VIROLOGY
186,475-480
(1992)
Differential
Interactions among Strains of Tomato Aspermy and Satellite RNAs of Cucumber Mosaic Virus
Virus
ENRIQUE MORIONES,* ISABEL DIAZ,t EMIL10 RODRIGUEZ-CEREZO,* AURORA FRAlLE,* AND FERNANDO GARCIA-ARENAL*!’ *Depto. de Patologia
Vegetal; and tDepto.
de Eioqulmica,
E. T.S.I. Agrhomos,
Received June 4, 199 1; accepted
September
C&dad Universitaria,
28040-Madrid,
Spain
30, 199 1
Tomato and tobacco plants were inoculated with either of two strains of tomato aspermy virus, I-TAV or V-TAV, and each of six isolates of cucumber mosaic virus satellite RNA (CMV-satRNA), Bl , 82, 83, G, Ix, or WL2. Ribonuclease protection assays, used to detect total satRNA and encapsidated satRNA, revealed that G-satRNA generated new satellite RNA not of the inoculated sequence. The other CMV-satRNAs were compared for their ability (1) to replicate, (2) to modulate symptoms, (3) to reduce TAV accumulation, and (4) to alter the extent of encapsidation of TAV genomic RNAs. The fraction of B2- and B8satRNAs encapsidated was greater for 1 -TAV than for V-TAV, although spread and accumulation of the satRNA were similar for both helper viruses. These results suggest that CMV-satRNA may spread in a nonencapsidated form. Accumulation of CMV-satRNA in systemically infected leaves was detected for all inoculum combinations except V-TAV and Ix-satRNA, for which the satellite RNA increased only in protoplasts and inoculated leaves of tobacco or tomato. In such inoculated leaves, Ix-satRNA was not detected in capsids. Thus the effectiveness of the TAV helpers of CMV-satRNAs may be controlled in at least some instances by the extent of satRNA spread or encapsidation rather than by the efficiency of satRNA replication. In contrast to infections initiated by inoculation of CMV and CMV-satRNA, inoculation of I-TAV or V-TAV and CMV-satRNA did not alter the relative amounts of viral Q 1992 Academic genomic RNAs encapsidated or result in accumulation of large amounts of double-stranded satRNA. Press.
Inc.
INTRODUCTION
helper of CMV-satRNA has not been characterized to the same extent as CMV, and comparison of the interactions TAV:CMV-satRNA and CMV:CMV-satRNA could illustrate the nature of the relationship between satRNAs and helper viruses. In this report we analyze the interaction of two strains of TAV with several CMVsatRNAs in two hosts. Our results show that this interaction is determined by the strain of TAV, the strain of satRNA, and the host plant. We also demonstrate that the observed differences in the ability of TAV strains to serve as effective helpers of CMV-satRNA may be controlled by the extent of satRNA spread or encapsidation rather than by the efficiency of satRNA replication.
Cucumber mosaic virus (CMV), the type member of the cucumovirus plant virus group, has a tripartite, plus sense RNA genome. CMV particles, in addition to the genomic RNAs 1,2, and 3 and the subgenomic RNA4, can also contain a small (340-380 nucleotides) satellite RNA (satRNA) that depends on CMV (helper virus) for both its replication and encapsidation. The replication of CMV RNA and the symptoms induced during CMV infections can be affected by the presence of the satRNA in a way that depends on the combination of strain of helper virus, strain of CMV-satRNA, and host plant (Jacquemond & Leroux, 1982; Kaper & Tousignant, 1977; Mossop & Francki, 1979). No satellite RNA has ever been found in field isolates of tomato aspermy virus (TAV), a cucumovirus closely related to CMV (Bernal et al., 1991; Moriones et al., 1991 a; O’Relly et al., 1991). However, TAV can act as a helper virus for CMV-satRNA (Gould et al., 1978; Mossop and Francki, 1979), although differences have been reported between the interactions of TAV and CMV with CMVsatRNA (Harrison et al., 1987; Jaegle et al., 1990; Lee & Kummert, 1985; Mossop & Francki, 1979). TAV as a ’ To whom correspondence dressed.
and reprint requests
MATERIAL
AND METHODS
Virus and satRNA strains and RNA preparations L,-CMV, Fny-CMV (Owen & Palukaitis, 1988) V-TAV (Habili & Francki, 1974), Bl-, B2-, B3-, WL2-, G-, (Garcia-Arena1 et al., 1987) and Ix-satRNAs (Moriones et a/., 1991 b) have been described. 1-TAV is a tomato isolate from Indiana, the gift of P. Palukaitis (Cornell University, NY). SatRNAs were multiplied in Nicotiana c/eve/and;; Gray using L,-CMV as helper virus. CMV and TAV were multiplied in N. clevelandi and purified as in Lot et al. (1972) and Habili and Francki (1974)
should be ad-
475
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476
MORIONES
ET AL
respectively. Encapsidated RNAs were extracted from virions with phenol/SDS and separated in preparative 6% polyacrylamide gels. Bands corresponding to satRNA were visualized by staining with 0.05% toluidine blue, excised, and the satRNA was purified as described by Palukaitis and Zaitlin (1984). Nucleic acids from infected leaves were extracted as in Palukaitis et al. (1983) and fractionated by precipitation in 2 and 4 M LiCl into fractions enriched for singlestranded (ss) and for double-stranded (ds) RNAs (DiazRuiz & Kaper, 1978). Biological
B1-
BZ-
B3-
G-
Ix-
WL2-
assays
Combinations of helper virus RNAs, with or without purified satRNAs, were inoculated onto the expanded third and fourth leaves of tobacco cv. Xanthi-nc, or onto the expanded cotyledons of tomato cv. Rutgers. The suspension used as inoculum (10 PI/plant) was 200 pg/ml helper RNA and 25 pg/ml satRNA in 0.1 n/l Na,HPO,. Plants were maintained in the greenhouse (20-25”, 16 hr light) and analyzed by dot-blot hybridization (Palukaitis et a/., 1985) for the presence of satRNA 7-10 days postinoculation. The symptoms were recorded for l-4 weeks postinoculation when virions and nucleic acids were extracted from leaves. Protoplasts from tobacco leaves were prepared as in Power and Chapman (1985) and 1.5 X 1O6protoplasts were inoculated with 40 pg of helper virus RNA with or without 2.5 pg of satRNA as in Power et al. (1989). At 30 hr postinoculation protoplasts were harvested and nucleic acids were extracted as in Dreher et al. (1989). RNA analyses Virion-encapsidated RNAs were analyzed in 1.2% agarose gels in TBE buffer (Rezaian & Symons, 1986). Viral and satRNAs were also detected and quantitated by ribonuclease protection analysis (RPA). RPA was performed as in Lopez-Galindez et a/. (1988) except that ribonuclease digestion of duplexes was performed with a mixture of RNases A and Tl , as in Winter et a/. (1985). cRNA probes transcribed from a clone with an insert of 3 19 nucleotides (nt) from B2-satRNA, or from a clone with a 1OOO-nt insert of V-TAV RNA3 were hybridized to different amounts of the RNA samples to be analyzed. Probes were labeled with [a3ZP]UTP; low activity probes (0.1 X 10” cpmlpg) or high activity probes (2.5 X 10” cpm/pg) were obtained by adding or omitting cold UTP to the transcription reaction (Sambrook et a/., 1989). Densitometric analyses of the autoradiograms of RPAs showed that the intensity of the bands resulting from ribonuclease digestions was linear for amounts of purified satRNA between 0.5 and 50 ng, using 200,000 cpm of the high activity probe, and be-
FIG. 1. Comparison of progeny CMV-satRNAs isolated from tobacco plants infected with I-TAV (lanes 2) or V-TAV (lanes 3) with the parental satRNAs preparations used for inoculation (lanes 1). RPAs of gel-purified satRNAs (Bl-, B2-, 83.. G-, Ix-, and WLZ-satRNAs) were performed with a cRNA probe to B2-satRNA. The products of ribonuclease digestion were separated in a 6% polyacrylamide-electrophoresis gel and visualized by autoradiography. (a) 15,000 cpm of undigested B2-satRNA probe; in addition to 3 19 nt complementary to B2aatRNA, the transcript has 28 extra nt at its 5’end. (b) Digested, unhybridized probe.
tween 5 ng and 500 ng, using 50,000 cpm of the low activity probe. Assays were routinely carried on using 0.15 pg of virion RNA, 1.5 pg of ssRNA from infected leaves, or total nucleic acids extracted from 0.5 X 1O6 protoplasts. When differences between the samples to be compared were not apparent, the assays were repeated using two fivefold dilutions of the above indicated amounts of RNA to make certain that the assay was performed within the range for linearity. RESULTS Replication
of CMV-satRNAs
by TAV
Dot-blot hybridization analyses of systemically infected leaves from tobacco and tomato inoculated with l-TAV, V-TAV, and any of the six strains of CMVsatRNA used in this work (Bl-, B2-, B3-, G-, lx-, and WL2-satRNA) showed the presence of progeny satRNA, except in the case of Ix-satRNA inoculated with V-TAV (not shown). The identity of progeny CMVsatRNA with the parental satRNAs was analyzed by RPA with a probe specific for a reference isolate, B2satRNA, using similar amounts of gel-purified parental and progeny satRNAs (Fig. 1). In all cases the pattern of mismatches was identical for the parental and the
TAV AS A HELPER TO CMV-satRNAS
A
B2- B3- Ix- WL2-82. Bl120121212121’2’1’2’
Ix-
6
Bl- 82.63.
Ix- WL2-B2-
Ix-
(Bl-, E32-, B3-, lx-, and FIG. 2. RPA quantitation of CMV-satRNAs WL2satRNAs) in virion RNA (A) or in the ssRNA enriched (B) fractions of nucleic acid extracts from tobacco (lanes O-2) or from tomato leaves (lanes 1’ and 2’). Helper viruses were Fny-CMV (lane O), 1-TAV (lanes 1 and 1’) and V-TAV (lanes 2 and 23. 0.15 pg of virion RNA or 1.5 rg of ssRNA-enriched faction used for each analysis.
progeny satRNA preparations, except for G-satRNA for which a variant from the original preparation was consistently supported by l- and V-TAV both in tobacco and in tomato. Because of this, G-satRNA was excluded from further analyses. The analysis of the efficiency of 1-TAV and V-TAV as helper viruses to CMV-satRNAs was first approached by comparing the relative amounts of satRNAs in TAV particles, as in previous reports (Harrison et al., 1987; Jaegle er al., 1990; Lee & Kummert, 1985; Mossop & Francki, 1979). Since both 1-TAV and V-TAV encapsidate two low molecular weight RNAs, one of them with an electrophoretic mobility similar to CMV-satRNAs (see Fig. 4 and Hull, 1976), satRNA quantitation was performed by RPA. In virions purified from tobacco plants, all five CMV-satRNAs accounted for a lower proportion of the RNA encapsidated by V-TAV (Fig. 2A, lanes 2) when compared with 1-TAV (Fig. 2A, lanes 1). The proportion of encapsidated satRNA was similar in 1-TAV and Fny-CMV virions (shown for B2-satRNA in Fig. 2A, lanes 0 and 1). Ix-satRNA was not detected in particles of V-TAV (Fig. 2A). RPA was performed in the ssRNA-enriched fraction of total nucleic acid extracts from systemically infected tobacco leaves to obtain an estimation of the total amount of satRNA in the plant. Three different situations were found (Fig. 2B): (a) B2and B3-satRNAs were found in similar amounts when either 1-TAV or V-TAV were used as helper viruses; (b) Bl- and WL2-satRNAs represented a higher percentage of the ssRNA fraction when 1-TAV was the helper
477
virus, as compared with V-TAV; and (c) Ix-satRNA was not detected in nucleic acid extracts of tobacco plants inoculated with V-TAV + Ix-satRNA. Similar anaylses were performed for B2-satRNA and Ix-satRNA in virion RNA and ssRNA fractions of extracts from tomato leaves (Fig. 2A and 2B, lanes 1’ and 2’). Again, IxsatRNA was not detected when V-TAV was the helper virus. In contrast, B2-satRNA was found to represent a similar amount of the encapsidated RNA and of ssRNA fraction when 1-TAV and when V-TAV were the helper strains. The relationship of B2-satRNA and Ix-satRNA with TAV, as representing the extreme cases of those described above was further analyzed. When the inoculated leaves of tobacco plants were extracted, lxsatRNA was found to be present in nucleic acid extracts from leaves inoculated with V-TAV, although in lower amounts than in leaves inoculated with l-TAV, while both TAV RNAs were found to similar levels. No Ix-satRNA was found, however, in V-TAV virions from these directly inoculated leaves (not shown). The replication of B2-satRNA and of Ix-satRNA by 1-TAV and by V-TAV was also analyzed in tobacco protoplasts to distinguish between effects due to replication or to spread-within-the-plant in the differential behavior of both satRNAs with both TAV strains. Both I-TAV and V-TAV did replicate to the same levels in tobacco protoplasts, as determined by the quantitation of RNA3 in protoplast’s extracts (Fig. 3B, lanes 1 and 2). Also, both B2-satRNA and Ix-satRNA were detected in similar amounts (Fig. 3A, lanes 1 and 2) in the extracts of protoplasts infected with I-TAV or with VTAV, indicating a similar ability of both TAV strains to replicate the two satRNAs. Effect of CMV-satRNA on TAV accumulation the symptoms induced by TAV
and on
The presence of CMV-satRNA did not affect the yield of V-TAV virions purified from tobacco (in the absence of satRNA 164.6 + 19.3 mg/kg fresh leaf, average of six experiments). The yield of I-TAV (689.5 f 223.5 mg/kg fresh leaf in the absence of satRNA) was reduced to about 50% when any of the six CMV-satRNAs was present in the inoculum (e.g., to 355.1 f 132.5 mg/kg fresh leaf for B2-satRNA and to 324.0 If: 110.4 mg/kg fresh leaf for Ix-satRNA). The yield of l-TAV or V-TAV purified from tomato plants in the absence of satRNA was similar (e.g., 118.0 -+ 16.8 mg/kg fresh leaf for 1-TAV in the absence of satRNA) and was not affected by the presence of any CMV-satRNA, including Ix-satRNA with necrotic tomato plants (yield of 1 1 1.6 f 6.9 mg/kg fresh leaf for 1-TAV in the presence of Ix-satRNA). The relative proportion of encapsidated
478
MORIONES
A a
B2bl
I3
Ix2
1
2
B2-
abl
Ix2
1
2
FIG. 3. Accumulation of viral and satellite RNA in tobacco protoplasts inoculated with 1-TAV RNA (lanes 1) or V-TAV RNA (lanes 2) plus B2-satRNA (629, or Ix-satRNA (Ix-). Total RNA extracted from 5 X 1O5 protoplasts 30 hr postinoculation was hybridized with ?labeled cRNA probes to EGsatRNA (A) or to V-TAV RNA3 (B) and subjected to RPA. (a) 200,000 cpm of undigested probes; (b) digested, unhybridized probes.
TAV RNAs 1, 2, 3, and 4 (Fig. 4, shown for TAV infections with !32- and WL2-satRNAs in tobacco), as well as of TAV ds RNAs 1-4 extracted from infected leaves (not shown) was not affected by any CMV-satRNA. The six strains of CMV-satRNAs used in this study either had no effect or attenuated to different degrees the symptoms induced by both l-TAV and V-TAV in tobacco plants. The effect of a particular satRNA was not the same on each TAV strain (Table 1). For example, B3-satRNA did not affect the symptoms induced by V-TAV but induced an extreme attenuation of those induced by l-TAV. B2-satRNA, which induces yellow chlorosis in tobacco when supported by the subgroup II of CMV strains (Palukaitis, 1988) attenuated the symptoms of both TAV strains. In tomato plants, the CMV-satRNAs that attenuated the symptoms of CMV also attenuated those induced by l-TAV and V-TAV. Ix-satRNA, which induces a lethal necrosis in tomato plants when supported by CMV strains of both subgroups I and II (Moriones eT al., 1991 b) also induced a lethal necrosis when supported by 1-TAV. DISCUSSION The amount of CMV-satRNA found in TAV particles depends on the virus and the satRNA strains and on the host plant: while in general more CMV-satRNA is
ET AL.
found in particles of l-TAV (at levels similar to CMV) than of V-TAV, Ix-satRNA being an extreme case, in tomato both TAVs encapsidate similar proportions of B2-satRNA. We have attempted to differentiate among the various possible components (satRNA replication, movement within the plant, and encapsidation) of the helper function. The similar levels of B2 and B3 CMVsatRNAs in ssRNA fractions from infected leaves show that their different amounts in 1-TAVvs V-TAV particles purified from tobacco can be attributed to an encapsidation effect. The reason why this differential encapsidation of B2-satRNA occurs in tobacco but not in tomato is unknown. Different amounts of Bl-satRNA, WL2-satRNA, and Ix-satRNA are found in both the particles of 1-TAV and V-TAV and in the ssRNA fractions from infected leaves, and may be due to differences between both TAVs in any of the components of the helper functions. The presence of Ix-satRNA in nucleic acid extracts of tobacco leaves directly inoculated with V-TAV, but in lower amounts than in leaves inoculated with I -TAV, indicates a different ability of both TAVs to assist the cell-to-cell movement, or the replication, of the satRNA. Since B2-satRNA and Ix-satRNA are replicated with similar efficiencies by l-TAV and V-TAV in tobacco protoplasts, the differences observed in planta must be due to differences in the ability of the strains of TAVs to mediate the movement within the plant of some (e.g., Ix-satRNA) but not all (e.g., B2satRNA) CMV-satRNAs. Our data do not exclude the possibility that different abilities to replicate some CMV-satRNAs may exist between different TAV strains, or between TAV and CMV, as pseudorecombinant experiments by Mossop and Francki (1979) seem to indicate. We have been unable to correlate the different behaviour observed for the different CMVsatRNAs with any feature of their nucleotide sequences. It has been suggested that viral assembly plays an important role in the systemic movement of CMV in infected plants (Suzuki et al., 1991). The data presented for B2-satRNA and Ix-satRNA could suggest rather than encapsidation and movement within the plant are different phenomena for the satRNAs, although the presence of Ix-satRNA in viral particles at undetectable levels cannot be excluded. The effect of CMV-satRNA on the helper virus differs for TAV and for CMV in several ways: (1) The modulation by a particular CMV-satRNA of the symptoms induced by CMV or TAV may be the same, or not: CMVsatRNAs that attenuate the symptoms induced by CMV in tomato or tobacco, or that are necrogenic with CMV for tomato, had similar effects with TAV (this work, Harrison et al., 1987; Lee & Kummert, 1985; Mossop & Francki, 1979) while satRNAs that induce
479
TAV AS A HELPER TO CMV-satRNAS TABLE 1 SYMPTOMS INDUCEDON TOBACCO ORTOMATO BYTwo STRAINSOF TOMATO ASPERMYVIRUS WITH OR WITHOUTDIFFERENTSATELLITERNAs OF CUCUMBER MOSAIC VIRUS Tomato
Tobacco 1-TAV
V-TAV
1-TAV
V-TAV
Severe mosaic, oak-leaf pattern Mosaic, oak-leaf pattern Very mild mosaic to symptomless Very mild mosaic to symptomless Mosaic, oak-leaf pattern Mosaic, oak-leaf pattern Mild mosaic
Severe mottle and ringspot Very mild mosaic to symptomless Very mild mosaic to symptomless Severe mottle and ringspot Mild mottle and ringspot Severe mottle and ringspotb Mild mottle and ringspot
Severe stunting, severe fern-leaf, mild mosaic NT”
Stunting, fern-leaf, severe mosaic NT
Mild stunting, mild fern-leaf NT
Mild stunting, mosaic NT
Mild stunting, fern-leaf, mild mosaic Systemic necrosis
Severe mosaic
Satellite RNAs None 81 -satRNA B2-satRNA B3satRNA G-satRNA Ix-satRNA WL2-satRNA
a NT, not tested. b No satRNA was detected
in the leaves for which the symptoms
chlorosis in tobacco with CMV attenuate TAV symptoms (this work, Jaegle et al., 1990). (2) For all reported CMV strain:CMV-satRNA combinations, in different host plants, the yield of CMV virions is variously reduced by the presence of CMV-satRNAs (Jacquemond & Leroux, 1982; Kaper & Tousignant, 1977; Mossop & Francki, 1979). This is also the case for some TAV strains in some host plants [B-TAV and l-TAV in tobacco (Lee & Kummert, 1985; this work)] but not for others [the Scotland strain of TAV and V-TAV in tobacco, V- and N-TAV in N. clevelandi;, l-TAV and VTAV in tomato (Harrison et al., 1987; Mossop & Francki, 1979; this work)], which indicates that the at-
-0
V-TAV
I-TAV 1
2
0
1
Fny-CMV 2
01
FIG. 4. Comparison of the RNAs encapsidated by 1-TAV, V-TAV. and Fny-CMV, when inoculated with no satRNA (lane 0), with B2satRNA (lanes l), or WL2satRNA (lanes 2) in tobacco plants. RNAs were purified from virions, separated in a 1.2% agarose electrophoresis gel, and visualized by ethidium bromide staining.
Mild stunting, fern-leaf
mild
Stunting, severe Stunting, severe
fern-leaf, mosaicb fern-leaf, mosaic
were assessed.
tenuation of TAV symptoms by CMV-satRNA is not related to a depression of virus yield. (3) While the presence of CMV-satRNAs results in a depression of relative amounts of the encapsidated RNAs 1 and 2 of CMV (Jacquemond & Leroux, 1982; Kaper & Tousignant, 1977; Mossop & Francki, 1979) this is never the case for TAV no matter what the effect of the satRNA on TAV yield or symptoms (this work, Gould er a/., 1978; Harrison et al., 1987; Mossop & Francki, 1979; Lee & Kummert, 1985). (4) dsCMV-satRNA becomes predominant in dsRNA fractions from infected leaves when CMV is the helper virus (Piazzola et a/., 1982). This is not the case for TAV in any of the interactions analyzed here. This indicates that in the cases when a depression of TAV virion yield is observed, competition of satRNA and helper virus RNA for replication is not likely to be the cause, as has been suggested for CMV. The results indicate that the interaction of TAV with the CMV-satRNAs is a phenomenon determined by the combination of strain of TAV, strain of CMV-satRNA, and species of host plant. This can help to explain conflicting reports on the ability of different strains of TAV to maintain different CMV-satRNAs (Harrison et al., 1987; Jaegle et a/., 1990; Lee & Kummert, 1985; Mossop & Francki, 1979) and parallels wtiat has been reported for the interaction of CMV with its satRNAs (Jacquemond & Leroux, 1982; Kaper &Tousignant, 1977). The specificity of the interaction between helper virus and satRNA is further shown by the selection of sequence variants of a satRNA by different helper viruses, as occurs for G-satRNA.
480
MORIONES ET AL.
ACKNOWLEDGMENTS We thank Peter Palukaitis, Cornell University, NY, for l-TAV. This work was in part supported by Grant PA86-0353 Comisi6n Interministerial de Ciencia y Tecnologfa, Spain; E.M.A. and E.R.C. were the recipients of fellowships from Ministerio de Educaci6n y Ciencia, Spain.
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MORIONES,E., ROOSSINCK, M. J., and GARCIA-ARENAL, F. (1991a). Nucleotide sequence of tomato aspermy virus RNA2. J. Gen. Viral. 72,779-783. MORIONES, E., FRAILE.A., and GARCIA-ARENAL, F. (1991 b). Host-associated selection of sequence variants from a satellite RNA from cucumber mosaic virus. Virology 184, 465-468. MOSSOP,D. W., and FRANCKI,R. I. B. (1979). Comparative studies of two satellite RNAs of cucumber mosaic virus. Virology 95. 395404. O’REILLY.D., THOMAS,C. J. R., and Courts, R. H. A. (1991). Tomato aspermy virus has an evolutionary relationship with other tripartite RNA plant viruses. J. Gen. Viral. 72, l-7. OWEN,J., and PALUKAITIS,P. (1988). Characterization of cucumber mosaic virus. 1. Molecular heterogeneity mapping of RNA 3 in eight CMV strains. Virology 168, 495-502. PALUKAITIS, P. (1988). Pathogenic&y regulation by satellite RNAs of cucumber mosaic virus: minor nucleotide sequence changes alter host response. Molec. Plant-Microbe Int. 1, 175-l 8 1. PALUKAITIS, P., and ZAITLIN,M. (1984). Satellite RNAs of cucumber mosaic virus: Characterization of two new satellites. Virology 132, 426-435. PALUKAITIS, P., GARCIA-ARENAL, F., SUIZINSKI,M. A., and ZAITLIN,M. (1983). Replication of tobacco mosaic virus. VII. Further characterization of single and double-stranded virus-related RNAs from TMV-infected plants. Virology 131, 533-545. PALUKAITIS, P., Cons, S., and ZAITLIN,M. (1985). Detection and identification of viroids and viral nucleic acids by ‘dot-blot’ hybridization. Acta Hort. 164, 109-l 18. PIAZZOL%P., TOUSIGNANT, M. E., and KAPER,1. M. (1982). Cucumber mosaic virus-associated RNA 5. IX. The overtaking of viral RNA synthesis by CARNA 5 and ds CARNA 5 in tobacco. Virology 122, 147-157. POWER, J. B., and CHAPMAN,J. V. (1985). Isolation culture and genetic manipulation of plant protoplasts. ln “Plant Cell Culture” (R. A. Dixon, Ed.), pp. 37-66. IRL Press, Oxford. POWER,J. B., DAVEY,M. R., MC LELLAN,M., and WILSON,D. (1989). “Laboratory Manual Plant Tissue Culture. Plant Genetic Manipulation Group.” Department of Botany. University of Nottingham, Nottingham, NG7 2RD, UK. REZAIAN,M. A., and SYMONS,R. H. (1986). Anti-sense regions in satellite RNA of cucumber mosaic virus form stable complexes with the viral coat-protein gene. Nucleic Acids Res. 14, 32293239. SAMBROOK, J., FRITSCH,E. F., and MANIATIS,T. (1989). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratoty. Cold Spring Harbor, NY. SUZUKI,M., KUWATA,S., KATAOKA,J., HASUTA,C., NITTA,N., and TAKANAMI,Y. (1991). Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system. Virology 183, 106-l 13. WINTER,E., YAMAMOTO,F., ALMOGUERA, C., and PERUCHO, M. (1985). A method to detect and characterize point mutations in transcribed genes: Amplification and overexpression of the mutant c-ki-ras allele in human tumor cells. Proc. Nat/. Acad. Sci. USA 82, 7575-7579.
186,475-480
(1992)
Differential
Interactions among Strains of Tomato Aspermy and Satellite RNAs of Cucumber Mosaic Virus
Virus
ENRIQUE MORIONES,* ISABEL DIAZ,t EMIL10 RODRIGUEZ-CEREZO,* AURORA FRAlLE,* AND FERNANDO GARCIA-ARENAL*!’ *Depto. de Patologia
Vegetal; and tDepto.
de Eioqulmica,
E. T.S.I. Agrhomos,
Received June 4, 199 1; accepted
September
C&dad Universitaria,
28040-Madrid,
Spain
30, 199 1
Tomato and tobacco plants were inoculated with either of two strains of tomato aspermy virus, I-TAV or V-TAV, and each of six isolates of cucumber mosaic virus satellite RNA (CMV-satRNA), Bl , 82, 83, G, Ix, or WL2. Ribonuclease protection assays, used to detect total satRNA and encapsidated satRNA, revealed that G-satRNA generated new satellite RNA not of the inoculated sequence. The other CMV-satRNAs were compared for their ability (1) to replicate, (2) to modulate symptoms, (3) to reduce TAV accumulation, and (4) to alter the extent of encapsidation of TAV genomic RNAs. The fraction of B2- and B8satRNAs encapsidated was greater for 1 -TAV than for V-TAV, although spread and accumulation of the satRNA were similar for both helper viruses. These results suggest that CMV-satRNA may spread in a nonencapsidated form. Accumulation of CMV-satRNA in systemically infected leaves was detected for all inoculum combinations except V-TAV and Ix-satRNA, for which the satellite RNA increased only in protoplasts and inoculated leaves of tobacco or tomato. In such inoculated leaves, Ix-satRNA was not detected in capsids. Thus the effectiveness of the TAV helpers of CMV-satRNAs may be controlled in at least some instances by the extent of satRNA spread or encapsidation rather than by the efficiency of satRNA replication. In contrast to infections initiated by inoculation of CMV and CMV-satRNA, inoculation of I-TAV or V-TAV and CMV-satRNA did not alter the relative amounts of viral Q 1992 Academic genomic RNAs encapsidated or result in accumulation of large amounts of double-stranded satRNA. Press.
Inc.
INTRODUCTION
helper of CMV-satRNA has not been characterized to the same extent as CMV, and comparison of the interactions TAV:CMV-satRNA and CMV:CMV-satRNA could illustrate the nature of the relationship between satRNAs and helper viruses. In this report we analyze the interaction of two strains of TAV with several CMVsatRNAs in two hosts. Our results show that this interaction is determined by the strain of TAV, the strain of satRNA, and the host plant. We also demonstrate that the observed differences in the ability of TAV strains to serve as effective helpers of CMV-satRNA may be controlled by the extent of satRNA spread or encapsidation rather than by the efficiency of satRNA replication.
Cucumber mosaic virus (CMV), the type member of the cucumovirus plant virus group, has a tripartite, plus sense RNA genome. CMV particles, in addition to the genomic RNAs 1,2, and 3 and the subgenomic RNA4, can also contain a small (340-380 nucleotides) satellite RNA (satRNA) that depends on CMV (helper virus) for both its replication and encapsidation. The replication of CMV RNA and the symptoms induced during CMV infections can be affected by the presence of the satRNA in a way that depends on the combination of strain of helper virus, strain of CMV-satRNA, and host plant (Jacquemond & Leroux, 1982; Kaper & Tousignant, 1977; Mossop & Francki, 1979). No satellite RNA has ever been found in field isolates of tomato aspermy virus (TAV), a cucumovirus closely related to CMV (Bernal et al., 1991; Moriones et al., 1991 a; O’Relly et al., 1991). However, TAV can act as a helper virus for CMV-satRNA (Gould et al., 1978; Mossop and Francki, 1979), although differences have been reported between the interactions of TAV and CMV with CMVsatRNA (Harrison et al., 1987; Jaegle et al., 1990; Lee & Kummert, 1985; Mossop & Francki, 1979). TAV as a ’ To whom correspondence dressed.
and reprint requests
MATERIAL
AND METHODS
Virus and satRNA strains and RNA preparations L,-CMV, Fny-CMV (Owen & Palukaitis, 1988) V-TAV (Habili & Francki, 1974), Bl-, B2-, B3-, WL2-, G-, (Garcia-Arena1 et al., 1987) and Ix-satRNAs (Moriones et a/., 1991 b) have been described. 1-TAV is a tomato isolate from Indiana, the gift of P. Palukaitis (Cornell University, NY). SatRNAs were multiplied in Nicotiana c/eve/and;; Gray using L,-CMV as helper virus. CMV and TAV were multiplied in N. clevelandi and purified as in Lot et al. (1972) and Habili and Francki (1974)
should be ad-
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0042.6822/92
$3.00
CopyrIght 0 1992 by Academvz Press, Inc All rights of reproduction I” any form reserved.
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MORIONES
ET AL
respectively. Encapsidated RNAs were extracted from virions with phenol/SDS and separated in preparative 6% polyacrylamide gels. Bands corresponding to satRNA were visualized by staining with 0.05% toluidine blue, excised, and the satRNA was purified as described by Palukaitis and Zaitlin (1984). Nucleic acids from infected leaves were extracted as in Palukaitis et al. (1983) and fractionated by precipitation in 2 and 4 M LiCl into fractions enriched for singlestranded (ss) and for double-stranded (ds) RNAs (DiazRuiz & Kaper, 1978). Biological
B1-
BZ-
B3-
G-
Ix-
WL2-
assays
Combinations of helper virus RNAs, with or without purified satRNAs, were inoculated onto the expanded third and fourth leaves of tobacco cv. Xanthi-nc, or onto the expanded cotyledons of tomato cv. Rutgers. The suspension used as inoculum (10 PI/plant) was 200 pg/ml helper RNA and 25 pg/ml satRNA in 0.1 n/l Na,HPO,. Plants were maintained in the greenhouse (20-25”, 16 hr light) and analyzed by dot-blot hybridization (Palukaitis et a/., 1985) for the presence of satRNA 7-10 days postinoculation. The symptoms were recorded for l-4 weeks postinoculation when virions and nucleic acids were extracted from leaves. Protoplasts from tobacco leaves were prepared as in Power and Chapman (1985) and 1.5 X 1O6protoplasts were inoculated with 40 pg of helper virus RNA with or without 2.5 pg of satRNA as in Power et al. (1989). At 30 hr postinoculation protoplasts were harvested and nucleic acids were extracted as in Dreher et al. (1989). RNA analyses Virion-encapsidated RNAs were analyzed in 1.2% agarose gels in TBE buffer (Rezaian & Symons, 1986). Viral and satRNAs were also detected and quantitated by ribonuclease protection analysis (RPA). RPA was performed as in Lopez-Galindez et a/. (1988) except that ribonuclease digestion of duplexes was performed with a mixture of RNases A and Tl , as in Winter et a/. (1985). cRNA probes transcribed from a clone with an insert of 3 19 nucleotides (nt) from B2-satRNA, or from a clone with a 1OOO-nt insert of V-TAV RNA3 were hybridized to different amounts of the RNA samples to be analyzed. Probes were labeled with [a3ZP]UTP; low activity probes (0.1 X 10” cpmlpg) or high activity probes (2.5 X 10” cpm/pg) were obtained by adding or omitting cold UTP to the transcription reaction (Sambrook et a/., 1989). Densitometric analyses of the autoradiograms of RPAs showed that the intensity of the bands resulting from ribonuclease digestions was linear for amounts of purified satRNA between 0.5 and 50 ng, using 200,000 cpm of the high activity probe, and be-
FIG. 1. Comparison of progeny CMV-satRNAs isolated from tobacco plants infected with I-TAV (lanes 2) or V-TAV (lanes 3) with the parental satRNAs preparations used for inoculation (lanes 1). RPAs of gel-purified satRNAs (Bl-, B2-, 83.. G-, Ix-, and WLZ-satRNAs) were performed with a cRNA probe to B2-satRNA. The products of ribonuclease digestion were separated in a 6% polyacrylamide-electrophoresis gel and visualized by autoradiography. (a) 15,000 cpm of undigested B2-satRNA probe; in addition to 3 19 nt complementary to B2aatRNA, the transcript has 28 extra nt at its 5’end. (b) Digested, unhybridized probe.
tween 5 ng and 500 ng, using 50,000 cpm of the low activity probe. Assays were routinely carried on using 0.15 pg of virion RNA, 1.5 pg of ssRNA from infected leaves, or total nucleic acids extracted from 0.5 X 1O6 protoplasts. When differences between the samples to be compared were not apparent, the assays were repeated using two fivefold dilutions of the above indicated amounts of RNA to make certain that the assay was performed within the range for linearity. RESULTS Replication
of CMV-satRNAs
by TAV
Dot-blot hybridization analyses of systemically infected leaves from tobacco and tomato inoculated with l-TAV, V-TAV, and any of the six strains of CMVsatRNA used in this work (Bl-, B2-, B3-, G-, lx-, and WL2-satRNA) showed the presence of progeny satRNA, except in the case of Ix-satRNA inoculated with V-TAV (not shown). The identity of progeny CMVsatRNA with the parental satRNAs was analyzed by RPA with a probe specific for a reference isolate, B2satRNA, using similar amounts of gel-purified parental and progeny satRNAs (Fig. 1). In all cases the pattern of mismatches was identical for the parental and the
TAV AS A HELPER TO CMV-satRNAS
A
B2- B3- Ix- WL2-82. Bl120121212121’2’1’2’
Ix-
6
Bl- 82.63.
Ix- WL2-B2-
Ix-
(Bl-, E32-, B3-, lx-, and FIG. 2. RPA quantitation of CMV-satRNAs WL2satRNAs) in virion RNA (A) or in the ssRNA enriched (B) fractions of nucleic acid extracts from tobacco (lanes O-2) or from tomato leaves (lanes 1’ and 2’). Helper viruses were Fny-CMV (lane O), 1-TAV (lanes 1 and 1’) and V-TAV (lanes 2 and 23. 0.15 pg of virion RNA or 1.5 rg of ssRNA-enriched faction used for each analysis.
progeny satRNA preparations, except for G-satRNA for which a variant from the original preparation was consistently supported by l- and V-TAV both in tobacco and in tomato. Because of this, G-satRNA was excluded from further analyses. The analysis of the efficiency of 1-TAV and V-TAV as helper viruses to CMV-satRNAs was first approached by comparing the relative amounts of satRNAs in TAV particles, as in previous reports (Harrison et al., 1987; Jaegle er al., 1990; Lee & Kummert, 1985; Mossop & Francki, 1979). Since both 1-TAV and V-TAV encapsidate two low molecular weight RNAs, one of them with an electrophoretic mobility similar to CMV-satRNAs (see Fig. 4 and Hull, 1976), satRNA quantitation was performed by RPA. In virions purified from tobacco plants, all five CMV-satRNAs accounted for a lower proportion of the RNA encapsidated by V-TAV (Fig. 2A, lanes 2) when compared with 1-TAV (Fig. 2A, lanes 1). The proportion of encapsidated satRNA was similar in 1-TAV and Fny-CMV virions (shown for B2-satRNA in Fig. 2A, lanes 0 and 1). Ix-satRNA was not detected in particles of V-TAV (Fig. 2A). RPA was performed in the ssRNA-enriched fraction of total nucleic acid extracts from systemically infected tobacco leaves to obtain an estimation of the total amount of satRNA in the plant. Three different situations were found (Fig. 2B): (a) B2and B3-satRNAs were found in similar amounts when either 1-TAV or V-TAV were used as helper viruses; (b) Bl- and WL2-satRNAs represented a higher percentage of the ssRNA fraction when 1-TAV was the helper
477
virus, as compared with V-TAV; and (c) Ix-satRNA was not detected in nucleic acid extracts of tobacco plants inoculated with V-TAV + Ix-satRNA. Similar anaylses were performed for B2-satRNA and Ix-satRNA in virion RNA and ssRNA fractions of extracts from tomato leaves (Fig. 2A and 2B, lanes 1’ and 2’). Again, IxsatRNA was not detected when V-TAV was the helper virus. In contrast, B2-satRNA was found to represent a similar amount of the encapsidated RNA and of ssRNA fraction when 1-TAV and when V-TAV were the helper strains. The relationship of B2-satRNA and Ix-satRNA with TAV, as representing the extreme cases of those described above was further analyzed. When the inoculated leaves of tobacco plants were extracted, lxsatRNA was found to be present in nucleic acid extracts from leaves inoculated with V-TAV, although in lower amounts than in leaves inoculated with l-TAV, while both TAV RNAs were found to similar levels. No Ix-satRNA was found, however, in V-TAV virions from these directly inoculated leaves (not shown). The replication of B2-satRNA and of Ix-satRNA by 1-TAV and by V-TAV was also analyzed in tobacco protoplasts to distinguish between effects due to replication or to spread-within-the-plant in the differential behavior of both satRNAs with both TAV strains. Both I-TAV and V-TAV did replicate to the same levels in tobacco protoplasts, as determined by the quantitation of RNA3 in protoplast’s extracts (Fig. 3B, lanes 1 and 2). Also, both B2-satRNA and Ix-satRNA were detected in similar amounts (Fig. 3A, lanes 1 and 2) in the extracts of protoplasts infected with I-TAV or with VTAV, indicating a similar ability of both TAV strains to replicate the two satRNAs. Effect of CMV-satRNA on TAV accumulation the symptoms induced by TAV
and on
The presence of CMV-satRNA did not affect the yield of V-TAV virions purified from tobacco (in the absence of satRNA 164.6 + 19.3 mg/kg fresh leaf, average of six experiments). The yield of I-TAV (689.5 f 223.5 mg/kg fresh leaf in the absence of satRNA) was reduced to about 50% when any of the six CMV-satRNAs was present in the inoculum (e.g., to 355.1 f 132.5 mg/kg fresh leaf for B2-satRNA and to 324.0 If: 110.4 mg/kg fresh leaf for Ix-satRNA). The yield of l-TAV or V-TAV purified from tomato plants in the absence of satRNA was similar (e.g., 118.0 -+ 16.8 mg/kg fresh leaf for 1-TAV in the absence of satRNA) and was not affected by the presence of any CMV-satRNA, including Ix-satRNA with necrotic tomato plants (yield of 1 1 1.6 f 6.9 mg/kg fresh leaf for 1-TAV in the presence of Ix-satRNA). The relative proportion of encapsidated
478
MORIONES
A a
B2bl
I3
Ix2
1
2
B2-
abl
Ix2
1
2
FIG. 3. Accumulation of viral and satellite RNA in tobacco protoplasts inoculated with 1-TAV RNA (lanes 1) or V-TAV RNA (lanes 2) plus B2-satRNA (629, or Ix-satRNA (Ix-). Total RNA extracted from 5 X 1O5 protoplasts 30 hr postinoculation was hybridized with ?labeled cRNA probes to EGsatRNA (A) or to V-TAV RNA3 (B) and subjected to RPA. (a) 200,000 cpm of undigested probes; (b) digested, unhybridized probes.
TAV RNAs 1, 2, 3, and 4 (Fig. 4, shown for TAV infections with !32- and WL2-satRNAs in tobacco), as well as of TAV ds RNAs 1-4 extracted from infected leaves (not shown) was not affected by any CMV-satRNA. The six strains of CMV-satRNAs used in this study either had no effect or attenuated to different degrees the symptoms induced by both l-TAV and V-TAV in tobacco plants. The effect of a particular satRNA was not the same on each TAV strain (Table 1). For example, B3-satRNA did not affect the symptoms induced by V-TAV but induced an extreme attenuation of those induced by l-TAV. B2-satRNA, which induces yellow chlorosis in tobacco when supported by the subgroup II of CMV strains (Palukaitis, 1988) attenuated the symptoms of both TAV strains. In tomato plants, the CMV-satRNAs that attenuated the symptoms of CMV also attenuated those induced by l-TAV and V-TAV. Ix-satRNA, which induces a lethal necrosis in tomato plants when supported by CMV strains of both subgroups I and II (Moriones eT al., 1991 b) also induced a lethal necrosis when supported by 1-TAV. DISCUSSION The amount of CMV-satRNA found in TAV particles depends on the virus and the satRNA strains and on the host plant: while in general more CMV-satRNA is
ET AL.
found in particles of l-TAV (at levels similar to CMV) than of V-TAV, Ix-satRNA being an extreme case, in tomato both TAVs encapsidate similar proportions of B2-satRNA. We have attempted to differentiate among the various possible components (satRNA replication, movement within the plant, and encapsidation) of the helper function. The similar levels of B2 and B3 CMVsatRNAs in ssRNA fractions from infected leaves show that their different amounts in 1-TAVvs V-TAV particles purified from tobacco can be attributed to an encapsidation effect. The reason why this differential encapsidation of B2-satRNA occurs in tobacco but not in tomato is unknown. Different amounts of Bl-satRNA, WL2-satRNA, and Ix-satRNA are found in both the particles of 1-TAV and V-TAV and in the ssRNA fractions from infected leaves, and may be due to differences between both TAVs in any of the components of the helper functions. The presence of Ix-satRNA in nucleic acid extracts of tobacco leaves directly inoculated with V-TAV, but in lower amounts than in leaves inoculated with I -TAV, indicates a different ability of both TAVs to assist the cell-to-cell movement, or the replication, of the satRNA. Since B2-satRNA and Ix-satRNA are replicated with similar efficiencies by l-TAV and V-TAV in tobacco protoplasts, the differences observed in planta must be due to differences in the ability of the strains of TAVs to mediate the movement within the plant of some (e.g., Ix-satRNA) but not all (e.g., B2satRNA) CMV-satRNAs. Our data do not exclude the possibility that different abilities to replicate some CMV-satRNAs may exist between different TAV strains, or between TAV and CMV, as pseudorecombinant experiments by Mossop and Francki (1979) seem to indicate. We have been unable to correlate the different behaviour observed for the different CMVsatRNAs with any feature of their nucleotide sequences. It has been suggested that viral assembly plays an important role in the systemic movement of CMV in infected plants (Suzuki et al., 1991). The data presented for B2-satRNA and Ix-satRNA could suggest rather than encapsidation and movement within the plant are different phenomena for the satRNAs, although the presence of Ix-satRNA in viral particles at undetectable levels cannot be excluded. The effect of CMV-satRNA on the helper virus differs for TAV and for CMV in several ways: (1) The modulation by a particular CMV-satRNA of the symptoms induced by CMV or TAV may be the same, or not: CMVsatRNAs that attenuate the symptoms induced by CMV in tomato or tobacco, or that are necrogenic with CMV for tomato, had similar effects with TAV (this work, Harrison et al., 1987; Lee & Kummert, 1985; Mossop & Francki, 1979) while satRNAs that induce
479
TAV AS A HELPER TO CMV-satRNAS TABLE 1 SYMPTOMS INDUCEDON TOBACCO ORTOMATO BYTwo STRAINSOF TOMATO ASPERMYVIRUS WITH OR WITHOUTDIFFERENTSATELLITERNAs OF CUCUMBER MOSAIC VIRUS Tomato
Tobacco 1-TAV
V-TAV
1-TAV
V-TAV
Severe mosaic, oak-leaf pattern Mosaic, oak-leaf pattern Very mild mosaic to symptomless Very mild mosaic to symptomless Mosaic, oak-leaf pattern Mosaic, oak-leaf pattern Mild mosaic
Severe mottle and ringspot Very mild mosaic to symptomless Very mild mosaic to symptomless Severe mottle and ringspot Mild mottle and ringspot Severe mottle and ringspotb Mild mottle and ringspot
Severe stunting, severe fern-leaf, mild mosaic NT”
Stunting, fern-leaf, severe mosaic NT
Mild stunting, mild fern-leaf NT
Mild stunting, mosaic NT
Mild stunting, fern-leaf, mild mosaic Systemic necrosis
Severe mosaic
Satellite RNAs None 81 -satRNA B2-satRNA B3satRNA G-satRNA Ix-satRNA WL2-satRNA
a NT, not tested. b No satRNA was detected
in the leaves for which the symptoms
chlorosis in tobacco with CMV attenuate TAV symptoms (this work, Jaegle et al., 1990). (2) For all reported CMV strain:CMV-satRNA combinations, in different host plants, the yield of CMV virions is variously reduced by the presence of CMV-satRNAs (Jacquemond & Leroux, 1982; Kaper & Tousignant, 1977; Mossop & Francki, 1979). This is also the case for some TAV strains in some host plants [B-TAV and l-TAV in tobacco (Lee & Kummert, 1985; this work)] but not for others [the Scotland strain of TAV and V-TAV in tobacco, V- and N-TAV in N. clevelandi;, l-TAV and VTAV in tomato (Harrison et al., 1987; Mossop & Francki, 1979; this work)], which indicates that the at-
-0
V-TAV
I-TAV 1
2
0
1
Fny-CMV 2
01
FIG. 4. Comparison of the RNAs encapsidated by 1-TAV, V-TAV. and Fny-CMV, when inoculated with no satRNA (lane 0), with B2satRNA (lanes l), or WL2satRNA (lanes 2) in tobacco plants. RNAs were purified from virions, separated in a 1.2% agarose electrophoresis gel, and visualized by ethidium bromide staining.
Mild stunting, fern-leaf
mild
Stunting, severe Stunting, severe
fern-leaf, mosaicb fern-leaf, mosaic
were assessed.
tenuation of TAV symptoms by CMV-satRNA is not related to a depression of virus yield. (3) While the presence of CMV-satRNAs results in a depression of relative amounts of the encapsidated RNAs 1 and 2 of CMV (Jacquemond & Leroux, 1982; Kaper & Tousignant, 1977; Mossop & Francki, 1979) this is never the case for TAV no matter what the effect of the satRNA on TAV yield or symptoms (this work, Gould er a/., 1978; Harrison et al., 1987; Mossop & Francki, 1979; Lee & Kummert, 1985). (4) dsCMV-satRNA becomes predominant in dsRNA fractions from infected leaves when CMV is the helper virus (Piazzola et a/., 1982). This is not the case for TAV in any of the interactions analyzed here. This indicates that in the cases when a depression of TAV virion yield is observed, competition of satRNA and helper virus RNA for replication is not likely to be the cause, as has been suggested for CMV. The results indicate that the interaction of TAV with the CMV-satRNAs is a phenomenon determined by the combination of strain of TAV, strain of CMV-satRNA, and species of host plant. This can help to explain conflicting reports on the ability of different strains of TAV to maintain different CMV-satRNAs (Harrison et al., 1987; Jaegle et a/., 1990; Lee & Kummert, 1985; Mossop & Francki, 1979) and parallels wtiat has been reported for the interaction of CMV with its satRNAs (Jacquemond & Leroux, 1982; Kaper &Tousignant, 1977). The specificity of the interaction between helper virus and satRNA is further shown by the selection of sequence variants of a satRNA by different helper viruses, as occurs for G-satRNA.
480
MORIONES ET AL.
ACKNOWLEDGMENTS We thank Peter Palukaitis, Cornell University, NY, for l-TAV. This work was in part supported by Grant PA86-0353 Comisi6n Interministerial de Ciencia y Tecnologfa, Spain; E.M.A. and E.R.C. were the recipients of fellowships from Ministerio de Educaci6n y Ciencia, Spain.
REFERENCES BERNAL,J. J., MORIONES, E., and GARCIA-ARENAL, F. (1991). Evolutionary relationships in the cucumoviruses: nucleotide sequence of tomato aspermy virus RNA1. 1. Gen. Viral. 72, 219 l-2 195. D~Az-RUIZ,J. R., and KAPER,J. M. (1978). Isolation of viral doublestranded RNAs using a LiCl fractionation procedure. Prep. Biothem. 8, 1-17. DREHER,T. W., RAO,A. L. N., and HALL,T. C. (1989). Replication in t&o of mutant brome mosaic virus RNAs defective in aminoacylation. J. Mol. Biol. 206, 425-438. GARCIA-ARENAL, F., ZAITLIN,M., and PALUKAITIS, P. (1987). Nucleotide sequence analysis of six satellite RNAs of cucumber mosaic virus: Primary sequence and secondary structure alterations do not correlate with differences in pathogenicity. Virology 158, 339-347. GOULD,A. R., PALUKAITIS,P., SYMONS,R. H., and MOSSOP,D. W. (1978). Characterization of a satellite RNA associated with cucumber mosaic virus. Virology 84, 443-455. HABILI,N., and FRANCKI,R. I. 6. (1974). Comparative studies on tomato aspermy and cucumber mosaic viruses. 1. Physical and chemical properties. Virology 57, 392-401. HARRISON,B. D., MAYO,M. A., and BAULCOMEE, D. C. (1987). Virus resistance in transgenic plants that express cucumber mosaic virus satellite RNA. Nature 328, 799-802. HULL,R. (1976). The behaviour of salt-labile plant viruses in gradients of cesium sulphate. Virology 75, 18-25. JACQUEMOND, M., and LEROUX,J. P. (1982). L’ARN satellite du virus de la mostiique du concombre. Il. Etude de la relation virus-ARN satellite chez divers h&es. Agronomie 2, 55-62. JAEGLE,M., DEVIC,M., LONGSTAFF, M., and BAULCOMBE,D. (1990). Cucumber mosaic virus satellite RNA (Y strain): analysis of sequences which affect yellow mosaic symptoms on tobacco. J. Gen. Virol. 71, 1905-1912. KAPER,J. M., and TOUSIGNANT, E. M. (1977). Cucumber mosaic virusassociated RNA5. 1. Role of host plant and helper virus in determining the amount of associated RNA5 with virions. Virology 80, 186-195. LEE,H. S., and KUMMERT, J. (1985). Induction of tomato necrosis by cucumoviruses, as related to specific interactions between genomic and satellite RNAs. Parasifica 41, 45-55. LOPEZ-GAL~NDU, C., LOPEZ,J. A., MELERO,J. A., DE LA FUENTE,L., MARTINEZ,C., ORTIN,J., and PERUCHO,M. (1988). Analysis of genetic variability and mapping of point mutations in influenza virus by the RNAse A mismatch cleavage method. Proc. Nat/. Acad. Sci. USA 85, 3522-3526. LOT,H., MARROUX, J., QUIOT,B., and ESVAN,C. (1972). Contribution a I’Btude du virus de la mosa’fque du concombre (CMV). II. MBthode de purification rapide du virus. Ann. Phytopathol. 4, 25-38.
MORIONES,E., ROOSSINCK, M. J., and GARCIA-ARENAL, F. (1991a). Nucleotide sequence of tomato aspermy virus RNA2. J. Gen. Viral. 72,779-783. MORIONES, E., FRAILE.A., and GARCIA-ARENAL, F. (1991 b). Host-associated selection of sequence variants from a satellite RNA from cucumber mosaic virus. Virology 184, 465-468. MOSSOP,D. W., and FRANCKI,R. I. B. (1979). Comparative studies of two satellite RNAs of cucumber mosaic virus. Virology 95. 395404. O’REILLY.D., THOMAS,C. J. R., and Courts, R. H. A. (1991). Tomato aspermy virus has an evolutionary relationship with other tripartite RNA plant viruses. J. Gen. Viral. 72, l-7. OWEN,J., and PALUKAITIS,P. (1988). Characterization of cucumber mosaic virus. 1. Molecular heterogeneity mapping of RNA 3 in eight CMV strains. Virology 168, 495-502. PALUKAITIS, P. (1988). Pathogenic&y regulation by satellite RNAs of cucumber mosaic virus: minor nucleotide sequence changes alter host response. Molec. Plant-Microbe Int. 1, 175-l 8 1. PALUKAITIS, P., and ZAITLIN,M. (1984). Satellite RNAs of cucumber mosaic virus: Characterization of two new satellites. Virology 132, 426-435. PALUKAITIS, P., GARCIA-ARENAL, F., SUIZINSKI,M. A., and ZAITLIN,M. (1983). Replication of tobacco mosaic virus. VII. Further characterization of single and double-stranded virus-related RNAs from TMV-infected plants. Virology 131, 533-545. PALUKAITIS, P., Cons, S., and ZAITLIN,M. (1985). Detection and identification of viroids and viral nucleic acids by ‘dot-blot’ hybridization. Acta Hort. 164, 109-l 18. PIAZZOL%P., TOUSIGNANT, M. E., and KAPER,1. M. (1982). Cucumber mosaic virus-associated RNA 5. IX. The overtaking of viral RNA synthesis by CARNA 5 and ds CARNA 5 in tobacco. Virology 122, 147-157. POWER, J. B., and CHAPMAN,J. V. (1985). Isolation culture and genetic manipulation of plant protoplasts. ln “Plant Cell Culture” (R. A. Dixon, Ed.), pp. 37-66. IRL Press, Oxford. POWER,J. B., DAVEY,M. R., MC LELLAN,M., and WILSON,D. (1989). “Laboratory Manual Plant Tissue Culture. Plant Genetic Manipulation Group.” Department of Botany. University of Nottingham, Nottingham, NG7 2RD, UK. REZAIAN,M. A., and SYMONS,R. H. (1986). Anti-sense regions in satellite RNA of cucumber mosaic virus form stable complexes with the viral coat-protein gene. Nucleic Acids Res. 14, 32293239. SAMBROOK, J., FRITSCH,E. F., and MANIATIS,T. (1989). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratoty. Cold Spring Harbor, NY. SUZUKI,M., KUWATA,S., KATAOKA,J., HASUTA,C., NITTA,N., and TAKANAMI,Y. (1991). Functional analysis of deletion mutants of cucumber mosaic virus RNA3 using an in vitro transcription system. Virology 183, 106-l 13. WINTER,E., YAMAMOTO,F., ALMOGUERA, C., and PERUCHO, M. (1985). A method to detect and characterize point mutations in transcribed genes: Amplification and overexpression of the mutant c-ki-ras allele in human tumor cells. Proc. Nat/. Acad. Sci. USA 82, 7575-7579.
