STUDIES QN THE VIRUS INQCULATION
0F DISSOCIATED LEAF
MESOPHYLL CELLS

”oasis for Ike Degree of DIL D.
MICHIGAN STATE UNIVERSITY
Roger M. Knutson

1965

 

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LIBRARY

Michigan State
University

THESIS

 
    
   

This is to certify that the

thesis entitled

STUDIES ON THE VIRUS INOCULATION
0F DISSOCIATED LEAF

MESOPHYLL CELLS

presented by

Roger M. Knutson

has been accepted towards fulfillment
of the requirements for

._Ph-_Q_-__degree mm; Plant Pathology

Zam/ yfiZAM/(AI/dvl”

flajor professor

 

2 , ~
Date 5-44;ng 2y "Iggy

 

0-169

ABSTRACT

STUDIES ON THE VIRUS INOCULATION
OF DISSOCIATED LEAF MBSOPHYLL CELLS

by Roger M. Knutson

The value of single-celled organisms in the study
of cellular physiological processes is widely recognized.
In the study of virus infection and biosynthesis, the
phage-Q. 32;; model has provided the basis for most pro-
posed mechanisms. The ideal system for study of virus
synthesis and related cell processes is a pOpulation of
single, functional cells acting simultaneously. The
study of higher plant virus infections at the cellular
level has been inhibited by the necessity of using the
leaf or portions of it as the unit of virus multiplication.

This study reports the infection of single, function-
al leaf-mesophyll cells. Such cells were obtained by the
action of pectinase enzymes on strips of leaf tissue, and
were maintained on a simple mineral nutrient medium plus
sucrose for experimental periods. Cells were inoculated
by the use of heat and violent agitation with an abrasive,
and virus in excess of the quantity used as inoculum was
detected in the cells by bioassay on a local lesion host
plant.

Mesophyll cells infected with tobacco mosaic virus

produced materials inhibitory to infection with that virus

Roger M. Knutson

on local lesion hosts. Use of acridine orange stains
and fluorescent antibody precipitations was unsuccessful

in determining specifically which cells were infected.

STUDIES Um Ind VIRUS INOCULATION
OF DISSOCIATED LEAF MESOPHYLL CELLS
By

0

3
Roger M? Knutson

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1965

Pu

ACKNOWLEDGMENTS

Although a single name appears as author, many people have
contributed, knowingly or unknowingly, to this work. Fore-
most among knowing contributors is Dr. H. H. Murakishi,
whose help has been most welcome and most essential. The
graduate students of the Department of Botany and Plant
Pathology have provided most of the immediate environment
in which I have worked for the past seven years. Their
help and that of the total department faculty is more dif-

ficult to evaluate, but no less important.

ii

DEDICATION

To my father, whose concern for the education of his
children made it possible, this thesis is most sin-

cerely dedicated.

iii

 

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TABLE OF

Introduction . . . . . .
Literature Review. . . .
Introduction. . . .
Cell Availability .
Virus Infection . .

Cellular Activity .

. Materials and Methods. .

Origin of Materials
Assay Method. . . .
Observations and Results

Cell Preparation. .

CONTENTS

Preliminary Experiments . . .

Inoculation Method.

Virus Synthesis . .

Inhibitor Formation .

Infected Cell Detection . . .

Discussion and Conclusions . . . .

Cell Maintenance. .

Cell Inoculation. .

Inhibitor Production.

Validity of Results
Conclusions . . . .

Literature Cited . . . .

iv

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10-

11-

LIST OF FIGURES

Relationship of degree of abrasion
to lesion numbers produced by rub-
bing with polyurethane pads . . . . .

Effect ofovarious periods of heat-
ing at 45 C. during the inoculation
procedure on quantity of virus re-
covered at 40 hours . . . . . . . . .

Use of the centrifuge as a means of
inoculation . . . . . . . . . . . . .

Effect of time of mixing with vortex
mixer and celite in the medium on
virus recovered at 40 hours . . . . .

Respiration of dissociated cells in
various media . . . . . . . . . . . .

Respiration of dissociated tomato
leaf mesophyll cells in various
stages of the inoculation procedure .

Virus recovered from cells frozen
and thawed before inoculation . . . .

Recovery of virus from cells inocu-
lated with and without heat and
vortex mixing . . . . . . . . . . . .

Separate effects of virus dilution
and cell dilution on virus recovered
at 24 hours . . . . . . . . . . . . .

Loss of infectivity from medium sup-
porting cells which produced virus. .

Loss of infectivity in cells isolated
from systemically infected tomato
leaves and incubated in Vickery!s
solution plus sucrose. . . . . . . . .

Increase of virus quantity with time .

Page

20

25

25

2’?

27

29

51

51

54

56

57
4o

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Figure 15- Results of three experiments showing
decline in virus recovered from cells
in the period from 12-24 hours after
inoculation . . . . . . . . . . . . .

Figure 14- Results showing a peak in virus
quantity at seven hours . . . . . . .

Figure 15— Plot of the high values obtained
at each time interval after inocu-
lation. O O O O O O O O O O O O O O 0

Figure 16- Plot of 22 comparable experiments in

which cells were inoculated and later
assayed for virus content . . . . . .

vi

 

 

 

IIST OF TABLES

page

Table 1- Inhibition of infection by cell and
culture fluid extracts. . . . . . . . . . . 47

vii

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INTRODUCTION

Plant viruses have been the subject of intensive in-
vestigation since early observations that certain maladies
of plants could not be associated with any microbial agent.
From the time of Stanley's and Bawden's crystallization of
tobacco mosaic virus and its recognition as a definable
chemical entity, investigators have been aware of the po-
tential of virus-host systems in the elucidation of cellular
physiological events. Understanding of higher plant virus
infection at the cellular level has been slow in coming be-
cause of the difficulty of studying the infection process
in single cells. Whole plants are the usual units, and
there has been little change in the induction of virus in-
fection since 1929 when Holmes demonstrated the efficacy of
mild abrasion in enhancing infection. Today, as then, virus
is rubbed on plant leaves with a variety of implements in
such a way as to cause slight injury to the leaf. A small
number of cells of the epidermis become infected, and the in-
fection gradually spreads from cell to cell until most, if
not all living cells of the leaf and eventually the plant,
contain virus which they themselves have produced.

If the whole plant is considered as the infected unit,
the process described above can demonstrate the effects of
environmental and physiological changes on virus replication

or symptom expression. The literature of virology contains

 

 

 

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tic

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many references to the effects of temperature, light and
other factors on virus quantity in the plant. Portions of
the plant, leaves or leaf discs, have been successfully
used in observations of the effects of various materials
or conditions on virus synthesis or virus quantity. As
the unit of study becomes smaller, it is increasingly less
certain that it contains even one of the original infected
cells of the leaf. Study of the very early stages of cel-
lular infection is most difficult with systems using
leaves or portions of leaves.

The functional unit of virus infection.is the single
cell. Furthermore, from what is known of virus synthesis,
we can consider pathogenesis as a genetic rather than a
metabolic phenomenon (Diener 1965). A virus particle en-
ters a single cell and causes its own replication in that
cell at the same time that infection is transferred to ad-
joining cells. The picture of virus infection at this level
is that of one cell infected with a virus and producing its
quota of new, complete virus particles. This does not oc—
cur in infected plants since adjoining cells become infected
before the first cell has produced its full complement of
virus. Study of cellular processes during virus infection
must be in single cells going through one cycle of infec-
tion without being in organic contact with other cells. Out-
lined in full, the requirements of the ideal system for study

of virus infection and replication at the cellular level are

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as follows. Single, physiOIOgically functional cells of
one kind must be available; they must be introduced to

the virus simultaneously; and all or a known proportion
must be infected. A single cycle of infection must occur,
and virus produced must be effectively measurable. The
similarity of the above requirements to the actuality of
the phage-bacterial system is not accidental. The basic
value of information obtained in many fields with the phage
system is a testimonial to how closely it approaches the
ideal.

I believe that an approach to such a system is possi-
ble with mechanically transmitted viruses of higher plants,
and the work reported here is dedicated to that belief.

Single cells of higher plants are available by tech-
niques of maceration or enzymatic digestion, and such
cells have at least some of their in-place physiological
activity. If leaf blades are used as the cell source, most-
ly mesophyll cells are obtained by enzymatic digestion.
Single cells obtained from callus tissue cultures often
demonstrate gross physiological and morphological differ-
ences. However, clones developed from single cells do not
present these problems (Muir, Hildebrandt and Riker 1954,
1958).

Since higher plant viruses have no intrinsic means of
bridging the cell wall, means of passing the wall or avoid-
ing it are a necessary part of any technique. Simultaneous

infection of cells can thus be relatively assured by the

 

 

neat

4

necessity of passing the cell wall.

The work presented here is not a study of the physio-
logical events of early virus infection, nor is it concerned
with the precise manner of virus entry into the cell, al-
though studies of both might easily develop from it. The
data and discussions involve only the demonstration that
the infection of single isolated plant cells by viruses is
possible, and that some cells are able to produce virus in
significant amounts while supported on simple artificial

media.

LITERATURE REVIEW

Introduction. The experimental work reported here is

 

concerned with infecting single plant cells with viruses
and observing the effects of such infection. There are
few reports of such work in the published literature, and
so far as I am aware, they have all utilized cells from
plant callus tissue cultures (Gautheret, 1959). Single
cell research as a model system for the study of cellular
physiology has great value. The elucidation of the reac-
tions and primary products of photosynthesis was not simple
using Chlorella and Scegsdesmus (Bassham, 1962), but it
would have been much more difficult if whole leaves of
higher plants had been the experimental units. The present
study of cellular virus replication is similarly complicated
by the use of leaves or portions of leaves as the units of
infection. White (1959) explicitly stated the point when
he said, "The cell is to biology what the molecule is to
chemistry, it is the lowest common denominator of all life."
This work is an attempt to reduce the study of plant viruses
to the level of that lowest common denominator.

gill availability. The first requirement if single
cells are to be the unit of study is some means of obtaining
then. Free-floating callus cells from tissue culture are

available with appropriate culture techniques (Steward 23 gl.,

5

1964; Northcroft, 1951). Quantities of physiologically
active cells have been produced by careful grinding of
leaves (Racusen and Aronoff, 1955), although the particular
cells observed lost photosynthetic capacity rapidly and
seemed to be deficient in protein synthesizing ability.

The knowledge that pectic materials and calcium ions
occupy much of the interlamellar volume in plant tissues
has been available for a long time, as has the knowledge
that certain fungi produced enzymes attacking these mater-
ials (Wood, 1960). The use of pectic enzymes of various
sorts, mostly pectin polygalacturonase (Devel and Stutz,
1958), for the gentle separation of cells was a logical
development from that knowledge. The earliest reference
to the use of pectic enzymes for cell separation is by
Emsweller and Stuart (1944) who macerated anther tissue
with fungus culture filtrates to facilitate observation
of meiotic figures. Later, other workers used similar
method to hydrolyze root tips and other plant materials
(Hohl, 1948; Chayen, 1949). Commercial preparations of
pectinases from Rhizopus tritici became available about
1950, and Orgell (1955) used such commercial materials to
prepare plant cuticle for studies of foliar absorption.
Tamaoki 23 gl. (1960) treated callus tissues from culture
with pectinase to help eliminate polysaccaride substances
from mitochondrial preparations.

Enzymatic separation of cells for the specific purpose

of studying virus replication within them was first accom-

 

 

 

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plished by Zaitlin in 1959. He obtained whole, individual
mesophyll cells from tobacco leaf tissue, and was able to
demonstrate that such cells from tobacco mosaic virus (TMV)

1“ labelled

infected plants, upon incubation, incorporated C
glycine or leucine into virus protein. Under some circum-
stances tissues do not separate easily with pectinase treat-
ment, and since ethylenediaminetetraacetic acid (EDTA) had
been shown to be effective in cell separation (Letham, 1960),
Zaitlin and Coltrin, @964) used EDTA in combination with
more highly purified enzymes to separate cells from more
refractory tissues. Earlier electron microscope observa-
tions (Wood 23 21., 1952) of pectinase separated cell walls
revealed that the fibrillar portion of the cell wall re-
mained intact and without holes.

The viability of cells obtained by any of the above
means is a problem. Racusen and Aronoff (1955) reported
that free cells produced no protein while excised leaf ma-
terial did. They also noted a drop of 80% in photosyn-
thetic capacity of single cells after one hour. In con-
trast with this are other observations indicating that some
cellular activities are continued for long periods of time
in dissociated cells. Chayen (1949) observed no mitotic
figures initially in root tip cells separated with pec-
tinase enzymes, but after four days in ringer's solution,
the cells had apparently regained mitotic capacity. As
previously noted, tobacco cells obtained by Zaitlin incor-

porated labelled amino acids into complete TMV. There are

 

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other observations of long continued prot0p1asmic stream-
ing in cells as an indication of metabolic activity (Wild-
man 33 gl., 1962; Hirai and Wildman, 1965). Previous work
indicates then, that single cells may be viable under quite
a variety of conditions. There is, furthermore, some in-
dication from whole leaf studies that ability to synthesize
virus is retained even after synthesisof normal protein

is suspended (Takahashi, 1941).

Ziggg infection. The manner of virus entry into the
cell is one of the questions of vital concern in the in—'
fection of single cells with plant viruses. When whole'
leaves or portions of leaves are inoculated with virus
preparations, the most common assumption has been that
virus enters the cells through small temporary wounds and
remains inside when the wound healing process is completed.
The cells so infected are epidermal cells covered with a
layer of cutin which must be at least locally interrupted
if virus is to enter living cells through the hypothesized
wounds. The leaf hairs have been considered another po-
tential site of entry, an idea lacking current, wide ac-
ceptance. Ectodesmata, protoplasmic strands extending
through the cell walls but not through the cutin layer,
are currently considered the most likely sites of attach-
ment and entry (Franks, 1961; Mundry, 1965; Brant, 1964).

Virus apparently can penetrate neither the tonoplast
or the plasmalemma of uninjured cells (Mundry, 1965), al-

though there are reports of infection occurring in single

 

 

leaf hair cells from the contact of cytoplasm with concen-
trated virus preparations (Zech, 1952). Tissue culture

cells incubated with sterile TMV have become infected with
and without artificial wounding (Kassanis 33 31., 1958; Wu
gt gl., 1960; Bergmann, 1959). There is then the possibility
of viruses establishing infection in individual cells without
abrasion. There is some evidence for the occurrence of pine—
cytosis (the uptake of small discrete volumes of material by
invagination of the cytoplasm) in plant cells (Jensen and
McLaren, 1960). Pinocytosis could provide a means for virus
entry into the cytoplasm by cell activity rather than by
lodging in wounded areas. Virus can be introduced by wounding
single cells as shown by Bends (1958) who inoculated single
cells of leaf hairs by scratching the surface with a single
grain of carborundum in the end of a fine glass rod.

Once virus has entered or been taken into a cell, a com-
plex series of reactions begins, leading to the final synthe-
sis of viral particles with a character determined by the
nucleic acid introduced. Since the concern of this thesis
is a single cycle of infection in one cell, the timing of
initial events in that first cell is important. The avail-
able information has come from several kinds of experiments.
The earliest and most obvious measurement was increase of
virus concentration in the infected plant. Later work con-
cerned changes in the character of the infectious unit as
determined by reaction to ultraviolet irradiation or HCl.

Finally, removal of the epidermis at various intervals

 

into

der:

 

 

 

10

after inoculation has given information on virus movement
into underlying cells or from underlying cells to the epi-
dermis.

Whole plant assays at intervals after infection show
a generally sigmoid increase in virus concentration. Such
results are, however, the product of two processes; the in-
crease in virus per cell, and the spread of virus from cell
to cell (Harrison, 1956). These two processes are insepa-
rable when the only information available is the total
quantity of virus present at some interval after the time
of inoculation.

Work reported on plant viruses in tissue culture deals
mostly with callus or crown gall tissue grown from virus
infected plants (Kassanis, 1957; Hildebrandt, 1958). There
have been few attempts to infect previously healthy tissue
cultures with TMV (Hildebrandt, 1958; Augier de Montgremier
g: g1., 1948; Kassanis 33 31., 1958; Bergmann, 1959). Sev-
eral papers (Kassanis gg,g1., 1958; Bergmann, 1959) report
the infection of cell suspensions in liquid media. The
incubation period used is long, and simultaneous infection
was not attempted. However, subculturing at various inter-
vals after inoculation, and subsequent assay of progeny may
give information on the time of virus entry and percentage
of cells infected. Studies of animal cells present no
problem of one cycle of infection since they, like bacteria,
release the mature virus into the medium (Ackerman, 1954).

Kassanis gt 31. (1958) seem to suggest that such release of

 

 

 

virus
evide

of su

ll

virus occurs in callus tissue infected with TMV, but their
evidence is not conclusive, and there is no other report
of such a phenomenon.

Post-inoculation changes in the response of virus
particles to altered environments have provided almost the
only information on early events following inoculation of
plants. Treatment with ribonuclease during the first hour
decreases the number of infections with TMV, while later
treatments have no effect.(Bawden and Harrison, 1955). Two
hours after inoculation, the virus is less readily inacti-
vated by ultraviolet irradiation. At about four hours the
UV inactivation curve departs from a first order slope, in-
dicating the presence of more than one infectious unit per
cell (Bawden and Harrison, 1955); a result confirmed for
TMV by Siegel and Wildman (1956). After six hours, UV ir-
radiation cannot prevent the further development of infec-
tion, indicating Spread of infectious units to cells below
the epidermis. The process suggested by these experiments
is one of rapid attachment to a temporary site on the cell
surface, penetration into the cell, loss of protein cover-
ing, multiplication in the cell and spread to adjacent
cells (Harrison, 1959). While results are indirect, and
the effect of UV or RNAase on the cells themselves is not
evaluated, movement into adjacent cells at six hours is
probably the most reliable measurement. Semal (1962) re-

ports a period of six to eight hours during which TMV in-

 

 

 

 

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fection sites on Nicotiana glutinosa are susceptible to

 

inactivation by treatment with 0.1 N HCl, indicating a
change in state of the infected cell or site at about
that time.

Since the large proportion of uninfected to infected
cells early in disease development is the major problem
in determining virus increase in leaf tissue, any re-
duction in this prOportion would be valuable in obtain-
ing information on virus increase in single cells. Dykstra
(1962) and Fry and Matthews (1965) have accomplished such
a reduction'by using the attached epidermis as the in-
fectible unit. ‘By their calculation, this gives about an
eight-fold increase in the proportion of initially in-
fected cells. Migration of infection into the mesophyll
occurs at about four hours after inoculation, and whole
virus is first noted in the epidermal cells at seven
hours. Tobacco mosaic virus-ribose nucleic acid
(TMV-RNA) was used as inoculum to eliminate the confusion
of residual virus on but not in the cells. This work pro-
vides the first good estimates of the time after inocula-
tion during which the plant cell produces infectious units
and whole virus. It also gives strong indications that
the cell to cell movement of infection in the leaf is by
RNA rather than whole virus.

Much slower movement of virus is revealed by the
system of Hirai and Wildman (1965). They detected no in-

crease in the infectivity of epidermal strips before the

 

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13

third day following inoculation. Residual virus could

have masked early increases, since intact TMV was their
inoculum for the underside of stem epidermal peels. Hair
cells removed from epidermal peels showed no increase in
infectivity prior to four days, which would indicate very
slow movement of virus compared to the results obtained by
Fry and Matthews. Nuclei in hair cells were observed to
move toward the base or infected area at three days indi-
cating some activity in these cells by 72 hours after virus
was applied. .

Cellular Activity. Activities of infected cells or
infected plants have been investigated by numerous workers.
Experiments involving direct observation of infected cells,
whether live or stained, and investigation of metabolic
activity or products have provided a basis for much spec-
ulation on the mode of virus synthesis. While direct cel-
lular observations present numerous interpretive problems,
and metabolic investigation of tissues is subject to errors
introduced by the variety of cell types and states, some
information so obtained has relevance to the study of
single cell infections.

The laboratory of J. G. Bald has been the source of
most recent cytological investigations of the virus in-
fected plant cell. Stains for RNA and intact virus have
been developed or modified (Bald, 1949, 1949a), and ob-
servations of live cells with phase microscopy have been

correlated with the results of staining procedures (Bald,

 

 

 

 

 

or C

and

hour

C02}

rang

14

1964). The greatest difficulty with such work involves
obtaining cells in early stages of infection when virus
is being actively synthesized. Cells are obtained from
tobacco plants three to five days after inoculation (Bald,
1964) and there is no certainty that the cells observed
have not been infected for some time. Bald and others
have made unquestioned observations of the early forma-
tion of crystalline inclusion bodies and the so-called
X-bodies (Solberg and Bald, 1962), but relating nucleahur
and nuclear observations to stages in virus synthesis is
difficult (Bald and Solberg, 1961). Cell RNA and virus
RNA cannot be differentiated by staining, and the effect
of virus presence in the cell on cellular RNA synthesis
or on cell ageing is not clearly understood. Since Fry
and Matthews (1965) report virus synthesis within six
hours after inoculation and Zach (1952) has observed what
is interpreted as RNA synthesis within 50 minutes of virus
introduction, the timing of cytological observations is
critical. Positive correlation of cytological observa-
tions with timed events in early virus synthesis would
make such observations extremely valuable.

The detection of virus within the cell requires
methods more specific than the usual cytological stains.
The preparation of flourescent dyes which can be attached
to globulin protein gives such specificity for some virus
components. Such dyes flouresce in the visible light

range when irradiated with ultraviolet of an appropriate

 

 

 

fee

recl

15

wave length, and can be detected in very small quantities
by microscopic observation. The labelling dyes can be
quickly and simply conjugated with serum containing anti-
bodies specific for almost any virus (Rinderknecht, 1962).
Early techniques developed with animal cells and their
viruses allowed investigators to count infected cells in
tissue cultures (Deibel and Hotchin, 1959). Successful
flourescent antibody staining has also been reported for
pectinase-separated plant mesophyll cells (Nagaraj, 1962).
The autoflourescence of plant cell walls and chlorophyll
reduce the value of the method somewhat as compared to
use with animal tissues, but it should be useful under
proper circumstances.

Experimental measurement of respiration in virus in—
fected tissues has been accomplished by many workers. A
recent review (Millard and Scott, 1962) summarizes most
of the available information on respiration of virus in-
fected tissues. Increases and/or decreases in overall
respiration have been detected, depending on condition of
plants, time after infection and numerous other unknown
factors. Owen (1955) reported increases in respiration
of tobacco leaves within an hour of inoculation with TMV,
but he could not correlate the increase with any other
manifestation of infection. Most such work has only em-
phasized that plant to plant variation is great enough to
obscure any virus induced change (Merritt 1960). Since

both wet and dry weight of infected tissue changes with

 

 

 

 

time

ment,
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16

time after infection, expression of respiration measure-
ments compared to a healthy control is difficult. The
energy for virus synthesis must come from increased met-
abolic activity, but the accurate measurement of that
activity is difficult.

Specific and non-specific inhibitors of virus in-
fection are among the products of virus infected cells.
Ross (1961, 1961a) reported systemic induced resistance
to virus infection, and considered the possibility that
the resistance was due to the movement of some inhibitory
material. The reaction.described may or may not be the
result of non-specific tissue necrosis (Bozarth gt g1.,
1962; Bozarth and Ross, 1964). Ioebenstein (1962,1965)
produced a similar resistance to virus infection by inocu-
lating plants with native virus protein, and later (Loe-
benstein and Ross, 1965) succeeded in isolating an inhib-
itory material from uninfected tiesues on a plant with
local lesions. The material is non-specific in that it
inhibits infection by several viruses. Yarwood (1955)
also described inhibition of infection on hypersensitive
hosts, and found inhibitory substances in extracts from
non-lesioned tissues. Sela and Applebaum (1962) discovered
and later (Sela 33 g1., 1964) partially purified a sub—
stance containing both protein and RNA, with inhibitory
properties similar to those described by others. With the
experimental systems used, it is impossible to determine

whether the inhibitory materials are the product of in-

 

 

 

time
me at
ener
abol

acti

feci
R05:
to 1

the

bane
Itor

leca

also
hosts
non-1

and 1

16

time after infection, expression of respiration measure-
ments compared to a healthy control is difficult. The
energy for virus synthesis must come from increased met-
abolic activity, but the accurate measurement of that
activity is difficult.

Specific and non—specific inhibitors of virus in-
fection are among the products of virus infected cells.
Ross (1961, 1961a) reported systemic induced resistance
to virus infection, and considered the possibility that
the resistance was due to the movement of some inhibitory
material. The reaction described may or may not be the
result of non-specific tissue necrosis (Bozarth gt g1.,
1962; Bozarth and Ross, 1964). Ioebenstein (1962,1965)
produced a similar resistance to virus infection by inocu—
lating plants with native virus protein, and later (Loe-
benstein and Ross, 1965) succeeded in isolating an inhib-
itory material from uninfected tissues on a plant with
local lesions. The material is non-specific in that it
inhibits infection by several viruses. Yarwood (1955)
also described inhibition of infection on hypersensitive
hosts, and found inhibitory substances in extracts from
non-lesioned tissues. Sela and Applebaum (1962) discovered
and later (Sela 33 31., 1964) partially purified a sub-
stance containing both protein and RNA, with inhibitory
properties similar to those described by others. With the
experimental systems used, it is impossible to determine:

whether the inhibitory materials are the product of in-

 

 

 

! .
,.
‘u—-"

is a
the 3
know;

stat«

large
If ti
allO'
be h-

at t]

17

fected cells or materials produced by the uninfected cells
stimulated by some non-specific product of the infection.
Potential inhibitors may act during any stage of the virus-
host cell interaction, making assay results difficult to
interpret. The subject of inhibitors of viral infection

is a most complicated one, and will only be clarified when
the precise mode of inhibition at the cellular level is
known; something far insthe future considering the present
state of knowledge.

The problems and areas of research discussed cover a
large part of the whole field of present day plant virology.
If there is any unifying principle it is that a system
allowing the simultaneous infection of single cells would
be helpful in the study of many aspects of virus infection

at the cellular level.

 

 

 

fec:

 

stir
Pots
host
inte
is a
the ‘
know.
state
large
If tI
allov
be he

at 2*

l7

fected cells or materials produced by the uninfected cells
stimulated by some non-specific product of the infection.
Potential inhibitors may act during any stage of the virus-
host cell interaction, making assay results difficult to
interpret. The subject of inhibitors of viral infection

is a most complicated one, and will only be clarified when
the precise mode of inhibition at the cellular level is
known; something far in3the future considering the present
state of knowledge.

The problems and areas of research discussed cover a
large part of the whole field of present day plant virology.
If there is any unifying principle it is that a system
allowing the simultaneous infection of single cells would
be helpful in the study of many aspects of virus infection

at the cellular level.

 

 

 

 

sect
spec

the

MATERIALS AND METHODS

Since many of the experiments performed here were
concerned with development and evaluation of what is ba-
sically a method, only general information relating to
the experiments as a whole, will be presented in this
section. Materials and methods as they relate to the
specific experiments will be presented and discussed with
the results.

Origin 21 Materials. The virus used for most exper-
iments was a rib grass strain of tobacco mosaic virus
(obtained by Dr. H. H. Murakishi from Dr. F. 0. Holmes
in 1958) known hereafter as HRG. The virus has been
maintained primarily in Nicotiana tobaccum var. White
Burley. When purified virus was needed it was prepared
from extracts of infected White Burley plants by the com-
mon methods of alternate low (10,000G) and high (95.000G)
speed centrifugation. When plant extracts were used di-
rectly as a source of inoculum, the leaves were frozen,
ground, strained through cheesecloth, centrifuged at low
speed and appropriately diluted. Tobacco Necrosis Virus
(TNV) used in several experiments was obtained from Dr.
William Hooker. It was increased in primary leaves of
bean and stored frozen until used.

Pinto bean plants used for bioassay of virus concen-

tration were grown in a greenhouse without strict tempera-

18

 

 

 

 

 

 

 

was
nine

uni f

for

SGeE
the 1
imat.
0n tI

rUDb:

l9

ture control. Most frequently, seeds were planted in a
1:1 sand—peat mixture at a rate of two seeds per three-
inch pot, although occasionally five or six plants were
grown in larger pots. Whenever possible inoculations were
made ten days after planting, although developmental stage
rather than precise age of the plants was used as a guide.
Plants with the primary leaves well expanded and with the
first trifoliate leaves still folded seem most susceptible
under our conditions. Seasonal variation in rate of growth
was noticeable, but plants were never inoculated prior to
nine days or after twelve days. Plants were selected for
uniformity just prior to inoculation.

Agggy method. The plants used for most assays were
Phaseolus vulgaris Var. Pinto. Variation in response of
individual plants of the same variety has been observed,
but the ease and speed with which large numbers of bean
plants can be grown more than compensates for this diffi-
culty. Since only the opposite primary leaves are used,
four nearly identical half leaves are available for inoc-
ulation and comparison on each plant.

A variety of implements have been effectively used
for inoculation. The experience of the operator would
seem to be more important than the apparatus. In most d?
the work reported here, pieces of polyurethane foam approx-
imately 1" X 1" X h" were used to distribute the inoculum
on the leaves. Leaves were not damaged, even by repeated

rubbing, and the distribution of the resulting lesions was

 

 

uni

ate

the

lee:

any

onl-

by t

 

a 6 - ‘1‘
I¢OdIOH Ho honld: Oluh0b<

CL

ing

on t

V

NOrs

 

20

uniform over the leaf surface. The pads were washed and
steam sterilized between uses. Since repeated rubbing of
the leaf'surface caused no microscopically visible injury,
an experiment was done to determine the relationship of
lesion numbers to degree of abrasion. The number of times
any one point on the leaf surface is rubbed should be the
only important variable since pressure is largely controlled

by the consistency of the foam pad.

 

    

lwmnuprumflnm'utlsmnww
9;

       

no 2! 30
Influu'eflwumoetdxhtnrumhltpud

ll

Fig.1.éRe1ationship of degree of abrasion to lesion
numbers produced by rubbing with polyurethane
pads. Twelve half-leaves per determination.

\

Since lesion numbers were not increased significantly
(P . .05) (Bailey 1959) by abrasion beyond 15 passes, cover—
ing the leaf about three times over, 10-15 rubs, depending
on the size of the leaf, were used for all determinations.

In order that results of experiments might be compared

more directly, the same arrangement of assay samples was

 

 

21

maintained whenever possible. Each sample to be tested
appeared on one half-leaf of six different plants, with
one check half—leaf on each plant. Lesion numbers were
totalled for the experimental and check inoculations on
the same plants, and results expressed as percent of
control. Lesions were counted at six to seven days since
all lesions do not develop simultaneously and few lesions
develop on Pinto bean after six days (Peacitelli and
Santilli, 1961).

Several experiments were conducted to assure that
the assay method would give reliable results. Bailey
(1959) is the source of all statistical formulae used in
the following evaluation.

The experimental design was such that the check
appeared most frequently on the right half of the leaf,
and since the operator is imperfectly bilaterally sym-
metrical, error could result from more abrasion on one
half than on the other. Accordingly, an experiment was
performed with 52 plants: 16 with the check on the right
half and 16 with the check on the left. Means of the two
sets are essentially identical, with no significant dif-
ference at the P = .01. Although these results indicate
that location in the four possible positions on the assay
plant has no effect on lesion numbers, a further test was
designed with ten plants. All 40 half leaves were care-
fully inoculated with the same concentration of a purified

virus preparation. lesions were read and tabulated for

22

analysis of variance. As might be expected, there were
significant (P = .05) differences between plants, but even
if the most disparate within-plant differences were selected
and six such plants compared, the differences between half
leaves on the same plant were not significant. The vari-
ance from sources other than between plant and between
half-leaves was high in all cases; a not unexpected result
considering the known sources of variability in the local
lesion bioassay for plant viruses.

As a final check on the validity of the assay method,
three dilutions of purified virus were made at 1-500, 1-1000
and 1—2000 from an original concentration determined to be
5 mg/ml by Takahashi's (1951) spectrophotometric method.
Pinto bean plants were inoculated with these dilutions;
each dilution appearing on every plant. Using the 1-1000
dilution as a base, the results expressed as proportion of
that dilution are l.84:1.0:0.58, which results are not
significantly different from 2:1:0.5. Bawden (1964) has
indicated that quantitative statements about amount of
virus must be made with great caution if lesion numbers
are the only quantitative information available. Accord-
ingly, most relative infectivity information is presented
in graphs showing trends and general relationships rather

than tables giving precise numbers.

OBSERVATIONS AND RESULTS

Cell preparation. The method used for preparation of

 

leaf mesophyll cells was essentially that of Zaitlin (1959),
modified somewhat for use with plants other than tobacco.
Zaitlin cut leaves in narrow strips and shook them in a
solution of .55 M (12%) sucrose, with .01% Pectinase (Nu-
tritional Biochemicals Co.) and 1/15 M Sorensen's buffer

pH 6-6.5. This concentration of sucrose works well for
tobacco leaves, but best separation of cells from tomato
leaves was obtained at about .25 M (8%) sucrose. Tomato
mesophyll cells are generally larger than tobacco cells

and more easily plasmolyzed, as determined by the effects
of various sucrose concentrations on strips of leaf ob-
served microscopically. Young succulent leaf tissue pro-
vides better yields of cells more quickly, and indeed,fully
hardened tissues frequently do not separate noticeably even
in double the usual pectinase concentration. The addition
of Ethylenediaminetetraacetate (EDTA) (.001 M) as used by
Zaitlin and Coltrin (1964) has proven beneficial for some-
what hardened tissues. Cells obtained by Zaitlin's method
or modifications of it were washed repeatedly by centri-
fugation in .18 M (6%) sucrose-buffer, pH 7, and used as
starting material for further experiments.

(Preliminary Experiments. Simple sucrose-buffer and

 

White's Solution with and without ferric citrate were used

25

24

as media in preliminary attempts to bring about detectable
virus infection and replication. The virus (HRG) was

added to the cell-medium mixture, which was centrifuged

and resuspended four times. Cells were harvested at 72
hours, frozen, ground and centrifuged. A trace of antigen
was demonstrated by microprecipitin tests (Ball, 1961), in
the buffer soluble contents of cells maintained in sucrose-
buffer. Various modifications were made, using microscopic
observation of cells and micropricipitin tests as evalua-
tion methods.

Since Yarwood (1961) had demonstrated that short per-
iods of heating to 45-5500. increased infections with TMV
on local lesion hosts, and increase in temperature could
be expected to accelerate most cell processes, various
periods of heating were added to the inoculation proce-
dure. The results of heating are shown in Fig. 2. With-
out heating, virus equal to 10% of control had been re-
covered; with heating,levels above 100% of control were
reached for the first time, indicating that replication
had definitely occurred. Since results are from a single
experiment, the only conclusion drawn is that heating, even
for short periods of time, increases virus recovery and
ten minutes at 45°C. apparently does not kill the cells.

Results from variation of centrifugal force applied
during the inoculation procedure are shown in Fig. 5.

The cells were centrifuged twice at each indicated G

force, incubated in Vickery's solution and sucrose for

Punumttnfeaflwel

PenumttflfmuanI

200

25

2 4 6 8 10
Minutes of heating

Fig. 2.éEffect of various periods of heating at 45°C. dur-

92-
800
700

600
500

500
200

100

ing the inoculation procedure on quantity of virus
recortaidat 40 hours. Results from a single exper-
iment.

1.2545678910111215
Gravity X 10'3

Fig. 5.-Use of the centrifuge as a means of inoculation.

Level of virus measured at 40 hours. Results from
two experiments.

26

40 hours, harvested and assayed on bean plants. The con-
trol for these experiments was a purified virus suspension,
centrifuged, diluted and incubated without cells.

Even though virus was recovered in excess of the a-
mount of inoculum when centrifugation was used as part of
the inoculation procedure, damage to cells is probable at
5000-5000G. Agitation on a Vortex mixer (Scientific In-
dustries, Inc.) was the most effective alternative method
of abrasion tried. Cells are rapidly agitated in the pres-
ence of an abrasive and the virus, then incubated, harvested,
and assayed as before. Figure 4 shows the results of one
experiment with vortex mixing and celite as the abrasive.
Timing of agitation is controlled precisely, and with nearly
uniform amounts of celite and cells, the degree of abrasion
should be reproducible.

Measurements with the Warburg respirometer indicate
relative activity of the cells in several media tested,
and show that respiratory activity of separated cells is
much lower than that of very roughly equivalent amounts of
leaf disc material (Fig. 5). Vickery's solution (KH2P04,
0.145 gm/l; Ca012, 0.255 gm/l; MgSO4'7H20, 0.875 gm/l;
(NH4)2804. 0.555 Sm/l) (Vickery 32 g1., 1957) has long been
used to maintain leaves and parts of leaves for extended
periods. It is a simple medium with known,chemically de-
fined constituents, and it provided higher virus recovery
than other materials tested in preliminary trials. Vickery's

solution plus .15 molar sucrose was used as incubation medium

27

600 -

500 -

200 I

Percent of control
xx
o
C)

100 - l

l I

 

 

 

 

2 4 6 8 10
Minutes of mixing

Fig. 4.-Effect of time of mixing with vortex mixer and
celite in the medium on virus recovered at 40
hours. Results from a single experiment.

 

 

60 159 140

Minutes

Fig. 5.-Respiration of dissociated cells in various media.
(3-White's mineral medium; El—Vickery's solution
plus sucrose; 15-Medium prepared from the solution
in which cells were separated;-—--tomato leaf discs.

 

28

in all following experiments.

Inoculation method. The inoculation method used at
this point included addition of virus-containing clarified
plant sap or purified virus to the cell medium mixture,
heating at 45°C., and vortex mixing with celite as the
abrasive. Carborundum was evaluated as an abrasive, but
‘ gave lower virus recovery than celite, and microscopically
appeared much too large and dangerous compared to the size
of the cells it was to abrade.

The respiratory response of dissociated cells to var-
ious steps in the inoculation procedure is shown in Fig. 6.
0n the basis of this data, agitation with celite increases
respiratory activity of cells, at least temporarily. 0n
the basis of preliminary data presented in Figs. 2,4,5 and
6, and other observations, the following procedure was
adopted for most succeeding experiments. Washed cells were
suspended in a minimum of medium, and mixed with purified
virus suspension. The resulting solution was heated 8-10
minutes at 45°C., celite was added, and the whole mixture
was agitated on the vortex mixer for 50 seconds. Aliquots
of this inoculated cell suspension were pipetted into pre-
viously prepared 45 ml quantities of Vickery's solution
plus sucrose in 125 ml flasks. The flasks were placed on
a reciprocating shaker at a speed sufficient to cause some
' formation of bubbles in the medium, and were maintained in
the dark at approximately 22°C for varying periods of time.

The cells were then harvested by centrifugation at 5500C,

40

30

)11 oxygen
N
c>

10

29

ODD

L L #
60 120 180
Minutes

Fig. 6.-Respiration of dissociated tomato leaf meSOphyll cells in

various stages of the inoculation procedure. C)-Vickery's
solution plus sucrose (V+S); £5=V+S plus ten minutes heating;
Dil+s plus heat plus vortex mixer (30 sec.);.cV+S plus heat
plus vortex plus celite;A-V+S plus heat: plus vortex plus
celite plus virus; I=V+S plus heat: plus vortex (two minutes)
plus celite.

 

‘4

 

50

.after washed, frozen and stored, as was a sample of the
incubation medium. Just prior to assay, cells were
thawed, ground in buffer and centrifuged. The superna-
tant of this centrifugation was the material indicated as
buffer soluble cell contents and used to inoculate assay
plants.

The presence in the medium of virus in excess of that
actually entering cells, renders interpretation of results
more difficult. Washing cells immediately after inocula-
tion, before incubation, provides the simplest solution.
Cells washed in four changes of medium immediately after
inoculation contained small amounts of virus after 24
hours, as did the medium in which they were incubated.
Such cells are injured by repeated washing after abrasion.

There are two major objections to leaving excess
virus in the medium, aside from the difficulty of detecting
early virus synthesis. First, the possibility that virus
might become passively attached to the dying cells and
give false indications of virus in the cells, and secondly
that infection may occur later without the necessity of
further heat or abrasion, giving erroneous ideas of the
time required for virus synthesis. Attempts were made to
eliminate these two latter possibilities.

Cells from healthy tomato plants were killed by
freezing and thawing, then run through the usual inocula-
tion procedure and incubated. Figure 7 shows results of

virus assays of incubation medium and buffer soluble cell

51

100

 

50

 

Percent of control

<>———-"""'43 ‘\\‘\\\:3-““~“‘~c>

 

 

-’-‘r A
l 4 14-
10 20 30

Hours after inoculation

Fig. 7.-Virus recovered from cells frozen and thawed before
inoculation. Each point is an average value from two
experiments. Z§=tirus from medium; C)=virus from cells.

80
6O '
40" A

A / \
Mr \A

RA
F DE‘ n l H
10 20 30 40
Hours after inoculation

Percent of control

 

Fig. 8.-Recovery of virus from cells inoculated with and without
heat and vortex mixing. A=with heat and mixing; D=without
heat and mixing. Results from a single experiment.

 

 

32

contents of these dead cells at various times after inocu-
,lation. Virus associated with buffer soluble cell contents
remains essentially constant for the total experimental
period (30 hours), while virus in the incubation medium
declines. lf virus were becoming attached to or associated
with non-living cell contents, increase of virus asSociated
with cell contents should correlate with decrease of virus
in the medium.

In another experiment, healthy cells were inoculated
either with or without heat and vortex mixing. Figure 8
shows the results of this experiment. Without heat and
mixing, buffer soluble cell contents contained practically
no infective virus. Further indication that virus is not
passively absorbed by dead cells, cell walls or the
celite left in the medium is given by the fact that only
traces of virus have ever been detected in the cell debris
ineluding celite, left after grinding and centrifuging
the cell samples prior to assay.

Passive absorption of virus by cells is also dis-
counted by experiments involving variation in numbers of
virus particles and numbers of cells. lf virus particles
are being absorbed or passively taken into cells without
replication, the amount of virus taken in and later assayed
should be directly proportional togthe number of cells in
the medium. However, if multiplication is occurring,
there should be an optimum number of cells for the amount

of medium used, at which cell concentration the recovery

: 15.x
I5

 

 

55

of virus would be greatest. Assuming that one virus par-
ticle is needed for an infection, at a constant cell con-
centration the virus recovered should be directly propor-
tional to the amount of virus used as inoculum, up to
some saturation point.

Cells are inoculated as before, but in one case
cell numbers in the inoculation mixture are varied while
virus concentration is kept constant, and in the other
case, cell numbers are kept constant and virus concen-
tration is varied. The results (Fig. 9) indicate that
the optimum cell concentration is not the maximum used,
and that recovered virus is proportional to the amount
used as inoculum over the range of cell concentrations
tested.

Another potential solution to the problem of residual
virus is the use of virus RNA as the inoculum. Several
attempts to inoculate cells with TMV—RNA prepared by the
heat-salt method (Lippincott, 1961) failed. Apparently
the RNA in the medium with cells was rapidly inactivated,
and.thus could not be used as inoculum in the present
system. ,

The above results suggest that l) passive attach-
ment of virus to cells and accidental infection after
'time of inoculation can'be discounted, and 2) RNA is not
extiseable inoculum. The use of a control level of in-
fectivity as a base line rather than a zero level is

therefore advisable. The control level is that infec-

54-

 

 

250'
200 r-

F. (J

o

H

4.)

c

o

o

“a

C)

t; 100

o

o

H

o

n.

C)
0 n
0.5 1.0 1.5 2.0
LOgml/dilution

Fig. 9.-Separate effects of virus dilution and cell dilution on

virus recovered at 24 hours. D=virus dilution; O=ce11 dilution.
Cell dilution data from two separate experiments.

1“

 

 

55

tivity recovered from a dilution of purified virus equal
to its dilution in the incubation medium.

Forty hours was selected as the period for virus
replication on the basis of my early results with serolo-
gical checks for virus presence. Later results showed
that virus is produced in the cells in a much shorter
time. However, the results at 40 hours may still have
value. Since data Fig. 10 indicate that most virus out-
side the cells is rendered non-infective within 24-30
hours of the usual incubation, virus present at 40 hours
after inoculation was probably produced by the cells after
time 0. The information in figure 10 was gathered from
numerous experiments in which infectivity above 100% of
control was found in buffer soluble cell contents.

Curves showing lass of virus activity in cells sep-
arated from systemically infected plants also have some
bearing on the detection of virus in cells 40 hours after
inoculation. Results of several experiments with such
previously infected cells are shown in Figure 11. Measured
infectivity is low by 22-50 hours after inoculation with
normal cell activity, but remains essentially unchanged
when cell and microorganism activity is suppressed by
merthiolate. Cells initially containing quantities of
virus may be less viable than those without initial virus,
but the difference is not likely to be great. Inactivation
of the virus in the medium is likely caused by metabolic

products of mesophyll cells or microorganisms. Virus

‘56

120
O\\\
/
80
60
40

O\
20 o

10 20 3O 40
Hours after inoculation

Percent of control

Fig. 10.-Loss of infectivity from medium supporting cells which

produced virus. Each point is the average of from 5-17
determinations.

57

200

100

Percent of initial infectivity

 

10 20 30 40 50
Hours after cell isolation

Fig. 11.-Loss of infectivity in cells isolated from systemically'
infected tomato leaves and incubated in vickery's solution
plus sucrose. £§=virus in medium; ()= virus in cells
(average of two experiments). ‘=virus in medium with 10
ppm merthiolate; .=virus in cells from that medium.

58

added as inoculum when merthiolate was not present, and
remaining outside the cells would not survive much beyond
24 hours, and thus most virus found in the medium or as-
sociated with the cell contents at 40 hours must have been
produced after time of inoculation.

The growth of microorganisms in the media presented
problems for long term experiments, since the leaves from
which cells were obtained could not be effectively ster-
ilized. Several additives to the media.were used in
attempts to control microorganism growth. Experiments
were done with merthiolate at one and ten parts per million
in the usual Vickery's plus sucrose incubation medium.
Virus quantities in inoculated cells never reached the
check level, or reached it only after 30-40 hours when
merthiolate was present. Respirometer measurements indi-
cated 75% reduction in 002 exchange,and loss of virus from
cells of systemically infected plants was negligible over
a 48 hour period in merthiolate medium (Fig. 11). All
the above would seem to indicate that while merthiolate
at ten parts per million can effectively control micro-
organism growth for more than 100 hours, it also suppresses
some cell activity when cells are exposed directly to it.

Penicillin and Chloromycetin (Parke-Davis Chloram-
phenicol) were also used in efforts to control bacterial
contamination, but neither seemed effective in suppressing
overall microorganism population in the medium.

Up to about 24 hours the growth of fungi and bacteria

59

is not sufficient to present problems of general competi-
tion or changes in the medium. After 50 hours medium pH
dropped slightly and the mesophyll cells were probably
affected. Barring some highly specific inhibitory inter-
action, the time period up to 24 hours should give results
not seriously complicated by the presence of microorganisms.

Eiggg synthesis. Many experiments were performed in
an attempt to determine the relationship of time after
inoculation to virus quantity in the cells. As reported
above, 40 hours was initially considered to be the earliest
time at which measurable increases in virus could be demon-
strated. Most frequently at this early stage of experimen-
tation, no samples were taken prior to 12 hours after inoc-
ulation, and some results (Fig. 12) suggested a virus in-
crease peak at about 20 hours.

As the processes of cell separation, inoculation and
incubation were repeated, the speed and efficiency'with
'which each step was accomplished increased. The result
was cells in a healthier or more lively condition at any
stage, even though no gross differences could be observed.
Infectivity was significantly above that of the control at
12 hours, the earliest time at which samples were taken,
but declined thereafter (Fig. 15). When samples were
'taken earlier than 12 hours, the peak was obtained at 7
'hours, followed by a decline (Fig. L4). The data shown
in Fig. 14 utilized an incubation medium other than

“Vickery's solution, but indications are that only quanti-

40

A 0/000\/0\
' A/A\

Percent of control

 

10 20 30 40 50
Hours after inoculation

Fig. 12.-Increase of virus quantity with time. Each point is an
average from two comparable experiments. l§=virus from
medium; CD-virus from cells.

I l I l J__

441

300

200

Percent of control

100

. 10 20 “ 30
Hours after inoculation

Fig. 13.-Resu1ts of three experiments showing decline in virus

recovered from cells in the period from 12-24 hours after
inoculation.

.L

 

‘42

O
300
H
O
H
u
r:
8 200
‘H
O
u
t:
G)
O
u
0.)
n.
100

l3r“"‘£>‘--“\‘~“
A

_—
10 20 30
Hours after inoculation

Fig. l4.-Resu1ts showing a peak in virus quantity at seven hours.
lsrvirus in medium;()=virus in cells. Each point is the
result of two replications of the inoculation and incubation.

45

tative differences in cell response are involved.

For experiments with TMV then, the earliest peak of
virus quantity observed was at seven hours, which is in
agreement with observations of Fry and Matthews (1964),
for the time required to replicate virus in cells func-
tioning as part of a whole leaf. Similar early high
values were obtained in several experiments with TNV,
but the small number of trials and some uncertainty in
the local lesion assay casts doubt on these results.

If the greatest virus quantities obtained from
inoculated cells at times of seven hours and after are
plotted on a time scale, the resulting line has the nega-
tive slope that would be expected for the measurement of
any product of a population of organisms declining in
overall health (Fig. 15). A decline in overall health
is believed to be involved in the leaf mesophyll cells
used in these experiments.

Cells were inoculated and assayed for TMV in 22
comparable experiments, and the results treated statis-
tically (Fig. 16). The curved line represents the 5%
significance level expressed as percent of control for
the various mean values of the control half leaves. Any
point above the curved line indicates that an amount of
virus significantly larger than that used as inoculum
'was found in buffer soluble cell contents. Results of
seventeen separate comparable experiments with cells

.from two plant genera indicate that intracdlular virus

Percent of control

 

10 20 30 40
Hours after inoculation

Fig. lS.-Plot of the high values obtained at each time interval after

inoculation. The negative slape indicates loss of capacity to
produce virus.

45

33.35 .82 26%
mommies.“ unwowmflawaw noumufivcfi as: noise .uzou outsoulo 3.300 ooowAOun. .ucounou
usug> now nozwnmu House use wouwanooafi muck uHaoo soana ca munuaauomxo ofinwummfioo «N no uona.0H .mwh

mo>uofinwawa xuwnu no uaoumoa HwooH no sense: new:
on or om ON 0a

1 d ‘ i I d

1013uoo go nuaozaa

 

 

46

multiplication ocCurred.

Inhibitor formation. Plants infected with viruses
have been shown to produce extractable materials which
are inhibitory to virus infection when mixed with the
virus prior to inoculation. An experiment was therefore
designed to determine if inhibitors were present in cells
infected as individual units. Cells were inoculated in
the usual way, and at 24-50 hours harvested, frozen,
ground and treated with hydrated calcium phosphate (HCP)
according to the method described by Sela and Applebaum
(1962). Extracts containing potential inhibitory mater-
ials were prepared from tobacco cells and tomato cells,
both inoculated and uninoculated, and from the medium in
which cells had been incubated. Purified virus prepara-
tions were diluted with-the solutions so prepared, and
the resulting inoculum was applied to Pinto bean, the
usual local lesion assay plant. The control was a dilu-
tion of the virus in .01 M phosphate buffer, pH 7. Re-
sults from two experiments with tomato cells are shown in
Table 1. Tobacco cells produce inhibitory materials that
tend to obscure the difference between inoailated and non-
inoculated cells. These results support the observations
(Zaitlin and Siegel, 1965) of a non-specific inhibitor from
pectinase separated tobacco cells. No serious attempt was
made to identify the inhibitory material or materials.
Without infection and intracellular virus production it

is unlikely there would be a difference in the amount of

47

inhibitor between inoculated and uninoculated cells.
inhibitory materials were produced in the presence of
the virus in greater amounts than were found in uninocu-

lated cells.

Table l.-Inhibition of infection by cell and culture
fluid extracts. Percent inhibition is cal—
culated with the buffer diluted purified
virus as the base. Results from two exper-
iments.

 

Material Percent inhibition
Medium alone 12 4

HCP treated
Frozen ground cells

Inoculated SO 6

Uninoculated 0 20
Incubation medium

Inoculated 67 25

Uninoculated ll 0

 

Infected cell detection. The proportion of infected

 

and non—infected cells in any experiment would be valuable
information; however no difference could be observed be-
tween inoculated and non-inoculated cells by direct micro-
scopic observation. Several means of demonstrating dif-
ferences were therefore attempted.

Flourescent antibody stains were prepared from rabbit
antiserum to the ribgrass strain of TMV (BBC) and flour-
esceinisothiocyanate by the method of Rinderkneckt (1962).
This is a simple,rapid method utilizing a column of "Sepha-
dex" for separation of dye-protein conjugate and unconjugated

dye. Cells from infected plants and healthy plants were

48

treated with the prepared flourescent antibody and ob-
served with a Zeiss flourescence microscope, using the
method outlined by Nagaraj (1962). Flouresence was ob-
served within cells from infected plants, but also within
some cells from healthy plants. No filter system used
could eliminate the flouresence from all healthy cells
and retain it in all infected cells. Isolated inoculated
cells compared to isolated healthy cells had similar pat-
terns of flouresence. Although Nagaraj reported he could
distinguish cells from healthy and TMV infected plants by
this method, in the present study the condition of any one
particular cell seems impossible to determine with his
method or several variations of it. Probably only a

small percentage of cells are infected by the inoculation
method described here, and without some means of deter-
mining the state of virtually every cell, information from
flourescent antibody staining is not conclusive.

A method utilizing acridine orange stain on killed
and fixed cells (Hooker and Sumanwar, 1964) gave similar
conflicting results with dissociated healthy and diseased
cells. It may well be that cells separated by pectinase
digestion and maintained in artificial media are suffi-
ciently different from cells in place on the leaf to in-
‘validate methods demonstrated effective with leaf tissue.

The problem of what percent of cells can be infected
by the method developed here remains unsolved, and is one

of the critical problems for future work. staining methods

49

may provide the answer, and would prove a much more rapid
means of evaluating variations in the inoculation proce-

dure; a task that is long and complicated when the amount
of virus recovered at some later time is the only indica-

tion of success or failure.

DISCUSSION AND CONCLUSIONS

The work presented here proposes to answer only one
question: Can individual meSOphyll cells obtained from
leaf tissue be infected with tobacco mosaic virus by the
methods described? Most of the experiments and observa-
tions reported, excluding those concerned directly with
methods, bear on this question.

Cell maintenance. The success achieved in maintain-
ing cells on simple mineral media supplemented with su-
crose indicates that for the short periods of time in-
volved, mesophyll cells are able to carry on some com—
plex activities requiring internal energy transformations.
More precise means of evaluating the responses of cells
to various media should make it possible to select more
effective nutrient mixtures.

Cell inoculation. The inoculation procedure developed
has the one outstanding quality of functionality, and
analogies to the whole plant inoculation procedure are
acknowledged. Improvements may require completely new
approaches. 'Without more precise knowledge of the means
by which plant viruses normally enter and infect cells,
improvements may have to be based on further guesses as
to what changes in treatment of cells will increase in—
fection. Some stimulation of the cells seems necessary.

Respiration measurements show increases in activity of

SO

 

/v

51

the cells after agitation with the abrasive Celite,

(Fig. 6) and the measurements of recovered infectivity

show the desirability of using heat as part of the stimu-

latory process. Whether the stimulated cell actively takes

in portions of the liquid environment including virus par-

ticles, or whether the virus itself is involved in attach-

ment and entry into the cytoplasm was not determined. Such

questions may however, be more easily answered in a single

cell inoculation system than in one using intact tissues.
Determination of the time period during which virus

is produced in populations of single cells is subject to

various errors in the system used. In the intact leaf

virus RNA apparently travels to adjacent cells before

whole virus is produced in the initially infected cell

(Fry and Matthews, 1964). It is not known whether such

movement out of the cell is still possible when the plas-

modesmata are not connected to adjacent cells, although

figures seen in tomato stem tissue (Livingstone, 1964)

show plasmodesmata extending through the walls of separ-

ated cells. Unless one assumes nearly 100% initial in-

fection, a quick buildup to a low carrying capacity and

an accelerating cell mortality, the progressive loss of

infectivity from the culture medium would argue against

release of stable infectious material. Virus RNA leaving

the cells would probably be inactivated quickly by

ribonucleases released from broken cells (Frisch-Niggemeyer

and Reddi, 1957), and even if not inactivated, the amount

52

would probably be too small to reflect in infectivity
measurements considering the low efficiency of TMV-RNA
as inoculum (Lippincott, 1961). A peak of virus quantity
for the population of cells was observed in buffer soluble
cell contents as early as seven hours after inoculation.
Assuming the validity ofthe assay method, the peak must
represent whole virus particles produced and retained in
the cell during those seven hours. In other experiments
peaks of activity were later, but since earliest values
are highest (Fig. 15), seven hours can be taken as a
figure approaching the minimum.

Inhibitor production. The experiments on inhibitor
production were not concerned directly with measurement
of infectivity, but rather with determining if cells were
infected at all. The basic assumption was that two groups
of cells treated identically except for the presence of
virus would differ in metabolic activity. The virus may
enter the cell and cause metabolic derangement resulting
in production of virus components. If this does not occur,
the plant cell continues normal function. If it does
occur, the redirection of cell metabolism would result
in the production of substances other than those directly
required for virus synthesis.

A significant difference in inhibition was observed
between materials associated with inoculated cells and
those associated with uninoculated cells. Specific in-

hibitory materials differing from normal cell products may

55

have been produced by infected cells, but the experiments
show only the amount of inhibition not its direct cause.
Production of more or different inhibitory materials by
inoculated cells is one of the best pieces of evidence
that virus synthesis or some part of the infection pro-
cess is occurring in inoculated cells.

Validity 9; results. All of the results expressed

 

in terms of infectivity stand or fall on the validity of
the assay method. This being the case, evaluation of the
method is a necessary part of the work. Numerous trials
indicated that the bean plants used were sufficiently
uniform for effective half—leaf assays. Several other
factors not precisely controlled are involved in the re-
sults obtained from assays. The direction of potential
error in all cases is such as to reduce rather than in-
crease difference between control and experimental results.
Three primary factors are responsible for the uncontrolled
variability: 1) production of inhibitory substances by
infected cells, 2) use of dilutions of purified virus for
controls, and 3) the loss from and inactivation of virus
in infected cells.

Judging from the results of experiments demonstrating
inhibitors, there is reason to suppose that all infected
cells produce materials which inhibit the demonstrable in-
fectivity of the virus they synthesize and contain. Such
inhibitors would be expected to lower infectivity results

and give indications of less virus than the cells had

54

actually produced. No attempt was made to study inhibitor
production for periods less than 24 hours after infection.
The use of direct dilutions of purified virus as con-
trol inoculum would give accurate indications of change in
infectivity of cell extracts only if we assume that no
virus is inactivated in the incubation medium during the
experimental period or during storage. The departure from
accuracy is in the direction of less virus over and above
that of the control rather than more. Kassanis gt El.
(1958) used virus added to aliquots of medium without
cells and incubated for the same length of time as the
control inoculum. Figure 4 shows an experiment indicating
875% of control virus level. This result was obtained with
a control similar to that used by Kassanis, and may be con-
trasted with a high of 524% of control obtained using di-
rect dilutions of purified virus as the control inoculum.
The loss of virus from cells with time is most clearly
shown when they are isolated from systemically infected
plants. With the culture system used, such cells show
rapid loss of demonstrable infectivity during 24 hours.
During that same period, cells isolated from healthy
plants and inoculated with TMV showed rapid increase in
contained infectivity. It is reasonable to suppose that
the observed increase masked the loss of some virus from
inoculated cells. A better indication of virus produced
would be obtained by adding the observed loss to the

observed gain and using this value for the capacity of

55

dissociated cells to synthesize virus.

The above factors combined with the normal loss of
virus known to occur when infected plant material is
macerated and centrifuged, allow the realistic supposi-
tion that virus synthesis did occur in the inoculated
single cells, and may be higher than the measured values
indicate.

It would be premature to state that the advantages
of a single cell inoculation system as outlined in the
introduction are available for the study of plant viruses.
Progress has been made, however. Single cells with at
least some synthetic capacity are available. Inoculation
procedures that infect at least a portion of a cell pop-
ulation have been developed. And most importantly, much
evidence indicates that those single cells actually pro-
duce whole, infective virus in measurable amounts. Means
of maintaining sterility in the system would be helpful,
but would present great technical problems without
wholesale changes in procedure. A means of determining
which cells in a mixture have been infected and are pro-
ducing virus is high on the priority list for future work.
In short some progress toward the ideal system outlined
in the introduction has been made.‘ Further effort should

be rewarding.

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