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Effects of two protein-defective strains of TMV on the stability of chloroplast ribosomal RNA
VIHOLOG’r
60,
592-594 (1974)
Effects of Two Protein-Defective Stability
of Chloroplast WOLFGANG
Max-Planck-Institut
fuer Biologic.
Strains of TMV on the Ribosomal
SCHUCH’
Abtl. Melchrrs.
Accepted
RNA
74 Tuebingen,
West German?,
May 9, 197-I
The effect of two protein-defective mutant strains ofTMY on rRNA contents of infected leaves of tobacco was investigated. PM, and DT-1 reduced drasticall? the amount of chloroplast rRNA after infertion. whereas the amount of cytoplasmic rRKA was not affected.
Hirai and Wildman (I) found that the synthesis of chloroplast. ribosomal RNA (c-rRNA) and chloroplast proteins is severely inhibited after infection of tobacco leaves with TMV vuigare. Fraser (2, 3) described the effect of vulgare and flavum strains of TMV on c-rRNA stability and synthesis. The severe yellow mosaic-producing strain flavum causes a greater breakdown of c-rRNA than the light-green dark-green mosaic strain vulgare; both strains inhibit c-rRNA synthesis; flavum, however, exerts a stronger inhibition. Both strains produce large amounts of viral coat protein and viral RNA (3). But part of the flavum protein is denatured. Thus, it might cause the physical disruption of chloroplasts which subsequently will lead to c-rRNA and chlorophyll degradation and the severe symptoms. Jockusch and Jockusch (4) argued that the relative proportion of insoluble coat protein causes the severity of the cytopathic effect. Fraser (2), however, argued that the appearance of symptoms might not be solely caused by the presence of insoluble coat protein and that the differing effects of vulgare and flavum on c-rRNA stability and synthesis, chlorophyll breakdown and symptom formation might result from dif’ Present address: Department of Zoology. University of Edinhurgh. West Mains Road. Edinhurgh EH9 3JT, Scotland.
fering metabolic control by the virus genames of the host cell metabolism. I have examined the possible influence of defective virus coat protein on c-rRNA stability by investigating the effects produced by two protein-defective mutants. PM, (5) and DT-1 (Dr. S. Sarkar, personal communication) produce small amounts of infective viral RNA. PM, synthesizes a soluble coat protein which has lost its capacity to associate with TMV RNA because of an aberrant mode of aggregation (6). DT-1 produces insoluble coat protein (Dr. S. Sarkar, personal communication). Both strains give rise to yellow symptoms on Nicotiana tobacum var. Samsun. The symptoms of DT-1 are more severe than those of PM, and appear about 2.5X days after inoculation under our conditions. PM, produces smaller yellow areas after about 4 days after infection. Systemic infections were performed on Nicotiana tabacum var. Samsun according to Sarkar (7). In order to get true random samples for RNA and chlorophyll determinations, leaves were frozen in solid CO,, then crumbled and aliquots taken for analysis. RNA was prepared from 0.2 g leaf material which was homogenized and extracted by a modification of a buffer-detergent-phenol method (8). This method gives a high reproducibility and efficiency of extraction. RNA samples were analyzed
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on 2.2% polyacrylamide gels for 3 hr (9). The areas under the peaks of the optical scans were determined and the RNA contents calculated from them. Chlorophyll content of leaves was determined according to Comar and Zscheile (10). Leaves were infected when they were about 8-10 cm long. They allow maximal rate of virus multiplication at this stage (3). Leaf content of cytoplasmic and chloroplast rRNA and chlorophyll were determined at various times after infection. Figure 1 a shows changes in c-rRNA content after inoculation. Control leaves lost c-rRNA; this is a normal consequence of leaf senescence (3). During the second day after infection, virus-infected leaves began to lose c-t-RNA faster than control leaves. The rate of c-rRNA breakdown was faster in DT-l-infected leaves than in PM,infected samples. Control leaves also lost cytoplasmic rRNA (Fig. 1 b). This, as well, is a normal event during senescence. Virus-infected leaves lost cytoplasmic rRNA at the same rate as control leaves. Figure 1 c shows changes in chlorophyll content of leaves after infection. The chlorophyll content of control leaves decreased as the leaves aged. The chlorophyll content of infected leaves was the same as that of the control leaves for 3 days after inoculation, but after the third day after infection, there was a rapid loss of chlorophyll in infected leaves. This coincided with the appearance of visible symptoms. In DT-linfected leaves, however, the symptoms occur at least half a day before the detection of the accelerated loss of chlorophyll. The effect of both PM, and DT-I strains of TMV on c-rRXA stability was more severe than the one exerted by vulgare and flavum strains of TMV (3), although the latter strains synthesize about 20-40 times more viral RNA. The rapid loss of c-rRNA in PM, and DT-1 infected leaves began 2 days before the onset of accelerated loss of chlorophyll. If chloroplast disruption was one of the first events to take place during symptom formation, c-rRNA degradation and accelerated loss of chlorophyll might be expected to occur simultaneously. It is
593
FIG. 1. Changes in c-rRNA (al, rRNA (b). and chlorophyll (c) content of tobacco leaves after inl’ection with either III-1 or PM, strains of’TMV. RNA and chlorophyll were extracted as described in the text. c-rRNA, rRNA, and chlorophyll content were calculated per gram fresh weight of leaf’ material. Control, uninfected (O--O); DT-l-inf’ected leaves (O---O); PM,-int’ected leaves (A--AI.
also unlikely that viral coat protein is involved in the physical disruption of chloroplasts at the time of comencement of c-rRNA breakdown, as at this time the bulk of the viral protein is not yet synthesized. While c-rRNA is broken down, cytoplasmic rRNA stability is not affected. The mechanism by which the viral genome might control the preferential breakdown of c-rRNA is not yet known. A final test of the extent to which viral protein is involved in symptom formation and crRNA breakdown would be possible with a mutant of TMV which completely fails to synthesize coat protein.
594
SHORT
COMMUNICATIONS
ACKNOWLEDGMENTS I thank Dr. R. S. S. Fraser for his help in preparing the manuscript. and the Max-Plan&Gesellschaft for financial support. REFERENCES 1. HIRAI. A.. and ~‘ILDMA~;, S. G.. Virology 38, 7X32 (1969). 2. FRASER, K. S. S.. Mol. Gen. Genet. 106, 73-79 (1969). 3. FRASER. K. S. S., Virology 47, 261-269 (197%).
4. JOCKUSCH, H., and JOCKUSCH, B., Mol. Gen. Genet. 102, 204-209 (1968). 5. SIEGEL, A.. ZAITLIN, M., and SEGHAL, 0. P., Froc. Nat. Acad. Sci. USA 48, 1845-1851 (1962). 6. ZAITLIN, M., and FERRIS, W. R., Science 143, 1451-1452 (1964). 7. SAHKAR. S.. Z. Vererbungsl. 97, 166-185 (1965). 8. FRASER, R. S. S., Virology 45, 804-807 (1971). 9. I,OENING. U. E:.. Hiochem. J. 107, 251-257 (1967). 10. COMAR, C. L., and ZSCHEIL~ F. P., Plant Physiol. 17, 198-209 (1942).
60,
592-594 (1974)
Effects of Two Protein-Defective Stability
of Chloroplast WOLFGANG
Max-Planck-Institut
fuer Biologic.
Strains of TMV on the Ribosomal
SCHUCH’
Abtl. Melchrrs.
Accepted
RNA
74 Tuebingen,
West German?,
May 9, 197-I
The effect of two protein-defective mutant strains ofTMY on rRNA contents of infected leaves of tobacco was investigated. PM, and DT-1 reduced drasticall? the amount of chloroplast rRNA after infertion. whereas the amount of cytoplasmic rRKA was not affected.
Hirai and Wildman (I) found that the synthesis of chloroplast. ribosomal RNA (c-rRNA) and chloroplast proteins is severely inhibited after infection of tobacco leaves with TMV vuigare. Fraser (2, 3) described the effect of vulgare and flavum strains of TMV on c-rRNA stability and synthesis. The severe yellow mosaic-producing strain flavum causes a greater breakdown of c-rRNA than the light-green dark-green mosaic strain vulgare; both strains inhibit c-rRNA synthesis; flavum, however, exerts a stronger inhibition. Both strains produce large amounts of viral coat protein and viral RNA (3). But part of the flavum protein is denatured. Thus, it might cause the physical disruption of chloroplasts which subsequently will lead to c-rRNA and chlorophyll degradation and the severe symptoms. Jockusch and Jockusch (4) argued that the relative proportion of insoluble coat protein causes the severity of the cytopathic effect. Fraser (2), however, argued that the appearance of symptoms might not be solely caused by the presence of insoluble coat protein and that the differing effects of vulgare and flavum on c-rRNA stability and synthesis, chlorophyll breakdown and symptom formation might result from dif’ Present address: Department of Zoology. University of Edinhurgh. West Mains Road. Edinhurgh EH9 3JT, Scotland.
fering metabolic control by the virus genames of the host cell metabolism. I have examined the possible influence of defective virus coat protein on c-rRNA stability by investigating the effects produced by two protein-defective mutants. PM, (5) and DT-1 (Dr. S. Sarkar, personal communication) produce small amounts of infective viral RNA. PM, synthesizes a soluble coat protein which has lost its capacity to associate with TMV RNA because of an aberrant mode of aggregation (6). DT-1 produces insoluble coat protein (Dr. S. Sarkar, personal communication). Both strains give rise to yellow symptoms on Nicotiana tobacum var. Samsun. The symptoms of DT-1 are more severe than those of PM, and appear about 2.5X days after inoculation under our conditions. PM, produces smaller yellow areas after about 4 days after infection. Systemic infections were performed on Nicotiana tabacum var. Samsun according to Sarkar (7). In order to get true random samples for RNA and chlorophyll determinations, leaves were frozen in solid CO,, then crumbled and aliquots taken for analysis. RNA was prepared from 0.2 g leaf material which was homogenized and extracted by a modification of a buffer-detergent-phenol method (8). This method gives a high reproducibility and efficiency of extraction. RNA samples were analyzed
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COMMUNICATIONS
on 2.2% polyacrylamide gels for 3 hr (9). The areas under the peaks of the optical scans were determined and the RNA contents calculated from them. Chlorophyll content of leaves was determined according to Comar and Zscheile (10). Leaves were infected when they were about 8-10 cm long. They allow maximal rate of virus multiplication at this stage (3). Leaf content of cytoplasmic and chloroplast rRNA and chlorophyll were determined at various times after infection. Figure 1 a shows changes in c-rRNA content after inoculation. Control leaves lost c-rRNA; this is a normal consequence of leaf senescence (3). During the second day after infection, virus-infected leaves began to lose c-t-RNA faster than control leaves. The rate of c-rRNA breakdown was faster in DT-l-infected leaves than in PM,infected samples. Control leaves also lost cytoplasmic rRNA (Fig. 1 b). This, as well, is a normal event during senescence. Virus-infected leaves lost cytoplasmic rRNA at the same rate as control leaves. Figure 1 c shows changes in chlorophyll content of leaves after infection. The chlorophyll content of control leaves decreased as the leaves aged. The chlorophyll content of infected leaves was the same as that of the control leaves for 3 days after inoculation, but after the third day after infection, there was a rapid loss of chlorophyll in infected leaves. This coincided with the appearance of visible symptoms. In DT-linfected leaves, however, the symptoms occur at least half a day before the detection of the accelerated loss of chlorophyll. The effect of both PM, and DT-I strains of TMV on c-rRXA stability was more severe than the one exerted by vulgare and flavum strains of TMV (3), although the latter strains synthesize about 20-40 times more viral RNA. The rapid loss of c-rRNA in PM, and DT-1 infected leaves began 2 days before the onset of accelerated loss of chlorophyll. If chloroplast disruption was one of the first events to take place during symptom formation, c-rRNA degradation and accelerated loss of chlorophyll might be expected to occur simultaneously. It is
593
FIG. 1. Changes in c-rRNA (al, rRNA (b). and chlorophyll (c) content of tobacco leaves after inl’ection with either III-1 or PM, strains of’TMV. RNA and chlorophyll were extracted as described in the text. c-rRNA, rRNA, and chlorophyll content were calculated per gram fresh weight of leaf’ material. Control, uninfected (O--O); DT-l-inf’ected leaves (O---O); PM,-int’ected leaves (A--AI.
also unlikely that viral coat protein is involved in the physical disruption of chloroplasts at the time of comencement of c-rRNA breakdown, as at this time the bulk of the viral protein is not yet synthesized. While c-rRNA is broken down, cytoplasmic rRNA stability is not affected. The mechanism by which the viral genome might control the preferential breakdown of c-rRNA is not yet known. A final test of the extent to which viral protein is involved in symptom formation and crRNA breakdown would be possible with a mutant of TMV which completely fails to synthesize coat protein.
594
SHORT
COMMUNICATIONS
ACKNOWLEDGMENTS I thank Dr. R. S. S. Fraser for his help in preparing the manuscript. and the Max-Plan&Gesellschaft for financial support. REFERENCES 1. HIRAI. A.. and ~‘ILDMA~;, S. G.. Virology 38, 7X32 (1969). 2. FRASER, K. S. S.. Mol. Gen. Genet. 106, 73-79 (1969). 3. FRASER. K. S. S., Virology 47, 261-269 (197%).
4. JOCKUSCH, H., and JOCKUSCH, B., Mol. Gen. Genet. 102, 204-209 (1968). 5. SIEGEL, A.. ZAITLIN, M., and SEGHAL, 0. P., Froc. Nat. Acad. Sci. USA 48, 1845-1851 (1962). 6. ZAITLIN, M., and FERRIS, W. R., Science 143, 1451-1452 (1964). 7. SAHKAR. S.. Z. Vererbungsl. 97, 166-185 (1965). 8. FRASER, R. S. S., Virology 45, 804-807 (1971). 9. I,OENING. U. E:.. Hiochem. J. 107, 251-257 (1967). 10. COMAR, C. L., and ZSCHEIL~ F. P., Plant Physiol. 17, 198-209 (1942).