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D RNA 3Yalpha (D3Yalpha), a defective (D) RNA 3 derived from the Y strain of cucumber mosaic virus (CMV-Y), was further characterized in combination with different helper viruses in the genus Cucumovirus. Interestingly, Nicotiana benthamiana plants inoculated with CMV-D8 and D3Yalpha developed systemic symptoms which were different from those induced by CMV-D8. To elucidate the potential effects of D RNA 3 on virus infection on the basis of the original combination of CMV-Y and D3Yalpha, a point mutation was made in the coat protein gene, which determined symptoms, of either CMV-Y RNA 3 (Y3) or D3Yalpha. Symptoms induced on N. benthamiana and N. tabacum plants, and semi-quantitative RT-PCR revealed that the ratio of RNA 3 to D RNA 3 was associated with the differences of symptoms in the leaf tissues. Furthermore, analysis of in situ hybridization suggested that there were spatial effects between coat proteins of Y3 and D3Yalpha in the infected leaves.

A defective (D) RNA 3 naturally generated from the Y strain of cucumber mosaic virus (CMV-Y) was characterized using a biologically active cDNA clone. Sequencing of the clone revealed that the D RNA 3, named D RNA 3Yalpha, was derived from CMV-Y RNA 3 and contained a 10 nt deletion in the 5' untranslated region and a 162 nt deletion within the 3a open reading frame. Co-inoculation of D RNA 3Yalpha with CMV-Y derived from in vitro transcripts facilitated propagation of CMV-Y containing D RNA 3Yalpha in Nicotiana benthamiana or Nicotiana tabacum plants. CMV-Y, however, did not produce deletion mutants upon serial mechanical passages in the plants.

Nucleotide (nt) sequencing has contributed to the identification of virus species and has also proved diagnostically useful in the control of tomato-infecting begomoviruses disease. We determined the complete nt sequences of the DNA-A genome and its cognate DNAbeta satellite molecules in isolates of Tobacco leaf curl Japan virus, Honeysuckle yellow vein mosaic virus, Eupatorium yellow vein virus in Japan. Pairwise comparison analyses based on the nt sequences of DNA-A from the genetic group of these viruses tentatively named as TbLCJV, HYVMV and EpYVV (TbJV/HYV/EpV) revealed that this group had a significance threshold of 84 % identity. Phylogenetic relationship analyses of the nt sequences of DNA-A and DNAbeta revealed that their isolates were separated into a discrete Far East Asian clade, distinct from all other begomoviruses. This clade was divided into two distinct clusters comprising the subgroups TbJV/HYV and EpV. Furthermore, recombination analysis revealed that members of the TbJV/HYV/EpV group had the genetic variation indicative of many recombination events. Our study demonstrates that this group forms a unique species complex, but that members have discrete lineages depending on their natural perennial host plants.

A virus causing chlorotic ringspot, yellow mosaic and vein clearing symptoms was prevalent on mungbean plants around Taean, Korea. The isolate caused mosaic on Chenopodium quinoa, Nicotiana benthamiana, Phaseolus vulgaris and Vida laba but no symptoms on peanut plants. Inclusion bodies such as scroll, pinwheel and laminated aggregates induced by the virus in the host cells were similar to those produced by members of the Potyvirus subdivision III. Multiple alignment as well as cluster dendrograms of the 709 nucleotide region comprising part of the coat protein gene and 3`untranslated region (UTR) showed that the isolate belongs to the BCMV-PSt subgroup. Altogether, these results support the identification of the causal virus as peanut stripe strain of Bean common mosaic virus (BCMV-PSt).

A virus causing vein banding, sometimes yellow mosaic and rugose symptoms on peanut was prevalent around Suwon, Korea. A survey conducted in the area found disease incidence, depending on cultivar, to range from 79 to . The virus was found to be seed-transmissible in all the five peanut cultivars tested with transmission rates ranging from 2 to . Host range analysis failed to differentiate 9 field isolates collected from different peanuts cultivars showing various symptoms. Inclusion bodies such as scroll, pinwheel and long laminated aggregates induced by the virus in host plant cells were similar to those induced by members of the Potyvirus subdivision III. The virus showed homology with Bean common mosaic virus (BCMV), BCMV-BICMV/AzMV strains and only with Desmodium mosaic virus. Based on biological characterization, electron microscopy and molecular analyses of a Korean isolate (Daewon 1), the virus was identified as peanut stripe strain of BCMV.

A virus isolate collected from infected paprika (Capsicum annuum var. grossum) was characterized as Potato virus Y (PVY) based on biological, serological, cytopathological, and molecular properties. In host range studies, the paprika isolate produced the mosaic symptom on some tobacco, tomato and pepper (Capsicum annuum). A new paprika isolate also infected potato cultivars which is different biological characteristic compared to the other popular potyvirus infecting paprika, Pepper mottle virus (PepMoV). Previously reported PVY strains, and did not infect pepper and typical PepMoV isolates did not infect potato. Distinctive inclusion patterns of the scroll, pinwheel, long laminated inclusions, and helper components in the cytoplasm of infected cells were also different to those observed by the typical PVY isolate infections. However, the paprika isolate reacted to the monoclonal antibody of strain with high absorbance readings. RT-PCR amplification, cloning, and sequencing of the 3` untranslated region and a part of coat protein gene also added additional evidence of the paprika isolate as the -related isolate. Multiple alignments as well as cluster dendrograms of PVY-paprika isolate revealed close phylogenetic relationship to the subgroup. Altogether, these results suggest that a new PVY isolate infecting paprika contained distinct characteristics compared to the other previously described PVY strains with closer relationship to the strain.

Kagoshima, Japan. The complete DNA-A Iike molecule of KG5Tob was determined and found to comprise 2762 nucleotides with a typical begomovirus genome organization. A comparison of the DNA-A sequences revealed that KG5Tob shared 87"/o with its closest relatives, Honeysuckle yellow vei7h mosaic virus (HWMV) and Tobacco leaf curl Yamaguchi virus (TbLCYV), however, on comparison of the intergenic region (IR) nt sequences, the isolate shared only 66 and 64"/o of the IR sequences with the former and the latter, respectively. Phylogenetic analysis of full length DNA-A component clustered KG5Tob in a lone cluster but close to HrvMV and TbLCrv. When the CPs were aligned, sintilar results were observed and KG5Tob was placed in a lone cluster. A comparison of the Other ORFs showed that different parts of the genome had different affinities, suggesting that recombination could have played a major role in the evolution of this isolate. Molecular data show that KG5Tob isolate is an isolate of HYVMV, although the isolate was obtained from tobacco.

mined and found to comprise 2763 and 1262 nucleotides (nt), respectively, with a typical begomovirus genome organization. The DNA~ molecule had the conserved ORF (~ C1) and satellite conserved region (SCR) typical of the Geminiviridae family containing a 5'-TMTATTAC-3' motif in the stem loop and the adenine-(A) rich region. Additionally, its entire sequence was interrupted at intervals wlth sequences that were not viral and whose origin was unknown. A comparison of the DNA-A sequences revealed that HY12 shared 740/0 with four other honeysuckle begomoviruses but 88-890/0 with tobacco begomoviruses found in Japan. The isolate shared up to 920/0 of the IR sequences with the honeysuckle begomoviruses but only 60-880/0 with those of tobacco. Phylogenetic analysis of full length DNA-A component as well as its CP clustered HY12 with the tobacco begomoviruses. uvl2 is an isolate of Howeysuckle yellow vein mosaic virus.

Two Cucumber mosaic virus (CMV) isolates, CMV-NA from Nicotiana affi?~is, and CMV-SM from Solawam mammosum (nipple fruit plant) inducing mild and severe systemic mosaic s~mptoms, respectively, on tobacco (Nicotiana tabacum cv. Xanthi-nc), were characterized on the basis of their nucleotide and amino acid seqtlences of RNA3, serology and host ranges. The complete nucleotide sequencing of RNA3s of CMV-NA and CMV-SM has been done. Multiple alignment analysis of the nucleotide sequences of RNA3 of the 5'noncoding region and amino acid sequences of the CP and MP revealed that, CMV-NA and CMV=SM belong to subgroup I. CMV-NA in'duced systemic infection on Cryptotae7~ia japbnica and

A survey of Potato virus Y (PVY) was conducted in farmers' fields in 9 Korean provinces, Gyeonggi-do, Gangwon-do, Gyeongsangnam-do, Gyeongsangbuk-do, Jeollanam-do, Jeollabuk~io, Chungchcongnam-do, Chungcheongbuk-do and Jeju-do from 1998 to 2000. One hundred and fifty nine samples from potato and tobacco were collected and analyzed. Based on ELISA results, three samples PVY-tob, PVY-potlii and PVY-pot-chw representing PVYN and PVYo, respectively, were further subjected to biological, serological and molecular analysis. Host range studies showed that PVY-pot-chw is symptomatically distinguishable from PVY-tob and PVY-pot-j isolates, both of which were symptomatically indistinguishable. However, all the three isolates failed to infect 10 capsicum varieties systemically. Isolates PVY-tob and PVY-pot-j reacted strongly wlth monoclonal antibody (MAb), anti-PVYN while PVY-pot-chw failed to react wlth the MAb but reacted strongly wlth anti-PVYo'c. Interestingly, both PVY-tob and PVY-pot-j isolates did not react with anti-Pvyo,C indicating that isolate PVY-pot-chw is serologically distinguishable from both PVY-tob and PVY-potlii. In order to ascertain the taxonomic status of the three isolates, the 3' untranslated terminal regions (3'-UTR) were sequenced. Alignment of the sequences revealed 990/0 sequence identity between PVY-tob and PVY-pot-j, both of which had 780/0 nt sequences identical to PVY-pot-chw. Phylogenetic analysis of the aligned 3'-UTRs clustered PVY-tob and PVY-pot-j in the PVY~ subgroup, and PVY-pot~:hw in the PVYO subgroup.

The dynamics of virus interference in Cucumber mosaic virus (CMV) infection in cowpea were investigated by tissue-blotting and in situ hybridization. Using co-inoculation assays, we discovered that spatial competition between CMV-LE (subgroup I) and CMV-m2 (subgroup II) occurred in the inoculated leaves. Interestingly, competitive interactions between the two viruses also could be observed in the non-inoculated upper leaf tissues of the plants. Furthermore, the pattern of exclusive distribution was observed between challenge and protecting viruses in the serially inoculated leaves. Taken together, it is suggested that the dynamics of competitive interactions between the two subgroups could be characterized by exclusive infection and multiplication of the individual viruses in cowpea plants.

A potyvirus, for which the name Japanese hornwort mosaic virus (JHMV) is proposed, was isolated from Japanese hornwort plants (Cryptotaenia japonica) with mosaic disease symptoms. The virus was used to inoculate mechanically 34 plants belonging to 33 species of 10 families. Of these species seven from two families were infected. Faint chlorotic spots appeared on the inoculated leaves of Chenopodium quinoa and C. amaranticolor, but no systemic infection occurred in these plants. JHMV systemically infected only Umbelliferae plants; they did not infect 26 other species in eight families. JHMV was transmitted in a nonpersistent manner by aphids (Myzus persicae). The virus was a flexuous rod-shaped particle about 750 nm in length. Sequencing the nucleotides in the 3′ terminal region of JHMV revealed that the coat protein contains 280 amino acids with a molecular mass of 32.2 kDa. The nucleotide sequence of the coat protein of JHMV had the highest similarity with that of Zantedeschia mosaic virus (83.3%) compared to those of other potyviruses (57.0%–64.9%). An antiserum against JHMV reacted strongly with JHMV and weakly with Potato virus Y. These results indicate that JHMV is a new potyvirus.

A strain of Cucumber mosaic virus (CMV-D8) systemically infects Japanese radish (Raphanus sativus), but the Y strain of CMV (CMV-Y) only infects the inoculated leaves. Both of these strains cause severe systemic mosaic on the plants after dual infection with Turnip mosaic virus (TuMV). Synergistic interactions on long-distance transport of CMV-Y and CMV-D8 with TuMV were analyzed using an immunobinding assay. Direct tissue blots probed with either anti-CMV-Y or anti-TuMV antiserum clearly showed that CMV-Y efficiently spread and accumulated in the tissues of noninoculated upper leaves and roots when co-inoculated with TuMV, and that long-distance movement of CMV-D8 was enhanced by the presence of TuMV.

The genetics of cucumber mosaic cucumovirus (CMV) and the pathogenicity of the virus for Raphanus sativus were analyzed using pseudorecombinants constructed from the infectious transcripts of two naturally occurring strains of cucumber mosaic cucumovirus (CMV-D8 and CMV-Y). CMV-D8, but not CMV-Y, could cause systemic infection of the plant. Viral accumulation and systemic movement in the plants was examined using immuno-tissue blot analysis, dot blot and Northern blot hybridization. Virus was equally distributed and CMV RNAs accumulated to similar levels in the inoculated cotyledons of radish irrespective of the pseudorecombinant, suggesting that there are no apparent differences in the ability of infection and viral accumulation between CMV-D8 and CMV-Y. We found, however, that both RNAs 2 and 3 of CMV-D8 are involved in determining the efficiency for the systemic infection of R. sativus. Co-operated interactions between genetic information of RNAs 2 and 3 would control the efficient translocation of virus from the inoculated leaves to the uninoculated upper leaves of radish plant.

We examined the correlation of the amino acid at position 129 in the coat protein (CP) of cucumber mosaic virus (CMV) with the phenotype of the viral pathology in tobacco by using CP mutants in which several amino acid substitutions had been introduced. An exchange between Ser129 in CMV-Y, a chlorosis-inducing strain, and Pro129 of CMV-O, a green-mosaic-inducing strain, reciprocally altered the phenotypes of those virus strains on tobacco. Replacement of either Ser129 in CMV-Y or Pro129 in CMV-O with a Leu, as is found in a chlorosis-inducing strain, CMV-M, resulted in veinal necrosis. Furthermore, we created mutants that have a Phe or a Gly at position 129. Two Phe129 mutants induced necrotic lesions on the inoculated leaves, and a Gly129 mutant induced green mosaic symptoms. In inoculated protoplasts, the mutant viruses and the wild-type virus all replicated RNA well, and accumulated CP; however, infection with the Leu129 and Phe129 mutants yielded few virions. The Phe129 mutants lacked the capacity to move systemically in tobacco; by 2 weeks post-inoculation, the Phe129 mutants occasionally gave rise to revertants that elicited chlorosis, green mosaic or veinal necrosis. Sequence analysis revealed that one had reverted to the parental Y strain, and the others had additional single amino acid changes (positions 138, 144 or 147). We suggest that amino acids at specific sites affect the whole structure of the CP and affect virus assembly, virus transport and symptom expression.

A satellite RNA (T73-satRNA) gave reduced symptom severity on tomato plants when coinoculated with an ordinary strain of cucumber mosaic virus (CMVO). cDNA for T73-satRNA was introduced into a binary vector (pTOK162) through a homologous recombination in an Agrobacterium tumefaciens cell, and then transferred to leaf disks of tomato. Stable integration and transcription of the cDNA in the regenerants were verified by Southern and northern blot hybridizations, respectively. Upon inoculation with CMV-O, the transformants exhibited very slight symptoms of CMV, grew normally, and finally set fruits, whereas untransformed wildtype tomato plants showed very severe symptoms, and their growth was retarded and formed few fruits. Agarose gel electrophoresis of total RNA from CMV-O-inoculated transformants detected RNA molecules corresponding to T73-satRNA.

A monomeric cDNA copy of cucumber mosaic virus (CMV) satellite RNA which is non-necrogenic to tomato was introduced into nuclear DNA of tobacco plants with the aid of the binary vector system of Agrobacterium tumefaciens. In order to check the stability of the introduced genes, kanamycin resistance and expression of biologically active satellite RNA upon infection with CMV were assayed up to the fifth generation of the homozygous progeny obtained from a primary transformant. The inserted cDNA in the progeny of the fifth generation was sequenced after cloning of the sequence amplified with the polymerase chain reaction. Neither loss nor mutations of the inserted genes were detected.

Four satellite RNA isolates of cucumber mosaic virus (CMV), S19-, T43-, T62- and T73-sat-RNAs were obtained from CMV-infected plants collected in Tochigi Prefecture of Japan. Their physical and biological properties were investigated. T43- and T62-satRNAs induced necrosis on tomato like Y-satRNA, whereas S19- and T73-satRNAs did not. T43-satRNA was found to be identical to T62-satRNA by nucleotide sequence determination. The sequences were compared with each other and with other satellite RNAs, revealing high degree of nucleotide sequence homology between S19-satRNA and Y-satRNA (91%) and between T43-satRNA and OY2-satRNA (94%). Further analyses of the three sequences indicate that no common open reading frame is present and thus, they do not seem to encode any protein products essential for their biological activity. Competition of the satellite RNAs with Y-satRNA was examined by mixed inoculations under the condition using tobacco as a host and CMV-O as a helper virus. The relative replication ability of T43-satRNA is superior to that of Y-satRNA while T73-satRNA is apparently similar to or a little better than Y-satRNA.

Accumulation of the coat proteins of three isolates of cucumber mosaic virus (CMV-D8 isolated from a Japanese radish plant showing mosaic symptoms, a melon isolate, CMV-KM and tobacco isolate, CMV-Y) in seven Japanese radish (Rhaphanus sativus) cultivars was compared with immunological detection by direct tissue blotting. In the inoculated leaves, viral antigen equally accumulated irrespective of CMV isolates and the radish cultivars. In contrast, viral antigen was detected in almost all of the upper leaves of the all cultivars inoculated with CMV-D8 whereas rarely detected in those of the plants inoculated with CMV-Y. Accumulation of CMV-KM in the upper leaves was intermediate between that of CMV-D8 and CMV-Y. The cultivars, Akidu-mari and Natsu were highly resistant against systemic infection with CMV-KM and CMV-Y, but Shijunichi and Shogoin were rather susceptible to systemic infection with the both isolates compared to the other radish cultivars.

To investigate the interactions between RNA3 and RNA4 from subgroups I and II in mixed infections, accumulation of CMV RNA were analyzed. In the mixed inoculation assays with CMV-LE (LE, subgroup I) and a reassortant LLm consisting of RNA1 and RNA2 from LE, and RNA3 from CMV-m2 (m2, subgroup II), LE RNA3 and RNA4 could systemically spread in the plants, whereas those of m2 could not. Furthermore, accumulation of virus short RNA and a cowpea-encoded RNA-directed RNA polymerase gene (VuRdRP1) mRNA were found in the plants, suggesting that VIGS and/or distinct antiviral responses (was) were activated by infection with CMV.

Momordica charantia L. plants systemically infected with Cucumber mosaic virus (CMV) were found in Oita Prefecture. The virus isolated from the host plant was characterized by biological, serological, and molecular biological methods. The purified virus was used to mechanically inoculate the host and produced green mottle, green mosaic, and/or chlorotic spots in the noninoculated upper leaves of the host. The virus was identified as an isolate of CMV containing genomic RNA3 derived from subgroup IA by several lines of evidence based on electron microscopy, serological detection, host range, symptoms, and the entire nucleotide sequence of RNA3.

Cucumber mosaic virus Y satellite RNA (Y-satRNA) induces distinctive yellow mosaic symptoms on tobacco, whereas S19 satellite RNA (S19-satRNA) causes an attenuated green mosaic on tobacco, although they show considerable sequence identity. Biological assays of infectious chimeric satellite RNA molecules synthesized from cDNA clones of Y-satRNA and S19-satRNA using common restriction sites showed that the determinant for the induction of yellow mosaic symptoms lies in the BstXI-NheI fragment, in which 14 nucleotide differences are found between the two satellite RNAs. To define more precisely the yellow mosaic determinant(s) in this fragment, several site-directed mutants of Y-satRNA were created. The replacement of AUU, at nucleotides 191 to 193 in Y-satRNA, with GC, which mimicks the S19-satRNA sequence at the corresponding site, abolished the ability of Y-satRNA to elicit a yellow mosaic. Conversely, a mutant RNA molecule derived from S19-satRNA in which GC at nucleotides 192 and 193 was changed to AUU induced the yellow mosaic symptoms. Thus, the phenotypes of two satellite RNAs on tobacco can be altered reciprocally by changing the sequences in this limited region.

Full-length DNA copies of RNAs 1, 2, and 3 of CMV Y strain (CMV-Y) were cloned downstream of modified phage T7 promoter sequences to obtain infectious RNA transcripts. The small number of extra nonviral nucleotides at the 5′ ends considerably decreased the specific infectivity of the transcripts of RNAs 1 and 2 but did not affect that of the RNA3 transcripts. Using the most infective transcripts, up to 45% of tobacco protoplasts could be infected. Various cDNA mutants were constructed from the full-length RNA3 cDNA to give RNA transcripts having deletions in the coding region of the 3a protein or the coat protein. These mutants replicated in tobacco protoplasts but did not produce systemic symptoms on tobacco when inoculated together with transcripts of RNAs 1 and 2. One of the mutants having a small in-frame deletion near the N-terminal region of the coat protein produced local lesions on cowpea and local chlorotic spots on the inoculated leaves of tobacco. These results suggest that both the 3a protein and the coat protein are involved in virus transport, and that viral assembly is associated with long-distance movement of CMV.

Cucumber mosaic virus Nucleotide sequence RNA 5

Five major RNAs were isolated by polyacrylamide gel electrophoresis from purified virions of the yellow strain of cucumber mosaic virus (CMV-Y), although the smallest RNA (RNA 5) was not detectable in RNAs of the ordinary strain of CMV (CMV-O). Synthesis of CMV-specific RNAs was investigated using tobacco mesophyll protoplasts inoculated with CMV in vitro . Incorporation of [ 3 H]uracil into RNAs was analyzed by electrophoresis in polyacrylamide gels. A combination of actinomycin D treatment and ultraviolet irradiation to protoplasts showed a marked inhibiting effect on host RNA synthesis, but little on viral RNA synthesis. Replication of the five major RNAs of CMV-Y occurred in the protoplasts, although RNAs 1 and 2 could not be resolved; RNAs 3 and 5 were especially predominant. In the case of CMV-O, RNA 5 was not synthesized in vivo . The infectivity of RNA extracted from CMV-infected tobacco protoplasts was detectable within 6 hr after inoculation, and reached its maximum at 18 hr; it then decreased rather sharply. The rate of viral RNA synthesis was highest at 12 to 18 hr after inoculation, and after that it gradually slowed. Double-stranded RNAs were also isolated from the infected protoplasts. Existence of five pairs of replicative form (RF) and replicative intermediate (RI) RNAs corresponding to the respective single-stranded CMV RNAs was assumed by examining their chromatographic behavior on a cellulose column, solubility in 1 M NaCl and susceptibility to ribonuclease treatment. The smallest RI, 5, was eluted from the cellulose column together with single-stranded RNAs with a buffer containing 15% ethanol. Much larger amounts of RF 3 and 5 were detected as compared with the other RF species, and the electrophoretic pattern of the RFs was very similar to that of single-stranded CMV RNAs syntheiszed in vivo except for the slower electrophoretic mobilities of the RFs.

In attempts to search polycations suitable for inoculation of tobacco mesophyll protoplasts with virus RNA, polyethyleneimine (PEI) was found to be the most competent on which markedly improved the efficiency of infection not only with cucumber mosaic virus (CMV) RNA and tobacco mosaic virus (TMV) RNA but also with CMV and tobacco necrotic dwarf virus (TNDV). For inoculation with virus RNAs and viruses, 25mM phosphate buffer, pH 5.0, and 10mM citrate buffer, pH 5.5, respectively, were found to be suitable. Over 90% of protoplasts could be constantly infected using CMV RNA and TMV RNA concentrations as low as 0.2 to 0.3μg/ml. The ratio of RNA and PEI was very important and excess amounts of RNA in inoculation mixtures lead to the drastic decrease in infection efficiency.

Full-length cDNA of a satellite RNA (Strain Y) which induces bright yellow symptoms on tobacco plants infected with cucumber mosaic virus (CMV) was cloned and sequenced. The published sequence of the satellite RNA was revised with three possible differences (residues 161, 167, and 173) and a nucleotide insertion at residue 234. The satellite cDNA was then inserted into a commercially available transcription vector. In vitro transcription products from the recombinant plasmid harbored 24 non-viral bases at their 5' ends and had very low infectivity when coinoculated with CMV. After removal of the extra 5' sequence of the transcripts with RNase H, the infectivity of the transcripts increased markedly. Analysis of the effects of extra 5' sequences of several lengths confirmed the importance of natural 5' ends for biological activity of the satellite. Trimming down to 6-9 extra bases at the 5' end enhanced the infectivity of the transcripts by 10-fold, although the specific activity of the natural satellite is still 100-fold higher. Dideoxynucleotide sequence analysis proved that the progeny satellite RNA did not retain the 24 non-viral bases at the 5' end of the transcript from pIBI 31-MC.

Activity of membrane-bound RNA-dependent RNA polymerase (M-RDRP) from cucumber mosaic virus (CMV)-infected tobacco plants increased prior to virus multiplication and that of soluble enzyme (S-RDRP) increased rapidly after concentration of CMV has reached its maximum. In vitro products of M-RDRP was mainly doublestranded CMV RNAs as replicative forms. M-RDRP was suggested to be associated with endoplasmic reticulum (ER) by subcellular fractionation of the membrane-fraction using Percoll discontinuous gradient-centrifugation and electron microscopic observation. The ER-associated enzyme had viral RNAs as endogenous templates and was firmly integrated into membrane structure, even after treated with detergent or Mg++-depletion or partially purified by Sepharose 4B chromatography. The purification procedures could effectively remove S-RDRP which synthesized small heterogeneous RNA in vitro. Joined with our previous study using CMV-infected tobacco protoplasts, it was suggested that S-RDRP is a host-encoded enzyme induced in leaf tissues but not in protoplasts as a consequence of virus infection process like “Pathogenesis-related proteins.” We speculate that the CMV RNA replication complex is composed at least of translation products of viral RNAs, ER membranes and viral template RNAs.

CMV RNAs 1 and 2 are considered to constitute the viral replicon. Tobacco plants were transformed with either RNA1 or RNA2 to produce plant lines V1 and V2, respectively. Plants homozygous for each of the RNAs were generated and crossed to produce V1V2 (V2V1) lines that expressed both RNA1 and RNA2. An RNase protection assay indicated that RNA1 and RNA2 multiplied in V1V2 (V2V1) plants. Surprisingly, V1V2 (V2V1) plants, unlike their parent lines, showed a remarkably high level of resistance to CMV; this resistance was more effective against RNA inoculation than against virion inoculation. Experiments using protoplasts showed that the resistance was expressed at the single cell level. All the data together suggested that the observed resistance does not fit the criteria for either ‘RNA-mediated’ or ‘replicase-mediated’ resistance.

The small RNA from cucumber mosaic virus (CMV) we had formerly referred to as RNA 5 was shown to have a satellite nature similar to CARNA 5 ( Kaper and Tousignant, 1977 , Virology 80, 186–195) and Sat-RNA ( Mossop and Francki, 1978 , Virology 86, 562–566). Our satellite RNA has a great deal in common with CARNA 5 and Sat-RNA in many respects, except that its replication causes a striking change in symptoms on tobacco and on some species of Nicotiana infected with CMV. Large amounts of satellite RNA were found in RNAs of naturally occurring CMV isolates which induced a unique brilliant yellow symptom on tobacco. When purified satellite RNA was inoculated onto tobacco after being mixed with CMV isolates which contained no satellite RNA and which normally produced ordinary green mosaic symptoms, all of the infected tobacco developed the brilliant yellow symptom along with some symptom attenuation. Virions isolated from those plants contained much satellite RNA. CMV could be freed from satellite RNA by local lesion transfer and selection. Satellite RNA had substantial stability in vivo and in vitro . It caused the severe necrotic symptom or ultimate death of tomato like CARNA 5 but unlike Sat-RNA.

We describe the use of biologically active cDNA clones to investigate genetic determinants of a satellite RNA that modulates symptoms normally induced by its helper virus, cucumber mosaic virus (CMV). For this purpose, we have investigated a CMV satellite RNA (Y-satRNA) that induces bright yellow symptoms on tobacco and necrosis on tomato. To determine the pathogenicity-modulating domain of Y-satRNA, several insertion and deletion mutants were created by using various restriction sites in the cDNA of Y-satRNA, and RNA transcripts derived from the clones were mixed with CMV and used to inoculate plants. Although the satellite RNA was able to tolerate small insertions (as much as 4 bases at present), small deletions were deleterious, indicating that the sequence requirements for viability of the satellite RNA are relatively inflexible. Biological activity assays of chimeric satellite RNAs between Y-satRNA and a non-necrogenic satellite RNA, T73-satRNA, suggested that only two (or at least one of two) specific bases (positions 318 and 325) in the 3' region direct the necrogenic property of Y-satRNA. Sequences involved in production of yellow symptoms were investigated by constructing chimeras between Y-sat cDNA and cDNA of a satellite RNA designated S19-satRNA. S19-satRNA has considerable homology to Y-satRNA but does not elicit yellow symptoms on tobacco. Chimeric clones were constructed by using a BstXI site that cuts within a stable secondary structure in the region between positions 100 and 200 (region Y). The results of infectivity tests with RNA transcripts suggest that formation of a secondary structure in region Y may be involved in induction of yellow symptoms as well as viability of Y-satRNA.