VIROLOGY
83,
413--416(1977)
SHORT COMMUNICATIONS Infection of Cowpea Mesophyll Protoplasts with Cucumber Mosaic Virus M. KOIKE,’
T. HIBI,2
K. YCBA
AND
Laboratory of Plant Pathology, Faculty of Agriculture,
University of Tokyo, Tokyo, Japan
Accepted July 27,1977 Mesophyll protoplasts isolated from cowpea primary leaves were inoculated with cucumber mosaic virus according to the same procedures as those developed for inoculating them with cowpea mosaic virus. Virus multiplication in the inoculated protoplasts was demonstrated by fluorescent-antibody staining, by electron microscopy of ultrathin sections, and by infectivity assay. Seventy to ninety-five percent of the inoculated protoplasm were infected. PolytL-ornithine) was not essential for CMV infection, but it had a stimulating effect. No sign of necrosis suggesting cell death was observed until 36 hr after inoculation.
Previously it was reported that meso- of darkness at 22” each day. Fully exphyll protoplasts from primary leaves of panded primary leaves were cut from locowpea could be infected with cowpea mo- to 12-day-old seedlings and mesophyll prosaic virus (CPMV) (1,2). Primary leaves toplasts were isolated according to the of 9- to lZday-old cowpea plants grown procedures described previously (1). The under appropriate conditions were most yellow strain isolate of CMV (4) was propsuitable for isolating protoplasts for. virus agated and purified according to the infection. This indicates that cowpea mes- method reported by Takanami and Toophyll protoplasts can be used as an excel- maru (5). Protoplasts were inoculated lent system for virus infection studies. In with the virus in the presence of poly(~this paper we describe the infection of ornithine) according to the same procecowpea mesophyll protoplasts with cucum- dures as those described for CPMV (1) ber mosaic virus (CMV). Cowpea plants except that CMV (final concentration of 5 infected with CMV exhibit local necrotic pg/ml) was used instead of CPMV in the symptoms, while those infected with inoculum. The inoculated protoplasts were CPMV show systemic symptoms without incubated at 25” as described previously (1). necrosis. Recently, tobacco mesophyll proAt various times after inoculation, protoplasts were reported to be infected with toplasts were collected by centrifugation CMV (3). Accordingly, it has become fea- and stained with the fluorescein isothiocysible to compare the infection process of anate-conjugated CMV-antibodies accordCMV in cowpea mesophyll protoplasts ing to the method reported by Otsuki and with that of CPMV and with that of CMV Takebe (31, except that 96% ethanol was in tobacco mesophyll protoplasts. used for fixation instead of acetone. The Cowpea plants (Vigna sesquepedalis cv. stained protoplasts were examined with Kurodane-Sanjaku) were grown in Hoag- a fluorescence microscope in a dark field land’s solution in a phytotron, which was through BV filters. The first specific yelkept under controlled conditions of 14 hr low-green fluorescence due to CMV antiof light (about 24,000 lx) at 27” and 10 hr gen was visible as weak fluorescent specks scattered in the protoplasts after 9-12 hr I To whom requests for reprints should be sent. of incubation. The fluorescent specks grad* Present address: Institute for Plant Virus Research, Tsukuba Science City, Yatabe, Ibaraki, 300- ually increased in size and number during further incubation. Large amorphous flu21, Japan. 413 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN
0042-6622
414
SHORT
COMMUNICATIONS
orescent masses like those found in CPMV-infected protoplasts (I) were not observed. After 36 hr of incubation, the fluorescence was distributed throughout the cytoplasm as a network among dark chloroplasts (Fig. 1). At this time 70-95% of the protoplasts showed fluorescence. When the protoplasts were inoculated with CMV in the absence of poly(L-ornithine), the percentage of protoplasts with specific fluorescence did not decrease, although the fluorescence intensity in the stained protoplasts slightly decreased. This indicates that poly(L-ornithine) is not essential for CMV infection, but that it has a stimulating action. The fluorescence was not observed in noninoculated protoplasts or in CMV-infected protoplasts treated previously with unconjugated CMV antibodies. For electron microscopy, inoculated protoplasts were collected by centrifugation after 36 hr of incubation, fixed, embedded, sectioned, and stained as described previously (6). A large number of virus particles were found aggregated in the cytoplasm of almost every protoplast. Virus particles were usually randomly distributed in the aggregates, but sometimes they formed large, crystalline arrays (Fig.
2). Virus aggregates were not detected in inoculated protoplasts at 0 hr or in noninoculated protoplasts after 36 hr of incubation. The cytopathic structures, consisting of many vesicles surrounded by electrondense material formed in the cytoplasm of CPMV-infected protoplasts (1 I, were never found in CMV-infected protoplasts. No virus particles were found in chloroplasts, in mitochondria, or in the central vacuole. The location of progeny CMV particles in cowpea protoplasts was similar to that reported for CMV in tobacco mesophyll protoplasts (7). No degeneration of subcellular organelles was observed in CMV-infected cowpea mesophyll protoplasts. The increase of CMV in inoculated protoplasts was further confirmed by assaying the infectivity of protoplast extracts on primary leaves of cowpea (cv. KurodaneSanjaku) (8) according to the method reported by Otsuki and Takebe (31, with some modifications, as follows: After 0 and 36 hr of incubation, protoplasts were collected by centrifugation, washed once in 0.6 M mannitol solution containing 10 mM CaCl,, and stored at -70”. After thawing, the protoplasts were suspended in 1 ml of 0.5 M citrate buffer, pH 6.5, containing
FIG. 1. Fluorescence micrograph of CMV-infected cowpea mesophyll protoplasta stained with fluorescent antibodies a&r 36 hr of incubation. Viral antigens are dispersed throughout the cytoplasm. The scale represents 25 pm.
SHORT COMMUNICATIONS
415
EPIG. 2. Electron micrograph of a CMV-infected cowpea mesophyll protoplast after 36 hr of incubal ;ion. CrYstalline aggregates of progeny virus particles (V) are in the cytoplasm; Cp, chloroplast. The escale represents 0.5 pm. TABLE
1
DEVELOPMENT OF INFECTIWTY IN PROTOPLASTS INOCULATED WITH CMV~
Experiment number
1 2
Number of lesions produced by extract of protoplasts 0 hr
36 hr
2.8 1.2
171.6 68.5
a Protoplasts inoculated with CMV were incubated in the incubation medium. Af%er 0 and 36 hr of incubation, 3.2 x 10’ protoplasts were harvested, extracted, and assayed for infectivity on six cowpea primary leaves. The results represent the number of lesions per cowpea leaf.
0.01 M EDTA, to which 1 ml of chloroform was added, The suspension was then homogenized in a Teflon homogenizer and centrifuged at 8000 g for 10 min. The supernatant fraction was aspirated with a pipet and mixed with 9 ml of 0.005 M borate but&r, pH 9.0, containing 0.005 M EDTA. It was then centrifuged at 105,000 g for 90 min and the resulting supernatant fraction was discarded. The pellet was resuspended in 1 ml of 0.005 M borate
buffer, pH 9.0, containing 0.005 M EDTA. Infectivity in the final suspension was assayed on primary leaves of six lo- to 12day-old cowpea plants (cv. Kurodane-Sanjaku). One of the two primary leaves of each plant was inoculated with the suspension, and the other was inoculated with 1 pglml of CMV as a control. The number of lesions was counted 2 days after inoculation. The results are shown in Table 1. A small amount of infectivity was found at the beginning of incubation which could be attributed to virus particles adsorbed to and taken up by protoplasts on inoculation. A large amount of infectivity was detected after 36 hr of incubation. These results show an increase in CMV in the inoculated protoplasts after 36 hr of incubation. It is concluded that cowpea mesophyll protoplasts can be infected with CMV when inoculated by the same procedures as those described for CPMV (I). As mentioned above, the fully expanded primary leaves of lo- to la-day-old cowpea plants were used for isolation of the protoplasts in this study. When younger or
416
SHORT
COMMUNICATIONS
older plants were used, protoplasts isolated from them showed a lower percentage of CMV infection and poorer survival. It has been reported that more local lesions are produced by CMV on fully expanded primary leaves of lo- to 12-day-old cowpea plants (cv. Kurodane-Sanjaku) grown at 25” and 4000 lx than on younger or older plants (8). Thus, the susceptibility of cowpea primary leaves to CMV corresponds to that of isolated mesophyll protoplasts, and the susceptibility to CMV infection changes during the plant growth in both systems. Cowpea mesophyll protoplasts can be infected with either CMV or CPMV (1) in the absence of poly(L-ornithine). By contrast poly(cornithine) has been shown to be essential for infection of tobacco mesophyll protoplasts with both CMV (3) and CPMV (9). No sign of necrosis suggesting cell death was detected in the cowpea mesophyll protoplasts 36 hr after inoculation, even though the yellow strain of CMV produces local necrotic lesions on cowpea primary leaves within 24 hr after inoculation (8). The same phenomenon has been reported with TMV in tobacco mesophyll protoplasts from tobacco varieties having TMV
hypersensitivity (101, suggesting that necrosis may be induced in a given cell through the cooperation of surrounding cells or tissues. ACKNOWLEDGMENT This work was partially supported by a grant from the Ministry of Agriculture and Forestry. REFERENCES 1. HIBI, T., REZELMAN, G., and VAN KAMMEN, A., Virology 64, 308-318 (1975). 2. BEIER, H., and BRUENING, G., ViroZogy 64, 272276 (1975). 3. OTSUKI, Y., and TAKEBE, I., Virology 52, 433438 (1973). 4. TOMARU, K., and HIDAXA, Z., Bull. Hatano Tobacco Eip. Sta. 46, 143-149 (1960). 5. TAKANAMI, Y., and TOMARU, K., Virology 37, 293-295 (1969). HIBI, T., and YORA, K., Ann. PhytopathoE. Sot. 6. Japan 38, 350-356 (1972). ‘. HONDA, Y., MATSUI, C., OTBUKI, Y., and TAIKEBE, I., PhytopathoZogy 64, 30-34 (1974). s, TOMARU, K., Bull. Hatano Tobacco Exp. Sta. 58, 1-18 (1967). 9. HUBER, R., REZELMAN, G., HIBI, T., and VAN KAMMEN, A., J. Gen. Virol. 34,315-323 (1977). lo. OTSUKI, Y., SHIMOMURA, T., and TAKEBE, I., Virology 50, 45-50 (1972).
83,
413--416(1977)
SHORT COMMUNICATIONS Infection of Cowpea Mesophyll Protoplasts with Cucumber Mosaic Virus M. KOIKE,’
T. HIBI,2
K. YCBA
AND
Laboratory of Plant Pathology, Faculty of Agriculture,
University of Tokyo, Tokyo, Japan
Accepted July 27,1977 Mesophyll protoplasts isolated from cowpea primary leaves were inoculated with cucumber mosaic virus according to the same procedures as those developed for inoculating them with cowpea mosaic virus. Virus multiplication in the inoculated protoplasts was demonstrated by fluorescent-antibody staining, by electron microscopy of ultrathin sections, and by infectivity assay. Seventy to ninety-five percent of the inoculated protoplasm were infected. PolytL-ornithine) was not essential for CMV infection, but it had a stimulating effect. No sign of necrosis suggesting cell death was observed until 36 hr after inoculation.
Previously it was reported that meso- of darkness at 22” each day. Fully exphyll protoplasts from primary leaves of panded primary leaves were cut from locowpea could be infected with cowpea mo- to 12-day-old seedlings and mesophyll prosaic virus (CPMV) (1,2). Primary leaves toplasts were isolated according to the of 9- to lZday-old cowpea plants grown procedures described previously (1). The under appropriate conditions were most yellow strain isolate of CMV (4) was propsuitable for isolating protoplasts for. virus agated and purified according to the infection. This indicates that cowpea mes- method reported by Takanami and Toophyll protoplasts can be used as an excel- maru (5). Protoplasts were inoculated lent system for virus infection studies. In with the virus in the presence of poly(~this paper we describe the infection of ornithine) according to the same procecowpea mesophyll protoplasts with cucum- dures as those described for CPMV (1) ber mosaic virus (CMV). Cowpea plants except that CMV (final concentration of 5 infected with CMV exhibit local necrotic pg/ml) was used instead of CPMV in the symptoms, while those infected with inoculum. The inoculated protoplasts were CPMV show systemic symptoms without incubated at 25” as described previously (1). necrosis. Recently, tobacco mesophyll proAt various times after inoculation, protoplasts were reported to be infected with toplasts were collected by centrifugation CMV (3). Accordingly, it has become fea- and stained with the fluorescein isothiocysible to compare the infection process of anate-conjugated CMV-antibodies accordCMV in cowpea mesophyll protoplasts ing to the method reported by Otsuki and with that of CPMV and with that of CMV Takebe (31, except that 96% ethanol was in tobacco mesophyll protoplasts. used for fixation instead of acetone. The Cowpea plants (Vigna sesquepedalis cv. stained protoplasts were examined with Kurodane-Sanjaku) were grown in Hoag- a fluorescence microscope in a dark field land’s solution in a phytotron, which was through BV filters. The first specific yelkept under controlled conditions of 14 hr low-green fluorescence due to CMV antiof light (about 24,000 lx) at 27” and 10 hr gen was visible as weak fluorescent specks scattered in the protoplasts after 9-12 hr I To whom requests for reprints should be sent. of incubation. The fluorescent specks grad* Present address: Institute for Plant Virus Research, Tsukuba Science City, Yatabe, Ibaraki, 300- ually increased in size and number during further incubation. Large amorphous flu21, Japan. 413 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN
0042-6622
414
SHORT
COMMUNICATIONS
orescent masses like those found in CPMV-infected protoplasts (I) were not observed. After 36 hr of incubation, the fluorescence was distributed throughout the cytoplasm as a network among dark chloroplasts (Fig. 1). At this time 70-95% of the protoplasts showed fluorescence. When the protoplasts were inoculated with CMV in the absence of poly(L-ornithine), the percentage of protoplasts with specific fluorescence did not decrease, although the fluorescence intensity in the stained protoplasts slightly decreased. This indicates that poly(L-ornithine) is not essential for CMV infection, but that it has a stimulating action. The fluorescence was not observed in noninoculated protoplasts or in CMV-infected protoplasts treated previously with unconjugated CMV antibodies. For electron microscopy, inoculated protoplasts were collected by centrifugation after 36 hr of incubation, fixed, embedded, sectioned, and stained as described previously (6). A large number of virus particles were found aggregated in the cytoplasm of almost every protoplast. Virus particles were usually randomly distributed in the aggregates, but sometimes they formed large, crystalline arrays (Fig.
2). Virus aggregates were not detected in inoculated protoplasts at 0 hr or in noninoculated protoplasts after 36 hr of incubation. The cytopathic structures, consisting of many vesicles surrounded by electrondense material formed in the cytoplasm of CPMV-infected protoplasts (1 I, were never found in CMV-infected protoplasts. No virus particles were found in chloroplasts, in mitochondria, or in the central vacuole. The location of progeny CMV particles in cowpea protoplasts was similar to that reported for CMV in tobacco mesophyll protoplasts (7). No degeneration of subcellular organelles was observed in CMV-infected cowpea mesophyll protoplasts. The increase of CMV in inoculated protoplasts was further confirmed by assaying the infectivity of protoplast extracts on primary leaves of cowpea (cv. KurodaneSanjaku) (8) according to the method reported by Otsuki and Takebe (31, with some modifications, as follows: After 0 and 36 hr of incubation, protoplasts were collected by centrifugation, washed once in 0.6 M mannitol solution containing 10 mM CaCl,, and stored at -70”. After thawing, the protoplasts were suspended in 1 ml of 0.5 M citrate buffer, pH 6.5, containing
FIG. 1. Fluorescence micrograph of CMV-infected cowpea mesophyll protoplasta stained with fluorescent antibodies a&r 36 hr of incubation. Viral antigens are dispersed throughout the cytoplasm. The scale represents 25 pm.
SHORT COMMUNICATIONS
415
EPIG. 2. Electron micrograph of a CMV-infected cowpea mesophyll protoplast after 36 hr of incubal ;ion. CrYstalline aggregates of progeny virus particles (V) are in the cytoplasm; Cp, chloroplast. The escale represents 0.5 pm. TABLE
1
DEVELOPMENT OF INFECTIWTY IN PROTOPLASTS INOCULATED WITH CMV~
Experiment number
1 2
Number of lesions produced by extract of protoplasts 0 hr
36 hr
2.8 1.2
171.6 68.5
a Protoplasts inoculated with CMV were incubated in the incubation medium. Af%er 0 and 36 hr of incubation, 3.2 x 10’ protoplasts were harvested, extracted, and assayed for infectivity on six cowpea primary leaves. The results represent the number of lesions per cowpea leaf.
0.01 M EDTA, to which 1 ml of chloroform was added, The suspension was then homogenized in a Teflon homogenizer and centrifuged at 8000 g for 10 min. The supernatant fraction was aspirated with a pipet and mixed with 9 ml of 0.005 M borate but&r, pH 9.0, containing 0.005 M EDTA. It was then centrifuged at 105,000 g for 90 min and the resulting supernatant fraction was discarded. The pellet was resuspended in 1 ml of 0.005 M borate
buffer, pH 9.0, containing 0.005 M EDTA. Infectivity in the final suspension was assayed on primary leaves of six lo- to 12day-old cowpea plants (cv. Kurodane-Sanjaku). One of the two primary leaves of each plant was inoculated with the suspension, and the other was inoculated with 1 pglml of CMV as a control. The number of lesions was counted 2 days after inoculation. The results are shown in Table 1. A small amount of infectivity was found at the beginning of incubation which could be attributed to virus particles adsorbed to and taken up by protoplasts on inoculation. A large amount of infectivity was detected after 36 hr of incubation. These results show an increase in CMV in the inoculated protoplasts after 36 hr of incubation. It is concluded that cowpea mesophyll protoplasts can be infected with CMV when inoculated by the same procedures as those described for CPMV (I). As mentioned above, the fully expanded primary leaves of lo- to la-day-old cowpea plants were used for isolation of the protoplasts in this study. When younger or
416
SHORT
COMMUNICATIONS
older plants were used, protoplasts isolated from them showed a lower percentage of CMV infection and poorer survival. It has been reported that more local lesions are produced by CMV on fully expanded primary leaves of lo- to 12-day-old cowpea plants (cv. Kurodane-Sanjaku) grown at 25” and 4000 lx than on younger or older plants (8). Thus, the susceptibility of cowpea primary leaves to CMV corresponds to that of isolated mesophyll protoplasts, and the susceptibility to CMV infection changes during the plant growth in both systems. Cowpea mesophyll protoplasts can be infected with either CMV or CPMV (1) in the absence of poly(L-ornithine). By contrast poly(cornithine) has been shown to be essential for infection of tobacco mesophyll protoplasts with both CMV (3) and CPMV (9). No sign of necrosis suggesting cell death was detected in the cowpea mesophyll protoplasts 36 hr after inoculation, even though the yellow strain of CMV produces local necrotic lesions on cowpea primary leaves within 24 hr after inoculation (8). The same phenomenon has been reported with TMV in tobacco mesophyll protoplasts from tobacco varieties having TMV
hypersensitivity (101, suggesting that necrosis may be induced in a given cell through the cooperation of surrounding cells or tissues. ACKNOWLEDGMENT This work was partially supported by a grant from the Ministry of Agriculture and Forestry. REFERENCES 1. HIBI, T., REZELMAN, G., and VAN KAMMEN, A., Virology 64, 308-318 (1975). 2. BEIER, H., and BRUENING, G., ViroZogy 64, 272276 (1975). 3. OTSUKI, Y., and TAKEBE, I., Virology 52, 433438 (1973). 4. TOMARU, K., and HIDAXA, Z., Bull. Hatano Tobacco Eip. Sta. 46, 143-149 (1960). 5. TAKANAMI, Y., and TOMARU, K., Virology 37, 293-295 (1969). HIBI, T., and YORA, K., Ann. PhytopathoE. Sot. 6. Japan 38, 350-356 (1972). ‘. HONDA, Y., MATSUI, C., OTBUKI, Y., and TAIKEBE, I., PhytopathoZogy 64, 30-34 (1974). s, TOMARU, K., Bull. Hatano Tobacco Exp. Sta. 58, 1-18 (1967). 9. HUBER, R., REZELMAN, G., HIBI, T., and VAN KAMMEN, A., J. Gen. Virol. 34,315-323 (1977). lo. OTSUKI, Y., SHIMOMURA, T., and TAKEBE, I., Virology 50, 45-50 (1972).
