[Show abstract][Hide abstract] ABSTRACT: Five isolates of Cucumber mosaic virus (CMV) from Lilium sp. (lily), which were isolated from specimens in Japan, Korea and Taiwan, were unable to support satellite RNA (satRNA) accumulation. In order to map the CMV sequences that are involved in satRNA support, HL-CMV (Japanese lily isolate), Y-CMV (ordinary strain) and Y-satellite RNA (Y-sat) were used as the source material. The pseudorecombinants between Y-CMV and HL-CMV revealed that RNA1 was essential for satRNA replication in lily. The results of chimeric constructs and various mutations showed that two amino acid residues (at positions 876 and 891) in the 1a protein were the determinants for the inability of HL-CMV to support a satRNA. Specifically, Thr at position 876 had a more pronounced effect than Met at position 891. Specific changes in RNA sequence were also detected in the 3' terminus of Y-sat and these particular alterations allowed it to be supported by HL-CMV. It is believed that, through evolution, the adaptation of CMV to lily resulted in the introduction of amino acid changes in the 1a protein, changes that coincidentally affected the ability of lily CMV to support satRNAs.
Journal of General Virology 09/2005; 86(Pt 8):2359-69. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The basis for differences in the timing of systemic symptom elicitation in zucchini squash between a pepper strain of Cucumber mosaic virus (Pf-CMV) and a cucurbit strain (Fny-CMV) was analysed. The difference in timing of appearance of systemic symptoms was shown to map to both RNA 2 and RNA 3 of Pf-CMV, with pseudorecombinant viruses containing either RNA 2 or RNA 3 from Pf-CMV showing an intermediate rate of systemic symptom development compared with those containing both or neither Pf-CMV RNAs. Symptom phenotype was shown to map to two single-nucleotide changes, both in codons for Ile at aa 267 and 168 (in Fny-CMV RNAs 2 and 3, respectively) to Thr (in Pf-CMV RNAs 2 and 3). The differential rate of symptom development was shown to be due to differences in the rates of cell-to-cell movement in the inoculated cotyledons, as well as differences in the rate of egress of the virus from the inoculated leaves. These data indicate that both the CMV 3a movement protein and the CMV 2a polymerase protein affect the rate of movement of CMV in zucchini squash and that these two proteins function independently of each other in their interactions with the host, facilitating virus movement.
Journal of General Virology 05/2005; 86(Pt 4):1213-22. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A filamentous virus was isolated from taro (Colocasia esculenta Schott) showing mosaic and chlorotic feather-ing symptoms in Chuncheon, Gangwon province in 2002. Based on ELISA, its appearance in electron microscope, serological relationships, and RT-PCR using specific primer and nucleotide sequence analysis of the CP gene, the isolated virus was identified as Dasheen mosaic virus (DsMV) and designated as Korean isolated (DsMV-Kr). DsMV was not serologically related to Zantedeschia mosaic virus (ZaMV), which has been reported to infect an Araceae plants. Since the coat protein revealed electrophoretic heterogeneity, about 42 kDa, 39 kDa and 31 kDa by SDS-PAGE, an improved purification method was established for the production of antisera against DsMV-Kr. The purification method used in this study may be effectively applied to the purification of other filamentous viruses. Dasheen mosaic virus (DsMV), a species of the genus Potyvirus in the family Potyviridae, is one of the major virus infecting taro in Korea and other countries (Chen et al., 2001; Shimoyama et al., 1992; Zettler et al., 1987; Zettler et al., 1990). DsMV is a flexuous filamentous virion with 750 nm in length and 13 nm in width and contains about 10kb positive-sense single-stranded RNA genome, has a covalently linked protein (VPg) at the 5'-terminus and is polyadenylated at the 3'-terminus. DsMV are transmitted by aphids in a non-persistent manner (Zettler et al., 1978). DsMV is the most important viral pathogen of cultivated aroid plants worldwide, including the genera Aglaonema, Caladium, Dieffenbachia, Epipremnum, Philodendron, Spathiphyllum, and Syngonium. In addition to these foliage plants, DsMV also infects the Araceae plants including the genera Cryptocoryne (commercially grown aquarium plant), Zantedeschia (Calla lily), and two high-carbohydrate tropical food crops known as dasheen or taro (Colocasia) and malanga (Xanthosoma). Taro (Colocasia esculenta Schott) originated from India and adjacent areas of southeast Asia is now widely cultivated in Asia and Oceania. In some Pacific Coast countries, this crop is one of the main sources of starch food. In Korea, the crop has been cultivated constantly over 4,000 tons/year. Taro leaves showing mosaic and chlorotic feathering symptoms from taro were collected in Chuncheon, Gangwon province in 2002. Although DsMV, which was discovered in 1969 (Zettler et al., 1970), has been reported from many countries, the relationships among isolates have not yet been studied systematically. Little information is available on the relation-ship between the Korean isolate and other known DsMV isolates. To establish the diagnostic system for virus-free taro stock production, we conducted an experiment to identity virus disease in taro showing mosaic and chlorotic feathering symptoms, and analyzed the biological, serological, and molecular characteristics in this study.
The plant pathology journal 01/2004; 20:135-141. · 0.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A lily strain of Cucumber mosaic virus (LK-CMV) was not able to systemically infect zucchini squash (Cucurbita pepo), while Fny strain of CMV (Fny-CMV) caused systemic mosaic and stunting symptom at 4 days post-inoculation on the same host species. The pathogenicity of LK-CMV in zucchini squash was investigated by reassortments of genomic RNAs of LK-CMV and Fny-CMV for infection, as well as by pseudorecombinants generated from biologically active transcripts of cDNA clones of LK-CMV and Fny-CMV, respectively. The assessments of pathogenicity for LK-CMV indicated that RNA2 of LK-CMV was responsible for systemic infection in zucchini squash. In the protoplast of zucchini squash, the RNA accumulations of all constructed pseudorecombinants were indistinguishable and LK-CMV replication was slightly lower than that of Fny-CMV, suggesting that the inability of LK-CMV to infect squash plants was responsible for the poor efficiency of virus movement, rather than the reduction of replication function.
Virus Research 12/2003; 97(1):1-6. · 2.75 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Zucchini squash (Cucurbita pepo) is a systemic host for most strains of the cucumovirus Cucumber mosaic virus (CMV), although the long-distance movement of the M strain of CMV (M-CMV) is inhibited in some cultivars. However, co-infection of zucchini plants with M-CMV and the potyvirus Zucchini yellow mosaic virus strain A (ZYMV-A) allowed M-CMV to move systemically, as demonstrated by tissue-print analysis. These doubly infected plants exhibited severe synergism in pathology. Infection of zucchini squash by M-CMV and an attenuated strain of ZYMV (ZYMV-AG) showed a milder synergy in pathology, in which ZYMV-AG also facilitated the long-distance movement of M-CMV similar to that promoted by ZYMV-A. Variation in the extent of synergy in pathology by the two strains of ZYMV did not correlate with differences in levels of accumulation of either virus. Thus, the extent of synergy in pathology is at least in part independent of the resistance-neutralizing function of the potyvirus.
Journal of General Virology 01/2003; 83(Pt 12):3173-8. · 3.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study examined the existence of genetically diverse population of Cucumber mosaic virus (CMV), known as quasispecies, from lily, Nicotiana benthamiana and from purified virions. Based on the conserved sequences of CMV lily isolates in intergenic region (IR) on RNA3, the genetic variation of IR from three different sources was investigated by a specific restriction endonuclease hydrolysis of amplified reverse transcription-polymer-ase chain reaction (RT-PCR) products using virus-specific primers, and was compared with IR sequences. The IR nucleotide sequences of CMV lily isolates were highly conserved, however, quasispecies was detected from all three sources in low level, containing sub-populations of RNA3. These subpopulations of RNA3 were inoculated onto zucchini squash by in vitro transcripts from corresponding full-length cDNA clones together with Fny RNA1 and 2 transcripts. The systemic symptom of zucchini plants infected by these quasispecies was chlorotic spotting, which was milder than severe mosaic and stunt symptom caused by Fny-CMV. The severity of symptom was correlated with RNA accumulation of viruses. These results suggest that the genome of CMV lily isolates consists of quasispecies populations. Viruses have extreme evolutionary capacities that have allowed adaptation to parasitize numerous host species. Among them, plant viruses have evolved into genetically diverse populations referred to as quasispecies, by means of genetic mode of RNA genome associated with error-prone replication, rapid rate of replication, and large population (Domingo et al., 2002; Rodriguez-Alvarado et al., 1995). Plant viral quasispecies are dynamic and complex distributions that react to environmental changes, resulting in a pool of mutants which could become a selective advantaged RNA species to new environment (Domingo et al., 1996; Roossinck and Palukaitis, 1990). Many reports suggested that the population diversity of Cucumber mosaic virus (CMV) has very different levels of variations in common hosts and commercial crops (Aranda et al., 1993; Kurath and Palukaitis, 1989; Palukaitis and Roossinck, 1995; Rodriguez-Alvarado et al., 1995; Schneider and Roossinck, 2001). CMV, the type species of the genus Cucumovirus in the family Bromoviridae, contains a tripartite genome of messenger-sense single-stranded RNA, designated as RNAs 1, 2 and 3 in order of decreasing size (Palukaitis et al., 1992). RNA3 encodes for two proteins involved in viral movement and encapsidation (Canto et al., 1997). Meanwhile, RNA2 encodes for the 2a protein, which is a RNA-dependent RNA polymerase of replication complex, whereas, RNA1 encodes for the 1a protein, another subunit of CMV replicase complex (Hayes and Buck, 1990). CMV has the largest host range encompassing over 1000 species of plants in 365 genera of 85 families (Palukaitis et al., 1992). Also, it has been suggested that a few CMV isolates have shown different biological and genetic properties from typical CMV strains. In particular, some CMV isolates from lily and alstroemeria have these variant properties. Chen et al. (2002) demonstrated that CMV subgroup II isolates from alstroemeria contained additional sequences of various lengths in the 3' noncoding region (NCR) that were generated by RNA recombination. In addition, some reports on lily isolates of CMV showed that most lily isolates could not infect tobacco plants and cucurbits unlike the typical CMV strains, but the genome of lily isolates was highly homologous (Chen et al., 2001; Jung et al., 2000; Masuta et al., 2002; Ryu et al., 2002). This report shows that RNA3 of CMV lily isolates exists in the subpopulation.
Plant Pathol. J. The Plant Pathology Journal The Korean Society of Plant Pathology. 01/2003; 19:210-216.
[Show abstract][Hide abstract] ABSTRACT: We analyzed the evolutionary histories of two lily strains of Cucumber mosaic virus (CMV) isolated in Japan and Korea (HL- and Ly2-CMVs). They share common biological characteristics in that their host ranges
are very restricted perhaps from a unique adaptation to lily plants. Although HL and Ly2 were isolated independently from
different lily species in separate countries, their RNA3 sequences had a very high sequence similarity (97%). The evolutionary
relationships between the two isolates were characterized by comparing their phylogenetic trees for the 3a and CP genes. The
two lily CMVs always formed a distinct cluster within subgroup IB in 3a, but within IA in CP. Together, the phylogenetic tree
topology and the sequence identity between the two lily CMVs suggest that they evolved from a common progenitor.
Journal of General Plant Pathology 04/2002; 68(2):163-168. · 0.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Infectious full-length cDNA clones of the Korean strain of tomato aspermy cucumovirus (KC-TAV) were constructed using a long-template reverse transcription-polymerase chain reaction. The in vitro RNA transcripts, which were produced using T7 RNA polymerase from full-length cDNAs, could systemically infect the Nicotiana tabacum cv. Xanti-nc plants and induce systemic symptoms on the upper leaves that are similar to the wildtype KC-TAV. The complete nucleotide sequences of genomic RNAs of KC-TAV were determined from the infectious full-length cDNA clones. RNA1 and RNA2 of KC-TAV contain 3412 nucleotides and 3074 nucleotides, respectively. RNA3 of KC-TAV, 2222 nucleotides long, encodes the 3a protein and coat protein (CP) that are separated by 295 nucleotides intergenic region. The overall sequence analysis of the whole genome of KC-TAV revealed a strong homology (99%) to the genome of the V-TAV strain, the only strain whose entire genomic nucleotide sequence was available in the database, and an overall 60% homology to those of other cucumber mosaic virus and peanut stunt virus strains. A sequence comparison analysis of the deduced amino acid sequences of cDNAs of KC-TAV RNA 1, 2, and 3 indicates that there is no genetic diversity in the TAV population, although the virus exists in different geographical distributions.
Molecules and Cells 03/2002; 13(1):52-60. · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT The complete nucleotide sequence of the Zucchini green mottle mosaic virus (ZGMMV), a new member of the genus Tobamovirus, has been determined. The genome of ZGMMV is 6,513 nucleotides long and contains four open reading frames coding for proteins of 131, 189, 28, and 17 kDa from the 5' to 3' end, respectively. The 5'- and 3'-non-translated regions consist of 59 and 163 residues, respectively. The sequences of the viral proteins exhibit high identity to the proteins of the members of the genus Tobamovirus and are distinct from other viruses within the subgroup of cucurbit-infecting tobamoviruses. Results from phylogenetic trees of the coding regions demonstrated that ZGMMV is a very close relative of Kyuri green mottle mosaic virus and Cucumber fruit mottle mosaic virus and is less similar to Cucumber green mottle mosaic virus. Full-length cDNA of ZGMMV was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) using the 5'-end primer containing a T7 RNA promoter sequence and 3'-end primer. Capped in vitro transcript from the RT-PCR products was infectious on zucchini squash, cucumber, and Nicotiana benthamiana plants. This cell-free system to produce infectious transcripts from uncloned cDNA copies is useful for quick assessment of infectivity of transcripts from plant RNA viruses prior to cloning. Synthesized capped transcript from a full-length cDNA clone of the virus was highly infectious. Progeny virus derived from infectious transcripts had the same biological and biochemical properties as wild-type virus. To our knowledge, this is the first report of a biologically active transcript from a cucurbit-infecting tobamovirus.
[Show abstract][Hide abstract] ABSTRACT: We have developed a new strategy for engineering resistance to multipleviruses in plants. The strategy exploits the human double stranded (ds)RNA-dependent protein kinase (PKR). PKR is one of theinterferon-induced enzymes. It confers viral resistance in mammals byinhibitingviral replication through the inactivation of the translational initiationfactor, eIF-2, upon activation by dsRNA. The humanPKR gene was fused to the promoter of theArabidopsis blue copper binding protein gene(BCB) that is induced rapidly in response to wounding. Thechimeric gene cassette was introduced into tobacco plants. Expression of thePKR gene in transgenic tobacco plants was demonstrated byRNA gel blot analysis and autophosphorylation assay of anM
r 68,000 protein. The transgenic plantsexpressing the PKR gene showed significantly reduced viralsymptoms or no viral symptoms at all, when challenged by different plant RNAviruses, such as Cucumber mosaic virus, Tobaccoetch virus, or Potato virus Y. Thus, expressionof a single component in the human interferon pathway, thePKR gene, can effectively confer resistance to multipleviruses in transgenic plants.