Arch Virol DOI 10.1007/s00705-014-2327-5
BRIEF REPORT
Complete genome sequence of nine isolates of canna yellow streak virus reveals its relationship to the sugarcane mosaic virus (SCMV) subgroup of potyviruses Ravendra P. Chauhan • Punsasi Rajakaruna Jeanmarie Verchot
•
Received: 13 October 2014 / Accepted: 20 December 2014 Ó Springer-Verlag Wien 2015
Abstract Complete genome sequences were obtained from nine isolates of canna yellow streak virus (CaYSV). CaYSV belongs to the sugarcane mosaic virus (SCMV) subgroup of potyviruses with johnsongrass mosaic virus (JGMV) as its closest relative. Multiple sequence alignments showed a pattern of amino acid substitutions in the CP sequences, which enabled us to relate these isolates to South East Asian or European isolates. Biological characterization of CaYSV identified Nicotiana benthamiana, Chenopodium quinoa and Phaseolus vulgaris as experimental hosts. Given the popularity and global trade of cannas, a clear picture of the genetic diversity of CaYSV is critical to disease management.
The family Potyviridae comprises more than 30 % of known plant virus species and the genus Potyvirus contains the largest collection of species within this family [10, 11, 22]. Within the genus Potyvirus, Gibbs and Ohshima [9] identified eleven potyvirus groups, nine of which reside within two divergent supergroups referred to as the potato virus Y (PVY) supergroup and the bean common mosaic virus (BCMV) supergroup [10, 11]. Canna yellow streak virus (CaYSV) is a major concern in the canna industry and was identified as the cause of severe streaking symptoms in European-grown canna
Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2327-5) contains supplementary material, which is available to authorized users. R. P. Chauhan P. Rajakaruna J. Verchot (&) Department of Entomology and Plant Pathology, Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA e-mail: [email protected]
varieties [16, 17]. The first full genome sequence of CaYSV was reported in 2010 [18]. Cannas belong to the plant family Cannaceae [14] and are a commercial crop producing edible rhizomes that are harvested in South America and South East Asia as a starch food for making chips or boiled to make noodles. Ornamental hybrids are grown worldwide for their colorful foliage and flowers. Cannas are produced and distributed through vegetative propagation, which makes the rhizomes a major source of virus spread through trade among growers. Canna rhizomes from the USA suspected of being infected with CaYSV were collected and grown in the greenhouse. Leaf extracts were taken from plants that showed obvious mosaic symptoms (Fig. S1a), tested by RT-PCR for CaYSV [20], and examined by electron microscopy to identify flexuous filamentous potyvirus particles (Fig. S1b). Leaf extracts were also subjected to 12.5 % SDS-PAGE and immunoblot analysis using a Pierce Fast Western Immunoblot Kit (Thermo Fisher Scientific). As an external control, leaf extracts from plants infected with bean yellow mosaic virus (BYMV) (another potyvirus that infects canna) were included. A commercial potyvirus group monoclonal (MAb) antiserum that detects most potyvirus coat proteins (CPs) (AC Diagnostics) detected both the 30.8-kDa BYMV CP and the 32.4-kDa CaYSV CP (Fig. S1c) but had greater reactivity with the BYMV CP than CaYSV CP [23]. The entire genome of a single isolate of CaYSV (CaYSV-OK) was amplified using total RNA extracted from a six-week-old canna ‘Wyoming’ plant that was grown in the greenhouse as described previously [4, 20]. RT-PCR was carried out using a One Step Superscript IIÒ RT-PCR System with Platinum TaqÒ polymerase (Life Technologies) and the oligonucleotide primers listed in Table S1. The genomes of eight more CaYSV isolates
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(named BZ1-3 or NC1-5) were also recovered by RT-PCR from individual plants grown in field plots in 2013. BZ-1, -2 and -3 isolates were derived from single ‘Australia’, ‘Cleopatra’ and ‘Red King Humbert’ plants, respectively. NC-1,-2,-3,-4 and -5 isolates were derived from single ‘Burning Ember’, ‘City of Portland’, ‘Firebird’, ‘King City Gold’, and ‘Red Futurity’ plants, respectively. Most PCR products were cloned into pCR2.1 or pGEM-T Easy Vector (Life Technologies and Promega Corp), and the plasmids were sequenced. In some cases, the PCR products were sequenced directly. The GenBank NCBI accession numbers for these CaYSV isolates are OK (KM882640), BZ-1 (KM882641), BZ-2 (KM882642), BZ-3 (KM882643), NC1 (KM882644), NC-2 (KM882645), NC-3 (KM882646), NC-4 (KM882647), and NC-5 (KM882648). Contig assembly was carried out using the CAP3 sequence assembly program [13]. BLASTn, BLASTp or LALIGN were used for pairwise comparisons of the deduced sequences with the GenBank-reported potyvirus sequences. MEGA version 6 was the software used for phylogenetic analysis [15]. The algorithm MUSCLE with 1000 bootstrap replications was used for alignments of the entire polyprotein and genome sequences [7]. The initial nucleotide (nt) sequence analysis confirmed the relatedness of CaYSV-OK and CaYSV reported from the UK (CaYSVUK) (Fig. S1d). Phylogenetic analysis was conducted to compare the polyprotein sequences of both the CaYSV-OK and CaYSV-UK isolates with 20 other potyviruses, a subset of three rymoviruses and two tritimoviruses (Fig. 1a). The same nodes were supported in the phenetic maximumlikelihood (ML) tree of polyproteins (Fig. 1a) and complete viral genome sequences (Fig. S2). Figure 1(a) shows the amino acid (aa) phylogeny with nodes that have 99-100 % bootstrap support and well represents the five divergent subgroups in the genus Potyvirus [10]: the sugarcane mosaic virus (SCMV) subgroup, the turnip mosaic virus (TuMV) subgroup, the bean yellow mosaic virus (BYMV) subgroup, the potato virus Y (PVY) subgroup, and the bean common mosaic virus (BCMV) subgroup. Onion dwarf mosaic virus is the sister group to all potyvirus subgroups (Fig. 1a) [10, 18]. The ML tree also demonstrates the close relationship of CaYSV-OK and CaYSV-UK to johnsongrass mosaic virus (JGMV) as its closest relative in the SCMV subgroup [9]. The nodes for the SCMV subgroup show 100 % nonparametric bootstrap support, although cocksfoot streak virus (CfSV) is associated at the base node with 91 % bootstrap support (Fig. 1a). To further validate the whole-genome analysis, a subset of individual JGMV protein-encoding regions (P1, HC-Pro, PIPO, CI, NIb, and CP) were compared with those of other members of the SCMV subgroup. For each gene or protein,
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the percent identity that CaYSV shared with JGMV stood out as significantly higher than with the other potyviruses (Figs. S3). CaYSV HC-Pro, PIPO, CI, NIb and CP showed between 59 % and 70 % nt sequence identity and between 49 to 76 % aa sequence identity to JGMV (Fig. S4). The P1 gene was the most divergent, with 54 % nt sequence identity and 37 % aa sequence identity. In contrast PenMV, MDMV, SCMV and CfSV appeared to cluster between 48 and 64 % nt sequence identity and 20 to 61 % aa sequence identity. Researchers sometimes argue that potyvirus phylogeny coincides with domestication of their hosts [10, 11]. The inclusion of CaYSV in the SCMV subgroup extends the host range of this subgroup to include species within the family Cannaceae as well as Poaceae (which includes Panicoideae and Pooideae) [3, 12, 21]. The data shown in Fig. 1(a) suggest that JGMV infecting sorghum and pearl millet (both members of the subfamily Panicoideae) and PenMV first diverged from CfSV infecting cocksfoot grass (a member of the subfamily Pooideae). These host species and were first domesticated in the same North African region. CaYSV appears to have emerged from JGMV, and its host, Canna, the only genus of the family Cannaceae, originated in the Americas [14]. It is easy to imagine that trade between Africa and the Americas contributed to the emergence of CaYSV [12, 14, 21]. To understand the genetic variability within the species Canna yellow streak virus, we analyzed the nine complete CaYSV sequences along with sequences previously published in GenBank. The BZ and NC isolates shared 94 to 98 % identity with the OK isolate (Table 1). Among the OK, BZ, and NC isolates, the coding sequence spanning from P3 to NIa-Pro shows 99-100 % identity, while NIb shows 94-97 % identity [1]. Interestingly, the P3 to NIaPro coding region of the UK isolate, encompassing a 4611-nt region of the genome (encoding 1538 aa) stood out as having 204 nt changes (encoding 58 aa). It is generally accepted that among species belonging to the genus Potyvirus, isolates generally show greater than 74 % identity in the coding region spanning from HC-Pro to the CP. Thus the greater than 94 % identity among isolates within this region points to the stability of the CaYSV genome [1]. The 50 UTR/P1/HC-Pro region spans 2250 nt and there are 240 nt changes in this region, relative to OK and BZ-1. The OK and BZ-1 isolates represent one similarity group, whose members share 99 to 100 % identity within this region. There are only two nt changes in the HC-Pro coding region, which result in one aa change. The second similarity group, containing all other isolates, showed 94 % identity within the 50 UTR, 78 % identity within the P1, and 95 % identity within the HC-Pro coding sequence. These eight isolates (BZ-2 and -3; NC-1 to -5, and UK) shared greater similarity to each other in this region
Genome sequence of canna yellow streak virus Fig. 1 Phylogenetic analysis of potyvirus polyproteins and the CaYSV coat protein. a) Phylogenetic tree generated by MEGA 6 software depicting relatedness of the complete polyproteins. Here, we show two groups in the genus Potyvirus that diverge at nodes A and B. The A node gives rise to the SCMV subgroup of viruses. The B node gives rise to four subgroups of viruses labeled B1-B4. The A subgroup is the SCMV subgroup that includes both the CaYSV-UK and the CaYSV-OK isolates. Subgroups B1 to B4 are the BYMV subgroup, TuMV subgroup, PVY subgroup, and BCMV subgroup, respectively. Five viruses belonging to the genera Rymovirus and Tritimovirus were included in the phylogenetic study. The nodes with 99 or 100 % bootstrap support are indicated by black rectangles. The scale bar represents 0.2 substitutions per site. (b) ML tree derived from comparisons of fifteen CaYSV CP aa sequences, showing three subgroups, A1, A2 and B. Subgroup A1 includes the OK isolate and four Thai isolates. Subgroup A2 represents five NC isolates. Group B represents BZ isolates along with UK and Israeli isolates. (c) The N-terminal sequences of CPs of these fifteen isolates of CaYSV, aligned using MUSCLE algorithm. There is a trypsin digestion site following the PKLK sequence that is conserved among CaYSV CPs. Alignment of CaYSV CP N-termini revealed the same subgroups (A1, A2 and B) as were obtained in the full-length CP aa phylogeny. Subgroup A1 aligns OK isolate with four Thai isolates. Subgroup A2 aligns five NC isolates together. Group B aligns BZ isolates along with UK and Israeli isolates. Thr and Ser residues are highlighted
(Table 1). The P1/HC-Pro polypeptide spans 699 aa with 70 aa changes. There were 64 aa positions that were not conserved in the P1 protein.
When comparing the CP and 30 UTR regions of CaYSV isolates, related sequences from isolates obtained in Thailand, Israel and the Netherlands were included (Table 1).
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The CP regions of the four Thai isolates share 98 to 99 % identity with that of the CaYSV-OK isolate. Interestingly, the CP sequences of the UK and Israeli isolates show less similarity to the OK isolate than the four Thai isolates (Table 1), suggesting that these viruses have a common origin. With regard to the 30 UTR, the sequences reported from Israel and the Netherlands share 96-99 % identity with the OK isolate. Although the BZ, NC, and OK isolates came from the USA, their CP sequences were not as closely related to each other as they were to European and South East Asian isolates. The ML tree generated based on alignments of the CP amino acid sequences showed two primary lineages (Fig. 1b). Group A contains isolates from Thailand, the OK isolate and NC isolates. Within group A, the NC isolates formed a separate subgroup from the Thai and OK isolates. The CP/30 UTR region of the UK isolate is 1088 nt long and shows 126 nt changes relative to the OK isolate. There are 27 aa changes in the CP compared to the OK isolate. Also, the 30 UTR of CaYSV-OK had one base less than the CaYSV-UK sequence (Fig. S1d). Group B contains the UK and Israeli isolates reported in GenBank as well as the BZ isolates. The BZ isolates showed greater similarity to the UK isolate. Evidence of South East Asian and European lineages suggests that geographic origin might contribute to the genetic variation within the CP. The N-terminus of the potyvirus CP contains a number of surface-located epitopes that are highly variable but sufficiently specific to be used to differentiate between members of distinct virus species [5, 22]. Such diversity can also be exploited to serologically characterize isolates and strains. With regard to specificity, the CP N-terminus has two defining features that are critical for subgroup comparisons. First, it can be separated from the core domain by trypsin digestion, and the PKLK motif that precedes the trypsin cleavage site is highly conserved (Fig. 1c). Second, it contains two motifs that are highly conserved among aphid-transmissible potyviruses: a) the DAG motif, which lies within 3-5 aa from the N-terminus, and b) the DA(V/T)G motif, nearer the trypsin digestion site [2, 8]. We identified the PKLK terminus of the tryptic fragment in the CaYSV CP for all isolates used in the ML tree. For CaYSV the N-terminal tryptic peptide is 70 aa long (Fig. 1c) and contains the highly conserved DAG and DTG motifs needed for virus aphid transmission (Fig. 1c). Furthermore, the N-terminal tryptic peptide shows more aa changes than the rest of the CaYSV CP (Fig. 1c). This is interesting given reports that single aa changes in the N-terminal tryptic peptide of the CP of other potyviruses can alter infectivity, RTM1 resistance, and O-glcN acylation of Thr residues [6]. For example, single aa changes near the DAG motif slowed potato virus A (PVA) movement [19]. Importantly, the subgroup A1, subgroup A2 and
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group B lineages appear to be defined in part by the specific patterns of Ser and Thr residues within this N-terminal tryptic fragment (Fig. 1c). While all isolates have Thr at positions 2, 58, 60 and 65, the members of subgroup A1, have four additional Ser residue at positions 22, 23, 36, and 50 and three Thr residues at positions 26, 29 and 35. Subgroup A2 contains the NC isolates that have the same three Ser residues in positions 22, 23, and 36 as the subgroup A1 isolates. However, they also contain a unique Ser at position 48 and Thr at position 30. The subgroup A2 isolates were the only ones that contained three Cys residues at positions 33, 41, and 51. Among group B isolates, the tryptic CP fragment of the CaYSV-OK and -UK isolates mostly contains highly similar or identical amino acids, but there were fifteen notable sequence changes resulting in dissimilar residues at the same aa positions (Fig. 1c). In particular, aa position 8 lies near the DAG motif and is either an isoleucine or valine residue. Amino acid positions 22 to 29 are also quite different among CaYSV-OK and -UK isolates (Fig. 1c). For CaYSV-OK, this sequence reads SSKNTGGT, and for CaYSV-UK, this sequence reads GNRSAGEA. In general, the group B isolates have four conserved Ser residues at positions 25, 34, 36, and 44 and two Thr residues at positions 35 and 49. The Ser-Thr at positions 34 and 35 is conserved among subgroup A1 and group B isolates. There are additional aa changes between aa positions 22 and 53 that help to differentiate these groups and subgroups. These data indicate that the patterns of Ser and Thr residues in the N-terminal domain of the CP coincide with relatedness of the CP reported in the phylogenetic tree. In summary, the N-terminus of the CP showed the greatest diversity and provides the hallmarks for the three clusters seen in Fig. 1 (b). The data in Fig. 1 (b) and (c) show that this region might be the most suitable for discriminating CaYSV isolates. Beyond diagnostic properties and phylogeny, it was important to identify additional biological properties that characterize members of the species Canna yellow streak virus, such as experimental host range. Sap inoculum of CaYSV-OK-infected tissue was prepared by grinding an infected leaf 1:5 (w/v) using 0.1 M phosphate buffer (pH 7.0) and 0.1 % sodium sulfite (w/v). Plants of the Tenderstar and Roma II varieties of P. vulgaris (runner type and bush type garden bean, respectively), N. benthamiana and C. quinoa were rub-inoculated with sap in three replicate experiments. Plants were monitored for up to 16 days for symptoms. Then, total RNA was isolated from the upper leaves of inoculated plants and tested for CaYSV infection by RT-PCR. Experiments were repeated three times to ensure reproducibility. Thirty percent (13 of 43 plants) of the inoculated N. benthamiana plants became systemically infected and
Genome sequence of canna yellow streak virus Fig. 2 N. benthamiana, C. quinoa and P. vulgaris plants two weeks after inoculation. The leaves of CaYSV-infected N. benthamiana curled downward. CaYSV-infected C. quinoa leaves showed vein clearing and chlorosis. CaYSVinfected P. vulgaris leaves also showed vein clearing early in infection. Symptoms matured to a mosaic pattern on the leaf. Infected leaves had an obvious feathery edge. Control plants were mock inoculated with phosphate buffer. The gel images next to each row of plants show the 695-bp RT-PCR products identifying CaYSVinfected systemic leaves (lanes 1-4), confirming a CaYSV infection of the test plants. Lane ‘‘L’’ contains the 1-kb ladder, lane ‘‘M’’ contains the RT-PCR product carried out using RNA derived from mock-inoculated plants. Lane ‘‘?’’ contains the PCR product derived using pYSOK7672 plasmid as a template. In lane ‘‘0’’, water was used instead of cDNA in the PCR reaction
tested positive for CaYSV infection. The primary symptom was downward curling of the leaves with no chlorosis or mosaic pattern (Fig. 2). Sixteen percent (5 of 30 plants) of the CaYSV-inoculated C. quinoa plants showed obvious systemic yellowing symptoms and tested positive for virus infection. Forty-six percent (12 of 26 plants) of the inoculated Tenderstar variety of P. vulgaris plants (runner-type bean) showed mosaic symptoms and a feathery edge and tested positive by RT-PCR for CaYSV infection (Fig. 2). Fifty-four percent (7 of 13 plants) of the inoculated Roma II variety of P. vulgaris plants (bush-type bean) tested
positive for CaYSV infection and showed an obvious mosaic in systemically infected leaves. In conclusion, robust phylogenetic analysis was carried out using the complete genome sequence of several isolates of CaYSV. The placement of CaYSV in the SCMV subgroup is strengthened by including the UK and OK isolates in this analysis and by the more focused analysis of P1, HC-Pro, PIPO, CI, NIb, and CP individually. When comparing CaYSV isolates, the greatest variability in aa sequence lies in the P1 and CP regions. Analysis of the viral CP revealed two distinct lineages of CaYSV.
123
123
94
93
94
94
CaYSV isolate NC-3 (accession: KM882646)
CaYSV isolate NC-4 (accession: KM882647)
CaYSV isolate NC-5 (accession: KM882648)
CaYSV isolate UK (accession: NC013261)
78 (73)
78 (73)
78 (73)
78 (74)
78 (74)
95 (99)
95 (99)
95 (99)
95 (99)
95 (99)
95 (99)
99 (99)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
99 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
94 (94)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 % (100 %)
92 (93)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 % (100 %)
6K2
88 (96)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 % (100 %)
VPg
99 (98)
99 (99)
99 (99)
99 (99)
99 (99)
99 (99)
99 (99)
99 (99)
99 % (99 %)
NIa Pro
97 (99)
94 (98)
95 (98)
95 (99)
97 (99)
95 (99)
97 (99)
97 (99)
97 % (98 %)
NIb
87 (91)
96 (93)
96 (93)
96 (93)
96 (93)
96 (93)
90 (91)
87 (91)
87 % (91 %)
CP
98 (98)
94
CaYSV isolate NC-2 (accession: KM882645)
78 (74)
95 (99)
100 (100)
100 % (100 %)
CI
CaYSV isolate K2, Clone 6-1 (Thai) (accession: HE774737)
94
CaYSV isolate NC-1 (accession: KM882644)
78 (74)
95 (99)
100 % (100 %)
6K1
99 (98)
94
CaYSV isolate BZ-3 (accession: KM882643)
78 (74)
99 % (99 %)
P3
CaYSV isolate PHC 478, 6C6 (Thai) (accession: HE774738)
94
CaYSV isolate BZ-2 (accession: KM882642)
100 % (100 %)
HC Pro
99 (99)
100 %
CaYSV isolate BZ-1 (accession: KM882641)
P1
CaYSV isolate PHC478, 9C4 (Thai) (accession: HE774739)
50 UTR
CaYSV isolate OK (accession: KM882640)
Table 1 Percent nucleotide (amino acid) sequence identities with Oklahoma isolate of CaYSV
92
100
100
100
100
100
100
93
93
30 UTR (%)
94 %
96 %
96 %
96 %
97 %
96 %
96 %
96 %
98 %
Complete genome
R. P. Chauhan et al.
99
96 89 (89)
99 (99)
Variability within the N-terminal domain of the CP can be used to differentiate isolates. This investigation also revealed characteristics of CaYSV that can contribute to the species definition, including identification of experimental host plants. In combination, these data will provide information that can be used to develop molecular diagnostic techniques to track the global spread of CaYSV, identify alternative hosts and assess molecular characteristics that are associated with differences in disease severity. Acknowledgments This project was funded by the Oklahoma Center for Advancement of Science and Technology AORS project number AR11.2-050 and the Oklahoma Department of Agriculture. We thank the Oklahoma State University Biochemistry and Molecular Biology Recombinant DNA and Protein Core Facility for providing the multiuser instruments and DNA sequencing. We also thank Ms. Yan Song and Dr. Peter Hoyt of the OSU Biochemistry and Molecular Biology Array and Bioinformatics Core Facility for bioinformatics support.
NIa Pro
NIb
CP
30 UTR (%)
Complete genome
Genome sequence of canna yellow streak virus
The authors declare that they have no com-
VPg
Conflict of interest peting interest.
CaYSVNetherlands (accession: EU042760)
CaYSV-Israel (accession: EF466138)
CaYSV isolate K2, Clone 5-4 (Thai) (accession: HE774736)
CaYSV isolate OK (accession: KM882640)
Table 1 continued
50 UTR
P1
HC Pro
P3
6K1
CI
6K2
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BRIEF REPORT
Complete genome sequence of nine isolates of canna yellow streak virus reveals its relationship to the sugarcane mosaic virus (SCMV) subgroup of potyviruses Ravendra P. Chauhan • Punsasi Rajakaruna Jeanmarie Verchot
•
Received: 13 October 2014 / Accepted: 20 December 2014 Ó Springer-Verlag Wien 2015
Abstract Complete genome sequences were obtained from nine isolates of canna yellow streak virus (CaYSV). CaYSV belongs to the sugarcane mosaic virus (SCMV) subgroup of potyviruses with johnsongrass mosaic virus (JGMV) as its closest relative. Multiple sequence alignments showed a pattern of amino acid substitutions in the CP sequences, which enabled us to relate these isolates to South East Asian or European isolates. Biological characterization of CaYSV identified Nicotiana benthamiana, Chenopodium quinoa and Phaseolus vulgaris as experimental hosts. Given the popularity and global trade of cannas, a clear picture of the genetic diversity of CaYSV is critical to disease management.
The family Potyviridae comprises more than 30 % of known plant virus species and the genus Potyvirus contains the largest collection of species within this family [10, 11, 22]. Within the genus Potyvirus, Gibbs and Ohshima [9] identified eleven potyvirus groups, nine of which reside within two divergent supergroups referred to as the potato virus Y (PVY) supergroup and the bean common mosaic virus (BCMV) supergroup [10, 11]. Canna yellow streak virus (CaYSV) is a major concern in the canna industry and was identified as the cause of severe streaking symptoms in European-grown canna
Electronic supplementary material The online version of this article (doi:10.1007/s00705-014-2327-5) contains supplementary material, which is available to authorized users. R. P. Chauhan P. Rajakaruna J. Verchot (&) Department of Entomology and Plant Pathology, Noble Research Center, Oklahoma State University, Stillwater, OK 74078, USA e-mail: [email protected]
varieties [16, 17]. The first full genome sequence of CaYSV was reported in 2010 [18]. Cannas belong to the plant family Cannaceae [14] and are a commercial crop producing edible rhizomes that are harvested in South America and South East Asia as a starch food for making chips or boiled to make noodles. Ornamental hybrids are grown worldwide for their colorful foliage and flowers. Cannas are produced and distributed through vegetative propagation, which makes the rhizomes a major source of virus spread through trade among growers. Canna rhizomes from the USA suspected of being infected with CaYSV were collected and grown in the greenhouse. Leaf extracts were taken from plants that showed obvious mosaic symptoms (Fig. S1a), tested by RT-PCR for CaYSV [20], and examined by electron microscopy to identify flexuous filamentous potyvirus particles (Fig. S1b). Leaf extracts were also subjected to 12.5 % SDS-PAGE and immunoblot analysis using a Pierce Fast Western Immunoblot Kit (Thermo Fisher Scientific). As an external control, leaf extracts from plants infected with bean yellow mosaic virus (BYMV) (another potyvirus that infects canna) were included. A commercial potyvirus group monoclonal (MAb) antiserum that detects most potyvirus coat proteins (CPs) (AC Diagnostics) detected both the 30.8-kDa BYMV CP and the 32.4-kDa CaYSV CP (Fig. S1c) but had greater reactivity with the BYMV CP than CaYSV CP [23]. The entire genome of a single isolate of CaYSV (CaYSV-OK) was amplified using total RNA extracted from a six-week-old canna ‘Wyoming’ plant that was grown in the greenhouse as described previously [4, 20]. RT-PCR was carried out using a One Step Superscript IIÒ RT-PCR System with Platinum TaqÒ polymerase (Life Technologies) and the oligonucleotide primers listed in Table S1. The genomes of eight more CaYSV isolates
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(named BZ1-3 or NC1-5) were also recovered by RT-PCR from individual plants grown in field plots in 2013. BZ-1, -2 and -3 isolates were derived from single ‘Australia’, ‘Cleopatra’ and ‘Red King Humbert’ plants, respectively. NC-1,-2,-3,-4 and -5 isolates were derived from single ‘Burning Ember’, ‘City of Portland’, ‘Firebird’, ‘King City Gold’, and ‘Red Futurity’ plants, respectively. Most PCR products were cloned into pCR2.1 or pGEM-T Easy Vector (Life Technologies and Promega Corp), and the plasmids were sequenced. In some cases, the PCR products were sequenced directly. The GenBank NCBI accession numbers for these CaYSV isolates are OK (KM882640), BZ-1 (KM882641), BZ-2 (KM882642), BZ-3 (KM882643), NC1 (KM882644), NC-2 (KM882645), NC-3 (KM882646), NC-4 (KM882647), and NC-5 (KM882648). Contig assembly was carried out using the CAP3 sequence assembly program [13]. BLASTn, BLASTp or LALIGN were used for pairwise comparisons of the deduced sequences with the GenBank-reported potyvirus sequences. MEGA version 6 was the software used for phylogenetic analysis [15]. The algorithm MUSCLE with 1000 bootstrap replications was used for alignments of the entire polyprotein and genome sequences [7]. The initial nucleotide (nt) sequence analysis confirmed the relatedness of CaYSV-OK and CaYSV reported from the UK (CaYSVUK) (Fig. S1d). Phylogenetic analysis was conducted to compare the polyprotein sequences of both the CaYSV-OK and CaYSV-UK isolates with 20 other potyviruses, a subset of three rymoviruses and two tritimoviruses (Fig. 1a). The same nodes were supported in the phenetic maximumlikelihood (ML) tree of polyproteins (Fig. 1a) and complete viral genome sequences (Fig. S2). Figure 1(a) shows the amino acid (aa) phylogeny with nodes that have 99-100 % bootstrap support and well represents the five divergent subgroups in the genus Potyvirus [10]: the sugarcane mosaic virus (SCMV) subgroup, the turnip mosaic virus (TuMV) subgroup, the bean yellow mosaic virus (BYMV) subgroup, the potato virus Y (PVY) subgroup, and the bean common mosaic virus (BCMV) subgroup. Onion dwarf mosaic virus is the sister group to all potyvirus subgroups (Fig. 1a) [10, 18]. The ML tree also demonstrates the close relationship of CaYSV-OK and CaYSV-UK to johnsongrass mosaic virus (JGMV) as its closest relative in the SCMV subgroup [9]. The nodes for the SCMV subgroup show 100 % nonparametric bootstrap support, although cocksfoot streak virus (CfSV) is associated at the base node with 91 % bootstrap support (Fig. 1a). To further validate the whole-genome analysis, a subset of individual JGMV protein-encoding regions (P1, HC-Pro, PIPO, CI, NIb, and CP) were compared with those of other members of the SCMV subgroup. For each gene or protein,
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the percent identity that CaYSV shared with JGMV stood out as significantly higher than with the other potyviruses (Figs. S3). CaYSV HC-Pro, PIPO, CI, NIb and CP showed between 59 % and 70 % nt sequence identity and between 49 to 76 % aa sequence identity to JGMV (Fig. S4). The P1 gene was the most divergent, with 54 % nt sequence identity and 37 % aa sequence identity. In contrast PenMV, MDMV, SCMV and CfSV appeared to cluster between 48 and 64 % nt sequence identity and 20 to 61 % aa sequence identity. Researchers sometimes argue that potyvirus phylogeny coincides with domestication of their hosts [10, 11]. The inclusion of CaYSV in the SCMV subgroup extends the host range of this subgroup to include species within the family Cannaceae as well as Poaceae (which includes Panicoideae and Pooideae) [3, 12, 21]. The data shown in Fig. 1(a) suggest that JGMV infecting sorghum and pearl millet (both members of the subfamily Panicoideae) and PenMV first diverged from CfSV infecting cocksfoot grass (a member of the subfamily Pooideae). These host species and were first domesticated in the same North African region. CaYSV appears to have emerged from JGMV, and its host, Canna, the only genus of the family Cannaceae, originated in the Americas [14]. It is easy to imagine that trade between Africa and the Americas contributed to the emergence of CaYSV [12, 14, 21]. To understand the genetic variability within the species Canna yellow streak virus, we analyzed the nine complete CaYSV sequences along with sequences previously published in GenBank. The BZ and NC isolates shared 94 to 98 % identity with the OK isolate (Table 1). Among the OK, BZ, and NC isolates, the coding sequence spanning from P3 to NIa-Pro shows 99-100 % identity, while NIb shows 94-97 % identity [1]. Interestingly, the P3 to NIaPro coding region of the UK isolate, encompassing a 4611-nt region of the genome (encoding 1538 aa) stood out as having 204 nt changes (encoding 58 aa). It is generally accepted that among species belonging to the genus Potyvirus, isolates generally show greater than 74 % identity in the coding region spanning from HC-Pro to the CP. Thus the greater than 94 % identity among isolates within this region points to the stability of the CaYSV genome [1]. The 50 UTR/P1/HC-Pro region spans 2250 nt and there are 240 nt changes in this region, relative to OK and BZ-1. The OK and BZ-1 isolates represent one similarity group, whose members share 99 to 100 % identity within this region. There are only two nt changes in the HC-Pro coding region, which result in one aa change. The second similarity group, containing all other isolates, showed 94 % identity within the 50 UTR, 78 % identity within the P1, and 95 % identity within the HC-Pro coding sequence. These eight isolates (BZ-2 and -3; NC-1 to -5, and UK) shared greater similarity to each other in this region
Genome sequence of canna yellow streak virus Fig. 1 Phylogenetic analysis of potyvirus polyproteins and the CaYSV coat protein. a) Phylogenetic tree generated by MEGA 6 software depicting relatedness of the complete polyproteins. Here, we show two groups in the genus Potyvirus that diverge at nodes A and B. The A node gives rise to the SCMV subgroup of viruses. The B node gives rise to four subgroups of viruses labeled B1-B4. The A subgroup is the SCMV subgroup that includes both the CaYSV-UK and the CaYSV-OK isolates. Subgroups B1 to B4 are the BYMV subgroup, TuMV subgroup, PVY subgroup, and BCMV subgroup, respectively. Five viruses belonging to the genera Rymovirus and Tritimovirus were included in the phylogenetic study. The nodes with 99 or 100 % bootstrap support are indicated by black rectangles. The scale bar represents 0.2 substitutions per site. (b) ML tree derived from comparisons of fifteen CaYSV CP aa sequences, showing three subgroups, A1, A2 and B. Subgroup A1 includes the OK isolate and four Thai isolates. Subgroup A2 represents five NC isolates. Group B represents BZ isolates along with UK and Israeli isolates. (c) The N-terminal sequences of CPs of these fifteen isolates of CaYSV, aligned using MUSCLE algorithm. There is a trypsin digestion site following the PKLK sequence that is conserved among CaYSV CPs. Alignment of CaYSV CP N-termini revealed the same subgroups (A1, A2 and B) as were obtained in the full-length CP aa phylogeny. Subgroup A1 aligns OK isolate with four Thai isolates. Subgroup A2 aligns five NC isolates together. Group B aligns BZ isolates along with UK and Israeli isolates. Thr and Ser residues are highlighted
(Table 1). The P1/HC-Pro polypeptide spans 699 aa with 70 aa changes. There were 64 aa positions that were not conserved in the P1 protein.
When comparing the CP and 30 UTR regions of CaYSV isolates, related sequences from isolates obtained in Thailand, Israel and the Netherlands were included (Table 1).
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The CP regions of the four Thai isolates share 98 to 99 % identity with that of the CaYSV-OK isolate. Interestingly, the CP sequences of the UK and Israeli isolates show less similarity to the OK isolate than the four Thai isolates (Table 1), suggesting that these viruses have a common origin. With regard to the 30 UTR, the sequences reported from Israel and the Netherlands share 96-99 % identity with the OK isolate. Although the BZ, NC, and OK isolates came from the USA, their CP sequences were not as closely related to each other as they were to European and South East Asian isolates. The ML tree generated based on alignments of the CP amino acid sequences showed two primary lineages (Fig. 1b). Group A contains isolates from Thailand, the OK isolate and NC isolates. Within group A, the NC isolates formed a separate subgroup from the Thai and OK isolates. The CP/30 UTR region of the UK isolate is 1088 nt long and shows 126 nt changes relative to the OK isolate. There are 27 aa changes in the CP compared to the OK isolate. Also, the 30 UTR of CaYSV-OK had one base less than the CaYSV-UK sequence (Fig. S1d). Group B contains the UK and Israeli isolates reported in GenBank as well as the BZ isolates. The BZ isolates showed greater similarity to the UK isolate. Evidence of South East Asian and European lineages suggests that geographic origin might contribute to the genetic variation within the CP. The N-terminus of the potyvirus CP contains a number of surface-located epitopes that are highly variable but sufficiently specific to be used to differentiate between members of distinct virus species [5, 22]. Such diversity can also be exploited to serologically characterize isolates and strains. With regard to specificity, the CP N-terminus has two defining features that are critical for subgroup comparisons. First, it can be separated from the core domain by trypsin digestion, and the PKLK motif that precedes the trypsin cleavage site is highly conserved (Fig. 1c). Second, it contains two motifs that are highly conserved among aphid-transmissible potyviruses: a) the DAG motif, which lies within 3-5 aa from the N-terminus, and b) the DA(V/T)G motif, nearer the trypsin digestion site [2, 8]. We identified the PKLK terminus of the tryptic fragment in the CaYSV CP for all isolates used in the ML tree. For CaYSV the N-terminal tryptic peptide is 70 aa long (Fig. 1c) and contains the highly conserved DAG and DTG motifs needed for virus aphid transmission (Fig. 1c). Furthermore, the N-terminal tryptic peptide shows more aa changes than the rest of the CaYSV CP (Fig. 1c). This is interesting given reports that single aa changes in the N-terminal tryptic peptide of the CP of other potyviruses can alter infectivity, RTM1 resistance, and O-glcN acylation of Thr residues [6]. For example, single aa changes near the DAG motif slowed potato virus A (PVA) movement [19]. Importantly, the subgroup A1, subgroup A2 and
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group B lineages appear to be defined in part by the specific patterns of Ser and Thr residues within this N-terminal tryptic fragment (Fig. 1c). While all isolates have Thr at positions 2, 58, 60 and 65, the members of subgroup A1, have four additional Ser residue at positions 22, 23, 36, and 50 and three Thr residues at positions 26, 29 and 35. Subgroup A2 contains the NC isolates that have the same three Ser residues in positions 22, 23, and 36 as the subgroup A1 isolates. However, they also contain a unique Ser at position 48 and Thr at position 30. The subgroup A2 isolates were the only ones that contained three Cys residues at positions 33, 41, and 51. Among group B isolates, the tryptic CP fragment of the CaYSV-OK and -UK isolates mostly contains highly similar or identical amino acids, but there were fifteen notable sequence changes resulting in dissimilar residues at the same aa positions (Fig. 1c). In particular, aa position 8 lies near the DAG motif and is either an isoleucine or valine residue. Amino acid positions 22 to 29 are also quite different among CaYSV-OK and -UK isolates (Fig. 1c). For CaYSV-OK, this sequence reads SSKNTGGT, and for CaYSV-UK, this sequence reads GNRSAGEA. In general, the group B isolates have four conserved Ser residues at positions 25, 34, 36, and 44 and two Thr residues at positions 35 and 49. The Ser-Thr at positions 34 and 35 is conserved among subgroup A1 and group B isolates. There are additional aa changes between aa positions 22 and 53 that help to differentiate these groups and subgroups. These data indicate that the patterns of Ser and Thr residues in the N-terminal domain of the CP coincide with relatedness of the CP reported in the phylogenetic tree. In summary, the N-terminus of the CP showed the greatest diversity and provides the hallmarks for the three clusters seen in Fig. 1 (b). The data in Fig. 1 (b) and (c) show that this region might be the most suitable for discriminating CaYSV isolates. Beyond diagnostic properties and phylogeny, it was important to identify additional biological properties that characterize members of the species Canna yellow streak virus, such as experimental host range. Sap inoculum of CaYSV-OK-infected tissue was prepared by grinding an infected leaf 1:5 (w/v) using 0.1 M phosphate buffer (pH 7.0) and 0.1 % sodium sulfite (w/v). Plants of the Tenderstar and Roma II varieties of P. vulgaris (runner type and bush type garden bean, respectively), N. benthamiana and C. quinoa were rub-inoculated with sap in three replicate experiments. Plants were monitored for up to 16 days for symptoms. Then, total RNA was isolated from the upper leaves of inoculated plants and tested for CaYSV infection by RT-PCR. Experiments were repeated three times to ensure reproducibility. Thirty percent (13 of 43 plants) of the inoculated N. benthamiana plants became systemically infected and
Genome sequence of canna yellow streak virus Fig. 2 N. benthamiana, C. quinoa and P. vulgaris plants two weeks after inoculation. The leaves of CaYSV-infected N. benthamiana curled downward. CaYSV-infected C. quinoa leaves showed vein clearing and chlorosis. CaYSVinfected P. vulgaris leaves also showed vein clearing early in infection. Symptoms matured to a mosaic pattern on the leaf. Infected leaves had an obvious feathery edge. Control plants were mock inoculated with phosphate buffer. The gel images next to each row of plants show the 695-bp RT-PCR products identifying CaYSVinfected systemic leaves (lanes 1-4), confirming a CaYSV infection of the test plants. Lane ‘‘L’’ contains the 1-kb ladder, lane ‘‘M’’ contains the RT-PCR product carried out using RNA derived from mock-inoculated plants. Lane ‘‘?’’ contains the PCR product derived using pYSOK7672 plasmid as a template. In lane ‘‘0’’, water was used instead of cDNA in the PCR reaction
tested positive for CaYSV infection. The primary symptom was downward curling of the leaves with no chlorosis or mosaic pattern (Fig. 2). Sixteen percent (5 of 30 plants) of the CaYSV-inoculated C. quinoa plants showed obvious systemic yellowing symptoms and tested positive for virus infection. Forty-six percent (12 of 26 plants) of the inoculated Tenderstar variety of P. vulgaris plants (runner-type bean) showed mosaic symptoms and a feathery edge and tested positive by RT-PCR for CaYSV infection (Fig. 2). Fifty-four percent (7 of 13 plants) of the inoculated Roma II variety of P. vulgaris plants (bush-type bean) tested
positive for CaYSV infection and showed an obvious mosaic in systemically infected leaves. In conclusion, robust phylogenetic analysis was carried out using the complete genome sequence of several isolates of CaYSV. The placement of CaYSV in the SCMV subgroup is strengthened by including the UK and OK isolates in this analysis and by the more focused analysis of P1, HC-Pro, PIPO, CI, NIb, and CP individually. When comparing CaYSV isolates, the greatest variability in aa sequence lies in the P1 and CP regions. Analysis of the viral CP revealed two distinct lineages of CaYSV.
123
123
94
93
94
94
CaYSV isolate NC-3 (accession: KM882646)
CaYSV isolate NC-4 (accession: KM882647)
CaYSV isolate NC-5 (accession: KM882648)
CaYSV isolate UK (accession: NC013261)
78 (73)
78 (73)
78 (73)
78 (74)
78 (74)
95 (99)
95 (99)
95 (99)
95 (99)
95 (99)
95 (99)
99 (99)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
99 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
94 (94)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 % (100 %)
92 (93)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 % (100 %)
6K2
88 (96)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 (100)
100 % (100 %)
VPg
99 (98)
99 (99)
99 (99)
99 (99)
99 (99)
99 (99)
99 (99)
99 (99)
99 % (99 %)
NIa Pro
97 (99)
94 (98)
95 (98)
95 (99)
97 (99)
95 (99)
97 (99)
97 (99)
97 % (98 %)
NIb
87 (91)
96 (93)
96 (93)
96 (93)
96 (93)
96 (93)
90 (91)
87 (91)
87 % (91 %)
CP
98 (98)
94
CaYSV isolate NC-2 (accession: KM882645)
78 (74)
95 (99)
100 (100)
100 % (100 %)
CI
CaYSV isolate K2, Clone 6-1 (Thai) (accession: HE774737)
94
CaYSV isolate NC-1 (accession: KM882644)
78 (74)
95 (99)
100 % (100 %)
6K1
99 (98)
94
CaYSV isolate BZ-3 (accession: KM882643)
78 (74)
99 % (99 %)
P3
CaYSV isolate PHC 478, 6C6 (Thai) (accession: HE774738)
94
CaYSV isolate BZ-2 (accession: KM882642)
100 % (100 %)
HC Pro
99 (99)
100 %
CaYSV isolate BZ-1 (accession: KM882641)
P1
CaYSV isolate PHC478, 9C4 (Thai) (accession: HE774739)
50 UTR
CaYSV isolate OK (accession: KM882640)
Table 1 Percent nucleotide (amino acid) sequence identities with Oklahoma isolate of CaYSV
92
100
100
100
100
100
100
93
93
30 UTR (%)
94 %
96 %
96 %
96 %
97 %
96 %
96 %
96 %
98 %
Complete genome
R. P. Chauhan et al.
99
96 89 (89)
99 (99)
Variability within the N-terminal domain of the CP can be used to differentiate isolates. This investigation also revealed characteristics of CaYSV that can contribute to the species definition, including identification of experimental host plants. In combination, these data will provide information that can be used to develop molecular diagnostic techniques to track the global spread of CaYSV, identify alternative hosts and assess molecular characteristics that are associated with differences in disease severity. Acknowledgments This project was funded by the Oklahoma Center for Advancement of Science and Technology AORS project number AR11.2-050 and the Oklahoma Department of Agriculture. We thank the Oklahoma State University Biochemistry and Molecular Biology Recombinant DNA and Protein Core Facility for providing the multiuser instruments and DNA sequencing. We also thank Ms. Yan Song and Dr. Peter Hoyt of the OSU Biochemistry and Molecular Biology Array and Bioinformatics Core Facility for bioinformatics support.
NIa Pro
NIb
CP
30 UTR (%)
Complete genome
Genome sequence of canna yellow streak virus
The authors declare that they have no com-
VPg
Conflict of interest peting interest.
CaYSVNetherlands (accession: EU042760)
CaYSV-Israel (accession: EF466138)
CaYSV isolate K2, Clone 5-4 (Thai) (accession: HE774736)
CaYSV isolate OK (accession: KM882640)
Table 1 continued
50 UTR
P1
HC Pro
P3
6K1
CI
6K2
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