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Confirmation of the constitution of alfalfa mosaic virus hybrid genomes by backcross experiments
VIROLOGY
69, 328-330
Confirmation
(1974)
of the Constitution Genomes
ANNEMIEKE
of Alfalfa
by Backcross
Mosaic
Virus
Hybrid
Experiments
DINGJAN-VERSTEEGH, LOUS VAN VLOTEN-DOTING AND E. M. J. JASPARS
Department
of Biochemistry, Leiden,
State University, The Netherlands
Accepted January
Wassenaarseweg 64,
16, 1974
Components of two types of hybrid strains of alfalfa mosaic virus were purified. Single lesions induced by inocula of each of these components, to which the two eomplementary components of the homologous parent strain were added, yielded progenies indistinguishable from the parents with regard to serology, component composition and symptoms on tobacco and bean. This strongly indicates that the RNAs of the hybrids had retained their identity during replication.
Alfalfa mosaic virus (AMV, cryptogram R/1:1.3 + 1.1 + 0.9/18:U/U:S/Ap) has a genome consisting of three pieces of RNA encapsulated in three different components, viz. bottom component (B), middle component (M), and top component b (Tb). The parts of the three partite genome of AMV are freely exchangeable between strains without loss of biological activity (1). In previous work (2), we showed that it was possible to construct new stable strains with hybrid character by exchanging nucleoprotein components of two different strains of AMV (AMV 425 and yellow spot mosaic virus, YSMV). The characteristics of the parent strains are extensively described in a previous publication (2). In Phase&s vulgaris L. var. “Berna” strain 425 induces only small pinpoint necrotic lesions on the inoculated leaves, whereas strain YSMV causes chlorosis, sometimes followed by necrosis on the inoculated as well as on the trifoliate leaves. In tobacco strain 425 causes chlorotic symptoms and strain YSMV causes yellow symptoms necrosing in a later stage, on both the inoculated and on the systematically infected leaves. Upon electrophoresis on polyacrylamide 328 Copyright All rights
0 1974 by Academic Preess, Inc. of reproduction in any form reserved.
gels of nucleoprotein material of both strains, a large number of peaks are found. The most characteristic difference between the two patterns is the ratio of peak 13 to 14. Peaks 13 and 14 represent together the Ta nucleoprotein. Furthermore the two strains differ serologically. The symptoms on bean are determined by M-RNA. All other known characteristics are localized on Tb-RNA. Hybrid strain Hl was constructed by combining B and Tb of AMV 425 with M of YSMV. Properties of this strain are identical to those of AMV 425 except that it causes symptoms on bean like those of YSMV. The components of this strain will be designated BEI~ , MH1 and Tbm . Hybrid strain H2 was constructed by combining B and Tb of YSMV with M of AMV 425. Properties of this strain are identical to those of YSMV except that it causes symptoms on bean which are like those of AMV 425. The components of this strain will be designated BHZ, MHz and Tbnz. The three components of the two hybrid strains were purified as described before (2). They had residual infectivities as is shown in Fig. 1. Each component of each of the hybrid strains Hl and H2 was combined with the
329
SHORT COMMUNICATIONS
Tb A. ’ /\
1 / B0 -1
130 -0
Tb ~25
T,b B. ’ 0
9 \
M
/\
/ B” -20-o
180
0 \ M
FIG. 1. Infectivity of combinations of components of the two hybrid strains of alfalfa mosaic virus. (A) Hl (originating from B,zs + Mvsuv -lTbrss) (B) H2 (originating from Bvsnv + Mb25 + Tbvsuv). Infectivities on Phaseolus vulgaris L. var. “Berna” of the component preparations are at the vertices of the triangles; infectivity of Tb at the top, of B at the bottom left, and of M at the bottom right. Infectivities of two-fold combinations are at the sides of the triangles. Infectivities of the three-fold combinations are indicated at the center of the triangles. Lesion numbers of one triangle are averages from one incomplete block. Concentration of all components was 0.7 yg/ml.
two complementary components of the strain to be reconstructed (Fig. 2). The preparations of the complementary components were the same as those used to construct the hybrid strains (2). Ten single lesions induced on tobacco by each of these inocula were propagated as described previously (9). As a control, two single lesions induced by a combination of the purified components from Hl and H2, respectively, were handled in the same way. All isolates originating from one particular combination of components showed similar symptoms on tobacco as well as on bean (Table 1). Mass cultures were made from three isolates of each backcross combination and from the two isolates of the reconstructed hybrids. Preparations obtained from these cultures were characterized with regard to serotype and component composition by the methods used before (L?).In all except one case strains indistinguishable from parent strains were obtained (Table 1). In the last few years an increasing number of plant virus genomes have been found to be composed of two or three RNA molecules. With a number of these viruses it was possi-
+
TbL2s
0 ysmv + Tb ysmv
M ~2s
MHZ +
BHZ
M ysmv + ;b ysmv
+
+
+ M125
B
~25
+ TbLzs
Bus
+ ML25
Tb ~srnv
Tb
HZ +
0 yrmv + M yrmv
FIG. 2. Scheme showing how the hybrids Hl and H2 of alfalfa mosaic virus were constructed and how their separated components were again combined with complementary components from the parent strains.
ble to construct hybrid strains by exchanging components between different viruses, strains or mutants (for review see 3). Characterization of most of these hybrids permits the conclusion that although the parts of the genome are completely dependent on each other for their replication they retain their own identity as judged by their phenotypic expression. This suggests that the RNAs are replicated true to type. However, little attention has been paid to one of the keystones of this hypothesis, namely the possibility to reconstruct parent strains starting from hybrids (4). In earlier work from our laboratory (5) it was shown that parent strains of AMV could be reconstructed from hybrids, but the genetic constitution of the hybrids was misinterpreted since at that time, only two components (B) and (Ta) were thought to be necessary for infection. Sanger (6) succeeded in construction of parent type isolates by combining long and short RNA components of two artificial hybrid strains of tobacco rattle virus. Incidentally, short remarks have been made in
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330
TABLE CHARACTERISTICS’
I
OF BACKCROSS AND CONTROL PROGENIES OBTAINED FROM HYBRIDS Hl AND H2 OF AMV STRAINS 425 ANn YSMV
Components constituting the inoculate B
M
Tb
Hl YSMV 425 H2 425 YSMV Hl H2
425 Hl 425 YSMV HZ YSMV Hl H2
425 YSMV Hl YSMV 425 H2 Hl H2
Symptoms on tobacco
Symptoms on bean
Serotype
Component composition
10A 1OY 10A 8Y” 10A 9Y” 2A 2Y
10A 1OY 10A 7Y lAb 10A 9Yb 2Y 2A
3A 3Y 3A 3Y 3A 3Y 2A 2Y
3A 3Y 3A 3Y 3A 3Y 2A 2Y
a When a characteristic of a backcross combination is similar to that of parent strain YSMV or 425 this is indicated by Y and A, respectively. Figures refer to number of isolates with a given characteristic. 6 During single lesion transfer one or two isolates were lost.
the literature about successful backcross experiments with comoviruses (7, 8), bromoviruses (9), and nepoviruses (10). In this report we present a quantitative and systematic approach to backcross experiments with a three partite genome involving four characteristics. In hybrid strains RNAs from diierent strains are mutually dependent for their replication, however, they appear to retain their own identity. ACKNOWLEDGMENTS We wish to thank Corrie Houwing, Marianne Kaashoek, Ing. Lyda Neeleman and Frans Brederode for their contributions to the experimental part of the work. This work was sponsored by the Netherlands Foundation for Chemical Research (S.O.N.), with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
REFERENCES 1. VAN VLOTEN-DOTING, L., DINGJAN-VERSTEEGH, A. M., and JASPARS, E. M. J.
Virology 40,419-430 (1970). 2. DINGJAN-VERSTEEGH, A. M., VAN VLOTENDOTING, L., and JASPARS, E. M. J. Virology
49, 716722 (1972). A. Ann. Rev. Phytopathol. 10, 125-250 (1972). 4. MENDEL, G. J. Verh. Naturjorsch. Ver. Brtinn 4, 3-47 (1866). 5. VAN VLOTEN-DOTING, L., KRUSEYAN, J., and JASPARS, E. M. J. Virology 34, 723-737
3. VAN KAMMEN,
(1968).
6. S~NCER, H. L. J. Viral. 3,394-312 (1969). 7. BRVENING, G. Virology 37, 577-584 (1969). 8. WOOD, H. A. Virology 49,592-598 (1972). 9. BANCROFT, J. B. J. Gen. Viral. 14, 223-228 (1972). 10. HARRISON, B. D., MURANT, A. F., and MAYO, M. A. Abstract of the 2nd International Congress of Plant Pathology, Minneapolis (1973).
69, 328-330
Confirmation
(1974)
of the Constitution Genomes
ANNEMIEKE
of Alfalfa
by Backcross
Mosaic
Virus
Hybrid
Experiments
DINGJAN-VERSTEEGH, LOUS VAN VLOTEN-DOTING AND E. M. J. JASPARS
Department
of Biochemistry, Leiden,
State University, The Netherlands
Accepted January
Wassenaarseweg 64,
16, 1974
Components of two types of hybrid strains of alfalfa mosaic virus were purified. Single lesions induced by inocula of each of these components, to which the two eomplementary components of the homologous parent strain were added, yielded progenies indistinguishable from the parents with regard to serology, component composition and symptoms on tobacco and bean. This strongly indicates that the RNAs of the hybrids had retained their identity during replication.
Alfalfa mosaic virus (AMV, cryptogram R/1:1.3 + 1.1 + 0.9/18:U/U:S/Ap) has a genome consisting of three pieces of RNA encapsulated in three different components, viz. bottom component (B), middle component (M), and top component b (Tb). The parts of the three partite genome of AMV are freely exchangeable between strains without loss of biological activity (1). In previous work (2), we showed that it was possible to construct new stable strains with hybrid character by exchanging nucleoprotein components of two different strains of AMV (AMV 425 and yellow spot mosaic virus, YSMV). The characteristics of the parent strains are extensively described in a previous publication (2). In Phase&s vulgaris L. var. “Berna” strain 425 induces only small pinpoint necrotic lesions on the inoculated leaves, whereas strain YSMV causes chlorosis, sometimes followed by necrosis on the inoculated as well as on the trifoliate leaves. In tobacco strain 425 causes chlorotic symptoms and strain YSMV causes yellow symptoms necrosing in a later stage, on both the inoculated and on the systematically infected leaves. Upon electrophoresis on polyacrylamide 328 Copyright All rights
0 1974 by Academic Preess, Inc. of reproduction in any form reserved.
gels of nucleoprotein material of both strains, a large number of peaks are found. The most characteristic difference between the two patterns is the ratio of peak 13 to 14. Peaks 13 and 14 represent together the Ta nucleoprotein. Furthermore the two strains differ serologically. The symptoms on bean are determined by M-RNA. All other known characteristics are localized on Tb-RNA. Hybrid strain Hl was constructed by combining B and Tb of AMV 425 with M of YSMV. Properties of this strain are identical to those of AMV 425 except that it causes symptoms on bean like those of YSMV. The components of this strain will be designated BEI~ , MH1 and Tbm . Hybrid strain H2 was constructed by combining B and Tb of YSMV with M of AMV 425. Properties of this strain are identical to those of YSMV except that it causes symptoms on bean which are like those of AMV 425. The components of this strain will be designated BHZ, MHz and Tbnz. The three components of the two hybrid strains were purified as described before (2). They had residual infectivities as is shown in Fig. 1. Each component of each of the hybrid strains Hl and H2 was combined with the
329
SHORT COMMUNICATIONS
Tb A. ’ /\
1 / B0 -1
130 -0
Tb ~25
T,b B. ’ 0
9 \
M
/\
/ B” -20-o
180
0 \ M
FIG. 1. Infectivity of combinations of components of the two hybrid strains of alfalfa mosaic virus. (A) Hl (originating from B,zs + Mvsuv -lTbrss) (B) H2 (originating from Bvsnv + Mb25 + Tbvsuv). Infectivities on Phaseolus vulgaris L. var. “Berna” of the component preparations are at the vertices of the triangles; infectivity of Tb at the top, of B at the bottom left, and of M at the bottom right. Infectivities of two-fold combinations are at the sides of the triangles. Infectivities of the three-fold combinations are indicated at the center of the triangles. Lesion numbers of one triangle are averages from one incomplete block. Concentration of all components was 0.7 yg/ml.
two complementary components of the strain to be reconstructed (Fig. 2). The preparations of the complementary components were the same as those used to construct the hybrid strains (2). Ten single lesions induced on tobacco by each of these inocula were propagated as described previously (9). As a control, two single lesions induced by a combination of the purified components from Hl and H2, respectively, were handled in the same way. All isolates originating from one particular combination of components showed similar symptoms on tobacco as well as on bean (Table 1). Mass cultures were made from three isolates of each backcross combination and from the two isolates of the reconstructed hybrids. Preparations obtained from these cultures were characterized with regard to serotype and component composition by the methods used before (L?).In all except one case strains indistinguishable from parent strains were obtained (Table 1). In the last few years an increasing number of plant virus genomes have been found to be composed of two or three RNA molecules. With a number of these viruses it was possi-
+
TbL2s
0 ysmv + Tb ysmv
M ~2s
MHZ +
BHZ
M ysmv + ;b ysmv
+
+
+ M125
B
~25
+ TbLzs
Bus
+ ML25
Tb ~srnv
Tb
HZ +
0 yrmv + M yrmv
FIG. 2. Scheme showing how the hybrids Hl and H2 of alfalfa mosaic virus were constructed and how their separated components were again combined with complementary components from the parent strains.
ble to construct hybrid strains by exchanging components between different viruses, strains or mutants (for review see 3). Characterization of most of these hybrids permits the conclusion that although the parts of the genome are completely dependent on each other for their replication they retain their own identity as judged by their phenotypic expression. This suggests that the RNAs are replicated true to type. However, little attention has been paid to one of the keystones of this hypothesis, namely the possibility to reconstruct parent strains starting from hybrids (4). In earlier work from our laboratory (5) it was shown that parent strains of AMV could be reconstructed from hybrids, but the genetic constitution of the hybrids was misinterpreted since at that time, only two components (B) and (Ta) were thought to be necessary for infection. Sanger (6) succeeded in construction of parent type isolates by combining long and short RNA components of two artificial hybrid strains of tobacco rattle virus. Incidentally, short remarks have been made in
SHORT COMMUNICATIONS
330
TABLE CHARACTERISTICS’
I
OF BACKCROSS AND CONTROL PROGENIES OBTAINED FROM HYBRIDS Hl AND H2 OF AMV STRAINS 425 ANn YSMV
Components constituting the inoculate B
M
Tb
Hl YSMV 425 H2 425 YSMV Hl H2
425 Hl 425 YSMV HZ YSMV Hl H2
425 YSMV Hl YSMV 425 H2 Hl H2
Symptoms on tobacco
Symptoms on bean
Serotype
Component composition
10A 1OY 10A 8Y” 10A 9Y” 2A 2Y
10A 1OY 10A 7Y lAb 10A 9Yb 2Y 2A
3A 3Y 3A 3Y 3A 3Y 2A 2Y
3A 3Y 3A 3Y 3A 3Y 2A 2Y
a When a characteristic of a backcross combination is similar to that of parent strain YSMV or 425 this is indicated by Y and A, respectively. Figures refer to number of isolates with a given characteristic. 6 During single lesion transfer one or two isolates were lost.
the literature about successful backcross experiments with comoviruses (7, 8), bromoviruses (9), and nepoviruses (10). In this report we present a quantitative and systematic approach to backcross experiments with a three partite genome involving four characteristics. In hybrid strains RNAs from diierent strains are mutually dependent for their replication, however, they appear to retain their own identity. ACKNOWLEDGMENTS We wish to thank Corrie Houwing, Marianne Kaashoek, Ing. Lyda Neeleman and Frans Brederode for their contributions to the experimental part of the work. This work was sponsored by the Netherlands Foundation for Chemical Research (S.O.N.), with financial aid from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).
REFERENCES 1. VAN VLOTEN-DOTING, L., DINGJAN-VERSTEEGH, A. M., and JASPARS, E. M. J.
Virology 40,419-430 (1970). 2. DINGJAN-VERSTEEGH, A. M., VAN VLOTENDOTING, L., and JASPARS, E. M. J. Virology
49, 716722 (1972). A. Ann. Rev. Phytopathol. 10, 125-250 (1972). 4. MENDEL, G. J. Verh. Naturjorsch. Ver. Brtinn 4, 3-47 (1866). 5. VAN VLOTEN-DOTING, L., KRUSEYAN, J., and JASPARS, E. M. J. Virology 34, 723-737
3. VAN KAMMEN,
(1968).
6. S~NCER, H. L. J. Viral. 3,394-312 (1969). 7. BRVENING, G. Virology 37, 577-584 (1969). 8. WOOD, H. A. Virology 49,592-598 (1972). 9. BANCROFT, J. B. J. Gen. Viral. 14, 223-228 (1972). 10. HARRISON, B. D., MURANT, A. F., and MAYO, M. A. Abstract of the 2nd International Congress of Plant Pathology, Minneapolis (1973).