Arch Virol DOI 10.1007/s00705-015-2471-6
ANNOTATED SEQUENCE RECORD
Complete genome sequence of a novel monopartite geminivirus identified in mulberry (Morus alba L.) Quan-You Lu1,2 • Zu-Jian Wu3 • Zhi-Song Xia1,2 • Lian-Hui Xie3
Received: 19 March 2015 / Accepted: 26 May 2015 Ó Springer-Verlag Wien 2015
Abstract The genome sequence of a novel geminivirus from mulberry samples exhibiting crinkle leaf symptoms is reported. The sequence consisted of 2952 nt, containing four open reading frames (ORFs) in the viral-sense strand and two ORFs in the complementary-sense strand. The size of the genome and the conserved origin of replication are similar to those of members of the family Geminiviridae, but the genomic organization, number of ORFs, and especially five contiguous GAAAAA repeats positioned upstream of ORF1 distinguish it from other geminiviruses. Phylogenetic analysis coupled with ORF analysis suggests that this is a novel virus that does not fit into the established seven genera of the family Geminiviridae. The virus, found in Zhenjiang, Jiangsu province, China, is tentatively named mulberry crinkle leaf virus isolate Jiangsu (MCLV-js).
& Lian-Hui Xie [email protected] Quan-You Lu [email protected] 1
Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China
2
Key Laboratory of Genetic Improvement of Silkworm and Mulberry, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangs 212018, China
3
Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
Geminiviruses, characterized by their twinned incomplete icosahedral capsid morphology, are plant viruses with single-stranded DNA genomes. The particles are about 30 9 18 nm in size. They infect numerous economically important crops, including tomato [1], cotton [2], and maize [3], and cause severe economic losses to agricultural production worldwide [4]. Four nucleotide sequence fragments were obtained from mulberry (Morus alba L.) by sequenceindependent amplification [5]. These four partially overlapping contigs, ranging from 272 to 542 nt in size, were assembled into a DNA sequence of 760 nt. BLASTx analysis showed that the amino acid sequence encoded by this fragment has homology to geminivirus-encoded proteins. We provide here the first report of the full-length sequence of a geminivirus genome from mulberry. Total DNA was extracted from mulberry leaves with crinkle leaf symptoms using a Rapid Plant Genomic DNA Isolation Kit (Sangon, Shanghai China), following the manufacturer’s instructions. The viral genome was amplified by PCR using a pair of adjacent specific primers of opposite polarity, PIF/PIR (PIF, 50 -TACATTATCATCGA ACCATGG-30 ; PIR, 50 -TTAGTTTATTACATGTCTGCT AGTG-30 ), based on a partial sequence. The PCR product was separated and recovered by electrophoresis on a 1.0 % agarose gel. The recovered DNA fragment (ca 3.0 kb) was ligated into pGEM-T Easy Vector (Promega) and sequenced using M13 primers and primer walking. Based on the first sequence of the complete viral genome, another pair of adjacent specific primers of opposite polarity, PIIF/ PIIR (PIIF, 50 -GCTCTGCTCTGAGATCAAGG-30 ; PIIR, 50 -TCCTGGTAATCGCCATTTAC-30 ), was synthesized to generate a second sequence of the complete viral genome and correct the first complete viral genome sequence. Primer synthesis and sequencing were performed by Shanghai Sangon, China. Two cycles of sequencing showed that the
123
Q.-Y. Lu et al.
complete nucleotide sequence of the viral genome was 2952 nt in size (GenBank accession number KR131749). Forty diseased mulberry leaf samples exhibiting crinkle leaf symptoms (Fig. 1, left) and fifteen healthy mulberry leaf samples (Fig. 1 right) were collected from an old mulberry field and a newly-developed mulberry field in Zhenjiang, Jiangsu Province, China, in September 2013 and were tested by PCR using the virus-specific primers MCV-DF (50 -CCATTTGACCCATTTTGACC-30 ) and MCVDR (50 -ATTAGCAGTCAACGTCACATTC-30 ). Thirty-nine of the symptomatic mulberry leaf samples were positive for geminivirus DNA, whereas none of the nonsymptomatic healthy samples contained geminivirus DNA. We presumed that the geminivirus was responsible for the crinkle leaf symptoms of the mulberry leaf. Thus, the geminivirus was tentatively named mulberry crinkle leaf virus isolate Jiangsu (MCLV-js). A second component DNA (DNA-B) and satellite molecules of MCLV-js were not detected by the rollingTM circle amplification (RCA) method [6] using a TempliPhi kit (GE Healthcare) combined with digestion assays with Hind III, Sal I, Xba I, Pac I, Sph I, Spe I, and Sac II endonucleases, the recognition sites for which were absent in the MCLV-js DNA molecule. These results suggest that MCLV-js is a monopartite geminivirus. There were six open reading frames (ORFs) in the MCLV-js DNA (Fig. 2B): four (V2, V3, V1, and V4) in the viral-sense strand and two (C1 and C2) in the 50 half of the complementary-sense strand. A large intergenic region (LIR) and a small intergenic region (SIR) were detected. The LIR, consisting of 346 nt, was located between C1 and V2. A conserved TAATATTAC sequence found in all other geminiviruses [7] was found in the MCLV-js DNA and positioned in the loop of a potential stem-loop structure within the LIR (Fig. 2A). The SIR, consisting of 37 nt, was located between V1 and V4. No TATA box was found upstream of V2 by computer-assisted search for putative transcription regulatory sequences. However, interestingly, five contiguous GAAAAA repeats were located in the upstream region of V2. The SIR contains a TATA box, which possibly functions in regulation of transcription V4. Analyzed by BLASTp, the amino acid sequences of V2 encoded by ORF1 and V4 encoded by ORF4 did not show similarity to those encoded by other geminiviruses. V1 encoded by ORF3, V3 encoded by ORF2, C1 encoded by ORF5, and C2 encoded by ORF6 were identified as the
123
putative coat protein (CP), the putative movement protein (MP), the viral replication-associated protein (Rep A), and the C1:C2-like Rep protein, respectively. The putative CP, MP, Rep A, and the C1:C2-like Rep protein of MCLV-js share maximum amino acid sequence identities of 40 %, 31 %, 45 %, and 63 % with those of citrus chlorotic dwarfassociated virus (CCDaV), an unclassified member of the family Geminiviridae [8]. Phylogenetic analysis based on the complete nucleotide sequence of the genome (Fig. 2C) and the amino acid sequences of the Rep A (Fig. 2D) and CP (not shown) showed that MCLV-js is closely related to CCDaV and clusters in a branch with CCDaV. However, MCLV-js does not fit into the established seven genera [7, 11] within the family Geminiviridae. The partial genomic sequence of a geminivirus, mulberry mosaic virus, found in mulberry plants cultivated near a cassava plantation, was available in GenBank. BLAST analysis showed that MCLV-js was different from mulberry mosaic virus (FJ827040 and HM138689). This is the first report of the complete genomic sequence of a geminivirus from mulberry. Further studies are in progress to estimate the infectivity, prevalence of MCLV-js, its impact on mulberry production, and the relationship between MCLV-js and expression of mulberry crinkle leaf symptoms.
Fig. 1 The diseased mulberry leaf with crinkle leaf symptom (left) and the healthy mulberry leaf (right)
Genome sequence of a monopartite geminivirus in mulberry
Fig. 2 A stem-loop structure containing a conserved TAATATT/AC sequence within the intergenic region (A), genomic organization of mulberry crinkle leaf virus isolate Jiangsu (MCLV-js) (B), and neighbor-joining (NJ) phylogenetic trees based on the alignment of the complete nucleotide sequence of the genome (C), and the amino acid sequences of the Rep A protein of MCLV-js and other representative geminiviruses with monopartite genomes (D). The solid arrows indicate the location and polarity of the predicted open reading frames (ORFs). The arrow in the stem-loop indicates the location of the DNA origin of replication, and the underlined A in the conserved TAATATT/AC was assigned as position 1. Phylogenetic trees were constructed using the NJ method in the program MEGA 3.1 [9]. The multiple sequence alignment of nucleotides and amino acids was performed using Clustal X [10]. The virus names, abbreviations and GenBank accession numbers are as follows:
MSV, maize streak virus (EF547097); SSEV-Asw, sugarcane streak Egypt virus isolate Asw (AF039528); WDV, wheat dwarf virus (EF536859); TbYDV, tobacco yellow dwarf virus (M81103); BCTVCA/Logan [US-Cal-85], beet curly top virus (M24597); HCTV [USSal-88], horseradish curly top virus (U49907); SpSCTV [US-AZSp09-10-09], spinach severe curly top virus (GU734126); TPCTV, tomato pseudo-curly top virus (X84735); BCTIV-Siv, beet curly top Iran virus isolate Siv (JX082259); SCTAV, spinach curly top Arizona virus (HQ443515); ECSV-A, Eragrostis curvula streak virus isolate A (FJ665632); ECSV-B (FJ665630); TCTV-A, turnip curly top virus isolate A (GU456689); TCTV-B (KC108896); TCTV-C (KC108905); CCDaV, citrus chlorotic dwarf associated virus (JQ920490); GCFaV, grapevine cabernet franc-associated virus (JQ901105); FBSLCV, French bean severe leaf curl virus (JX094280); mMCLV-js (this study)
Acknowledgments This study was supported by funds from the National Basic Research Program of China (973 Program) (2014CB138402) and Natural Science Foundation of Jiangsu Province (No. BK2006084). The authors thank James L. Starr (Texas A&M University) for his editorial assistance in the preparation of this manuscript.
2. Sattar MN, Kvarnheden A, Saeed M, Briddon RW (2013) Cotton leaf curl disease—an emerging threat to cotton production worldwide. J Gen Virol 94:695–710 3. Shepherd DN, Martin DP, Van Der Walt E, Dent K, Varsani A, Rybicki EP (2010) Maize streak virus: an old and complex ‘emerging’ pathogen. Mol Plant Pathol 11:1–12 4. Rojas MR, Hagen C, Lucas WJ, Gilbertson RL (2005) Exploiting chinks in the plant’s armor: evolution and emergence of geminiviruses. Annu Rev Phytopathol 43:361–394 5. Bohlander SK, Espinosa R, Le Beau MM, Rowley JD, Dı´az MO (1992) A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics 13:1322–1324
References 1. Czosnet H, Laterrot H (1997) A worldwide survey of tomato yellow leaf curl viruses. Arch Virol 142:1391–1406
123
Q.-Y. Lu et al. 6. Shepherd DN, Martin DP, Lefeuvre P, Monjane AL, Owor BE, Rybicki EP, Varsani A (2008) A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. J Virol Methods 149:97–102 7. Brown JK, Fauquet CM, Briddon RW, Zerbini M, Moriones E, Navas-Castillo J (2012) Family Geminiviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses. Elsevier/Academic Press, Amsterdam, pp 351–373 8. Loconsole G, Saldarelli P, Doddapaneni H, Savino V, Martelli GP, Saponari M (2012) Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae. Virology 432:162–172
123
9. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163 10. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25(24):4876–4882 11. Varsani A, Navas-Castillo J, Moriones E, Herna´ndez-Zepeda C, Idris A, Brown JK, Zerbini FM, Martin DP (2014) Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus. Arch Virol 159:2193–2203
ANNOTATED SEQUENCE RECORD
Complete genome sequence of a novel monopartite geminivirus identified in mulberry (Morus alba L.) Quan-You Lu1,2 • Zu-Jian Wu3 • Zhi-Song Xia1,2 • Lian-Hui Xie3
Received: 19 March 2015 / Accepted: 26 May 2015 Ó Springer-Verlag Wien 2015
Abstract The genome sequence of a novel geminivirus from mulberry samples exhibiting crinkle leaf symptoms is reported. The sequence consisted of 2952 nt, containing four open reading frames (ORFs) in the viral-sense strand and two ORFs in the complementary-sense strand. The size of the genome and the conserved origin of replication are similar to those of members of the family Geminiviridae, but the genomic organization, number of ORFs, and especially five contiguous GAAAAA repeats positioned upstream of ORF1 distinguish it from other geminiviruses. Phylogenetic analysis coupled with ORF analysis suggests that this is a novel virus that does not fit into the established seven genera of the family Geminiviridae. The virus, found in Zhenjiang, Jiangsu province, China, is tentatively named mulberry crinkle leaf virus isolate Jiangsu (MCLV-js).
& Lian-Hui Xie [email protected] Quan-You Lu [email protected] 1
Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China
2
Key Laboratory of Genetic Improvement of Silkworm and Mulberry, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangs 212018, China
3
Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China
Geminiviruses, characterized by their twinned incomplete icosahedral capsid morphology, are plant viruses with single-stranded DNA genomes. The particles are about 30 9 18 nm in size. They infect numerous economically important crops, including tomato [1], cotton [2], and maize [3], and cause severe economic losses to agricultural production worldwide [4]. Four nucleotide sequence fragments were obtained from mulberry (Morus alba L.) by sequenceindependent amplification [5]. These four partially overlapping contigs, ranging from 272 to 542 nt in size, were assembled into a DNA sequence of 760 nt. BLASTx analysis showed that the amino acid sequence encoded by this fragment has homology to geminivirus-encoded proteins. We provide here the first report of the full-length sequence of a geminivirus genome from mulberry. Total DNA was extracted from mulberry leaves with crinkle leaf symptoms using a Rapid Plant Genomic DNA Isolation Kit (Sangon, Shanghai China), following the manufacturer’s instructions. The viral genome was amplified by PCR using a pair of adjacent specific primers of opposite polarity, PIF/PIR (PIF, 50 -TACATTATCATCGA ACCATGG-30 ; PIR, 50 -TTAGTTTATTACATGTCTGCT AGTG-30 ), based on a partial sequence. The PCR product was separated and recovered by electrophoresis on a 1.0 % agarose gel. The recovered DNA fragment (ca 3.0 kb) was ligated into pGEM-T Easy Vector (Promega) and sequenced using M13 primers and primer walking. Based on the first sequence of the complete viral genome, another pair of adjacent specific primers of opposite polarity, PIIF/ PIIR (PIIF, 50 -GCTCTGCTCTGAGATCAAGG-30 ; PIIR, 50 -TCCTGGTAATCGCCATTTAC-30 ), was synthesized to generate a second sequence of the complete viral genome and correct the first complete viral genome sequence. Primer synthesis and sequencing were performed by Shanghai Sangon, China. Two cycles of sequencing showed that the
123
Q.-Y. Lu et al.
complete nucleotide sequence of the viral genome was 2952 nt in size (GenBank accession number KR131749). Forty diseased mulberry leaf samples exhibiting crinkle leaf symptoms (Fig. 1, left) and fifteen healthy mulberry leaf samples (Fig. 1 right) were collected from an old mulberry field and a newly-developed mulberry field in Zhenjiang, Jiangsu Province, China, in September 2013 and were tested by PCR using the virus-specific primers MCV-DF (50 -CCATTTGACCCATTTTGACC-30 ) and MCVDR (50 -ATTAGCAGTCAACGTCACATTC-30 ). Thirty-nine of the symptomatic mulberry leaf samples were positive for geminivirus DNA, whereas none of the nonsymptomatic healthy samples contained geminivirus DNA. We presumed that the geminivirus was responsible for the crinkle leaf symptoms of the mulberry leaf. Thus, the geminivirus was tentatively named mulberry crinkle leaf virus isolate Jiangsu (MCLV-js). A second component DNA (DNA-B) and satellite molecules of MCLV-js were not detected by the rollingTM circle amplification (RCA) method [6] using a TempliPhi kit (GE Healthcare) combined with digestion assays with Hind III, Sal I, Xba I, Pac I, Sph I, Spe I, and Sac II endonucleases, the recognition sites for which were absent in the MCLV-js DNA molecule. These results suggest that MCLV-js is a monopartite geminivirus. There were six open reading frames (ORFs) in the MCLV-js DNA (Fig. 2B): four (V2, V3, V1, and V4) in the viral-sense strand and two (C1 and C2) in the 50 half of the complementary-sense strand. A large intergenic region (LIR) and a small intergenic region (SIR) were detected. The LIR, consisting of 346 nt, was located between C1 and V2. A conserved TAATATTAC sequence found in all other geminiviruses [7] was found in the MCLV-js DNA and positioned in the loop of a potential stem-loop structure within the LIR (Fig. 2A). The SIR, consisting of 37 nt, was located between V1 and V4. No TATA box was found upstream of V2 by computer-assisted search for putative transcription regulatory sequences. However, interestingly, five contiguous GAAAAA repeats were located in the upstream region of V2. The SIR contains a TATA box, which possibly functions in regulation of transcription V4. Analyzed by BLASTp, the amino acid sequences of V2 encoded by ORF1 and V4 encoded by ORF4 did not show similarity to those encoded by other geminiviruses. V1 encoded by ORF3, V3 encoded by ORF2, C1 encoded by ORF5, and C2 encoded by ORF6 were identified as the
123
putative coat protein (CP), the putative movement protein (MP), the viral replication-associated protein (Rep A), and the C1:C2-like Rep protein, respectively. The putative CP, MP, Rep A, and the C1:C2-like Rep protein of MCLV-js share maximum amino acid sequence identities of 40 %, 31 %, 45 %, and 63 % with those of citrus chlorotic dwarfassociated virus (CCDaV), an unclassified member of the family Geminiviridae [8]. Phylogenetic analysis based on the complete nucleotide sequence of the genome (Fig. 2C) and the amino acid sequences of the Rep A (Fig. 2D) and CP (not shown) showed that MCLV-js is closely related to CCDaV and clusters in a branch with CCDaV. However, MCLV-js does not fit into the established seven genera [7, 11] within the family Geminiviridae. The partial genomic sequence of a geminivirus, mulberry mosaic virus, found in mulberry plants cultivated near a cassava plantation, was available in GenBank. BLAST analysis showed that MCLV-js was different from mulberry mosaic virus (FJ827040 and HM138689). This is the first report of the complete genomic sequence of a geminivirus from mulberry. Further studies are in progress to estimate the infectivity, prevalence of MCLV-js, its impact on mulberry production, and the relationship between MCLV-js and expression of mulberry crinkle leaf symptoms.
Fig. 1 The diseased mulberry leaf with crinkle leaf symptom (left) and the healthy mulberry leaf (right)
Genome sequence of a monopartite geminivirus in mulberry
Fig. 2 A stem-loop structure containing a conserved TAATATT/AC sequence within the intergenic region (A), genomic organization of mulberry crinkle leaf virus isolate Jiangsu (MCLV-js) (B), and neighbor-joining (NJ) phylogenetic trees based on the alignment of the complete nucleotide sequence of the genome (C), and the amino acid sequences of the Rep A protein of MCLV-js and other representative geminiviruses with monopartite genomes (D). The solid arrows indicate the location and polarity of the predicted open reading frames (ORFs). The arrow in the stem-loop indicates the location of the DNA origin of replication, and the underlined A in the conserved TAATATT/AC was assigned as position 1. Phylogenetic trees were constructed using the NJ method in the program MEGA 3.1 [9]. The multiple sequence alignment of nucleotides and amino acids was performed using Clustal X [10]. The virus names, abbreviations and GenBank accession numbers are as follows:
MSV, maize streak virus (EF547097); SSEV-Asw, sugarcane streak Egypt virus isolate Asw (AF039528); WDV, wheat dwarf virus (EF536859); TbYDV, tobacco yellow dwarf virus (M81103); BCTVCA/Logan [US-Cal-85], beet curly top virus (M24597); HCTV [USSal-88], horseradish curly top virus (U49907); SpSCTV [US-AZSp09-10-09], spinach severe curly top virus (GU734126); TPCTV, tomato pseudo-curly top virus (X84735); BCTIV-Siv, beet curly top Iran virus isolate Siv (JX082259); SCTAV, spinach curly top Arizona virus (HQ443515); ECSV-A, Eragrostis curvula streak virus isolate A (FJ665632); ECSV-B (FJ665630); TCTV-A, turnip curly top virus isolate A (GU456689); TCTV-B (KC108896); TCTV-C (KC108905); CCDaV, citrus chlorotic dwarf associated virus (JQ920490); GCFaV, grapevine cabernet franc-associated virus (JQ901105); FBSLCV, French bean severe leaf curl virus (JX094280); mMCLV-js (this study)
Acknowledgments This study was supported by funds from the National Basic Research Program of China (973 Program) (2014CB138402) and Natural Science Foundation of Jiangsu Province (No. BK2006084). The authors thank James L. Starr (Texas A&M University) for his editorial assistance in the preparation of this manuscript.
2. Sattar MN, Kvarnheden A, Saeed M, Briddon RW (2013) Cotton leaf curl disease—an emerging threat to cotton production worldwide. J Gen Virol 94:695–710 3. Shepherd DN, Martin DP, Van Der Walt E, Dent K, Varsani A, Rybicki EP (2010) Maize streak virus: an old and complex ‘emerging’ pathogen. Mol Plant Pathol 11:1–12 4. Rojas MR, Hagen C, Lucas WJ, Gilbertson RL (2005) Exploiting chinks in the plant’s armor: evolution and emergence of geminiviruses. Annu Rev Phytopathol 43:361–394 5. Bohlander SK, Espinosa R, Le Beau MM, Rowley JD, Dı´az MO (1992) A method for the rapid sequence-independent amplification of microdissected chromosomal material. Genomics 13:1322–1324
References 1. Czosnet H, Laterrot H (1997) A worldwide survey of tomato yellow leaf curl viruses. Arch Virol 142:1391–1406
123
Q.-Y. Lu et al. 6. Shepherd DN, Martin DP, Lefeuvre P, Monjane AL, Owor BE, Rybicki EP, Varsani A (2008) A protocol for the rapid isolation of full geminivirus genomes from dried plant tissue. J Virol Methods 149:97–102 7. Brown JK, Fauquet CM, Briddon RW, Zerbini M, Moriones E, Navas-Castillo J (2012) Family Geminiviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy: classification and nomenclature of viruses: ninth report of the international committee on taxonomy of viruses. Elsevier/Academic Press, Amsterdam, pp 351–373 8. Loconsole G, Saldarelli P, Doddapaneni H, Savino V, Martelli GP, Saponari M (2012) Identification of a single-stranded DNA virus associated with citrus chlorotic dwarf disease, a new member in the family Geminiviridae. Virology 432:162–172
123
9. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163 10. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 25(24):4876–4882 11. Varsani A, Navas-Castillo J, Moriones E, Herna´ndez-Zepeda C, Idris A, Brown JK, Zerbini FM, Martin DP (2014) Establishment of three new genera in the family Geminiviridae: Becurtovirus, Eragrovirus and Turncurtovirus. Arch Virol 159:2193–2203
