University of Montana
ScholarWorks at University of Montana
Theses, Dissertations, Professional Papers
Graduate School
1987
Characterization of intercellular wash fluid from
tobacco mosaic virus infected pinto bean primary
leaves
Susan M. Geske
The University of Montana
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Geske, Susan M., "Characterization of intercellular wash fluid from tobacco mosaic virus infected pinto bean primary leaves" (1987).
Theses, Dissertations, Professional Papers. Paper 7413.
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19 87
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Characterization of I n t e r c e l l u l a r Wash Fluid from
Tobacco Mosaic Virus Infected Pinto Bean Primary Leaves
by
Susan M
- Geske
B.S., B.A. U n i v e r s i t y of Montana, 1984
Presented in p a r t i a l f u l f i l l m e n t of the requirement fo r
the degree of
Masters of Arts
University of Montana
1987
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Geske,
Susan M
. , M.A* ,
March
13,
1987
Botany
C h a r a c t e r i z a t i o n of i n t e r c e l l u l a r wash f l u i d from tobacco
mosaic v ir u s infected p in to bean primary leaves.
{ 66 pp.).
Director:
Dr.
Meyer Chessin
I n t e r c e l l u l a r wash f l u i d (IWF) was o b t a i n e d from tobacco
mosaic v i r u s (TMV) i n f e c t e d primary l e a v e s of Ph a s e o l u s
v u l g a r i s var. Pinto, on selected days a f t e r inoculation, v ia
in vacuo i n f i l t r a t i o n of phosphate b uffer and cen trif u g atio n .
Tests for the presence of the i n t r a c e l l u l a r enzyme glucose-6P - d e h y d r o g e n a se i n t h e IWF e x t r a c t s showed t h a t t h e
e x t r a c t i o n procedure did not cause major c e l l damage.
As an i n h i b i t o r of v i r u s e s t a b l i s h m e n t IWF had l i m i t e d
e f f e c t when mixed with v ir u s p r i o r to inoculation.
But when
i n f i l t r a t e d 6-9 h rs . a f t e r TMV i n o c u l a t i o n ,
IWF reduced
l e s i o n numbers by 60-69% and d i m in i s h e d the appearance of
l a r g e r l e s i o n size class es in d ica tin g i t a c t s as an i n h i b i t o r
of v ir u s r e p l i c a t i o n .
IWF p r o t e i n s produced d i s t i n c t peaks i n c o n c e n t r a t i o n
d u r i n g a 10 day p e r i o d a f t e r v i r u s i n o c u l a t i o n .
Peaks
o c c u r r e d 12 h o u r s ,
4 days, 7 days,
and 10 days a f t e r
infection.
There a l s o appears to be a s l i g h t p e r i o d i c i t y
over time i n the l e v e l s of i n t e r c e l l u l a r p r o t e i n s a f t e r
wounding only one-half of a single primary l e a f with b uffer
and C e l i t e .
IWF e x t r a c t i o n s taken 7 days a f t e r i n o c u l a t i o n
contained the g r e a te s t p ro te in concentrations.
IWF samples were d ia l y z e d , in washed tubing, f o r 24 hr s.
before t e s t i n g i n h i b i t o r y a c t i v i t y against v ir u s e s t a b l i s h ­
ment or r e p l i c a t i o n .
I n h i b i t i o n r e s u l t s confirm t h a t a
s u b s ta n c e r e s p o n s i b l e f o r v i r u s i n h i b i t i o n , g r e a t e r than
10,000-14,000 d molecular weight, i s present in IWF e x tr a c ts .
I n c r e a s e d q u a n t i t i e s of 8 IWF p r o t e i n s were the d i r e c t
r e s u l t of v i r u s i n f e c t i o n .
These p r o t e i n bands s t a i n e d
darker than other p ro teins with Coomassie b r i l l i a n t blue on
SDS-PAGE g e l s .
The a p p a r e n t m o le c u la r w e ig h ts of t h e s e
p r o t e i n s were:
36,500 d; 34,000 d; 33,000 d; 28,750 d;
26,500 d; 18,200 d; 16,800 d; and 15,200 d, SDS-PAGE and
immuno-blot experiments indicate the three lower molecular
weight enhanced p r o t e i n s were produced
novo, but the
others were not.
High performance l i q u i d chromatography t e s t s of Day 7 IWF
samples shows the appearance of a new p r o t e i n i n the TMV
i n f e c t e d h a l f - l e a f and, to a l e s s e r e x t e n t , i n the a d j a c e n t
h alf.
The p ro te in peak i s detectable s l i g h t l y or not a t a l l
in the opposite primary leaf.
IWF can function in i n h i b i t i n g v ir u s r e p l i c a t i o n , although
a specific a n tiv ira l
p r o t e i n has not been i s o l a t e d i n t h i s
study.
The p re senc e of enhanced, s p e c i f i c p r o t e i n s i n IWF
from i n f e c t e d t i s s u e i n d i c a t e s t h a t c e l l s are s e l e c t i v e l y
r e l e a s i n g or a l l o w i n g p r o t e i n s to e n t e r the i n t e r c e l l u l a r
spaces a f t e r TMV i n f e c t i o n . General c e l l l e a k i n e s s a f t e r
v ir u s i n f e c t i o n apparently did not seem to have occurred.
ii
ACKNOWLEDGEMENT
I s i n c e r e l y thank Dr. Meyer Chessin f o r h i s generous
help and i n s i g h t during t h i s study.
I wish to thank Dr. R.C.
Judd f o r h is patience and help in teaching me e l e c t r o p h o r e si s
techniques and fo r running the HPLC samples.
In addition, I
would l i k e t o thank Dr s. D.E. B i l d e r b a c k and G.P. Mel 1 f o r
t h e i r support as members of my graduate committee.
I am e s p e c i a l l y g r a t e f u l f o r Allan Z i p f ' s and Amitava
Mitra's encouragement and knowledge in helping me throughout
t h i s r e s e a r c h work.
F i n a l l y I would l i k e to thank Dave
Dorward, Lisa Wentz, and other f r i e n d s f o r t h e i r laboratory
a s s i s t a n c e and Dr. R.J. F e s s e n d e n f o r t h e use of h i s
computer.
This p r o j e c t was su pp orted i n p a r t by a U S - I s r a e l BARD
r e s e a r c h g r a n t and by the U n i v e r s i t y of Montana Botany
Department.
iix
TABLE OF CONTENTS
ABSTRACT
ü
ACKNOWLEDGEMENT
iü
TABLE OF CONTENTS
iv
LIST OF TABLES
v
LIST OF ILLUSTRATIONS
vi
INTRODUCTION
1
MATERIALS AND METHODS
Chemicals
Plants
Virus
Inoculation and Extraction of IWF
IWF Tests
Presence of Glucose 6-P Dehydrogenase
IWF as
I n h i b i t o r of Virus Establishment
IWF as
I n h i b i t o r of Virus Replication
IWF D i a l y z i b i l i t y
Determination of Protein Contents
SDS-PAGE Gels
Immuno-Blot Analysis
High Performance Liquid Chromatography
18
18
18
18
19
21
21
22
22
22
23
23
26
26
RESULTS AND DISCUSSION
IWF Tests
Glucose-6-Phosphate Dehydrogenase
IWF as
I n h i b i t o r of Establishment
IWF as
I n h i b i t o r of Replication
D ializibility
Determination of Protein Contents
SDS-PAGE Protein P r o f i l e s
Immuno-Blot Analysis
High Performance Liquid Chromatography
28
28
28
28
31
35
35
37
42
43
SUMMARY AND CONCLUSIONS
47
REFERENCES
58
IV
LIS T OF TABLES
Table 1.
Chemical Constituents of a 15% SDS-PAGE Gel.
p . 25
Table 2.
Presence of Glucose-6-Phosphate-Dehyrogenase
in Leaf Homogenates and I n t e r c e l l u l a r Fluid.
p . 29
Table 3.
Percent I n h i b i t i o n of Virus Establishment by
IWF Extracts Taken a t Selected I n t e r v a l s
After Inoculation.
p . 30
Table 4.
Comparison of Average Lesion Number and
Diameters Using Day 7 IWF Extracts Applied
6-9 Hours After Inoculation with TMV.
p . 33
Table 5.
Lesion Size-Class Frequency Percentages Using p . 34
Day 7 IWF E xtracts I n f i l t r a t e d in Pinto Bean
Leaves 6-9 Hours After TM
V Inoculations.
Table 6.
Comparison of
IWF Protein Contents
p . 36
1
(ug X 10 /ml) Taken a t Selected Times After
TM
V Inoculation.
2
IWF Protein Contents (mg xlO /ml) of Buffer
Inoculated Pinto Bean Primary Leaves.
—
Table 7.
p . 38
LIST OF ILLUSTRATIONS
Figure 1.
Representation of Pinto bean primary
leaves showing treatment d i v i s i o n s .
p . 20
Figure 2.
15% SDS-PAGE gel showing eigh t enhanced
p r o t e i n s from a Day 7 IWF e x t r a c t i o n .
p . 40
Figure 3.
Comparison of p ro teins from IWF
e x t r a c t i o n s taken a t selec ted days a f t e r
TM
V infection.
p . 41
Figure 4*
Comparison of HPLC samples fromside
and side #3 Day 7 IWF e x t r a c t s .
#1
p . 45
Figure 5.
Comparison of HPLC samples fromside
and side #4 Day 7 IWF e x t r a c t s .
#2
p . 46
VI
INTRODUCTION
V i r u s e s and p l a n t s have long been a s s o c i a t e d w i t h one
another*
P l a n t v i r u s e s a r e known t o i n f e c t n e a r l y a l l
»
s p e c i e s of h i g h e r p l a n t s , t h a t have been s t u d i e d to d a t e ,
e i t h e r n a t u r a l l y or a r t i f i c i a l l y
Mosaic V irus
Angiosperm
(CMV) can i n f e c t
fam ilies,
(Hammond 1982).
p lan ts
and A l f a l f a
in
Mosaic V irus
i n f e c t 393 d ic o t and 8 monocot species
1972).
found
Cucumber
alm o st
all
(AMV)
can
(Beczner and Schmelzer
Because v i r u s e s do not have a f r e e l i v i n g phase i n
th eir
life
around.
cycles,
they
must
rely
on l i v i n g
plants
year
In many c a s e s crop d e s t r o y i n g v i r u s e s can s u r v i v e
a d verse c o n d i t i o n s
or
overw inter
i n weed s p e c i e s w i t h o u t
causing harm or noticeable symptoms; y et when introduced i n t o
a s u s c e p t i b l e crop,
y ield s
Virus
can be reduced d r a m a t i c a l l y ;
X,
disease
there
they can cause e x t e n s i v e damage.
reduced p o t a t o
symptoms
is
a great
y ie ld s
in
w ill
the case of
not
be
for v iru s
Potato
indicated
on th e above ground p l a n t
p otential
Crop
p arts.
by
Since
induced damage
to
a
v a r i e t y of crops and a l a c k of chemical means t o c o n t r o l such
diseases,
it
i s imperative to study how p la n t s are capable of
defending
themselves
naturally.
Enhancing a p l a n t ' s
defense mechanisms may then be the e f f e c t i v e
viral
n atu ral
way to control
diseases.
Plant Responses to Virus
Plants
ways.
since
respond
to v i r u s
Few i n f e c t i o n s r e s u l t
infection
in t o t a l
in
a
few d i s t i n c t
d e s t r u c t i o n of a plan t,
t h i s could lead to the disappearance of t h a t p a r t i c u l a r
v i r u s by el i m i n a t in g or reducing the spread or appearance
of
the host p l a n t species.
More often subliminal,
l o c a l i z e d i n f e c t i o n s can occur,
systemic,
to a l e s s detrimental
and
degree,
depending on the s p e c i f i c h o s t - v i r u s combination.
According t o S u l z i n s k i and Z a i t l i n
in fectio n s
are
restricted
to
only
(1982),
a few
believed t h a t w ith in a population of p lan t
are
c a p ab le
sublim inal
infection
of
carrying
out
virus
su blim inal
cells.
cells,
It
is
most c e l l s
rep licatio n .
Yet,
in
i n f e c t i o n s t h e v i r u s i s unable t o move from the
are
infecte d
contain v i r u s which i s r e p l i c a t i n g a t a normal r a t e ,
the host
a p p ea r s
center.
to
be
resistan t.
Although
asymptomatic.
New evidence
the
cells
The
th at
plant,
(Taliansky
et
in
al.
a
sense,
1982)
is
support
S u l z i n k i ' s and Z a i t l i n ' s h y p o t h e s i s t h a t t h e h o s t p l a n t has
an a b i l i t y
v iru s,
to destroy a " t r a n s i ocation protein",
which allows c e l l
to c e l l movement.
coded by the
An a d d i t i o n of a
"helper" v ir u s to a host p lant can induce the systemic spread
of a subliminal
may suppress
the host p l a n t ' s natural
Another
resp o n se.
i n f e c t i o n by another v ir u s.
reaction
From
th e
to
v iru s
original
defense.
infectio n
site
The helper v ir u s
of
is
a
in fectio n,
m u l t i p l i e s and spreads throughout the e n t i r e plant.
sy s t e m ic
a v iru s
Mottling
and mosaic symptoms are o ften the r e s u l t of a systemic spread
(Sela
1981
c).
Green
islan d s,
w hich
are
areas
of
c y t o l o g i c a l l y normal c e l l s t h a t contain low concentrations of
v iru s,
are
surrounded
t i t e r s of v i r u s .
v irus
in fectio n
by c h l o r o t i c
areas
containing
high
These i s l a n d s a r e r e s i s t a n t to subs equent
(Chessin 1983) and a r e thought t o be unable
to support TMV r e p l i c a t i o n
h ost-v iru s
infectio n
{Atkinson and Matthews 197 0).
co m bin ation w i l l
w ill
lead
to
d e te r m in e whether a s y s t e m ic
stunted
p r e d i s p o s i t i o n to other diseases
The other
is
(Sela
is
1981
response.
is
common response
the l o c a l i z a t i o n of v ir u s
localization
This
is
Resistance
of p la n t s
particles
to v ir u s
the N-gene.
tobacco
y ield s
or
a
to v i r u s
infection
by the plant.
as
a
of
Often
leaf
tissue
hypersensitive
in the genus Nicotiana
(Sela 1981 and Otsuki
et
al.
The h ypersensitive response i s b est
in
(N i c o t i a n a
occurs
in many p la n t species.
r es p o n se
to
spread
gene
known
non-specific
low
(Matthews 19 81).
referred
c o n f e r r e d by a s i n g l e
197 2) c a l l e d
growth,
accompanied by n e c r o t i z a t i o n
c).
The
spp.)
with
TMV,
although
it
Hypersensitivity i s a general,
to d ise as e agents,
does have a t e m p e r a t u r e s e n s i t i v e s t e p .
but
the re s p o n s e
Tobacco v a r i e t i e s
known to c a r r y the dominant a l l e l e f o r h y p e r s e n s i t i v i t y do
not develop n e c r o t i c
This
suggests
c e ll's
that
lesio n s at
the
inherent a b i l i t y
N-gene
28 C (Otsuki
itse lf
does
et
not
al.
1972).
control
to c o n t r i b u t e to TMV s y n t h e s i s .
a
It
a l s o does not appear to a f f e c t c e l l t o c e l l v i r u s movement by
altering cell
Various
v iral
(Helms
membrane
in
there
mitochondrial
al.
cellu lar
infectio n
Generally,
site s
structural
1986).
a
h y p ersensitiv ely
starch
of
mitochondrial
rough
Reunov a l s o
1962)
n oticed
that
an d
partial
endoplasmic
the o n s e t
responding
a c c u m u l a t io n s
M cInty re
numbers,
amounts
p roperties.
changes do occur a f t e r
are
and
or physiological
near
ly tic
plant.
infectio n
in creases
volume,
reticulum
of
in
and in
(Reunov
processes
et
were
stim u lated
along w i t h
and a decrease
in ten sified
in c e l l
wall
i s h e i g h t e n e d as a r e s u l t
eventual
g o l g i apparatus a c t i v i t y
synthesis.
Cellular
of TMV i n f e c t i o n ,
d e s t r u c t i o n of v ir u s p a r t i c l e s
c e l l s undergoing a u t o l y s i s .
any case,
cellu lar
lesions.
centers,
a b y - p r o d u c t of
There
co llap se
There i s s t i l l
tends
to
but
the
edges
encompassing
the
lesio ns,
the p r o c e s s
follows
be very
contain
l e a d i n g to the
by hydrolases w ith in
phenols and toxic quinones are responsible
or a r e j u s t
metabolism
d e b a t e whether
for n e c r o t i z a t i o n
(Sela 1981 c).
In
and produces n e c r o t i c
little
high
no v i r u s
virus
tite rs.
is
in
In
d etected
lesion
tissu es
(Yarwood
1960) .
Virus p a r t i c l e s
d u r i n g th e
in itial
m u l t i p l y and s p r e a d a t
phases of t h e i r
life
sim ilar
c y c l e w h ether
p a r t i c l e s are in systemic or hypersensitive p la n t s
a l.
197 2).
Lesion size and shape,
by p h y s i o l o g i c a l
heterogenous c e l l s found in a l e a f
Lesions reportedly
et al.
that v irus
in v ir u s
in
15.5 bean epidermal
one-fifth
that
(Helms and McIntyre 1962).
m u ltip licatio n
a h y p ersen sitiv e
spread in a systemic host.
cells
number
(Otsuki e t
d i f f e r e n c e s between the
1972; and Yarwood 1960).
spread
the
may be determined
stop enlarging a few days a f t e r
s i g n a l l i n g a decline
Otsuki
however,
and morphological
rates
It
infection
(Sela
1981c;
is a t t h i s point
plant
d iffers
from
Average l e s i o n s may involve only
and microlesions may involve
(Helms and McIntyre 1962).
tobacco p la n t s may be somewhat l a r g e r .
Thus,
v ir u s
about
Lesions in
spread i s
e f f e c t i v e l y h a l t e d s i n c e only a small p o r t i o n of l e a f c e l l s
are
involved.
Sela
(1981 c) claims t h a t l o c a l i z a t i o n of v i r u s
i s a means of l i m i t i n g v i r u s r e p l i c a t i o n and t h a t necrosis of
leaf
tissu e
One e f f e c t
is
a resu lt
associated
phenomenon
of
im m ediately
local
of l o c a l i z a t i o n and not t h e cause.
w ith
local
acquired
su r r o u n d in g
lesion
formation
resistance
local
lesio n s
(LAR).
are
is
the
Tissues
resistan t
to
subsequent v i r u s i n f e c t i o n t o v a r y i n g d e g r e e s (Loebenstein
1972).
The o t h e r e f f e c t ,
is
sy stem ic
which may j u s t be a v a r i a t i o n of LAR
acquired
Systemic r e s i s t a n c e i s
resistan ce
(SAR)
1981
c).
induced by v i r u s i n f e c t i o n in one p a r t
of the p la n t which causes another p a r t
subsequent v i r u s
(Sela
in fectio n.
to become r e s i s t a n t
to
Even l e a v e s a t a c o n s i d e r a b l e
d i s t a n c e from the o r i g i n a l i n f e c t i o n s i t e become r e s i s t a n t
(Roberts
1961).
1984;
Ross
Wounding
suggested
1966;
Bozarth and Ross 1964;
treatm en ts
do
not
induce
and Ross
SAR.
Ross
t h a t one or more s u b s t a n c e s formed d u r i n g l o c a l
l e s i o n production,
moved systemically to uninfected areas and
a c t i v a t e d mechanisms t h a t norm ally l i m i t e d l e s i o n s i z e or
lo ca lize d v iru s
in fectio n .
Applebaum
demonstrated t h a t
(1962)
F u r t h e r work done by Sela and
a substance i n h i b i t o r y
to
v i r u s i n f e c t i o n was p r e s e n t i n v i r u s i n f e c t e d t i s s u e s . They
called
a
this
number
substance an a n t i v i r a l
of
v iru s
in h ib ito rs
factor.
have
Since
been
that
iso la te d
time,
and
c h a r a c t e r i z e d from diverse p la n t species.
A n tiv ir al
Substances
During the l a s t 25 years,
from v i r u s
many substances were e x tr a c te d
i n f e c t e d and h e a l t h y p l a n t s
t h a t appeared t o be
antiviral
in nature.
Various workers,
using d i f f e r e n t p la n t
h o s t - s p e c i e s and diverse e x t r a c t i o n procedures,
demonstrate
present
(197 8)
that
an a n t i v i r a l
i n both.
found
R agetli
endogenous
substance (s)
and Weintraub
inhibitors
in
and Phytolacca americana along with
Even though endogenous i n h i b i t o r s
agents,
were able
of
some s o r t
(1962)
Dianthus
13 other
endogenous
was
and Grasso
caryophyl1us
species
t ested .
are e f f e c t iv e a n t i v i r a l
they tend not to be p r o t e c t i v e of the h o s t
which produces them.
to
species
The p o t e n t i a l f o r a v i r i c i d e based on
inh ibitors
is
slim
because
most
endogenous
i n h i b i t o r s have not been adequately shown to be i n h i b i t o r s of
v iru s re p lic a tio n .
Since sy st e m i c i n f e c t i o n s a r e the most
common response of crop p la n t s to v ir u s infec tio n,
which i n h i b i t s
adequate
m o stly v i r u s
p ro te ctio n
inh ibition
is
to
attained.
a
estab lishm en t
susceptib le
More
would not
p lant
important
a viricide
are
unless
the
i n h i b i t o r s of v i r u s e s t a b l i s h m e n t or r e p l i c a t i o n ,
are
substances
that
n atu rally
defend
the
give
host
100%
induced
f o r they
species
in
which they a r e found and from which p o s s i b l e v i r i c i d e s may
eventually be made.
Induced
a n tiv ira l
Chenopodium amaranticolor
substances
have
var.
Prince
Samsun
(Mitra 1985;
e n tirely
found
(Faccioli and Capponi 19 83),
s t r a mo niu m (Loebenstein and Ross 1963),
var.
b een
in
Datura
Phaseolus v u l g a r i s
(Kimmins 1969), N ic o t i a n a g l u t i n o s a , N. ta b a c u m
NN,
and
other
hypersensitive
Loebenstein and Gera 1981;
understood,
these
Nicotiana
spp.
and Sela 19 81 b).
substances
can
in h ib it
Not
v iru s
establishment,
Perhaps
the
one
m u l t i p l i c a t i o n or spread.
the f i r s t
iso la te d
g lu tino sa
by
antiviral
substance
Sela
Applebaum
and
to be studied was
(1962)
and a p p r o priately c a l l e d an a n t i v i r a l
from
factor
N.
(AVF).
Sela and h is co-workers have since p u r i f i e d and chara cteriz ed
AVF (Mozes e t a l . 1978; Antignus e t a l . 1977; and Sela e t a l .
1966).
AVF,
tobacco,
ex tracted
was
found
from
to
be
hom o gen iz ed
a
TMV-
in fected
phosphogl y c o p r o t e i n
with
a
m o l e c u l a r weight of 22,000 d. I t was s t a b l e i n SDS and a t pH
2.5.
AVF does not complex w i t h TMV p a r t i c l e s
affect
local
the uncoating of the v ir u s i t s e l f .
l e s i o n formation,
yet
it
certainly
nor does i t
AVF does not cause
i s present
in those
tobacco s p e c i e s which respond i n a h y p e r s e n s i t i v e manner;
i.e.,
containing
the N-gene.
A mode of AVF a c t i o n has been proposed by Gat-Edelbaum
et al.
(1983) and Sela
(1981 b ) . T heir working model p l a c e s
AVF i n th e r o l e of a messenger of a n t i v i r a l
messenger stimulator)
A series
production i s
of
the
which in turn confer
upon the c e l l s .
s t e p s have been o u t l i n e d
stimulated.
quickly a f t e r v ir u s
there is
(or a
which i s passed on to uninfected c e l l s .
AVF may then t r i g g e r defense mechanisms,
v ir u s r e s i s t a n c e
activ ity
First,
in oculation
to show how AVF
because AVF i s
released
(Gat-Edelbaum e t a l .
so
1983)
thought t o be a p r e - e x i s t i n g AVF p r e c u r s o r w i t h i n
cell.
The
N-gene
is
resp on sible
for
coding
for
processing enzymes which convert pre-AVF i n t o ac t i v e AVF in a
coupled
reaction
component.
with enzyme phosphorylation of
someunknown
Phosphorylation can be t r i g g e r e d by TMV or other
v iral
infectio ns,
p o l y a c ry l i c acid,
as" poly I:
1983,
fungal
ethylene,
poly C,
infections,
dsRNA,
TMV c o a t
s y n th e tic nucleotides such
and tRNA among others
(Gat-Edelbaum e t al.
Sela 1981 b and Kassanis 1980).
There
is
evidence
th at
the
infectin g
t u r n s on the p l a n t ' s d e f e n s e mechanisms.
only
protein,
be
shown
after
1
or
2
d ays
v iru s
actu ally
AVF a c t i v i t y
a fte r
can
in o cu latio n .
Presumably t h i s allows time to accumulate enough TMV-dsRNA to
s t i m u l a t e AVF p r o d u c t i o n .
then
tran sp o rted
elucidated
to
Once in i t s a c t i v e form,
h ealth y
how t r a n s i oca t i o n
happens once AVF i s present
A second a n t i v i r a l
c ells.
takes
Sela
place
has
or
AVF i s
not
ex actly
yet
what
in an uninfected c e l l .
s u bs ta nce ,
d istin ct
from AVF,
been i s o l a t e d from v i r u s i n f e c t e d N. ta b a c u m v ar .
has
Samsun NN
p r o t o p l a s t medium and has been given the name IVR (I n h i b it o r
of
V iral
R ep licatio n )
L o e b e n s t e in
(1983),
th is
AVF.
u np ublished d a t a ) ,
and Gera e t
al.
(1983)
s u b s ta n c e and d i f f e r e n c e s
General
p rin cip les
substance.
of
(1985,
(Loebenstein
IVR p r o p e r t ie s
-
one
a
26,000
1981).
and c h a r a c t e r i z e d
were noted between IVR and
and
2 biologically
the
oth er
a
ac tive
57,000
d
The 57,000 d s u b s t a n c e may of course be a dimer
the s m a l l e r su b s ta n c e .
but whether i t
ascertained.
been d e t e c t e d
virus
Gera
Gera and L o e b e n s t e i n
p u rified
include
d
and
is
It
IVR i s
thought t o be a p r o t e i n ,
a phosphorylated glycoprotein has not been
is
s t a b l e to pH 2.5 and a t 80 C. IVR has not
in
in tact
p lan ts,
replication
w ithin
intact
8
although
p la n t s
it
does
inh ibit
(Gera and Loebenstein
1983
).
IVR i n h i b i t o r y
activ ity
is
not
restricted
to
th e
i n d u c i n g v i r u s as Cucumber Mosaic Virus and P o t a t o Virus X
were
two other v ir u s e s
that
IVR i n h i b i t e d
in
the p r o t o p l a s t
studies.
IVR
is
thought
s t i m u l a t i o n of v i r u s
alter
TMV d i r e c t l y
number,
indicating
the c e l l
v iru s
rep lic a tio n
l o c a l i z i n g mechanisms.
nor
that
does
it
affect
phy sically
a new a n t i v i r a l
TMV i n f e c t e d
1985).
suppress
v ia
IVR does not
th e
plasmodesmata
restricting
virus
w ithin
i s not the mode of ac t i o n of IVR.
Recently,
from
to
glutinosa
Two s u b s t a n c e s ,
a n tiv iral
s u b s ta n c e has been i s o l a t e d
protoplast
medium
(Mitra
d e s i g n a t e d AVS-30 and AVS-65,
p ro p erties.
AVS-30
was
determ ined
have
to
be
endogenous; whereas, AVS-65 was an induced s u b s ta n c e which
showed extreme potency a g a i n s t v i r a l
sy nth esis.
AVS-65
is
sim ilar
to
RNA-directed p r o t e i n
AVF i n
that
it
is
a
phosphorylated glycoprotein with a molecular weight of 24,000
d.
I t i s a l s o h e a t s t a b l e to 64 C.
demonstrated
that
AVS-65
is
Mitra*s e x t e n s i v e study
more i n h ib i t o r y
than AVS-30 and
t h a t AVS-65 can induce f u r t h e r production of AVS-65
cells.
cell.
Thus,
resistance
can be
transferred
from c e l l
to
AVS-65 i s a s s o c i a t e d w i t h d r a m a t i c d e c r e a s e s i n TMV
coat p r o t e i n synthesis in i nfected p r o t o p l a s t s ,
not
in other
been
d e te rm i n e d
to
be
th e
immediate
but s t i l l
cause
of
has
th at
decrease.
AVS-65 i s an en d - p ro d u ct of a s p e c i f i c methodology and
it
is
uncertain
h ypersensitive
if
the
v arieties
substances
of
tobacco
extracted
are
from
indeed
the
three
same.
However
tests
have
been made
d i f f e r e n t p lan t species.
i 9^ more
effectiv e
p rotoplasts
than
presence of
cell
65 access.
It
at
in
using AVS-65 o b t a i n e d
AVS-65 i s o l a t e d from N. gl u t i n o s a
in h ib itin g
whole
walls
v iru s
plants,
rep licatio n
possibly
i s also b e t t e r a t
to
the
inhibiting virus replicatio n
the AVS-65s obtained from
N. Nicot iana v a r s. Samsun NN and Xanthi nc.
c u ltiv ar
due
in
in i n t a c t p l a n t s which may l i m i t AVS-
in p r o t o p l a s t s when compared to
tobacco
from
Samsun NN,
however,
An AVS-65 from
works
b e tte r
at
i n h i b i t i n g v ir u s establishment compared to the other two.
All
have
three a n t i v i r a l
been
compared
substances
amongst
mammalian in te r f er o n .
(AVF,
them selv es
IVR and AVF are
IVR,
as
and AVS-65)
w ell
as
with
s e r o l o g i c a l l y weakly
cross r e a c t i v e but IVR and i n t e r f e r o n are not r e a c t i v e a t a l l
(L oebenstein 1985,
unpub lish ed d a t a ) .
t e m p e r a t u r e and pH s t a b i l i t y ,
acetate.
In addition,
with v ir u s p a r t i c l e s .
S im ilarities
include
and p r e c i p i t a t i o n w i t h z i n c
AVF and AVS-65 do not complex d i r e c t l y
These molecules are
a c tiv e
inhibitors
a t extremely low concentrations. AVS-65 has not been detected
by common p r o t e i n a s s a y s ,
a l t h o u g h an tib o d y p r o d u c t i o n to
AVS-65 has not been undertaken.
(Mitra 1985).
cell
Yet,
AVF may be a c t i v e a t
it
is
highly p otent
only a few molecules per
(Sela 1981 a) as compared t o IVR and i n t e r f e r o n which
may also work a t such r e l a t i v e l y low concentrations
Loebenstein 1983 and Orchansky e t al.
P ierpoint
(1983),
and
(1983),
Rosenberg
Chessin
et
10
al.
(Gera and
1982).
(1983),
(1985)
Reichman
describe
et
al.
current
sim ilarities
substances
and
found
d ifferen ces
in
p lants
between
and
induced
interferon.
an tiv iral
S im ilarities
include :
1.
2.
3.
4.
5.
proteinaceous nature
s i m i l a r molecular weights
an tiv iral activ ity
release d by v i r u s i nfec ted t i s s u e s
dependence on host t r a n s c r i p t i o n and t r a n s l a t i o n f o r
activity
dsRNA presence i s mandatory for a c t i v i t y
6.
An i n t e r e s t i n g r e p o r t
purified
by Rosenberg e t
al.
(1985)
states
that
human leukocyte i n t e r f e r o n i n h i b i t s TMV r e p l i c a t i o n
i n tobacco p r o t o p l a s t s and t h a t
tobacco was 10-1000
t im es
more r e s p o n s i v e to i n t e r f e r o n than t h e animal c e l l systems
tested.
p la n t
to
However,
an tiv iral
inhibit
this
i s only one i s o l a t e d
incidence,
and
substances have not been conclusively proven
animal v i r u s e s
in animal
cell
systems
(Sela
19 81
are
routinely
a) .
Because
p lan t
and
animal
thought of as comparable,
and p la n t
an tiv iral
systems
not
the d ifferen ce s between i n t e r f e r o n
substances
could be extremely important
i n d e l i n e a t i n g the u n i v e r s a l i t y of a n t i v i r a l m a t e r i a l s .
stated
before,
AVS-65 can
induce more AVS-65 but
can not induce more i n t e r f e r o n in other c e l l s .
species s p e c i f i c in t h a t i t
in
its
interferon
Interferon is
a c t s ag ainst a l l v ir u s es to which
a p a r tic u la r species is susceptible.
only
As
source
species.
there
a p p ear s
In te rfero n is
AVF and IVR a r e
not
active
species
specific.
Overall,
a n tiv ira l
substances
th at
p l a n t s exposed t o v i r u s ,
to
be more
than
are
induced
in
much l i k e
11
one
class
of
hypersensitive
the m u l t i p l e c l a s s e s of
an tiv iral
substances
found
i n mammals -
the
interferons.
C e r t a i n l y the p l a n t s p e c i e s used and the type of e x t r a c t i o n
pro c e d u r e
f o l l o w ed
substance
found.
p la y s
A p lan t
mechanisms to help i t
Pathogenesis Related
Pathogenesis
discovery.
strictly
a
role
in
probably
(b)
(PR) p ro t e i n s
These p r o t e i n s
were
a s s o c ia te d with v i r a l
1976),
in v ir u s
for
or
at
least
system ic
it
multiplication.
intercellu lar
PR,
PS,
d
conditions
and
mobile
PAGE g e l s
(Antoniw and White 1983).
al.
or
are
forming
mechanisms,
in h ib itio n
throughout
from
of
v iru s
Europe and in
and physical make-up of
hom o gen ized
tissu es,
as they a r e v a r i o u s l y c a l l e d ,
They
under
generally not exceeding
are
resistan t
so lub le
1985 and Abu-Jawdah 1983,
12
under
acid
to a range of proteases,
n o n -d e n a t u r i n g
They appear a t
l e s i o n formation caused by v ir u s
et
lesion
localizin g
substances,
1983).
(pH 2.5 - 3.0),
in
be
and p ro t o p l a s t s .
p or b p r o t e i n s ,
(Redolfi
to
new p r o t e i n s
local
the chemical
are low molecular weight
18,000
with
work done
ex trac te d
fluids,
these
infected,
resistan ce
Further
p ro tein s
thought
new
was believed t h a t they were responsible
Canada revealed some of
these
are a r e l a t i v e l y
1978; G i a n in a z z i e t a l .
Since
associated
acquired
defense
or fungal pathogen induced
1977;
tobacco c u l t i v a r s ,
many
originally
(Antoniw e t a l .
produced
h as
an tiv iral
Proteins
d i s e a s e s i n tobacco
readily
actual
l o c a l i z e v ir u s infe ctio ns.
related
and Van Loon
th e
or v i r o i d
co nd itio ns
the onset of
infection
1982); however,
local
(Fortin
they a l s o
are
induced by a wide d i v e r s i t y of c h em icals or by n a t u r a l
ageing s t r e s s e s
such as f l o w e r i n g or l e a f
Loon 1983 and Fraser and Clay 1983).
produced only
(Asselin e t
for
de
a l.
novo
Conej ero e t
that
green
1985).
tissues,
There i s
p ro d u c t i o n
al.
1983;
of
not
roots
still
b
or
inner
inconclusive
proteins
and Van Loon 1983),
evidence
(DeTapia
but i t
stems
1986;
i s agreed
but th e tobacco s p e c i e s i n which they a r e found
carry
the N -
gene;
manner to TMV i n f e c t i o n
ring
They are reported to be
Proteins have not been proven to have any a n t i v i r a l
activ ities,
1985,
all
(Van
these p ro te in s are coded for by the host plant.
b -
also
in
senescence
1982).
near
respond
in a h y p e rs e n s itiv e
(Asselin e t a l .
1985 and Ahl e t a l .
T he ir h i g h e s t c o n c e n t r a t i o n can be found i n a
lesion
temperature
i.e.
margins.
sensitive.
A h yp ersensitiv e
Above 3 0 C,
formed and n e i t h e r are b -p r o tein s
n ecrotic
re s p o n s e
is
l e s i o n s a re not
(Van Loon 19 85).
Ahl and h er co-workers (1985) have e x t e n s i v e l y s t u d i e d
the presence of b - p ro te in s
for
the
genus
in tobacco to devise a phylogeny
NA^o^_iana.
The
p h y lo g e n y
p ro d u c e d
is
remarkably s i m i l a r to other phylogenies based on chromosomal
and cytological
bl
is
sexually
ab ility
is
studies.
in d icate
that
protein
t r a n s m i t t e d v ia a monogenic inheritance.
The
to synthesize b - p ro t e i n s in healthy tobacco hybrids
associated
w ith
high
N e c r o s i s V irus i n f e c t i o n
on to
Her r e s u l t s
resistan ce
to
TMV and
Tobacco
(Ahl e t a l . 1983) and can be passed
offspring.
Induced,
b
-
p ro tein
lik e
su b sta n ces
d e m o n s t r a t e d i n a v a r i e t y of p l a n t s p e c i e s .
13
have
b een
Because of the
complexity
of
ex tractin g,
induced p ro teins,
each s e t
names to the p r o t e i n s
outlines
17 p la n t
Van Loon e t
al.
analyzing,
and comparing
of researc hers
they have found.
species and t h e i r
has
given d i s t i n c t
Redolfi e t al.
unique
these
(1983)
induced proteins.
(1983) d id compare a few of
these p r o te in s
w i t h a n t i s e r a t o tobacco c u l t i v a r Xanthi nc and Samsun NN b p ro te in s.
It
was found t h a t
the b - p ro t e i n s from Nicotiana
species are s e r o l o g ic a l l y r e l a t e d ,
b - p ro te in s
from a l l
sero log ically
from
other p lan t
reactiv e.
d ifferen t
and perhaps i d e n t i c a l ,
s p e c i e s t e s t e d were not
They concluded t h a t b -
genera
are
but
su fficien tly
proteins
d ifferen t
to
be
considered genus s p e c i f i c .
Conceivably,
defense
fluid s,
PR or b p r o t e i n s
mechanisms.
where,
it
They
w alls
be
found
i s hypothesized,
changes i n th e chemical
cell
can
a r e n e c e ssa ry
(Parent
et
in
in p lan t
in tercellu lar
they i n i t i a t e
enzymatic
co m po sition or environment of
al.
1984).
Others b e l i e v e
they
the
are
a f f i l i a t e d w i t h t h e r e c o v e r y phenomenon of d i s e a s e d p l a n t s
(Nuecken and Nienhaus 1983),
(Antoniw e t a l .
1980 and Coutts 1978) or a r e j u s t
phytopathoiogical
resp o n s e
symptom development
Kassanis
a c q u i r e d r e s i s t a n c e phenomena
of
the
plant
to
(Comacho-Henriquez e t al.
(1983),
nevertheless,
questions
a general
in fe c tio n
and
19 83).
the
importance
of i n - d e p t h s t u d i e s of PR (b) p r o t e i n s as a way to s o l v e the
question of how n atural
r e s i s t a n c e in p l a n t s i s a t t a i n e d .
In
many c a s e s PR (b) p r o t e i n s can be induced by non-p atho gen ic
means
and r e s i s t a n c e
to d i s e a s e
14
is
not
n ormally produced
after
the
realize
proteins
th at
carrying
in fected
are
present.
because b p r o t e i n s
p la n ts
and
p la n ts,
mechanism pathway,
in
they
the
But
are
is
im portant
found i n a l l
in tercellu lar
may i n d e e d
even i f
it
N -
spaces
gene
of v i r u s
of
a defense
they are not a n t i v i r a l
themselves.
15
be p a r t
to
Research Goals
Research f o r
th is
work was d e signed i n such a way to
show w h ether or not an a n t i v i r a l
travel
s u b s ta n c e could p o s s i b l y
i n the i n t e r c e l l u l a r spaces of tobacco mosaic v i r u s
i n f e c t e d p ri m a r y l e a v e s of p i n t o beans.
addressed
(1985,
only
r e c e n t ly
u np ub lish ed
research
into
This id ea has been
by Wierenga-Brants
data)
an tiv iral
with
tobacco
substances
(1983)
cu ltiv ars.
has
centered
p la n t homogenates or p r o t o p l a s t s to e x tr a c t
t h o ug h
it
has
n eig hb orin g
b een
proposed
cells,
in tercellu la r
little
fluid s
and
substances may be present
a
chemical
signal
is
acquired r e s i s t a n c e
1961).
If
so,
it
th at
work
the
AVF may
has
It
responsible
Past
on u sin g
inhibitors.
Even
trav el
been
p o ssib ility
there.
and Mitra
done
that
to
w ith
an tiv iral
has been suggested t h a t
for
inducing
sy stem ic
(Chessin, p e r s o n a l communication; Ross
i s p ro b a b le t h a t such a s i g n a l may t r a v e l
through the i n t e r c e l l u l a r spaces t o r e a c h u n i n f e c t e d p l a n t
tissues.
can
be
It
i s a l s o of
found
pre sence
of
in
b
-
interest
p in to
b ean
p ro tein s
to determine i f b - p ro teins
in tercellu lar
in
flu id s.
in tercellu lar
flu id s
The
may
s u b s t a n t i a t e t h e i r p ossib le r o l e in plant defense mechanisms.
The aim was n o t
rather,
as
to
to
its
to
characterize
biological
iso la te
the
a specific
entire
su bsta nce,
intercellular
p r o p e r t i e s and i t s
but
wash f l u i d
protein profile.
The
p r o t e i n s in the i n t e r c e l l u l a r wash f l u i d were to be compared
to
the pathogenesis
- related
(b) proteins.
P i n t o beans were chosen because of the convenience of
their
fast
growth r a t e
and because l i t t l e
16
work has been done
wi t h t h i s
species as a source of
17
IWF,
MATERIALS AND METHODS
I.
Chemicals
All c h e mi c a l s f o r IWF e x t r a c t i o n ,
e l e c t r o b l o t t i n g and
SDS-PAGE, with the exception of SDS, were obtained from Sigma
Chemical
Co.
(St.
- Schlesinger
Louis,
Chem.
MO).
Mfg.
SDS was obtained from Gallard
Corp.
(Carle
Place,
NY).
Molecular
weight markers and the k i t f or p r ot ei n anal ysi s were obtained
from Bio - Rad Laboratories
I I.
(Richmond,
CA).
Plants
Phaseolus v ul ga r i s var.
e x p e r i me n t s
extracts.
with
a
as
as
bei ng
the
single
application
Corp.,
source
of
a
gener al
Dansville, NY 14437).
a c o nt r ol l ed
i n t h e greenhouse.
were
a l t hough
for
IWF
t h r e e per 4 “ pot,
plant
fertilizer
Plants were kept
in
chamber or
Temperature remained a t 20 -
ma i n t a i ne d ,
bioassay
plant
growth room or environmental
t he p h o t o p e r i o d was 16 hours per day.
conditions
used in a l l
Seeds were sown i n J i f f y mix,
(RA.PID.GRO.
either
wel l
Pinto was
23 C,
and
Standard greenhouse
t e mp e r a t u r e
r anges
f l u c t u a t e d more during some summer days.
P r i m a r y l e a v e s of
day
pinto
extraction.
bean
plants
M eristematic
c o m p a r a b l e s i z e d e l e v e n to t w e l v e
were
used
tissue
and
for
virus
the
assay
trifoliate
or
IWF
leaf
bud
of each plant wa s pinched off prior to treatment.
III.
Virus
Tobacco
method
of
mosaic
Gooding
virus
and
was
Hebert
i n f e c t e d N i c o t i a n a tabacum var .
18
isolated
(1967)
a c cor di ng
from
to
the
systemically
Samsun l e a v e s and from two
infect ed,
hyper sensi tive
Xanthi -
nc and
tobacco species
t a ba c u m var.
was d i l u t e d w i t h b u f f e r
--
tabacum var.
Samsun NN.
Vi rus inoculum
(see below)
so t h a t
100 - 200 l esi ons per Pinto bean h a l f - l e a f
IV.
an average of
were produced.
Inoculati on and Extraction of IWF
The
divided
manner
two p ri mar y
into
four
leaves
halves
of
a
typical
and i n o c u l a t e d
bean
in
plant
t he
was
following
(see Fig. 1).
Side #1 - TMV + Cel i t e
Side #2 - No rubbing
Side #3 - Buffer + Cel it e
Side #4 - No rubbing
The b u f f e r was a 0.067M, pH 7.0 Na-K phosphat e s o l u t i o n
which was kept a t 4 C.
TM
V sol ut i ons a t
were r i n s e d
Cel i t e was added to both b u f f e r and
10 mg/ml t o t a l volume as an abrasive.
Leaves
i n lukewarm t ap w a t e r w i t h i n 5 minut es
i n o c u l a t i o n and t h e n pl a c e d
i n the greenhouse,
growth room or environmental
chamber u n t i l
after
controlled
ext r ac t i ons
were
made.
Intercellular
intervals
after
extraction
day
various sources
1982,
fluid
was
i nocul at i on from a t
u s i ng
a mo d i f i e d
extracted
least
selected
compiled
per
from
de Wit and Spikeman
Leaves were r i n s e d ,
in room temperature
at
18 - 24 plant s
procedure
(Parent and Asselin 1984,
and Klement 1965).
ext r act i on,
dry.
wash
distilled
j u s t p r i o r to
water
and b l o t t e d
The mid - v e i n s were removed and t he h a l f - l e a v e s t hen
placed
Glass
dur i ng
in
cold
and rubber
buffer
with
weights
evacuation.
0.1%
prevented
Vacuum
19
(v/v)
2-mercaptoethanol.
floatation
infiltration
of
of
the leaves
buffer
was
Fig. 1.
Representation of pint o bean primary leaves showing
treatment divi si ons.
20
achieved using a Nevaco,
Pump Co.,
Berkeley,
model 911 vacuum pump (Nelson Vacuum
CA)
connect ed
Leaves were under vacuum f o r
to
a
glass
2.5 mi nut es,
a f t e r which the
vacuum was released,
over a 25-30 second period,
buffer
the pl ant
were
to
infiltrate
quickly
polycarbonate
b lo tted
centrifuge
intercellular
dry,
rolled
t ubes
(Nalge
dessicator.
allowing the
spaces.
and
Leaves
placed
Co.,
Rochester,NY).
The t ub es were spun a t 2800 -2900 rpm (1000 -
1150 g) f o r 8
t o 10 mi nut es i n a S e r v a l 1 Angle T a b l e - t o p c e n t r i f u g e
Sorvall,
Inc.,
Norwalk,
CN).
into
The f l u i d was v i s u a l l y
(Ivan
checked
f o r c l a r i t y and e i t h e r used i mmedi at el y or s t o r e d i n g l a s s ,
10 X 75mm disposable c u l t u r e tubes a t -17 C f or f uture use.
V.
IWF Tests
A.
Presence of Glucose 6-P Dehydrogenase
To determine whether c e l l u l a r damage occurred due to the
e x t r a c t i o n pr ocedur e i t s e l f ,
gl ucose 6-P dehydrogenase was
measured i n f r e s h IWF samples and crushed,
400 ul
IWF or cr ushed l e a f
healthy leaves.
samples were assayed f o r enzyme
a c t i v i t y usi ng a pr ocedur e modi f i ed from Sigma Di a g n o s t i c s
Kit No. 345-UV.
intervals
Absorbance readings were taken at one minute
for f i ve minutes immediately a f t e r
G-6-P dehydrogenase
Spectronic
1001 s p l i t
substrate
solution.
the addi t i on of
A Bausch and Lomb
beam spectrophotometer
(Rochester,
NY)
was used.
Leaf homogenate was prepared mixing 2.3 8 grams of t i s s u e
and 20 ml of b u f f e r s o l u t i o n w i t h a mo r t a r and p e s t l e .
The
m i x t u r e was spun a t 1150 g f o r 20 mi nut es and the s u p e r n a t a n t
was assayed.
21
B.
IWF as I n h i b i t o r of Virus Establishment
I n t e r c e l l u l a r wash f l u i d
to
10 days
ratio
after
with
Celi t e .
taken a t
inoculation
TMV inoculum,
Mi xt ur es
st ood
was mixed
wi t h
for
sel ect ed times from 0
the
in
a 1:1
addition
5 mi nutes
of
or
1:0,5
10 mg/ml
before
inoculation
u s i ng t he p ri mar y l e a f d i v i s i o n s s t a t e d above.
Inoculated
leaves were r i ns ed in tap water a f t e r 24 hours.
days,
C.
After 5 to 7
l e s i o ns were counted,
IWF as I n h i b i t o r of Virus Replication
Whole
pinto
bean
primary
leaves
inoculated with TMV 6 to 9 hours p r i o r
wer e
completely
to treatment.
Leaves
were p r e p a r e d as f o r IWF e x t r a c t i o n and randomly p l ac ed i n
beaker s
c o n t a i n i n g Day 7 IWF e x t r a c t s
ratio.
Control
Infiltration
of
leaves
the mixtures
stated.
Infiltrated
covered,
and p l a c e d
days.
Lesion
were
leaves
placed
in
were
put
were
then
i n a 1:4
buffer
was accomplished as
in a controlled
numbers
and b u f f e r
on paper
only.
previously
towelling,
growth chamber f o r
counted
and
5
diameters
measured,
D.
IWF D i a l y z i b i l i ty
Day 7 e x t r a c t s were dialyzed against d i s t i l l e d water for
24
hours
at
Industries,
4 C using
Inc.,
Spec t r o p o r
L, A.,
m o l e c u l a r wei ght c u t -
CA)
4
( Spectrum
dialysis
of f p o i n t of 10 -
tubing
14 kd.
Medi cal
with
a
The t u b i n g
was prepared in a b o i l i n g water bath using sodium bicarbonate
followed by washing in d i s t i l l e d water.
was checked by t e s t i n g
Biological
activity
the e x t r a c t s as i n h i b i t o r s of v i r a l
22
establishment.
agai ns t v i r u s
E.
Day 10 e x t r a c t s
were
tested
for
activity
replication.
Determination of Protein Contents
Protein
contents
of
IWF
extracts
determined using the p r o t e i n assay k i t
- Rad Labs,
standard
that
assay
employs
t he
pr ocedur e
was
albumin used to determine
taken from p l a n t s
were
routinely
supplied from the Bio
Br adford method
f ol l o we d
with
the standard curve.
(197 6).
bovine
The
serum
IWF e x t r a c t s
in which only one-half of a si ngl e primary
l e a f had been rubbed with buff er and Celi te were also assayed
f or
p r ot ei n contents.
In t h i s
from 3 to 7 days a f t e r
F.
SDS -
IWF was extracted dai l y
inoculation.
PAGE Gels
Extracts
against
case,
were d i a l y z e d ,
d istilled
water
i n pr e par ed d i a l y s i s
overnight,
after
t ubi ng,
which they were
concentrated in a Speed Vac Concentrator
(Savant Instruments,
Inc.,
dry.
Farmingdale,
pellet
was
(wt/vol)
50 ul
SDS,
NY) a t 45 C heat u n t i l
of
solubilizing
10% (vol/vol)
8% 2 - mercaptoethanol.
solution
glycerol,
Added to each
containing
O.IM Tris
(pH 6.8),
10%
and
Samples were vortexed and heated in
100 C water f o r 5 minutes.
SDS-PAGE was
o ut l i ned by Laemmli
were
generally
carried
out
(197 0)
15%
and Judd
17.5% g e l s
acrylamide.
we r e
also
Table 1 l i s t s
to
(19 82).
a c r y 1 am i de
methylenebisacrylamide r a t i o ,
and
a c c o r di n g
the
procedure
Separating gels
(a c r y 1 a m i d e / N, N'
30:0.8), a l t ho ug h 10%, 12.5%,
used.
Stacking
gels
wer e
3%
the chemical c o ns t i t ue nt s of a 15%
gel.
23
One hundred t o 150 ug of IWF sample p r o t e i n were added
t o each l a n e .
wei ght
markers
Phosphor yl ase
ovalbumin
inhibitor
done a t
I n c l u d e d i n each gel were t he low mo l e c u l a r
from Bio
B
{92.5K),
-
Rad Labs.
bovine
and lysozyme
(31K),
(14.3K).
albumin
soybean t r y p s i n
Electrophoresis was
7% a c e t i c acid overnight,
with 0.25% Coomassie b r i l l i a n t blue
weights
of
Gels
stai ned
in f i xi ng sol ut i on for
f o l l o we d by d e s t a i n i n g i n f i x i n g s o l u t i o n .
molecular
were
(66.2K),
10 wat t s with constant power for 2 to 3 hours.
were fi xed i n 40% methanol,
hour,
serum
(45K) , c a r b o n i c anhydrase
(21.5K),
These markers
1
Apparent
pr ot ei n bands were ascertained by band
l oc a t i o n in r e l a t i o n to low molecular weight markers.
24
Table 1.
Chemical
Constituents of a 15% SDS-PAGE Gel.
Stacking Gel
Acrylamide:BIS
IM Tris (pH 6.8)
0.2M EDTA
Water
TEMED
Ammonium p e r s u l f a t e
(10% wt/vol)
2.5 ml
1.88 ml
0.15 ml
10.3 ml
0.0075 ml
0.15 ml
Separating Gel
AcrylamideiBIS
1.875M Tris (pH 8.8)
0.2M EDTA
Water
TEMED
Ammonium p e r s u l f a t e (10% wt/vol)
25
15.0 ml
6.0 ml
0.3 ml
8.4 ml
0.015 ml
0.3 ml
G.
Immuno-Blot Analysis
1.
Preparation of Antiserum
•
A New Zealand r a b b i t was b l e d p r i o r
to 4 subcutaneous
injections
of a dialyzed and concentrated Day 7,
extraction
in
Complete
Freund's
adjuvant.
side #1 IWF
Intramuscular
and
intravenous i n j e c t i o n s were then made for two months with Day
7,
side
#1
IWF e x t r a c t i o n s
phosphate b u f f e r ,
pH 7.4,
resuspended
w i t h 0,85% NaCl),
in
PBS
{ O.OIM
Antiserum was
c o l l e c t e d 4 times.
2,
Electroblot ti ng
I n t e r c e l l u l a r wash f l u i d samples were run on a 15% SDS-
PAGE gel
and e l e c t r o b l o t t e d onto n i t r o c e l l u l o s e
Millipore;
Mi l 1i p o r e Corp.,
paper
South San Francisco,
{HAHY-
CA) a t 25
v o l t s and 1 amp. f o r 1,5 hours usi ng a method modi f i ed from
Judd
(1986),
The n i t r o c e l l u l o s e paper b l o t s were blocked for
1 hour in Dulbecco's modified PBS (dPBS)
0.5% Tween 20,
f o r 1 hour.
lOOmM PBS,
pH 7,4 , and t hen exposed to IWF a n t i b o d i e s
Two washes in dPBS -
followed antibody
lOul
- Tween,
exposure.
Tween f o r 10 minut es each
Blots
were then incubated with
p r o t e i n A - horseradish peroxidase in 50ml dPBS - Tween
f o r 1 hour to r e v e a l anti body bi ndi ng,
TBS ( 20mM T r i s ,
SOOmM NaCl,
A 20 minute wash i n
peroxidase buffer,
preceeded p r o t e i n band development.
pH 7.2
)
Developing sol ut i on was
SOmg 4-chloro-l-napht hol in 20ml methanol added to 100 ml TBS
with
100
ul
of
30% hydrogen
per oxi de.
stopped development and increased contrast,
H,
High Performance Liquid Chromatography
26
D istilled
water
High perfor mance l i q u i d chromatography was performed
using
a
Perkin
d e l i v e r y system.
-
El mer
(Norwalk,
CT)
Series
An HPLC g r a d i e n t p r o f i l e ,
4 solvent
as d e s c r i b e d by
«
Judd
(1987),
A lltech
was employed t o
r e v e r s e - p h a se
Deerfield,
IL).
separate
Cl 8 column
the
proteins
(Alltech
in
an
Associates,
E l u t i o n t i mes were r ecor ded on a Perki n -
Elmer LCI-lOO computing i n t e g r a t o r .
Fi f ty m i c r o l i t e r samples
of f r e s h Day 7 IWF e x t r a c t s from a l l 4 t r e a t m e n t s i d e s were
examined.
The r e s u l t i n g p r o t e i n f r a c t i o n s were not i s o l a t e d
because of
time co n s t r a i n t s .
27
RESULTS AND DISCUSSION
A.
G1ucose-6--Phosphate Dehydrogenase
R esults
from
testing
dehydrogenase a c t i v i t y
enzyme i s
present
for
show t hat
g lu c o s e ~ 6- p h o s p h a t e
only a small
amount of
the
in i n t e r c e l l u l a r f l u i d s as compared to the
amount w i t h i n l e a f c e l l s
(Table 2).
Although t h e s e r e s u l t s
a r e s i m i l a r t o t h o s e found by Rathmell and Sequei r a (1974),
they
reported
protein)
of
intercellular
a
difference
in
specific
enzyme
between
fluids,
indicating
Dehydrogenase were p r e s e n t .
enzyme s p e c i f i c a c t i v i t i e s ,
activity
homogeni zed
two
indicate
leaves
isozymes
My r e s u l t s ,
(mU/mg
of
based on
and
G-6-Psimilar
t h a t t he same G-6-P-
Dehydrogenase i s present both wi t hi n and outside of the c e l l .
Many d i f f e r e n c e s
the d i f f er ences
Sequeira
this
however,
cause
in enzyme a c t i v i t i e s
(1974)
study
reported.
Rathmell
t e s t e d tobacco c u l t i v a r Bottom Special
P i nt o
indicate
major
i n t e s t i n g systems coul d account f o r
cell
bean
that
was
tested.
t he
These
extraction
breakdown.
What
results
procedur e
trace
amount
and
and in
still,
does
of
not
cell
l eak age t h a t may occur due t o c u t l e a v e s ,
would not account
for
activity,
the l a r g e
differences
concentrations,
four
B.
protein
and p r o t e i n p r o f i l e s found in IWF from the
i nocul at i on treatments.
IWF as I n h i b i t o r of Establishment
IWF,
ol d
in b io l o g i c a l
when mixed with TMV and inoculated onto 11 - 12 day
P i nt o beans,
produced v a r i a b l e
virus establishment
(Table 3).
28
results
At most,
in
inhibiting
a 50% r e d u c t i o n i n
Table 2.
Presence of Glucose-6-Phosphate-Dehydrogenase
Homogenates and I n t e r c e l l u l a r Fluid.
Sample
IWF- Buffer/
c e l i t e rubbed
IWF- No rubbing
Healthy Leaf
Homogenate
a -
G-6-P--DH
Protein
(mg/ml )
in
Leaf
Specific Act i vi t y
(mU/mg protein)
a
1.09 mU
2.14
0.51
0.93 mU
2.07
0.45
30.60 mU
46.70
0.65
mU=mill i u n i t s. One I n t e r n a t i o n a l Unit (U) = t he amount of
enzyme a c t i v i t y t h a t w i l l convert 1 micromole of s ubs t r at e
per minute a t 23 C.
29
Table 3.
P er cent I n h i b i t i o n of Vi rus E s t a b l i s h me n t
Ext ract s Taken a t Selected I nt e r v a l s After Inoculation.
by
IWF
a
Extracti on Day
Percent
I nh i bi t i on
b
Day
0
+2 0 (n=3 )
Day
2
-14
(n=2 )
Day
3
-29
(n=3 )
Day
4
- 9 (n=6 )
Day
5
+2 0 (n=4)
Day
7
- 5 (n=9 )
Day
8
+ 8 (n=2)
Day
9
-44
(n=3 )
- 3 (n=5)
Day 10
a - Based on the l e s i o n number r a t i o 1:2/3:4.
A p o s i t i v e number
indicates
a
s t i mu l a t i o n of l e s i o n
production.
A negative
number i ndi cat es percent i nhi bi t i on .
b - n = Number of experiments performed per extraction day.
30
l e s i o n numbers was observed,
and
w i t h IWF e x t r a c t s taken t h r e e
ni ne days a f t e r i n o c u l a t i o n c o n s i s t e n t l y producing a t
le*ast
30% v i r u s
increased.
inhibition.
Usi ng
Day
7
Often
IWF
lesion
numbers
extracts,
four
were
of
nine
e x p e r i me n t s produced an average i n h i b i t i o n of 26% whereas,
f i ve
of
13%.
the nine experiments produced an average increase
The
low
percentage
of
inhibition
varied
of
widely
depending on which day the IWF was e x t r a c t e d , but t h e r e was
generally
no
P i nt o bean
trend.
These p a r t i c u l a r
IWF has l i t t l e ,
establishment.
i f no,
their
and Spi egel
methods
ability
for
(1983),
(1985,
testing
Mitra
that
a r e the r e s u l t s
(19 85,
unpubl i shed d a t a ) .
inhibitory
infer
to i n h i b i t v i r u s
Contrasting these r e s u l t s ,
obtained by Wieringa-Brants
data),
results
activity
unpublished
Even though
agai nst
virus
establishment and r e p l i c a t i o n were d i f f e r e n t from my methods,
and they used only IWF from tobacco c u l t i v a r s Samsun NN and
Xanthi
nc,
all
were a b l e
t o produce a range of 30% -
100%
reducti on in l es i o n numbers.
A l a g time may be n e c e s s a r y f o r an i n h i b i t o r i n IWF t o
st i mul at e
IWF i s
not
cell
defense mechanisms in Pinto bean.
introduced a t
act
quickly
replication.
Ther ef or e,
Thus,
the same time with TMV inoculum,
enough
or
at
a
rate
faster
when
it
than
may
TMV
This may explain the varied i n h i b i t i o n r e s u l t s .
Pi nt o
bean
IWF
is
not
an
inhibitor
of
virus
establishment.
C.
IWF as Inhibitor of Replication
Overall,
l e s i o n numbers were decr eas ed 60 -
31
69% by t he
four
treatment e x tr a c ts
compared t o t he c o n t r o l
Average l e s i o n d i a m e t e r s changed l i t t l e
control,
(Table 4).
as compared t o the
a l t h o u g h t he appearance of the l a r g e r l e s i o n s i z e
c l a s s e s was d i m i n i s h e d i n the t r e a t m e n t s usi ng s i d e #1 and
s i d e #2 IWF e x t r a c t s
IWF e x t r a c t s
inhibitor
(Table 5).
from v i r u s
This s t r o n g l y i m p l i e s t h a t
infected
Pint o bean l e a v e s
is
an
of v i r u s r e p l i c a t i o n .
Interestingly,
IWF e x t r a c t i o n s
showed some
bi ol ogi cal a c t i v i t y in i n h i b i t i n g l esi on numbers
when IWF i s
infiltrated
6-9
all
hours
evidence t h a t by a t
s u bs t a nc e
spaces
non-infected
whet her an a n t i v i r a l
inf ect ed s i t e s
signal
has
after
least
antiviral
of
f our
is
7 days a f t e r v i r u s
located
plant
within
tissues.
Thi s
is
inoculation,
an
the
It
intercellular
is
yet
unknown
s u bs t a nce has a c t u a l l y moved from the
to the uninfected areas or whether a chemical
moved to
the uninfected areas and st i mulat ed
production of an a n t i v i r a l
It
TMV i n o c u l a t i o n .
is possible
that
substance.
IWF may i n h i b i t v i r u s r e p l i c a t i o n
even though average l esi on diameters changed only s l i g h t l y
this
experiment.
Infiltrating
p l a n t c e l l s t o an a n t i v i r a l
inhibitor
if
it
of
was
replication
introduced
m u l t i p l i c a t i o n cycle,
of
non-visible
development
of
intercellular
IWF i nt o leaves
could
at
a
still
effect
crucial
point
in
exposes many
i n h i b i t o r a t the same time.
l es i o n
in
An
numbers
the
virus
which may then r e s u l t in the production
m icrolesions.
necrotic
wash
the
fluid
lesions
would
decrease in l es i o n numbers.
32
Perhaps
by
an
account
inhibiting
inhibitor
for
the
in
the
t he
drastic
Table 4.
Comparison of Average Lesion Number and Diameters Using
Day 7 IWF Ext racts Applied 6-9 Hours After Inoculation with TMV.
Control
Average Lesion
Number (a)
Average Lesion
Diameter
—
1
( X 10
mm)
(a)
1
Treatment
2
3
4
60.0
22.1
23.9
18.4
18.3
3.3
3.2
2.9
3.5
3,9
- See Table 5 f or experimental
33
sample s i z e s .
Table 5.
Lesion Size-Class Frequency Percentages Using Day 7 IWF
E x t r a c t s I n f i l t r a t e d i n Pi nt o Bean Leaves 6-9 Hours A f t e r TMV
Inoculat ions,
Size Class
—
1
( X 10 mm)
Treatment
Control
1
2
3
4
1
11.9
16.6
10.9
11.6
5.4
2
26.7
22.1
32.7
22 .3
17 .3
3
20.0
23.5
32,3
26.5
26.9
4
11.5
14.7
11.7
12.1
16 .5
5
10.7
13.4
8.2
13.5
19.2
6
5.2
5.1
2.7
7.4
5.4
7
6.3
3.2
0.8
2.3
5.0
8
3.3
1.4
0.4
2.3
1.5
94-
4.4
0.0
0.4
1.9
2.7
Average of 6 t r i a l s .
Total leaves used in a l l 6 t r i a l s :
Control (N=36), Treatments
(N=47-48)
Total l e s i o n diameters measured in a l l 6 t r i a l s :
Control (N=27 0)
Treatments (N=215-260).
34
D.
Dialyzibility
Biological
activity
24* hours d i a l y s i s .
dialyzed
was ret ai ned by IWF sol ut i ons a f t e r
This experiment did not d i r e c t l y
IWF
extracts
wi t h
the
results
for
although
establishment
was
reduced
non-dialyzed
both
are
29% wi t h
e x t r a c t compared to a side #3 e x t r a c t .
on v i r a l
IWF
extracts,
sim ilar.
a Day 7,
compare
Virus
side
#1
IWF
Testing for an e f f e c t
r e p l i c a t i o n w i t h a Day 10 s i d e #1 e x t r a c t ,
lesion
numbers were d e c r e a s e d by 73% b ut d i a m e t e r s were de c r e a s e d
only by 15%.
These r e s u l t s
greater
10
than
-
14
indicate
kd mo l e c u l a r
t h a t an i n h i b i t o r
weight
is
of
present
in
measured
at
i n t e r c e l l u l a r wash f l ui d s .
E.
Determination of Protein Contents
Protein
different
contents
intervals
of
IWF
extracts
a f te r virus
were
inoculation.
i n p r o t e i n content occurred in the 12 hour.
Day 10 e x t r a c t s .
contained
t he
Treatment
greatest
Peak increases
Day 4,
Day 7,
and
#1 of Day 7 IWF e x t r a c t s usual ly
amount
of
total
protein
(Table 6).
Total p r o t e i n markedly decreased from Day 0 to 12 hours a f t e r
i noculat ion.
Preliminary
protein
taken three
to
pinto
plants
bean
Cel i t e
time
gave
still
seven days
that
interesting
appears
in
a f t e r wounding j u s t hal f
The r e s u l t s
content
after
were
inocul at i on
only
results.
the
studies
levels
of
IWF e x t r a c t s
treatments
rubbed w i t h
A slight
of
buffer
periodicity
intercellular
from
and
over
pr o t e i ns
of a singl e primary l eaf on a plant.
r e p r e s e n t j u s t one t r i a l
35
usi ng 12 to 15 p l a n t s
1
(ug xlO /ml)
Table 6 .
Comparison of IWF P r o t e i n Contents
Taken a t Selected Times After TM
V Inoculation.
Extr act i on
Time
Treatment
2
3
1
4
1:3
Percent Difference
1:2
2:4
3:4
+
2
0 hrs(5)
38.6
32.6
37 .7
50 .9
12 hrs(2)
14.4
15.2
9.2
9.5
24 hrs(2)
12.3
---------
-----
12.0
10.2
+
36 hrs(2)
16.3
13.5
20.8
15.1
48 hrs(2)
15.2
17.9
23.0
4 d a ys (5)
57.0
36.7
5 days (1)
26.9
7 d a y s (11)
+ 18
- 36
-25
-
+ 60
- 3
---- —
---- —
-13
- 22
+ 21
- 11
+3 8
13.6
- 34
- 15
+ 32
+69
15.7
10.5
+263
+ 55
+249
+4 9
28.1
28.6
17 .5
-
-
4
+ 61
+6 3
63.0
34.7
30.0
19.5
+110
+ 82
+ 78
+54
8 days(1)
35.2
23 .5
21.4
22.5
+ 64
+ 50
+
4
- 5
9 d a y s (1)
35.8
18.5
17.3
16.1
+107
+ 94
+ 15
+ 7
10 days(6)
55.1
19 .7
18.0
14.3
+2 06
+180
+ 38
+26
(x)= Number of t r i a l s .
e x t r a c t i o n day.
+ 57
3
6
5
IWF extr act ed from 18-24 pl ant s per
36
per
e x t r a c t i o n day.
and d cor respond
Treatment
to
the
letters,
same s i d e s
of
i.e.,
pinto
si d e a,
Side c was the only side rubbed
Both e x p e r i me n t a l r e s u l t s
2,
3,
and 4
(Table 7).
s uppor t the h y p o t h e s i s t h a t
p r o t e i n s a r e bei ng made by t he h o s t p l a n t a f t e r a s t r e s s it
viral
since i t
or wounding.
be
These f i n d i n g s a r e a p p a r e n t l y novel
has not been previously reported t hat
t h r e e day c y c l i c i t y
c,
bean pri mary
leaves as in the ot her experiments using sides 1,
respect i vel y.
b,
in p r o t e i n l e v e ls .
pl ant s have a
I t i s not known why
p l an t s would produce bur s t s of prot ei ns and r el eas e them i nt o
the i n t e r c e l l u l a r
spaces.
Perhaps i t
can be r e l a t e d t o t he
r e p l i c a t i o n cycle of the v i r u s or the development of lesions.
But t h a t would not e x p l a i n the p r o t e i n c y c l e shown i n non­
infected plants.
(1983),
Rosenberg e t
and Orchansky et al.
maintain c e l l s
in a s t a t e
al.
(1982)
(1985),
of r e s i s t a n c e
beans may a l s o be produci ng an a n t i v i r a l
other
def ens e
intercellular
proteins,
and
al.
have noticed t h a t AVF can
supplemental doses a f t e r vi r u s i nf ect i on.
as
Reichman e t
l on ge r i f
given i n
TMV infect ed Pinto
s u bs t a nc e , as wel l
releasing
them
into
t he
spaces t hr oughout t he course of i n f e c t i o n t o
remain i n a s t a t e of higher r es i s t ance.
SDS-PAGE Protein P r o f i l e s
IWF p r o t e i n p r o f i l e s were el ucidat ed by running samples
through
an
SDS-PAGE
gel.
Eight
bands
showed
p r o t e i n l e v e l s in lane 1, containing side #1 IWF,
all
of
the other lanes
(Fig.
the eight bands were:
2).
compared to
The apparent molecular weights
36,500 d;
37
increased
34,000 d;
33,000 d;
Table 7.
IWF Protein Contents
Pinto Bean Primary Leaves.
Extracti on
Time
a
—2
(mg xlO /ml)
Treatment
b
c
d
of Buffer Inoculated
Percent Dif f erence
a :d
a :c
3 days
312
239
314
277
- 1
+ 13
4 days
379
408
342
249
+11
+ 52
5 days
363
309
405
366
-10
-
6 days
376
401
206
159
+83
+136
7 days
198
2 89
154
273
+29
- 27
Side "c" was rubbed only.
IWF ext r act ed from 12-15 pl a n t s per e x t r a c t i o n day.
38
1
28,7 50
d;
26, 500
Occasionally,
show
up
in
d;
18,200
d;
16, 800 ;
t he
gels
in
l ane
1,
composed of two p r o t e i n bands,
The singl e
was composed of
attained
however,
there
d.
would
were
no
The 36,500 d band was
both of
which i n c r e a s e d i n
band representi ng
the 33,00 0 d prot ei n
t h r e e p r o t e i n bands.
This i n f o r m a t i o n was
by u n d e r l o a d i n g SDS-PAGE gel
samples.
15,200
a band n ear 10,000 d mo l e c u l a r weight
comparable bands i n the o t h e r l a n e s .
st ai ni ng.
and
lanes
with
the
IWF
These bands m i g r a t e s i m i l a r l y i n an SDS-PAGE gel
indicating that
conclusions
t h e i r mo l e c u l a r we i g ht s ar e a l i k e ,
can be drawn about
s u b u n i t s of the same p r o t e i n .
whether
but
no
they are polypeptide
All enhanced bands,
produced
with IWF e x t r a c t s taken three or more days a f t e r inoculation,
had c o r r e s p o n d i n g
However,
t ho s e
were
barely v is ib l e ,
extracts
bands
appear
either
that
t he
t he
stained
SDS-PAGE g e l s
revealed
in
three
lanes.
lightly
or were
run w i t h Day 0 or
12 hour
five
more
other
larger
mo l e c u l a r
weight
p r o t e i ns were not produced de novo a f t e r v i r u s i nf ect i on,
the t h r e e s m a l l e r mo l e c u l a r wei ght
p r o t e i n s may have been.
The evidence was not conclusive. Bands
decrease
in
extraction
staining
(Fig.
3).
intensity
tended to increase or
depending
In a d d i t i o n ,
but
on
t he
day
a 20,500 d p r o t e i n
of
band
was a p p a r e n t i n onl y l ane #1 i n
Day 0 and 12 hour e x t r a c t s
but i t s
Day 4.
appearance diminishes by
These
results
are
similar
in
r e s u l t s o b t a i n e d by Abu-Jawdah (1983,
the v i r u s
18,500
some
respects
1982).
to
the
He found t h a t
induced p r o t e i n pi had a molecular weight of about
dal t ons
under
denaturing
39
conditions.
The
18,200
D A Y- 7
MW
2
3
92.5
66.2
45.0
31.0
21.5
14.3
Fi gure 2.
15% SDS-PAGE gel showing e i g h t enhanced p r o t e i n s
from a Day 7 IWF extract i on.
40
D A Y - 4
MW
1 2
3 4 1
D A Y - 7
2
3
D A Y - 8
4
1
2
3
DAY-IO
4
1
2
3
4
DAY-11
1
2
3
4
* 2. 5
6 6.2
Figure 3.
Comparison of p r o t e i n s from IWf e x t r a c t i o n s
at sel ect ed days a f t e r TMV i nf e c t i o n.
41
taken
dal t on p r o t e i n
pi protein.
Wax and
this
study may correspond to Abu-Jawdah's
Bean c u l t i v a r s
used i n h i s
Immuna and he used an a c i d
homogenates
Redolfi
to obtain
and
proteins,
600
in
Cantisani
all
dal tons
only
(1984)
from
acid
little
in
this
PS p r o t e i n s
prot ei ns.
study
but
possibility,
induced protei ns
from them.
also
f ound
only
four
PS
has
been
done
leaves
of
that
w ith
were not
This
does
bean
None of the
could be automat ical ly
pH 7-0.
though,
homogenized
p r o te in contents.
extractions
at
of
Saxa.
research
because
conditions,
from l e a f
extracts
i n t e r c e l l u l a r f l u i d and i t s
p rot ei ns
extraction
of which had a mo l e c u l a r weight of 13 ,900 +/ -
Phaseolus v ul ga r i s var.
Ver y
four
study were B r i t t l e
identified
made under
not
preclude
as
acid
the
t h e s e p r o t e i n s may indeed be PS
These pr o t ei ns were produced in a pl ant exhi bi t i ng
the hypersensi tive response to v i r u s
i n f e c t i o n and were also
seen,
i n l e a v e s which showed
a l b e i t i n low c o n c e n t r a t i o n s ,
systemic acquired r esi s t a n c e .
I nf ec t i on with TMV may be causing the plant
t he manuf act ure
of
proteins,
for
defense,
or
to i ncrease
selectively
r e l e a s e c e l l u l a r p r o t e i n s i nt o the i n t e r c e l l u l a r spaces.
gels
show
that
protein
staining
in
l a ne
1
is
The
increased
s e l e c t i v e l y f or s p e c i f i c prot ei ns compared to the s t ai n i n g in
the ot her t hr ee lanes;
is
not
r e p or t s
occurring
suggesting t h a t general
after
TMV i n f e c t i o n ,
(Matthews 19 81).
Immuno-Blot Analysis
42
cell
contrary
leakiness
to
some
Immuno-blots were produced from Day 0 and Day 10 IWF
extracts.
at
the
Protein bands appeared on the n i t r o c e l l u l o s e paper
locations
where
t he f i v e ,
larger
mo l e c u l a r
weight
enhanced pr ot ei ns appeared on SDS-PAGE gels in the Day 0 IWF
extracts.
All
inoculation
four lanes,
treatm ents,
nitrocellulose
all
r e p r e s e n t i n g the f our d i f f e r e n t
paper.
showed
protein
bands
on
the
The 20,500 d prot ei n al so appeared in
l a n e s of the Day 0 immuno-blot,
al t hough the bands were
very l i g h t i n samples from t r e a t m e n t s #2,
#3, and #4.
Only
the three smaller molecular weight protei ns did not appear in
t he
Day 0 immuno-blots
proteins
were
not
indicating
present
in
that
t hose
healthy
a ppear s ,
then,
t h a t t he 18,200 d,
p r ot ei ns
were produced de novo a f t e r
leaf
particular
tissue.
It
16,800 d, and the 15,200 d
i nf ect i on with
tobacco
mosaic virus.
Ten days
proteins
leaves.
are
after
virus
present
t hroughout
Immuno-blots
p r o t e i n bands,
each sample.
immuno-blot
infection,
of
Pinto
the
enhanced
bean
primary
taken of Day 10 IWF e x t r a c t s
representi ng a l l
of
The presence of a l l
would
the
all
seem
to
the enhanced proteins,
in
prot ei n bands in the Day 10
indicate
capable of producing antibodies
showed
that
the
rabbit
to IWF prot eins and t h a t
was
the
l a c k of a n t i b o d i e s was not t he r e a s o n f o r the l ack of p r o t e i n
band
development
of
t he
three
proteins
in
the
Day 0 IWF
extracts.
High Performance Liquid Chromatography
High per for mance l i q u i d chromatography of IWF samples
produced unexpected r e s u l t s .
43
Fewer t o t a l
prot ei n peaks show
up
in
this
exper i ment
as
opposed
to
p r o t e i n bands found in SDS-PAGE gels,
some of
t he
total
number of
perhaps i ndi cat i ng t h a t
the st ai ned p r o t e i n bands in SDS-PAGE gels represent
polypeptide
subunits.
Comparing s i d e #1 to s i d e #3 t h e r e a ppear s t o be only an
increase
i n one p r o t e i n
in
side
#1,
but
a diminishing
complet e l o s s of two t o f o u r o t h e r p r o t e i n s
comparing Fig.
4 t o Fig.
5,
(Fig. 4).
there is a noticeable
or
When
trend in
the p r o t e i n peak which has a r e t e n t i o n time of 4.228 minutes
i n the s i d e #1 IWF e x t r a c t .
I t i s a t i t s h i g h e s t i n s i d e #1
and gradually decreases to where i t
the
side #4 IWF e x t r a c t .
i s almost undetectable in
This may i ndi cat e
that
the pr ot ei n
i s i n c r e a s e d by TMV i n f e c t i o n w i t h i n a l e a f and may be a p a r t
of
the d ef ens e mechanism of
appear s
to happen w i t h
the p l a n t .
Just
the o p p o s i t e
the p r o t e i n peaks w i t h a r e t e n t i o n
time of 3.684 mi nut es and 6.756 mi nut es i n s i d e
peaks are undetectable in side #1.
However,
these
results
do s uppor t
It
#3.
These
i s unkown why t h i s
t he
other
is.
e x p e r i me n t a l
evidence presented t h a t show there are p r o t e i n changes in the
i n t e r c e l l u l a r spaces in plant s
44
i nfect ed with TMV.
A
i.m
F=y3T
SOI
-
2 .773
SIDE
1
I.no
3-544
11.030
DE 3
Fi gur e 4.
Comparison of HPLC samples from si d e #1 and si d e
#3 Day 7 IWF ex t r ac t s .
45
SIDE
Fi gure 5.
Comparison of HPLC samples from si d e #2 and s i d e
#4 Day 7 IWF ext r ac t s .
46
SUMMARY AND CONCLUSIONS
Extracting
infiltration
intercellular
and cent ri f ugat i on,
proteins,
via
_in vacuo
has many advantages.
It
is
a si mpl e pr ocedur e and avoi ds some of the d i s a d v a n t a g e s of
leaf
homogenizat ion.
The techni que i s g e n t l e t o the p l a n t
c e l l s and does not cause major c e l l
though,
major
to r o u t i n e l y
cell
damage i s
undergoing mu l t i p l e
other
test
workers
i nspecti on of
the general
occurring,
infiltrations
do.
damage.
Many
especially
prior
if
leaves
ar e
to c e n t r i f u g a t i o n as
depend
on v i s u a l
t h e i r e x t r a c t i o n samples to determine c e l l u l a r
deWit and Spikeman 1982).
Even low c o n c e n t r a t i o n s of c h l o r o p h y l l ,
in
i s necessary,
technique to assure no
researchers
damage ( Parent and A s s e l i n 1984;
damage,
It
i n d i c a t i v e of c e l l
may be masked by tannins or other substances present
the sample.
Based on the dif fer ence between glucose-6- P-
dehydrogenase found in the i n t e r c e l l u l a r wash f l u i d s and l eaf
homogenates,
the
si ngle
infiltration
technique
used i n
this
study did not cause detect abl e damage.
A virus
inhibitor
intercellular
Although
variable
spaces
inhibition
when
is
apparently
of
TMV i n f e c t e d
of
virus
IWF was
mixed
present
Pi nt o
bean
establishment
with
in
was
TMV inoculum
the
leaves.
highly
prior
to
inocul at ing,
IWF did s i g n i f i c a n t l y reduce l es i o n numbers when
infiltrated
6-9 hours a f t e r
that
IWF c o n t a i n s
According
virus
an
TMV i n f e c t i o n .
inhibitor
t o Loebens t ei n and Gera
replication is
of
This s u g g e s t s
virus
(1981),
replication.
an i n h i b i t o r of
act i ve when added 5-12 hours post vi r u s
47
i noculation,
place.
which ensures t h a t vi r us establishment has taken
Additional
p r o vi d e d
by
fr equency
evidence for an e f f e c t
IWF which
of
small
produced
lesion
size
a
on r e p l i c a t i o n was
slight
classes
increase
and
appear ance of t he l a r g e r l e s i o n s i z e c l a s s e s .
from
TMV i n f e c t e d
greatest
Pi nt o
effects,
inoculation
but
multiplication will
effects
on l e s i o n
experiments,
half-leaves
extracts
treatments
Ge n e r a l l y an a n t i v i r a l
bean
also
from
showed
the
in
the
a d i mi ni s h ed
IWF e x t r a c t s
produced
three
antiviral
the
other
activity.
subs t a nce which a c t s a g a i n s t v i r u s
have an e f f e c t
on l esi on diameters,
numbers a r e much more v a r i a b l e .
but
In my
l es i o n numbers were a l t e r e d the most.
I t appear s t h a t t he i n h i b i t o r i n my e x t r a c t s i s l a r g e r
than 10,000-14,000 d molecular weight based upon the r e s u l t s
obtained
from
dialysis
determined whether any of
PAGE gels
is
in
the
Pi nt o
levels
It
the induced pr ot ei ns
the i n h i b i t o r ,
TMV i n f e c t e d
cyclicity
experiments.
has
not
been
shown in SDS-
though.
beans
of
showed a s t r a n g e
their
three
intercellular
day
proteins.
The l a r g e s t di f f er ence in p r o t e i n contents between TMV rubbed
leaves
and
extracts,
content.
contained
buffer
which
Day
the
IWF e x t r a c t .
response
in
7
rubbed
leaves
o c c ur r e d
showed an i n c r e a s e
IWF
greatest
extracts
amounts
Wounding
protein
also
levels,
from
of
though
48
Day
263% t o t a l
to
4
leaves
per m i l l i l i t e r
induce
the
a
IWF
protein
TMV i n f e c t e d
pr ot ei n
tended
s h o r t e r - general ly only two days,
of
in
of
cyclical
periodicity
was
and the percent di f f er ence
between e x t r a c t s from v i r u s and buf f er rubbed leaves was much
smaller.
Eight
enhanced
proteins
were
detected
in
the
i n t e r c e l l u l a r spaces of TMV i n f e c t e d Pi nt o bean l e a v e s .
these,
Of
t he f i v e l a r g e s t mol e c ul a r weight p r o t e i n s were not
produced
de
experiments,
novo;
it
but
according
to
the
i mmu n o - b l o t
appears t h a t the three smaller pr ot ei ns were.
A 20,500 d p r o t e i n was p r e s e n t i n SDS-PAGE g e l s usi ng Day 0
and
12
hour
extracts,
tissues
{ s i de
staining
of
#1).
this
slightly visible.
but
only
By t h r e e
band
was
from
days
reduced
TMV i n f e c t e d
after
plant
inoculation,
a l t hough
it
was
still
From Day 3 to Day 10 t he 20,500 d p r o t e i n
was o c c a s s i o n a l l y found i n IWF e x t r a c t s from b u f f e r rubbed
leaves
( side #3), but agai n,
band was very
light.
the s t a i n i n g i n t e n s i t y of t h i s
Some of
the
enhanced p r o t e i n bands
i n c r e a s e d i n s t a i n i n g i n t e n s i t y over time,
peaking a t Day 4
or Day 7 depending on the protei n.
High
per formance
liquid
chromatography
e x pe r i me n t s
r ei nf or ced the previous evidence which showed q u a l i t a t i v e and
quantitative
protein
changes
in
Pi nt o
bean
leaves
after
inocul at i on with virus.
It
is
cellular
is
wel l
would
assumed
from c e l l
be
evidence
proteins
plants
undergo many changes
p r o t e i n l e v e l s when diseased
generally
travel
known t h a t
more
are
to c e l l
energy
(Fortin
that
many
via
increased
in
these
(Matthews
1985)
since
1981).
suggests
tobacco i n t e r c e l l u l a r
49
and i t
substances
the piasmodesmat a
efficient
and A s s e l i n
of
(Van Loon 1982)
that
in
can
this
Recent
certain
spaces a f t e r
exposure
to
virus
or
other
stress
a d d i t i o n t o t he above study,
shown
that
protein
producing
profiles
prot ei ns,
spaces
In
my study w i t h Pi nt o beans has
change
in
the
spaces over t i me a f t e r v i r u s i n f e c t i o n .
intercellular
ag ent s .
by d i r e c t
viral
intercellular
Travel through t he
inhibitors
or
other
which may be involved in such defense mechanisms as
wound responses,
s t i mu l a t i on of
systemic acquired r es i s t an ce
or i n d u c t i o n of o t h e r t o x i c s u b s t a n c e s ,
may be j u s t a n o t h e r
mechanism by which the p l a n t can e f f e c t i v e l y b a t t l e v i r u s
infections.
My r e s u l t s
from v i r u s
is
indicate
that
the
i n t e r c e l 1uar wash f l u i d
i nf ect ed bean leaves has an a n t i v i r a l
inhibitory
toward v i r u s
It
The more s t u d i e d
antiviral
substances,
antiviral
p r o p e r t y a l t hough AVF i s u s u a l l y a s s o c i a t e d wi t h
inhibition
of
replication.
AVF,
replication.
nature.
virus
IVR, and AVS-65,
establishm ent
Because
of
the
also
rather
e x p e r i me n t a l
show t h i s
than
viral
differences
in
e x t r a c t i o n procedures used in my study and those used by Sela
(1981 c),
Gera and L oe b e ns t e i n (1983), and Mi t r a
inhibitory
activity
present
directly
related
extracts
were n e i t h e r
e x p e r i me n t s
to the other
testing
establishment
as
any
of
IWF e x t r a c t s
antiviral
concentrated
nor
can
not
substances.
purified
t he
be
IWF
prior
to
i n h i b i t i o n of v i r u s m u l t i p l i c a t i o n or
the
respective a n tiv iral
inhibitor
in
(1985),
other
workers
substances.
have
done
with
their
This may e x p l a i n why the
in IWF did not produce i n h i b i t i o n exceeding 7 5% in
t he e x pe r i me n t s as have t he o t h e r known a n t i v i r a l
50
substances.
Although W i e r i n g a - B r a n t s* (19 83)
(1985,
unpubl i shed
fluids
from v i r u s
p u r i f i e d IWF,
of
virus
larger
work w i t h
infected
leaves
establishment
and
present
Asselin
from the p o s s i b i l i t y
lesions
hypersensitive
Co n c e n t r a t i o n s
expand
continuously,
of
for
the
albeit
they
sl owl y,
appear
to
in
stop
substances
found
spaces may be much lower
thus making t r avel
naturally
than t hose
through i n t e r c e l l u l a r
secondary importance.
TMV i n f e c t e d
bean l e a f
advantageous
and
for
produce pr o t ei ns
these
comparable
a
it
of p r o t e i n l e v e l s produced i n
intercellular
No other research,
findings
is
virus
I am aware of,
not
known
infected
enhanced
not
or
to
be
has report ed
this
stressed
proteins,
would
be
plant
to
at
least
The pathogenesis
serologically related
found i n bean p l a n t s
they may a l l
why
appear
in bur st s.
eight
are
spaces
that
to a known PS protein.
p r o t e i ns
proteins
whereas
inhibitory
The t h r e e day c y c l i c i t y
(b)
account
The difference may also stem
plants,
intercellular
Of
may
intercellular
3-4 days i n h y p e r s e n s i t i v e tobacco p l a n t s .
found wi t hi n c e l l s ,
s uch
tobacco
the
t h a t P i nt o bean p l a n t s a r e i n h e r e n t l y
bean
expanding a f t e r
unique.
in
Perhaps
i n t h e i r def ense mechanisms than tobacco p l a n t s .
Necrotic
space of
was not based on usi ng
1984)
di f f er ence between our st udi es.
within
intercellular
and m u l t i p l i c a t i o n .
amounts
(Parent
different
tobacco
they were able to achieve up to 100% i n h i b i t i o n
protein
fluids
data)
and M i t r a ' s
one
is
related
to the induced PS
(Van Loon e t
al.
1983),
but
be r e p r e s e n t a t i v e s of a l a r g e r c l a s s of defense
51
p r o t e i n s with each genus producing a unique set.
It
is
thought
proteinaceous
resistance
that
a
in nature,
chemical
signal,
may play a r o l e
phenomenon of
virus
infected
p e r s o n a l communication; Ryan 1984).
in
possibly
the
plants
At f i r s t ,
acquired
(Chessin,
t he 20,500 d
p r o t e i n p r e s e n t i n Day 0 and 12 hour e x t r a c t s may appear t o
be such a s i g n a l .
However,
the SDS-PAGE band r e p r e s e n t i n g
t h i s p r o t e i n diminishes gr e a t l y over time.
Systemic acquired
r e s i s t a n c e can be demonstrated in non-infected leaves 10 days
after
If
i n o c u l a t i o n of o t h e r l e a v e s on the p l a n t
t he 20,500 d p r o t e i n
s y s t e mi c
resistance,
apparent in a l l
Matthews
is
it
a signal
would be
p r o t e i n which induces
expected
Day 10 IWF ext r a c t s .
(1981)
after
composed
of
readily
has r e p o r t e d t h a t v i r u s p a r t i c l e s and
washing
17,500
to be
I t was not.
even some v i r u s coat p r o t e i n may s t i l l
surface
(Ross 1961).
with
wat er .
d mo l e c u l a r
be l e f t
TMV coat
weight
on the l e a f
protein
sub-units.
It
is
is
p o s s i b l e t h a t t he 20,500 d and t he 17,500 d p r o t e i n s a r e the
same but
t h a t would not explain the presence of
the 20,50 0 d
p r o t e i n in leaves t h a t were not infected.
Ryan (1984)
f or
al so r e p or t s
inducing pr ot ei nas e
t hat
wound signals
r esponsi bl e
i n h i b i t o r s have molecular weights of
5,000 - 10,000 d , but
these would not be apparent in my SDS-
PAGE g e l s
samples
since
electrophoresed.
all
were
Ryan's wound signal s
dialyzed
were
before
smaller
bei ng
than the
m o l e c u l a r wei ght c u t - o f f p o i n t of the d i a l y s i s t ubi ng.
likens
t he
proteinase
inhibitor
52
proteins
to
t he
He
induced
tobacco PR (b) p rot ei ns on the basi s of molecular weights and
stability
under
acid
or
proteolytic
conditions.
Proteinase
i n h i b i t o r s have sub-unit molecular weights of 8,000-12,000 d.
Bean IWF p r o t ei n p r o f i l e s r a r e l y
in
the
showed any induced protei ns
10,000 -14,000 d range.
Another i n t e r e s t i n g
that
general
cell
are
inoculated
from SDS-PAGE gels
leakage i s not occurring a f t e r
with
s p e c i f i c prot ei ns
result
virus.
The p r e s e n c e
of
in IWF from i nfected t i s s u e
i ndi cat es
plant
cells
enhanced,
i ndi cat es
t hat
c e l l s are s e l e c t i v e l y rel easi ng or allowing pr ot ei ns i nt o the
intercellular
it
is
unknown
i nvolved
in
def ense
mechanisms or a r e j u s t t he r e s u l t of p h y s i o l o g i c a l
changes
whet her
spaces
these
after
proteins
the pl ant i s undergoing.
increased
cell
membrane
leakage
detriment al
the plant
hydrol yti c
enzymes but
are
directly
Past studi es
Non-specific
to
TMV infecti on.
leakiness
of
(Matthews 1981) r epor t
after
proteins
not
But
virus
from
only because
because
a general
infection.
cells
may
be
they contain many
exodus of
prot ei ns
may e f f e c t c e l l u l a r metabolism.
In c o n c l u s i o n ,
present
i n the
bean l e a v e s .
present,
Vi r us
spaces.
spaces of
TMV i n f e c t e d
Based on v i r u s r e p l i c a t i o n s t u d i e s i t
to a l e s s e r exte nt,
t hrough
infection
quantitative
i n h i b i t o r of m u l t i p l i c a t i o n i s
intercellular
systemic r e s i s t a n c e
travel
a virus
t he
intercellular
protein
is also
i n l e a v e s which have a c q u i r e d
i n f e r r i n g t hat an a n t i v i r a l
is
Pi nt o
responsible
changes
spaces
for
found
of
t he
in
substance can
plant
leaves.
qualitative
t he
and
intercellular
Although these pr o t ei ns have not been d i r e c t l y shown
53
t o be a n t i v i r a l
plant
defense
resistance.
alternate
proteins
route
for
to t r a v e l .
fluids
species,
mechani sms
Intercellular
similarities,
and
i n t hemsel ves,
they may f u n c t i o n in o v e r a l l
such
spaces
antiviral
as
systemic
may simply
substances
acquired
serve
or
as an
associated
This study has e l u c i d a t e d some of the
differences,
obtained
and p r o p e r t i e s
from
various
between p r o t e i n s
virus
infected
plant
b u t more r e s e a r c h needs t o be done t o expand upon
the r e s u l t s obtained thus f ar.
bFuture Studies
To f u r t h e r u nd e r s t a nd t he i n t e r - r e l a t i o n s h i p s between
vi r u s
i nf ect i on,
protein
induced i n h i b i t o r s ,
production
experimental
by
Pinto
acquired res i s t ance,
bean
host
plants,
procedures
ar e
necessary
technique would be t r u l y helpful
whet her
intercellular
d ef ens e mechanisms.
for
glucose-6-
spaces
to
acet one
increase
or
are
cell
organic
i nvol ved
in
in
IWF samples
solvent,
membrane permeabili ty but
d r a s t ic in t h e ir effects
extracts
det er mi ne
if
this
antiviral
This could be accomplished by t e s t i n g
P-dehydrogenase
other
dur i ng IWF
in answering the question of
leaves which have been t r e a t e d with heat
as
more
research i s needed.
Experiments which t e s t membrane i n t e g r i t y
extraction
and
from v a r i o u s l y
or chemicals,
that
ar e
such
known
to
which are much l e s s
than homogenization.
treated
t aken from
By t e s t i n g
P i n t o bean l e a v e s ,
IWF
a more
a c c u r a t e c o n c l u s i o n could be drawn about r e l a t i v e membrane
damage incurred by the e x t r a c t i o n procedure presented i n t h i s
54
study.
Further
t e s t i n g of
replication
should
IWF samples as an i n h i b i t o r of vir us
include
r e s p on di n g h o s t p l a n t s ,
testing
them
in
systemically
f ol l o wed by an assay of v i r u s t i t e r
e i t h e r by ELISA methods or a b i o a s s a y on hyper sens i t i v e l y
r es p on di n g
indication
directly
number
plants.
of
in
and
physical
This
inhibitory
a
"double
activity
hypersensitive
size.
In
virus replication
gives
than t e s t i n g
plant
addition,
p r o p e r t i e s of
assay"
a
better
IWF e x t r a c t s
and measuring
exper i ment s
IWF i n r e s p e c t
to
lesion
test
the
t o i t s a c t i v i t y as a
i n h i b i t o r would allow comparisons t o be
made between IWF and the three other characterized a n t i v i r a l
substances - AVF,
IVR,
and AVS-65.
An a c i d e x t r a c t i o n of Pi nt o bean i n t e r c e l l u l a r f l u i d s
may
d e mo n s t r a t e
cultivar
that,
the
as
determine whether
or
if
they
may
pre s enc e
yet,
has
of
been
PS p r o t e i n s
untested.
PS prot ei ns are s t r i c t l y
travel
in
the
in
Thi s
cellular
intercellular
a
bean
would
prot ei ns
spaces.
The
pr e s enc e or absence of PS p r o t e i n s i n IWF e x t r a c t s may have
im plications
resistance,
for
the
role
of
PS p r o t e i n s
in
acquired
since PS pr o t ei ns have only been detected in l eaf
homogenates so f ar .
The
question
periodicity
interest.
contents
c o mp l e t e l y
periodicity
why
of p r o t e i n
physiological
protein
of
of
healthy,
Pinto
levels
It
bean
plants
after virus
would be
IWF samples,
infection
necessary
on a d a i l y
unrubbed bean p l a n t s
i s inherent.
u nd e r go
is
a
of
to
assay
basis,
from
to d e t e r mi n e
if
Complementary e x per i ment s would
55
include assaying p ro t e i n l e v e l s
from
which
IWF e x t r a c t s
had
of
the homogenized l e a v e s
already
p r o t e i n p e r i o d i c i t y within the c e l l s
been
taken
to
show
and assaying TMV l evel s
of i nf e c t e d leaves over time.
Many p o s s i b i l i t i e s e x i s t for study of the prot ei ns found
in
the
intercellular
spaces
p l ant s .
Radioactive
tracers
de
production
of
novo
of
virus
infected
Pi nto
bean
could be employed to determine
proteins.
Ext ensi ve
immuno-blot
experiments may al so produce more conclusive evidence f or de
novo
production
protein
as
p r o mi s i n g
Basic
proteins.
band which appears
activity
signal
of
an
inducer
results
may be
research
i nvol ved
of
in
is
Each p r ot ei n
bands p r e s e n t e d
antiviral
ability
in
resistance
20,500
may
the q u e s t i o n of
wash f l u i d s
composed of
shown
the
acquired
d
its
yield
whether a
resistance.
could al so be accomplished with the pr ot ei ns
compare the bands
peak
virus
s yst emi c
could be
extr acted
t o not only d e mon s t r a t e
polypeptide
subunits,
but
produced w i t h the SDS-PAGE gel
in t h i s
study.
Additionally,
HPLC e x pe r i me n t s
activity
and elucidat ed with HPLC
peak f r a c t i o n
and run on an SDS-PAGE gel
protein
of
in SDS-PAGE gels and t e s t i n g
i n answeri ng
found i n i n t e r c e l l u l a r
experiments.
Isolation
against
virus
could
be
also
the
to
protein
each p r o t e i n
tested
replication
if
for
its
and f o r
its
to induce r es i s t ance.
High performance l i q u i d chromatography experiments could
a l s o be done w i t h f r e s h IWF e x t r a c t s
t aken on a d a i l y b a s i s
a f t e r TMV i n f e c t i o n to show trends in concentration l e v e l s of
56
(distinct
pr ot ei ns
in order
to give a b e t t e r understanding of
how prot ei ns are changing in response to v i r u s
i nfection.
Much knowledge has a l r e a d y been gained i n the l a s t 20
y e a r s about a c q u i r e d r e s i s t a n c e
with
disease
agents.
However,
in p l a n t s
more
after
studies
infection
need
to
be
undertaken in order to produce or lead to the production of a
marketable v i r i c i d e
or
enhance a p l a n t ' s natural
i nducer
of
resistance
defense against v i r u s
57
which would
i nfection.
REFERENCES
Abu - Jawdah, Y. 19 83.
Induction of pathogenesis r e l a t e d (b)
p ro t e i n s in
Phaseol us v u l g a r i s upon fungal or v i r a l
i n f e c t i o n or a f t e r chemical i n j u r y .
Neth. J. PI. Path.
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