (known as infection spots) at 6–8 days post-inoculation (dpi), and the chlorosis spread gradually across the leaves; Fig. 1a–c shows representative images from 03 to 336. By 8 dpi, the top leaf of susceptible plants became yellowish and chlorotic from its base (Fig. 1a, b). Furthermore, CMV-Y was detected in the top leaves of symptomatic plants at 5 and 10 dpi (Fig. 1d). These results suggested that lines 03-336, 03-258, and 03-263 were susceptible to CMV-Y and a systemic infection had been established. By contrast, the three cultivars and NIL-M neither exhibited symptoms of CMV-Y infection nor developed the lesions typical of the HR, although they showed minor physical damage caused by rub-inoculation (Table 2; Fig. 1a–c). Line 03–009 contained both susceptible and resistant plants (Fig. 1c). RT-PCR analysis did not detect cDNA from CMV-Y in NIL-M (Fig. 1d) or the cultivars following inoculation, indicating these lines were resistant to CMV-Y infection.

Fig. 1

Representative images of plants inoculated with CMV-Y and of RT-PCR analyses to detect CMV-Y infections. (a-b) Plants from NIL-M and line 03-336 at 8 days post-inoculation (dpi). (c) Plants from NIL-M, line 03-336, and line 03–009 at 15 dpi. (d) RT-PCR analysis of CMV-Y infections in NIL-M and line 03-336. (e) F₁ plants from line 03-336 and NIL-M at 11 dpi. Arrows indicate physical damage caused by rub-inoculation. (f) RT-PCR analysis of CMV-Y infections in F₁ plants. (g) F₂ progeny derived from plants shown in (e) at 14 dpi. (h) RT-PCR analysis of CMV-Y infections in F₂ progeny derived from plants shown in (e). GAPDH was amplified as a positive control in RT-PCR analyses (d, f, h). R: resistant (asymptomatic); S: susceptible (symptomatic); M: mock-inoculation control; I: inoculated

To establish a model for studying CMV resistance in spinach, line 03-336 and NIL-M were selected as representative susceptible and resistant lines. Unlike the cultivars, these lines result from many generations of self-fertilization and thus each is a homogeneous population suitable for genetic study. Examination of over 100 plants from each line confirmed that 03-336 and NIL-M were true-breeding for CMV susceptibility and resistance, respectively (Table 2). Line 03–009, a dioecious line produced by sib-crossing over many generations that contains both susceptible and resistant individuals, was also subjected to further analysis. Examination of 64 plants from line 03–009 revealed they segregated for susceptibility and resistance in a 1:1 ratio (Table 2). This line will be particularly useful in identifying the gene(s) responsible for resistance since it showed clear segregation for the trait but has high genetic homogeneity.

Genetic characterization of CMV resistance in lines NIL-M and 03–009

The F₁ and F₂ progeny resulting from a cross between the CMV-resistant line NIL-M and the CMV-susceptible line 03-336 were examined to determine the genetic nature of CMV resistance in spinach. Following CMV-Y inoculation, 12 F₁ plants showed no lesions or other symptoms of infection, although some exhibited physical damage from rub-inoculation, and no CMV-Y was detected (Table 2; Fig. 1e–f). This suggested that dominant factors were responsible for CMV resistance in spinach.

Next, 180 F₂ progeny were inoculated with CMV-Y and their resistance determined. This F₂ population contained 41 symptomatic and 139 asymptomatic plants (Table 2; Fig. 1g). To confirm that the presence and absence of symptoms judged by visual observation properly represented susceptibility and resistance, the top leaves of randomly selected individual F₂ plants, as well as the parental lines (NIL-M and 03-336), were sampled at 14 dpi and subjected to an RT-PCR assay designed to amplify a fragment of cDNA produced from viral RNA3. This fragment was detected in line 03-336 (the susceptible parent) and in symptomatic F₂ plants, but not in NIL-M (the resistant parent) or in asymptomatic F₂ plants (Fig. 1h). Such a result indicated that F₂ plants with symptoms were susceptible, and developed systemic infections with CMV-Y, while F₂ plants without symptoms were resistant. The numbers observed in the F₂ population (139 resistant and 41 susceptible plants) were consistent with a segregation ratio of 3:1, which would be expected if a dominant allele at a single locus controlled resistance (Table 2). Given that NIL-M was derived from a cross between lines 03-336 and 03–009 [13], it is reasonable to assume that the resistance shown by NIL-M originated in dioecious line 03–009, which was observed to segregate into resistant and susceptible individuals (Table 2).

To confirm whether resistance to CMV in line 03–009 was also regulated by a single factor, the responses to CMV-Y inoculation were monitored in five sib-crossed progeny populations (#10, #11, #13, #14, and #16) derived from 03 to 009 (Table 2). Populations #11 and #13 consisted only of susceptible plants, thus these populations were likely to result from crosses between two homozygous parents. By contrast, ~ 90 individual plants from populations #10 and #16 inoculated with CMV-Y segregated into resistant and susceptible individuals in a 3:1 ratio. This result suggested that populations #10 and #16 each resulted from a cross between two heterozygous parents, lending further support to the assumption that resistance was determined by a single locus. Finally, of 30 plants from population #14 inoculated with CMV-Y, 16 displayed susceptibility and the other 14 showed resistance, which was consistent with a 1:1 segregation ratio (Table 2). This result can again be explained by assuming that a single pair of dominant and recessive alleles determined resistance, and would be expected if one parent was homozygous and the other heterozygous. Taken together, these results indicated that CMV resistance in NIL-M and 03–009 shared a common origin and was controlled by a single gene. Hereafter, this gene is referred to as SRCm1 (Spinach Resistance to CMV 1).

SRCm1 resistance was found to be dominantly inherited, similar to the resistance identified by Pound and Cheo [4] in the Virginia Savoy variety. However, our preliminary tests did not indicate the same temperature sensitivity, as Virginia Savoy’s resistance deteriorates at 28 °C. To confirm SRCm1’s stability under different environmental conditions and CMV strains, further studies involving fine mapping and allelism tests are required to determine whether SRCm1 represents a new locus or shares a gene with Virginia Savoy. In this study, HR was not observed in either the traditional blight-resistant cultivars or in NIL-M, which carries SRCm1. Based on these observations, the resistance mechanisms appear similar, but further analysis is necessary to confirm if they share the same gene or allele as NIL-M.

Efficient identification of CMV resistance genes in spinach relies on genomic sequence data and well-characterized plant materials. Recently, high-quality pseudomolecules for the six chromosomes of line 03–009 (97.0% BUSCO completeness) became available. NIL-M shares a nearly identical genetic background with line 03–009, and whole genome sequencing reads for line 03-336 (DRX401202) are also accessible [13]. Our preliminary mapping suggests that SRCm1 resides in a ~ 3-Mb region in the distal portion of chromosome 1, where only genes encoding NLR proteins were identified as potential candidates. Given that SRCm1 likely encodes an NLR protein and HR was absent in NIL-M and 03–009, the resistance may result from extreme resistance (ER). However, dominant resistance conferred by non-NLR genes, such as Tm-1 or JAX1 [17, 18], which also do not induce HR, could theoretically explain the resistance. While our data suggest that SRCm1 likely encodes an NLR protein, the possibility of it being a non-NLR gene conferring dominant resistance cannot be ruled out. Further experimental verification is needed to confirm this hypothesis. Additionally, since SRCm1 exhibits dominant inheritance, resistance due to mutations in essential host factors (e.g., translation initiation factors), which typically confer recessive resistance without HR, is unlikely [19]. As resistant plants did not exhibit HR, unlike Arabidopsis ecotype C24, these three lines offer insight into a novel mechanism of CMV resistance. Such knowledge will improve the design of spinach breeding programs aimed at developing resistance against CMV.

Limitations

Because spinach virus diseases are usually not accompanied by severe, distinct disease symptoms, there have not been many studies worldwide that have targeted virus disease resistance factors for spinach breeding. Despite the mild symptoms, it is crucial to breed for virus disease resistance to enhance spinach quality. Seed companies often protect spinach varieties and breeding lines with valuable genotypes, such as disease resistance. As a result, there are not many opportunities to obtain useful information on CMV resistance factors from past papers and commercially available spinach seeds. Therefore, it may be up to our own future efforts to determine how practical the SRCm1 gene, we have discovered here, is for breeding.