J. gen. Virol. (1987), 68, 1801-18ll.
Printed in Great Britain
1801
Key words: MStpV/plant and insect hosts/protein level detection
Differences in Levels of Detection for the Maize Stripe Virus Capsid and
Major Non-capsid Proteins in Plant and Insect Hosts
By B. W. F A L K , 1. J. H. T S A I 2 AND S. A. L O M M E L 3
1Department of Plant Pathology, University of California, Davis, Californ& 95616, ZFort
Lauderdale Research and Education Center, Fort Lauderdale, Florida 33314 and 3Department of
Plant Pathology, Kansas State University, Manhattan, Kansas 66506, U.S.A.
(Accepted 16 March 1987)
SUMMARY
Antisera to the Mr 32000 (32K) capsid and Mr 16500 (16"5K) major non-capsid
proteins were used for immunological analyses of extracts from maize stripe virus
(MStpV)-infected maize (Zea mays) plants and from inoculative Peregrinus maidis, the
MStpV planthopper vector. The 32K protein was easily detected in extracts of both
MStpV-infected plants and inoculative P. maidis by ELISA and by immunological
analysis of Western blots. In a time course study, no 32K protein was detected in P.
maidis until 8 days after the beginning of a 5 day acquisition access period on MStpVinfected plants. The percentage of MStpV-positive P. maidis increased with time
indicating multiplication of MStpV in P. maMis. The 32K protein was detected only in
individual P. maidis that also transmitted MStpV to plant hosts. The 16.5K protein was
also detected in MStpV-infected plant hosts but not in extracts of groups or of
individual MStpV-inoculative P. rnaidis. In vitro translation of MStpV virion RNAs in
rabbit reticulocyte lysates showed that both the 32K and 16.5K proteins were present in
the translation products. The ready detection of the MStpV-coded 32K protein in both
plant and insects and detection of the 16.5K protein in only plant hosts is discussed.
INTRODUCTION
Maize stripe virus (MStpV) infects several plant species of the Poaceae and is transmitted by
the maize planthopper, Peregrinus maidis Ashmead. MStpV has several properties in common
with a group of other planthopper-borne viruses, including rice hoja blanca virus (RHBV)
(Morales & Niessen, 1983), rice stripe virus (RSV) (Toriyama, 1982; Gingery et al., 1983),
European wheat striate mosaic virus (Ammar et al., 1985) and rice grassy stunt virus (Hibino et
al., 1985). These viruses have been tentatively grouped in the RSV group (Gingery, 1987).
Based on their virus-vector relationships all members of the RSV group are believed to infect
their planthopper vectors. All exhibit a 10 to 20 day incubation period in their vectors between
virus acquisition and subsequent transmission, and transovarial transmission by viruliferous
females to a high percentage of their progeny has been shown for MStpV and RHBV (Tsai &
Zitter, 1982; Morales & Niessen, 1983). Also, once an individual insect (e.g.P. maidis) begins to
transmit a given virus (e.g. MStpV), it retains the ability to transmit for the remainder of its life.
Taken together, these data suggest that these viruses have persistent-propagative relationships
with their insect vectors. However, proof for MStpV multiplication in P. maidis has not yet been
demonstrated.
Two virus-specific proteins, the virion capsid (Mr 32000, 32K) and a major non-structural
protein (16.5K), have been detected in MStpV-infected plants (Gingery et al., 1981; Falk &
Tsai, 1983; Ammar et al., 1985). The 16-5K protein appears to be a major component of MStpVspecific inclusion bodies in infected maize (Zea mays L.) (Ammar et aI., 1985), and the protein
accumulates to very high concentrations in MStpV-infected Z. mays as well as other plant hosts
(Gingery et al., 1981 ; Falk & Tsai, 1983). Although the 16.5K protein can be readily detected in
0000-7466 © 1987 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
1802
B. W. FALK, J. H. TSAI AND S. A. LOMMEL
M S t p V - i n f e c t e d plants, we were unable to detect it previously in M S t p V - i n o c u l a t i v e P. maidis
(Falk & Tsai, 1983). T h a t the 16.5K protein is also virus-coded has been q u e s t i o n e d ( G o r d o n ,
1984; J o n e s et al., 1984).
W e now p r o v i d e serological d a t a w h i c h support the c o n t e n t i o n that M S t p V replicates in its
vector, P. maidis. Also, we show that both the 16.5K and 32K proteins are d e t e c t a b l e in M S t p V
R N A - d i r e c t e d in vitro translation products and are therefore e n c o d e d by the viral g e n o m e .
H o w e v e r , only the 32K protein is readily detectable in extracts o f M S t p V - i n o c u l a t i v e (infected)
P. maidis.
METHODS
Virus and vector maintenance. The MStpV and P. maidis isolates were the Florida, U.S.A. isolates used
previously (Falk & Tsai, 1983, 1984; Tsai & Zitter, 1982). Z. mays L. was used as the maintenance host for both
MStpV and P. maidis as described (Tsai & Zitter, 1982). For experiments analysing the MStpV-specific proteins,
P. maidis were given a 48 to 120 h acquisition access period (AAP) on MStpV-infected Z. mays. P. maidis were then
transferred to healthy Z. mays every 3 to 4 days and were harvested at 25 days post-AAP. MStpV-infected Z. mays
was harvested 10 to 20 days after inoculation, when plants showed symptoms typical of MStpV infection.
To assay P. maidis at intervals after AAP on MStpV-infected Z. mays, 500 P. maidis were given a 5 day AAP on
MStpV-infected Z. mays. Insects were then transferred to healthy plants every 2 to 3 days, Twelve to 15 P. maidis
were collected on each sampling date (day 0, before AAP; day 5 at end of AAP, and daily from day 6 to the end of
the experiment). Healthy P. maidis were also collected at each sampling date. Each sample was stored at - 2 0 °C
until all P. maidis could be individually tested for the MStpV 32K protein by ELISA. Individual P. maidis were
considered positive for the 32K protein when their A~05values were greater than three times the S.D. plus the mean
of healthy controls.
Individual inoculative P. maidis were compared for the ability to transmit MStpV and for the presence of
MStpV proteins by allowing a group of 40 P. maidis a 5 day AAP to MStpV-infected Z. mays. These were then
transferred as groups of I0 P. maidis per plant every 3 to 4 days to healthy Z. mays. After 17 days on the healthy Z.
mays, 24 P. maidis survived. These were separately caged, each on a single Z. mays seedling, for a 96 h inoculation
access period. The 20 surviving P. maidis were removed, numbered and individually stored at - 20 °C until they
were tested for MStpV proteins. The 20 test Z. mays were numbered correspondingly and observed for symptom
development indicative of infection by MStpV.
Serological detection of MStpV proteins. MStpV 32K protein was recovered by purification of MStpV
nucleoprotein from infected Z. mays as described previously (Falk & Tsai, 1984). The 16.5K protein was purified
from infected Z. mays by differential pH precipitation and electro-elution from SDS-polyacrylamide gels (Falk &
Tsai, 1983). Antisera were produced by intramuscular injection of purified proteins into New Zealand white
rabbits following the protocol used previously (Falk & Tsai, 1983). We emulsified 1 mg of purified protein with
Freund's complete adjuvant (Difco) for the first injection, and with Freund's incomplete adjuvant for the second
and third injections. Bleedings began 10 days after the third injection.
ELISA tests were done for the MStpV 32K protein using the double antibody sandwich method as described by
Clark & Adams (1977). Microtitre plates (Immulon II; Dynatech Laboratories, Alexandria, Va, U.S.A.) were
coated with purified immunoglobulins at 1 ~tg/ml and alkaline phosphatase-conjugated antibodies were used at a
1 : 1000 dilution. Samples were extracted in PBS (0-05 M-phosphate pH 7.4, 0.15 M-NaCI) containing 0-05 ~ Tween
20 and 2 ~ polyvinylpyrrolidone 40. Individual P. maidis (approx. 1.7 mg) were prepared for ELISA by trituration
in 1 ml of buffer.
Immunological analysis of Western blots was done by the method of Burnette (198 I). Blots were washed for 15
min in PBST (0-02 M-phosphate, 0-15 M-NaCI, pH 7-4, 0-3~ Tween 20) containing 3 ~ bovine serum albumin,
followed by two 20 min washes in PBST. Antiserum dilutions of 1/500 and 1/375 in PBST for the 32K and 16-5K
antisera, respectively, were added to the washed blots which were incubated with shaking for 1.5 h. Blots were
given two washes of 20 rain in PBST and then incubated in Protein A conjugated with horseradish peroxidase
(Boehringer) diluted 1/2000 in PBST for 1 h. Blots were again washed, 5 to I0 ml of substrate (0.1 mg/ml ophenylenediamine in 0-0006~ hydrogen peroxide) was added and blots were kept in the dark for 30 to 60 min.
Blots were removed from the substrate, briefly rinsed in PBST, allowed to dry and then photographed.
SDS-PAGE and Western blotting. Initially proteins were extracted from uninoculated and MStpV-infected Z.
mays and P. maidis by two different methods. Proteins were extracted after the method of White & Brakke (1983)
as modified by Falk et al. (1987). This was done to determine whether the 16.5K protein was associated with the
virion-containing fraction of plant sap, or if it was a soluble protein. The differential pH extraction method
(Gingery et al., 1981; Falk & Tsai, 1983) was used when attempting to concentrate the MStpV 16.5K protein
selectively. Two g of plant tissue or 10 to 20 P. maidis were used for each extraction.
To assay individual P. maidis for MStpV proteins, each insect was triturated directly in electrophoresis sample
buffer (one insect per 200 I~1),Samples were then immediately heated at 100 °C for 2 to 3 rain, centrifuged at 12 000
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
M S t p V proteins
1803
g (max.) for 5 rain and further processed for SDS-PAGE. Proteins from 1/10th to 1/20th of an individual insect
were applied to a single gel line. As the mean weight of a single P. maidis was 1.7 rag, to obtain equivalent samples
of Z. mays tissue, a single disc was taken from a leaf using a no. 2 cork borer and processed for SDS-PAGE as
described for single P. maidis.
Proteins were fluorescently labelled before electrophoresis analysis with 2-methoxy-2,4-diphenyl-3(2H)
furanone or they were analysed as unlabelled proteins and subsequently detected by silver staining (Morrisey,
1981) or by immunological analysis of Western blots. Gels consisted of 5 % and 12% (stacking and resolving gels,
respectively) polyacrylamide, or were linear gradient gels of 10 to 20 ~ acrylamide with a 3 ~ stacking gel using the
buffer system of Laemmli (1970). Proteins were visualized by exposing the gel to u.v. illumination (302 nm) or by
silver staining (Morrisey, 1981). Fluorescently labelled proteins were photographed using Polaroid P/N 665 film
and a Wratten 9 filter.
Western blotting of proteins separated by SDS-PAGE was done according to Burnette (1981). Proteins were
transferred from SDS gels to 0-2 p.m pore size nitrocellulose (BA83, Schleicher & Schiill) using an electroblotting
chamber. Transfer was at 10 V/cm for 2 h, or 5 V/cm overnight, followed by 10 V/cm for 15 rain. All transfers were
done at 4 °C. Blots were then immunologically analysed for MStpV proteins.
Analysis of MStp V RNA in vitro translationproducts. RNAs were extracted from a mixture of the four sucrose
density gradient-purified MStpV nucleoprotein components as described previously (Falk & Tsai, 1984). Virion
RNA extracted from southern bean mosaic virus (SBMV) was used as a control. Rabbit reticulocyte lysate was
obtained from Green Hectares (Oregon, Wisconsin, U.S.A.). The lysate was cleared of endogenous mRNA by
treatment with micrococcal nuclease and calcium chloride (Pelham & Jackson, 1976).
A 100 ~tl lysate mixture was prepared according to Dougherty & Hiebert (1980a) with 6 to 8 l.tg of MStpV or
SBMV RNA and 4 ~tCiof [35S]methionine (500 to 800 Ci/mmol). The reaction mixture was incubated at 30 °C for
1 h. Samples of the reaction mixture were treated with ribonuclease A and then adjusted to contain 10~ glycerol,
2~ SDS and 5~ 2-mercaptoethanol. Samples were placed in a boiling water bath for 5 min and then analysed by
SDS-PAGE in an 8 to 15~o acrylamide gradient gel. Products were detected by fluorography.
Translation products were immunologically analysed by selective immunoprecipitation using antisera to the
MStpV 32K and 16-5K proteins by the method of Dougherty & Hiebert (1980b). Antigen-antibody complexes
were reacted with Staphylococcus aureus cell wall proteins (Sigma) and collected by centrifugation. Precipitates
were analysed by SDS-PAGE and fluorography.
RESULTS
Detection o f M S t p V proteins in Z. mays and P. maidis
E L I S A analysis of P. maidis after a 5 day A A P on MStpV-infected Z. mays showed that the
percentage of ELISA-positive P. maidis per sampling date increased with time (Table 1). In
three separate experiments, each using 12 to 15 P. maidis per sampling date, no insects were
positive for the 32K protein until at least 8 days after the beginning of the A A P , which was 3
days after removal of the P. maidis from the MStpV-infected acquisition source plants. The
number of 32K-positive P. maidis per sample averaged around 35 ~ from day 12 to the end of the
sampling date. The A405 values for the individual ELISA-positive P. maidis at any given
sampling date were variable, e.g. those from day 10 in experiment 2 ranged from 0.075 to 0.41.
Uninfected P. maid& from the same test plate averaged 0-011.
Previous attempts to detect the MStpV 16.5K protein in P. maidis by indirect E L I S A gave
unclear results and double antibody sandwich E L I S A is not an efficient way to detect the 16.5K
protein, even in plant samples (Falk & Tsai, 1983). W h e n P. maidis were tested by indirect
ELISA, both healthy and MStpV-inoculative P. maid& gave strong non-specific reactions and
could not be differentiated. Therefore, we used S D S - P A G E and immunological analysis of the
S D S - P A G E - s e p a r a t e d proteins to assay unequivocally both the MStpV 32K and 16.5K proteins
in the same samples of Z. mays and P. maidis.
Both the 32K and 16-5K proteins were detected when protein preparations from MStpVinfected Z. mays were analysed by S D S - P A G E (Fig. 1). N o similar proteins were seen in
preparations from uninoculated Z. mays. Proteins extracted from uninoculated and MStpVviruliferous P. maidis contained too m a n y proteins to determine visually whether the 32K or
16.5K proteins were present in the preparations. Serological analysis of the protein extracts after
their transfer to nitrocellulose m e m b r a n e s showed that the 32K protein was detected primarily
in the 145000 g pellet fractions of both MStpV-infected Z. mays and MStpV-inoculative P.
maidis (Fig. 2). Somewhat less 32K protein was also detected in the 145000 g supernatant
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
1804
B. W. FALK, J. H. TSAI AND S. A. LOMMEL
Z. mays
M r ( X l 0 -3)
I
HS HP MS MP
P. maMis
I
C
NC
I
MP MS HP
HS
I
92
67
45
31
21
14
Fig. 1. SDS-polyacrylamide gel showing proteins extracted from healthy and MStpV-infected Z. mays
and P. maidis samples. Proteins were extracted and fractionated by centrifugation at 145000 g for 90
rain. The supernatant and pellet fractions were saved and analysed separately. Proteins were
fluorescently labelled before electrophoresis and visualized after electrophoresis was complete by
exposing the gel to u.v. illumination. HS and MS refer to healthy and MStpV-infected supernatant
fraction proteins, respectively, and HP and MP refer to healthy and MStpV-infected 145000 g pellet
fraction proteins, respectively. The first lane shows protein markers. Lanes designated C and NC show
purified MStpV 32K capsid and 16.5K non-capsid proteins respectively.
T a b l e 1. E L I S A detection o f the M S t p V 32K protein over time in P. m a i d i s after acquisition
access to MStpV-infected Z. m a y s *
No. of P. maidis in which 32K protein was
detected/no, in group
A
Dayt
0
5
6
Expt. 1~
0/12
0/12
0/12
Expt. 2
0/12
0/12
0/12
Expt. 3
0/12
0/12
-
8
0/12
1/12
0/12
0/12
2/15
9
10
11
12
13
14
15
16
17
18
5/12
3/12
2/12
5/12
4/12
6/12
2/12
5/12
3/12
4/12
2/12
1/12
3/12
7/12
4/12
7/12
-
0/12
1/12
3/12
5/12
6/13
5/15
3/i2
5/15
-
7
-
* Groups of P. ma/d/s were given a 5 day AAP on MStpV-infected Z. mays.
t Shows day of collection relative to the start of the AAP.
The results of three separate experiments are shown. Fraction shows the number of positive P. maidis over the
number tested.
fraction f r o m M S t p V - i n o c u l a t i v e P. maidis and a strong r e a c t i o n for the 32K p r o t e i n was seen
for the 145000 g s u p e r n a t a n t f r a c t i o n f r o m M S t p V - i n f e c t e d Z. mays. I n contrast, the 16.5K
protein was d e t e c t e d entirely in the 145 000 g s u p e r n a t a n t f r a c t i o n f r o m M S t p V - i n f e c t e d plants
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
M S t p V proteins
1805
Z. mays
P. maidis
'1
HS ~.HP MS MP NC
I
C
MS
MP HS HP
C~
D~
NC~
LL
Fig. 2. Immunologicallyanalysed Western blot of SDS-PAGE-separated total proteins (see Fig. 1).
Upper blot was probed with antiserum to the MStpV 32K capsid protein. Lower blot was probed with
antiserum to the MStpV 16.5Knon-capsidprotein. C showslocation of capsid protein reaction. NC and
D show locations of non-capsid protein monomer and dimer reactions, respectively.
and not in the virion fraction. It can also be seen that the 16.5K protein represents a significant
proportion of the proteins from the MStpV-infected plants. Lane MS of the Z. rnays samples in
Fig. 1 and 2 is adjusted so that the proteins represent only 50 ~tg of fresh weight starting material.
All other Z. mays lanes (MP, HS and HP) represent the proteins from 500 ixg starting material.
Lanes MP, MS, HP and HS of the P. maidis samples all represent proteins from equivalent
amounts (350 ~tg) of sample with respect to the starting material.
The 16.5K protein was not detected in the 145000 g protein extracts from pooled MStpVinoculative P. maidis (Fig. 2), nor was it detected when attempts were made to concentrate this
protein by differential precipitation (data not shown).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
1806
B.W. FALK, J. H. TSAI AND S. A. LOMMEL
The serological reaction labelled D for the lanes MS and NC for the blot in Fig. 2 that was
probed with antisera to the MStpV 16-5K protein represents the 16-5K dimer. This was a
consistent reaction and was slightly above the reaction for the 32K protein. We interpret this as
being a 16.5K dimer because it is about twice the mol. wt. of the 16-5K protein, and the original
purified 16.5K protein used for antiserum production and for the 16.5K control (lane NC) on
this gel was purified by electro-elution from SDS gels, and only samples with a mol. wt. of 16000
to 18 000 were electro-eluted.
Because the 16.5K protein was not detected by us in pooled P. maidis samples, we analysed
individual P. maidis for both the 16.5K and 32K proteins. Individual P. maidis were first tested
for their ability to transmit MStpV as an indicator of MStpV multiplication in P. maidis. These
same individuals were then tested for the presence of the MStpV 32K and 16-5K proteins by
immunological analysis of SDS-PAGE-separated total proteins. Eleven of the 20 P. maidis
tested transmitted MStpV to Z. mays. When total protein extracts from all 20 of these P. maidis
were immunologically probed for the 32K and 16-5K proteins, the 32K protein was readily
detected only in extracts from the inoculative P. maMis. Results for nine of the transmitters (V)
and three non-transmitters (Nv) along with four healthy P. maidis not given an AAP on MStpVinfected Z. mays are shown in Fig. 3. The nine transmitters show positive reactions for the
MStpV 32K protein. None of the P. maidis gave a reaction for the 16.5K protein.
We quantitatively compared the levels of detection of the 32K and the 16-5K proteins in total
proteins extracted from Z. mays and P. maidis samples of similar weight. When Z. mays and P.
maidis samples representing 100 btg were analysed, the 32K protein was readily detected in both,
and no reactions were observed for healthy samples. Based on the intensity of reactions for
known amounts of the 32K protein, MStpV-infected Z. mays contained about 250 ng of 32K
protein per 100 btg, and P. maidis had about 50 ng per 100 txg (Fig. 4). In contrast, the 16.5K
protein was estimated to be about 10~o of the total proteins in MStpV-infected plant samples or
about 10 btg of the 100 ~tg sample. The 16.5K protein was easily detected when plant samples
equivalent to only 2 ~tg of starting material were loaded onto the gel. The 16.5K protein was not
detectable in P. maidis samples which were positive for the 32K protein. In some samples, a
reaction was obtained just below the 16.5K region of the gels, but this occurred in a few healthy
and in some MStpV 32K-positive P. maidis samples. This reaction also occurred when blots
were probed with antisera to tobacco mosaic virus, beet yellows virus and SBMV and therefore
is not a 16.5K reaction (data not shown).
Analysis of the MStpV RNA in vitro translation products
When the mixture of MStpV virion RNAs was incubated with the rabbit reticulocyte lysate in
vitro, four MStpV RNA-specific prominent proteins of Mr larger than about 15 000 were seen in
the products (Fig. 5). (The prominent protein at 46 000 is the endogenous globin mRNA product
and can also be seen in the control lane.) Two of these co-electrophoresed with purified MStpV
32K (C) and 16.5K (NC) proteins. The protein co-electrophoresing with purified 32K protein
was specifically immunoprecipitated with antiserum to MStpV 32K protein. Similarly, the
translation product that co-electrophoresed with purified MStpV 16-5K protein was specifically
immunoprecipitated by antiserum to MStpV 16.5K protein. No MStpV translation products
were immunoprecipitated by antiserum to SBMV. Similarly, antiserum to the MStpV 32K
protein did not immunoprecipitate any of the SBMV translation products.
DISCUSSION
The data shown here provide evidence that MStpV multiplies in its vector P. maidis, that
both the 32K and the 16.5K proteins are encoded by the MStpV virion RNAs, and that the 32K
and 16.5K proteins are readily detectable in MStpV-infected Z. mays, yet only the 32K protein is
detectable in MStpV-inoculative P. maidis. The ELISA detection of the 32K protein over time in
P. maidis after AAP on MStpV-infected Z. mays supports the biological data suggesting the
multiplication of MStpV in P. maidis. No 32K protein was detectable immediately after AAP,
and in the blotting experiment (Fig. 3), the 32K protein was detectable only in transmitters.
Despite the sensitive, specific detection of the MStpV 16.5K protein from MStpV-infected Z.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
1807
MStp V proteins
(a) V V N v V
V
V V V V V N v N v H H H H MS
M r ( X 10 -3)
92
67
45
31
21
14
(b)
~~
mC
(c)
--NC
Fig. 3. Total protein extracts from individual P. maidis after analysis by SDS PAGE and Western
blotting. Individual P. maidis were exposed to MStpV and individually tested for their ability to
transmit MStpV as described in the text, V indicates that the individual transmitted MStpV and NV
that it did not. H are healthy individual P. maidis that were not exposed to MStpV. (a) SDS-PAGEseparated proteins. (b) The above proteins after electroblotting to nitrocellulose and probing with
antiserum to the MStpV 32K capsid protein antiserum. Arrow shows location of 32K protein
serological reaction. (c) Proteins after electroblotting and probing with antiserum to the MStpV 16.5K
non-capsid protein. Arrow shows location of 16-5Kprotein serological reaction. MS shows purified 32K
and 16.5K MStpV proteins. Locations of protein standards are shown at the right.
mays, this protein was not detected in inoculative P. maidis, even by immunological analysis of
total proteins on Western blots. In 'spiked' experiments we added small quantities of the
partially purified 16.5K protein (2 ~tg) to individual insects during extraction of total proteins.
Proteins representing 1/10 of the individual insects were analysed in a given gel lane and the
16.5K protein was consistently recovered and detected as well as when the 16.5K protein alone
was tested (data not shown). This represents strong detection of only 200 ng protein. We could
consistently detect as little as 100 ng of the 16.5K protein in spiked P. maidis extracts. In
contrast, we estimated the amount of the 16.5K protein in symptomatic Z. mays leaves to be
about 10 ~tg/100 ~tg of leaf tissue.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
1808
B. W. FALK, J. H. TSAI AND S. A. LOMMEL
~)
(b)
1
2
4
MMM
. . . . .
H H
u
~
1 2
H
u
3
4
MM
M HH
H
u
NC
H
(c)
(a)
4
3
2
1
MM
M
H H H
4
i
3
. . . . . . . . . . .
2
m
1
•
M
.
M M H
•
.
.
.
.
.
.
.
H H
.
Fig. 4. Immunologically analysed Western blots for SDS-PAGE-separated total proteins from 1~)0lag
samples of Z. mays and P. maidis. Both (a) and (b) show results for healthy (H) and MStpV-infected (M)
Z. mays. Aliquots from the same samples were used for blots (a) and (b). Blots (c) and (d) show results for
healthy (H) and MStpV-infected (M) P. maidis. Lanes 1, 2, 3 and 4 are healthy Z. mays (a and b) or
healthy P. maidis (c and d) samples which contain 200, 100, 50 and 25 p-g of purified 32K plus 16.5K
proteins, respectively. Antisera were to 32K (a, c) or 16-5K (b, d) proteins.
The 32K protein was estimated to be present in MStpV-infected Z. mays at 250 ng per 100 pg,
while in P. maidis the 32K protein was present at about 50 ng per 100 ~tg. Therefore, the ratio of
the 16-5K: 32K proteins in MStpV-infected Z. mays is about 40:1. As the 32K protein is fivefold
less by weight in P. maidis as opposed to Z. mays, if the 16-5K protein were present in P. maidis in
similar amounts relatNe to the 32K protein, we could expect 2 ~tg of the 16-5K protein in a 100 ~tg
P. maidis sample. But since the 16-5K protein is not detectable in P. maidis and the limit of
detection is 100 ng in our system, this shows that if the 16-5K protein is present at all in P. maidis
the ratio of 32K: 16.5K protein is at least 20-fold less than it is in Z. mays. By weight the amount
of 16.5K protein in P. maidis as opposed to Z. mays would be at least 100-fold less.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
1809
MStp V proteins
1
3
2
4
5
6
7
8
M r ( × 10 -3)
105
75
46
C--
~
m
e
29
NC-v,
Fig. 5. Analysis of MStpV RNA and SBMV RNA in vitro translation products. [35S]Methioninelabelled products were separated in an 8 to 15% SDS-polyacrylamidegel and detected by fluorography.
Lane 1, MStpV translation products immunoprecipitated by MStpV 32K capsid protein antiserum;
lane 2, MStpV products untreated; lane 3, MStpV products immunoprecipitated by MStpV 16.5Knoncapsid protein antiserum; lane 4, SBMV translation products irnmunoprecipitated by SBMV capsid
protein antiserum; lane 5, MStpV translation products immunoprecipitated by SBMV capsid protein
antiserum; lane 6, no exogenous RNA; lane 7, SBMV translation products immunoprecipitated by
MStpV 32K capsid protein antiserum; lane 8, total products of SBMV translation. C and NC show
locations of MStpV 32K capsid and 16-5K non-capsid proteins, respectively.
These data would not be surprising if, as has been suggested (Jones et al., 1984), the 16.5K
protein is a host-coded protein induced to high levels in Z. mays by MStpV infection. However,
our data provide strong evidence that both the 32K and 16-5K proteins are coded for by the
MStpV genome. A mixture of all the MStpV RNAs was used for the in vitro translations and
therefore no assignment of the 32K or 16-5K proteins to a specific R N A can be made. The
translation efficiency of the MStpV RNAs was poor relative to the control SBMV RNA. Similar
results were obtained for the related RSV (Toriyama, 1986). Also, it is very unlikely that the
proteins detected by our in vitro translation represent the total proteins which are encoded by the
MStpV genome. However, the specific immunoprecipitation of proteins which co-electrophoresed with the 16.5K and 32K proteins from the translation products strongly suggests that the
16.5K and 32K proteins are virus-coded and not virus-induced host-coded proteins.
These data then raise the question: if the 16.5K and 32K proteins are encoded by the MStpV
genome and MStpV infects both plants and insects (P. maidis), why then is the 16.5K protein so
prominent in infected plant hosts and not detectable in MStpV-inoculative (infected) P. maidis?
One possibility is that the 16.5K protein is present in MStpV-infected P. maidis, but in a much
lower concentration, relative to the 32K protein, than it is in MStpV-infected plant hosts, and it
was undetected by us. Another is that the 16.5K protein is not produced during MStpV infection
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
1810
B. W . F A L K , J. H. TSAI A N D S. A. L O M M E L
of P. maidis, but is produced during MStpV infection of plant hosts. At this point, it is not clear
which of these possibilities, if either, is correct.
The differences seen here between detection of the MStpV-specific proteins in MStpVinfected insect (P. maidis) and plant hosts are interesting but perhaps not unique. There has been
little research on virus-specific proteins, other than capsid proteins, for many of the viruses that
infect plant hosts as well as their insect vectors. Wound tumour virus, a member of the
reoviridae which infects both plant and insect hosts, has some isolates which can only infect
plants (Liu et al., 1975; Reddy & Black, 1977; Nuss & Summers, 1984). These transmissiondefective isolates have deletions in specific genomic RNA segments that are required for
infection of insect hosts but not plant hosts. Similarly Sindbis virus, an alphatogavirus that
infects vertebrate hosts and its invertebrate vector, matures differently and establishes different
types of infections in the two cell types (Gliedman et al., 1975). Also, actinomycin D inhibits the
maturation of the virus in invertebrate cells but not in vertebrate cells (Scheefers-Borchel et aL,
1981). If MStpV replicates differently in invertebrate and plant hosts, we might then expect to
see differences between the MStpV-specific proteins made in the different hosts.
The excellent technical assistance of Ms G. Echenique is gratefully acknowledged. We thank Drs T. J. Morris
and G. Bruening for their comments on the manuscript and Dr F. Ponz and C. Chay for help with the in vitro
translation. This research was supported in part by U S D A grants 83-CRSR-2-2151, 84-CRSR-2-2507, and a grant
from the American Seed Research Foundation.
REFERENCES
AMMAR,E. D., GINGERY, R. E. & NAULT, L. R. (1985). Two types of inclusions in maize infected with maize stripe
virus. Phytopathology 75, 84-89.
BURNETTE, W. N. (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate
polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and
radioiodinated Protein A. Analytical Biochemistry 112, 195-203.
CLARK, M. F. & ADAMS,A. N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent
assay for the detection of plant viruses. Journal of General Virology 34, 475-483.
DOUGrIERTY, W. G. & HIEBERT, E. (1980a). Translation of potyvirus R N A in a rabbit reticulocyte lysate:
identification of nuclear inclusion proteins as products of tobacco etch virus R N A translation and cylindrical
inclusion protein as a product of the potyvirus genome. Virology 104, 174-182.
DOUGHERTY, W. G. & HIEBERT, E. (1980b). Translation of potyvirus R N A in a rabbit reticulocyte lysate: cell-free
translation strategy and a genetic m a p of the potyviral genome. Virology 104, 183 194.
FALK, B. W. & TSAI, J. H. (1983). Assay for maize stripe virus-infected plants by using antiserum produced to a
purified noncapsid protein. Phytopathology 73, 1259 1262.
FALg, B. W. & TSAI, J. H. (1984). Identification of single- and double-stranded R N A s associated with maize stripe
virus. Phytopathology 74, 909-915.
FALK, B. W., MORALES,F. J., TSAI,J. H. & NIESSEN, A. 1. (1987). Serological and biochemical analysis of the capsid and
major noncapsid proteins of maize stripe virus, rice hoja blanca virus and Echinochloa hoja blanca virus.
Phytopathology 77, 196-201.
GINGERY, R. E. (1987). The rice stripe virus group. In The Filamentous Plant Viruses. Edited by R. G. Milne. N e w
York: Plenum Press (in press).
GINGERY, R. E., NAULT, L. R. & BRADFUTE,O. E. (1981). Maize stripe virus: characteristics of a m e m b e r of a new
virus class. Virology 112, 99 108.
GINGERY, R. E., NAULT,L. R. & YAMASHITA,S. (1983). Relationship between maize stripe virus and rice stripe virus.
Journal of General Virology 64, 1765-1770.
GLIEDMAN, J. B., SMITrt, I. F. & BROWN, D. T. (1975). Morphogenesis of Sindbis virus in cultured Aedes albopictus
cells. Journal of Virology 16, 913-926.
GORDON, D. T. (1984). C o m m e n t s of the chairperson of the international working group on maize virus diseases.
Maize Virus Diseases Newsletter l, 1-11.
HIBINO, H., USUGI, T., OMURAT., TSUCHIZASKI,T., SHOHARA, K. & IWASAKI, M. (1985). Rice grassy stunt virus: a
planthopper-borne circular filament. Phytopathology 75, 894-899.
JONES, P., CARPENTER, J. M. & AUTREY, L. J. C. (1984). Investigations into the association between a non-capsid
protein and virus infection in maize. Maize Virus Diseases Newsletter 1, 52-53.
LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature, London 227, 680-685.
LIU, H. Y., KIMURA,I. & BLACK,L. M. (1975). Specific infectivity of different wound tumor virus isolates. Virology
52, 320-326.
MORALES, F. J. & NIESSEN, A. I. (1983). Association of spiral filamentous virus-like particles with rice hoja blanca.
Phytopathology 73, 971-974.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44
M S tp V proteins
1811
r~ORRISEY, J. n. (1981). Silver stain for protein in polyacrylamide gels. A modified procedure with enhanced
uniform sensitivity. Analytical Biochemistry 117, 307-310.
NUSS,D. L. & SU~,IMERS,D. (1984). Variant ds-RNAs associated with transmission-defective isolates of wound tumor
virus represent terminally conserved remnants of genome segments. Virology 133, 276-286.
PELHAM, H. R. B. & JACKSON, R. J. (1976). An efficient mRNA-dependent translation system from reticulocyte
lysates. European Journal of Biochemistry 67, 247 256.
REDDY, D. V. R. & BLACK,L. M. (1977). Isolation and replication of mutant populations of wound tumor virions
lacking certain genome segments. Virology 80, 336-346.
SCHEEFERS-BORCHEL, U., SCHEEFERS, H., EDWARDS, J. & BROWN, D. T. (1981). Sindbis virus maturation in cultured
mosquito cells is sensitive to actinomycin D. Virology 110, 292-301.
TORIYhMA, S. (1982). Characterization of rice stripe virus: a heavy component carrying infectivity. Journal of
General Virology 61, 187-195.
TORIYhMA,S. (1986). An RNA-dependent RNA polymerase associated with the filamentous nucleoproteins of rice
stripe virus. Journal of General Virology 67, 1247-1255.
TSAI,J. ft. & zI'rTER, T. A. (1982). Characteristics of maize stripe virus transmission by the corn delphacid. Journalof
Economic Entomology 75, 397--400.
WHITE,L L. & BRAKKE,M. K. (1983). Protein changes in wheat infected with wheat streak mosaic virus and in barley
infected with barley stripe mosaic virus. Physiological Plant Pathology 22, 87-100.
(Received 22 September 1986)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 31 Jul 2017 15:34:44