VIROLOGY 2, 820-827 (1956)

Infection of Nicotiana glutinosa L. Following Injection of Two Strains of Tobacco Mosaic Virus

into a Single Cell

G. T. A. BENDA’

Virus Laboratory, University of California, Berkeley, California,

Accepted September 18, 1966

When a mixture of the ordinary strain and the yellow aucuba strain of tobacco mosaic virus was injected into a single hair cell of a seedling leaf of Nicotiana glutinosa L., either or both strains were subsequently detected in the lesion. Of the 20 lesions examined, the ordinary strain alone was found in 14 lesions, the yellow aucuba strain alone in 1 lesion, and both strains in 5 lesions. With a single exception, lesions have appeared only on leaves on which the inoculated hair remained alive for at least one day,

INTRODUCTION

Kunkel (1934a) first demonstrated that more than one strain of virus can be isolated from a single primary lesion. He isolated both yellow aucuba and the “ordinary” tobacco mosaic virus (TMV) from lesions on leaves of Nieoliana lungsdorfii Weinm. inoculated with the mixture. This was confirmed by Lauffer and Price (1945) whose results indicated that the fraction of mixed lesions (lesions from which more than one strain can be isolated) depended on the total virus concentration of the inoculum, the two strains having been mixed at concentrations of equal infectivity.

These results do not answer unequivocally the question whether the two strains entered through a single cell, for a local lesion may well be initiated by the infection of several cells close to one another. If the inoculated strains entered through separate cells, then the mixed lesion could simply be a composite of partial lesions of the two strains; if the two strains entered through one cell, then the evidence is clear that the cell of initial infection does not have an effective mechanism for excluding more than one strain.

1 Public Health Service R&search Fellow of the National Cancer Institute.

820

STRAIN3 OF TMV IS A SINGLE (‘ELL 821

To resolve the question whether more than one strain can enter through a single cell, one rell per leaf was directly inoculated with a mixture of two strains of TMV. The results of this experiment, are the subject of this paper.

The technical problem of inoculating single hair cells has Beck ap- proached previously (Sheffield, 1936; Hildebrand, 1913; Zech, 1952). The method here adopted is an extensive modification of that employed by Miss Sheffield.

Ma4TERIALS ANI> METHOI>S

In brief, the inoculum was introduced into a leaf hair eel1 of S. gltc- tinosa by puncturing the cell through a drop of virus solution. The resulting lesion was analyzed for the presence of each of t*hc two st’rains of TMV used.

Purified preparations of both t,he ordinary strain and the yellow aueuba strain of TMV were used. The inoculum contained 11 mg of each of the strains and 0.35 ml of glycerine per milliliter (t,he partial pressure of water vapor from a glycerine solution of this conc*entration approximates that of air at a relative humidity of 78 ‘?A at 25”). A single preparat,ion of inoculum was used throughout> these studies.

Plants of N. g2ulinosa were grown from seed in &inch pots ill the greenhouse. The seeds were sown in vermiculite which overlaid a sandy loam. The first leaf was inoculated when it was roughly 3 to 5 mm long, a size which corresponds approximately to $i to s4 of its final lengt’h. Plants were pretreated by placing the pot in a bowl containiug wat,er at a temperature between 35” and 40”, covering with a beaker, and incu- bating for 4 hours at 36”. Then the covered pot was brought, into the laboratory, placed under fluorescent lights, and uncovered after 2 hours. The leaves were detached and placed on filter paper 1vhic.h had been moistened with distilled water, and which was supported on a glass slide. The tip of one hair per leaf was marked with petroleum jelly mixed wif,h lampblac~k prepared from benzene. The hair was inoculated from 3; to :< hours aft’er t’he leaf was detached.

A4fter inoculation, the leaves were placed on moist. filter paper in \~entilated dishes of transparent plastic. Light was supplied by a bank of fluorescent lights from 10 a.m. to 2 a.m. Leaves remained green for at least’ a week under these conditions. The day after inoculation, t,he hair which had been marked and inoculated was checked; if it, was upright,, it was considered “alive”, and if prone as “collapsed”. .\fter 2 t,0 :Z da,vs

822 G. T. A. BENDA

the lesions became clearly defined. The lesions were necrotic, circular, and approximately 1 mm in diameter. On the average, less than one lesion occurred for every fifteen hairs inoculated.

The micromanipulation. The inoculation required the positioning under the microscope of the hair, the pipette, and the probe. The oper- ation was observed under a reflection microscope, the Leitz Ultropak, with objective and ocularlenses magnifying 11 and 20 times, respectively. The leaf was supported on moist filter paper overlying a plastic wedge which had an angle with the horizontal of 18”. The leaf with the hair to be inoculated was moved in and out of the field of the microscope with the movable stage. The probe was moved by a de Fonbrune pneumatic micromanipulator and the pipette by a Prior mechanical micromanipu- lator. After the initial adjustment, the microtools remained, with the exception of vertical adjustments, in the field of the microscope for an entire set of leaves.

In the inoculation, the following procedure was observed. The hair which consists of a single row of cells was brought into the field of the microscope. An area of the hair suitable for inoculation was selected, i.e., a portion of the hair that was fairly rigid, that was not too close to the leaf surface, and that was higher than any other cell in the area which the microtools must traverse. The microtools were lowered to this level of the hair. A drop was deposited with the pipette, and the pipette was raised again. The cell was then punctured with the probe through the drop, and the probe was raised. The leaf was withdrawn, removed with forceps, and replaced with another leaf.

To determine whether a cell had been punctured, indirect criteria were required since the observation with the reflection microscope is limited to surface detail. The deposited drop, due to the hydrophobic surface of the hair, had a high contact angle. The lens effect of the drop, the refraction and interference of light, gave to the drop a bright, colored appearance. If the cell had been pierced, a drop of cell content was extruded and apparently retracted (Hildebrand, 1943). The change of size of the drop and the rapidly changing colors were the two criteria of puncture.

Various precautions were observed to avoid contamination. Special care was taken in selecting the part of the hair to be inoculated in order that no other cell would be touched by pipette or probe accidentally. Throughout the entire operation the leaves were handled only with sterilized forceps. Healthy leaves, handled like the treated leaves except

STHAINS OF TMV IN A SINGLE CELL 8%

for inoculation, never developed lesions. The large class of leaves with hairs which had died within a day of inoculation have, with a single exception, not developed symptoms and constitute a very discriminating control. These observations indicate that the precautions do avoid contamination.

The microtools. A satisfactory pipette must be able to deliver a drop of controllable volume. The force required to set the virus inoculum int,o motion, to create additional surface, and to counteract, the capillary attraction of glass and inoculum, exceeds the force required to keep the liquid in motion. To control the flow of the liquid, therefore, a brake ill the form of a constriction was introduced near the tip of the pipett’e. The diameter of the constriction was of the order of the tip itself.

The pipette was prepared essentially by the method of de P’onbrunr (1949). Capillary glass tubing (Kimball quality, 0.75-1.0 mm diameter) was inserted into a metal pipette holder with sealing wax, the end of the tubing was drawn out over an alcohol flame, and the tip was sealed and hooked. The pipette was then completed with the adjustable heat from an electric filament in the field of a dissection microscope. A J-gm weight was hung on the hooked portion, the tubing was bent; t.o an angle of 20” with the body of the pipette, and the tubing was extended and t,hinned a few millimeters below the bend. The weight was then removed and the tubing was constricted in the thinner portion by applying heat, locally. With the constriction completed, the weight was then returned and the point was finished off just below the constriction by heating very slowly during the final thinning of the tube to assure an even break. The outside surface of the pipette was made hydrophobic by exposing the pipette to the vapors of Dri-Film (a General Electric Co. silicone).

In the manipulation, the pipette was operated by a pneumatic system consisting of a syringe, plastic tubing, and the pipett’e holder with pi- pette. The pipette was initially filled with a portion of the inoculum estimated to be of the order of 1CF ml. The drop deposited by the pipette was less than lo-* ml. After the inoculations were completed, the pipet,te was (*leaned by repeatedly rinsing with distilled wat’er.

The probe was prepared from 2-mm Pyrex rod by progressively draw- ing out t,he tip portion over variously sized flames, and finally by heat,ing with an electric filament as described for the pipette. As wit,h the pipette, t,he rod was bent to an angle of 20” just above where the tip was to be, and the rod was drawn out first with a 2-gm weight, then with a >$-gm weight’. The final point) was prepared by et8ching the probe for 30 seconds

824 G. T. A. BENDA

in the vapors of hydrofiuoric acid and rinsing in water, a method sug- gested by Dr. Lester Goldstein.

The determination of TMV strain. The strain or strains responsible for the lesion observed following inoculation were determined by the stand- ard host reaction method for these strains (Kunkel, 19348). The method depends on the differential symptoms on N. sylvestris Spegaz. and Comes, which shows local lesions with yellow aucuba and only systemic symp- toms with the ordinary strain of TMV.

The leaf with lesion was ground up 4 days after inoculation between a ground-glass spatula and a ground-glass depression slide in two drops of distilled water. The macerate was applied with the spatula to one plant of Turkish tobacco (N. tabacum L.), to two plants of N. sylvestris, and to one plant of N. glutinosa. The leaves of these plants had been previously dusted with 400-mesh Carborundum, and the leaves were rinsed immedi- ately after inoculation. During inoculation the leaves were supported on clean paper toweling or on sterilized pot labels. The depression slide, spatula, and forceps were, of course, sterile.

In those cases where local lesions developed on N. sylvestris leaves, the virus from five local lesions was separately isolated from N. glutinosa and passed to N. sylvestris in the manner described. Furthermore, inocula from some local lesions were also passed from N. sybestris to healthy N. sylvestris.

The st,rain or strains responsible for a lesion were determined by the following criteria: a lesion was considered to have resulted from infection with the ordinary strain if only systemic symptoms appeared on N. sylvestris and the systemic disease on Turkish tobacco was a green mottle. il lesion was considered to have resulted from the yellow aucuba strain if local lesions appeared on N. sylvestris, the systemic disease on Turkish tobacco was a yellow mottle, and the virus from single lesions of N. glutinosa transferred to N. sylvestris evoked local lesions. A lesion was considered to have resulted from a mixture of both strains if the symp- toms on the inoculated leaves of N. sylvestris were necrotic lesions, and on the systemically infected leaves, a green mottle.

RESULTS

The results reported are those for twenty consecutive lesions obtained in a period of about 30 days. The lesions were assayed and the strains determined as described above. The lesions were found to have resulted from infection by ordinary TMV in 14 cases, from yellow aucuba in one case, and from both strains in five cases.

STRAINS OF TMV IN 4 HISGLIC (‘ELL 825

Under these conditions, the amount of virus deposited with the pi- pette generally did not produce symptoms when the macerated first leaves were inoculated to N. glutinosa and to N. syhestris. Thus, in tests of twenty-six leaves which had been injected but which had not devel- oped lesions, only one infection resulted, a local lesion 011 N. ghtinosa. In a further control, where the juice from each of ten leaves was inocau- lated undiluted to plants of N. sylvestris, one plant, showed systemic sympt,oms only.

Five lesions were considered to have resulted from infection by a mixt>ure of both strains. Virus from all five when inoculated direct’ly t’o N. sylnestris gave local lesions characteristic of infection by yellon aucuba and syst’emic symptoms characteristic of infection by the ordi- nary strain. The possibility that a single new strain caused the nom- bination of symptoms noted was excluded by recovering in each case one or bot,h of the strains inoculated. Virus from the local lesions 011 N. sybwstris was transferred by isolating the lesions and inoculat’ing the juice t’o healthy N. sylvestris. In one of the five cases, where virus from the local lesions was used as inoculum 10 days after inoculation, the only infections obtained were the systemic symptoms typical of t,he ordinary strain. In the other four cases, the lesions were tested within a week and local lesions were again obtained. For the three cases where the isolation was attempted, the yellow aucuba strain was obtained from the lesions and passed to Turkish ttobacco where it gave the bright yellow mot,tle characteristic of infection with it. When virus from plant,s of A;. syhstris with both systemic and local symptoms was t,aken from fragments of leaves secondarily infected and inoculated into healthy N. syhsfris plants, only systemic symptoms developed in the four (asses t.ricd.

In each of the five cases of mixed infection, virus from earh of a number of single lesions 011 the leaves of N. glutinosa was also transferred to single plants of N. syhestris. Of the twent,y-iline local lesions thus tested, nine plants gave no symptoms whatsoever, and the remainder systemic sympt’oms caharacteristir of infection with the ordinary strain. One of these plants developed a single local lesion, virus from which on further passage in N. syloestris again produced local lesions.

The experiments also gave further evidence concerning the relatiorl of the inoculated hair to its lesion. With one except,ion, lesions followed only when t,ha inoculated hair remained alive for at least one day after inoculat,ion. A total of 1157 hairs was inoculated wit,h this inoculum preceding and including the set discussed. Of t,he 694 hairs which re- mained alive for one day afber inoculation, 70 subsequently de\-(>loped

826 G. T. A. BENDA

lesions, whereas, among the 463 hairs which had collapsed within one day, only a single lesion developed.

The inoculated hair appeared to be approximately at the center of any lesion which developed. The lesion was generally first noted on the second day, and the inoculated hair usually collapsed by the third day. Occasionally, the inoculated hair in the center of the fully developed lesion was alive on the fourth day. This observation indicates that the death of the hair is not a direct effect of inoculation and infection.

DISCUSSION

The formation of mixed lesions in these tests provides unequivocal evidence that two strains can enter through a single epidermal cell, and that both can subsequently multiply. The evidence indicates that for this system the inoculated cell does not have a mechanism whereby infection can be restricted to one strain, or to one initiating particle. Recently, Siegel and Wildman (1956) have proposed that one and no more than one particle of the ordinary strain of TMV can initiate a lesion on the leaves of N. glutinosa. The authors take cognizance of the findings of Lauffer and Price (1945) on mixed lesions and suggest either that no exclusion mechanism is operative between strains or “that the exclusion mechanism is imperfect and that at high concentrations of applied virus a low level of multiple infection takes place” which was not detected in their experiment. For the data here presented, the amount of virus entering the cell is uncertain. The drop deposited on the cell contains approximately lo6 particles, but presumably only a small portion of these enter. The data thus cannot decide unambiguously between the proposed alternatives.

The results presented do not necessarily throw any light on the question of the simultaneous multiplication of more than one strain of virus in a single cell. It is possible, for example, that the cell into which the mixture of virus strains is introduced acts solely as distributor, and that the strains are separated as they pass to the cells adjacent to the hair by a chance of distribution. A consequence of such a distribution may be that in mixed lesions, the entire lesion may be divided into parts each of which contains one or the other strain exclusively.

The data indicate that one in four lesions in these experiments is a mixed lesion. This is a minimum figure which doubtless reflects more the sensitivity of N. sylvestris to a mixed inoculum than the actual fraction of mixed lesions. The assay depends upon the assumption that the char-

STRAINS OF TMV IN A SINGLE CKLL 827

acteristk symptoms of each strain are independent of the concentration of the other strain. Sadasivan (1940) has shown that a concentration of the ordinary strain in excess of yellow aucuba tends to inhibit the forma- tion of local lesions by the latter, the inhibition increasing w&h t.he excess in concentration of the former.

ACKNOWLEDGMENTS

I wish t.o thank particularly Professor W. M. Stanley for making the facilities of the Virus Laboratory available throughout the work, Dr. It. C. Backus and 1)r. Roger Hart for their valuable discussions, Dr. C. A. Knight for the gift of purified virus, and Mrs. Winnifred Elliott and Mr. J. McNultp for their friendly help.

REFERENCES

I)@: FONBRUSE, P. (1949). ‘I’echnique de Micromanipulation. Masson, Paris. HILDEBI~ND, E. M. (1943). Micrurgy and botany with special reference to phyto-

pathology. In Micrurgical and Germ-free Techniques (a. A. Reyniers, ed.), pp. 72-92. C. C Thomas, Baltimore.

KUSKIm L. 0. (1934a). Abstract: Tobacco and aucuba-mosaic infections by single 1mits of virus. Phylopathology 24, 13.

KUNKEL, I,. 0. (1934b). Studies on acquired immunity with tobacco and aucuba mosaics. Phytopathology 24, 437466.

IAUFFER, IN. A. and PRICE, W. C. (1945). Infection by viruses. .-I wh. Biochem. 8, 449-468.

SADASIVAN, T. S. (1940). A quantitative study of the interaction of viruses in plants. .4nn. Appl. Biol. 27, 359-367.

SHEFFIELD, F. M. L. (1936). The susceptibility of the plant cell to virus disease. i2nn. Appl. Biol. !U, 498-505.

SIEGEL, .4., and WILDMAN, S. G. (1956). The inactivation of the infectious centers of tobacco mosaic virus by ultraviolet light. virology 2, 69-82.

ZECH, H. (1952). Untersuchungen iiber den Infektionsvorgang und die Wanderung des Ts.bakmosaik-virus im Pflanzenkgrper. Planta 40, 461-514.