Arch Virol (2014) 159:791–795 DOI 10.1007/s00705-013-1891-4

BRIEF REPORT

Yellow tailflower mild mottle virus: a new tobamovirus described from Anthocercis littorea (Solanaceae) in Western Australia Stephen J. Wylie • Hua Li • Michael G. K. Jones

Received: 2 August 2013 / Accepted: 8 October 2013 / Published online: 19 October 2013 Ó Springer-Verlag Wien 2013

Abstract The complete genome sequence of a tobamovirus was determined from a wild plant of yellow tailflower (Anthocercis littorea, family Solanaceae) that exhibited mild mottling and chlorosis on the leaves. The virus induced severe symptoms including systemic necrosis when inoculated to plants of three other solanaceous species. The viral genome was resequenced after passage in Nicotiana benthamiana. The two genomes were 6379 nucleotides in length, and they differed by three nucleotides. Phylogenetic analysis and the deduced architecture of the genome place the virus, provisionally named yellow tailflower mild mottle virus, with other tobamoviruses that infect solanaceous hosts.

In 1886 in Wageningen, The Netherlands, Adolf Mayer (1843-1942) described the first plant virus, which he named tobacco mosaic virus (TMV) [11]. TMV is the type member of the genus Tobamovirus, family Virgaviridae. The family is named for the rod-like shape of the virions, from the Latin virga = rod. There are currently 33 species of tobamoviruses recognized by the International Committee on Taxonomy of Viruses [9]. The tobamoviruses are thought to be an ancient lineage of viruses that co-evolved with the angiosperms 120-140 million years ago [6]. The genomes are of single-stranded, positive-sense RNA that

Electronic supplementary material The online version of this article (doi:10.1007/s00705-013-1891-4) contains supplementary material, which is available to authorized users. S. J. Wylie (&)  H. Li  M. G. K. Jones Australian Plant Virology, Western Australian State Agricultural Biotechnology Centre, School of Veterinary and Life Sciences, Murdoch University, Perth 6150, Australia e-mail: [email protected]

lack a poly-(A) tail. They are transmitted mechanically [1]. The genomes are approximately 6.4 kb in size, arranged in four open reading frames (ORF). The two ORFs located at the 5’ end of the genome constitute the replicase. ORF1 reads through a stop codon to generate a protein of about 183 kDa. ORF2 is a truncated version of the replicase protein encoded by ORF1; it terminates at the internal stop codon to yield a protein of 126 kDa. The two remaining ORFs encode a movement protein (MP) of about 30 kDa and a coat protein (CP) of about 17 kDa [3]. The structure of the 3’ untranslated region (UTR) resembles a t-RNAHIS. Traditionally, there have been three recognized tobamovirus subgroups, distinguished by their sequence phylogeny and by other characteristics of biology and genomic architecture. Subgroup I members infect members of the family Solanaceae and do not have overlapping MP and CP genes. Subgroup II members infect legumes, cucurbits and others and have slightly overlapping MP and CP genes. Subgroup III members infect brassicas, asterids and others, and their MP and CP genes overlap to a greater extent than do members of subgroup II [1, 12]. More recently, a reassessment of the group showed that at least five multitypic and two monotypic clades exist that usually correlate with host lineages. Those tobamoviruses infecting members of the Solanales (subgroup I), and Lamiales (subgroup III) grouped closely and were subtended by a monotypic clade consisting of cactus mild mottle virus (host Caryophyllales). The tobamoviruses infecting the Cucurbitales (subgroup II), the Fabales and Malpighiales, and the Malvales formed close but distinct clades that were subtended by the monotypic clade consisting of frangipani mosaic virus (host Gentianales) [15]. The tobamoviruses TMV and tobacco mild green mosaic virus (TMGMV) are reported in Australia on the exotic weed N. glauca [4]. Clitoria yellow mottle virus

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(CYMV) is described only from Australia, but only on the exotic legume Clitoria ternatea (butterfly pea) in northern Australia [6]. Until now, no tobamoviruses have been described from the Australian indigenous flora. Here, we describe a new tobamovirus from yellow tailflower (Anthocercis littorea Labill.) within the family Solanaceae. Anthocercis is a genus of shrubs endemic to southern temperate Australia with the center of distribution in the southwest of Australia, a region of exceptionally rich species diversity [8]. Yellow tailflower grows to 1-3 metres high on deep calcareous sandy soils along the coast. It is a plant that commonly colonizes dunes after disturbances such as fire, when dense stands of it emerge and dominate for 5-10 years. It stabilizes dunes and acts as a nursery plant under which other species become established [13]. Leaves from a yellow tailflower plant were collected from a sparse stand of them in sand dunes (GPS coordinates -30.652238, 115.145273) south of the village of Cervantes in southwestern Australia. Some leaves displayed mild mottling and chlorosis. Sap from these leaves was inoculated to seedlings of Nicotiana glutinosa, Chenopodium quinoa, C. amaranticolor, and Australian indigenous solanaceous species N. benthamiana [7] and N. umbratica, in phosphate buffer (pH 7.0) using silicaceous powder as an abrasive. Total RNA from leaves of the original yellow tailflower plant from which the newly identified virus was derived, and from a symptomatic plant of N. benthamiana that was inoculated with sap of the original host plant, was extracted using a PowerplantÒ RNA isolation kit (Mo Bio LaboraTM tories). cDNA was synthesized with GoScript reverse transcriptase (Promega Corporation) using the random primer VRT (5’-CGT ACA GTT AGC AGG CNN NNN NNN NNN N-3’, where N is any nucleotide). The use of primer VRT caused an adaptor to be incorporated sequence at the 5’ ends of cDNA molecules. A single unique PCR primer was used for each sample. PCR primers BC1 (5’AGG ACG TAC AGT TAG CAG GC-3’) and BC2 (5’TGG TCG TAC AGT TAG CAG GC-3’), each of which contained a unique 4-nucleotide tag at the 5’ end, annealed to the complement of the adaptor sequence added to cDNA molecules synthesized from double-stranded RNA (dsRNA) molecules and were used to amplify the cDNA by TM PCR. Amplification using GoTaq DNA polymerase (Promega Corporation) was performed under cycling conditions of 95 °C for 10 s, 50 °C for 20 s, and 72 °C for 30 s over 35 cycles. The resulting amplicons were purified directly using QIAquickÒ PCR purification columns (QIAGEN). The amplified DNA was quantified, and 0.5 ug of each sample was pooled in one tube for sequencing. Eighty-six other PCR samples from experiments not associated with this virus, and each similarly tagged with a unique 4-nucleotide sequence, were pooled with them. The

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Australian Genome Research Facility, Melbourne, Australia, did library preparation and sequencing of paired ends over 100 cycles on an Illumina HiSeq2000 machine. Joining of paired sequences, separating of tagged sequence reads, de novo assembly of contigs, editing where required, and calculation of the genome architecture were done primarily using the CLC Genomics Workbench v6.0.5 (CLC Bio) and Geneious Pro v6.1.6 (Biomatters) packages. Parameters for de novo assembly of contigs were minimum overlap of 50 % of read length, 10 % maximum gaps per read. Three assemblies were done for each dataset using minimum overlap identities of 80 %, 90 %, and 95 %. Consensus sequences were compared with sequences in the GenBank database using Blastx. Open reading frames and identities of deduced proteins were predicted by homology to annotated virus sequences available on GenBank, at the Conserved Domain Database (CDD) within NCBI, and InterProScan (Zdobnov & Apweiler 2001) accessed at http://www.ebi.ac.uk/Tools/pfa/iprscan/. The substitution model with the lowest Bayesian information criterion score was calculated within MEGA5 and used for estimating evolutionary relationships between sequences. Estimates of evolutionary relationships were calculated from global alignments of aa sequences within MEGA5 using the statistical methods neighbor-joining (NJ), maximum parsimony, and maximum likelihood, and Mr Bayes within Geneious Pro. Internal settings were cost matrix of 65 %, gap open penalty of 12 and gap extension penalty of 3. Bootstrap analysis of 1,000 replicates was used to assess support for relationships. The NJ trees presented were congruent with other analyses. Small necrotic local lesions developed on the inoculated leaves of C. amaranticolor and C. quinoa five days after inoculation. N. glutinosa plants displayed a severe systemic response that was visible within four days of inoculation. Upper leaves became pale yellow or bleached of color and curled downwards. Inoculated leaves abscised, and the whole plant died within 10-15 days of inoculation. On N. benthamiana and N. umbratica plants, systemic necrosis occurred within 15 days when young seedlings were inoculated at the four-leaf stage. When more mature plants of these species were inoculated, they exhibited initial symptoms of leaf distortion, pallor, and flower abortion within seven days. Leaves often exhibited inter-veinal bleaching (Fig. 1a). Later symptoms were leaf distortion, mosaic including protruding dark-green islands on the leaves, flower distortion (Fig. 1b) and abortion, but rarely systemic necrosis. When sap extracted from symptomatic plants was used to inoculate other Nicotiana seedlings, the same symptoms developed on them. Yellow tailflower cDNA yielded approximately 3.02 million reads after sequencing. Following de novo assembly and BLAST analysis of contigs, one was identified with

Yellow tailflower mild mottle virus

Fig. 1 (a) Nicotiana benthamiana leaf infected with yellow tailflower mild mottle virus (YTMMV) showing inter-veinal clearing of chlorophyll. (b) Mature N. benthamiana plant showing advanced symptoms of YTMMV infection, including leaf and flower deformation, and dark-green islands on leaves

sequence identity to genomes of tobamoviruses. Of the total reads, approximately 15,300 (0.50 %) of them mapped to the tobamovirus genome sequence (isolate Cervantes) of 6379 nucleotides (nt). Maximum coverage was 4171-fold, minimum coverage was 18-fold, and mean coverage was 256-fold. N. benthamiana cDNA yielded 12.20 million reads, of which approximately 164,800 (1.35 %) mapped to a tobamovirus-like sequence of 6379 nt (isolate Nb). Maximum coverage was 40,170-fold, minimum coverage was 129-fold, and mean coverage was 3110-fold. Pairwise alignment of the two genome sequences revealed that they were almost identical; they differed at only three nucleotides. A synonymous G[A transition occurred at nt 2686 within the replicase. A G[A transition occurred at nt 6282, and a U[A transversion at nt 6329, both within the 3’ UTR region. The 5’UTR was 70 nt in length, comparable in size to those of other tobamoviruses. The 5’ proximal nucleotides were GUAAUUUUU, which shared high identity with the 5’ proximal GUAUUUUU of several other tobamoviruses, including TMV (GenBank accession NC_001367), bell pepper mottle virus (NC_009642) and odontoglossum ringspot virus (NC_001728). The sizes and positions of the estimated four ORFs conformed to subgroup I tobamoviruses, i.e., the predicted MP and CP genes did not overlap. The two ORFs at the 5’ end of the genome were predicted to encode replicase proteins. As with other tobamoviruses [1], ribosomes translating ORF1 (nt 71-4912) are predicted to read through an amber (UAG) stop codon (nt 3428-3430) and terminate at an ochre (UAA) stop codon (4913-4915). The genes of both ORFs started in the context CAACAAUGGC, and the mass of the large ‘readthrough’ protein was calculated to be 183 kDa. It encompassed methyltransferase (nt 218-1378), helicase (nt 2558-3334), and RNA-dependent RNA polymerase (RdRp) (nt 4220-4699) domains. The highly conserved RdRp motif

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S/UGx3 Ux3 NS/Ux22 GDD motif occurred at nt 4409-4516 as SGx3 Ux3 NUx22 GDD. ORF2 (nt 71-3427) is predicted to encode a truncated version of the large replicase encoded by ORF1. In this case, protein synthesis is terminated at the amber stop codon located at nt 3428-3430. The calculated mass of the protein was 126 kDa, which is in keeping with those of other tobamoviruses. ORF3 (nt 4924-5721) was predicted to encode the MP. The start codon was within the context UCCAAUGGC in reading frame ?2, and the gene terminated at an ochre stop codon. The protein mass was calculated at 30 kDa, the typical size of tobamovirus MPs. ORF4 (5727-6200) in reading frame ?1 was predicted to encode a CP calculated to be 17.8 kDa, in line with other tobamovirus CPs. The AUG start codon was in the context AAUUAUGUC and terminated at an ochre stop codon (nt 6201-6203). The 3’ UTR was 179 nt long (nt 6201-6379), and like those of other tobamoviruses, the proximal 3’ nts were GCCCA. The complete genome sequences of YTMMV isolates Cervantes and Nb (isolate from N. benthamiana plant) were assigned GenBank accession codes KF495564 and KF495565, respectively. The cDNA synthesis method used, whereby cDNA was primed randomly while an adaptor sequence was added to the 5’ end of each new cDNA strand, was sufficient to Table 1 Percent identity of the nucleotide (nt) sequence of the complete genome and deduced amino acid (aa) sequences of the large replicase (rep) protein, the movement protein (MP), and coat protein (CP) of yellow tailflower mild mottle virus, GenBank accession no. KF495564, with those of some other tobamoviruses for which complete genome sequences are available Virus

Subgroup

Complete genome (nt)

Rep (aa)

MP (aa)

CP (aa)

Obuda pepper virus NC_003852 Paprika mild mottle virus NC_004106

I

62

71

63

65

I

62

71

66

64

Tomato virus NC_002692

I

60

67

54

63

Pepper mild mottle virus NC_003630 Brugmansia mild mottle virus, NC_010944

I

59

65

61

60

I

59

66

59

64

Turnip vein-clearing virus NC_001873

III

53

59

37

53

Zucchini green mottle mosaic virus NC_003878

II

46

43

28

47

Frangipani mosaic virus NC_014546

II

45

42

27

48

Clitoria yellow mottle virus NC_016519

II

46

41

19

48

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detect the tobamovirus sequences. dsRNA is generated during the replicative phase of single-stranded RNA (ssRNA) viruses. When this method is used, cDNA derived from dsRNA species should be preferentially amplified because the PCR primers anneal to the complementary sequence of the adaptor. This complementary sequence should be synthesised only in the presence of dsRNA, when cDNA strands are synthesised from each complementary strand. Analysis of the resulting sequence data revealed that 0.5-1.35 % of the reads were derived from the viral genome. The remaining reads were of plant origin, about 45 % of which were derived from the chloroplast genome, signifying that cDNA of ssRNA of plant origin was also amplified. The complete genome nucleotide sequence of YTMMV was aligned in a pairwise manner with genomes of other tobamoviruses (Fig. S1). Sequence identity ranged from 46 % with subgroup III viruses such as cactus mild mottle virus (NC_011803) to 70 % with subgroup I viruses including paprika mild mottle virus (NC_004106) and Obuda pepper virus (NC_003852). Deduced amino acid sequences of the large readthrough replicase protein, MP

S. J. Wylie et al.

and CP revealed the same pattern of relatedness—that YTMMV was most closely related to subgroup I tobamoviruses, all of which have been isolated from solanaceous host plants (Table 1). With other solanaceous-infecting tobamoviruses, YTMMV shared 66-71 % amino acid sequence identity in the replicase gene and 60-65 % identity in the CP gene (Table 1, Fig. 2, Fig. S2). Adams and colleagues [1] showed that isolates of different tobamovirus species share less than 93.8 % amino acid sequence identity between replicases, and less than 93 % identity between CPs. These demarcation criteria clearly distinguish yellow tailflower mild mottle virus as a member of a distinct species within the genus Tobamovirus. Its solanaceous host, its genome architecture, and the sequence similarity of its proteins to homologous proteins of other tobamoviruses that infect members of the Solanaceae place YTMMV within subgroup I of the genus Tobamovirus. Although members of the genus Tobamovirus have been described from exotic plants in Australia, none have previously been isolated from indigenous host species. Not surprisingly, YTMMV was not closely related to the only

subgroup I

subgroup III

subgroup II

Monotypic subgroup Monotypic subgroup Malvales subgroup Fabales, Malpighiales subgroup

Fig. 2 Neighbour-joining tree showing predicted evolutionary relationships of amino acid sequences of the 186-kDa replicases of isolates of yellow tailflower mild mottle virus, indicated by a filled circle, and those of some other tobamoviruses representing described subgroups

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Yellow tailflower mild mottle virus

other tobamovirus isolated only from Australia, CYMV, which infects an exotic leguminous weed and may be a member of the subgroup that infects members of the Fabales and Malpighiales, although it does not cluster closely with them (Fig. 2). The existence of these two potentially indigenous tobamoviruses is a tantalising indication of the presence of others on the Australian continent. The identification and roles of viruses that have long-standing relationships with wild-plant communities is a poorly studied area, yet it is a significant one for a number of reasons, one of which is that it is from wild plants that new pathogenic viruses emerge to threaten agricultural and horticultural crops. Tobamoviruses have an international distribution, and some are responsible for considerable economic loss [2, 5, 14, 16]. It is worth noting that Koch’s postulates were not tested to determine if YTMMV infection induced the faint mottling symptoms observed on the leaves of the original natural host plant of Anthocercis littorea. It is relevant that YTMMV induced severe symptoms on the other solanaceous plants tested. The recently opened Indian Ocean Drive, which runs past the original collection site, may provide opportunities for a mechanically-transmitted tobamovirus to extend its range to other hosts, potentially to the commercially produced solanaceous crops of tomatoes and capsicums that are cultivated in areas surrounding the greater Perth metropolitan region nearby. Australia has a large cohort of endemic solanaceous plants [10] that should be tested for tobamoviruses and other viruses in their natural habitats. Acknowledgments This study was funded by Australian Research Council Linkage Grant LP110200180, with financial support from the Western Australian Botanic Gardens and Parks Authority and the Australian Orchid Foundation.

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Yellow tailflower mild mottle virus: a new tobamovirus described from Anthocercis littorea (Solanaceae) in Western Australia.

The complete genome sequence of a tobamovirus was determined from a wild plant of yellow tailflower (Anthocercis littorea, family Solanaceae) that exh...
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