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Differential replication in zucchini squash of a cucumber mosaic virus satellite RNA maps to RNA 1 of the helper virus
VIROLOGY
181, 371-373
(1991)
Differential
Replication in Zucchini Squash of a Cucumber Mosaic Virus Satellite RNA Maps to RNA 1 of the Helper Virus MARILYN Department
J. ROOSSINCK
of Plant Pathology,
Received September
AND PETER PALUKAITIS’
Cornell University,
2 1I 1990; accepted
Ithaca. New York 14853 November
6, 1990
Cucumber mosaic virus (CMV) supports the replication and encapsidation of its satellite RNA, both in solanaceous and cucurbit host plants: however, different strains of CMV support the replication of satellite RNAs with different efficiency. In addition, replication of satellite RNA is very efficient in solanaceous host plants and generally poor in cucurbit host plants. The WL,-satellite (WL,-sat) RNA is an exception, and replicates to high levels in both solanaceous and curcubit host plants with most CMV strains as the helper virus. Two strains of CMV were used in this study: Fny-CMV, which replicates the WL,-sat RNA efficiently in all hosts tested; and Sny-CMV, which does not replicate the WL,-sat RNA to detectable levels in zucchini squash (Cucurbita pepo), but does replicate WL,-sat RNA efficiently in other hosts. Using pseudorecombinants constructed between Fny-CMV and Sny-CMV we have mapped to RNA 1 the o 1991 Academic PMS. I~C. ability to support the efficient replication of WL,-sat RNA in zucchini squash.
effects are dependent on both the helper virus and the species of the infected plant. The majority of satellite RNAs found associated with various strains of CMV replicate with the helpervirus to high levels in solanaceous plants, such as tobacco. However, these satellite RNAs generally replicate poorly in cucurbits. A notable exception is the WL,-satellite (WL,-sat) RNA ( 73). With most helper virus strains of CMV, including the Fny-CMV strain, the WL,-sat RNA replicates efficiently in cucurbits (( 73) and our unpublished results). By contrast, the Sny-strain of CMV does not support the replication of WL,-sat RNA to a detectable level. In a previous study we described the construction of pseudorecombinants of the Fny- and Sny-CMV strains (74) which were used to map two pathogenicity domains to RNA 1. In this studywe report the use of these pseudorecombinants to map the domain responsible for the efficient replication of the WL,-sat RNA in zucchini squash (Cucurbita pepo, cv Black Beauty). Zucchini squash plants were inoculated at the cotyledon stage, using 100 @g/ml of viral RNA, with orwithout the addition of 5 pg/ml of gel-purified WL,-sat RNA (75) in 50 ml\/l sodium phosphate buffer, pH 9.2. A single preparation of each pseudorecombinant virus was used to inoculate plants, which were maintained in an environmentally controlled chamber with day temperatures of 23”, night temperatures of 19”, and a 16hr day. Leaf tissue was assessed by dot-blot hybridization for the presence of satellite RNA 10 to 14 days after
Cucumber mosaic cucumovirus (CMV) is a tripartite, RNA plant virus with a single stranded plus-sense genome. CMV infects a wide range of plants, encompassing nearly 800 species, and is of worldwide economic importance (7,Z). The CMV genome consists of three RNAs and four open reading frames (ORFs). RNAs 1 and 2 each contain one ORF, encoding proteins of 11 1 kDa (the la protein) and 97 kDa (the 2a protein), and are the only viral requirements for replication of the viral RNAs (3). These RNAs contain conserved regions found in all members of the Sindbis-like virus supergroup (4-6). RNA 3 contains two ORFs: the 5’ half of RNA 3 encodes a 30-kDa protein (the 3a protein) thought to be involved in viral movement (7) and the 3’half of RNA 3 generates a subgenomic RNA, RNA 4, which acts as a messenger for the viral coat protein (8, 9). In addition to the encapsidated genomic RNAs and the subgenomic RNA, virus particles often contain a satellite RNA of approximately 330 to 380 nucleotides (for review see Ref. (70)). By definition, the satellite RNA is dependent on the helper virus for both its replication and encapsidation. The CMV satellite RNA does not contain any demonstrably functional ORFs, nor does it show any extensive homology with the helper viral RNAs. Nevertheless, the presence of satellite RNA can result in a dramatic alteration in viral symptoms in CMV-infected plants, ranging from necrosis and death (7 7) to amelioration of disease (72). These symptom ’ To whom correspondence
should be addressed 371
0042-6822191
$3.00
Copyright 0 1991 by Academic Press. Inc All rights of reproduction I” any form reserved.
SHORT COMMUNICATIONS
372 TABLE 1
REPLICATIONOF THE WL, SATELLITERNA BY PSEUDORECOMBINANTS OF FNY- AND SNY-CMV RNAs Pseudorecombinante FSS SFF FSF SFS FFS SSF FFF sss
Number of sat (+) plants* (%) 12/12 3/68 12/12 5/l 00 12/l 2 O/60 18/18 l/65
(100) (4.4) (100) (5) (100) (0) (100) (1.5)
’ FSS indicates RNA 1 from Fny-CMV, RNA 2 from Sny-CMV, and RNA 3 from Sny-CMV. Other strains follow similar designations. b Number of plants which tested positive for CMV satellite RNA by dot-blot hybridization, as described in the text, over the total number of plants tested. The data are derived from two (SFS), three (FSS, FSF, FFS, and SSF), four (SFF), or five (SSS and FFF) seperate experiments.
inoculation. Tissue samples were taken from upper leaves, ground and extracted as previously described (16), and spotted onto nitrocellulose filters. Subsequently, the filters were either baked at 80” for 90 min or uv cross-linked by exposure to shortwave uv light for 45 set (Fotodyne UV transilluminator). Filters were then prehybridized as described (16), hybridized with approximately 4 X 1O6 cpm of 32P-labeled cDNA made to gel-purified G-sat RNA, in 4.5 ml of buffer (hybridization buffer II in Ref. (16)), and washed, priorto autoradiography ( 17). As indicated in Table 1, plants infected with pseudorecombinant strains containing RNA 1 from FnyCMV and RNAs 2 and 3 from either strain (i.e., FSS, FSF, FFS, and FFF), and WL,-sat RNA all showed 1OOYoinfection with WL,-sat RNA. Plants infected with RNA 1 from Sny-CMV and RNAs 2 and 3 from either strain (i.e., SFF, SFS, SSF, and SSS) generally contained no detectable WL,-sat RNA. However, in plants infected with either the SFS/WL,-sat RNA or SFFiWL,sat RNA combination, WL,-sat RNA was detected about 5% of the time, and one plant out of 65 infected with the SSSNVL,-sat RNA combination was also positive for satellite RNA by dot-blot hybridization. In the initial studies of WL,-sat RNA replication using these pseudorecombinants, we detected WL,-sat RNA about 309/o of the time with the SFF/WL,-sat RNA pseudorecombinant. When we purified viral RNA (18) from those plants which did not contain detectable WL,-sat RNA, we were able to segregate out an SFFCMV isolate which did not support these levels of WL-1 sat RNA replication. This isolate was used for the studies described here. Although it is possible that the original SFF-CMV combination contained low levels of RNA 1 from Fny-CMV, it seems more likely that the ability to
support WL,-sat RNA replication by the SFF-CMV pseudorecombinant was due to the heterogeneity of the original CMV-241 strain from which Sny-CMV was derived (14). The CMV-241 strain was able to support WL,-sat RNA replication in a low percentage of plants (data not shown). No detectable Fny-CMV RNA 1 was present in either SFF or SFS (14). Furthermore, the timing and severity of symptoms induced in zucchini squash by pseudorecombinants SFF- and SFS-CMV were not affected by repeated passage, as would have been expected if Fny-CMV RNA 1 was contaminating the strains. Since the detection of satellite RNA was limited by the sensitivity of the dot-blot hybridization technique, we could not be certain that there was no WL,-sat RNA replication occurring in the plants which tested negative for satellite RNA. To further test the possible replication of satellite RNA in zucchini squash plants inoculated with these strains, virus was purified from zucchini squash plants (19) that had been inoculated with SSF/WL,-sat RNA and SSS/WL,-sat RNA and were satellite RNA negative as determined by dot-blot hybridization. These viruses subsequently were inoculated onto young tobacco plants (Nicotiana tabacum cv Xanthi nc) at a concentration of 50 pg/ml in sodium phosphate buffer, pH 7.0. In tobacco plants even very low levels of satellite RNA are likely to be amplified. Ten days after inoculation tobacco plants inoculated with purified virus from the SSF/WL,-sat RNA- and SSS/WL,-sat RNA-infected zucchini squash plants tested positive for CMV satellite RNA by dot-blot hybridization (data not shown), indicating that the replication of WL,-sat RNA had been supported by these strains in zucchini squash, albeit at levels below the sensitivity of the dotblot detection system. To confirm that the satellite RNA from the infected tobacco plants was indeed WL,-sat RNA, these RNAs were analyzed by RNase protection assays (Fig. l), as previously described (20). The probe for the assay was a 32P-labeled, full-length, minussense, WL-sat RNA transcript, using EcoRI-digested pWLsat47 as template DNA, and T3 RNA polymerase; plus-sense control transcript was generated from the same plasmid, using Smal-digested DNA and T7 RNA polymerase as previously described (2 1). We have shown here that the WL,-sat RNA can replicate efficiently with Sny-CMV in tobacco, but not in zucchini squash. The poor replication of WL,-sat RNA in squash maps to RNA 1 of Sny-CMV. This implies a host-virus interaction involving RNA 1. Although RNA 1 is known to be required for replication of the viral RNAs and the satellite RNA (3) and contains both the conserved motifs of a helicase (22) as well as the properties of a nucleotide binding protein (23) in the carboxy terminal third of the 1a ORF, its precise role in replication is unknown. The domain(s) comprising approxi-
SHORT COMMUNICATIONS
373
REFERENCES SSFIWL
Tr
FIG. 1. Polyacrylamide gel electrophoretic analysis of RNase protection of CMV satellite RNAs. Each lane represents the protected fragments of the antisense WL,-sat probe, after annealing to viral RNA from plants infected with the indicated virus. Strains are as designated in footnote a to Table 1; Tr, in vitro generated plus sense transcript; C, control, no RNA.
mately two-thirds of the la protein at the amino terminus does not contain any significant homology to proteins which have had functions assigned to them; however, the amino terminal third of the la protein shows a high degree of homology with analogous proteins of the supergroup of Sindbis-like viruses (4). Although the efficiency of WL,-sat RNA replication maps to RNA 1, just as the previously described symptom differences between Fny- and Sny-CMVs mapped to RNA 1 (Id), the domain responsible for satellite RNA replication efficiency is most likely a different domain; i.e., several related strains of CMV either display rapidly appearing, severe symptoms, or slowly developing, mild symptoms, but the inefficiency of WL,-sat RNA replication is a property unique to the Sny-CMV strain. With the availability of cDNA clones of the Fny-CMV strain from which infectious RNA transcripts can be generated (24, we should be able to more precisely map this differential ability to replicate WL,-sat RNA by constructing recombinant viruses between the Fnyand Sny-CMV strains. ACKNOWLEDGMENTS This work was supported by grant No. 88-37263-3806 from the US Department of Agriculture CGO, and grant No. DE-FG0286ER13505 from the US Department of Energy.
1. DOUINE. L., QUIOT, J. B., MARCHOUX, G., and ARCHANGE, P., Ann. Phytopathol. 11) 439-475 (1979). 2. KAPER, J. M., and WATERWORTH, H. E., ln “Handbook of Plant Virus Infection and Comparative Diagnosis” (E. Kurstack, Ed.), pp. 257-332. Elsevier/North-Holland Biomedical, Amsterdam, 1981, 3. NITTA, N., TAKANAMI, Y., KUWATA, S., and Kueo, S., 1. Gen. Viral. 69, 2695-2700 (1988). 4. AHLQUIST, P., STRAUSS, E. G., RICE, C. M., STRAUSS,J. H., HASELOFF, J., and ZIMMERMAN, D., J. Viral. 53, 536-542 (1985). 5. REZAIAN, M. A., WILLIAMS, R. H. V., GOULD, A. R., and SYMONS, R. H., fur. J. Biochem. 143, 277-284 (1984). 6. REZAIAN, M. A., WILLIAMS, R. H. V., and SYMONS, R. H., fur. J. Biochem. 150, 331-339 (1985). 7. STUSSI-GARAUD,C., GARAUD,J. C., BERNA,A., and GODEFROY-COLBURN, T.. J. Gen. viral. 68, 1779-1784 (1987). 8. SCHWINGHAMER, M. W., and SYMONS, R. H., Virology 63, 252262 (1975). 9. GOULD, A. R., and SYMONS, R. H., fur. J. Biochem. 91,269-278 (1978). 10. FRITSCH, C., and MAYO, M. A., ln “Plant Viruses,” Vol. 1, “Structure and Replication” (C. L. Mandahar, Ed.), pp. 289-321. CRC Press, Boca Raton, FL, 1989. 11. KAPER, J. M., and WATERWORTH,H. E., Science 196, 429-431 (1977). 12. MOSSOP, 0. W., and FRANCKI, R. I. B., Virology 95, 395-404 (1979). 13. PALUKAITIS.P., Mol. Plant-Microbe Interact. 1, 175-l 81 (1988). 14. ROOSSINCK,M. J., and PALUKAITIS, P., Mol. P/ant-Microbe Interact. 3, 188-l 92 (1990). 15. GARCIA-ARENAL, F., ZAITLIN, M., and PALUKAITIS,P., Vjrology 158, 339-347 (1987). 16. PALUKAITIS. P., Corrs, S., and ZAITLIN, M., Acta Hortic. 154, 109-l 18 (1985). 17. PALUKAITIS,P., In “Methods in Enzymology” (A. Weissbach and H. Weissbach, Eds.), Vol. 118, pp. 723-742. Academic Press, San Diego, 1986. 18. PALUKAITIS,P., and ZAITLIN, M., Virology 132, 426-435 (1984). 19. LOT, H., MARROU, J., QUIOT, J. B.. and ESVANS, C., Ann. Phytopathol. 4, 25-38 (1972). 20. KURATH, G., and PALUKAITIS,P., Virology 173, 231-240 (1989). 21. KURATH, G., and PALUKAITIS,P., Mol. Plant-Microbe Interact. 2, 91-96 (1989). 22. HODGMAN, T. C., Nature (London) 333, 22-23 (1988). 23. GORBALENYA,A. E., and KOONIN, E. V., Nucleic Acids Res. 17, 8413-8440 (1989). 24. Rino, T. M., and PALUKAITIS,P., Mol. Gen. Genet. 222,249-256 (1990).
181, 371-373
(1991)
Differential
Replication in Zucchini Squash of a Cucumber Mosaic Virus Satellite RNA Maps to RNA 1 of the Helper Virus MARILYN Department
J. ROOSSINCK
of Plant Pathology,
Received September
AND PETER PALUKAITIS’
Cornell University,
2 1I 1990; accepted
Ithaca. New York 14853 November
6, 1990
Cucumber mosaic virus (CMV) supports the replication and encapsidation of its satellite RNA, both in solanaceous and cucurbit host plants: however, different strains of CMV support the replication of satellite RNAs with different efficiency. In addition, replication of satellite RNA is very efficient in solanaceous host plants and generally poor in cucurbit host plants. The WL,-satellite (WL,-sat) RNA is an exception, and replicates to high levels in both solanaceous and curcubit host plants with most CMV strains as the helper virus. Two strains of CMV were used in this study: Fny-CMV, which replicates the WL,-sat RNA efficiently in all hosts tested; and Sny-CMV, which does not replicate the WL,-sat RNA to detectable levels in zucchini squash (Cucurbita pepo), but does replicate WL,-sat RNA efficiently in other hosts. Using pseudorecombinants constructed between Fny-CMV and Sny-CMV we have mapped to RNA 1 the o 1991 Academic PMS. I~C. ability to support the efficient replication of WL,-sat RNA in zucchini squash.
effects are dependent on both the helper virus and the species of the infected plant. The majority of satellite RNAs found associated with various strains of CMV replicate with the helpervirus to high levels in solanaceous plants, such as tobacco. However, these satellite RNAs generally replicate poorly in cucurbits. A notable exception is the WL,-satellite (WL,-sat) RNA ( 73). With most helper virus strains of CMV, including the Fny-CMV strain, the WL,-sat RNA replicates efficiently in cucurbits (( 73) and our unpublished results). By contrast, the Sny-strain of CMV does not support the replication of WL,-sat RNA to a detectable level. In a previous study we described the construction of pseudorecombinants of the Fny- and Sny-CMV strains (74) which were used to map two pathogenicity domains to RNA 1. In this studywe report the use of these pseudorecombinants to map the domain responsible for the efficient replication of the WL,-sat RNA in zucchini squash (Cucurbita pepo, cv Black Beauty). Zucchini squash plants were inoculated at the cotyledon stage, using 100 @g/ml of viral RNA, with orwithout the addition of 5 pg/ml of gel-purified WL,-sat RNA (75) in 50 ml\/l sodium phosphate buffer, pH 9.2. A single preparation of each pseudorecombinant virus was used to inoculate plants, which were maintained in an environmentally controlled chamber with day temperatures of 23”, night temperatures of 19”, and a 16hr day. Leaf tissue was assessed by dot-blot hybridization for the presence of satellite RNA 10 to 14 days after
Cucumber mosaic cucumovirus (CMV) is a tripartite, RNA plant virus with a single stranded plus-sense genome. CMV infects a wide range of plants, encompassing nearly 800 species, and is of worldwide economic importance (7,Z). The CMV genome consists of three RNAs and four open reading frames (ORFs). RNAs 1 and 2 each contain one ORF, encoding proteins of 11 1 kDa (the la protein) and 97 kDa (the 2a protein), and are the only viral requirements for replication of the viral RNAs (3). These RNAs contain conserved regions found in all members of the Sindbis-like virus supergroup (4-6). RNA 3 contains two ORFs: the 5’ half of RNA 3 encodes a 30-kDa protein (the 3a protein) thought to be involved in viral movement (7) and the 3’half of RNA 3 generates a subgenomic RNA, RNA 4, which acts as a messenger for the viral coat protein (8, 9). In addition to the encapsidated genomic RNAs and the subgenomic RNA, virus particles often contain a satellite RNA of approximately 330 to 380 nucleotides (for review see Ref. (70)). By definition, the satellite RNA is dependent on the helper virus for both its replication and encapsidation. The CMV satellite RNA does not contain any demonstrably functional ORFs, nor does it show any extensive homology with the helper viral RNAs. Nevertheless, the presence of satellite RNA can result in a dramatic alteration in viral symptoms in CMV-infected plants, ranging from necrosis and death (7 7) to amelioration of disease (72). These symptom ’ To whom correspondence
should be addressed 371
0042-6822191
$3.00
Copyright 0 1991 by Academic Press. Inc All rights of reproduction I” any form reserved.
SHORT COMMUNICATIONS
372 TABLE 1
REPLICATIONOF THE WL, SATELLITERNA BY PSEUDORECOMBINANTS OF FNY- AND SNY-CMV RNAs Pseudorecombinante FSS SFF FSF SFS FFS SSF FFF sss
Number of sat (+) plants* (%) 12/12 3/68 12/12 5/l 00 12/l 2 O/60 18/18 l/65
(100) (4.4) (100) (5) (100) (0) (100) (1.5)
’ FSS indicates RNA 1 from Fny-CMV, RNA 2 from Sny-CMV, and RNA 3 from Sny-CMV. Other strains follow similar designations. b Number of plants which tested positive for CMV satellite RNA by dot-blot hybridization, as described in the text, over the total number of plants tested. The data are derived from two (SFS), three (FSS, FSF, FFS, and SSF), four (SFF), or five (SSS and FFF) seperate experiments.
inoculation. Tissue samples were taken from upper leaves, ground and extracted as previously described (16), and spotted onto nitrocellulose filters. Subsequently, the filters were either baked at 80” for 90 min or uv cross-linked by exposure to shortwave uv light for 45 set (Fotodyne UV transilluminator). Filters were then prehybridized as described (16), hybridized with approximately 4 X 1O6 cpm of 32P-labeled cDNA made to gel-purified G-sat RNA, in 4.5 ml of buffer (hybridization buffer II in Ref. (16)), and washed, priorto autoradiography ( 17). As indicated in Table 1, plants infected with pseudorecombinant strains containing RNA 1 from FnyCMV and RNAs 2 and 3 from either strain (i.e., FSS, FSF, FFS, and FFF), and WL,-sat RNA all showed 1OOYoinfection with WL,-sat RNA. Plants infected with RNA 1 from Sny-CMV and RNAs 2 and 3 from either strain (i.e., SFF, SFS, SSF, and SSS) generally contained no detectable WL,-sat RNA. However, in plants infected with either the SFS/WL,-sat RNA or SFFiWL,sat RNA combination, WL,-sat RNA was detected about 5% of the time, and one plant out of 65 infected with the SSSNVL,-sat RNA combination was also positive for satellite RNA by dot-blot hybridization. In the initial studies of WL,-sat RNA replication using these pseudorecombinants, we detected WL,-sat RNA about 309/o of the time with the SFF/WL,-sat RNA pseudorecombinant. When we purified viral RNA (18) from those plants which did not contain detectable WL,-sat RNA, we were able to segregate out an SFFCMV isolate which did not support these levels of WL-1 sat RNA replication. This isolate was used for the studies described here. Although it is possible that the original SFF-CMV combination contained low levels of RNA 1 from Fny-CMV, it seems more likely that the ability to
support WL,-sat RNA replication by the SFF-CMV pseudorecombinant was due to the heterogeneity of the original CMV-241 strain from which Sny-CMV was derived (14). The CMV-241 strain was able to support WL,-sat RNA replication in a low percentage of plants (data not shown). No detectable Fny-CMV RNA 1 was present in either SFF or SFS (14). Furthermore, the timing and severity of symptoms induced in zucchini squash by pseudorecombinants SFF- and SFS-CMV were not affected by repeated passage, as would have been expected if Fny-CMV RNA 1 was contaminating the strains. Since the detection of satellite RNA was limited by the sensitivity of the dot-blot hybridization technique, we could not be certain that there was no WL,-sat RNA replication occurring in the plants which tested negative for satellite RNA. To further test the possible replication of satellite RNA in zucchini squash plants inoculated with these strains, virus was purified from zucchini squash plants (19) that had been inoculated with SSF/WL,-sat RNA and SSS/WL,-sat RNA and were satellite RNA negative as determined by dot-blot hybridization. These viruses subsequently were inoculated onto young tobacco plants (Nicotiana tabacum cv Xanthi nc) at a concentration of 50 pg/ml in sodium phosphate buffer, pH 7.0. In tobacco plants even very low levels of satellite RNA are likely to be amplified. Ten days after inoculation tobacco plants inoculated with purified virus from the SSF/WL,-sat RNA- and SSS/WL,-sat RNA-infected zucchini squash plants tested positive for CMV satellite RNA by dot-blot hybridization (data not shown), indicating that the replication of WL,-sat RNA had been supported by these strains in zucchini squash, albeit at levels below the sensitivity of the dotblot detection system. To confirm that the satellite RNA from the infected tobacco plants was indeed WL,-sat RNA, these RNAs were analyzed by RNase protection assays (Fig. l), as previously described (20). The probe for the assay was a 32P-labeled, full-length, minussense, WL-sat RNA transcript, using EcoRI-digested pWLsat47 as template DNA, and T3 RNA polymerase; plus-sense control transcript was generated from the same plasmid, using Smal-digested DNA and T7 RNA polymerase as previously described (2 1). We have shown here that the WL,-sat RNA can replicate efficiently with Sny-CMV in tobacco, but not in zucchini squash. The poor replication of WL,-sat RNA in squash maps to RNA 1 of Sny-CMV. This implies a host-virus interaction involving RNA 1. Although RNA 1 is known to be required for replication of the viral RNAs and the satellite RNA (3) and contains both the conserved motifs of a helicase (22) as well as the properties of a nucleotide binding protein (23) in the carboxy terminal third of the 1a ORF, its precise role in replication is unknown. The domain(s) comprising approxi-
SHORT COMMUNICATIONS
373
REFERENCES SSFIWL
Tr
FIG. 1. Polyacrylamide gel electrophoretic analysis of RNase protection of CMV satellite RNAs. Each lane represents the protected fragments of the antisense WL,-sat probe, after annealing to viral RNA from plants infected with the indicated virus. Strains are as designated in footnote a to Table 1; Tr, in vitro generated plus sense transcript; C, control, no RNA.
mately two-thirds of the la protein at the amino terminus does not contain any significant homology to proteins which have had functions assigned to them; however, the amino terminal third of the la protein shows a high degree of homology with analogous proteins of the supergroup of Sindbis-like viruses (4). Although the efficiency of WL,-sat RNA replication maps to RNA 1, just as the previously described symptom differences between Fny- and Sny-CMVs mapped to RNA 1 (Id), the domain responsible for satellite RNA replication efficiency is most likely a different domain; i.e., several related strains of CMV either display rapidly appearing, severe symptoms, or slowly developing, mild symptoms, but the inefficiency of WL,-sat RNA replication is a property unique to the Sny-CMV strain. With the availability of cDNA clones of the Fny-CMV strain from which infectious RNA transcripts can be generated (24, we should be able to more precisely map this differential ability to replicate WL,-sat RNA by constructing recombinant viruses between the Fnyand Sny-CMV strains. ACKNOWLEDGMENTS This work was supported by grant No. 88-37263-3806 from the US Department of Agriculture CGO, and grant No. DE-FG0286ER13505 from the US Department of Energy.
1. DOUINE. L., QUIOT, J. B., MARCHOUX, G., and ARCHANGE, P., Ann. Phytopathol. 11) 439-475 (1979). 2. KAPER, J. M., and WATERWORTH, H. E., ln “Handbook of Plant Virus Infection and Comparative Diagnosis” (E. Kurstack, Ed.), pp. 257-332. Elsevier/North-Holland Biomedical, Amsterdam, 1981, 3. NITTA, N., TAKANAMI, Y., KUWATA, S., and Kueo, S., 1. Gen. Viral. 69, 2695-2700 (1988). 4. AHLQUIST, P., STRAUSS, E. G., RICE, C. M., STRAUSS,J. H., HASELOFF, J., and ZIMMERMAN, D., J. Viral. 53, 536-542 (1985). 5. REZAIAN, M. A., WILLIAMS, R. H. V., GOULD, A. R., and SYMONS, R. H., fur. J. Biochem. 143, 277-284 (1984). 6. REZAIAN, M. A., WILLIAMS, R. H. V., and SYMONS, R. H., fur. J. Biochem. 150, 331-339 (1985). 7. STUSSI-GARAUD,C., GARAUD,J. C., BERNA,A., and GODEFROY-COLBURN, T.. J. Gen. viral. 68, 1779-1784 (1987). 8. SCHWINGHAMER, M. W., and SYMONS, R. H., Virology 63, 252262 (1975). 9. GOULD, A. R., and SYMONS, R. H., fur. J. Biochem. 91,269-278 (1978). 10. FRITSCH, C., and MAYO, M. A., ln “Plant Viruses,” Vol. 1, “Structure and Replication” (C. L. Mandahar, Ed.), pp. 289-321. CRC Press, Boca Raton, FL, 1989. 11. KAPER, J. M., and WATERWORTH,H. E., Science 196, 429-431 (1977). 12. MOSSOP, 0. W., and FRANCKI, R. I. B., Virology 95, 395-404 (1979). 13. PALUKAITIS.P., Mol. Plant-Microbe Interact. 1, 175-l 81 (1988). 14. ROOSSINCK,M. J., and PALUKAITIS, P., Mol. P/ant-Microbe Interact. 3, 188-l 92 (1990). 15. GARCIA-ARENAL, F., ZAITLIN, M., and PALUKAITIS,P., Vjrology 158, 339-347 (1987). 16. PALUKAITIS. P., Corrs, S., and ZAITLIN, M., Acta Hortic. 154, 109-l 18 (1985). 17. PALUKAITIS,P., In “Methods in Enzymology” (A. Weissbach and H. Weissbach, Eds.), Vol. 118, pp. 723-742. Academic Press, San Diego, 1986. 18. PALUKAITIS,P., and ZAITLIN, M., Virology 132, 426-435 (1984). 19. LOT, H., MARROU, J., QUIOT, J. B.. and ESVANS, C., Ann. Phytopathol. 4, 25-38 (1972). 20. KURATH, G., and PALUKAITIS,P., Virology 173, 231-240 (1989). 21. KURATH, G., and PALUKAITIS,P., Mol. Plant-Microbe Interact. 2, 91-96 (1989). 22. HODGMAN, T. C., Nature (London) 333, 22-23 (1988). 23. GORBALENYA,A. E., and KOONIN, E. V., Nucleic Acids Res. 17, 8413-8440 (1989). 24. Rino, T. M., and PALUKAITIS,P., Mol. Gen. Genet. 222,249-256 (1990).