- Email: [email protected]
Corrigendum to “Complexity and recombination analysis of novel begomovirus associated with Spinach yellow vein disease in India”. [Plant Gene 13 (2018) 42–49]
Plant Gene 17 (2019) 100170
Contents lists available at ScienceDirect
Plant Gene journal homepage: www.elsevier.com/locate/plantgene
Corrigendum
Corrigendum to “Complexity and recombination analysis of novel begomovirus associated with Spinach yellow vein disease in India”. [Plant Gene 13 (2018) 42–49]☆ Anurag Kumar Sahua, Rakesh Kumar Vermab, R.K. Gaurb, Neeti Sanan-Mishraa, a b
T
⁎
International Centre for Genetic Engineering & Biotechnology, New Delhi 110067, India Mody University of Science & Technology, Laxmangarh, Rajasthan 332311, India
A B S T R A C T
Spinach (Spinacia oleracea) leaves with vein yellowing were observed in Rajasthan province of India. The plants were severely stunted in growth and exhibited symptoms typical of begomovirus infection. Analysis of collected samples indicated that the infected plants contained a novel begomovirus (showing < 91% nucleotide sequence similarity with the known viruses) and its associated betasatellite. Comprehensive analysis on the complexity and recombination suggested that it was probably derived from Papaya leaf curl virus and may have evolved to infect Spinach in absence of the main host. The nucleotide diversity (π) and nucleotide substitution rates for the DNA-A of SYVSV are 0.10382 and 2.436 × 10–3 substitutions site-1 year-1, respectively. Thus mutation and recombination seem to be the driving forces for the emergence and evolution of new begomoviruses. This suggests the existence of a complex recombination pattern to form a “gene pool” of the crop-infecting virus–betasatellite complexes and strengthens the hypothesis for increase of host range of begomovirus, thus raising concern about its spread to other crops.
1. Introduction The Geminiviridae family contains virus that exhibit characteristic twinned geminate particle morphology with circular, single-stranded DNA (ssDNA) genomes (Cui et al., 2004; Cui et al., 2005). Based upon their host range, insect vectors and genome organization, they are classified into seven genera viz. Begomovirus, Becurtovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus (Varsani et al., 2014). The genus Begomovirus includes virus with bipartite genome, where the genes are resident on two different circular ssDNA molecules (DNA-A and DNA-B), of about 2.6 to 2.8 kb each (Brown et al., 2012) or monopartite genome with all genes resident on one (DNA-A-like) ssDNA of about 2.8 kb. The begomoviruses are notorious pathogens of crops that are transmitted by whiteflies (Bemisia tabaci), throughout the tropical and subtropical regions of the world (Markham et al., 1994; Hanley-Bowdoin et al., 1999). Majority of the monopartite begomoviruses are associated with betasatellites, which affect the replication of their respective virus and may alter the symptoms in host plants. Betasatellites typically contain a Satellite Conserved Region (SCR), an adenine-rich region and a βC1
open reading frame (ORF). The βC1 ORF has been characterized as a pathogenicity determinant that suppresses host antiviral silencing and promotes virus movement in plants (Cui et al., 2005; Yang et al., 2011; Li et al., 2014). The diversity and host range of the betasatellite and the associated viruses are increasing (Leke et al., 2011; Leke et al., 2012). Recombination, during the replication, has been reported to play a major role in the emergence and evolution of begomoviruses (Padidam et al., 1999; Lefeuvre & Moriones, 2015). In addition nucleotide substitutions that occur during evolution also contribute towards genetic variation (Singh et al., 2012). Thus mutation, pseudo-recombination and recombination are driving forces for the emergence and evolution of new begomoviruses. This stresses the importance of recombination studies when analyzing new viruses. Spinach (Spinacia oleracea) is an important green-leafy vegetable of Amaranthaceae family that is cultivated extensively in India. It is often recognized as one of the functional foods for its nutritional values like high soluble dietary fiber and antioxidants as lutein, zea-xanthin. It is thus recommended in cholesterol controlling and weight reduction programs. S. oleracea is also infected by members of the begomovirus family. In the present investigation symptomology, molecular biology
DOI of original article: https://doi.org/10.1016/j.plgene.2018.01.001 The authors regret that the above-mentioned publication contains incorrect Fig. 2a and inaccurate description at legends of Fig. 2 and Fig. 5. Sections of Table 1 and Table 5 also contain errors. Moreover at several places (including abstract) there are language related problems, which mislead the appropriate meaning. Hence, we are respectfully requesting to publish a corrigendum to replace the discrepancies with proper description. ⁎ Corresponding author at: Plant RNAi Biology Lab, International Centre for Genetic Engineering & Biotechnology, New Delhi 10067, India. E-mail address: [email protected] (N. Sanan-Mishra). ☆
https://doi.org/10.1016/j.plgene.2019.100170
Available online 03 January 2019 2352-4073/ © 2019 Elsevier B.V. All rights reserved.
Plant Gene 17 (2019) 100170
A.K. Sahu et al.
Fig. 2. Line diagram to represent (a) the genome of SYVV-[Sik]. The right and left orientations have been marked. Accordingly the iteron regions (IR) have been named as RIR (Right IR) and LIR (Left IR). The number of amino acids (aa) encoded by each ORF is also indicated. The color coding of strand indicate their orientation on the genome. Individual ORFs on the viral (+) strand have been marked in yellow color and those on the complementary (−) strand in blue color. (b) βsatellite consists of single ORF βC1 followed by an A-rich region and satellite conserved region (SCR). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
begomoviruses, using default parameters. From the results first 34 hits were used to perform the recombination and mutation analysis. Maximum-likelihood (ML) phylogenetic trees for each data set were generated by using PAUP* 4.0 (Swofford, 2003) with tree-bisectionrecombination branch-swapping in each case. The best-fit model of nucleotide substitution for each data set was determined using MODELTEST (Posada and Crandall, 1998) (Table 2). To assess the support for individual nodes on the phylogenies, a bootstrap analysis was performed utilizing 1000 replicate neighbor-joining trees under the ML substitution model in each case. 34 full-length DNA-A sequences were used to evaluate recombination in SYVSV genome using the recombination detection program (RDP v3.44), which is based on a pair wise scanning approach (http:// darwin.uvigo.es/rdp/rdp.html) that includes GENECONV, MaxChi, Chimaera, Bootscan and 3Seq with the Bonferronicorrected P value cutoff of 0.01 (Martin et al., 2010). The default detection thresholds were employed in each case.
and computational biology approaches were used to analyze the genetic diversity and distribution of a novel begomovirus, Spinach yellow vein Sikar virus (SYVSV) and its betasatellite component associated with leaf yellow vein disease of S. oleracea in India. 2. Methods 2.1. Isolation, cloning and sequencing of full-length genomes The infected samples were collected from vegetable fields in Rasidpura, Rajasthan province, India, Latitude: 27.29° E 80′ 41.94′ ′ and Longitude: 74.67°E 23′14.23′ ′. Nearly 10–15% of Spinach plants showed yellow veining and leaf yellowing. To investigate the type of infecting virus, total DNA was extracted from leaves of twenty infected plants using the cetyl trimethyl ammonium bromide (CTAB) method (Manen et al., 2005) and subjected to PCR using a pair of degenerate primers specific to the coat protein region of begomovirus using conditions described earlier (Bela-ong and Bajet, 2007). The forward primer sequence was GGRTTDGARGCATGHGTACATG (AC 1048) and the reverse primer sequence was GCCYATR TAYAGRAAGCCMAG (AV 494) (Bela-ong and Bajet, 2007). These primers have been used previously in screening of variety of begomoviruses such as Tomato leaf curl virus (JN009664), Croton yellow vein mosaic Hissar virus (JN000701), Radish leaf curl virus (JN998450), Chili leaf curl virus (JN000700), Cotton leaf curl virus (JQ693143), etc. PCR amplification of ~750 bp from highly conserved coat protein region confirmed the presence of begomovirus infection. To validate the monopartite/bipartite nature of the virus, the whole viral genome was amplified by Rolling Circle Amplification (RCA) using TempliPhi DNA amplification kit (GE Healthcare). The RCA products were digested with restriction endonucleases, viz. BamHI, KpnI, HindIII or SalI and resultant fragments of ~2.75 kb and ~1.3 kb in size (Fig. 1) were cloned into the pTZ57R/T vector (Fermentas) linearized with the respective restriction enzyme. The cloned fragments were rechecked and processed for sequencing in both directions. The consensus sequence was generated by overlapping both the results and aligning them using the available begomovirus sequences. Using the CLUSTAL W method in SDT v. 1.0 (Muhire et al., 2014) percentage pairwise identity of the cloned sequences with the representative sequences in the NCBI database were generated.
2.3. Infectivity assay To assess the infectivity of SYVSV, an agroinfectious clone of the DNA molecule of the begomovirus isolate was generated. RCA products were partially digested with HindIII and a linearized 5.6-kb band (Fig. 1c) was eluted. This fragment was ligated into pCAMBIA2301 vector. The resulting dimeric clone (pCASY-A) of SYVSV DNA-A and the head-to-tail tandem repeat clone (pCASY-β) of the betasatellite were prepared for use as the infectious clones. The two clones were mobilized separately into strain AGL1 of Agrobacterium tumefaciens by the freeze–thaw method (Holsters et al., 1978). Positive Agrobacterium colonies were used to obtain cultures with OD600 = 0.8. The cells were pelleted and resuspended in 2-Morpholino EthaneSulfonic acid (MES) buffer (pH 5.6). Twenty plants of N. benthamiana at the 2- to 4-leaf stage were agroinoculated three or four times using a needleless syringe. Different combinations of clones i.e., pCAMBIA, pCASY-A, pCASY-β and pCASY-A + pCASY-β were introduced to check the efficacy of inoculation. Following agroinoculation, plants were incubated in a growth chamber at 25 °C. The symptom induction was monitored till 25 dpi. 3. Results 3.1. Identification of novel begomovirus (SYVSV) and associated satellite
2.2. Phylogenetic and recombination analysis Positive PCR reaction using degenerate CP primers confirmed the presence of begomovirus infection in the Spinach samples (data not
The sequences obtained were subjected to NCBI Blast analysis with 2
Plant Gene 17 (2019) 100170
13.05
8.32
31.41
14.08
11.29
Papaya leaf curl virus (CAW31017) 99% Papaya leaf curl virus (CAD24673) 100% Papaya leaf curl virus (ACT82446) 78% Papaya leaf curl virus (CAD24676) 79% Papaya leaf curl virus (not specified) Papaya leaf curl virus (NP689462) 100% Papaya leaf curl betasatellite 15.47
28.23
Predicted highest amino acids identities (%) Predicted molecular weight (kDa)
119 557 201
75 2455 2228
294 2612 1728
134 1624 1220
102 1383 1075
256 1078 308
134 456 52
The DNA-A (GenBank Accession No KF660223) of 2753 nt contained six predicted open reading frames (ORFs) viz. AV1, AV2, AC1, AC2, AC3, and AC4. These ORFs are normally conserved among the begomoviral DNA-A genome and the same was observed for SYVSV (Table 1, Supplementary File 1). The SYVSV DNA-A sequences had 78–88% nucleotide sequence identity with other begomoviruses reported from India (GQ200446, GQ200448, KX302713, KM525657, JN807765, Y15934, HM143914, JN135233, JQ954859, FJ593629 and FN678906) and Pakistan (AJ436992, LT009399, FM955601, FM955602, LT009395, HE580234, JX524173, LN906593, LN906594, LT009397, LT009398, LN845913, LN845919, LT009400, LN845915, LN845917, LT009396 LN845914 and LN845916) (Fig. 3a). It was observed that majority of the reported sequences were begomoviruses associated with Papaya (Fig. 4a). The SYVSV DNA-A sequence was used in a phylogenetic analysis based on alignment with selected full-length DNA-A sequences of begomovirus isolates available in the NCBI database. The selection included majority of begomovirus species identified so far mostly in Asian subcontinent. ML Phylogenetic analysis for the full genome DNA-A sequences of Spinach begomovirus and other selected complete DNA-A sequences indicated that Sikar isolate clusters with the isolates of Papaya leaf curl virus from Pakistan. In addition to main genome (DNA-A), the associated β-satellite, of 1367 nt (GenBank Accession No. KF425298) was also identified. ML Phylogenetic analysis based on complete DNA-β sequences demonstrates that it clusters with Papaya leaf curl betasatellite (HM101173) from India (Fig. 3b). Further nucleotide sequence comparison with other betasatellites showed 77–94% nt sequence identity to other Indian isolates which include betasatellite of Papaya leaf curl and Tomato leaf curl (Fig. 4b). Maximum composite likelihood estimates the pattern of nucleotide substitution and the analysis showed that the rates of different transitional substitutions varied from 10.64 to 15.81 and transversional substitutions varied from 4.94 to 7.35 and the overall transition/ transversion bias is R = 1.05. Sequence comparison of DNA-A and betasatellite with the analyzed sequences revealed average evolutionary divergence was 0.082 ± 0.002 and 0.034 ± 0.006, respectively. Therefore, the existence of novel identified and previously reported begomoviruses clearly indicated a high degree of diversity.
Complement strand
Complement strand
Complement strand
AC2
AC1
AC4
C1 Symptoms
Complement strand
Complement strand AC3
Replication Enhancer protein Transcriptional activator protein Replication associated protein C4 protein
3.3. Detection of recombination among Spinach Yellow Vein Disease associated begomoviruses
DNA-β
Sense strand AV1 Coat protein
DNA-A
Sense strand AV2 Pre-coat protein
Third frame (+) Second frame (+) First frame (+) Third frame (−) Second frame (−) Third frame (−) Second frame (−)
Predicted size (no. of amino acid) Stop codon (nucleotide coordinates) Start codon (nucleotide coordinates) Frame ORFs
Strand
shown). Rolling circle amplification (RCA) using ɸ29 polymerase yielded DNA fragments of ~2.75 kb and ~1.3 kb size. These two fragments were independently cloned (Fig. 1) in to pTZ57R/T vector (Fermentas) and sequenced. NCBI Blast analysis of the resulting 2753 nt sequence (GenBank Accession No. KF660223) indicated that it showed < 89% similarity to the DNA-A component of the begomoviral genomes. It contained six predicted open reading frames (ORFs AV1, AV2, AC1, AC2, AC3, and AC4) (Fig. 2a, Supplementary File 1). Based on the DNA-A sequence comparison and the ICTV species demarcation at 91% nucleotide sequence identity (Brown et al., 2015) the virus isolate identified in this study was categorized as a novel begomovirus species and named as Spinach yellow vein Sikar virus (SYVSV). The 1367 nt sequence (GenBank Accession No. KF425298) showed similarity to DNA-β. It contained an adenine (A)-rich region, a satelliteconserved region (SCR) and an ORF βC1 located on the complementary sense strand encoding a protein of 119 amino acids (Fig. 2b). Further nucleotide sequence comparison indicated that this betasatellite had 77–94% nt sequence identity with other Indian betasatellite isolates (Table 1). According to the demarcation criterion, it emerged as an isolate of Papaya leaf curl virus betasatellite. 3.2. Diversity and phylogenetic analysis of SYVSV
Description Components
Table 1 Positions and coding capacity of predicted genes for the genome of begomovirus and its satellite molecules isolated from Spinach, and their highest amino acid sequence identities.
A.K. Sahu et al.
Recombination is known to play a major role in the emergence and 3
Plant Gene 17 (2019) 100170
A.K. Sahu et al.
Table 5 Infectivity of cloned viral components on N. benthamiana. Host plant
Viral construct
Number of symptomatic plants/number of inoculated plants
Incubation period (Days)*
Type of symptoms on inoculated plants
PCR DNA-A (CP) β-satellite
Nicotiana benthamiana
pCAMBIA pCASY-A pCASY-A+ pCASY- β
20/20 20/20 20/20
25 25 25
− LC LC
− + +
− − +
(0.035 ± 0.006) as compared to dS (0.017 ± 0.006), concomitantly the DNA-A genome showed higher dS (0.127 ± 0.009) than dN (0.104 ± 0.005). SYVSV and its betasatellite showed high degree of genetic variability (π > 0.08) at 0.10382, 0.033, respectively (Table 4). It is known that although recombination is the major but not the only driving force for emergence and evolution of new begomoviruses, the nucleotide substitutions occurring during evolution also contribute to genetic variation.
evolution of geminiviruses (George et al., 2015) which is most likely to increase genetic variation. To determine whether the begomoviruses identified in this study show evidence of recombination, RDP4 analysis was conducted based on alignments with full-length sequences of selected begomoviruses available in the database and results are presented in Table 2. It was observed that isolates from Papaya [IN:Moh01] (KX302713), Papaya [IN:Lko-01] (KM525657) and Papaya [PK:Bha] (LT009395) were overall very similar to SYVSV. The next set of similarity was with the isolates of Croton yellow vein mosaic virus (CYVMV) [IN:Del-01] (FJ592936) and CYMV [PK:Lah:01] (HE580234). PepLCLV, which was identified from capsicum spp., showed recombination pattern similar to SYVSV. In contrast recombination pattern shown by CheTLCV, which was identified from cherry tomato spp., Pak Lahore, was different (Table 2). Overall the analysis showed that SYVSV had highly recombinant origin, which was distinct from that of other begomoviruses. Phylogenetic tree of DNA-A showed that the recombinant sequences of SYVSV clustered with their respective contributors, which included mainly papaya and croton. Thus, recombination analysis for the SYVSV clones indicated that the virus probably infected alternate host in absence of its main host. This may have resulted in enhanced recombination patterns causing the emergence of the new isolates. In addition it was observed that most of the Spinach yellow vein disease associated begomovirus contain both interspecies and obvious intraspecies recombination events (Table 2).
3.5. Discussion (Para 5) The SYVSV isolates have high mutation rates, just like other begomoviruses (Duffy and Holmes, 2009; Kumar et al., 2010; Saleem et al., 2016). The data set was determined to be under statistically significant negative selection: (−) 1.3023, with no sites under positive selection (P < .05), indicating that most fixations were likely to be neutral. The analysis revealed that the mean substitution rate (Table 3) for βC1 gene were considerably higher (3.635 × 10−5) than those for complete DNA-A of SYVSV (2.436 × 10−3 subs site−1 year−1). The high nucleotide substitution rate for the satellite suggested that they were evolving rapidly. Betasatellites are important pathogenicity determinant for monopartite begomoviruses (Cui et al., 2004; Saunders et al., 2004; Li et al., 2014; Bhattacharyya et al., 2015) and may be responsible for induction of severe disease symptoms. Fig.5 Representative pictures to show symptoms induced on Nicotiana benthamiana by infectious constructs of Spinach yellow vein Sikar virus (SYVSV) at 25 dpi. The infectious clones were infiltrated on the abaxial side of tobacco leaves (a) Control (pCAMBIA) empty vector, (b) pCASY-A and (c) pCASY-A + pCASY-β. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Genetic structure and substitution rate of begomoviruses and betasatellites The mean rate of nucleotide substitution was determined using relaxed molecular clock model rather than strict molecular clock model. This estimated the mean substitution rate for DNA-A full genome at 2.436 × 10−3 substitution site-1 year-1 while the mean substitution rate in the βC1gene was higher at 3.635 × 10−5 substitution site-1 year-1 (Table 3). The high nucleotide substitution rate for betasatellite suggests that they are evolving rapidly. Nucleotide diversity at nonsynonymous (dN) and at synonymous positions (dS) were also estimated using Pamilo-Bianchi-Li method. βC1 gene exhibited higher dN
Acknowledgments AKS is thankful to Department of Biotechnology, Govt. of India for RA Fellowship. The authors thank Prof Rob Briddon for critically assessing the manuscript. All authors apologise for any inconvenience caused.
4
Contents lists available at ScienceDirect
Plant Gene journal homepage: www.elsevier.com/locate/plantgene
Corrigendum
Corrigendum to “Complexity and recombination analysis of novel begomovirus associated with Spinach yellow vein disease in India”. [Plant Gene 13 (2018) 42–49]☆ Anurag Kumar Sahua, Rakesh Kumar Vermab, R.K. Gaurb, Neeti Sanan-Mishraa, a b
T
⁎
International Centre for Genetic Engineering & Biotechnology, New Delhi 110067, India Mody University of Science & Technology, Laxmangarh, Rajasthan 332311, India
A B S T R A C T
Spinach (Spinacia oleracea) leaves with vein yellowing were observed in Rajasthan province of India. The plants were severely stunted in growth and exhibited symptoms typical of begomovirus infection. Analysis of collected samples indicated that the infected plants contained a novel begomovirus (showing < 91% nucleotide sequence similarity with the known viruses) and its associated betasatellite. Comprehensive analysis on the complexity and recombination suggested that it was probably derived from Papaya leaf curl virus and may have evolved to infect Spinach in absence of the main host. The nucleotide diversity (π) and nucleotide substitution rates for the DNA-A of SYVSV are 0.10382 and 2.436 × 10–3 substitutions site-1 year-1, respectively. Thus mutation and recombination seem to be the driving forces for the emergence and evolution of new begomoviruses. This suggests the existence of a complex recombination pattern to form a “gene pool” of the crop-infecting virus–betasatellite complexes and strengthens the hypothesis for increase of host range of begomovirus, thus raising concern about its spread to other crops.
1. Introduction The Geminiviridae family contains virus that exhibit characteristic twinned geminate particle morphology with circular, single-stranded DNA (ssDNA) genomes (Cui et al., 2004; Cui et al., 2005). Based upon their host range, insect vectors and genome organization, they are classified into seven genera viz. Begomovirus, Becurtovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus and Turncurtovirus (Varsani et al., 2014). The genus Begomovirus includes virus with bipartite genome, where the genes are resident on two different circular ssDNA molecules (DNA-A and DNA-B), of about 2.6 to 2.8 kb each (Brown et al., 2012) or monopartite genome with all genes resident on one (DNA-A-like) ssDNA of about 2.8 kb. The begomoviruses are notorious pathogens of crops that are transmitted by whiteflies (Bemisia tabaci), throughout the tropical and subtropical regions of the world (Markham et al., 1994; Hanley-Bowdoin et al., 1999). Majority of the monopartite begomoviruses are associated with betasatellites, which affect the replication of their respective virus and may alter the symptoms in host plants. Betasatellites typically contain a Satellite Conserved Region (SCR), an adenine-rich region and a βC1
open reading frame (ORF). The βC1 ORF has been characterized as a pathogenicity determinant that suppresses host antiviral silencing and promotes virus movement in plants (Cui et al., 2005; Yang et al., 2011; Li et al., 2014). The diversity and host range of the betasatellite and the associated viruses are increasing (Leke et al., 2011; Leke et al., 2012). Recombination, during the replication, has been reported to play a major role in the emergence and evolution of begomoviruses (Padidam et al., 1999; Lefeuvre & Moriones, 2015). In addition nucleotide substitutions that occur during evolution also contribute towards genetic variation (Singh et al., 2012). Thus mutation, pseudo-recombination and recombination are driving forces for the emergence and evolution of new begomoviruses. This stresses the importance of recombination studies when analyzing new viruses. Spinach (Spinacia oleracea) is an important green-leafy vegetable of Amaranthaceae family that is cultivated extensively in India. It is often recognized as one of the functional foods for its nutritional values like high soluble dietary fiber and antioxidants as lutein, zea-xanthin. It is thus recommended in cholesterol controlling and weight reduction programs. S. oleracea is also infected by members of the begomovirus family. In the present investigation symptomology, molecular biology
DOI of original article: https://doi.org/10.1016/j.plgene.2018.01.001 The authors regret that the above-mentioned publication contains incorrect Fig. 2a and inaccurate description at legends of Fig. 2 and Fig. 5. Sections of Table 1 and Table 5 also contain errors. Moreover at several places (including abstract) there are language related problems, which mislead the appropriate meaning. Hence, we are respectfully requesting to publish a corrigendum to replace the discrepancies with proper description. ⁎ Corresponding author at: Plant RNAi Biology Lab, International Centre for Genetic Engineering & Biotechnology, New Delhi 10067, India. E-mail address: [email protected] (N. Sanan-Mishra). ☆
https://doi.org/10.1016/j.plgene.2019.100170
Available online 03 January 2019 2352-4073/ © 2019 Elsevier B.V. All rights reserved.
Plant Gene 17 (2019) 100170
A.K. Sahu et al.
Fig. 2. Line diagram to represent (a) the genome of SYVV-[Sik]. The right and left orientations have been marked. Accordingly the iteron regions (IR) have been named as RIR (Right IR) and LIR (Left IR). The number of amino acids (aa) encoded by each ORF is also indicated. The color coding of strand indicate their orientation on the genome. Individual ORFs on the viral (+) strand have been marked in yellow color and those on the complementary (−) strand in blue color. (b) βsatellite consists of single ORF βC1 followed by an A-rich region and satellite conserved region (SCR). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
begomoviruses, using default parameters. From the results first 34 hits were used to perform the recombination and mutation analysis. Maximum-likelihood (ML) phylogenetic trees for each data set were generated by using PAUP* 4.0 (Swofford, 2003) with tree-bisectionrecombination branch-swapping in each case. The best-fit model of nucleotide substitution for each data set was determined using MODELTEST (Posada and Crandall, 1998) (Table 2). To assess the support for individual nodes on the phylogenies, a bootstrap analysis was performed utilizing 1000 replicate neighbor-joining trees under the ML substitution model in each case. 34 full-length DNA-A sequences were used to evaluate recombination in SYVSV genome using the recombination detection program (RDP v3.44), which is based on a pair wise scanning approach (http:// darwin.uvigo.es/rdp/rdp.html) that includes GENECONV, MaxChi, Chimaera, Bootscan and 3Seq with the Bonferronicorrected P value cutoff of 0.01 (Martin et al., 2010). The default detection thresholds were employed in each case.
and computational biology approaches were used to analyze the genetic diversity and distribution of a novel begomovirus, Spinach yellow vein Sikar virus (SYVSV) and its betasatellite component associated with leaf yellow vein disease of S. oleracea in India. 2. Methods 2.1. Isolation, cloning and sequencing of full-length genomes The infected samples were collected from vegetable fields in Rasidpura, Rajasthan province, India, Latitude: 27.29° E 80′ 41.94′ ′ and Longitude: 74.67°E 23′14.23′ ′. Nearly 10–15% of Spinach plants showed yellow veining and leaf yellowing. To investigate the type of infecting virus, total DNA was extracted from leaves of twenty infected plants using the cetyl trimethyl ammonium bromide (CTAB) method (Manen et al., 2005) and subjected to PCR using a pair of degenerate primers specific to the coat protein region of begomovirus using conditions described earlier (Bela-ong and Bajet, 2007). The forward primer sequence was GGRTTDGARGCATGHGTACATG (AC 1048) and the reverse primer sequence was GCCYATR TAYAGRAAGCCMAG (AV 494) (Bela-ong and Bajet, 2007). These primers have been used previously in screening of variety of begomoviruses such as Tomato leaf curl virus (JN009664), Croton yellow vein mosaic Hissar virus (JN000701), Radish leaf curl virus (JN998450), Chili leaf curl virus (JN000700), Cotton leaf curl virus (JQ693143), etc. PCR amplification of ~750 bp from highly conserved coat protein region confirmed the presence of begomovirus infection. To validate the monopartite/bipartite nature of the virus, the whole viral genome was amplified by Rolling Circle Amplification (RCA) using TempliPhi DNA amplification kit (GE Healthcare). The RCA products were digested with restriction endonucleases, viz. BamHI, KpnI, HindIII or SalI and resultant fragments of ~2.75 kb and ~1.3 kb in size (Fig. 1) were cloned into the pTZ57R/T vector (Fermentas) linearized with the respective restriction enzyme. The cloned fragments were rechecked and processed for sequencing in both directions. The consensus sequence was generated by overlapping both the results and aligning them using the available begomovirus sequences. Using the CLUSTAL W method in SDT v. 1.0 (Muhire et al., 2014) percentage pairwise identity of the cloned sequences with the representative sequences in the NCBI database were generated.
2.3. Infectivity assay To assess the infectivity of SYVSV, an agroinfectious clone of the DNA molecule of the begomovirus isolate was generated. RCA products were partially digested with HindIII and a linearized 5.6-kb band (Fig. 1c) was eluted. This fragment was ligated into pCAMBIA2301 vector. The resulting dimeric clone (pCASY-A) of SYVSV DNA-A and the head-to-tail tandem repeat clone (pCASY-β) of the betasatellite were prepared for use as the infectious clones. The two clones were mobilized separately into strain AGL1 of Agrobacterium tumefaciens by the freeze–thaw method (Holsters et al., 1978). Positive Agrobacterium colonies were used to obtain cultures with OD600 = 0.8. The cells were pelleted and resuspended in 2-Morpholino EthaneSulfonic acid (MES) buffer (pH 5.6). Twenty plants of N. benthamiana at the 2- to 4-leaf stage were agroinoculated three or four times using a needleless syringe. Different combinations of clones i.e., pCAMBIA, pCASY-A, pCASY-β and pCASY-A + pCASY-β were introduced to check the efficacy of inoculation. Following agroinoculation, plants were incubated in a growth chamber at 25 °C. The symptom induction was monitored till 25 dpi. 3. Results 3.1. Identification of novel begomovirus (SYVSV) and associated satellite
2.2. Phylogenetic and recombination analysis Positive PCR reaction using degenerate CP primers confirmed the presence of begomovirus infection in the Spinach samples (data not
The sequences obtained were subjected to NCBI Blast analysis with 2
Plant Gene 17 (2019) 100170
13.05
8.32
31.41
14.08
11.29
Papaya leaf curl virus (CAW31017) 99% Papaya leaf curl virus (CAD24673) 100% Papaya leaf curl virus (ACT82446) 78% Papaya leaf curl virus (CAD24676) 79% Papaya leaf curl virus (not specified) Papaya leaf curl virus (NP689462) 100% Papaya leaf curl betasatellite 15.47
28.23
Predicted highest amino acids identities (%) Predicted molecular weight (kDa)
119 557 201
75 2455 2228
294 2612 1728
134 1624 1220
102 1383 1075
256 1078 308
134 456 52
The DNA-A (GenBank Accession No KF660223) of 2753 nt contained six predicted open reading frames (ORFs) viz. AV1, AV2, AC1, AC2, AC3, and AC4. These ORFs are normally conserved among the begomoviral DNA-A genome and the same was observed for SYVSV (Table 1, Supplementary File 1). The SYVSV DNA-A sequences had 78–88% nucleotide sequence identity with other begomoviruses reported from India (GQ200446, GQ200448, KX302713, KM525657, JN807765, Y15934, HM143914, JN135233, JQ954859, FJ593629 and FN678906) and Pakistan (AJ436992, LT009399, FM955601, FM955602, LT009395, HE580234, JX524173, LN906593, LN906594, LT009397, LT009398, LN845913, LN845919, LT009400, LN845915, LN845917, LT009396 LN845914 and LN845916) (Fig. 3a). It was observed that majority of the reported sequences were begomoviruses associated with Papaya (Fig. 4a). The SYVSV DNA-A sequence was used in a phylogenetic analysis based on alignment with selected full-length DNA-A sequences of begomovirus isolates available in the NCBI database. The selection included majority of begomovirus species identified so far mostly in Asian subcontinent. ML Phylogenetic analysis for the full genome DNA-A sequences of Spinach begomovirus and other selected complete DNA-A sequences indicated that Sikar isolate clusters with the isolates of Papaya leaf curl virus from Pakistan. In addition to main genome (DNA-A), the associated β-satellite, of 1367 nt (GenBank Accession No. KF425298) was also identified. ML Phylogenetic analysis based on complete DNA-β sequences demonstrates that it clusters with Papaya leaf curl betasatellite (HM101173) from India (Fig. 3b). Further nucleotide sequence comparison with other betasatellites showed 77–94% nt sequence identity to other Indian isolates which include betasatellite of Papaya leaf curl and Tomato leaf curl (Fig. 4b). Maximum composite likelihood estimates the pattern of nucleotide substitution and the analysis showed that the rates of different transitional substitutions varied from 10.64 to 15.81 and transversional substitutions varied from 4.94 to 7.35 and the overall transition/ transversion bias is R = 1.05. Sequence comparison of DNA-A and betasatellite with the analyzed sequences revealed average evolutionary divergence was 0.082 ± 0.002 and 0.034 ± 0.006, respectively. Therefore, the existence of novel identified and previously reported begomoviruses clearly indicated a high degree of diversity.
Complement strand
Complement strand
Complement strand
AC2
AC1
AC4
C1 Symptoms
Complement strand
Complement strand AC3
Replication Enhancer protein Transcriptional activator protein Replication associated protein C4 protein
3.3. Detection of recombination among Spinach Yellow Vein Disease associated begomoviruses
DNA-β
Sense strand AV1 Coat protein
DNA-A
Sense strand AV2 Pre-coat protein
Third frame (+) Second frame (+) First frame (+) Third frame (−) Second frame (−) Third frame (−) Second frame (−)
Predicted size (no. of amino acid) Stop codon (nucleotide coordinates) Start codon (nucleotide coordinates) Frame ORFs
Strand
shown). Rolling circle amplification (RCA) using ɸ29 polymerase yielded DNA fragments of ~2.75 kb and ~1.3 kb size. These two fragments were independently cloned (Fig. 1) in to pTZ57R/T vector (Fermentas) and sequenced. NCBI Blast analysis of the resulting 2753 nt sequence (GenBank Accession No. KF660223) indicated that it showed < 89% similarity to the DNA-A component of the begomoviral genomes. It contained six predicted open reading frames (ORFs AV1, AV2, AC1, AC2, AC3, and AC4) (Fig. 2a, Supplementary File 1). Based on the DNA-A sequence comparison and the ICTV species demarcation at 91% nucleotide sequence identity (Brown et al., 2015) the virus isolate identified in this study was categorized as a novel begomovirus species and named as Spinach yellow vein Sikar virus (SYVSV). The 1367 nt sequence (GenBank Accession No. KF425298) showed similarity to DNA-β. It contained an adenine (A)-rich region, a satelliteconserved region (SCR) and an ORF βC1 located on the complementary sense strand encoding a protein of 119 amino acids (Fig. 2b). Further nucleotide sequence comparison indicated that this betasatellite had 77–94% nt sequence identity with other Indian betasatellite isolates (Table 1). According to the demarcation criterion, it emerged as an isolate of Papaya leaf curl virus betasatellite. 3.2. Diversity and phylogenetic analysis of SYVSV
Description Components
Table 1 Positions and coding capacity of predicted genes for the genome of begomovirus and its satellite molecules isolated from Spinach, and their highest amino acid sequence identities.
A.K. Sahu et al.
Recombination is known to play a major role in the emergence and 3
Plant Gene 17 (2019) 100170
A.K. Sahu et al.
Table 5 Infectivity of cloned viral components on N. benthamiana. Host plant
Viral construct
Number of symptomatic plants/number of inoculated plants
Incubation period (Days)*
Type of symptoms on inoculated plants
PCR DNA-A (CP) β-satellite
Nicotiana benthamiana
pCAMBIA pCASY-A pCASY-A+ pCASY- β
20/20 20/20 20/20
25 25 25
− LC LC
− + +
− − +
(0.035 ± 0.006) as compared to dS (0.017 ± 0.006), concomitantly the DNA-A genome showed higher dS (0.127 ± 0.009) than dN (0.104 ± 0.005). SYVSV and its betasatellite showed high degree of genetic variability (π > 0.08) at 0.10382, 0.033, respectively (Table 4). It is known that although recombination is the major but not the only driving force for emergence and evolution of new begomoviruses, the nucleotide substitutions occurring during evolution also contribute to genetic variation.
evolution of geminiviruses (George et al., 2015) which is most likely to increase genetic variation. To determine whether the begomoviruses identified in this study show evidence of recombination, RDP4 analysis was conducted based on alignments with full-length sequences of selected begomoviruses available in the database and results are presented in Table 2. It was observed that isolates from Papaya [IN:Moh01] (KX302713), Papaya [IN:Lko-01] (KM525657) and Papaya [PK:Bha] (LT009395) were overall very similar to SYVSV. The next set of similarity was with the isolates of Croton yellow vein mosaic virus (CYVMV) [IN:Del-01] (FJ592936) and CYMV [PK:Lah:01] (HE580234). PepLCLV, which was identified from capsicum spp., showed recombination pattern similar to SYVSV. In contrast recombination pattern shown by CheTLCV, which was identified from cherry tomato spp., Pak Lahore, was different (Table 2). Overall the analysis showed that SYVSV had highly recombinant origin, which was distinct from that of other begomoviruses. Phylogenetic tree of DNA-A showed that the recombinant sequences of SYVSV clustered with their respective contributors, which included mainly papaya and croton. Thus, recombination analysis for the SYVSV clones indicated that the virus probably infected alternate host in absence of its main host. This may have resulted in enhanced recombination patterns causing the emergence of the new isolates. In addition it was observed that most of the Spinach yellow vein disease associated begomovirus contain both interspecies and obvious intraspecies recombination events (Table 2).
3.5. Discussion (Para 5) The SYVSV isolates have high mutation rates, just like other begomoviruses (Duffy and Holmes, 2009; Kumar et al., 2010; Saleem et al., 2016). The data set was determined to be under statistically significant negative selection: (−) 1.3023, with no sites under positive selection (P < .05), indicating that most fixations were likely to be neutral. The analysis revealed that the mean substitution rate (Table 3) for βC1 gene were considerably higher (3.635 × 10−5) than those for complete DNA-A of SYVSV (2.436 × 10−3 subs site−1 year−1). The high nucleotide substitution rate for the satellite suggested that they were evolving rapidly. Betasatellites are important pathogenicity determinant for monopartite begomoviruses (Cui et al., 2004; Saunders et al., 2004; Li et al., 2014; Bhattacharyya et al., 2015) and may be responsible for induction of severe disease symptoms. Fig.5 Representative pictures to show symptoms induced on Nicotiana benthamiana by infectious constructs of Spinach yellow vein Sikar virus (SYVSV) at 25 dpi. The infectious clones were infiltrated on the abaxial side of tobacco leaves (a) Control (pCAMBIA) empty vector, (b) pCASY-A and (c) pCASY-A + pCASY-β. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Genetic structure and substitution rate of begomoviruses and betasatellites The mean rate of nucleotide substitution was determined using relaxed molecular clock model rather than strict molecular clock model. This estimated the mean substitution rate for DNA-A full genome at 2.436 × 10−3 substitution site-1 year-1 while the mean substitution rate in the βC1gene was higher at 3.635 × 10−5 substitution site-1 year-1 (Table 3). The high nucleotide substitution rate for betasatellite suggests that they are evolving rapidly. Nucleotide diversity at nonsynonymous (dN) and at synonymous positions (dS) were also estimated using Pamilo-Bianchi-Li method. βC1 gene exhibited higher dN
Acknowledgments AKS is thankful to Department of Biotechnology, Govt. of India for RA Fellowship. The authors thank Prof Rob Briddon for critically assessing the manuscript. All authors apologise for any inconvenience caused.
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