Arch Virol DOI 10.1007/s00705-015-2367-5

BRIEF REPORT

Detection and characterization of the first North American in switchgrass

Bright O. Agindotan • Leslie L. Domier • Carl A. Bradley

Received: 30 July 2014 / Accepted: 10 February 2015 Ó Springer-Verlag Wien 2015

Abstract infections have the potential to reduce 79 %, 79 %, and 87 % identical to each other, respectively, biomass yields in energy crops, including Panicum virgatum and 46–48 %, 31 %, 31 %, and 42–48 % identical to those of (switchgrass). As a first step towards managing virus-induced the closest mastrevirus relatives. Based on a genome-wide biomass reduction, deep sequencing was used to identify identity threshold of 75 % set by the International Committee associated with mosaic symptoms in switchgrass. Two on Taxonomy of Viruses and phylogenetic analyses, the two sequences with homology to mastreviruses were identified. virus sequences appear to represent a new mastrevirus spe- Total DNA extracted from switchgrass varieties ‘Dewey cies. The mastrevirus is tentatively named switchgrass mo- Blue’ and ‘Cloud Nine’ was used as template to amplify saic-associated virus 1 (SgMaV-1) and is the first mastrevirus mastrevirus DNA by the rolling-circle method. Complete reported from North America. mastrevirus genome sequences were obtained from cloned amplicons. The two nucleotide sequences were 88 % identi- Keywords Bioenergy crop Panicum virgatum cal to each other but only 56–57 % identical to the closest Biomass crop Mastrevirus DNA virus relatives in the Mastrevirus. Predicted amino acid se- quences of the coat protein, replication-associated protein A, Switchgrass (Panicum virgatum L.) is a perennial, warm- replication-associated protein, and putative movement protein season, C-4 grass native to North America with potential as encoded by the two mastrevirus-like sequences were 95 %, a cellulosic crop [8]. Presently, knowledge of pathogens that may reduce biomass yield in switchgrass is incomplete. To identify previously uncharacterized viruses GenBank accession numbers: SgMaV-1cn: KF806701; SgMaV-1db: in switchgrass, we deep-sequenced DNase-digested total KF806702; SgMaV-1cn: KJ957193; SgMaV-2: KJ957192. RNA extracted from pooled symptomatic leaves of switchgrass on an Illumina HiSeq 2000 (San Diego, CA). B. O. Agindotan Energy Biosciences Institute, University of Illinois, Unexpectedly, two contigs with homology to single-s- Urbana-Champaign, IL 61801, USA tranded DNA mastreviruses (Mastrevirus-seq-1, 401 nt; GenBank accession no. KJ957192 and Mastrevirus-seq-2, Present Address: 1159 nt; GenBank accession no. KJ957193) were identified B. O. Agindotan (&) Department of Plant Sciences and Plant Pathology, Montana in Blastx searches [3]. Both contigs contained partial coat State University, Bozeman, MT 59717, USA protein (CP) coding sequences. Mastrevirus-seq-1 and e-mail: [email protected] Mastrevirus-seq-2 share 36 % (at 80 % coverage) and 49 % (at 93 % coverage) amino acid sequence identity L. L. Domier United States Department of Agriculture, Agricultural Research with the CPs of chickpea chlorosis virus E (AFD63057) Service and Department of Crop Sciences, University of Illinois, and sporobolus striate 2 (SSMV-2; Urbana-Champaign, IL 61801, USA YP_006666530), respectively. Mastrevirus-seq-2 also showed 67 % amino acid sequence identity (at 32 % cov- C. A. Bradley Department of Crop Sciences and Energy Biosciences Institute, erage) with the movement protein (MP) of dragonfly-as- University of Illinois, Urbana-Champaign, IL 61801, USA sociated mastrevirus (DfasMV; YP_007004036). 123 B. O. Agindotan et al.

To further characterize the putative switchgrass mas- varieties collected (Fig. 2a), using both partially purified treviruses discovered by deep sequencing and to determine virion DNA and total DNA as templates. All leaves sam- their prevalence in the field, one pool of leaf samples was pled had mosaic symptoms of varying severity except used from each of 18 varietal stands of switchgrass (17 ‘Heavy Metal’, which was asymptomatic. Mastrevirus varieties were symptomatic and one variety, ‘Heavy met- DNA was detected in all switchgrass varieties with mosaic al’, was asymptomatic). The symptomatic varieties were symptoms except ‘Shenandoah’. ‘Alamo’, ‘Cave-in-Rock’, ‘Cloud Nine’, ‘Dallas Blue’, Because mastreviruses have circular genomes, Phi29 ‘Dewey Blue’, ‘Heavy Metal’, ‘K7A45’, ‘Kanlow’, DNA polymerase (Illustra TempliPhiTM, GE, Healthcare, ‘Northwind’, ‘Prairie Sky’, ‘Rehbraun’, ‘Rotstrahlbusch’, USA) was used to amplify complete genomes by the roll- ‘Ruby Ribbons’, ‘Shenandoah’, ‘Squaw’, and ‘Warrior’ ing-circle amplification (RCA) method using templates (See Fig. 1 for symptoms on ‘Cloud Nine’ and ‘Dewey obtained from partially purified virus preparations from Blue’). The switchgrass varieties were sampled in 2012 leaf samples of ‘Cloud Nine’ and ‘Dewey Blue’ with from an experimental plot established in 2008 at the mosaic symptoms (Fig. 1). Products were screened for University of Illinois Energy Farm near Urbana, IL. Leaf restriction endonuclease sites that also were present in samples also were collected in the same field and pooled pUC19. The restriction endonuclease enzymes BamH1, from Panicum amarum, a related species. EcoR1, NdeI, PstI, SmaI, and XbaI were tested. Only PstI DNA was extracted using a DNeasy Extraction Kit generated a product for both RCA amplicons with the size (QIAGEN, Valencia, CA, USA) from partially purified (2.7 kb) expected for a full-length mastrevirus genome virus preparations [1] or from leaf tissues. Primer pair (Fig. 2b). In contrast, NdeI and XbaI generated a genome- B358-RP (50-CCAAACGCTGAACGAACCTCTTGA-30) length product for the ‘Dewey Blue’ RCA amplicon but did and B359FP (50-ACCCAGCTTGCAGGTCATCAACTA- not cleave the ‘Cloud Nine’ RCA product (data not shown). 30) was designed (based on and specific to the Mastrevirus- The enzyme SmaI generated a genome-length product for seq-2 sequence) to target a region within the CP coding the ‘Dewey Blue’’ RCA amplicon but generated three sequence. Primer pair B358-RP/B359FP was used to screen fragments from the ‘Cloud Nine’ RCA product (data not each of the 18 switchgrass varieties by polymerase chain shown). Production of a single band upon digestion of the reaction (PCR). rolling-circle amplification product by PstI suggests that Amplification products of the expected size (*500 bp) the DNA template had a closed circular conformation. were detected in 17 of 18 (94.4 %) of the switchgrass However, direct detection of circular DNA forms via Southern hybridization or recovery from virions is required to verify episomal status of the sequence. The PstI-digested RCA products obtained from the two switchgrass mastrevirus isolates were ligated into PstI-di- gested pUC19 and used to transform Escherichia coli. Plasmid DNA was extracted from five clones per virus amplicon, and the inserts were fully sequenced by primer walking. A primer pair, B599-FP (CCAGATCTAGGTA- CACATACAAGAG) and B600-RP (TTCTGTCCTTGCT GGAAGTC), based on the sequenced genome, was used to amplify a 1.1-kb product that corresponded to the CP coding region and extended across the PstI cleavage site in the virus genome, confirming that sequences flanking the PstI site of the virus genome were not lost during cloning. The closed circular DNA genome of the switchgrass mastrevirus ‘Cloud Nine’ amplicon was 2,734 bp (Gen- Bank accession number: KF806701). The ‘Dewey Blue’ amplicon was 2,739 bp (GenBank accession number: KF806702). The complete genome sequences of the two switchgrass-associated viruses shared 88 % nucleotide se- quence identity, which indicated that the two sequences represent strains of the same species. The genome orga- nization was typical of mastreviruses [4]. Each switchgrass Fig. 1 Mosaic symptoms on switchgrass leaf samples from which a new mastrevirus was identified. A) Switchgrass variety ‘Dewey mastrevirus genome encoded two large open reading Blue’. B. Switchgrass variety ‘Cloud Nine’ frames (ORFs) on the virus-sense strand (movement 123 Detection of a mastrevirus in switchgrass

Fig. 2 PCR amplification of a partial coat protein gene and restriction endonuclease digestion of rolling-circle products of a switchgrass mastrevirus. A) PCR amplification of a partial coat protein gene region from the switchgrass (Panicum virgatum) varieties Squaw (lane 1), ‘Kanlow’ (lane 2), ‘Cloud Nine’ (lane 3), ‘Warrior’ (lane 4),: ‘Northwind’ (lane 5), ‘K7A45’ (lane 6), ‘Dewey Blue’ (lane 7), ‘Alamo’ (lane 8), ‘Ruby Ribbons’ (lane 9), ‘Cave-In- Rock’ (lane 10), ‘Rotstrahlbusch’ (lane 11), Panicum amarum (lane 12), ‘Prairie Sky’ (lane 13), ‘Rehbraun’ (lane 14), ‘Dallas Blue’ (lane 15), ‘Heavy Metal’ (lane 16), ‘Shenandoah ‘ (lane 17), and ‘Squaw’ (lane 18). Water was used as a control in lane 19. DNA was obtained from partially purified virus preparations. M, GeneRuler 1 kb Plus DNA Ladder (Fermentas, Waltham, MA). B) Restriction digestion of rolling-circle products from switchgrass DNA samples. 1, switchgrass variety ‘Cloud Nine’; 2, switchgrass variety ‘Dewey Blue’; M, GeneRuler 1 kb Plus DNA Ladder. Electrophoresis was carried out in three different 1.2 % gels for different lengths of time

protein [MP] and CP), and two ORFs on the virus-antisense the virus is unknown. The Rep amino acid sequences of the strand (replication protein A [RepA] and replication pro- two isolates were 80.5 % identical. tein [Rep]). A splice site for the transcript coding Rep was Nucleotide and predicted amino acid sequences of the predicted using NetGene 2 (www.cbs.dtu.dk/services/Net two switchgrass mastrevirus genomes were aligned to Gene2/)[6]. The CP, MP, RepA, and Rep amino acid se- mastrevirus sequences available in GenBank using MUS- quences of the isolates shared 95 %, 87 %, 79 %, and CLE [5]. Pairwise nucleotide sequence identity scores were 79 % identity, respectively. calculated from the alignments with the Species Demar- The Rep amino acid sequence predicted for the ‘Cloud cation Tool [9]. The full genome sequences of the Nine’ genome was shorter than the Rep amino acid se- switchgrass mastrevirus were most closely related to quence for ‘Dewey Blue’ due to different splice sites in the DfasMV and sporobolus striate mosaic virus (SSMV-1), at complementary-sense transcript. Both donor and acceptor 56 % and 57 % global pairwise nucleotide sequence splice sites showed significant variation. As a result, 42 identity, respectively (Fig. 3a). The amino acid sequences amino acids were deleted in ‘Cloud Nine’ Rep compared to of CP, MP, RepA, and Rep of the two switchgrass mas- ‘Dewey Blue’. The impact of this deletion on infectivity of trevirus genomes were 46 %–48 %, 23 %–27 %, 31 %,

123 B. O. Agindotan et al.

Fig. 3 Phylogenetic analysis of coat, movement, and RepA proteins (ESV), streak Reunion virus (MSRV), of mastreviruses, including the two genomes of switchgrass mosaic- (MSV), miscanthus streak virus (MiSV), oat dwarf virus (ODV), associated virus 1 (SgMaV-1). A) Genome. B) Movement protein. C) panicum streak virus (PanSV), Paspalum dilatatum striate mosaic Coat protein. D) Replication-associated protein (RepA). The viruses virus (PDSMV), paspalum striate mosaic virus (PSMV), saccharum and acronyms used are as follows: Axonopus compressus streak virus streak virus (SacSV), sporobolus striate mosaic virus 1 (SSMV-1), (ACSV), bean yellow dwarf virus (BeYDV), Bromus catharticus sporobolus striate mosaic virus 2 (SSMV-2), streak Egypt striate mosaic virus (BCSMV), chickpea chlorosis Australia virus virus (SSEV), sugarcane streak Reunion virus (SSRV), sugarcane (CpCAV), chickpea chlorosis virus-A (CpCV-A), chickpea chlorosis streak virus (SSV), tobacco yellow dwarf virus (TYDV), urochloa virus-B (CpCV-B), chickpea chlorotic dwarf virus (CpCDV), chick- streak virus (UroSV), wheat dwarf India virus (WDIV), wheat dwarf pea redleaf virus (CpRLV), chloris striate mosaic virus (CSMV), virus (WDV), and (BCTV). BCTV, a curtovirus, Digitaria ciliaris striate mosaic virus (DCSMV), was used as an out-group. Shown nodes are those with bootstrap striate mosaic virus (DDSMV), digitaria streak virus (DSV), dragon- threshold values C50 %. Nodes with lower bootstrap values were fly-associated mastrevirus (DfasMV), Eragrostis curvula streak virus collapsed to polytomies (ECSV), eragrostis minor streak virus (EMSV), eragrostis streak virus and 42 %–48 % identical to those of the closest relatives, species (88 % identity) but qualify as belonging to a new DfasMV (NC019497.1) and SSMV-1 (JQ48052). Max- species (with only 56 %–57 % identity to any other mas- imum-likelihood phylogenetic trees were constructed from trevirus). Because the virus genome was detected in most alignments of the genome nucleotide sequences and CP, switchgrass with mosaic symptoms, we tentatively as- MP, RepA amino acid sequence alignments using MEGA5 signed the name switchgrass mosaic-associated virus [12] (Fig. 3). Beet curly top virus (BCTV, strain Loghan), (SgMaV-1). However, infectivity and pathogenicity tests genus Curtovirus, was used as an out-group for the gen- are required to establish that the mastrevirus genomes ome, MP, CP, and RepA phylogenetic trees (Fig. 3). represent functional and not defective virus genomes. The For the phylogenetic tree, the switchgrass mastrevirus two strains of the virus are designated ‘Dewey Blue’ genomes formed a distinct clade (Fig. 3a). The most (SgMaV-1db) and ‘Cloud Nine’ (SgMaV-1cn). closely related viruses to switchgrass mastrevirus genomes Mastrevirus-seq-2, a partial nucleotide sequence ob- are DfasMV, SSMV-1, and SSMV-2. Interestingly, tained from switchgrass by deep sequencing, shared DfasMV, SSMV-1, and SSMV-2 are mastreviruses found 99.6 % nucleotide sequence identity with SgMaV-1cn and in Australia [7, 11]. Based on the suggested threshold of is therefore the same virus. It is referenced as SgMaV-1 \75 % genome-wide nucleotide sequence identity for the (KJ957193). The second partial sequence, Mastrevirus-seq- mastrevirus species demarcation threshold [4], the 1, did not align significantly with the nucleotide sequences switchgrass mastrevirus genomes represent the same of the two SgMaV-1 strains. The predicted amino acid

123 Detection of a mastrevirus in switchgrass sequence of Mastrevirus-seq-1 shared 34.8 % and 37.0 % 3. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) amino acid sequence identity with the CPs of SgMaV-1db Basic local alignment search tool. J Mol Biol 215:403–410 4. Brown JK, Fauquet CM, Briddon RW, Zerbini M, Moriones E, and SgMaV-1cn (its closest relatives), respectively. Thus, Navas-Castillo J (2012) Family . In: King AMQ, Mastrevirus-seq-1 may represent a second mastrevirus Adams MJ, Carstens EB, Lefkowitz EJ (eds) Virus taxonomy: species and needs to be characterized. It is tentatively classification and nomenclature of viruses; ninth report of the named switchgrass mosaic-associated virus -2 (SgMaV-2: International Committee on Taxonomy of Viruses. Elsevier Academic Press, Waltham, pp 1029–1033 KJ957192) because it was the second mastrevirus detected 5. Edgar RC (2004) MUSCLE: multiple sequence alignment with in switchgrass with mosaic symptoms. high accuracy and high throughput. Nucleic Acids Res Detection of SgMaV-1 in 17 of 18 switchgrass varieties 32:1792–1797 suggests vector transmission, as no mastrevirus is reported 6. Hebsgaard SM, Korning PG, Tolstrup N, Engelbrecht J, Rouze P, Brunak S (1996) Splice site prediction in Arabidopsis thaliana to be transmitted through seed. are known pre-mRNA by combining local and global sequence information. vectors of mastreviruses [4, 10]. Abundant Nucleic Acids Res 24:3439–3452 species observed on and in the experimental plot included 7. Kraberger S, Thomas JE, Geering ADW, Dayaram A, Stainton D, Flexamia atlantica, Graminella aureovittata and G. mohri. Hadfield J, Walters M, Parmenter KS, van Brunschot S, Collings DA, Martin DP, Varsani A (2012) Australian monocot-infecting While G. aureovittata is known to transmit switchgrass mastrevirus diversity rivals that in Africa. Virus Res 169:127–136 mosaic virus (SwMV) [2], the other two leafhopper species 8. Lewandowski I, Scurlock JMO, Lindvall E, Christou M (2003) may acquire SwMV but have not been shown to transmit The development and current status of perennial rhizomatous this virus. Detection of the first mastrevirus in North grasses as energy crops in the US and Europe. Biomass Bioenerg 25:335–361 America opens opportunities for further research into the 9. Muhire B, Martin DP, Brown JK, Navas-Castillo J, Moriones E, biology of SgMaV-1, including its infectivity, host range, Zerbini FM, Rivera-Bustamante R, Malathi VG, Briddon RW, pathogenicity, epidemiology, and vector transmission. Al- Varsani A (2013) A genome-wide pairwise-identity-based pro- so, SgMaV-2 (KJ957192) requires complete genome se- posal for the classification of viruses in the genus Mastrevirus (family Geminiviridae). Arch Virol 158:1411–1424 quencing and the study of its biology. 10. Poojari S, Alabi OJ, Fofanov VY, Naidu RA (2013) A Leafhopper-transmissible DNA virus with novel evolutionary Acknowledgments This work was funded by the Energy Bio- lineage in the family Geminiviridae implicated in grapevine sciences Institute. redleaf disease by next-generation sequencing. PLoS ONE 8:e64194 11. Rosario K, Padilla-Rodriguez M, Kraberger S, Stainton D, Martin References DP, Breitbart M, Varsani A (2013) Discovery of a novel mas- trevirus and alphasatellite-like circular DNA in dragonflies (Epiprocta) from Puerto Rico. Virus Res 171:231–237 1. Agindotan BO, Ahonsi MO, Domier LL, Gray ME, Bradley CA 12. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2010) Application of sequence-independent amplification (SIA) (2011) MEGA5: Molecular evolutionary genetics analysis using for the RNA viruses in bioenergy crops. J Virol Methods maximum likelihood, evolutionary distance, and maximum par- 169:119–128 simony methods. Mol Biol Evol 28:2731–2739 2. Agindotan BO, Prasifka JR, Gray ME, Dietrich CH, Bradley CA (2013) Transmission of Switchgrass mosaic virus by Graminella aureovittata. Can J Plant Pathol 35:384–389

123