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A temperature-sensitive mutant of TMV selected at high temperatures
VIROLOGY
41, 389-391
(1970)
A Temperature-Sensitive
Mutant
of TMV Selected
Earlier work in this laboratory indicated that defective strains may be generated or selected from tobacco mosaic virus (TMV)infected tobacco plants grown at high temperatures (1). In this study a strain, HT-4, was selected which proved to be temperature-sensitive. This communication describes the method of isolation and some characterist,ics of this strain. One or two leaves of a number of young tobacco (Sicotiana tabacum L. var. Samsun) plants were inoculated with a purified solution of TMV U1 strain (2) at lOA mg/ ml. After 1 week the plants were transferred to a temperature-controlled growth chamber maintained at approximately 35”. Two to three weeks after moving to the higher temperature, some of the uninoculat’ed leaves near the apex developed a yellow oak-leaf pattern similar to that described for defective strains (3). Tissue showing good oak-leaf patterns was used to separate free viral RNA from virus rods. This was done since the chances of isolating a defective strain are greater if the free RNA is used for isolation purposes. It is known that defective strains of TnIV do not produce virus particles in infected tissue since the defective coat protein synthesized is unable to coat the RNA (3), and many temperature-sensitive strains produce coat prot,eins incapable of coating RNA in infected plants grown at high temperatures (4, 5). The method of Diener (6) was used with modifications to separate free viral RSA from virus particles. The separated RNA was used in attempts to isolate defective strains. Twenty grams of tissue showing good oak-leaf patterns was ground in 3 volumes of GPS buffer (0.1 M glycine, 0.05 M KH, POa, 0.3 d/ sodium chloride, adjusted to pH 9.5) (6) and 3 volumes of chloroform. After a low speed centrifugation, the aqueous supernatant solution was centrifuged at 105,OOOgfor GO minutes and
at High Temperatures
the pellets resuspended in 1 ml of GPS buffer. This was clarified at 60009 for 10 minutes and the pellet resuspended in 2 ml GPS buffer and centrifuged again at SOOOg for 10 minutes. The two 60009 supernatant solutions, which at this stage contained both virus and free viral RXA, were combined. To separate the free infectious RNA from the virus, 0.7 ml of this solution was centrifuged through a 3.9.ml cushion of 20 % sucrose at 105,OOOg for 60 minutes. The virus pelleted, and the other materials, including free infectious RNA, hardly sedimented even a short distance into the SUcrose layer. The top 1 ml of the centrifuged solution, containing 0.7 ml of the solution applied on the gradient and 0.3 ml of sucrose solution, was mixed with 2 ml of Trisphosphate buffer (TP) pH 8.6 (7) and rubbed on Xicotiana tabacum 1,. var. Santhi CC. No doubt the material from the centrifuged solution contained material other than viral RSA, but only the infectious RNA would produce lesions on the local lesion host. The lesions that developed were cut out soon after they had appeared, ground in 2-3 drops of TP buffer (7), and transferred to young tobacco plants. It has been observed that infectivity in lesions due to defective strains can also be transferred from young lesions (J. J. Hubert, unpublished data). A total of 101 lesions were transferred from two such experiments. Of these, 40 plant’s had symptoms similar to that induced by the common strain, 14 had different symptoms, and the rest appeared uninfected. Three of the 14 that were different, HT-4, HT-11, and HT-13, showed yellowing, vein clearing, and oakleaf patterns. The coat protein composition of the three isolates was the same, and hence these are all probably different isolates of the same strain. The two lower leaves of 10 young tobacco plants were inoculated with HT-4 at 1W4 mg/ml, and five were maintained at 23’ 389
390
SHORT
COMMUNICATIONS
and five at 35”. Three and 7 days after inoculation, 2-3 g of the inoculated leaves mere ground in 10 ml of 0.066 dl phosphate bufer pH 7.0. The extract was tested for infectivity by local lesion assay on X. tabacum L. var. Xanthi n.c. Assays were done at different dilutions. The lesion numbers were converted for dilution and expressed as lesions per half-leaf. HT-4 produced large amounts of virus when the plants were grown at 23”. However, at 35” the infected plants produced very little virus as tested by infectivity measurements (Table 1). It has already been demonstrated that with the common strain at 35” about 50% as much virus is produced as at 23” (4), and this has also been our own experience. But, it is evident that HT-4 produced only about. 2% as much virus at 35” as at 23” when assayed 3 days after infection and only 0.8 % as much when assayed 7 days after infection (Table 1). Virus (HT-4) from plants grown at 23” was purified by the polyethylene glycol procedure (8). The virus protein prepared by the acetic acid procedure (9) was analyzed for amino acid composition by methods described earlier (10). The coat protein composition shows three changes from strain G-TAMV (11,12) and two changes from the strains isolated after chemical treatment of the common strain at the Virus Laboratory, University of California, Berkeley (12) (Table 2). The temperature sensitivity of the HT-4 coat protein was determined on the basis TABLE
1
EFFECT OF TEMPERATURE ON INFECTIVITY OF HT-4 EXPRESSED AS LESION NUMBERS PER HALF-LEAF OF Xc&hi rz.c., AVERAGE OF 12 HALF-LEAVES Lesion numbers Tested 23” 3 Days after inoculat,ion temperature treatment 7 Days after inoculation temperature treatment
l/l
a Lesion numbers tissue dilution.
35”
and
400
8
and
1212
10
expressed
after
correction
to
TABLE
2
AMINO ACID COMPOSITION OF HT-4 PROTEISn Moles amino acid per mole protein *-e& 24.HOW '/Z-Hour Integral TMV GTAM\;b residue hydrolysis hydrolysis value sty&, Asp Thr Ser Glu Pro GUY Ala CYS Val Met Ile Leu Tyr Trp Phe Aw LYS
22.7 18.4 9.04 16 9.31 4.82 16.76 0.820 11.32 1.61 6.54 10.86 5.8G
20.7 16.2 7.67 16 10.13 4.59 15.88 10.92 1.90 5.77 10.45 5.61
7.96 8.03 1.06
7.48 7.58 1.00
23 20c 1oc lBd 10 5 17 le 11 2 7 11 6 2J 8 8 1
18 16 16 16 8 G 14 1 14 9 12 4 3 8 11 2
22 19 10 10 10 4 18 1 12 2 8 11 G 2 8 8 1
$tg 223
22 19 10 16 10 5 17 1 12 2 8 11 0 2 8 8 1
a The values represent average of three replicates. b From Tsugita (1962). c Obtained by extrapolation to zero time of hydrolysis. d Values based on 16 for Glu. e From separate runs of performic acid-oxidized protein. f Determined from UV absorbance spectrophotometry.
of the ease with which the protein denatured on heating at 35”. The purified coat protein in 0.066 M pH 7.0 phosphate buffer at about 1 mg/ml was heated at 35” for different periods of time. The samples were centrifuged at 12,OOOgfor 10 minutes when the flocculated, heat-denatured protein pelleted and the supernatant liquids containing undenatured coat protein were mixed with 4 volumes of 0.1 i\r NaOH. The protein content in the supernatant liquid was determined by spectrophotometry and expressed as percentage of the total. Controls were run with coat protein of the common strain U1. The temperature-sensitive nature of HT.4 coat protein is evident (Fig. 1). The isolation of temperature-sensitive strains from plants grown at high tempera-
SHORT
I
15
I
30
I
60
I
I
120
-
U,'A'
PROTEIN
-
HT'4'
PROTEIN
180 TIME
FIG.
391
COMMUNICSTIONS
ACKNOWLEDGMENTS I thank Dr. Milton Zaitlin and Dr. Albert Siegel for encouragement and critical perusal of t~he manllscript. The competent assistance of Fran Almli and Ruth Smith is gratefully appreciated. These studies were supported by a grant, from the Nat,ional Science Foundation and by Atomic Energy Commission Contract AT(ll-l-873. Arizona Agricultural Experiment Station Technical Paper No. 1607.
1
300
IN MINUTES
1. Effect of temperature (35”) on the denaturation of HT4
tures requires some comment. It is natural to expect that selection would be against such strains at high temperatures. However, the method of selection used here would pick up strains which produce free viral RNA in infected tissue. Many temperaturesensitive strains essentially produce only free viral RNA at nonpermissive temperatures (4). The similarity of the coat protein composition of HT-4 with G-TAMV and other strains (11, 12) suggests that these strains may also prove to be temperature-sensitive. Like G-TAMV, HT-4 produces local lesions on ,J;. sylvestris and does not infect tomato plants.
I
240
coat protein as a function of time. REFERENCES
1. HARIHARASUBRAMANIAN,
V., and ZAITLIN, M., Virology 36, 521623 (1968). 2. SIEGEL, A., and WILDMAN, S. G., Phytopathology 44, 277-282 (1954). 3. SIEGEL, A., ZAITLIN, M., and SEHGAL, 0. P., Proc. Nat. Acad. Sci. U.S. 48, 1845-1851 (1962). .4. JOCKUSCH, H., Virology 35, 94-101 (1968). 5. H.~RIHARA~UBRAMANIAN, V., ZAITLIN, M., and SIEGEL, A., Virology 40, 579-589 (1970). 6. DIENER, T. O., Virology 16, 140-146 (1962). 7. SARICAR, S., Virology 20, 185-193 (1963). 8. GOODING, G. V., JR., and HEBERT, T. T., Phytopathology 57, 1285 (1967). 9. FRSENKEL-CONRAT, H., viirology 4, l-4 (1957). 10. Z~ITLIN, M.,and MCCAUGHEY, W.F., Virology 26, 500-503 (1965). 11. KNIGHT, C. A., SILVA, D. M., D~HL, D., and TSUGIT.~, A., Virology 16, 236-243 (1962). 12. TSUGITA, A., J. ;lfol. Biol. 5, 293-300 (1962). v. HARIHIRASUBRA3fANIAN Department of Agricultural Biochemistry University of Arizona Tucson, Arizona 867’21 Accepted April 11, 19’70
41, 389-391
(1970)
A Temperature-Sensitive
Mutant
of TMV Selected
Earlier work in this laboratory indicated that defective strains may be generated or selected from tobacco mosaic virus (TMV)infected tobacco plants grown at high temperatures (1). In this study a strain, HT-4, was selected which proved to be temperature-sensitive. This communication describes the method of isolation and some characterist,ics of this strain. One or two leaves of a number of young tobacco (Sicotiana tabacum L. var. Samsun) plants were inoculated with a purified solution of TMV U1 strain (2) at lOA mg/ ml. After 1 week the plants were transferred to a temperature-controlled growth chamber maintained at approximately 35”. Two to three weeks after moving to the higher temperature, some of the uninoculat’ed leaves near the apex developed a yellow oak-leaf pattern similar to that described for defective strains (3). Tissue showing good oak-leaf patterns was used to separate free viral RNA from virus rods. This was done since the chances of isolating a defective strain are greater if the free RNA is used for isolation purposes. It is known that defective strains of TnIV do not produce virus particles in infected tissue since the defective coat protein synthesized is unable to coat the RNA (3), and many temperature-sensitive strains produce coat prot,eins incapable of coating RNA in infected plants grown at high temperatures (4, 5). The method of Diener (6) was used with modifications to separate free viral RSA from virus particles. The separated RNA was used in attempts to isolate defective strains. Twenty grams of tissue showing good oak-leaf patterns was ground in 3 volumes of GPS buffer (0.1 M glycine, 0.05 M KH, POa, 0.3 d/ sodium chloride, adjusted to pH 9.5) (6) and 3 volumes of chloroform. After a low speed centrifugation, the aqueous supernatant solution was centrifuged at 105,OOOgfor GO minutes and
at High Temperatures
the pellets resuspended in 1 ml of GPS buffer. This was clarified at 60009 for 10 minutes and the pellet resuspended in 2 ml GPS buffer and centrifuged again at SOOOg for 10 minutes. The two 60009 supernatant solutions, which at this stage contained both virus and free viral RXA, were combined. To separate the free infectious RNA from the virus, 0.7 ml of this solution was centrifuged through a 3.9.ml cushion of 20 % sucrose at 105,OOOg for 60 minutes. The virus pelleted, and the other materials, including free infectious RNA, hardly sedimented even a short distance into the SUcrose layer. The top 1 ml of the centrifuged solution, containing 0.7 ml of the solution applied on the gradient and 0.3 ml of sucrose solution, was mixed with 2 ml of Trisphosphate buffer (TP) pH 8.6 (7) and rubbed on Xicotiana tabacum 1,. var. Santhi CC. No doubt the material from the centrifuged solution contained material other than viral RSA, but only the infectious RNA would produce lesions on the local lesion host. The lesions that developed were cut out soon after they had appeared, ground in 2-3 drops of TP buffer (7), and transferred to young tobacco plants. It has been observed that infectivity in lesions due to defective strains can also be transferred from young lesions (J. J. Hubert, unpublished data). A total of 101 lesions were transferred from two such experiments. Of these, 40 plant’s had symptoms similar to that induced by the common strain, 14 had different symptoms, and the rest appeared uninfected. Three of the 14 that were different, HT-4, HT-11, and HT-13, showed yellowing, vein clearing, and oakleaf patterns. The coat protein composition of the three isolates was the same, and hence these are all probably different isolates of the same strain. The two lower leaves of 10 young tobacco plants were inoculated with HT-4 at 1W4 mg/ml, and five were maintained at 23’ 389
390
SHORT
COMMUNICATIONS
and five at 35”. Three and 7 days after inoculation, 2-3 g of the inoculated leaves mere ground in 10 ml of 0.066 dl phosphate bufer pH 7.0. The extract was tested for infectivity by local lesion assay on X. tabacum L. var. Xanthi n.c. Assays were done at different dilutions. The lesion numbers were converted for dilution and expressed as lesions per half-leaf. HT-4 produced large amounts of virus when the plants were grown at 23”. However, at 35” the infected plants produced very little virus as tested by infectivity measurements (Table 1). It has already been demonstrated that with the common strain at 35” about 50% as much virus is produced as at 23” (4), and this has also been our own experience. But, it is evident that HT-4 produced only about. 2% as much virus at 35” as at 23” when assayed 3 days after infection and only 0.8 % as much when assayed 7 days after infection (Table 1). Virus (HT-4) from plants grown at 23” was purified by the polyethylene glycol procedure (8). The virus protein prepared by the acetic acid procedure (9) was analyzed for amino acid composition by methods described earlier (10). The coat protein composition shows three changes from strain G-TAMV (11,12) and two changes from the strains isolated after chemical treatment of the common strain at the Virus Laboratory, University of California, Berkeley (12) (Table 2). The temperature sensitivity of the HT-4 coat protein was determined on the basis TABLE
1
EFFECT OF TEMPERATURE ON INFECTIVITY OF HT-4 EXPRESSED AS LESION NUMBERS PER HALF-LEAF OF Xc&hi rz.c., AVERAGE OF 12 HALF-LEAVES Lesion numbers Tested 23” 3 Days after inoculat,ion temperature treatment 7 Days after inoculation temperature treatment
l/l
a Lesion numbers tissue dilution.
35”
and
400
8
and
1212
10
expressed
after
correction
to
TABLE
2
AMINO ACID COMPOSITION OF HT-4 PROTEISn Moles amino acid per mole protein *-e& 24.HOW '/Z-Hour Integral TMV GTAM\;b residue hydrolysis hydrolysis value sty&, Asp Thr Ser Glu Pro GUY Ala CYS Val Met Ile Leu Tyr Trp Phe Aw LYS
22.7 18.4 9.04 16 9.31 4.82 16.76 0.820 11.32 1.61 6.54 10.86 5.8G
20.7 16.2 7.67 16 10.13 4.59 15.88 10.92 1.90 5.77 10.45 5.61
7.96 8.03 1.06
7.48 7.58 1.00
23 20c 1oc lBd 10 5 17 le 11 2 7 11 6 2J 8 8 1
18 16 16 16 8 G 14 1 14 9 12 4 3 8 11 2
22 19 10 10 10 4 18 1 12 2 8 11 G 2 8 8 1
$tg 223
22 19 10 16 10 5 17 1 12 2 8 11 0 2 8 8 1
a The values represent average of three replicates. b From Tsugita (1962). c Obtained by extrapolation to zero time of hydrolysis. d Values based on 16 for Glu. e From separate runs of performic acid-oxidized protein. f Determined from UV absorbance spectrophotometry.
of the ease with which the protein denatured on heating at 35”. The purified coat protein in 0.066 M pH 7.0 phosphate buffer at about 1 mg/ml was heated at 35” for different periods of time. The samples were centrifuged at 12,OOOgfor 10 minutes when the flocculated, heat-denatured protein pelleted and the supernatant liquids containing undenatured coat protein were mixed with 4 volumes of 0.1 i\r NaOH. The protein content in the supernatant liquid was determined by spectrophotometry and expressed as percentage of the total. Controls were run with coat protein of the common strain U1. The temperature-sensitive nature of HT.4 coat protein is evident (Fig. 1). The isolation of temperature-sensitive strains from plants grown at high tempera-
SHORT
I
15
I
30
I
60
I
I
120
-
U,'A'
PROTEIN
-
HT'4'
PROTEIN
180 TIME
FIG.
391
COMMUNICSTIONS
ACKNOWLEDGMENTS I thank Dr. Milton Zaitlin and Dr. Albert Siegel for encouragement and critical perusal of t~he manllscript. The competent assistance of Fran Almli and Ruth Smith is gratefully appreciated. These studies were supported by a grant, from the Nat,ional Science Foundation and by Atomic Energy Commission Contract AT(ll-l-873. Arizona Agricultural Experiment Station Technical Paper No. 1607.
1
300
IN MINUTES
1. Effect of temperature (35”) on the denaturation of HT4
tures requires some comment. It is natural to expect that selection would be against such strains at high temperatures. However, the method of selection used here would pick up strains which produce free viral RNA in infected tissue. Many temperaturesensitive strains essentially produce only free viral RNA at nonpermissive temperatures (4). The similarity of the coat protein composition of HT-4 with G-TAMV and other strains (11, 12) suggests that these strains may also prove to be temperature-sensitive. Like G-TAMV, HT-4 produces local lesions on ,J;. sylvestris and does not infect tomato plants.
I
240
coat protein as a function of time. REFERENCES
1. HARIHARASUBRAMANIAN,
V., and ZAITLIN, M., Virology 36, 521623 (1968). 2. SIEGEL, A., and WILDMAN, S. G., Phytopathology 44, 277-282 (1954). 3. SIEGEL, A., ZAITLIN, M., and SEHGAL, 0. P., Proc. Nat. Acad. Sci. U.S. 48, 1845-1851 (1962). .4. JOCKUSCH, H., Virology 35, 94-101 (1968). 5. H.~RIHARA~UBRAMANIAN, V., ZAITLIN, M., and SIEGEL, A., Virology 40, 579-589 (1970). 6. DIENER, T. O., Virology 16, 140-146 (1962). 7. SARICAR, S., Virology 20, 185-193 (1963). 8. GOODING, G. V., JR., and HEBERT, T. T., Phytopathology 57, 1285 (1967). 9. FRSENKEL-CONRAT, H., viirology 4, l-4 (1957). 10. Z~ITLIN, M.,and MCCAUGHEY, W.F., Virology 26, 500-503 (1965). 11. KNIGHT, C. A., SILVA, D. M., D~HL, D., and TSUGIT.~, A., Virology 16, 236-243 (1962). 12. TSUGITA, A., J. ;lfol. Biol. 5, 293-300 (1962). v. HARIHIRASUBRA3fANIAN Department of Agricultural Biochemistry University of Arizona Tucson, Arizona 867’21 Accepted April 11, 19’70