Arch Virol DOI 10.1007/s00705-014-2149-5
BRIEF REPORT
bC1 is a pathogenicity determinant: not only for begomoviruses but also for a mastrevirus Jitendra Kumar • Jitesh Kumar • Sudhir P. Singh Rakesh Tuli
•
Received: 13 March 2014 / Accepted: 8 June 2014 Ó Springer-Verlag Wien 2014
Abstract bC1 proteins, encoded by betasatellites, are known to be pathogenicity determinants, and they are responsible for symptom expression in many devastating diseases caused by begomoviruses. We report the involvement of bC1 in pathogenicity determination of a mastrevirus. Analysis of field samples of wheat plants containing wheat dwarf India virus (WDIV) revealed the presence of a full-length and several defective betasatellite molecules. The detected betasatellite was identified as ageratum yellow leaf curl betasatellite (AYLCB). No begomovirus was detected in any of the samples. The fulllength AYLCB contained an intact bC1 gene, whereas the defective molecules contained complete or partial deletions of bC1. Agroinoculation of wheat with the full-length AYLCB and WDIV or of tobacco with ageratum enation virus enhanced the pathogenicity and accumulation of the respective viruses, whereas the defective molecules could not. This study indicates that bC1 is a pathogenicity determinant for WDIV and can interact functionally not only with begomoviruses but also with a mastrevirus. Keywords AYLCB WDIV AEV Trans-replication Virus accumulation Betasatellites are single-stranded DNA molecules associated with begomoviruses (family Geminiviridae) [1–3]. They are approximately half the size (*1.3 kb) of their Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2149-5) contains supplementary material, which is available to authorized users. J. Kumar J. Kumar S. P. Singh R. Tuli (&) Department of Biotechnology, National Agri-Food Biotechnology Institute, Mohali 160071, Punjab, India e-mail: [email protected]
helper viruses and encode a single protein, bC1, which is a pathogenicity determinant [4–6]. bC1 suppresses silencing, up-regulates viral DNA levels in plants, helps in virus movement, and modulates virus symptoms and host range [4, 6–13]. Betasatellites share only the nonanucleotide (TAATATTAC) sequence with the helper begomovirus, which is predicted to serve as the origin of replication [1– 3]. Betasatellites depend on their helper viruses for replication, encapsidation and transmission by the insect vector and are often required by their helper viruses for symptom induction in their original hosts [14, 15]. Some monopartite begomoviruses are either poorly infectious or induce atypical symptoms in the absence of the betasatellite [6–8, 12, 14]. The association of betasatellites with monopartite begomoviruses has been reported in numerous diseases of plants [1–3], and in select cases, with bipartite begomoviruses [16–18]. In a recent study, the association of a betasatellite, ageratum yellow leaf curl betasatellite (AYLCB), with a mastrevirus, wheat dwarf India virus (WDIV), which infects wheat, was detected [19]. AYLCB enhanced WDIV accumulation and symptom (dwarfing) severity and remained capable of interacting with ageratum enation virus (AEV) [19]. The present study shows that AYLCB with a full-length bC1 gene enhanced virus (WDIV or AEV) accumulation and symptom severity, whereas defective molecules with bC1 deleted could not. Leaf samples from 50 symptomatic wheat plants were collected during 2011–12 from fields at Mohali, India. The symptomatic plants were selected on the basis of dwarfing and yellowing (Fig. 1A-C). Leaves from five asymptomatic plants were taken as negative controls. Total DNA was isolated using a DNeasy Plant Mini Kit column (QIAGEN GmbH, Germany). WDIV and satellite DNAs were amplified by rolling-circle amplification (RCA) and
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Fig. 1 Wheat plants suspected of being infected with WDIV and detection of the virus and satellite DNAs. Wheat plants (red circle) showing dwarfing with yellowing (A, B) and reddening with yellowing (C). NdeI-digested RCA products showing the fragments of *1.3, *1.6, *2.8 and *5.6 kb in the diseased wheat samples (D). PCR amplicon of *2.8 kb representing the genome size of WDIV (E). PCR products of the betasatellite showing multiple
amplicons of *700, *900 bp and *1.3 kb (F). Lanes 1, 2 and 3 in panels D-F represent the symptomatic samples of panels A, B and C, whereas lane 4 represents an asymptomatic plant sample. M, EcoRI/ HindIII-digested kDNA. The dimeric (WD) and monomeric (WM) forms of WDIV and the partial digestion products of WDIV (WP), betasatellite (beta) and defective betasatellite (M beta) are shown
polymerase chain reaction (PCR). RCA was performed using a TempliPhi Amplification Kit (GE Healthcare, USA), and the RCA products were partially digested with NdeI restriction endonuclease to obtain DNA fragments of WDIV and the satellite. The RCA products were also digested separately with the BamHI, EcoRI, HindIII, KpnI and PstI restriction endonucleases to detect the presence of a begomovirus. The results showed identical products for all samples and thus suggested the presence of identical viruses in all diseased plant samples (data not shown). MF1_FOR/REV was used in PCR assays to amplify WDIV DNA from wheat samples, whereas b01/04 was used to amplify betasatellite DNA (Supplementary Table 1) as described earlier [19]. The RCA and PCR assays were performed on all 55 wheat samples (50 symptomatic and 5 asymptomatic). Both RCA and PCR yielded fragments of *2.7 and 1.3 kb, the sizes expected for WDIV and betasatellite, respectively, whereas the asymptomatic plants did not (Fig. 1D-F). In addition to a fragment of *1.3 kb,
smaller amplicons of *657 and *900 bp were obtained using the betasatellite-specific primers (Fig. 1F). Sequencing of the RCA and PCR products revealed the presence of WDIV (accession no. JF781306) and AYLCB (accession no. KC305085) in the symptomatic wheat samples. Additional fragments of smaller sizes were detected using the betasatellite-specific primers. These represented defective molecules of AYLCB with smaller size (accession nos. KC305086, KC305087, and KC305089-KC305091). Detailed analysis of the sequence revealed that all of the betasatellite molecules, including the full-length (*1.3 kb) and smaller (less than *1.3 kb in length) molecules, retained three highly conserved features: a predicted stem-loop structure with the loop sequence TAATATTAC as the origin of replication (the nonanucleotide sequence), a region of high sequence similarity known as the ‘‘satellite conserved region’’ (SCR), and an adenine (A)-rich region. The full-length betasatellite molecule (1365 bp) contained the positionally and
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Fig. 2 Representation of the genomic organization of the full-length and the defective molecules of AYLCB. The sequences deleted in defective AYLCB molecules are indicated by dashed lines within the solid lines. Size variants of AYLCBs are indicated, with their length
given in bp: 657, 891, 929, 1046, 1084 and 1365 bp. The positions of the bC1 gene, the A-rich region and the satellite conserved region (SCR), are also shown. The region suggested to be responsible for trans-replication of a betasatellite [20] is also shown
sequence-conserved full-length bC1 gene, while the defective molecules contained partial deletions of bC1, except the 657-bp molecule, in which all of bC1 was absent (Fig. 2). Infectious clones of WDIV, AEV and AYLCB (fulllength as well as defective AYLCB) were prepared in the binary vector pCAMBIA1301 (CAMBIA, Canberra, Australia) as described previously [19]. The full-length and defective AYLCBs of 1365, 1084, 1046, 929, 891 and 657 bp are represented by AYLCB_1365, AYLCB_1084, AYLCB_1046, AYLCB_929, AYLCB_891 and AYLCB_657, respectively. Derivatives of the binary vector pCAMBIA1301, containing the infectious constructs were introduced separately into Agrobacterium tumefaciens strain GV3101 by transformation. Leaves of seven-day-old seedlings of 200 wheat plants (25 plants for each of the eight constructs; control, WDIV, WDIV and AYLCB_1365, WDIV and AYLCB_1084, WDIV and AYLCB_1046, WDIV and AYLCB_929, WDIV and AYLCB_891, WDIV and AYLCB_657) at the two- to three-leaf stage were agroinoculated using a needleless syringe. A total of 48 Nicotiana tabacum plants (six plants for each of the eight constructs: control, AEV, AEV and AYLCB_1365, AEV and AYLCB_1084, AEV and AYLCB_1046, AEV and AYLCB_929, AEV and AYLCB_891, AEV and AYLCB_657) at the four-leaf stage were inoculated. The inoculated wheat and tobacco plants were maintained in separate plant growth chambers (PGR14, Conviron, Canada) at 22 and 25 °C, respectively. The presence of WDIV or AEV in the agroinoculated wheat or tobacco plants was investigated using the primer pairs CP01/02 or CPB1/2 (Supplementary Table 1),
respectively, as described previously [19]. The presence of the full-length or defective AYLCB in the agroinoculated, infected plants was investigated using the BSCRF/R primer pair (Supplementary Table 1), which amplifies the sequence (Supplementary Fig. 1) conserved in all of the betasatellites without discriminating between the fulllength and defective molecules. One hundred fifty wheat plants out of the 200 infectious-clone-inoculated plants showed the presence of WDIV (Fig. 3K) and/or AYLCB (Fig. 3N). A total of 38 N. tabacum plants out of the 48 infectious-clone-inoculated plants, showed the presence of AEV (Fig. 3L) and/or AYLCB (Fig. 3O). The PCRamplified products from the inoculated plant samples were sequenced to confirm their identity to the sequence of the initially inoculated clones. Wheat plants harboring WDIV showed dwarfing in comparison to the mock-inoculated control (Fig. 3A, B), whereas the plants containing WDIV and full-length AYLCB (1365 bp; Fig. 2) showed more-severe stunting (Fig. 3E). Wheat plants with WDIV and defective molecules of AYLCB showed a phenotype similar to that of the plants inoculated with WDIV in the absence of AYLCB (Fig. 3B, C, D). The average height of five wheat plants at 42 days postinfection (dpi), in the case of the control and those infected with WDIV and full-length AYLCB, WDIV and defective AYLCB and WDIV, alone was 70.3 ± 2.51, 35.3 ± 1.52, 53.3 ± 3.05 and 54.3 ± 1.52 cm, respectively. Tobacco plants containing AEV showed leaf curling and stunting in comparison to mock-inoculated control (Fig. 3F, G). The viral symptoms, leaf curling and stunting, were more severe in the presence of AEV and full-length AYLCB (Fig. 3J). Plants with AEV and defective AYLCB
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Fig. 3 Effects of the AYLCB on the symptom severity and accumulation of WDIV and AEV in wheat and tobacco, respectively. Mock-inoculated wheat plants and those inoculated with (A), WDIV (B), WDIV and 1084-bp AYLCB (C), WDIV and 657-bp AYLCB (D) and WDIV and 1365-bp AYLCB (E) at 35 dpi are shown. Mockinoculated tobacco plants (A), and those inocuated with AEV (B), AEV and 1084-bp AYLCB (C), AEV and 657-bp AYLCB (D), and AEV and 1365-bp AYLCB (E) at 30 dpi are shown. Semi-quantitative PCR (K, L) and real-time qPCR (M, N) – based on three replicates – were used for analysis of WDIV (K, M) or AEV (L, N) from agroinoculated, infected wheat and tobacco plants,
respectively. The CP genes of WDIV (WCP) and AEV (ACP) were amplified by semi-quantitative PCR (K, L) and real-time qPCR (M, N). The samples used for semi-quantitative PCR (K, L) and real-time PCR (M, N) were as follows: virus alone (WDIV or AEV; lane 1), virus and 657-bp AYLCB (lane 2), virus and 891-bp AYLCB (lane 3), virus and 929-bp AYLCB (lane 4), virus and 1046-bp AYLCB (lane 5), virus and 1084-bp AYLCB (lane 6), virus and 1365-bp AYLCB (lane 7). The results of semi-quantitative PCR-based quantification of AYLCB in wheat (O) and tobacco (P) are shown. The same samples used for panels (K) and (L) were used for the quantification of AYLCB (O, P)
molecules showed viral symptoms, leaf curling and stunting, similar to the plants inoculated with AEV in the absence of AYLCB (3G-I). The average height of three tobacco plants at 35 dpi, in the case of the control and those infected with AEV and full-length AYLCB, AEV and defective AYLCB and AEV alone, was 50.41 ± 3.51, 12.4 ± 3.02, 28.15 ± 4.05 and 30.2 ± 4.44 cm, respectively. The coat protein genes of WDIV and AEV were amplified by real-time qPCR and semi-quantitative PCR
for quantification of virus accumulation in the presence of full-length and defective AYLCB molecules. The concentration of total DNA from the inoculated, infected plants was adjusted to 50 ng ll-1, and the primer pairs CP01/02 and CPB1/2 (Supplementary Table 1) were used for WDIV and AEV, respectively. Elongation factor-1 alpha (EF1afor/rev; Supplementary Table 1) and actin (TbAct.F/R; Supplementary Table 1) genes were amplified as internal controls for wheat and tobacco, respectively. Three biological replicates were amplified separately in real-time
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PCR assays for each satellite-virus combination, as described earlier [19]. Semi-quantitative and real-time qRT PCR revealed that the accumulation of WDIV or AEV was highest in plants inoculated with WDIV and AYLCB_1365 or AEV and AYLCB_1365, (Fig. 3K-N). Accumulation of WDIV or AEV was lower in the plants inoculated with a defective AYLCB molecule together with WDIV or AEV, similar to the plants inoculated with WDIV or AEV alone (Fig. 3K-N). Detection and sequencing of full-length (1365 bp) and defective AYLCB molecules (657, 891, 929, 1046 and 1084 bp) from systemically infected wheat and tobacco leaves showed that they were maintained by WDIV as well as AEV in their respective hosts. The region predicted to be responsible for the trans-replication of a betasatellite [20] was present in all the betasatellites – full-length as well as defective molecules. Conservation of the trans-replication region among the AYLCB sequences (Fig. 2) and the maintenance of bC1-less/truncated defective AYLCB molecules by WDIV and AEV (Fig. 3O and P), indicates that the region between the A-rich region and the SCR, as predicted earlier [20], is responsible for trans-replication of the betasatellite. This also suggests that the bC1 gene is not required for trans-replication of betasatellite with the helper viruses, AEV and WDIV. However, more-severe viral disease symptoms (Fig. 3A-J) and the higher accumulation of helper viruses (WDIV and AEV) in the presence of full-length AYLCB in comparison to the defective AYLCB (Fig. 3K-N) show that bC1 is a pathogenicity determinant and helps in virus accumulation. The presence of defective AYLCB molecules in a field population of wheat plants is presumably the result of errors during DNA replication (polymerase ‘jumping’) or some other mechanism [21]. The fact that the defective AYLCB detected by us are maintained in the infection, suggests that the deletions are not in the region essential for replication. In fact, in some cases, deletions have been predicted to give a replicative advantage to such defective molecules [22]. The defective molecules have been suggested to compete with full-length molecules for replication and, as a consequence, lead to delay or attenuation of symptoms [22–24]. bC1 is known as a pathogenicity determinant for begomoviruses in infection of permissive host plants [4–6]. However, this is the first study in which the involvement of bC1 in the determination of pathogenicity of a mastrevirus has been reported. The trans-replication ability and involvement of a betasatellite, AYLCB, in symptom severity of a mastrevirus can have serious implications for the economic impact of the virus on crop yield. Acknowledgements The authors are grateful to the Department of Biotechnology, Government of India for supporting the present work
at National Agri-Food Biotechnology Institute, Mohali, India; to the Council of Scientific and Industrial Research for Senior Research Fellowship to JK and JK; and to the Department of Science and Technology, Government of India, for the JC Bose Fellowship to RT.
References 1. Briddon RW, Bull SE, Amin I, Idris AM, Mansoor S, Bedford ID, Dhawan P, Rishi N, Siwatch SS, Abdel-Salam AM, Brown JK, Zafar Y, Markham PG (2003) Diversity of DNAb a satellite molecule associated with some monopartite begomoviruses. Virology 312:106–121 2. Briddon RW, Stanley J (2006) Subviral agents associated with plant single stranded DNA viruses. Virology 344:198–210 3. Nawaz-ul-Rehman MS, Fauquet CM (2009) Evolution of geminiviruses and their satellites. FEBS Lett 583:1825–1832 4. Saeed M, Behjatnia SAA, Mansoor S, Zafar Y, Hasnain S, Rezaian MA (2005) A single complementary-sense transcript of a geminiviral DNA b satellite is determinant of pathogenicity. Mol Plant Microbe Interact 18:7–14 5. Tahir MN, Mansoor S (2011) bC1 of chili leaf curl betasatellite is a pathogenicity determinant. Virol J 8:509 6. Saunders K, Norman A, Gucciardo S, Stanley J (2004) The DNA b satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein (bC1). Virology 324:37–47 7. Cui X, Tao X, Xie Y, Fauquet CM, Zhou X (2004) A DNAb associated with Tomato yellow leaf curl China virus is required for symptom induction. J Virol 78:13966–13974 8. Li Z, Xie Y, Zhou X (2005) Tobacco curly shoot virus DNA b is not necessary for infection but intensifies symptoms in a hostdependent manner. Phytopathology 95:902–908 9. Cui X, Li G, Wang D, Hu D, Zhou X (2005) A begomovirus DNAb-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus. J Virol 79:10764–10775 10. Eini O, Dogra SC, Dry IB, Randles JW (2010) Silencing suppressor activity of a begomovirus DNAb encoded protein and its effect on heterologous helper virus replication. Virus Res 167:97–101 11. Saeed M, Zafar Y, Randles JW, Rezaian MA (2007) A monopartite begomovirus-associated DNA b satellite substitutes for the DNA B of a bipartite begomovirus to permit systemic infection. J Gen Virol 88:2881–2889 12. Nawaz-ul-Rehman MS, Mansoor S, Briddon RW, Fauquet CM (2009) Maintenance of an old world betasatellite by a new world helper begomovirus and possible rapid adaptation of the betasatellite. J Virol 83:9347–9355 13. Patil BL, Fauquet CM (2010) Differential interaction between cassava mosaic geminiviruses and geminivirus satellites. J Gen Virol 91:1871–1882 14. Briddon RW, Mansoor S, Bedford ID, Pinner MS, Saunders K, Stanley J, Zafar Y, Malik KA, Markham PG (2001) Identification of DNA components required for induction of cotton leaf curl disease. Virology 285:234–243 15. Jose J, Usha R (2003) Bhendi yellow vein mosaic disease in India is caused by association of a DNA b satellite with a begomovirus. Virology 305:310–317 16. Rouhibakhsh A, Malathi VG (2005) Severe leaf curl disease of cowpea—a new disease of cowpea in northern India caused by Mungbean yellow mosaic India virus and a satellite DNAb. Plant Pathol 54:259
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J. Kumar et al. 17. Jyothsna P, Haq QM, Singh P, Sumiya KV, Praveen S, Rawat R, Briddon RW, Malathi VG (2013) Infection of Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus with betasatellites, results in enhanced level of helper virus components and antagonistic interaction between DNA B and betasatellites. Appl Microbiol Biotechnol 97:5457–5471 18. Sivalingam PN, Varma A (2012) Role of betasatellite in the pathogenesis of a bipartite begomovirus affecting tomato in India. Arch Virol 157:1081–1092 19. Kumar J, Kumar J, Singh SP, Tuli R (2014) Association of satellites with a mastrevirus in natural infection: complexity of Wheat dwarf India virus disease. J Virol 88:7093–7104. doi:10.1128/JVI.02911-13 20. Saunders K, Briddon RW, Stanley J (2008) Replication promiscuity of DNA-beta satellites associated with monopartite begomoviruses; deletion mutagenesis of the Ageratum yellow vein virus DNA-beta satellite localizes sequences involved in replication. J Gen Virol 89:3165–3172
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21. Stenger DC, Stevenson MC, Hormuzdi SG, Bisaro DM (1992) A number of subgenomic DNAs are produced following agroinoculation of plants with beet curly top virus. J Gen Virol 73:237–242 22. Stanley J, Frischmuth T, Ellwood S (1990) Defective viral DNA ameliorates symptoms of geminivirus infection in transgenic plants. Proc Nat Acad Sci USA 87:6291–6295 23. Stanley J, Saunders K, Pinner MS, Wong SM (1997) Novel defective interfering DNAs associated with ageratum yellow vein geminivirus infection of Ageratum conyzoides. Virology 239:87–96 24. Frischmuth T, Stanley J (1994) Beet curly top virus symptom amelioration in Nicotiana benthamiana transformed with a naturally occurring viral subgenomic DNA. Virology 200:826–830
BRIEF REPORT
bC1 is a pathogenicity determinant: not only for begomoviruses but also for a mastrevirus Jitendra Kumar • Jitesh Kumar • Sudhir P. Singh Rakesh Tuli
•
Received: 13 March 2014 / Accepted: 8 June 2014 Ó Springer-Verlag Wien 2014
Abstract bC1 proteins, encoded by betasatellites, are known to be pathogenicity determinants, and they are responsible for symptom expression in many devastating diseases caused by begomoviruses. We report the involvement of bC1 in pathogenicity determination of a mastrevirus. Analysis of field samples of wheat plants containing wheat dwarf India virus (WDIV) revealed the presence of a full-length and several defective betasatellite molecules. The detected betasatellite was identified as ageratum yellow leaf curl betasatellite (AYLCB). No begomovirus was detected in any of the samples. The fulllength AYLCB contained an intact bC1 gene, whereas the defective molecules contained complete or partial deletions of bC1. Agroinoculation of wheat with the full-length AYLCB and WDIV or of tobacco with ageratum enation virus enhanced the pathogenicity and accumulation of the respective viruses, whereas the defective molecules could not. This study indicates that bC1 is a pathogenicity determinant for WDIV and can interact functionally not only with begomoviruses but also with a mastrevirus. Keywords AYLCB WDIV AEV Trans-replication Virus accumulation Betasatellites are single-stranded DNA molecules associated with begomoviruses (family Geminiviridae) [1–3]. They are approximately half the size (*1.3 kb) of their Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2149-5) contains supplementary material, which is available to authorized users. J. Kumar J. Kumar S. P. Singh R. Tuli (&) Department of Biotechnology, National Agri-Food Biotechnology Institute, Mohali 160071, Punjab, India e-mail: [email protected]
helper viruses and encode a single protein, bC1, which is a pathogenicity determinant [4–6]. bC1 suppresses silencing, up-regulates viral DNA levels in plants, helps in virus movement, and modulates virus symptoms and host range [4, 6–13]. Betasatellites share only the nonanucleotide (TAATATTAC) sequence with the helper begomovirus, which is predicted to serve as the origin of replication [1– 3]. Betasatellites depend on their helper viruses for replication, encapsidation and transmission by the insect vector and are often required by their helper viruses for symptom induction in their original hosts [14, 15]. Some monopartite begomoviruses are either poorly infectious or induce atypical symptoms in the absence of the betasatellite [6–8, 12, 14]. The association of betasatellites with monopartite begomoviruses has been reported in numerous diseases of plants [1–3], and in select cases, with bipartite begomoviruses [16–18]. In a recent study, the association of a betasatellite, ageratum yellow leaf curl betasatellite (AYLCB), with a mastrevirus, wheat dwarf India virus (WDIV), which infects wheat, was detected [19]. AYLCB enhanced WDIV accumulation and symptom (dwarfing) severity and remained capable of interacting with ageratum enation virus (AEV) [19]. The present study shows that AYLCB with a full-length bC1 gene enhanced virus (WDIV or AEV) accumulation and symptom severity, whereas defective molecules with bC1 deleted could not. Leaf samples from 50 symptomatic wheat plants were collected during 2011–12 from fields at Mohali, India. The symptomatic plants were selected on the basis of dwarfing and yellowing (Fig. 1A-C). Leaves from five asymptomatic plants were taken as negative controls. Total DNA was isolated using a DNeasy Plant Mini Kit column (QIAGEN GmbH, Germany). WDIV and satellite DNAs were amplified by rolling-circle amplification (RCA) and
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Fig. 1 Wheat plants suspected of being infected with WDIV and detection of the virus and satellite DNAs. Wheat plants (red circle) showing dwarfing with yellowing (A, B) and reddening with yellowing (C). NdeI-digested RCA products showing the fragments of *1.3, *1.6, *2.8 and *5.6 kb in the diseased wheat samples (D). PCR amplicon of *2.8 kb representing the genome size of WDIV (E). PCR products of the betasatellite showing multiple
amplicons of *700, *900 bp and *1.3 kb (F). Lanes 1, 2 and 3 in panels D-F represent the symptomatic samples of panels A, B and C, whereas lane 4 represents an asymptomatic plant sample. M, EcoRI/ HindIII-digested kDNA. The dimeric (WD) and monomeric (WM) forms of WDIV and the partial digestion products of WDIV (WP), betasatellite (beta) and defective betasatellite (M beta) are shown
polymerase chain reaction (PCR). RCA was performed using a TempliPhi Amplification Kit (GE Healthcare, USA), and the RCA products were partially digested with NdeI restriction endonuclease to obtain DNA fragments of WDIV and the satellite. The RCA products were also digested separately with the BamHI, EcoRI, HindIII, KpnI and PstI restriction endonucleases to detect the presence of a begomovirus. The results showed identical products for all samples and thus suggested the presence of identical viruses in all diseased plant samples (data not shown). MF1_FOR/REV was used in PCR assays to amplify WDIV DNA from wheat samples, whereas b01/04 was used to amplify betasatellite DNA (Supplementary Table 1) as described earlier [19]. The RCA and PCR assays were performed on all 55 wheat samples (50 symptomatic and 5 asymptomatic). Both RCA and PCR yielded fragments of *2.7 and 1.3 kb, the sizes expected for WDIV and betasatellite, respectively, whereas the asymptomatic plants did not (Fig. 1D-F). In addition to a fragment of *1.3 kb,
smaller amplicons of *657 and *900 bp were obtained using the betasatellite-specific primers (Fig. 1F). Sequencing of the RCA and PCR products revealed the presence of WDIV (accession no. JF781306) and AYLCB (accession no. KC305085) in the symptomatic wheat samples. Additional fragments of smaller sizes were detected using the betasatellite-specific primers. These represented defective molecules of AYLCB with smaller size (accession nos. KC305086, KC305087, and KC305089-KC305091). Detailed analysis of the sequence revealed that all of the betasatellite molecules, including the full-length (*1.3 kb) and smaller (less than *1.3 kb in length) molecules, retained three highly conserved features: a predicted stem-loop structure with the loop sequence TAATATTAC as the origin of replication (the nonanucleotide sequence), a region of high sequence similarity known as the ‘‘satellite conserved region’’ (SCR), and an adenine (A)-rich region. The full-length betasatellite molecule (1365 bp) contained the positionally and
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Fig. 2 Representation of the genomic organization of the full-length and the defective molecules of AYLCB. The sequences deleted in defective AYLCB molecules are indicated by dashed lines within the solid lines. Size variants of AYLCBs are indicated, with their length
given in bp: 657, 891, 929, 1046, 1084 and 1365 bp. The positions of the bC1 gene, the A-rich region and the satellite conserved region (SCR), are also shown. The region suggested to be responsible for trans-replication of a betasatellite [20] is also shown
sequence-conserved full-length bC1 gene, while the defective molecules contained partial deletions of bC1, except the 657-bp molecule, in which all of bC1 was absent (Fig. 2). Infectious clones of WDIV, AEV and AYLCB (fulllength as well as defective AYLCB) were prepared in the binary vector pCAMBIA1301 (CAMBIA, Canberra, Australia) as described previously [19]. The full-length and defective AYLCBs of 1365, 1084, 1046, 929, 891 and 657 bp are represented by AYLCB_1365, AYLCB_1084, AYLCB_1046, AYLCB_929, AYLCB_891 and AYLCB_657, respectively. Derivatives of the binary vector pCAMBIA1301, containing the infectious constructs were introduced separately into Agrobacterium tumefaciens strain GV3101 by transformation. Leaves of seven-day-old seedlings of 200 wheat plants (25 plants for each of the eight constructs; control, WDIV, WDIV and AYLCB_1365, WDIV and AYLCB_1084, WDIV and AYLCB_1046, WDIV and AYLCB_929, WDIV and AYLCB_891, WDIV and AYLCB_657) at the two- to three-leaf stage were agroinoculated using a needleless syringe. A total of 48 Nicotiana tabacum plants (six plants for each of the eight constructs: control, AEV, AEV and AYLCB_1365, AEV and AYLCB_1084, AEV and AYLCB_1046, AEV and AYLCB_929, AEV and AYLCB_891, AEV and AYLCB_657) at the four-leaf stage were inoculated. The inoculated wheat and tobacco plants were maintained in separate plant growth chambers (PGR14, Conviron, Canada) at 22 and 25 °C, respectively. The presence of WDIV or AEV in the agroinoculated wheat or tobacco plants was investigated using the primer pairs CP01/02 or CPB1/2 (Supplementary Table 1),
respectively, as described previously [19]. The presence of the full-length or defective AYLCB in the agroinoculated, infected plants was investigated using the BSCRF/R primer pair (Supplementary Table 1), which amplifies the sequence (Supplementary Fig. 1) conserved in all of the betasatellites without discriminating between the fulllength and defective molecules. One hundred fifty wheat plants out of the 200 infectious-clone-inoculated plants showed the presence of WDIV (Fig. 3K) and/or AYLCB (Fig. 3N). A total of 38 N. tabacum plants out of the 48 infectious-clone-inoculated plants, showed the presence of AEV (Fig. 3L) and/or AYLCB (Fig. 3O). The PCRamplified products from the inoculated plant samples were sequenced to confirm their identity to the sequence of the initially inoculated clones. Wheat plants harboring WDIV showed dwarfing in comparison to the mock-inoculated control (Fig. 3A, B), whereas the plants containing WDIV and full-length AYLCB (1365 bp; Fig. 2) showed more-severe stunting (Fig. 3E). Wheat plants with WDIV and defective molecules of AYLCB showed a phenotype similar to that of the plants inoculated with WDIV in the absence of AYLCB (Fig. 3B, C, D). The average height of five wheat plants at 42 days postinfection (dpi), in the case of the control and those infected with WDIV and full-length AYLCB, WDIV and defective AYLCB and WDIV, alone was 70.3 ± 2.51, 35.3 ± 1.52, 53.3 ± 3.05 and 54.3 ± 1.52 cm, respectively. Tobacco plants containing AEV showed leaf curling and stunting in comparison to mock-inoculated control (Fig. 3F, G). The viral symptoms, leaf curling and stunting, were more severe in the presence of AEV and full-length AYLCB (Fig. 3J). Plants with AEV and defective AYLCB
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Fig. 3 Effects of the AYLCB on the symptom severity and accumulation of WDIV and AEV in wheat and tobacco, respectively. Mock-inoculated wheat plants and those inoculated with (A), WDIV (B), WDIV and 1084-bp AYLCB (C), WDIV and 657-bp AYLCB (D) and WDIV and 1365-bp AYLCB (E) at 35 dpi are shown. Mockinoculated tobacco plants (A), and those inocuated with AEV (B), AEV and 1084-bp AYLCB (C), AEV and 657-bp AYLCB (D), and AEV and 1365-bp AYLCB (E) at 30 dpi are shown. Semi-quantitative PCR (K, L) and real-time qPCR (M, N) – based on three replicates – were used for analysis of WDIV (K, M) or AEV (L, N) from agroinoculated, infected wheat and tobacco plants,
respectively. The CP genes of WDIV (WCP) and AEV (ACP) were amplified by semi-quantitative PCR (K, L) and real-time qPCR (M, N). The samples used for semi-quantitative PCR (K, L) and real-time PCR (M, N) were as follows: virus alone (WDIV or AEV; lane 1), virus and 657-bp AYLCB (lane 2), virus and 891-bp AYLCB (lane 3), virus and 929-bp AYLCB (lane 4), virus and 1046-bp AYLCB (lane 5), virus and 1084-bp AYLCB (lane 6), virus and 1365-bp AYLCB (lane 7). The results of semi-quantitative PCR-based quantification of AYLCB in wheat (O) and tobacco (P) are shown. The same samples used for panels (K) and (L) were used for the quantification of AYLCB (O, P)
molecules showed viral symptoms, leaf curling and stunting, similar to the plants inoculated with AEV in the absence of AYLCB (3G-I). The average height of three tobacco plants at 35 dpi, in the case of the control and those infected with AEV and full-length AYLCB, AEV and defective AYLCB and AEV alone, was 50.41 ± 3.51, 12.4 ± 3.02, 28.15 ± 4.05 and 30.2 ± 4.44 cm, respectively. The coat protein genes of WDIV and AEV were amplified by real-time qPCR and semi-quantitative PCR
for quantification of virus accumulation in the presence of full-length and defective AYLCB molecules. The concentration of total DNA from the inoculated, infected plants was adjusted to 50 ng ll-1, and the primer pairs CP01/02 and CPB1/2 (Supplementary Table 1) were used for WDIV and AEV, respectively. Elongation factor-1 alpha (EF1afor/rev; Supplementary Table 1) and actin (TbAct.F/R; Supplementary Table 1) genes were amplified as internal controls for wheat and tobacco, respectively. Three biological replicates were amplified separately in real-time
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Role of betasatellite bC1 in mastrevirus pathogenicity
PCR assays for each satellite-virus combination, as described earlier [19]. Semi-quantitative and real-time qRT PCR revealed that the accumulation of WDIV or AEV was highest in plants inoculated with WDIV and AYLCB_1365 or AEV and AYLCB_1365, (Fig. 3K-N). Accumulation of WDIV or AEV was lower in the plants inoculated with a defective AYLCB molecule together with WDIV or AEV, similar to the plants inoculated with WDIV or AEV alone (Fig. 3K-N). Detection and sequencing of full-length (1365 bp) and defective AYLCB molecules (657, 891, 929, 1046 and 1084 bp) from systemically infected wheat and tobacco leaves showed that they were maintained by WDIV as well as AEV in their respective hosts. The region predicted to be responsible for the trans-replication of a betasatellite [20] was present in all the betasatellites – full-length as well as defective molecules. Conservation of the trans-replication region among the AYLCB sequences (Fig. 2) and the maintenance of bC1-less/truncated defective AYLCB molecules by WDIV and AEV (Fig. 3O and P), indicates that the region between the A-rich region and the SCR, as predicted earlier [20], is responsible for trans-replication of the betasatellite. This also suggests that the bC1 gene is not required for trans-replication of betasatellite with the helper viruses, AEV and WDIV. However, more-severe viral disease symptoms (Fig. 3A-J) and the higher accumulation of helper viruses (WDIV and AEV) in the presence of full-length AYLCB in comparison to the defective AYLCB (Fig. 3K-N) show that bC1 is a pathogenicity determinant and helps in virus accumulation. The presence of defective AYLCB molecules in a field population of wheat plants is presumably the result of errors during DNA replication (polymerase ‘jumping’) or some other mechanism [21]. The fact that the defective AYLCB detected by us are maintained in the infection, suggests that the deletions are not in the region essential for replication. In fact, in some cases, deletions have been predicted to give a replicative advantage to such defective molecules [22]. The defective molecules have been suggested to compete with full-length molecules for replication and, as a consequence, lead to delay or attenuation of symptoms [22–24]. bC1 is known as a pathogenicity determinant for begomoviruses in infection of permissive host plants [4–6]. However, this is the first study in which the involvement of bC1 in the determination of pathogenicity of a mastrevirus has been reported. The trans-replication ability and involvement of a betasatellite, AYLCB, in symptom severity of a mastrevirus can have serious implications for the economic impact of the virus on crop yield. Acknowledgements The authors are grateful to the Department of Biotechnology, Government of India for supporting the present work
at National Agri-Food Biotechnology Institute, Mohali, India; to the Council of Scientific and Industrial Research for Senior Research Fellowship to JK and JK; and to the Department of Science and Technology, Government of India, for the JC Bose Fellowship to RT.
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