[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. DOI:10.1099/vir.0.81059-0 · 3.18 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. DOI:10.1099/vir.0.80744-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A filamentous virus was isolated from taro (Colocasia esculenta Schott) showing mosaic and chlorotic feathering 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.
[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. DOI:10.1016/S0168-1702(03)00215-6 · 2.32 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. DOI:10.1099/0022-1317-83-12-3173 · 3.18 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-polymerase 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 subpopulations 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.
[Show abstract][Hide abstract] ABSTRACT: This study was conducted to determine the causal virus that naturally infected hollyhock (Althaea rosea) plant showing mild mosaic symptom in 1999. Flexuous virus particles were found in the cytoplasm of plant tissue from infected hollyhock under transmissible electron microscopy. A virus from the genus Potyvirus under the family Potyviridae was isolated and was maintained on Chenopodium quinoa for three passages. Chlorotic local legions were used to inoculate 20 species of indicator plants. The virus infected all the tested cucurbit plants, but failed to infect Nicotiana benthamiana. Based on the host range test and RT-PCR analysis, the potyvirus was identified as a strain of Zucchini yellow mosaic virus-A (ZYMV-A), one of the major pathogens of cucurbits. Infectivity analysis showed that ZYMV-A induced faster systemic symptom than ZYMV-Cu on squash and other cucurbit plants, suggesting that ZYMV-A was a more severe strain. To better characterize ZYMV-A, Western blot assay was carried out to the coat protein (CP) of the virus using ZYMV-specific antiserum with ZYMV-Cu and other potyviruses. The CP of the virus reacted strongly with the antiserum against ZYMV, and other tested antisera did not react with the CP of ZYMV-A. Results strongly suggest that the potyvirus infecting hollyhock was a novel strain of ZYMV. This is the first report on ZYMV as the causal virus infecting hollyhock in Korea. Zucchini yellow mosaic virus (ZYMV), a species of the genus Potyvirus in the family Potyviridae, was first reported in Italy and France by Lisa et al. (1981). The virus is transmitted from infected plants to healthy ones by several kinds of aphids in a non-persistent manner (Gal-On et al., 1995). ZYMV has caused devastating epidemics in a number of commercial cucurbits worldwide, and several distinct biological strains of ZYMV have been described (Desbiez and Lecoq, 1997; Lecoq and Pitat, 1984; Provvidenti et al., 1984). Like other potyviruses, ZYMV has a positive sense single-stranded RNA genome translated as one polyprotein precursor which is cleaved subsequently to yield eight to nine functional proteins by virus-encoded protein proteases: P1, HC-Pro, and NIa. P1 and HC-Pro proteinases are located at the N-terminal region of the protein and act as suppression factor to post-transcriptional gene silencing (PTGS) (Brigneti et al., 1998). NIa interacts with NIb containing the activity of viral RNA-dependent RNA polymerase (Hong et al., 1995), while VPg is one of the determinant factors in pathogenicity. Other potyvirus-encoded proteins include that of CI protein acting as ATPase/ RNA helicase, and coat protein (CP) of potyvirus which has multifunctional activities involved in aphid transmission, cell-to-cell movement, systemic movement, encapsidation of the viral RNA, and regulation of viral RNA amplification (Gal-On et al., 1992; Dolja et al., 1993; Varrelamann and Maiss, 2000; Hong et al., 1995). To date, there have been several reports on different strains of ZYMV (Lee and Wong, 1998; Wisler et al., 1995; Kundu et al., 1997; Yoon and Choi, 1998). However, all the known strains (or isolates) of ZYMV were originally isolated from cucurbit plants such as squash and cucumber. In this report, biological properties of a novel ZYMV strain isolated from a hollyhock (Althaea rosea) plant were investigated.
[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. DOI:10.1007/PL00013070 · 0.97 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: 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. · 2.09 Impact Factor
[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.
[Show abstract][Hide abstract] ABSTRACT: Rice black-streaked dwarf virus (RBSDV) and Maize rough dwarf virus (MRDV) are closely related viruses. Since the two viruses produce identical symptoms on maize, barley, and wheat, diagnosis of infected plants is difficult. Previously, we reported that partial cDNA clones of RBSDV S5 and S6 from the Japanese isolate (RBSDV-H) have lower sequence homology to MRDV than do cDNA clones from other genomic segments. In order to test whether cDNA clones of RBSDV-H S5 and S6 can be used for molecular diagnosis, RBSDV field isolates from Korea and from Hokkaido, Japan were tested in dot blot hybridizations probed with RBSDV-H S5 and S6 cDNA clones. Hybridization with these probes was more intense against the RBSDV genome than against the MRDV genome. Therefore, RBSDV-H S5 and S6 cDNA clones can be used to differentiate between the two viruses. Furthermore, RBSDV-H S5 and S6 clones reacted more strongly against the viruses from stunted maize plants from Korean fields than to MRDV, indicating that RBSDV may be the causal dis-ease agent in maize plants in Korea. Plant reoviruses have been classified into three genera: Phytoreovirus, Fijivirus, and Oryzavirus (Van Regenmortel et al., 2000). The genus Fijivirus includes the largest num-ber of related virus species (Boccardo and Milne, 1984). In particular, Rice black-streaked dwarf virus (RBSDV) and Maize rough dwarf virus (MRDV) are considered very closely related (Azuhata et al., 1993; Isogai et al., 1995a; MacMahon et al., 1999). They are serologically related and have common plant hosts (maize, wheat, and barley), in which both produce similar symptoms (Luisoni et al., 1973). Both RBSDV and MRDV are transmitted by a com-mon plant hopper vector, Laodelphax striatellus (Milne and Lovisolo, 1977; Shinkai, 1962). However, some differences do exist; the major differences are the symptoms produced on rice. RBSDV naturally infects rice and induces tumors as well as a distinctive stunting effect, while MRDV cannot naturally infect rice. If MRDV is forced to infect rice exper-imentally, only mild symptoms are induced (Milne and Lovisolo, 1977). Additionally, MRDV is transmitted through the eggs of the insect vector, unlike RBSDV. Nucleotide sequence comparison reveals that the viruses are closely related, but distinct (Azuhata et al., 1992; Azuhata et al., 1993; Isogai et al., 1998; Marzachí et al., 1991; Marzachí et al., 1995; Marzachí et al., 1996; Uyeda et al., 1990). There-fore, correct identification of RBSDV and MRDV is diffi-cult, and previous identification methods based on biological properties, such as host range and symptom anal-ysis, should be reexamined. Previously, we showed that all MRDV genomic seg-ments have some sequence homology to their counterparts in the RBSDV-H genome (Isogai et al., 1995a). However, partial cDNA clones of RBSDV-H genomic segments S1, S4, S5, and S6 showed low signal intensity against the MRDV genome in dot blot analysis. In particular, the signal intensities suggested that RBSDV-H S5 and S6 cDNAs have lower homology to MRDV than do RBSDV-H cDNAs from other genomic segments. In this respect, par-tial cDNA clones from RBSDV-H S5 and S6 are promising candidates for distinguishing between RBSDV and MRDV. In this study, we tested the use of molecular hybridization using RBSDV-H S5 and S6 cDNA to distinguish RBSDV from MRDV. We also used these cDNAs to analyze whether field virus isolates collected from diseased maize samples in Korea were RBSDV or MRDV.
[Show abstract][Hide abstract] ABSTRACT: Reverse transcription and polymerase chain reaction (RT-PCR) was used for detection and identification of three cucumoviruses (cucumber mosaic virus, CMV; peanut stunt virus, PSV; tomato aspermy virus, TAV) in various plants sources with a single pair of primers, designed as CPTALL-3 and CPTALL-5. The pair of cucumovirus genus-specific primers that flank the coat protein gene were designed and used to amplify a DNA fragment of approximately ranging from 938 to 966 bp. The RT-PCR with the set of primers specifically amplified the target size of DNA fragment in all the tested cucumoviruses (CMV S-IA, S-IB and S-II, PSV and TAV). No DNA product of any length was produced when brome mosaic virus or tobacco mosaic virus RNA was used as templates. The cucumoviruses examined were differentiated by PCR-restriction fragment length polymorphism with different enzymes. This indicates that the designed primers are only specific for the cucumoviruses and useful for reliable information of identification of members of the Cucumovirus genus.