Virus Research 178 (2013) 478–485
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Molecular characterization and pathogenicity of a carrot (Daucus carota) infecting begomovirus and associated betasatellite from India Jitendra Kumar a,∗,1 , Samatha Gunapati b,1 , Sudhir P. Singh a , Rekha Gadre b , Naresh C. Sharma c , Rakesh Tuli a a
National Agri-Food Biotechnology Institute, Mohali 160071, Punjab, India Department of Biochemistry, Devi Ahilya Vishwavidyalaya, Indore, India c Department of Biochemistry and Genetics, Barkatullah University, Bhopal, India b
a r t i c l e
i n f o
Article history: Received 2 June 2013 Received in revised form 8 October 2013 Accepted 12 October 2013 Available online 25 October 2013 Keywords: Geminivirus variants Synergism Virus host range Viral genome Silencing suppression
a b s t r a c t The yellow mosaic pattern and shortening of leaf petiole are common disease symptoms associated with begomovirus infection in carrot. DNA from field infected carrot leaves was analyzed by rolling circle amplification and sequencing. The results established the presence of ageratum enation virus (AEV), which is referred to here as ageratum enation virus-carrot (AEV-Car). Symptomatic ageratum (Ageratum conyzoides) plants, growing adjacent to the carrot fields, also showed the presence of AEV (AEV-Age). Ageratum yellow leaf curl betasatellite (AYLCB) was also detected in the AEV infected carrot and ageratum samples. AEV-Car and AEV-Age are 95–97% identical in their DNA sequences, represent groups of isolates from the respective plant hosts (carrot and ageratum). Agroinoculation using infectious clones of AEVCar plus AYLCB or AEV-Age plus AYLCB in carrot, ageratum, tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) produced yellow mosaic and curling symptoms in leaves of inoculated plants. Agroinoculation of the two isolates together, along with the betasatellite (AEV-Car plus AEV-Age plus AYLCB) resulted in the enhancement of symptoms in comparison to the plants inoculated with single isolate. Plants with more severe symptoms showed a higher level of viral DNA accumulation, suggesting synergistic interactions between the two isolates of AEV. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Carrot (Daucus carota) is one of the important vegetable crops in India, with an annual production of 350,000 metric tons (http://www.carrotmuseum.co.uk/statistics.html). Several RNA viruses namely alfalfa mosaic virus, carrot latent virus, carrot mottle virus, carrot red leaf virus, carrot yellow leaf virus, celery mosaic virus, curly top virus, carrot virus Y, carrot thin leaf potyvirus, carrot mottle dwarf virus and cucumber mosaic virus cause major yield loss worldwide (Murant et al., 1985; Latham and Jones, 2004; Howell and Mink, 1976; Stubbs, 1952; Afreen et al., 2009). Recently, a DNA virus, tomato leaf curl New Delhi virus (ToLCNDV; genus Begomovirus), was detected on carrot plants exhibiting yellow mosaic symptoms in several parts of India (Sivalingam et al., 2011). Begomoviruses (family Geminiviridae) are plant viruses with a single-stranded, circular DNA genome encapsidated in twinned
∗ Corresponding author. Tel.: +91 172 2290131; fax: +91 172 4604888. E-mail addresses: [email protected], jit [email protected] (J. Kumar). 1 These authors contributed equally to the article. 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.10.010
icosahedral particles (Brown et al., 2011). Begomoviruses are transmitted by whiteflies (Bemisia tabaci Genn.), and infect a wide range of plants. They have either bipartite (DNA-A and DNA-B) or monopartite (one component equivalent to DNA-A) genome, where each component accounts for approximately 2.6–2.8 kb size (Brown et al., 2011). The DNA-A component of bipartite begomoviruses encodes proteins required for replication, control of gene expression and encapsidation, whereas DNA-B encodes proteins required for movement of the virus. The genome of monopartite begomovirus is homologous to the DNA-A of bipartite begomovirus. It encodes the proteins for viral replication, gene expression, encapsidation and movement (Brown et al., 2011). The majority of monopartite begomoviruses in the Old World are associated with a class of ssDNA satellites known as betasatellites. Betasatellites are half the begomovirus genome in size and encode a single protein, called, C1. The protein C1 mediates up-regulation of viral DNA levels in plants by suppressing the anti-viral defense system of the host plant (Saunders et al., 2000; Briddon et al., 2001; Jose and Usha, 2003; Kumar et al., 2013). Mixed infection of host is common for plant viruses. Co-infecting viruses interact either synergistically or antagonistically (Mascia et al., 2010; Rentería-Canett et al., 2011; Syller, 2012). Synergistic interactions facilitate enhanced replication and accumulation of
J. Kumar et al. / Virus Research 178 (2013) 478–485
the participating viruses, resulting in symptom enhancement in the host plant (Murphy and Bowen, 2006; Rentería-Canett et al., 2011; Syller, 2012). In contrast, in antagonistic interaction, one or both of the viruses interfere with replication and symptom induction of the other virus or each other (Wintermantel et al., 2008; Power, 1996). Exchange of genetic material occurs often during mixed infection via homologous and non-homologous recombination. The recombination events depend on population size, frequency and distribution of the variants (viral quasispecies population) and host defense mechanism (Padidam et al., 1999; Froissart et al., 2005; Mubin et al., 2009). AEV is a monopartite begomovirus with associated betasatellite that helps in the accumulation of AEV and manifestation of disease symptoms (Saunders et al., 2000; Mubin et al., 2009,2010). Earlier reports on AEV infecting ageratum, Pointed Guard (Trichosanthes dioica), Cat’s Whiskers (Cleome gynandra) and Crassocephalum crepidioides (Kumar et al., 2011; Pandey et al., 2011; Raj et al., 2010, 2011) indicate wide host range of the virus. This work broadens the host range further, by reporting that the AEV infects carrots also. To our knowledge, this is the first detailed study on a begomovirus, infecting carrot and the synergistic interaction between the isolates from carrot (AEV-Car) and those from ageratum (AEV-Age), as well as their associated betasatellite.
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2.3. Sequence and phylogenetic analysis DNA sequence similarity searches were performed using BLAST (Altschul et al., 1990). Pairwise global sequence alignment was used to determine the nucleotide sequence identity between the begomoviruses and betasatellite identified in this study with other known sequences (Needleman and Wunsch, 1970). Phylogenetic trees were constructed using distance-based algorithm (NeighborJoining) with 1000 bootstrap replicates. The evolutionary distances were computed, using the maximum composite likelihood method in MEGA4 (Tamura et al., 2007). 2.4. Prediction of genomic recombinations The probable recombination fragments in virus and satellite genome were detected using recombination detection program version 3 (RDP 3) at default setting with a P-value cutoff of 0.05 (Martin et al., 2010). The modules, RDP, GENECONV, Bootscan, MaxChi, Chimera, SiScan and 3Seq were used to scan the recombination events and identify probable parental sequences and recombination breakpoints. 2.5. Testing of infectivity and co-infectivity
Symptomatic carrot plants, exhibiting yellow mosaic, mild leaf curling and reduced leaf lamina, were collected during 2009–2010 from the fields of Hajipur (latitude: 25◦ 41 North; longitude: 85◦ 13 East), Bihar and Mohali (latitude: 30◦ 47 North; longitude: 76◦ 41 East), Punjab (Fig. 1). Three carrot fields were selected from each location. A total of twelve carrot samples, two each from six carrot fields were collected. A total of twelve ageratum plants, showing leaf curling and yellow mosaic (Fig. 1) and growing adjacent to carrot field were collected from both the locations. Six asymptomatic plants each of carrot and ageratum were collected from the two locations. One asymptomatic carrot plant from each field was also collected. Genomic DNA was isolated from all the samples, using DNAeasy plant mini kit column (Qiagen GmbH, Germany).
Dimeric head-to-tail tandem repeats of begomovirus genomes (carrot, JF728867 and ageratum, JF728864) were produced by partial digestion of RCA products with BamHI restriction endonuclease. The resulting fragments were cloned into the binary vector pCAMBIA1301 (Cambia, Canberra, Australia). Dimeric infectious clone of betasatellite (JF728869) was made similarly in pCAMBIA1301, verified by sequencing and transformed into Agrobacterium tumefaciens GV3101. Healthy young plantlets (4–5 leaf stage) of Nicotiana tabacum, Solanum lycopersicum, Daucus carota and Ageratum conyzoides were agroinoculated with the infectious clones of (i) carrot isolate of begomovirus, (ii) ageratum isolate of begomovirus, (iii) carrot isolate of begomovirus plus the betasatellite, (iv) ageratum isolate of begomovirus plus the betasatellite, (v) carrot isolate of begomovirus plus ageratum isolate of begomovirus and (vi) carrot isolate of begomovirus plus ageratum isolate of begomovirus plus the betasatellite. A total of sixty plants were inoculated for each of the four species (N. tabacum, S. lycopersicum, D. carota and A. conyzoides). An A. tumefaciens strain containing empty pCAMBIA1301 was used as control. Agroinoculated plants were maintained at 25 ◦ C in 16 h light and 8 h dark.
2.2. Amplification of virus and satellite DNA
2.6. Detection and quantification of viral DNA in agroinoculated plants
The full-length begomoviral genome was amplified in sequence non-specific manner using genomic DNA as template and rolling circle amplification (Wu et al., 2008). Rolling circle amplification (RCA) was accomplished using templiPhi DNA amplification kit (GE Healthcare, USA). The RCA products were digested by HindIII restriction endonuclease and analyzed by gel electrophoresis. RCA products were also digested with several other restriction endonucleases (BamHI, KpnI, NdeI, NcoI, SalI and XbaI) to detect mixed infections with other begomoviruses and the presence of alpha- and betasatellites. The presence of both the satellites was investigated by PCR using specific primers (‘nanofor’/‘nanorev’ and 01/04; Kumar et al., 2010). A high fidelity PCR enzyme mix (Thermo Scientific, EU) was used in all PCR assays. The RCA restriction fragments and the PCR products expected to represent the virus and satellite genome were cloned in pDrive cloning vector (QIAGEN GmbH, Germany). Five clones from each sample were sequenced, using automated sequencer (3730xl DNA analyzer, Applied Biosystem, USA).
Genomic DNA was isolated from the systemically infected sixth leaf of the inoculated carrot, ageratum, tobacco and tomato plants. The AgeF/AgeR (Supplementary Table 1) and 01/04 (Kumar et al., 2010) primers were used in PCR assays for detection of the virus and betasatellite, respectively. The PCRs were carried out at 95 ◦ C for 4 min for denaturation, followed by 35 cycles of 95 ◦ C for 20 s, 50 ◦ C for 30 s, 72 ◦ C for 90 s, and final extension at 72 ◦ C for 5 min. For quantification of the viruses in agroinoculated plants, the concentration of DNA was adjusted to 50 ng/L in all the cases. AgeF/AgeR primers were used to quantify the viral DNA using semiquantitative PCR assay on the inoculated plants. Plant encoded actin gene was used as an internal control (Act.F/R; Supplementary Table 1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2013. 10.010. Southern hybridization was performed to investigate virus accumulation in tobacco plants (Southern, 1975). Five micrograms
2. Materials and methods 2.1. Virus source
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Fig. 1. Symptoms of AEV infection on Carrot and Ageratum. Healthy plants of carrot (A) and ageratum (C) showing green leaves with wide leaf lamina. Infected plants of carrot (B) showing yellow mosaic and mild leaf curling with reduced leaf lamina and plants of ageratum showing (D) only leaf curling and (E) yellow mosaic and severe leaf curling.
of genomic DNA from each sample were gel electrophoresed and transferred to Hybond-N+ Nylon membrane (Amersham Biosciences, USA), using SSC buffer. Coat protein gene of the AEV-Age was labeled with [alpha-32 P]-dCTP and used as probe. The hybridization signal was captured using phosphorimager (BioRad, CA). All procedures were performed using standard conditions (Sambrook et al., 1989). Differential accumulation of the two viral isolates (ageratum and carrot) in the inoculated tobacco plants was investigated using primer pairs AgeRTfor/AgeRTrev for the ageratum isolate and CarRTfor/CarRTrev for the carrot isolate (Supplementary Table 1) in real-time PCR assay. Fifty nanograms of DNA template for each sample were applied in Platinum SYBR Green chemistry (Life Technologies, USA) and Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems, USA). Tobacco actin gene was amplified with TbAct.F/R primers (Supplementary Table 1) and used as an internal control. Three biological and two technical replicates were used for each combination. Isolate specific primers AEV-Cfor/rev and AEV-Afor/rev (Supplementary Table 1) were used for the amplification and sequencing of AEV-Age and AEV-Car from the agroinoculated plants.
3. Results 3.1. Amplification of virus and satellite DNA Leaves from ten out of twelve plants of carrot, and all twelve symptomatic ageratum plants (Fig. 1) yielded a DNA product of ∼2.8 kb upon digestion of RCA products with BamHI, HindIII, NdeI, NcoI, SalI and XbaI. The HindIII digested RCA product was cloned and sequenced. No such fragment was obtained from asymptomatic plant samples. A fragment of ∼1.3 kb was obtained after KpnI digestion of the RCA product, indicating the presence of satellites in all the begomovirus positive samples. On using the betasatellite specific primers, a PCR amplicon of ∼1.3 kb was obtained, whereas no amplification was noticed when alphasatellite-specific primers were used.
3.2. AEV and AYLCB were detected in carrot and ageratum The begomovirus genomes isolated from carrot and ageratum exhibited a size of 2760 and 2748 bp, respectively. Nucleotide sequences of two clones from carrot (accession nos. JF728865 and JF728867) and six from ageratum (accession nos. JF728860, JF728861, JF728862, JF728863, JF728864 and JF728866), representing full-length begomovirus genomes, were submitted to GenBank. Sequence analysis revealed a genome organization typical of monopartite begomovirus with two open reading frames (ORFs; V1 and V2) on the virion strand and four ORFs (C1, C2, C3 and C4) on the complementary strand. The global sequence alignment showed that the virus isolated from carrot and ageratum were highly identical (96–98% identity) to ageratum enation virus at nucleotide level (Table 1). Different clones sequenced from field samples of carrot and ageratum revealed a nucleotide identity of 95–97% between isolates from the two plant species (Supplementary Table 2). All AEV sequences (including the virus under study) made one cluster in phylogenetic tree (Fig. 2A). On the basis of the nucleotide sequence identity and phylogenetic analysis, the two groups of virus isolates (ageratum and carrot) are supposed to be isolates of AEV, and hence referred to here as AEV-Car (carrot isolate) and AEV-Age (ageratum isolate). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2013. 10.010. The RCA and PCR products of ∼1.3 kb indicated the presence of betasatellites and the sizes were of 1372 and 1359 bp from carrot and ageratum, respectively. The nucleotide sequences of one betasatellite from ageratum (accession no. JF728868) and one from carrot (accession no. JF728869) were submitted to GenBank. Structural features including A-rich region, ˇC1 ORF and a highly conserved region (satellite conserved region) resembled other known betasatellites. The betasatellites detected from ageratum and carrot showed highest identity of 94% to Digera arvensis yellow vein betasatellite (DAYVB; AM494977), and ageratum yellow leaf curl betasatellite (AYLCB; AM412239), respectively (Table 1). The two betasatellite isolates (ageratum and carrot) showed an identity of 98% between them. Both the betasatellite isolates clustered together in the phylogenetic tree (Fig. 2B). Based on the
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Table 1 AEV and AYLCB clones from Ageratum conyzoides and Daucus carota. The other begomovirus and betasatellite clones with which the AEV and AYLCB clones under study showed highest nucleotide identity. S. no.
AEV clones
Host
Begomoviruses
Identity (%)
1 2 3 4 5 6 7 8
JF728860 JF728861 JF728862 JF728863 JF728864 JF728866 JF728865 JF728867
Ageratum Carrot
AEV-[IN:Pal:UwuA:10] (FN794201) AEV-[IN:Pal:AgrA:10] (FN794198) AEV-[IN:Pal:UwuA:10] (FN794201) AEV-[IN:Pal:AgrA:10] (FN794198) AEV-[PK:Lah:06] (AM261836)
97 97 97 97 98 97 96 96
S. no.
AYLCB clones
Host
Betasatellites
Identity (%)
9 10
JF728868 JF728869
Ageratum Carrot
DAYVB-[PK:Fai:07] (AM494977) AYLCB-[PK:Lah:05] (AM412239)
94 94
Same host () and same clone ().
nucleotide sequence identity and phylogenetic analysis, the two betasatellite isolates (ageratum and carrot) were identified as isolates of AYLCB, and hence referred to here as AYLCB-Car (carrot isolate) and AYLCB-Age (ageratum isolate). The begomovirus and betasatellite genomic components of the carrot isolate were 12 and 13 nucleotides longer in comparison to that of ageratum. No alphasatellites were detected in the symptomatic samples of carrot and ageratum. 3.3. Recombinant origin of the AEV and AYLCB A recombination fragment of 823 nucleotides was detected in AEV-Age with the major and minor parents resembling AEV-[IN:Lko:Cle:08] (FJ177031) and AEV-[NP:SB:02] (AJ437618), respectively. The breakpoints were determined at nucleotides 231 and 1054 with average probability value of 1.488 × 10−09 .
A recombination fragment of 1210 nucleotides was detected in AEV-Car with the major parent resembling AEV-[PK:Lah:06] (AM698011) and the minor parent resembling pedilanthus leaf curl virus, PedLCV [PK:Mul:07] (AM712436). The breakpoints were predicted at nucleotides 1809 and 259 with average probability value of 8.530 × 10−13 . Recombination analysis of viral genomes revealed that a 12 nucleotides region (Fig. 3) in AEV-Car was donated by the minor parent resembling PedLCV [PK:Mul:07]. Multiple sequence alignment showed that the 12 nucleotide regions of AEV-Car was similar to the corresponding region of PedLCV (Fig. 3). This further confirmed recombinant origin of the 12 nucleotide regions. AYLCB-Car and AYLCBAge also had the recombination fragment and the breakpoints were at nucleotides 1025 and 1373 with average probability value of 1.124 × 10−35 . The major and minor parents resembled ageratum yellow leaf curl betasatellite (AM412239; AYLCB
Fig. 2. Phylogenetic trees showing the relationship of AEV-Car (red color font) and AEV-Age (green color font) with other closely related begomoviruses (A) and of AYLCB (red and green color font) with other known betasatellites (B). The percent bootstrap scores (1000 replicates) are shown next to the nodes. The evolutionary history was inferred and the evolutionary distances computed as described in Section 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. Alignment of AEV-Car and AEV-Age sequences with probable major and minor parents. The alignment starts from the origin of virus replication. The 12-nt region of the AEV-Car and minor parent is shown in red color. An ageratum isolate of AEV (AEV-Age), carrot isolate of AEV (AEV-Car), major and minor parents of AEV-Age (M&M AEV-Age), and major and minor parents of AEV-Car (M&M AEV-Car) are shown. The binding sites of ‘AgeRTfor’ and ‘CarRTfor’ primers are indicated. These primers were used in real-time PCR assay to assess accumulation of specific isolate in agroinoculated tobacco plants.
[PK:Lah:06]) and tomato leaf curl betasatellite (AJ542489; ToLCB [BD:Gaz:03]). 3.4. Inoculation of both the AEV clones along with AYLCB induced severe symptoms Inoculated plants were observed for the symptoms of virus infection at 21 days post inoculation (dpi). The plants inoculated with AEV-Car plus AYLCB or AEV-Age plus AYLCB, caused leaf curling in tobacco and tomato (Fig. 4B and E), and yellow mosaic in carrot and ageratum (Fig. 4H and K). The plants inoculated with AEV-Car plus AEV-Age plus AYLCB produced more severe leaf curling in tobacco and tomato and yellow mosaic in carrot and ageratum (Fig. 4C, F, I and L; indicated by arrow) in comparison to any other combination. The plants inoculated with single isolates or both the isolates (AEV-Car plus AEV-Age) in the absence of the betasatellite exhibited mild symptoms (Supplementary Fig. 1). Symptoms induced by AEV-Car or AEV-Age in carrot were similar. The rate of the viral infection under laboratory conditions was very high. A many as 80% plants of each species (tobacco, tomato, carrot and ageratum) inoculated with one construct or combinations of the constructs showed viral symptoms. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2013. 10.010. 3.5. Both the clones could infect systemically The plants inoculated with any of the AEV isolates along with the AYLCB yielded an amplicon of ∼2.8 kb, representing the viral genome and another amplicon of ∼1.3 kb representing the betasatellite DNA. These could be recovered from distantly located upper leaves of the plant (systemically infected sixth leaf). Sequencing of the amplified PCR products confirmed the respective AEV isolate and AYLCB in each of the inoculated plants that showed the viral disease symptoms. 3.6. Higher accumulation of AEV was detected in plants inoculated with both the isolates of AEV along with AYLCB Semi-quantitative PCR assay revealed that the virus accumulation was the lowest in AEV-Age or AEV-Car inoculated plants and highest in plants inoculated with AEV-Car plus AEV-Age plus AYLCB (Fig. 5A). The virus accumulation in AEV-Car plus AEV-Age inoculated plants was lower in comparison to plants inoculated with
AEV-Car plus AEV-Age plus AYLCB but higher in comparison to all other combinations (data not shown). Southern blot analysis, using total genomic DNA of tobacco as template, showed highest accumulation of the virus in plants inoculated with AEV-Car plus AEV-Age plus AYLCB, in comparison to the other two combinations, i.e., AEV-Age plus AYLCB or AEV-Car plus AYLCB (Fig. 5B). Real-time PCR using isolate specific primers (binding site shown in Fig. 3) indicated that the two viral isolates accumulated at a high level in plants inoculated with AEV-Car plus AEV-Age plus AYLCB, followed by those inoculated with AEV-Car plus AEV-Age (Fig. 5C). Relative viral DNA levels are shown for each construct and the combinations of the constructs. 3.7. AEV recovered from inoculated plants showed mutations DNA sequences of the full-length genomes of AEV-Car and AEV-Age retrieved from the inoculated plants at 21 dpi were compared with the sequences of the infectious clones of the virus, used for agroinoculation. Mutations were noticed in case of both AEV-Car and AEV-Age recovered from inoculated plants. These were distributed at various positions across the AEV genome. Single nucleotide changes at 6–12 positions were observed when the AEV-Age was recovered from carrot, tobacco and tomato, and AEV-Car from ageratum, tobacco and tomato. Fewer single nucleotide changes (at 2–3 positions only) were found in AEV-Age recovered from ageratum and AEV-Car from carrot. The AEV-Car and AEV-Age genomic sequences retrieved from the plants inoculated with AEV-Car plus AEV-Age or plants inoculated with AEV-Car plus AEV-Age plus AYLCB showed 3–6 nucleotide changes. Some of the nucleotide changes were within the coding regions which reflected amino acid substitutions. 4. Discussion Begomoviruses are widely distributed in India and affect a number of vegetable crops (Prasanna et al., 2010; Reddy et al., 2005). ToLCNDV was reported in carrot with an incidence of 30–40% (Sivalingam et al., 2011). We did not find ToLCNDV but detected AEV in carrot fields growing at two distant locations in India. Comparison of the coat protein gene sequence of ToLCNDV (EU224371) with that of AEV, revealed an identity of 80% and 95% at nucleotide and amino acid sequence level respectively. Though the two isolates of AEV (AEV-Car and AEV-Age) have some variation in their genomic sequences, they
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Fig. 4. Virus host interaction in severity of symptom formation. Mock vector (pCAMBIA1301) inoculated tobacco (A), tomato (D), carrot (G) and ageratum (J) plants; AEV-Car plus AYLCB inoculated tobacco (B), tomato (E), carrot (H) and ageratum (K) and AEV-Car plus AEV-Age plus AYLCB inoculated tobacco (C), tomato (F), carrot (I) and ageratum (L). Symptoms such as curling, reduced leaf lamina and yellow mosaic are indicated by arrow.
gave similar phenotypes on inoculation of carrot plants. Multiple sequence alignment revealed that the genome of AEV-Car was 12 nucleotides longer than AEV-Age (Fig. 3) in recombinant region. Mixed inoculation using the two isolates together
(AEV-Car plus AEV-Age) along with AYLCB, resulted into severe disease phenotype and enhanced virus accumulation in comparison to inoculation with a single isolate of AEV. This suggests possible differences in host-pathogen interaction mechanisms
Fig. 5. Semi-quantitative PCR (A), Southern hybridization (B) and real-time PCR (C) showing quantification of AEV in agroinoculated plants. Lanes 1–4, AEV-Car inoculated; lanes 5–8, AEV-Car plus AYLCB inoculated and lanes 9–12, AEV-Car plus AEV-Age plus AYLCB inoculated tobacco, tomato, carrot and ageratum plants (A). Similar results were found for the AEV-Age and AEV-Age plus AYLCB inoculated plants. Actin gene amplicon used as internal control is shown. M = DNA digested by HindIII/EcoRI. Lanes 13–15, Southern hybridization, using DNA from agroinoculated tobacco plants. Coat protein gene of AEV-Age was used as probe with the genomic DNA isolated from plants inoculated with AEV-Car plus AYLCB (lane 13), AEV-Age plus AYLCB (lane 14) and AEV-Car plus AEV-Age plus AYLCB (lane 15). Different forms of viral DNA are labeled as OC (open circular), SC (supercoiled), SS (single stranded), and Lin (linear). Each lane was loaded with 4 g total genomic DNA and the gel stained with ethidium bromide (EtBr) is shown as the loading control (B). Graphical representation of relative viral DNA levels in the agroinoculated plants using real-time PCR analysis (C). Each bar represents mean value of three plants for the levels of AEV-Car or AEV-Age in N. tabacum. X-axis shows names of the inoculated constructs; Y-axis shows relative virus accumulation (relative to the tobacco actin gene). Error bars show standard errors.
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of AEV-Car and AEV-Age which synergises to enhance virus accumulation and symptom formation (Vanitharani et al., 2004; Syller, 2012). Genomic sequences of AEV-Car and AEV-Age recovered from the inoculated plants at 21 dpi showed that such recycling led to nucleotide changes in the genome. The viral genomic DNAs with changes in nucleotide sequence (2–3 nts or 6–12 nts mutations, depending upon the host) represent the formation of quasispecies populations of AEV-Car and AEV-Age in the inoculated hosts. We speculate that the virus may be subjected to selection upon mutations within the coding and non-coding regions. Such changes may determine synergistic and antagonistic interactions between the invading viruses. In a recent report, amino acid substitutions in viral proteins have been reported to lead to synergistic and antagonistic interactions between East African cassava mosaic Cameroon virus and African cassava mosaic virus (Reddy et al., 2012). The C2/AC2 and C4/AC4 proteins of begomoviruses are known as transcription activators and silencing suppressors respectively. They play an important role in synergism between two begomoviruses (Vanitharani et al., 2004; Rentería-Canett et al., 2011). The expression of AC2 protein of East African cassava mosaic Cameroon virus (EACMCV) was reported to enhance the concentration of African cassava mosaic virus-Cameroon (ACMV-[CM]). The AC4 protein of ACMV-[CM] also increased the concentration of EACMCV (Vanitharani et al., 2004). Thus the C2/AC2 and C4/AC4 proteins are potential candidates for influencing synergism either due to their silencing suppressor activity or differences in virus-host interaction mechanism (Vanitharani et al., 2004). The identity (Supplementary Table 2) among C2 (89%) and C4 (90%) proteins of the two isolates (AEV-Car and AEV-Age) indicates the possible differences in their interactions with the hosts. Further studies are required to investigate the role of C2 and C4 proteins of the two isolates or other unknown factors in synergism. The differences between sequences of AEV-Car and AEV-Age isolates, obtained from the field grown carrot and ageratum plants and further mutations that appeared after recycling of the virus through different hosts, suggest the need to understand the mechanisms underlying the interactions between AEV isolates, the hosts and the production of quasispecies populations. Such mutations and recombination between the two isolates (AEV-Car and AEV-Age) may generate virus variants with better fitness and enhanced virulence. This is the first detailed study on the association of begomovirus and betasatellite in leaf yellowing disease of carrot in India. Considering that AEV infects ornamental, vegetable, legume and weed plants (Kumar et al., 2011; Pandey et al., 2011; Raj et al., 2010, 2011) and has ability to evolve rapidly (Khan et al., 2012), there is a need to devise approaches to limit the spread of such viruses. Acknowledgement Authors are thankful to Department of Biotechnology, Government of India for financial support. RT is thankful to DST, Govt of India for JC Bose Fellowship. Thanks to Dr. Birendra Singh (Skåne University Hospital, Sweden) for critical comments on the manuscript. References Afreen, B., Khan, A.A., Naqvi, Q.A., Kumar, S., Pratap, D., Snehi, S.K., Raj, S.K., 2009. Molecular identification of a Cucumber mosaic virus subgroup II isolate from carrot (Daucus carota) based on RNA3 genome sequence analyses. J. Plant Dis. Prot. 116 (5), 193–199. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215 (3), 403–410. Briddon, R.W., Mansoor, S., Bedford, I.D., Pinner, M.S., 1 Saunders, K., Stanley, J., Zafar, Y., Malik, K.A., Markham, P.G., 2001. Identification of DNA components required for induction of cotton leaf curl disease. Virology 285, 234–243.
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Ageratum conyzoides and its role in begomoviral epidemics; Ageratum enation virus: an emerging threat in India. Vegetos 25 (2), 20–28. Kumar, J., Kumar, A., Roy, J.K., Tuli, R., Khan, J.A., 2010. Identification and molecular characterization of begomovirus and associated satellite DNA molecules infecting Cyamopsis tetragonoloba. Virus Genes 41, 118–125. Kumar, Y., Hallan, V., Zaidi, A.A., 2011. First report of Ageratum enation virus infecting Crassocephalum crepidioides (Benth.) S. Moore and Ageratum conyzoides L. in India. J. Gen. Plant Pathol. 77, 214–216. Kumar, J., Singh, S.P., Kumar, A., Khan, J.A., Tuli, R., 2013. Detection and characterization of a new betasatellite: variation in disease symptoms of Tomato leaf curl Pakistan virus-India due to associated betasatellite. Arch. Virol. 158, 257–261. Latham, L.J., Jones, R.A.C., 2004. Carrot virus Y symptoms, losses, incidence, epidemiology and control. Virus Res. 100, 89–99. 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Molecular characterization and pathogenicity of a carrot (Daucus carota) infecting begomovirus and associated betasatellite from India Jitendra Kumar a,∗,1 , Samatha Gunapati b,1 , Sudhir P. Singh a , Rekha Gadre b , Naresh C. Sharma c , Rakesh Tuli a a
National Agri-Food Biotechnology Institute, Mohali 160071, Punjab, India Department of Biochemistry, Devi Ahilya Vishwavidyalaya, Indore, India c Department of Biochemistry and Genetics, Barkatullah University, Bhopal, India b
a r t i c l e
i n f o
Article history: Received 2 June 2013 Received in revised form 8 October 2013 Accepted 12 October 2013 Available online 25 October 2013 Keywords: Geminivirus variants Synergism Virus host range Viral genome Silencing suppression
a b s t r a c t The yellow mosaic pattern and shortening of leaf petiole are common disease symptoms associated with begomovirus infection in carrot. DNA from field infected carrot leaves was analyzed by rolling circle amplification and sequencing. The results established the presence of ageratum enation virus (AEV), which is referred to here as ageratum enation virus-carrot (AEV-Car). Symptomatic ageratum (Ageratum conyzoides) plants, growing adjacent to the carrot fields, also showed the presence of AEV (AEV-Age). Ageratum yellow leaf curl betasatellite (AYLCB) was also detected in the AEV infected carrot and ageratum samples. AEV-Car and AEV-Age are 95–97% identical in their DNA sequences, represent groups of isolates from the respective plant hosts (carrot and ageratum). Agroinoculation using infectious clones of AEVCar plus AYLCB or AEV-Age plus AYLCB in carrot, ageratum, tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum) produced yellow mosaic and curling symptoms in leaves of inoculated plants. Agroinoculation of the two isolates together, along with the betasatellite (AEV-Car plus AEV-Age plus AYLCB) resulted in the enhancement of symptoms in comparison to the plants inoculated with single isolate. Plants with more severe symptoms showed a higher level of viral DNA accumulation, suggesting synergistic interactions between the two isolates of AEV. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Carrot (Daucus carota) is one of the important vegetable crops in India, with an annual production of 350,000 metric tons (http://www.carrotmuseum.co.uk/statistics.html). Several RNA viruses namely alfalfa mosaic virus, carrot latent virus, carrot mottle virus, carrot red leaf virus, carrot yellow leaf virus, celery mosaic virus, curly top virus, carrot virus Y, carrot thin leaf potyvirus, carrot mottle dwarf virus and cucumber mosaic virus cause major yield loss worldwide (Murant et al., 1985; Latham and Jones, 2004; Howell and Mink, 1976; Stubbs, 1952; Afreen et al., 2009). Recently, a DNA virus, tomato leaf curl New Delhi virus (ToLCNDV; genus Begomovirus), was detected on carrot plants exhibiting yellow mosaic symptoms in several parts of India (Sivalingam et al., 2011). Begomoviruses (family Geminiviridae) are plant viruses with a single-stranded, circular DNA genome encapsidated in twinned
∗ Corresponding author. Tel.: +91 172 2290131; fax: +91 172 4604888. E-mail addresses: [email protected], jit [email protected] (J. Kumar). 1 These authors contributed equally to the article. 0168-1702/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.virusres.2013.10.010
icosahedral particles (Brown et al., 2011). Begomoviruses are transmitted by whiteflies (Bemisia tabaci Genn.), and infect a wide range of plants. They have either bipartite (DNA-A and DNA-B) or monopartite (one component equivalent to DNA-A) genome, where each component accounts for approximately 2.6–2.8 kb size (Brown et al., 2011). The DNA-A component of bipartite begomoviruses encodes proteins required for replication, control of gene expression and encapsidation, whereas DNA-B encodes proteins required for movement of the virus. The genome of monopartite begomovirus is homologous to the DNA-A of bipartite begomovirus. It encodes the proteins for viral replication, gene expression, encapsidation and movement (Brown et al., 2011). The majority of monopartite begomoviruses in the Old World are associated with a class of ssDNA satellites known as betasatellites. Betasatellites are half the begomovirus genome in size and encode a single protein, called, C1. The protein C1 mediates up-regulation of viral DNA levels in plants by suppressing the anti-viral defense system of the host plant (Saunders et al., 2000; Briddon et al., 2001; Jose and Usha, 2003; Kumar et al., 2013). Mixed infection of host is common for plant viruses. Co-infecting viruses interact either synergistically or antagonistically (Mascia et al., 2010; Rentería-Canett et al., 2011; Syller, 2012). Synergistic interactions facilitate enhanced replication and accumulation of
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the participating viruses, resulting in symptom enhancement in the host plant (Murphy and Bowen, 2006; Rentería-Canett et al., 2011; Syller, 2012). In contrast, in antagonistic interaction, one or both of the viruses interfere with replication and symptom induction of the other virus or each other (Wintermantel et al., 2008; Power, 1996). Exchange of genetic material occurs often during mixed infection via homologous and non-homologous recombination. The recombination events depend on population size, frequency and distribution of the variants (viral quasispecies population) and host defense mechanism (Padidam et al., 1999; Froissart et al., 2005; Mubin et al., 2009). AEV is a monopartite begomovirus with associated betasatellite that helps in the accumulation of AEV and manifestation of disease symptoms (Saunders et al., 2000; Mubin et al., 2009,2010). Earlier reports on AEV infecting ageratum, Pointed Guard (Trichosanthes dioica), Cat’s Whiskers (Cleome gynandra) and Crassocephalum crepidioides (Kumar et al., 2011; Pandey et al., 2011; Raj et al., 2010, 2011) indicate wide host range of the virus. This work broadens the host range further, by reporting that the AEV infects carrots also. To our knowledge, this is the first detailed study on a begomovirus, infecting carrot and the synergistic interaction between the isolates from carrot (AEV-Car) and those from ageratum (AEV-Age), as well as their associated betasatellite.
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2.3. Sequence and phylogenetic analysis DNA sequence similarity searches were performed using BLAST (Altschul et al., 1990). Pairwise global sequence alignment was used to determine the nucleotide sequence identity between the begomoviruses and betasatellite identified in this study with other known sequences (Needleman and Wunsch, 1970). Phylogenetic trees were constructed using distance-based algorithm (NeighborJoining) with 1000 bootstrap replicates. The evolutionary distances were computed, using the maximum composite likelihood method in MEGA4 (Tamura et al., 2007). 2.4. Prediction of genomic recombinations The probable recombination fragments in virus and satellite genome were detected using recombination detection program version 3 (RDP 3) at default setting with a P-value cutoff of 0.05 (Martin et al., 2010). The modules, RDP, GENECONV, Bootscan, MaxChi, Chimera, SiScan and 3Seq were used to scan the recombination events and identify probable parental sequences and recombination breakpoints. 2.5. Testing of infectivity and co-infectivity
Symptomatic carrot plants, exhibiting yellow mosaic, mild leaf curling and reduced leaf lamina, were collected during 2009–2010 from the fields of Hajipur (latitude: 25◦ 41 North; longitude: 85◦ 13 East), Bihar and Mohali (latitude: 30◦ 47 North; longitude: 76◦ 41 East), Punjab (Fig. 1). Three carrot fields were selected from each location. A total of twelve carrot samples, two each from six carrot fields were collected. A total of twelve ageratum plants, showing leaf curling and yellow mosaic (Fig. 1) and growing adjacent to carrot field were collected from both the locations. Six asymptomatic plants each of carrot and ageratum were collected from the two locations. One asymptomatic carrot plant from each field was also collected. Genomic DNA was isolated from all the samples, using DNAeasy plant mini kit column (Qiagen GmbH, Germany).
Dimeric head-to-tail tandem repeats of begomovirus genomes (carrot, JF728867 and ageratum, JF728864) were produced by partial digestion of RCA products with BamHI restriction endonuclease. The resulting fragments were cloned into the binary vector pCAMBIA1301 (Cambia, Canberra, Australia). Dimeric infectious clone of betasatellite (JF728869) was made similarly in pCAMBIA1301, verified by sequencing and transformed into Agrobacterium tumefaciens GV3101. Healthy young plantlets (4–5 leaf stage) of Nicotiana tabacum, Solanum lycopersicum, Daucus carota and Ageratum conyzoides were agroinoculated with the infectious clones of (i) carrot isolate of begomovirus, (ii) ageratum isolate of begomovirus, (iii) carrot isolate of begomovirus plus the betasatellite, (iv) ageratum isolate of begomovirus plus the betasatellite, (v) carrot isolate of begomovirus plus ageratum isolate of begomovirus and (vi) carrot isolate of begomovirus plus ageratum isolate of begomovirus plus the betasatellite. A total of sixty plants were inoculated for each of the four species (N. tabacum, S. lycopersicum, D. carota and A. conyzoides). An A. tumefaciens strain containing empty pCAMBIA1301 was used as control. Agroinoculated plants were maintained at 25 ◦ C in 16 h light and 8 h dark.
2.2. Amplification of virus and satellite DNA
2.6. Detection and quantification of viral DNA in agroinoculated plants
The full-length begomoviral genome was amplified in sequence non-specific manner using genomic DNA as template and rolling circle amplification (Wu et al., 2008). Rolling circle amplification (RCA) was accomplished using templiPhi DNA amplification kit (GE Healthcare, USA). The RCA products were digested by HindIII restriction endonuclease and analyzed by gel electrophoresis. RCA products were also digested with several other restriction endonucleases (BamHI, KpnI, NdeI, NcoI, SalI and XbaI) to detect mixed infections with other begomoviruses and the presence of alpha- and betasatellites. The presence of both the satellites was investigated by PCR using specific primers (‘nanofor’/‘nanorev’ and 01/04; Kumar et al., 2010). A high fidelity PCR enzyme mix (Thermo Scientific, EU) was used in all PCR assays. The RCA restriction fragments and the PCR products expected to represent the virus and satellite genome were cloned in pDrive cloning vector (QIAGEN GmbH, Germany). Five clones from each sample were sequenced, using automated sequencer (3730xl DNA analyzer, Applied Biosystem, USA).
Genomic DNA was isolated from the systemically infected sixth leaf of the inoculated carrot, ageratum, tobacco and tomato plants. The AgeF/AgeR (Supplementary Table 1) and 01/04 (Kumar et al., 2010) primers were used in PCR assays for detection of the virus and betasatellite, respectively. The PCRs were carried out at 95 ◦ C for 4 min for denaturation, followed by 35 cycles of 95 ◦ C for 20 s, 50 ◦ C for 30 s, 72 ◦ C for 90 s, and final extension at 72 ◦ C for 5 min. For quantification of the viruses in agroinoculated plants, the concentration of DNA was adjusted to 50 ng/L in all the cases. AgeF/AgeR primers were used to quantify the viral DNA using semiquantitative PCR assay on the inoculated plants. Plant encoded actin gene was used as an internal control (Act.F/R; Supplementary Table 1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2013. 10.010. Southern hybridization was performed to investigate virus accumulation in tobacco plants (Southern, 1975). Five micrograms
2. Materials and methods 2.1. Virus source
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Fig. 1. Symptoms of AEV infection on Carrot and Ageratum. Healthy plants of carrot (A) and ageratum (C) showing green leaves with wide leaf lamina. Infected plants of carrot (B) showing yellow mosaic and mild leaf curling with reduced leaf lamina and plants of ageratum showing (D) only leaf curling and (E) yellow mosaic and severe leaf curling.
of genomic DNA from each sample were gel electrophoresed and transferred to Hybond-N+ Nylon membrane (Amersham Biosciences, USA), using SSC buffer. Coat protein gene of the AEV-Age was labeled with [alpha-32 P]-dCTP and used as probe. The hybridization signal was captured using phosphorimager (BioRad, CA). All procedures were performed using standard conditions (Sambrook et al., 1989). Differential accumulation of the two viral isolates (ageratum and carrot) in the inoculated tobacco plants was investigated using primer pairs AgeRTfor/AgeRTrev for the ageratum isolate and CarRTfor/CarRTrev for the carrot isolate (Supplementary Table 1) in real-time PCR assay. Fifty nanograms of DNA template for each sample were applied in Platinum SYBR Green chemistry (Life Technologies, USA) and Applied Biosystems 7500 fast real-time PCR system (Applied Biosystems, USA). Tobacco actin gene was amplified with TbAct.F/R primers (Supplementary Table 1) and used as an internal control. Three biological and two technical replicates were used for each combination. Isolate specific primers AEV-Cfor/rev and AEV-Afor/rev (Supplementary Table 1) were used for the amplification and sequencing of AEV-Age and AEV-Car from the agroinoculated plants.
3. Results 3.1. Amplification of virus and satellite DNA Leaves from ten out of twelve plants of carrot, and all twelve symptomatic ageratum plants (Fig. 1) yielded a DNA product of ∼2.8 kb upon digestion of RCA products with BamHI, HindIII, NdeI, NcoI, SalI and XbaI. The HindIII digested RCA product was cloned and sequenced. No such fragment was obtained from asymptomatic plant samples. A fragment of ∼1.3 kb was obtained after KpnI digestion of the RCA product, indicating the presence of satellites in all the begomovirus positive samples. On using the betasatellite specific primers, a PCR amplicon of ∼1.3 kb was obtained, whereas no amplification was noticed when alphasatellite-specific primers were used.
3.2. AEV and AYLCB were detected in carrot and ageratum The begomovirus genomes isolated from carrot and ageratum exhibited a size of 2760 and 2748 bp, respectively. Nucleotide sequences of two clones from carrot (accession nos. JF728865 and JF728867) and six from ageratum (accession nos. JF728860, JF728861, JF728862, JF728863, JF728864 and JF728866), representing full-length begomovirus genomes, were submitted to GenBank. Sequence analysis revealed a genome organization typical of monopartite begomovirus with two open reading frames (ORFs; V1 and V2) on the virion strand and four ORFs (C1, C2, C3 and C4) on the complementary strand. The global sequence alignment showed that the virus isolated from carrot and ageratum were highly identical (96–98% identity) to ageratum enation virus at nucleotide level (Table 1). Different clones sequenced from field samples of carrot and ageratum revealed a nucleotide identity of 95–97% between isolates from the two plant species (Supplementary Table 2). All AEV sequences (including the virus under study) made one cluster in phylogenetic tree (Fig. 2A). On the basis of the nucleotide sequence identity and phylogenetic analysis, the two groups of virus isolates (ageratum and carrot) are supposed to be isolates of AEV, and hence referred to here as AEV-Car (carrot isolate) and AEV-Age (ageratum isolate). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2013. 10.010. The RCA and PCR products of ∼1.3 kb indicated the presence of betasatellites and the sizes were of 1372 and 1359 bp from carrot and ageratum, respectively. The nucleotide sequences of one betasatellite from ageratum (accession no. JF728868) and one from carrot (accession no. JF728869) were submitted to GenBank. Structural features including A-rich region, ˇC1 ORF and a highly conserved region (satellite conserved region) resembled other known betasatellites. The betasatellites detected from ageratum and carrot showed highest identity of 94% to Digera arvensis yellow vein betasatellite (DAYVB; AM494977), and ageratum yellow leaf curl betasatellite (AYLCB; AM412239), respectively (Table 1). The two betasatellite isolates (ageratum and carrot) showed an identity of 98% between them. Both the betasatellite isolates clustered together in the phylogenetic tree (Fig. 2B). Based on the
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Table 1 AEV and AYLCB clones from Ageratum conyzoides and Daucus carota. The other begomovirus and betasatellite clones with which the AEV and AYLCB clones under study showed highest nucleotide identity. S. no.
AEV clones
Host
Begomoviruses
Identity (%)
1 2 3 4 5 6 7 8
JF728860 JF728861 JF728862 JF728863 JF728864 JF728866 JF728865 JF728867
Ageratum Carrot
AEV-[IN:Pal:UwuA:10] (FN794201) AEV-[IN:Pal:AgrA:10] (FN794198) AEV-[IN:Pal:UwuA:10] (FN794201) AEV-[IN:Pal:AgrA:10] (FN794198) AEV-[PK:Lah:06] (AM261836)
97 97 97 97 98 97 96 96
S. no.
AYLCB clones
Host
Betasatellites
Identity (%)
9 10
JF728868 JF728869
Ageratum Carrot
DAYVB-[PK:Fai:07] (AM494977) AYLCB-[PK:Lah:05] (AM412239)
94 94
Same host () and same clone ().
nucleotide sequence identity and phylogenetic analysis, the two betasatellite isolates (ageratum and carrot) were identified as isolates of AYLCB, and hence referred to here as AYLCB-Car (carrot isolate) and AYLCB-Age (ageratum isolate). The begomovirus and betasatellite genomic components of the carrot isolate were 12 and 13 nucleotides longer in comparison to that of ageratum. No alphasatellites were detected in the symptomatic samples of carrot and ageratum. 3.3. Recombinant origin of the AEV and AYLCB A recombination fragment of 823 nucleotides was detected in AEV-Age with the major and minor parents resembling AEV-[IN:Lko:Cle:08] (FJ177031) and AEV-[NP:SB:02] (AJ437618), respectively. The breakpoints were determined at nucleotides 231 and 1054 with average probability value of 1.488 × 10−09 .
A recombination fragment of 1210 nucleotides was detected in AEV-Car with the major parent resembling AEV-[PK:Lah:06] (AM698011) and the minor parent resembling pedilanthus leaf curl virus, PedLCV [PK:Mul:07] (AM712436). The breakpoints were predicted at nucleotides 1809 and 259 with average probability value of 8.530 × 10−13 . Recombination analysis of viral genomes revealed that a 12 nucleotides region (Fig. 3) in AEV-Car was donated by the minor parent resembling PedLCV [PK:Mul:07]. Multiple sequence alignment showed that the 12 nucleotide regions of AEV-Car was similar to the corresponding region of PedLCV (Fig. 3). This further confirmed recombinant origin of the 12 nucleotide regions. AYLCB-Car and AYLCBAge also had the recombination fragment and the breakpoints were at nucleotides 1025 and 1373 with average probability value of 1.124 × 10−35 . The major and minor parents resembled ageratum yellow leaf curl betasatellite (AM412239; AYLCB
Fig. 2. Phylogenetic trees showing the relationship of AEV-Car (red color font) and AEV-Age (green color font) with other closely related begomoviruses (A) and of AYLCB (red and green color font) with other known betasatellites (B). The percent bootstrap scores (1000 replicates) are shown next to the nodes. The evolutionary history was inferred and the evolutionary distances computed as described in Section 2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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Fig. 3. Alignment of AEV-Car and AEV-Age sequences with probable major and minor parents. The alignment starts from the origin of virus replication. The 12-nt region of the AEV-Car and minor parent is shown in red color. An ageratum isolate of AEV (AEV-Age), carrot isolate of AEV (AEV-Car), major and minor parents of AEV-Age (M&M AEV-Age), and major and minor parents of AEV-Car (M&M AEV-Car) are shown. The binding sites of ‘AgeRTfor’ and ‘CarRTfor’ primers are indicated. These primers were used in real-time PCR assay to assess accumulation of specific isolate in agroinoculated tobacco plants.
[PK:Lah:06]) and tomato leaf curl betasatellite (AJ542489; ToLCB [BD:Gaz:03]). 3.4. Inoculation of both the AEV clones along with AYLCB induced severe symptoms Inoculated plants were observed for the symptoms of virus infection at 21 days post inoculation (dpi). The plants inoculated with AEV-Car plus AYLCB or AEV-Age plus AYLCB, caused leaf curling in tobacco and tomato (Fig. 4B and E), and yellow mosaic in carrot and ageratum (Fig. 4H and K). The plants inoculated with AEV-Car plus AEV-Age plus AYLCB produced more severe leaf curling in tobacco and tomato and yellow mosaic in carrot and ageratum (Fig. 4C, F, I and L; indicated by arrow) in comparison to any other combination. The plants inoculated with single isolates or both the isolates (AEV-Car plus AEV-Age) in the absence of the betasatellite exhibited mild symptoms (Supplementary Fig. 1). Symptoms induced by AEV-Car or AEV-Age in carrot were similar. The rate of the viral infection under laboratory conditions was very high. A many as 80% plants of each species (tobacco, tomato, carrot and ageratum) inoculated with one construct or combinations of the constructs showed viral symptoms. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.virusres.2013. 10.010. 3.5. Both the clones could infect systemically The plants inoculated with any of the AEV isolates along with the AYLCB yielded an amplicon of ∼2.8 kb, representing the viral genome and another amplicon of ∼1.3 kb representing the betasatellite DNA. These could be recovered from distantly located upper leaves of the plant (systemically infected sixth leaf). Sequencing of the amplified PCR products confirmed the respective AEV isolate and AYLCB in each of the inoculated plants that showed the viral disease symptoms. 3.6. Higher accumulation of AEV was detected in plants inoculated with both the isolates of AEV along with AYLCB Semi-quantitative PCR assay revealed that the virus accumulation was the lowest in AEV-Age or AEV-Car inoculated plants and highest in plants inoculated with AEV-Car plus AEV-Age plus AYLCB (Fig. 5A). The virus accumulation in AEV-Car plus AEV-Age inoculated plants was lower in comparison to plants inoculated with
AEV-Car plus AEV-Age plus AYLCB but higher in comparison to all other combinations (data not shown). Southern blot analysis, using total genomic DNA of tobacco as template, showed highest accumulation of the virus in plants inoculated with AEV-Car plus AEV-Age plus AYLCB, in comparison to the other two combinations, i.e., AEV-Age plus AYLCB or AEV-Car plus AYLCB (Fig. 5B). Real-time PCR using isolate specific primers (binding site shown in Fig. 3) indicated that the two viral isolates accumulated at a high level in plants inoculated with AEV-Car plus AEV-Age plus AYLCB, followed by those inoculated with AEV-Car plus AEV-Age (Fig. 5C). Relative viral DNA levels are shown for each construct and the combinations of the constructs. 3.7. AEV recovered from inoculated plants showed mutations DNA sequences of the full-length genomes of AEV-Car and AEV-Age retrieved from the inoculated plants at 21 dpi were compared with the sequences of the infectious clones of the virus, used for agroinoculation. Mutations were noticed in case of both AEV-Car and AEV-Age recovered from inoculated plants. These were distributed at various positions across the AEV genome. Single nucleotide changes at 6–12 positions were observed when the AEV-Age was recovered from carrot, tobacco and tomato, and AEV-Car from ageratum, tobacco and tomato. Fewer single nucleotide changes (at 2–3 positions only) were found in AEV-Age recovered from ageratum and AEV-Car from carrot. The AEV-Car and AEV-Age genomic sequences retrieved from the plants inoculated with AEV-Car plus AEV-Age or plants inoculated with AEV-Car plus AEV-Age plus AYLCB showed 3–6 nucleotide changes. Some of the nucleotide changes were within the coding regions which reflected amino acid substitutions. 4. Discussion Begomoviruses are widely distributed in India and affect a number of vegetable crops (Prasanna et al., 2010; Reddy et al., 2005). ToLCNDV was reported in carrot with an incidence of 30–40% (Sivalingam et al., 2011). We did not find ToLCNDV but detected AEV in carrot fields growing at two distant locations in India. Comparison of the coat protein gene sequence of ToLCNDV (EU224371) with that of AEV, revealed an identity of 80% and 95% at nucleotide and amino acid sequence level respectively. Though the two isolates of AEV (AEV-Car and AEV-Age) have some variation in their genomic sequences, they
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Fig. 4. Virus host interaction in severity of symptom formation. Mock vector (pCAMBIA1301) inoculated tobacco (A), tomato (D), carrot (G) and ageratum (J) plants; AEV-Car plus AYLCB inoculated tobacco (B), tomato (E), carrot (H) and ageratum (K) and AEV-Car plus AEV-Age plus AYLCB inoculated tobacco (C), tomato (F), carrot (I) and ageratum (L). Symptoms such as curling, reduced leaf lamina and yellow mosaic are indicated by arrow.
gave similar phenotypes on inoculation of carrot plants. Multiple sequence alignment revealed that the genome of AEV-Car was 12 nucleotides longer than AEV-Age (Fig. 3) in recombinant region. Mixed inoculation using the two isolates together
(AEV-Car plus AEV-Age) along with AYLCB, resulted into severe disease phenotype and enhanced virus accumulation in comparison to inoculation with a single isolate of AEV. This suggests possible differences in host-pathogen interaction mechanisms
Fig. 5. Semi-quantitative PCR (A), Southern hybridization (B) and real-time PCR (C) showing quantification of AEV in agroinoculated plants. Lanes 1–4, AEV-Car inoculated; lanes 5–8, AEV-Car plus AYLCB inoculated and lanes 9–12, AEV-Car plus AEV-Age plus AYLCB inoculated tobacco, tomato, carrot and ageratum plants (A). Similar results were found for the AEV-Age and AEV-Age plus AYLCB inoculated plants. Actin gene amplicon used as internal control is shown. M = DNA digested by HindIII/EcoRI. Lanes 13–15, Southern hybridization, using DNA from agroinoculated tobacco plants. Coat protein gene of AEV-Age was used as probe with the genomic DNA isolated from plants inoculated with AEV-Car plus AYLCB (lane 13), AEV-Age plus AYLCB (lane 14) and AEV-Car plus AEV-Age plus AYLCB (lane 15). Different forms of viral DNA are labeled as OC (open circular), SC (supercoiled), SS (single stranded), and Lin (linear). Each lane was loaded with 4 g total genomic DNA and the gel stained with ethidium bromide (EtBr) is shown as the loading control (B). Graphical representation of relative viral DNA levels in the agroinoculated plants using real-time PCR analysis (C). Each bar represents mean value of three plants for the levels of AEV-Car or AEV-Age in N. tabacum. X-axis shows names of the inoculated constructs; Y-axis shows relative virus accumulation (relative to the tobacco actin gene). Error bars show standard errors.
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of AEV-Car and AEV-Age which synergises to enhance virus accumulation and symptom formation (Vanitharani et al., 2004; Syller, 2012). Genomic sequences of AEV-Car and AEV-Age recovered from the inoculated plants at 21 dpi showed that such recycling led to nucleotide changes in the genome. The viral genomic DNAs with changes in nucleotide sequence (2–3 nts or 6–12 nts mutations, depending upon the host) represent the formation of quasispecies populations of AEV-Car and AEV-Age in the inoculated hosts. We speculate that the virus may be subjected to selection upon mutations within the coding and non-coding regions. Such changes may determine synergistic and antagonistic interactions between the invading viruses. In a recent report, amino acid substitutions in viral proteins have been reported to lead to synergistic and antagonistic interactions between East African cassava mosaic Cameroon virus and African cassava mosaic virus (Reddy et al., 2012). The C2/AC2 and C4/AC4 proteins of begomoviruses are known as transcription activators and silencing suppressors respectively. They play an important role in synergism between two begomoviruses (Vanitharani et al., 2004; Rentería-Canett et al., 2011). The expression of AC2 protein of East African cassava mosaic Cameroon virus (EACMCV) was reported to enhance the concentration of African cassava mosaic virus-Cameroon (ACMV-[CM]). The AC4 protein of ACMV-[CM] also increased the concentration of EACMCV (Vanitharani et al., 2004). Thus the C2/AC2 and C4/AC4 proteins are potential candidates for influencing synergism either due to their silencing suppressor activity or differences in virus-host interaction mechanism (Vanitharani et al., 2004). The identity (Supplementary Table 2) among C2 (89%) and C4 (90%) proteins of the two isolates (AEV-Car and AEV-Age) indicates the possible differences in their interactions with the hosts. Further studies are required to investigate the role of C2 and C4 proteins of the two isolates or other unknown factors in synergism. The differences between sequences of AEV-Car and AEV-Age isolates, obtained from the field grown carrot and ageratum plants and further mutations that appeared after recycling of the virus through different hosts, suggest the need to understand the mechanisms underlying the interactions between AEV isolates, the hosts and the production of quasispecies populations. Such mutations and recombination between the two isolates (AEV-Car and AEV-Age) may generate virus variants with better fitness and enhanced virulence. This is the first detailed study on the association of begomovirus and betasatellite in leaf yellowing disease of carrot in India. Considering that AEV infects ornamental, vegetable, legume and weed plants (Kumar et al., 2011; Pandey et al., 2011; Raj et al., 2010, 2011) and has ability to evolve rapidly (Khan et al., 2012), there is a need to devise approaches to limit the spread of such viruses. Acknowledgement Authors are thankful to Department of Biotechnology, Government of India for financial support. RT is thankful to DST, Govt of India for JC Bose Fellowship. Thanks to Dr. Birendra Singh (Skåne University Hospital, Sweden) for critical comments on the manuscript. References Afreen, B., Khan, A.A., Naqvi, Q.A., Kumar, S., Pratap, D., Snehi, S.K., Raj, S.K., 2009. Molecular identification of a Cucumber mosaic virus subgroup II isolate from carrot (Daucus carota) based on RNA3 genome sequence analyses. J. Plant Dis. Prot. 116 (5), 193–199. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment search tool. J. Mol. Biol. 215 (3), 403–410. Briddon, R.W., Mansoor, S., Bedford, I.D., Pinner, M.S., 1 Saunders, K., Stanley, J., Zafar, Y., Malik, K.A., Markham, P.G., 2001. Identification of DNA components required for induction of cotton leaf curl disease. Virology 285, 234–243.
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