Background
Chrysanthemum (
Chrysanthemum morifolium) is among the most economically important floricultural crops in
Asteraceae worldwide [
1,
2]. Chrysanthemum flowers are widely used in ornamentation, food, and tea owing to their high ornamental or edible value [
3]. In China, chrysanthemum flowers have been used in traditional medicine considering their antioxidative, anti-inflammatory, antitumor, and hypolipidemic effects [
4,
5]. Chrysanthemum plants are susceptible to virus invasion during cultivation, especially when grown on a large scale [
6]. Virus-like symptoms such as foliar mosaic, stunting, and flower variegations, are commonly observed in chrysanthemum [
6,
7]. To date, more than 20 viruses and viroids are reported to infect chrysanthemum; in China, the most problematic of which are chrysanthemum virus B (CVB), chrysanthemum virus R (CVR), cucumber mosaic virus (CMV), tomato aspermy virus (TAV), tobacco mosaic virus (TMV), potato virus Y (PVY), chrysanthemum stunt viroid (CSVd), and chrysanthemum chlorotic mottle viroid (CChMVd) [
6,
8,
9].
CVB, which is transmitted by aphids and/or sap inoculation, is a major pathogen of chrysanthemum worldwide and causes symptoms ranging from leaf mottling to severe mosaic or flower malformations [
10,
11]. It is a single-stranded positive RNA virus that belongs to the genus
Carlavirus within the family
Betaflexiviridae, and has a filamentous particle that is approximately 680 × 12 nm in size [
11,
12]. The genome of CVB is approximately 9.0 kb, with a with a 5′-cap structure and a 3′-poly(A) tail, and encompasses six open reading frames (ORFs) [
11,
13]. ORF1 encodes a large protein implicated in viral replication, ORF2 to ORF4 encode triple gene block (TGB) proteins that are implicated in viral movement, ORF5 encodes a coat protein (CP), and ORF6 encodes a 12 kD cysteine-rich protein (CRP), whose zinc finger domain can bind single-stranded and double-stranded nucleotides in vitro [
14]. The heterologous expression of CRP in
Nicotiana tabacum cv. Samsun or
Nicotiana benthamiana seedlings using a potato virus X (PVX) vector could induce a hypersensitive response [
14,
15]. Moreover, CRP can also function as a plant transcription factor and regulates cell and proliferation size in tobacco species [
16]. Recently, CRP was demonstrated to be a symptom determinant [
17] and a viral suppressor of RNA silencing during viral infection [
18].
To date, 10 full-length genome sequences of CVB isolates from different regions and countries have been obtained. Of these, the full-length genome sequence of Japanese isolate (CVB-S) was the first to be obtained [
11]. Subsequently, an infectious cDNA clone of CVB-S, pCVB, was constructed; however, no visible symptoms were observed in chrysanthemum seedlings infiltrated with pCVB [
19]. In 2012, complete genome sequences of CVB-TN, CVB-PB, CVB-UP, and CVB-UK isolates from India were reported [
13]. Sequence analysis showed that all the four Indian isolates were similar to each other and had a genome organization quite similar to that of CVB-S, with which they shared 70–73% sequence identity at the genome level [
13]. More recently, using next-generation sequencing (NGS) technology, complete genome sequences of two Chinese isolates (CVB-HZ1 and CVB-HZ2) and three Russian isolates (CVB-GS1, CVB-GS2 and CVB-FY) were obtained from
Gynura japonica and chrysanthemum, respectively [
20,
21]. These genome sequences of CVB isolates from different hosts and countries are valuable and diverse materials for the study of pathogenicity mechanisms and functional genes of
Carlavirus.
In recent years, although substantial research on genome characterization and pathogenesis of CVB has been conducted, studies on the mechanisms underlying the response of host plants to CVB are scarce. In this study, an integrated NGS and comparative transcriptomic analysis of chrysanthemum leaves was carried out to elucidate the mechanisms underlying the response of plants against CVB-CN infection at the molecular level. These findings improve our understanding of the critical aspects of the molecular mechanisms underlying the defense responses of chrysanthemum to Carlavirus.
Discussion
In the past two decades, the NGS technology has been frequently used to assist in the discovery of new virus species [
71‐
74]. In this study, the NGS analysis of the
C. morifolium leaves demonstrated the presence of a virus related to the genus
Carlavirus within the family
Betaflexiviridae [
11,
13]. Furthermore, the complete viral genome, which is 9014 nt in length, was obtained using RT-PCR and 5′- and 3′-RACE-PCR (Fig.
1b). Based on the bioinformatics analysis of the genomic features and phylogeny, the CVB-CN was determined to be a Chinese isolate of CVB that was closer to CVB-GS1 (Fig.
1c). Subsequently, comparative transcriptomic analysis of leaves was carried out to explore the mechanisms underlying the response of chrysanthemum to CVB-CN.
In the past decade, the availability of transcriptome data from host plants in response to RNA viruses has dramatically expanded. For example, data sets from the leaves of
N. tabacum systemically infected with the M strain of CMV (M-CMV) with 8513 DEGs were generated by Lu et al. [
75]; from the leaves of
D. grandiflorum in response to CMV, tomato spotted wilt virus (TSWV), and PVX with 124 DEGs were produced by Choi et al. [
76]; from the leaves of
L. regale in response to CMV with 1346 DEGs were reported by Sun et al. [
64]; from the leaves of
P. vulgaris in response to soybean mosaic virus with 3548 DEGs were identified by Zhang et al. [
77]; and from stems of
C. annuum in response to three tobacco etch virus strains HAT, Mex21, and N having 24, 1190, and 4010 DEGs, respectively, were published by Murphy et al. [
78]. However, to the best of our knowledge, transcriptome data on the mechanisms underlying the response of chrysanthemum to CVB are still not available.
In this study, the RNA sequencing-based comparative transcriptomic analysis of CVB-CN-infected and control
C. morifolium leaves was carried out to further elucidate the mechanisms underlying the response of chrysanthemum to CVB-CN infection. A total of 4934 SDEGs, which contained 4097 upregulated and 837 downregulated genes, were screened in
C. morifolium leaves following CVB-CN infection using three biological replicates (Fig.
3). This result indicates that a large quantity of transcriptional changes occurred during CVB-CN infection. This finding corroborates with those of previous studies wherein it was observed that host plants extensively alter their gene expression in response to viral infections [
49,
75,
78]. GO enrichment analysis revealed that the CVB-CN responsive SDEGs were predominantly involved in the “Unsaturated fatty acid biosynthetic process”, “Secondary metabolite biosynthetic process”, “Transcription”, “Actin filament organization”, “Translation”, and “Regulation of transcription” in the biological process category (Fig.
4a,b), suggesting that these GO terms might be involved in the responses of chrysanthemum to CVB-CN. Among the top 25 enriched KEGG pathways, “Ribosome”, “Phenylpropanoid biosynthesis”, “Flavonoid biosynthesis”, and “Plant hormone signal transduction” were the four most significantly changed pathways in chrysanthemum in response to CVB-CN (Fig.
4c), indicating that the enhancement of these pathways might conduce to chrysanthemum responses and associated defense mechanisms to CVB-CN. These findings are in line with previous studies which showed that host metabolism, transcription, translation, and plant hormone signal transduction are frequently implicated in the defense responses of plants to viral infections [
49‐
52,
55,
76,
77].
Besides its roles in plant development, fruit ripening, and organ senescence, ethylene is also a crucial modulator of plant responses to abiotic and biotic stimuli, including viral infections [
65,
66,
79,
80]. Ethylene has been shown to modulate host defenses against viral infections in both positive and negative manners [
39,
66,
80,
81]. For example, in
N. tabacum, ethylene pathway has a crucial role in systemic resistance to Chilli veinal mottle virus (ChiVMV), and silencing of the ethylene biosynthetic and signaling components accelerate ChiVMV-induced cell death [
81]. More recently, it was shown that ethylene can regulate the
NbMYB4L gene to mediate resistance against TMV, and overexpression of
NbMYB4L induces significant resistance to TMV in
N. benthamiana [
66]. In contrast, Zhao et al. [
80] reported that ethylene plays a negative role in rice resistance to rice dwarf virus (RDV), and promotion of ethylene production results in elevated susceptibility to RDV. In
N. benthamiana, the ethylene pathway had been reported to mediate the susceptibility to turnip mosaic virus (TuMV) and exogenous application of the ethylene precursor, ACC, enhances TuMV accumulation in infected plants [
39]. Thus, ethylene has a complex role in the defenses of plants to viruses. In the present study, based on the KEGG database and previous studies on the model plant species
Arabidopsis, 20 SDEGs were directly mapped to the ethylene biosynthesis- and signaling pathway (Fig.
6d). Interestingly, 17 SDEGs of them, which covered not only the ethylene biosynthesis pathway but also the ethylene signaling pathway, were upregulated in
C. morifolium during infection with the CVB-CN (Fig.
6d and Additional file
12). It is important to notice that three
CTR1 genes were also upregulated following the CVB-CN infection (Fig.
6d and Additional file
12). We speculated that CTR1, a negative regulator of ethylene signaling, was were upregulated probably to reduce the damage caused by the excessive accumulation of virus-induced ethylene to normal plant growth and development, because that silencing of
CTR1 in tomato can enhance the upregulation of a set of defense-related genes and increase host resistance against tomato leaf curl virus infection [
82]. To further investigate how the ethylene pathway is involved in plant resistance to CVB-CN, we examined the precise role of this pathway in
N. benthamiana using VIGS and PVX-expressing CVB-CN CRP. Compared with the TRV:GFP-infiltrated plants,
N. benthamiana plants with silenced ethylene pathway components displayed more severe downward leaf curling and hypersensitive response and had higher accumulation of CRP mRNA (Fig.
7). These results suggest that the ethylene pathway plays an essential role in plant resistance to CVB-CN. This finding will increase our understanding of the role of ethylene in plant defense responses and its associated mechanisms.
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