David North Plant Research Centre, Bureau of Sugar Experiment Stations, PO Box 86, Q 4068 Indooroopilly, Australia1
Cooperative Research Centre for Tropical Plant Pathology, The University of Queensland, Q 4070 St Lucia, Australia2
Department of Plant Pathology, University of Minnesota, MN 55108 St Paul, USA3
Research School of Biological Sciences, Australian National University, GPO Box 475, ACT 2601 Canberra, Australia4
Author for correspondence: Grant Smith (at David North Plant Research Centre). Fax +61 7 3871 0383. e-mail GSmith{at}bses.org.au
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() |
---|
An isolate of SCYLV was obtained from sugarcane cultivar CP65-357 from Florida, which had been maintained in glasshouses on the St Paul Campus, University of Minnesota. Viral particles were purified as described by Scagliusi & Lockhart (2000) and RNA was extracted by treatment with Proteinase K and SDS. First strand cDNA was synthesized using MMLV-Superscript II (Gibco-BRL), second strand with RNaseH, E. coli DNA polymerase and E. coli DNA ligase. The products were fractionated on a CL-4B spun column (Pharmacia), blunt-end ligated into pBluescript SK(-) (Stratagene) and sequenced. Rapid amplification of cDNA ends (RACE) was performed using a 5'/3' RACE kit (Boehringer) and the DNA fragments were cloned into plasmid pCR2.1 (Invitrogen), and sequenced. The entire 5895 nucleotide sequence of the SCYLV-F genome was determined in both orientations from two or more independently generated clones (EMBL accession no. AJ249447).
There were no detectable similarities between the 5' and 3' untranslated regions (UTRs) and any sequences in the databases. The 5' UTR starts with the sequence ACAAAA, which is consistent with the 5'-terminal motif of many poleroviruses. The 3' UTR is 217 nucleotides long, which is slightly longer than most polerovirus 3' UTRs but considerably shorter than the 600+ nucleotides of the 3' UTRs of luteoviruses.
The SCYLV-F genome is arranged like those of the poleroviruses. SCYLV-F ORF0 begins at the first AUG codon in the sequence and encodes a 30·2 kDa protein (Table 1). No significant similarities were detected when the databases were searched with the SCYLV-F ORF0 amino acid sequence using the programs BLASTX and BLASTP (Altschul et al., 1997
). It has been noted that the equivalent genes in polerovirus genomes are similarly poorly conserved (Miller et al., 1995
). SCYLV-F ORF1 encodes a 72·5 kDa protein which was found to be most similar to the equivalent genes in the genomes of the poleroviruses (Table 1
) and Pea enation mosaic virus-1 (PEMV-1; Enamovirus). ORF1 of Potato leaf roll virus (PLRV; Polerovirus) has been shown to be a serine protease (Hulanicka et al., 1999
) and a protease-motif, HX2934[D/E]X6263TXKGYSG (Gorbalenya et al., 1989
), is conserved in all polerovirus ORF1 amino acid sequences and the equivalent sequence carried by PEMV-1. SCYLV-F also encodes the protease-motif and hence it is likely that SCYLV-F ORF1 encodes a protease. SCYLV-F ORF2 was found to be most similar to the RNA-dependent RNA polymerase (RdRp) genes of the poleroviruses (Table 1
), sobemoviruses, barnaviruses and PEMV-1. The 5' terminus of the SCYLV-F RdRp overlaps ORF1 in the -1 reading frame by 460 nucleotides. There is a likely frameshifting slippery heptamer, 5' GGGAAAC 3', in this overlap at position 1753, and sequence on the 3' side of the heptamer motif could form a pseudoknot (Fig. 1
). This arrangement with a slippery heptamer and a pseudoknot in the overlap of ORFs 1 and 2 is conserved in the genomes of the poleroviruses and PEMV-1, and permits -1 ribosomal frameshifting from ORF1 to the RdRp gene (Prüfer et al., 1992
). The heptamer and the pseudoknot probably have the same role in the SCYLV-F genome and thus ORFs 1 and 2 of SCYLV-F may be translated together by frameshifting to produce a single 120·6 kDa fusion protein (Table 1
). The pseudoknot predicted in the SCYLV-F sequence has a similar structure to those of the polerovirus and PEMV-1 genomes. However, the sequence that forms the pseudoknot in the SCYLV-F genome includes only 12 of the 19 conserved nucleotides identified by Miller et al. (1995)
in polerovirus and PEMV-1 sequences (Fig. 1
). Furthermore, the likely 3' stem in the SCYLV-F pseudoknot is the shortest so far predicted for a luteovirid, consisting of only 3 base pairs. Together, these differences suggest that the selective forces acting on the SCYLV-F frameshifting site are probably somewhat different from those acting on the equivalent sites in polerovirus genomes.
|
|
To test the significance of the contrasting database search results (Table 1), multiple alignments of the RdRp, CP and RT protein gene sequences were constructed and analysed (Fig. 2
). The program SiScan (M. J. Gibbs, J. S. Armstrong & A. J. Gibbs, Australian National University; http://life.anu.edu.au/molecular/software/siscan/) was used to calculate total pair-wise identity within a window that was passed over a nucleotide alignment of SCYLV-F, BYDV-PAV, PEMV-1 and PLRV sequences. SiScan was also used to calculate Z scores from the identity scores using 100 equivalent sets of randomized sequences. The raw scores (Fig. 2A
) and Z scores (Fig. 2B
) confirmed that recombination had taken place and suggested that at least two inter-species recombinations contributed to the evolution of SCYLV-F. Direct inspection of the alignments showed that SCYLV-F ORF2 is most similar to the RdRp gene of PLRV over its entire length and database searches using the program BLASTP (Altschul et al., 1997
) showed that the arginine-rich N-terminal domain of the SCYLV-F CP is also most similar to its counterpart in the PLRV CP. The affinities of the SCYLV-F CP change at about amino acid residue 40. Most of the remainder of the CP sequence is closest to that of BYDV-PAV. A closer affinity to the PEMV-1 sequence becomes apparent in the last 15 amino acid residues of the CP or in the short proline-rich stretch at the N terminus of the RT protein. Alignments also suggested that the internal UTR sequence of SCYLV-F is more closely related to that of PLRV than to that of BYDV-PAV and we identified two short stretches of identity in the alignment of the SCYLV-F and PLRV sequences (5' UAGCGGG; 5' CGCAAUCCC).
|
Minimum evolution and maximum parsimony trees were found for a subset of luteovirus sequences (Fig. 2DF
) by heuristic searching with the program PAUP version 4d64 (kindly supplied by David L. Swofford). Minimum evolution trees were calculated using the JukesCantor correction. Bootstrap values were calculated from neighbour-joining trees that were found using 1000 bootstrapped samples of the data. As the trees illustrate (Fig. 2D
, E
), no other luteovirid so far characterized has a polerovirus-like RdRp gene and a CP gene most closely related to those of viruses of the newly defined Luteovirus genus. Thus, it is clear that our new evidence of recombination at a site between the RdRp and CP genes cannot be explained by any of the previously identified recombinations that map to this region in luteovirid genomes (Veidt et al., 1988
; Rathjen et al., 1994
; Guilley et al., 1995
). Similarly, the evidence of recombination at a site close to the boundary between the CP and RT protein genes cannot be explained by the recombinational event previously identified close to this site in the evolution of Cucurbit aphid-borne yellows virus (CABYV; Gibbs & Cooper, 1995
). Phylogenetic analyses show that the RdRp and CP genes of SCYLV-F and CABYV have different affinities (Fig. 2D
, E
), and hence the ancestors of these viruses must have diverged before the respective recombinational events and those events were independent.
The fact that the inter-species recombinations evident within the SCYLV-F sequence map to the same genomic locations as other previously described recombinations suggests that selection against recombination occurs at other sites, or operates in favour of certain new combinations of genes. One possibility is that the recombination events have been linked to changes in the combination of hosts and vectors used by the luteovirids (Gibbs, 1994 ) as determined by new combinations of RdRp, MP and RT protein genes. Alternatively, there may be strong selection against recombination at other sites.
Clearly inter-species recombination has occurred very frequently in the evolution of luteovirids. For this reason, it is difficult to distinguish the recombinant and parental lineages. We cannot assume that SCYLV-F is a recombinant simply because its sequence provides new evidence of recombination. The evidence depends on the availability of sequences from several species and the order in which the sequences were obtained could influence its interpretation. However, given that we have evidence of not one, but two, recombinational events that appear to have connected sequences from a polerovirus, a luteovirus and an enamovirus, which are otherwise largely distinct lineages, we think it is likely that SCYLV-F is a recombinant.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() |
---|
Anon. (1995). Yellow leaf syndrome. Mauritius Sugar Industry Research Institute Annual Report 1995, p. 51.
Bailey, R. A., Bechet, G. R. & Cronjé, C. P. R. (1996). Notes on the occurrence of yellow leaf syndrome of sugarcane in southern Africa.Proceedings of the South African Sugar Technologists Association 70, 3-6.
Bork, P. & Gibson, T. J. (1996). Applying motif and profile searches.Methods in Enzymology 266, 162-183.[Medline]
Comstock, J. C., Irvine, J. E. & Miller, J. D. (1994). Yellow leaf syndrome appears on the United States mainland. Sugar Journal, March 1994, 3335.
Gibbs, M. J. (1994). Risks in using transgenic plants.Science 264, 1650-1651.[Medline]
Gibbs, M. J. & Cooper, J. I. (1995). A recombinational event in the history of luteoviruses probably induced by base-pairing between the genomes of two distinct viruses.Virology 206, 1129-1132.[Medline]
Gorbalenya, A. E., Koonin, E. V., Blinov, V. M. & Donchenko, A. P. (1989). Sobemovirus genome appears to encode a serine protease related to cysteine proteases of picornaviruses.FEBS Letters 236, 287-290.
Guilley, H., Richards, K. E. & Jonard, G. (1995). Nucleotide sequence of beet mild yellowing virus RNA.Archives of Virology 140, 1109-1118.[Medline]
Hulanicka, M. D., Sadowy, E., Juszczuk, M. & Gronenborn, B. (1999). ORF1 of potato leafroll virus encodes a serine proteinase indispensable for viral replication. Abstract VW15.04, XIth International Congress of Virology, p. 48.
Irey, M. S., Baucum, L. E., Derrick, K. S., Manjunath, K. L. & Lockhart, B. E. (1997). Detection of the luteovirus associated with yellow leaf syndrome of sugarcane (YLS) by a reverse transcriptase polymerase chain reaction and incidence of YLS in commercial varieties in Florida. Proceedings of the ISSCT 5th Pathology and 2nd Molecular Biology Workshop.
Miller, W. A. & Rasochová, L. (1997). Barley yellow dwarf viruses.Annual Review of Phytopathology 35, 167-190.[Medline]
Miller, W. A., Dinesh-Kumar, S. P. & Paul, C. P. (1995). Luteovirus gene expression.Critical Reviews in Plant Science 14, 179-211.
Prüfer, D., Tacke, E., Schmitz, J., Kull, B., Kaufmann, A. & Rohde, W. (1992). Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.EMBO Journal 11, 1111-1117.[Abstract]
Rathjen, J. P., Karageorgos, L. E., Habili, N., Waterhouse, P. M. & Symons, R. H. (1994). Soybean dwarf luteovirus contains the third variant genome type in the luteovirus group.Virology 198, 671-679.[Medline]
Scagliusi, S. M. & Lockhart, B. E. (1997). Transmission, characterization and serology of sugarcane yellow leaf luteovirus. Proceedings of the ISSCT 5th Pathology and 2nd Molecular Biology Workshop.
Scagliusi, S. M. & Lockhart, B. E. (2000). Transmission, characterization and serology of a luteovirus associated with yellow leaf syndrome of sugarcane.Phytopathology 90, 120-124.
Schenck, S. (1990). Yellow leaf syndrome a new sugarcane disease. Experiment Station Hawaiian Sugar Planters Association Annual Report 1990, p. 38.
Smith, G. R., Fraser, T. A., Braithwaite, K. S. & Harding, R. M. (1995). RTPCR amplification of RNA from sugarcane with yellow leaf syndrome using luteovirus group-specific primers. Proceedings of the 10th Australasian Plant Pathology Society Conference, p. 84.
Vega, J., Scagliusi, S. M. M. & Ulian, E. C. (1997). Sugarcane yellow leaf disease in Brazil: evidence of association with a luteovirus.Plant Disease 81, 21-26.
Veidt, I., Lot, H., Leiser, D., Scheidecker, D., Guilley, H., Richards, K. & Jonard, G. (1988). Nucleotide sequence of beet western yellows virus RNA.Nucleic Acids Research 16, 9917-9932.[Abstract]
Received 10 January 2000;
accepted 3 March 2000.