VIROLOGY
65, 583-587
Quantity
(197%
of Microinjected Infection ROBERT
Department
of Phnt
Tobacco
of Single Cultured
S. HALLIWELL
Sciences,
Mosaic
Texas
AND
Agricuhd
Accepted
Tobacco
WILLIAM
Experiment
Station,
Virus Required
Station,
for
Cells
S. GAZAWAY’ Texas
A&M
hiuersity,
College
Texas 77843 February
10, 1975
An average volume of 1.11 pl of a standard TMV suspension was microinjected into single cultured tobacco cells using a glass capillary microneedle. The volume of virus suspension injected was calculated from 20 microneedles. The average number of characteristic TMV particles (290-310 nm) delivered was determined by direct count of the particles on slotted EM grids. All cells produced crystalline virus inclusions when injected with 620 or more TMV particles. One of two cells was infected after injection with 310 TMV particles and only one of four cells became infected after injection of 72 particles. Cell injections at lower concentrations produced no signs of infection. Injected cells producing crystalline inclusions (the criterion for infection) were infectious when assayed on Nicotiam glutinosa L. Uninoculated controls and cells bioassayed immediately after injection with the standard and lowest infectious concentration gave a negative assay.
The ability to assay infectious viruses quantitatively is fundamental to an understanding of their properties. Among the various assay methods developed for plant viruses, the local-lesion assay is the most sensitive and gives a linear response. However, under optimum conditions, the locallesion assay requires about 50,000 virus particles to initiate a single infection (I). This does not approach the assay sensitivity of bacterial and some animal viruses. According to Matthews (2), there are three possible reasons for the low level of sensitivity of assay with plant viruses: 1) The inefficiency of the inoculation process, 2) the presence of noninfectious virus particles in the inoculum, and 3) the need for two or more particles with multiparticulate viruses. Although many virus particles must be applied to a leaf surface to produce a single lesion by mechanical means, it has been demonstrated indirectly that TMV is highly infectious, and probably only one ‘Present address: Standard Limon, Costa Rica, C. A.
Fruit Co., Apartado
N, 583
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
virus particle is required to initiate infection (3-5). However, evidence that more than one infectious particle is involved also has been reported by several researchers (1, 6-8). Kleczkowski (8) suggested that areas on a leaf vary in susceptibility so that each area requires different concentrations of virus particles to cause infection. The information available indicates that either a large number of TMV particles constitutes one ‘infectious unit when purified by present techniques or very few infectious units come in contact with infection sites by the assay methods used. Nims and Halliwell (9, 10) injected purified TMV into cultured Nicotiana tabacum cv. Samsun NN callus cells, a local-lesion variety, and obtained infection in all cells. Using similar procedures with N. tabacum cv. Samsun, Russell and Halliwell (II) substantiated these findings. In these reports, cytoplasmic vesiculation was followed by crystal formation in the inoculated cells, which served as an indication of successful infection (9, 11). The single-cell microinjection method used by these workers has the important advantage that
584
SHORT
COMMUNICATIONS
known numbers of virus particles can be injected into a single cell, but the minimum number of particles required to establish infection of a single cell has not been determined. The following is a report on the minimum number of TMV particles required to cause infection in cultured tobacco cells inoculated via microinjection. Preparation of tobacco cell culture. Healthy tobacco (Nicotiana tabacum L. cv. Samsun) cells and cell-chains derived from surface-sterilized petiole sections were produced in a modified culture medium of Murashige and Skoog (12). The modifications included the addition of coconut milk (150 ml/l) and the deletion of kinetin. The medium was adjusted to pH 5.6 by adding 0.1 N NaOH. Aliquots of medium were poured into slit-bottom Bellco flasks, plugged with cotton, covered with a stainless-steel cap, and autoclaved for 20 min at 1.05 kg/cm*. The flasks were then placed on a gyrotory shaker at 150 cycles/min for 6 hr to allow resuspension of precipitates. Cell injection. Preparation of microculture chambers, microneedles, and the method of single cell injection followed the techniques of Nims et al. (9, IO). Three cells were used in each of the first four experiments. One cell was injected with a predetermined concentration of infective TMV (Fig. 1). A second cell was
FIG. 1. Injection
injected with autoclaved TMV to determine the cell response to foreign material and the effect of mechanical damage. Untreated controls were also observed in all experiments. The cell’s activities in response to injection were monitored continuously. During the period of observation, particular emphasis was placed upon: 1) The formation of callose at the injection site, 2) the rate of cyclosis, 3) cytoplasmic vesiculation, 4) the response of the nucleus, 5) time to formation of virus crystal inclusions, and 6) time of cell death as determined by termination of cyclosis. Bioassay of inoculated cells. The contents of all inoculated cells, including controls and those with crystalline inclusions, were bioassayed on a local-lesion host plant. The microculture chamber was placed under a dissecting microscope and the mineral oil enveloping the microculture was removed with a hypodermic syringe. The glass coverslip was inverted and the cell transferred with a micropipet to a drop of 0.01 M potassium phosphate (pH 8.0). The cell was then crushed with a sterile glass rod. The final volume was brought to 0.5 ml with the phosphate solution and mixed thoroughly. The preparation was then assayed by rubbing a leaf of N. tubucum cv. Samsun NN previously dusted with Carborundum. Other cells injected with similar quan-
of cultured
tobacco cell.
SHORT
tities of infectious virus were bioassayed immediately after injection to determine if the inoculum contributed to the production of local lesions. Preparation of TMV. TMV was obtained from infected tobacco leaves by the PEG method of Gooding and Hebert (13). The preparation was further purified by differential centrifugation. The final preparation was passed through a Millipore filter (0.45 lrn) and dispersed in sterile serum vials. The contents of each vial were tested on nutrient media to assure the absence of microorganisms before use. A local-lesion assay was employed to determine the relative infectivity of TMV at various dilutions. TMV suspensions were serially diluted by a factor of ten with 0.01 M phosphate buffer (pH 7.0). The greatest dilution where infectivity was detected by the local-lesion assay constituted the initial (stock) concentration of TMV to be injected into the individual single cells. By spectrophotometry, the concentration of the stock virus was estimated to be 15.4 mg of virus/l. Determination of volume injected. An average volume of inoculum injected into cells was determined by calculating the volume of 20 different microneedles. A standard quantity of the virus preparation drawn into the needle was measured with a micrometer under a microscope at 100x.
FIG. 2. TMV
crystals
585
COMMUNICATIONS
(arrows)
Measurements were taken from the needle tip to the base of the meniscus (virus-oil interface). The average volume was calculated to be 1.1 pl. Determination of quantity of TMV injected. The actual number of virus particles delivered during microinjection was determined by direct count. A drop of virus suspension was placed on a clean glass slide and the normal volume drawn into the needle. A small drop of 0.01 M potassium phosphate solution containing 0.1% albumin was placed on a Formvar-coated, carbon-reinforced slot grid with a micropipet. The grid containing the droplet was placed under a light microscope and the virus suspension in the microneedle was injected into the droplet and allowed to air dry. The grid was shadowed with a platinum-palladium alloy (80:20) and observed with the electron microscope. All of the characteristic virus particles (i.e., those particles having a length of 290-310 nm exclusive of fragments) on the grid were counted. Virus particles on four grids were counted, and an average per injection was determined. Crystal formation. The development of virus crystals as reported by Nims et al. (9) and Russell and Halliwell (11) was a reliable index of infection. Virus crystals first appeared as dark gray areas in the cytoplasm under phase contrast. As the crys-
in an inoculated
tobacco cell.
586
SHORT
COMMUNICATIONS
tals thickened, the angularity became very prominent. Fully developed TMV crystals displayed the typical hexagonal configuration (Fig. 2) described by previous workers (9, 11, 14). Virus crystals were observed as early as 45 hr and as late as 89 hr after injection. Cell injection. A total of 30 cells was inoculated with various concentrations of infectious TMV. Four control cells were injected with heat-denatured TMV. The highest concentration of virus injected was the greatest dilution that produced local lesions when assayed on a local-lesion host. This comprised the stock suspension. Subsequent injections were made with the stock suspension and more dilute virus suspensions until no infections were observed. All cells injected with stock virus and a ten-fold dilution became infected and produced virus crystals. One of two cells injected with 5 x 10d2 stock dilution produced virus crystals. The 1 x 10m2 stock dilution was the lowest concentration at which cells became infected. Only one of four cells injected produced virus crystals. Four cells inoculated with the 1 x 10msstock dilution did not become infected. Quantity of TMV injected. When all TMV particles were counted on slot grids using the electron microscope, an average of 72 TMV particles was found in each 1.11 pl of the 1 x 10e2 dilution of virus stock. Bioassay. The number of lesions resulting from bioassays of cells containing TMV crystals varied from 4-8.75 lesions/halfleaf. When control cells were microinjected with the stock suspension or any dilutions thereof and then were bioassayed immediately, no lesions resulted. Fraenkel-Conrat et al. (7) and Steere (I) demonstrated that a minimum of 50,000-75,000 TMV particles was required to cause a single infection on intact leaves. In their inoculation techniques, much of the virus inoculum was ineffective since many of the TMV particles never entered a cell. Motoyoshi et al. (15) treated tobacco protoplasts with isotope-labeled cowpea chlorotic mottle virus and determined that approximately 760 virions were required to
cause 52% infection of the protoplasts after 44 hr. By microinjection the number of viruses for infection of 100% of the cells was 620. Seventy-two virus particles were required to infect 25% of the injected cells. Although the results clearly demonstrate that only a few particles are necessary to cause an infection, the data presented in this paper are not sufficient to determine that one TMV particle alone can cause an infection, When injecting such small quantities of virus into a cell, there is no way to determine accurately the number of infectious particles actually present in any given dosage. To do so, a large number of cells would have to be inoculated with a particular concentration. For this reason, the number of virus particles injected into a cell is given within a range of possible error. Unfortunately, the microinjection technique is time consuming and is not well suited for the inoculation of the large numbers of cells that are required for statistical interpretation. Cell variability could present a problem in attempts to determine the validity of the theory that one virus particle can cause an infection. Kleczkowski (8) suggested that cells located in different regions of the leaf varied in their susceptibility of virus infection. Atkinson and Matthews (16) noted a differential response of tobacco leaf cells to TMV infection. They reported that some cells became infected while other cells remained free of the virus. However, this differential susceptibility which exists in intact plant tissues (8, 16) may not be present in cell cultures. Murakishi et al. (17) infected 94% of cultured cells by adding TMV (83 pg/ml) to a suspension of cells and vibrating the mixture with a Vortex mixer. Using the microinjection technique, all cells injected with the stock suspension or a 10-l dilution became infected. On the basis of these results and previous reports (g-11), the variability of cultured N. tabacum cv. Samsun cells to TMV infection is apparently of minor importance. The small percentage of cells that become infected when inoculated with lower concentrations of TMV could be the result
SHORT
COMMUNICATIONS
of several factors. After the virus particles have been injected into the cell, they encounter physical and biochemical barriers before they reach the infection or replicative site. Many of these potentially infectious TMV particles could conceivably be trapped in vacuoles, subject to proteolytic and RNase digestion, or could become lodged in other areas of the cytoplasm and never reach the infection site. TMV-RNA is vulnerable to degradation by ribonucleases once the particle has shed its protein coat. Considering these barriers to virus infection, it is reasonable to assume that the greater the number of virus particles present in the cell, the greater the chance of establishing an infection. This does not necessarily mean that one virus particle cannot cause an infection. On the contrary, a single TMV particle probably can cause an infection (4). If a virus particle could be placed in close proximity to the intracellular infection site, one infectious particle would probably be sufficient to establish infection and replication. REFERENCES 1. STEERE, R. L., Phytopathology 46, 196-208 (1955). 2. MATHEWS, R. E. F., “Plant Virology.” Academic
587
Press, New York and London, 1970. 3. FURUMOTO, W. A., and MICKEY, R. Virology 32, 216-233 (1967). 4. KUNKEL, L. O., Phytopathology 24, 13 (Abstract) (1934). 5. LAUFFER, M. A. and PRICE, W. C. Arch. Biochem. 8, 449-468 (1945). 6. FRAENKEL-CONRAT, H., In “The Viruses.” (F. M. Burnet and W. M. Stanley, eds.), Vol. 1, pp. 439-457. Academic Press, New York, 1950. 7. FRAENKEL-CONRAT, H., VELDEE, S., and Woo, J., Virology 22, 432-433 (1964). 8. KLECZKOWSKI, A. Virology 34, 186 (1968). 9. NIMS, R. C., HALLIWELL, R. S., and ROSBERG, D. W., Cytologia 32, 224-235 (1967). IO. NIMS, R. C., HALLIWELL, R. S., and ROSBERG,D. W., Protoplasma 64, 305-314 (1967). 11. RUSSELL, T. E., and HALLIWELL, R. S., Phytopathology 64,1520-1526 (1974). 12. MURASHIGE, T. and SKOOC, F. Physiol. Plant. 15, 473-497 (1962). 13. GOODING, G. V., and HEBERT, T. T., Hzytopathology 57, 1285 (1967). 14. HANSEN, A. J. and HILDEBRANDT, A. C., Virology 28, 15-21 (1966). 15. MOTOYOSHI, F., BANCROFT, J. B., and WARS, J. W., Virology 21, 159-161 (1973). 16. ATKINSON, P. H., and MATHEWS, R. E. F., Virology 32, 171-173 (1967). 17. MURAKISHI, H. H., HARTMANN, J. X., PELCHER, L. E., and BEACHY, R. N., Virology 41, 365-367 (1970).
65, 583-587
Quantity
(197%
of Microinjected Infection ROBERT
Department
of Phnt
Tobacco
of Single Cultured
S. HALLIWELL
Sciences,
Mosaic
Texas
AND
Agricuhd
Accepted
Tobacco
WILLIAM
Experiment
Station,
Virus Required
Station,
for
Cells
S. GAZAWAY’ Texas
A&M
hiuersity,
College
Texas 77843 February
10, 1975
An average volume of 1.11 pl of a standard TMV suspension was microinjected into single cultured tobacco cells using a glass capillary microneedle. The volume of virus suspension injected was calculated from 20 microneedles. The average number of characteristic TMV particles (290-310 nm) delivered was determined by direct count of the particles on slotted EM grids. All cells produced crystalline virus inclusions when injected with 620 or more TMV particles. One of two cells was infected after injection with 310 TMV particles and only one of four cells became infected after injection of 72 particles. Cell injections at lower concentrations produced no signs of infection. Injected cells producing crystalline inclusions (the criterion for infection) were infectious when assayed on Nicotiam glutinosa L. Uninoculated controls and cells bioassayed immediately after injection with the standard and lowest infectious concentration gave a negative assay.
The ability to assay infectious viruses quantitatively is fundamental to an understanding of their properties. Among the various assay methods developed for plant viruses, the local-lesion assay is the most sensitive and gives a linear response. However, under optimum conditions, the locallesion assay requires about 50,000 virus particles to initiate a single infection (I). This does not approach the assay sensitivity of bacterial and some animal viruses. According to Matthews (2), there are three possible reasons for the low level of sensitivity of assay with plant viruses: 1) The inefficiency of the inoculation process, 2) the presence of noninfectious virus particles in the inoculum, and 3) the need for two or more particles with multiparticulate viruses. Although many virus particles must be applied to a leaf surface to produce a single lesion by mechanical means, it has been demonstrated indirectly that TMV is highly infectious, and probably only one ‘Present address: Standard Limon, Costa Rica, C. A.
Fruit Co., Apartado
N, 583
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
virus particle is required to initiate infection (3-5). However, evidence that more than one infectious particle is involved also has been reported by several researchers (1, 6-8). Kleczkowski (8) suggested that areas on a leaf vary in susceptibility so that each area requires different concentrations of virus particles to cause infection. The information available indicates that either a large number of TMV particles constitutes one ‘infectious unit when purified by present techniques or very few infectious units come in contact with infection sites by the assay methods used. Nims and Halliwell (9, 10) injected purified TMV into cultured Nicotiana tabacum cv. Samsun NN callus cells, a local-lesion variety, and obtained infection in all cells. Using similar procedures with N. tabacum cv. Samsun, Russell and Halliwell (II) substantiated these findings. In these reports, cytoplasmic vesiculation was followed by crystal formation in the inoculated cells, which served as an indication of successful infection (9, 11). The single-cell microinjection method used by these workers has the important advantage that
584
SHORT
COMMUNICATIONS
known numbers of virus particles can be injected into a single cell, but the minimum number of particles required to establish infection of a single cell has not been determined. The following is a report on the minimum number of TMV particles required to cause infection in cultured tobacco cells inoculated via microinjection. Preparation of tobacco cell culture. Healthy tobacco (Nicotiana tabacum L. cv. Samsun) cells and cell-chains derived from surface-sterilized petiole sections were produced in a modified culture medium of Murashige and Skoog (12). The modifications included the addition of coconut milk (150 ml/l) and the deletion of kinetin. The medium was adjusted to pH 5.6 by adding 0.1 N NaOH. Aliquots of medium were poured into slit-bottom Bellco flasks, plugged with cotton, covered with a stainless-steel cap, and autoclaved for 20 min at 1.05 kg/cm*. The flasks were then placed on a gyrotory shaker at 150 cycles/min for 6 hr to allow resuspension of precipitates. Cell injection. Preparation of microculture chambers, microneedles, and the method of single cell injection followed the techniques of Nims et al. (9, IO). Three cells were used in each of the first four experiments. One cell was injected with a predetermined concentration of infective TMV (Fig. 1). A second cell was
FIG. 1. Injection
injected with autoclaved TMV to determine the cell response to foreign material and the effect of mechanical damage. Untreated controls were also observed in all experiments. The cell’s activities in response to injection were monitored continuously. During the period of observation, particular emphasis was placed upon: 1) The formation of callose at the injection site, 2) the rate of cyclosis, 3) cytoplasmic vesiculation, 4) the response of the nucleus, 5) time to formation of virus crystal inclusions, and 6) time of cell death as determined by termination of cyclosis. Bioassay of inoculated cells. The contents of all inoculated cells, including controls and those with crystalline inclusions, were bioassayed on a local-lesion host plant. The microculture chamber was placed under a dissecting microscope and the mineral oil enveloping the microculture was removed with a hypodermic syringe. The glass coverslip was inverted and the cell transferred with a micropipet to a drop of 0.01 M potassium phosphate (pH 8.0). The cell was then crushed with a sterile glass rod. The final volume was brought to 0.5 ml with the phosphate solution and mixed thoroughly. The preparation was then assayed by rubbing a leaf of N. tubucum cv. Samsun NN previously dusted with Carborundum. Other cells injected with similar quan-
of cultured
tobacco cell.
SHORT
tities of infectious virus were bioassayed immediately after injection to determine if the inoculum contributed to the production of local lesions. Preparation of TMV. TMV was obtained from infected tobacco leaves by the PEG method of Gooding and Hebert (13). The preparation was further purified by differential centrifugation. The final preparation was passed through a Millipore filter (0.45 lrn) and dispersed in sterile serum vials. The contents of each vial were tested on nutrient media to assure the absence of microorganisms before use. A local-lesion assay was employed to determine the relative infectivity of TMV at various dilutions. TMV suspensions were serially diluted by a factor of ten with 0.01 M phosphate buffer (pH 7.0). The greatest dilution where infectivity was detected by the local-lesion assay constituted the initial (stock) concentration of TMV to be injected into the individual single cells. By spectrophotometry, the concentration of the stock virus was estimated to be 15.4 mg of virus/l. Determination of volume injected. An average volume of inoculum injected into cells was determined by calculating the volume of 20 different microneedles. A standard quantity of the virus preparation drawn into the needle was measured with a micrometer under a microscope at 100x.
FIG. 2. TMV
crystals
585
COMMUNICATIONS
(arrows)
Measurements were taken from the needle tip to the base of the meniscus (virus-oil interface). The average volume was calculated to be 1.1 pl. Determination of quantity of TMV injected. The actual number of virus particles delivered during microinjection was determined by direct count. A drop of virus suspension was placed on a clean glass slide and the normal volume drawn into the needle. A small drop of 0.01 M potassium phosphate solution containing 0.1% albumin was placed on a Formvar-coated, carbon-reinforced slot grid with a micropipet. The grid containing the droplet was placed under a light microscope and the virus suspension in the microneedle was injected into the droplet and allowed to air dry. The grid was shadowed with a platinum-palladium alloy (80:20) and observed with the electron microscope. All of the characteristic virus particles (i.e., those particles having a length of 290-310 nm exclusive of fragments) on the grid were counted. Virus particles on four grids were counted, and an average per injection was determined. Crystal formation. The development of virus crystals as reported by Nims et al. (9) and Russell and Halliwell (11) was a reliable index of infection. Virus crystals first appeared as dark gray areas in the cytoplasm under phase contrast. As the crys-
in an inoculated
tobacco cell.
586
SHORT
COMMUNICATIONS
tals thickened, the angularity became very prominent. Fully developed TMV crystals displayed the typical hexagonal configuration (Fig. 2) described by previous workers (9, 11, 14). Virus crystals were observed as early as 45 hr and as late as 89 hr after injection. Cell injection. A total of 30 cells was inoculated with various concentrations of infectious TMV. Four control cells were injected with heat-denatured TMV. The highest concentration of virus injected was the greatest dilution that produced local lesions when assayed on a local-lesion host. This comprised the stock suspension. Subsequent injections were made with the stock suspension and more dilute virus suspensions until no infections were observed. All cells injected with stock virus and a ten-fold dilution became infected and produced virus crystals. One of two cells injected with 5 x 10d2 stock dilution produced virus crystals. The 1 x 10m2 stock dilution was the lowest concentration at which cells became infected. Only one of four cells injected produced virus crystals. Four cells inoculated with the 1 x 10msstock dilution did not become infected. Quantity of TMV injected. When all TMV particles were counted on slot grids using the electron microscope, an average of 72 TMV particles was found in each 1.11 pl of the 1 x 10e2 dilution of virus stock. Bioassay. The number of lesions resulting from bioassays of cells containing TMV crystals varied from 4-8.75 lesions/halfleaf. When control cells were microinjected with the stock suspension or any dilutions thereof and then were bioassayed immediately, no lesions resulted. Fraenkel-Conrat et al. (7) and Steere (I) demonstrated that a minimum of 50,000-75,000 TMV particles was required to cause a single infection on intact leaves. In their inoculation techniques, much of the virus inoculum was ineffective since many of the TMV particles never entered a cell. Motoyoshi et al. (15) treated tobacco protoplasts with isotope-labeled cowpea chlorotic mottle virus and determined that approximately 760 virions were required to
cause 52% infection of the protoplasts after 44 hr. By microinjection the number of viruses for infection of 100% of the cells was 620. Seventy-two virus particles were required to infect 25% of the injected cells. Although the results clearly demonstrate that only a few particles are necessary to cause an infection, the data presented in this paper are not sufficient to determine that one TMV particle alone can cause an infection, When injecting such small quantities of virus into a cell, there is no way to determine accurately the number of infectious particles actually present in any given dosage. To do so, a large number of cells would have to be inoculated with a particular concentration. For this reason, the number of virus particles injected into a cell is given within a range of possible error. Unfortunately, the microinjection technique is time consuming and is not well suited for the inoculation of the large numbers of cells that are required for statistical interpretation. Cell variability could present a problem in attempts to determine the validity of the theory that one virus particle can cause an infection. Kleczkowski (8) suggested that cells located in different regions of the leaf varied in their susceptibility of virus infection. Atkinson and Matthews (16) noted a differential response of tobacco leaf cells to TMV infection. They reported that some cells became infected while other cells remained free of the virus. However, this differential susceptibility which exists in intact plant tissues (8, 16) may not be present in cell cultures. Murakishi et al. (17) infected 94% of cultured cells by adding TMV (83 pg/ml) to a suspension of cells and vibrating the mixture with a Vortex mixer. Using the microinjection technique, all cells injected with the stock suspension or a 10-l dilution became infected. On the basis of these results and previous reports (g-11), the variability of cultured N. tabacum cv. Samsun cells to TMV infection is apparently of minor importance. The small percentage of cells that become infected when inoculated with lower concentrations of TMV could be the result
SHORT
COMMUNICATIONS
of several factors. After the virus particles have been injected into the cell, they encounter physical and biochemical barriers before they reach the infection or replicative site. Many of these potentially infectious TMV particles could conceivably be trapped in vacuoles, subject to proteolytic and RNase digestion, or could become lodged in other areas of the cytoplasm and never reach the infection site. TMV-RNA is vulnerable to degradation by ribonucleases once the particle has shed its protein coat. Considering these barriers to virus infection, it is reasonable to assume that the greater the number of virus particles present in the cell, the greater the chance of establishing an infection. This does not necessarily mean that one virus particle cannot cause an infection. On the contrary, a single TMV particle probably can cause an infection (4). If a virus particle could be placed in close proximity to the intracellular infection site, one infectious particle would probably be sufficient to establish infection and replication. REFERENCES 1. STEERE, R. L., Phytopathology 46, 196-208 (1955). 2. MATHEWS, R. E. F., “Plant Virology.” Academic
587
Press, New York and London, 1970. 3. FURUMOTO, W. A., and MICKEY, R. Virology 32, 216-233 (1967). 4. KUNKEL, L. O., Phytopathology 24, 13 (Abstract) (1934). 5. LAUFFER, M. A. and PRICE, W. C. Arch. Biochem. 8, 449-468 (1945). 6. FRAENKEL-CONRAT, H., In “The Viruses.” (F. M. Burnet and W. M. Stanley, eds.), Vol. 1, pp. 439-457. Academic Press, New York, 1950. 7. FRAENKEL-CONRAT, H., VELDEE, S., and Woo, J., Virology 22, 432-433 (1964). 8. KLECZKOWSKI, A. Virology 34, 186 (1968). 9. NIMS, R. C., HALLIWELL, R. S., and ROSBERG, D. W., Cytologia 32, 224-235 (1967). IO. NIMS, R. C., HALLIWELL, R. S., and ROSBERG,D. W., Protoplasma 64, 305-314 (1967). 11. RUSSELL, T. E., and HALLIWELL, R. S., Phytopathology 64,1520-1526 (1974). 12. MURASHIGE, T. and SKOOC, F. Physiol. Plant. 15, 473-497 (1962). 13. GOODING, G. V., and HEBERT, T. T., Hzytopathology 57, 1285 (1967). 14. HANSEN, A. J. and HILDEBRANDT, A. C., Virology 28, 15-21 (1966). 15. MOTOYOSHI, F., BANCROFT, J. B., and WARS, J. W., Virology 21, 159-161 (1973). 16. ATKINSON, P. H., and MATHEWS, R. E. F., Virology 32, 171-173 (1967). 17. MURAKISHI, H. H., HARTMANN, J. X., PELCHER, L. E., and BEACHY, R. N., Virology 41, 365-367 (1970).
