- Email: [email protected]
High-throughput Sequencing Reveals vsiRNAs Derived from Cucumber green mottle mosaic virus-infected Watermelon
Accepted Manuscript Title: High-Throughput Sequencing Reveals vsiRNAs Derived From Cucumber Green Mottle Mosaic Virus-Infected Watermelon Author: SUN Yuyan, NIU Xiaowei, CUI Di, FAN Min PII: DOI: Reference:
S2468-0141(17)30146-2 http://dx.doi.org/doi: 10.1016/j.hpj.2017.03.002 HPJ 57
To appear in:
Horticultural Plant Journal
Received date: Revised date: Accepted date:
8-10-2016 20-1-2017 21-3-2017
Please cite this article as: SUN Yuyan, NIU Xiaowei, CUI Di, FAN Min, High-Throughput Sequencing Reveals vsiRNAs Derived From Cucumber Green Mottle Mosaic Virus-Infected Watermelon, Horticultural Plant Journal (2017), http://dx.doi.org/doi: 10.1016/j.hpj.2017.03.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
High-throughput Sequencing Reveals vsiRNAs Derived from Cucumber green mottle mosaic virus-infected Watermelon SUN Yuyan, NIU Xiaowei, CUI Di, and FAN Min* Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China Received 8 October 2016; Received in revised form 20 January 2017; Accepted 21 March 2017
* Corresponding author.
1
E-mail address: [email protected]
2468-0141 ©2017 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)
Abstract Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus, and is a serious pathogen of Cucurbitaceae crops. Virus-derived small interfering RNAs (vsiRNAs), which are processed by Dicer-like and Argonaute proteins as well as RNA-dependent RNA polymerase, mediate the silencing of viral genomic RNA and host transcripts. To identify the CGMMV derived vsiRNAs and reveal interactions between CGMMV and watermelon host plant, deep sequencing technology was used to identify and characterize the vsiRNAs derived from CGMMV in infected watermelon plants in present study. A total of 10 801 368 vsiRNA reads representing 71 583 unique sRNAs were predicted in CGMMV-inoculated watermelon plants. The CGMMV vsiRNAs were mostly 21 or 22 nt long. The majority of the CGMMV vsiRNAs (i.e., 91.7%) originated from the viral sense strand. Additionally, uracil was the predominant 5′-terminal base of vsiRNAs. Furthermore, the putative targets and functions of some of the CGMMV vsiRNAs were predicted and investigated. The results enhance our understanding of the interaction between CGMMV and the host watermelon and provide molecular basis for CGMMV resistance improvement in watermelon and other Cucurbitaceae crops. Keywords: Citrullus lanatus; Cucumber green mottle mosaic virus; high-throughput sequencing; vsiRNAs
1. Introduction Watermelon (Citrullus lanatus L.) is a member of the family Cucurbitaceae, which includes several other important crops, such as cucumber, melon, pumpkin, and bottle gourd. Watermelon fruits contain several important nutrients for humans, including vitamins, minerals, carbohydrates, and dietary fiber (Perkins-Veazie et al., 2006; Collins et al., 2007). Approximately 97 million tons of watermelon are produced worldwide every year, with China being the leading producer (FAO, 2013). However, the Cucumber green mottle mosaic virus (CGMMV) is a major cucurbit pathogen belonging to the genus Tobamovirus, and has been responsible for considerable watermelon yield losses (Tesoriero et al., 2016). Infected plants exhibit severe mosaic symptoms with discoloration and deformation (Kim et al., 2010). CGMMV consists of a single-stranded, positive-sense RNA genome within a 300 nm × 18 nm viral particle (Li et al., 2016). Furthermore, the CGMMV genome encodes 4 proteins, namely the 186 kD read-through protein, 129 kD RNA-dependent polymerase, 29 kD movement protein, and 17.3 kD coat protein (Kim et al., 2004; Li et al., 2015).
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Virus-induced gene silencing is a common antiviral strategy used by plants. Previous studies revealed that virus-derived small RNA (sRNA) fragments accumulate in infected host plants (Yan et al., 2010; Li et al., 2016), indicating that the host RNA-silencing machinery targets viral RNA (Hamilton and Baulcombe, 1999). The production of virus-derived small interfering RNAs (vsiRNAs) is the most significant characteristic of virus-induced gene silencing (Hamilton and Baulcombe, 1999; Donaire et al., 2008). Viral RNA can be processed into specifically-sized vsiRNAs by distinct Dicer-like (DCL) proteins, including DCL1–4, which process siRNAs that are 21–24 nt long (Jiang et al., 2012; Zhu and Guo, 2012). Other key components, including the Argonaute (AGO) protein and RNA-dependent RNA polymerase, are also required for vsiRNA biogenesis (Jiang et al., 2012; Zhu and Guo, 2012). The vsiRNAs are then recruited to host RNA-induced silencing complex which target and inhibit viral gene expression and protein translation. High-throughput sequencing of vsiRNAs has revealed the origin and potential role of vsiRNAs in virus–host interactions (Yan et al., 2010; Visser et al., 2014; Li et al., 2016). For instance, deep sequencing of sRNAs from Rice stripe virus (RSV) -infected rice leaves revealed that siRNAs were derived almost equally from virion and the complementary RNA strands, and the length of siRNAs were mostly 20–22 nt long (Yan et al., 2010). A next-generation sequencing approach identified siRNAs associated with Apple stem grooving virus (ASGV) infections in apple plants (Visser et al., 2014). Furthermore, the vsiRNA profiles associated with an ASGV genetic variant and the involvement of tRNA-derived sRNAs in plant–virus interactions have also been described (Visser et al., 2014). Moreover, the CGMMV-derived vsiRNAs and their potential targets were identified in cucumber plants (Li et al., 2016). Thus, in this study, we identified and characterized the CGMMV vsiRNAs from infected watermelon plants using deep sequencing technology. The length and origin of vsiRNAs, as well as their targets and KEGG pathway were analyzed. The results of this study increase our understanding of the interaction between CGMMV and the host watermelon and provide molecular basis for CGMMV resistance improvement in watermelon and other Cucurbitaceae crops.
2. Materials and methods 2.1. Plant material inoculation and sample collection Seeds of watermelon advanced inbred line ‘JJZ-M’ were planted in plastic pots considering soil, vermiculite and organic fertilizer incubated in a greenhouse of Zhejiang Academy of Agricultural Sciences at December of 2015. At the two true-leaf stage, plants were mechanically inoculated. The virus inoculum was prepared by grinding virus-infected bottle gourd leaves in sodium phosphate buffer (1:5, w/v). Control plants were mock-inoculated with sodium phosphate buffer. Leaf samples used for deep sequencing were harvested at 25 days post inoculation. The presence of CGMMV was confirmed based on the appearance of mosaic symptoms and the results of a reverse transcription polymerase chain reaction analysis. Leaf samples from 3 plants were combined in equal amounts. The collected samples were stored at −80 °C.
2.2. Total RNA isolation and library construction Total RNA was extracted from each sample using RNAiso reagent (TaKaRa, Japan). The quantity and quality of RNA were assessed using a spectrophotometer and by agarose gel electrophoresis. The sRNA libraries were then constructed. First, total RNA was isolated and purified by polyacrylamide gel electrophoresis. Using T4 RNA ligase, sRNAs (i.e., 18–30 nt) were ligated to a pair of adapters at their 5′- and 3′-ends. The ligated products were selected by size-fractionation and purified from gels. The adapter-ligated fractions were then amplified using a primer pair specific for the adapters to produce the sequencing libraries. The purified amplicons were directly sequenced by 1GENE Technology (Hangzhou, China) using an Illumina HiSeq 4 000 system. Sequencing data were deposited at NCBI-SRA under the accession numbers of SRR3318267 for CGMMV-infected sample and SRR3318270 for mock-inoculated sample.
2.3. CGMMV vsiRNA identification and bioinformatic analyses 2
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After removing the adapter sequences and low-quality reads, the remaining 18–30 nt sRNAs were aligned with the CGMMV genome sequence using SOAP2 software (Li et al., 2009). Reads with no mismatches with the CGMMV reference sequence were considered as valid vsiRNAs. The vsiRNAs underwent further bioinformatic analyses, including an assessment of vsiRNA length distributions, biases of 5′-terminal base, polarity distributions, and genome coverage.
2.4. Target gene predictions for CGMMV vsiRNAs and functional analyses Putative targets of vsiRNAs with > 200 reads were identified using RNAhybrid software (Rehmsmeier et al., 2004) and the watermelon genome as a reference (Guo et al., 2013). The following criteria were used to identify viable vsiRNA/target duplexes: no more than four mismatches between the vsiRNA and target sequences (G–U bases were considered 0.5 mismatches); no more than two adjacent mismatches in the vsiRNA/target duplex; no adjacent mismatches in positions 2–12 of the vsiRNA/target duplex (5′ end of vsiRNA); no mismatches in positions 10 or 11 of the vsiRNA/target duplex; no more than 2.5 mismatches in positions 1–12 of the vsiRNA/target duplex (5′ end of miRNA); minimum free energy of the vsiRNA/target duplex ≥ 74% of the vsiRNA bound to its perfect complement; and energy threshold ≤ −29. The target genes were functionally annotated and categorized using Blast2GO software with default parameters (Götz et al., 2008). Furthermore, the target genes were used as queries to search the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for gene mapping to KEGG reference metabolic pathways (Kanehisa and Goto, 2008).
3. Results 3.1. Symptoms of watermelon plants inoculated with CGMMV At 25 days post inoculation, characteristic symptoms of a CGMMV infection (e.g., appearance of mosaic patterns on leaves, shriveling of veins, and slight growth inhibition) were observed in virus-inoculated plants, while the mock-inoculated plants exhibited no disease symptoms (Fig. 1).
3.2. Identification of CGMMV vsiRNAs A total of 34 325 694 and 28 831 544 sRNAs representing 28 132 773 and 26 131 989 clean reads were obtained from the CGMMV-infected and mock-inoculated watermelon plants, respectively. In the CGMMV-infected library, there were significantly more 21- and 22-nt reads, but considerably fewer 24-nt reads compared with the mock-inoculated library (Fig. 2, A). The CGMMV siRNAs were predominantly 21- and 22-nt long, accounting for 37.33 % and 43.55 % of the total reads, respectively (Fig. 2, B). Moreover, 10 801 368 vsiRNA reads representing 71 583 unique sRNAs were predicted for the CGMMV-inoculated watermelon plants, accounting for 38.39% of the clean reads. In contrast, only 290 reads representing 270 unique sRNAs were identified as CGMMV vsiRNAs in the mock-inoculated library, accounting for only 0.001% of the total clean reads.
3.3. Analysis of CGMMV vsiRNA polarity distribution and genome coverage According to the results of the SOAP2 analysis, the majority of CGMMV siRNAs were derived from the sense strand (91.70%) 3
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of the viral RNA. Additionally, CGMMV siRNAs almost covered the whole virus genome (Fig. 3). Two hotspots were detected at positions 600–1 000 bp and 5 400–5 700 bp of the CGMMV genome (Fig. 3).
3.4. 5′-terminal nucleotides of siRNAs Previous studies concluded that the 5′-terminal nucleotides of siRNAs are important for directing the siRNAs to specific AGO complexes (Hutvagner and Simard, 2008; Mi et al., 2008). The CGMMV siRNAs with a uracil (U) at the 5′ terminal were the most abundant, accounting for 38.95% of the total vsiRNAs, followed by siRNAs with cytosine (C; 25.45%), adenine (A; 24.28%), and guanine (G; 11.32%) bases at the 5′ terminal (Fig. 4). Uracil was the most common 5′-terminal nucleotide for 21-, 22-, 23-, and 24-nt CGMMV siRNAs (Fig. 4).
3.5. Target gene predictions for CGMMV vsiRNAs Bioinformatic analyses used to predict the potential host transcripts for vsiRNAs with > 200 reads identified 326 target genes. These genes encoded a variety of transcription factors and functional proteins, including a zinc finger, MYB, squamosa promoter-binding-like protein 8, WD40, bHLH, auxin response factor, ethylene-responsive transcription factor, eIF-4, cytochrome P450, nucleotide binding site–leucine-rich repeats, IAA-amino acid hydrolase, cinnamyl alcohol dehydrogenase 9, lipoxygenase, and cellulose synthase. The predicted targets affect diverse biological processes, such as metabolic processes, cellular processes, localization, single-organism processes, biological regulatory activities, and responses to stimuli. The targets of the CGMMV-derived siRNAs were used to search the KEGG database (Fig. 5). The predominant pathways associated with the CGMMV targets included ribosome biogenesis in eukaryotes (ko03008), RNA transport (ko03013), RNA degradation (ko03018), peptidoglycan biosynthesis (ko00550), nicotinate and nicotinamide metabolism (ko00760), folate biosynthesis (ko00790), ethylbenzene degradation (ko00642), PPAR signaling pathway (ko03320), and carbon fixation in photosynthetic organisms (ko00710). These findings suggested that the vsiRNAs may be important for CGMMV infections because of interactions with their targets.
4. Discussion Our data indicated that 21- and 22-nt vsiRNAs were highly abundant in CGMMV-infected watermelon plants, accounting for 37.33% and 43.55% of all vsiRNAs, respectively. These results suggest that DCL4 and DCL2 may be the predominant DCL ribonucleases influencing watermelon CGMMV vsiRNA biogenesis (Garcia-Ruiz et al., 2010). The DCL4 protein is the primary ribonuclease responsible for processing 21-nt vsiRNAs, while DCL2 produces 22-nt vsiRNAs (Jiang et al., 2012). Arabidopsis thaliana plants infected with Turnip crinkle virus (TCV) only accumulate DCL2-dependent 22-nt vsiRNAs (Bouche et al., 2006). In contrast, plants infected with the TCV ΔP38 mutant (i.e., lacks the viral suppressor P38) contain only DCL4-dependent 21-nt 4
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vsiRNAs (Fusaro et al., 2006). In the present study, CGMMV siRNAs with a U at the 5′ terminal were the most abundant, accounting for 38.95% of all vsiRNAs. This observation is consistent with the results of a study conducted by Qi et al. (2009) who reported that vsiRNAs more commonly have an A or U as the initiation base than a G or C. Additionally, A. thaliana AGOs exhibit a bias toward the 5′-terminal nucleotides when recruiting endogenous siRNAs (Takeda et al., 2008). Two AGOs (i.e., AGO1 and AGO2) select vsiRNAs based on sequence length and the identity of the 5′-terminal nucleotide. The AGO1 and AGO2 proteins preferentially bind to 21-nt vsiRNAs with a U or A as their 5′-terminal nucleotide, respectively (Mi et al., 2008). We observed a clear preference for U as the 5′-terminal nucleotide, indicating that CGMMV vsiRNAs have a high binding affinity for the AGO1 homolog of watermelon. A considerable majority (i.e., 91.70%) of the CGMMV siRNAs in infected watermelon plants were derived from the viral genome sense strand. This is similar to the distribution of siRNAs from Cymbidium ringspot virus (CRV) and Tobacco rattle virus (TRV) (Molnár et al., 2005; Donaire et al., 2008). Molnár et al. (2005) observed that the production of vsiRNAs exhibited a strong bias toward the sense strand. The vsiRNAs from the TRV, which belongs to the same family as CGMMV, were reported to be derived predominantly from positive-strand RNA fragments (Donaire et al., 2008). However, our results differed from another study that determined that in CGMMV-infected cucumber plants, only 54.62% of the vsiRNAs were derived from the sense strand (Li et al., 2016). The mechanism underlying these contradictory results remains to be elucidated. Although many transcripts that are potentially targeted by vsiRNAs have been detected based on bioinformatic analyses (Qi et al., 2009; Li et al., 2016), only a few studies have verified the targeting of host genes during vsiRNA-mediated RNA silencing. For example, a siRNA derived from the Y satellite of the Cucumber mosaic virus (CMV) targets the host CHL1 gene, leading to a dramatic down-regulation of CHL1 expression (Wang et al., 2011). Additionally, the transformation of tobacco plants with a CHL1-targeting RNAi vector resulted in Y satellite-like symptoms (Wang et al., 2011). Shimura et al. (2011) revealed the molecular mechanism regulating the yellowing symptom induced by the Y satellite. In the present study, we predicted the potential transcripts for CGMMV vsiRNAs. These predicted targets encode many transcriptional factors and functional enzymes involved in a broad range of KEGG pathways, such as those related to ribosome biogenesis in eukaryotes, RNA transport, RNA degradation, peptidoglycan biosynthesis, folate biosynthesis, and PPAR signaling. Additional studies on viruses and vsiRNAs may lead to a better understanding of how hosts and viruses interact with each other to ensure co-evolutionary survival.
Acknowledgments This work was granted from Project funded by China Postdoctoral Science Foundation (2016M601973), the National Natural Science Foundation of China (31572145 and 31272188), Zhejiang Major Agricultural Science and Technology Projects for New Varieties Breeding (2016C02051-4-2).
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Fig. 1
Symptoms of watermelon leaves infected with CGMMV
Fig. 2
Size distribution of total sRNAs in mock-inoculated and CGMMV-infected libraries (A) and vsiRNAs in the CGMMV-infected library (B)
Fig. 3
Genome coverage of vsiRNAs (21–24 nt) in CGMMV-inoculated watermelon plants
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Fig. 4
Relative frequency of nucleotides at the 5′-terminal of vsiRNAs
Fig. 5
Top 20 KEGG pathways associated with targets of CGMMV vsiRNAs
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S2468-0141(17)30146-2 http://dx.doi.org/doi: 10.1016/j.hpj.2017.03.002 HPJ 57
To appear in:
Horticultural Plant Journal
Received date: Revised date: Accepted date:
8-10-2016 20-1-2017 21-3-2017
Please cite this article as: SUN Yuyan, NIU Xiaowei, CUI Di, FAN Min, High-Throughput Sequencing Reveals vsiRNAs Derived From Cucumber Green Mottle Mosaic Virus-Infected Watermelon, Horticultural Plant Journal (2017), http://dx.doi.org/doi: 10.1016/j.hpj.2017.03.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
High-throughput Sequencing Reveals vsiRNAs Derived from Cucumber green mottle mosaic virus-infected Watermelon SUN Yuyan, NIU Xiaowei, CUI Di, and FAN Min* Institute of Vegetables, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China Received 8 October 2016; Received in revised form 20 January 2017; Accepted 21 March 2017
* Corresponding author.
1
E-mail address: [email protected]
2468-0141 ©2017 Chinese Society for Horticultural Science (CSHS) and Institute of Vegetables and Flowers (IVF), Chinese Academy of Agricultural Sciences (CAAS)
Abstract Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus, and is a serious pathogen of Cucurbitaceae crops. Virus-derived small interfering RNAs (vsiRNAs), which are processed by Dicer-like and Argonaute proteins as well as RNA-dependent RNA polymerase, mediate the silencing of viral genomic RNA and host transcripts. To identify the CGMMV derived vsiRNAs and reveal interactions between CGMMV and watermelon host plant, deep sequencing technology was used to identify and characterize the vsiRNAs derived from CGMMV in infected watermelon plants in present study. A total of 10 801 368 vsiRNA reads representing 71 583 unique sRNAs were predicted in CGMMV-inoculated watermelon plants. The CGMMV vsiRNAs were mostly 21 or 22 nt long. The majority of the CGMMV vsiRNAs (i.e., 91.7%) originated from the viral sense strand. Additionally, uracil was the predominant 5′-terminal base of vsiRNAs. Furthermore, the putative targets and functions of some of the CGMMV vsiRNAs were predicted and investigated. The results enhance our understanding of the interaction between CGMMV and the host watermelon and provide molecular basis for CGMMV resistance improvement in watermelon and other Cucurbitaceae crops. Keywords: Citrullus lanatus; Cucumber green mottle mosaic virus; high-throughput sequencing; vsiRNAs
1. Introduction Watermelon (Citrullus lanatus L.) is a member of the family Cucurbitaceae, which includes several other important crops, such as cucumber, melon, pumpkin, and bottle gourd. Watermelon fruits contain several important nutrients for humans, including vitamins, minerals, carbohydrates, and dietary fiber (Perkins-Veazie et al., 2006; Collins et al., 2007). Approximately 97 million tons of watermelon are produced worldwide every year, with China being the leading producer (FAO, 2013). However, the Cucumber green mottle mosaic virus (CGMMV) is a major cucurbit pathogen belonging to the genus Tobamovirus, and has been responsible for considerable watermelon yield losses (Tesoriero et al., 2016). Infected plants exhibit severe mosaic symptoms with discoloration and deformation (Kim et al., 2010). CGMMV consists of a single-stranded, positive-sense RNA genome within a 300 nm × 18 nm viral particle (Li et al., 2016). Furthermore, the CGMMV genome encodes 4 proteins, namely the 186 kD read-through protein, 129 kD RNA-dependent polymerase, 29 kD movement protein, and 17.3 kD coat protein (Kim et al., 2004; Li et al., 2015).
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Virus-induced gene silencing is a common antiviral strategy used by plants. Previous studies revealed that virus-derived small RNA (sRNA) fragments accumulate in infected host plants (Yan et al., 2010; Li et al., 2016), indicating that the host RNA-silencing machinery targets viral RNA (Hamilton and Baulcombe, 1999). The production of virus-derived small interfering RNAs (vsiRNAs) is the most significant characteristic of virus-induced gene silencing (Hamilton and Baulcombe, 1999; Donaire et al., 2008). Viral RNA can be processed into specifically-sized vsiRNAs by distinct Dicer-like (DCL) proteins, including DCL1–4, which process siRNAs that are 21–24 nt long (Jiang et al., 2012; Zhu and Guo, 2012). Other key components, including the Argonaute (AGO) protein and RNA-dependent RNA polymerase, are also required for vsiRNA biogenesis (Jiang et al., 2012; Zhu and Guo, 2012). The vsiRNAs are then recruited to host RNA-induced silencing complex which target and inhibit viral gene expression and protein translation. High-throughput sequencing of vsiRNAs has revealed the origin and potential role of vsiRNAs in virus–host interactions (Yan et al., 2010; Visser et al., 2014; Li et al., 2016). For instance, deep sequencing of sRNAs from Rice stripe virus (RSV) -infected rice leaves revealed that siRNAs were derived almost equally from virion and the complementary RNA strands, and the length of siRNAs were mostly 20–22 nt long (Yan et al., 2010). A next-generation sequencing approach identified siRNAs associated with Apple stem grooving virus (ASGV) infections in apple plants (Visser et al., 2014). Furthermore, the vsiRNA profiles associated with an ASGV genetic variant and the involvement of tRNA-derived sRNAs in plant–virus interactions have also been described (Visser et al., 2014). Moreover, the CGMMV-derived vsiRNAs and their potential targets were identified in cucumber plants (Li et al., 2016). Thus, in this study, we identified and characterized the CGMMV vsiRNAs from infected watermelon plants using deep sequencing technology. The length and origin of vsiRNAs, as well as their targets and KEGG pathway were analyzed. The results of this study increase our understanding of the interaction between CGMMV and the host watermelon and provide molecular basis for CGMMV resistance improvement in watermelon and other Cucurbitaceae crops.
2. Materials and methods 2.1. Plant material inoculation and sample collection Seeds of watermelon advanced inbred line ‘JJZ-M’ were planted in plastic pots considering soil, vermiculite and organic fertilizer incubated in a greenhouse of Zhejiang Academy of Agricultural Sciences at December of 2015. At the two true-leaf stage, plants were mechanically inoculated. The virus inoculum was prepared by grinding virus-infected bottle gourd leaves in sodium phosphate buffer (1:5, w/v). Control plants were mock-inoculated with sodium phosphate buffer. Leaf samples used for deep sequencing were harvested at 25 days post inoculation. The presence of CGMMV was confirmed based on the appearance of mosaic symptoms and the results of a reverse transcription polymerase chain reaction analysis. Leaf samples from 3 plants were combined in equal amounts. The collected samples were stored at −80 °C.
2.2. Total RNA isolation and library construction Total RNA was extracted from each sample using RNAiso reagent (TaKaRa, Japan). The quantity and quality of RNA were assessed using a spectrophotometer and by agarose gel electrophoresis. The sRNA libraries were then constructed. First, total RNA was isolated and purified by polyacrylamide gel electrophoresis. Using T4 RNA ligase, sRNAs (i.e., 18–30 nt) were ligated to a pair of adapters at their 5′- and 3′-ends. The ligated products were selected by size-fractionation and purified from gels. The adapter-ligated fractions were then amplified using a primer pair specific for the adapters to produce the sequencing libraries. The purified amplicons were directly sequenced by 1GENE Technology (Hangzhou, China) using an Illumina HiSeq 4 000 system. Sequencing data were deposited at NCBI-SRA under the accession numbers of SRR3318267 for CGMMV-infected sample and SRR3318270 for mock-inoculated sample.
2.3. CGMMV vsiRNA identification and bioinformatic analyses 2
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After removing the adapter sequences and low-quality reads, the remaining 18–30 nt sRNAs were aligned with the CGMMV genome sequence using SOAP2 software (Li et al., 2009). Reads with no mismatches with the CGMMV reference sequence were considered as valid vsiRNAs. The vsiRNAs underwent further bioinformatic analyses, including an assessment of vsiRNA length distributions, biases of 5′-terminal base, polarity distributions, and genome coverage.
2.4. Target gene predictions for CGMMV vsiRNAs and functional analyses Putative targets of vsiRNAs with > 200 reads were identified using RNAhybrid software (Rehmsmeier et al., 2004) and the watermelon genome as a reference (Guo et al., 2013). The following criteria were used to identify viable vsiRNA/target duplexes: no more than four mismatches between the vsiRNA and target sequences (G–U bases were considered 0.5 mismatches); no more than two adjacent mismatches in the vsiRNA/target duplex; no adjacent mismatches in positions 2–12 of the vsiRNA/target duplex (5′ end of vsiRNA); no mismatches in positions 10 or 11 of the vsiRNA/target duplex; no more than 2.5 mismatches in positions 1–12 of the vsiRNA/target duplex (5′ end of miRNA); minimum free energy of the vsiRNA/target duplex ≥ 74% of the vsiRNA bound to its perfect complement; and energy threshold ≤ −29. The target genes were functionally annotated and categorized using Blast2GO software with default parameters (Götz et al., 2008). Furthermore, the target genes were used as queries to search the Kyoto Encyclopedia of Genes and Genomes (KEGG) database for gene mapping to KEGG reference metabolic pathways (Kanehisa and Goto, 2008).
3. Results 3.1. Symptoms of watermelon plants inoculated with CGMMV At 25 days post inoculation, characteristic symptoms of a CGMMV infection (e.g., appearance of mosaic patterns on leaves, shriveling of veins, and slight growth inhibition) were observed in virus-inoculated plants, while the mock-inoculated plants exhibited no disease symptoms (Fig. 1).
3.2. Identification of CGMMV vsiRNAs A total of 34 325 694 and 28 831 544 sRNAs representing 28 132 773 and 26 131 989 clean reads were obtained from the CGMMV-infected and mock-inoculated watermelon plants, respectively. In the CGMMV-infected library, there were significantly more 21- and 22-nt reads, but considerably fewer 24-nt reads compared with the mock-inoculated library (Fig. 2, A). The CGMMV siRNAs were predominantly 21- and 22-nt long, accounting for 37.33 % and 43.55 % of the total reads, respectively (Fig. 2, B). Moreover, 10 801 368 vsiRNA reads representing 71 583 unique sRNAs were predicted for the CGMMV-inoculated watermelon plants, accounting for 38.39% of the clean reads. In contrast, only 290 reads representing 270 unique sRNAs were identified as CGMMV vsiRNAs in the mock-inoculated library, accounting for only 0.001% of the total clean reads.
3.3. Analysis of CGMMV vsiRNA polarity distribution and genome coverage According to the results of the SOAP2 analysis, the majority of CGMMV siRNAs were derived from the sense strand (91.70%) 3
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of the viral RNA. Additionally, CGMMV siRNAs almost covered the whole virus genome (Fig. 3). Two hotspots were detected at positions 600–1 000 bp and 5 400–5 700 bp of the CGMMV genome (Fig. 3).
3.4. 5′-terminal nucleotides of siRNAs Previous studies concluded that the 5′-terminal nucleotides of siRNAs are important for directing the siRNAs to specific AGO complexes (Hutvagner and Simard, 2008; Mi et al., 2008). The CGMMV siRNAs with a uracil (U) at the 5′ terminal were the most abundant, accounting for 38.95% of the total vsiRNAs, followed by siRNAs with cytosine (C; 25.45%), adenine (A; 24.28%), and guanine (G; 11.32%) bases at the 5′ terminal (Fig. 4). Uracil was the most common 5′-terminal nucleotide for 21-, 22-, 23-, and 24-nt CGMMV siRNAs (Fig. 4).
3.5. Target gene predictions for CGMMV vsiRNAs Bioinformatic analyses used to predict the potential host transcripts for vsiRNAs with > 200 reads identified 326 target genes. These genes encoded a variety of transcription factors and functional proteins, including a zinc finger, MYB, squamosa promoter-binding-like protein 8, WD40, bHLH, auxin response factor, ethylene-responsive transcription factor, eIF-4, cytochrome P450, nucleotide binding site–leucine-rich repeats, IAA-amino acid hydrolase, cinnamyl alcohol dehydrogenase 9, lipoxygenase, and cellulose synthase. The predicted targets affect diverse biological processes, such as metabolic processes, cellular processes, localization, single-organism processes, biological regulatory activities, and responses to stimuli. The targets of the CGMMV-derived siRNAs were used to search the KEGG database (Fig. 5). The predominant pathways associated with the CGMMV targets included ribosome biogenesis in eukaryotes (ko03008), RNA transport (ko03013), RNA degradation (ko03018), peptidoglycan biosynthesis (ko00550), nicotinate and nicotinamide metabolism (ko00760), folate biosynthesis (ko00790), ethylbenzene degradation (ko00642), PPAR signaling pathway (ko03320), and carbon fixation in photosynthetic organisms (ko00710). These findings suggested that the vsiRNAs may be important for CGMMV infections because of interactions with their targets.
4. Discussion Our data indicated that 21- and 22-nt vsiRNAs were highly abundant in CGMMV-infected watermelon plants, accounting for 37.33% and 43.55% of all vsiRNAs, respectively. These results suggest that DCL4 and DCL2 may be the predominant DCL ribonucleases influencing watermelon CGMMV vsiRNA biogenesis (Garcia-Ruiz et al., 2010). The DCL4 protein is the primary ribonuclease responsible for processing 21-nt vsiRNAs, while DCL2 produces 22-nt vsiRNAs (Jiang et al., 2012). Arabidopsis thaliana plants infected with Turnip crinkle virus (TCV) only accumulate DCL2-dependent 22-nt vsiRNAs (Bouche et al., 2006). In contrast, plants infected with the TCV ΔP38 mutant (i.e., lacks the viral suppressor P38) contain only DCL4-dependent 21-nt 4
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vsiRNAs (Fusaro et al., 2006). In the present study, CGMMV siRNAs with a U at the 5′ terminal were the most abundant, accounting for 38.95% of all vsiRNAs. This observation is consistent with the results of a study conducted by Qi et al. (2009) who reported that vsiRNAs more commonly have an A or U as the initiation base than a G or C. Additionally, A. thaliana AGOs exhibit a bias toward the 5′-terminal nucleotides when recruiting endogenous siRNAs (Takeda et al., 2008). Two AGOs (i.e., AGO1 and AGO2) select vsiRNAs based on sequence length and the identity of the 5′-terminal nucleotide. The AGO1 and AGO2 proteins preferentially bind to 21-nt vsiRNAs with a U or A as their 5′-terminal nucleotide, respectively (Mi et al., 2008). We observed a clear preference for U as the 5′-terminal nucleotide, indicating that CGMMV vsiRNAs have a high binding affinity for the AGO1 homolog of watermelon. A considerable majority (i.e., 91.70%) of the CGMMV siRNAs in infected watermelon plants were derived from the viral genome sense strand. This is similar to the distribution of siRNAs from Cymbidium ringspot virus (CRV) and Tobacco rattle virus (TRV) (Molnár et al., 2005; Donaire et al., 2008). Molnár et al. (2005) observed that the production of vsiRNAs exhibited a strong bias toward the sense strand. The vsiRNAs from the TRV, which belongs to the same family as CGMMV, were reported to be derived predominantly from positive-strand RNA fragments (Donaire et al., 2008). However, our results differed from another study that determined that in CGMMV-infected cucumber plants, only 54.62% of the vsiRNAs were derived from the sense strand (Li et al., 2016). The mechanism underlying these contradictory results remains to be elucidated. Although many transcripts that are potentially targeted by vsiRNAs have been detected based on bioinformatic analyses (Qi et al., 2009; Li et al., 2016), only a few studies have verified the targeting of host genes during vsiRNA-mediated RNA silencing. For example, a siRNA derived from the Y satellite of the Cucumber mosaic virus (CMV) targets the host CHL1 gene, leading to a dramatic down-regulation of CHL1 expression (Wang et al., 2011). Additionally, the transformation of tobacco plants with a CHL1-targeting RNAi vector resulted in Y satellite-like symptoms (Wang et al., 2011). Shimura et al. (2011) revealed the molecular mechanism regulating the yellowing symptom induced by the Y satellite. In the present study, we predicted the potential transcripts for CGMMV vsiRNAs. These predicted targets encode many transcriptional factors and functional enzymes involved in a broad range of KEGG pathways, such as those related to ribosome biogenesis in eukaryotes, RNA transport, RNA degradation, peptidoglycan biosynthesis, folate biosynthesis, and PPAR signaling. Additional studies on viruses and vsiRNAs may lead to a better understanding of how hosts and viruses interact with each other to ensure co-evolutionary survival.
Acknowledgments This work was granted from Project funded by China Postdoctoral Science Foundation (2016M601973), the National Natural Science Foundation of China (31572145 and 31272188), Zhejiang Major Agricultural Science and Technology Projects for New Varieties Breeding (2016C02051-4-2).
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Fig. 1
Symptoms of watermelon leaves infected with CGMMV
Fig. 2
Size distribution of total sRNAs in mock-inoculated and CGMMV-infected libraries (A) and vsiRNAs in the CGMMV-infected library (B)
Fig. 3
Genome coverage of vsiRNAs (21–24 nt) in CGMMV-inoculated watermelon plants
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Fig. 4
Relative frequency of nucleotides at the 5′-terminal of vsiRNAs
Fig. 5
Top 20 KEGG pathways associated with targets of CGMMV vsiRNAs
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