Download Early embryo invasion as a determinant in pea of the seed

Journal of General Virology (1992), 73, 1615-1620.
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Printed in Great Britain
Early embryo invasion as a determinant in pea of the seed transmission of
pea seed-borne mosaic virus
Daowen Wang and Andrew J. Manle*
Department of Virus Research, John lnnes Institute, John lnnes Centre, Colney Lane, Norwich NR4 7UH, U.K.
Seed transmission of an isolate of pea seed-borne
mosaic virus (PSbMV) in several pea genotypes has
been studied. Cross-pollination experiments showed
that pollen transmission of P S b M V did not occur and
accordingly, virus was not detected in pollen grains by
ELISA or electron microscopy. Comparative studies
between two pea cultivars, one with a high incidence of
seed transmission and one with none, showed that
PSbMV infected the floral tissues (sepals, petals,
anther and carpel) of both cultivars, but was not
detected in ovules prior to fertilization. Virus was
detected equally well in seed coats of the progeny in
both cultivars. Analysis of virus incidence and concen-
tration in pea seeds of different developmental stages
demonstrated that in the cultivar with a high incidence
of seed transmission, P S b M V directly invaded immature embryos, multiplied in the embryonic tissues and
persisted during seed maturation. In contrast, the
cultivar without seed transmission did not show
invasion of immature embryos by the virus; there was
no evidence for virus multiplication or persistence
during embryo development and seed maturation.
Hence seed transmission of PSbMV resulted from
direct invasion of immature pea embryos by the virus
and the block to seed transmission in the nonpermissive cultivar probably occurred at this step.
Introduction
which are compatible with all the aforenoted host
changes are seed-transmissible, and (ii) the complexity of
the process provides many stages at which seed
transmission may be regulated.
There are many examples which illustrate the influence of host and viral genotypes on seed transmission
and the regulation of seed transmission at various
developmental steps but there are few examples of
systematic analyses of the seed transmission process in a
well-defined genetic system. General mechanisms which
regulate the efficiency of seed transmission in many
host-virus interactions have not emerged from these
studies.
We have chosen to study seed transmission of the
potyvirus pea seed-borne mosaic virus (PSbMV) in pea
as it is a well-defined genetic and developmental system,
and constitutes a problem of major importance in plant
pathology (Khetarpel & Maury, 1988). Pea cultivars
have been identified which vary from a high frequency of
seed transmission to zero (Stevenson & Hagedorn, 1969,
1973; Wang et al., 1992b) and there is a substantial
understanding of the molecular and physiological aspects
of pea embryology (Casey, 1990). The complete sequence
of PSbMV has been published (Johansen et al., 1991).
Furthermore, seeds of many other major food legume
species such as bean, cowpea, lentil, peanut and soybean
Since the identification (Reddick & Steward, 1919) of
seed transmission of viruses evidence has accumulated
that indicates that the phenomenon is the end result of a
complex interaction between the host and the virus
which may be influenced by a variety of environmental
factors (for reviews, see Bennett, 1969; Shepherd, 1972;
Bos, 1977; Carroll, 1981). This is illustrated by the fact
that a seed-transmitted virus often has variants that are
not seed-transmitted, that different genotypes of the
same host species can differ in their efficiencies of
transmission of a single isolate and that the transmission
efficiency can be affected by temperature and daylength.
To accomplish the process of seed transmission, the
virus must initiate an infection during the vegetative
growth of its host, establish itself in the developing
embryos, remain stable during seed dessication and
storage and eventually be reactivated during, or after,
seed germination. While the genetic structure of the virus
remains the same throughout, the host changes genetically from diploid to haploid and back to diploid, and
physiologically from vegetative growth to reproductive
growth and back to vegetative growth. This developmental progression reveals two important aspects of plant
virus seed transmission: (i) only those viral genotypes
00014)900 © 1992 SGM
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D. W a n g and A . J. M a u l e
also t r a n s m i t p o t y v i r u s e s . S o m e studies o n t h e s e i n t e r a c tions h a v e b e e n c a r r i e d out, m o s t n o t a b l y b e a n c o m m o n
m o s a i c virus ( B C M V ) / P h a s e o l u s vulgaris ( M e d i n a &
G r o g a n , 1961; S c h i p p e r s , 1963; P h a t a k , 1974), p e a n u t
stripe m o s a i c virus ( P S t V ) / p e a n u t (Xu et al., 1991) a n d
s o y b e a n m o s a i c virus ( S M V ) / s o y b e a n (Bowers & G o o d m a n , 1979; I r w i n & G o o d m a n , 1981). I d e n t i f i c a t i o n o f a
g e n e r a l p r i n c i p l e i n v o l v e d in d e t e r m i n i n g p o t y v i r u s seed
t r a n s m i s s i o n in t h e i r l e g u m e hosts w o u l d be a m a j o r
a d v a n c e in our u n d e r s t a n d i n g o f this i m p o r t a n t process.
Methods
PSbMV isolate andpea genatypes. An isolate of PSbMV, PSbMV-28,
originally derived from a PSbMV-infected seed of Pisum sativum cv.
Waverex (Wang et al., 1992b) was maintained on, and purified (Wang
et al., 1992a) from P. sativum cv. Dark Skinned Perfection. Combining
pea cvs. Bunting and Progreta 0ear-type, marrowfat type), Helka
(semi-leafless [afila]-type, non-marrowfat type), Birte and Vedette
(leaf-type, non-marrowfat type) were manually inoculated with a
suspension of virus in 50 raM-sodiumphosphate buffer pH 7.0. Healthy,
buffer-inoculated and infected plants were maintained in a greenhouse
at 18 to 22 °C with a light period of about 14 h.
Cross-pollination experiments. Healthy plants of cvs. Bunting, Helka,
Birte and Vedette were cross-pollinated with pollen from infected
plants of the homologous genotype and grown to give mature seed. In
the case of cv. Progreta which was shown to lack seed transmission in a
previous study (Wang et aL, 1992b), pollen grains from the infected
plants were used to pollinate healthy plants of cv. Vedette in order to
resolve whether there was any PSbMV in Progreta pollen grains. Seeds
from cross-pollinated plants and infected pollen-donor plants were
harvested and germinated to assess pollen transmission and embryo
transmission rates. Seed-borne infection was determined from the
appearance of symptoms 3 weeks after germination and by indirect
ELISA (Cockbain et al., 1988) using polyclonal antibodies raised to
PSbMV-28.
Detection of PSbMV in inflorescence samples. Sepal, petal, pollen,
anther and carpel samples were collected from infected plants of
Progreta and Vedette and assayed for PSbMV using indirect ELISA
(Cockbain et al., 1988) or processed for electron microscopy (Wang et
al., 1991). Pollen grains from single flowers were washed several times
by mild sonication in extraction buffer (20 raM-sodium phosphatebuffered saline, pH 7.4, containing 0.05% v/v Tween 20, 2% polyvinyl
pyrrolidone and 0.1% Triton X-100) and centrifugation, and finally
ground by mortar and pestle to be used as individual samples in indirect
ELISA. This washing procedure removed PSbMV attached to the
surface of a pollen grain as well as PSbMV-infected tissue debris
derived from anther epidermal tissues. Ovules dissected from a single
flower were combined and treated as a single sample in indirect
ELISA.
Detection of PSbMV in embryos of different developmental stages.
Seeds representative of five different developmental stages (20, 40, 80,
200 and 280 mg embryo fresh weight) before dessication and three
stages after dessication (24 seeds for each stage) were separately
analysed for the incidence of virus infection. Seeds were dissected into
testa, embryo and, for the two earliest stages, embryo sac fluid. When
dissecting immature embryos in which contamination by PSbMV
derived from the embryo sac fluid might be a problem, embryos were
washed several times in distilledwater before grinding. The embryo sac
fluid from each immature seed was drawn off in a capillary tube and
diluted in 300 I11 extraction buffer prior to assay. Samples from all
stages were scored for the presence or absence of PSbMV and for the
concentration of virus present [absorbance at 405 nm (-4405)obtained
from the indirect ELISA (Cockbain et al., 1988).
Northern blot hybridization analyses of PSbMV in immature embryos.
Genomic RNA of PSbMV-28 was prepared and reverse-transcribed as
described in Wang et al. (1992a). The eDNA was cloned into EcoRIdigested pBluescript (Stratagene). One clone, no. 14 with an insert size
of 4 kbp, specifically hybridized to PSbMV RNA and was used to
prepare a 32p-labelled probe by the method of Feinberg & Vogelstein
(1983). Limited sequence analysis at the ends oftbe clone showed that it
corresponded to nucleotides (nt) 5928 to 9899 on the published
sequence of PSbMV (Johansen et al., 1991). For Northern blot
hybridization analysis of PSbMV in immature embryos, total RNA
was extracted from individual embryos according to Casey et al. (1985)
and electrophoresed through 1% agarose containing formaldehyde
(Sambrook et al., 1989). Separated RNAs were vacuum-transferred
onto nylon membrane (Hybond-N, Amersham) and cross-linked by
u.v. illumination. The filter was hybridized, washed and exposed to Xray film as described in Sambrook et al. (1989).
Results
Pollen transmission o f P S b M V
F o r these e x p e r i m e n t s , five c u l t i v a r s o f p e a were c h o s e n
to include p e a s o f the leaf- a n d afila-types, o f t h e
marrowfat and non-marrowfat type and of variable
efficiency for seed t r a n s m i s s i o n . C u l t i v a r s P r o g r e t a ,
Bunting, H e l k a , Birte a n d V e d e t t e h a d b e e n s h o w n
p r e v i o u s l y to h a v e seed t r a n s m i s s i o n o f 0, 2, 37, 49 a n d
74%, r e s p e c t i v e l y w i t h P S b M V - 2 8 ( W a n g et al., 1992b).
A f t e r t r a n s f e r o f p o l l e n f r o m P S b M V - i n f e c t e d p l a n t s to
h e a l t h y p l a n t s n o n e o f the r e c i p i e n t s s h o w e d virusi n d u c e d s y m p t o m s a n d similarly, n o n e o f t h e i r p r o g e n y
seeds c a r r i e d a s e e d - b o r n e i n f e c t i o n w h e n t e s t e d b y
E L I S A . D o n o r p l a n t s e x h i b i t e d seed t r a n s m i s s i o n
efficiencies s i m i l a r to those o b s e r v e d p r e v i o u s l y ( W a n g
et al., 1992b).
Detection o f P S b M V in f l o r a l tissues
A more thorough investigation of the distribution of
P S b M V was c a r r i e d out b y E L I S A a n d e l e c t r o n
m i c r o s c o p y o f the floral p a r t s o f two c u l t i v a r s ( P r o g r e t a
a n d V e d e t t e ) w h i c h were r e p r e s e n t a t i v e o f t h e e x t r e m e s
o f the r a n g e o f seed t r a n s m i s s i o n efficiency. Sepal, petal,
a n t h e r a n d c a r p e l s a m p l e s were collected f r o m i n f e c t e d
p l a n t s 1 o r 2 d a y s before f e r t i l i z a t i o n a n d t e s t e d b y
E L I S A ( T a b l e 1). A l l t h e inflorescence p a r t s were
infected. T h i s was c o n f i r m e d b y e l e c t r o n m i c r o s c o p y
w h e r e t y p i c a l P S b M V - a s s o c i a t e d structures, i n c l u d i n g
p i n - w h e e l inclusions s i m i l a r to those seen in l e a f
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PSbMV seed transmission in pea
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Table 1. Virus incidence in the inflorescenceparts of cvs Progreta and Vedette
Progreta
Flower
part
Incidence of
virus invasion*
Sepal
Petal
Anther
Carpel
Ovule
Pollen
30]30
30/30
30/30
30/30
0/20
0]10
Vedette
ELISA
(A40s)i"
0.411
0.651
1.358
0.204
Incidence of
virus invasion*
___ 0.331
_ 0-212
+ 0.240
+ 0-047
ND~
ND
ELISA
(A4os)t
30]30
30]30
30/30
30/30
0/20
0/10
0.613
0.532
1.132
0.128
+ 0.312
+ 0-165
+ 0.253
+ 0.059
NO
NO
* No. of positive samples/no, tested.
t A4os from indirect ELISA; average value of infected samples (background value from healthy
samples was 0.04 to 0-05).
:~ ND, Not determined.
Table 2. Incidence of PSbMV in the tissues of developing seeds from cvs Progreta and Vedette
Virus invasion (%)
Testa
Pea
cultivar*
Seed
transmission (%)
Progreta
Vedette
0
100
60-80
100
it
ii
iii
Embryonic sac fluid
iv
v
i
Embryo
ii
iii
iv
v
i
ii
iii
iv
v
0
12
-:~
-
-
4
12
0
8
4
75
45
-
4
0
54
67
75
* Twenty-four immature seeds from each developmental stage were dissected and assayed for virus incidence by indirect ELISA.
t Developmental stages: i, ii, iii, iv and v.
:~ (-) Not obtained.
mesophyll cells (Wang et al., 1991), were always found in
sections of sepal, petal, anther-epidermal tissues and
carpel. Virus particles were also observed, most frequently as bundles of particles (Wang et al., 1991). In
contrast, none of these structures were seen inside pollen
grains or ovules from either cultivar; funicle tissue
adjacent to the ovule showed some cells containing
PSbMV. To confirm these observations, pollen grains
and ovules from individual flowers were tested by ELISA
as described in Methods. Neither pollen grains nor ovule
samples from infected Progreta orVedette plants gave a
positive reaction with antibody to PSbMV (Table 1).
Detection of PSbMV in embryos of different
developmental stages
If virus invasion of the ovule occurred post-fertilization,
it was important to determine whether this happened
early or late in seed development, whether all parts of the
seed became infected at the same time and whether two
pea cultivars, permissive and non-permissive for seed
transmission, behaved similarly. The strategy followed
was to test by ELISA, seeds of different developmental
status for PSbMV incidence after dissection into testa,
embryo and embryo sac fluid, taking precautions where
possible to avoid cross-contamination of the samples.
The developmental stages chosen were from late embryo
histogenesis to the mature seed stage just before
dessication. The results are summarized in Table 2.
Virus antigen was detected by ELISA in the testa
tissues of all immature seeds from both cultivars
indicating that the ovule was invaded early in seed
development. In contrast, virus was never detected in
100% of the embryo or embryo sac fluid samples for
either cultivar. The maximum incidence was in Vedette
(75% of stage v embryos and 75% of stage i embryo sac
fluid samples), a figure which corresponded with the
overall rate of seed transmission in this cultivar. Embryo
sac fluid could be obtained only from stage i and ii seeds.
In Vedette, virus was readily detected in the embryo sac
fluid at stage i and appeared to decline in abundance by
stage ii. Very little virus was present in these samples or
whole embryos at all five stages from Progreta. PSbMV
was barely detectable in the two earliest stage embryos of
Vedette but was then detected with increasing frequency
to a maximum at stage v. The patterns of virus
accumulation in embryos of Vedette and Progreta
differed not only in the frequency of detection but also in
the concentration of virus (Fig. 1), indicating that
PSbMV multiplied in embryos of Vedette.
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D. Wang and A. J. Maule
50
I
-
I
I
I
I
I
Stability of the virus during seed maturation and
dehydration has been shown to be a determinant of the
efficiency of seed transmission in SMV-infected soybean
(Bowers & Goodman, 1979; Irwin & Goodman, 1981).
Mature seeds from PSbMV-infected Progreta and
Vedette plants were harvested during natural seed
dessication, dissected into testa and embryo (cotyledons
plus axis) and analysed for the incidence of PSbMV
(Table 3). Virus antigen was detected in all the testa
samples from both cultivars at all stages, and in 50 to
80% of Vedette embryos. There was no reduction in the
incidence of virus infection, or virus concentration (data
not shown), with time.
I
~(b)
(a)
40
-7
30
-~ 20
10
0
....
i
11
0 0"2
~0-8-1-0
~
1
A4o5 ~
iii
iv
0"2-0,4 ~
1"0-1-2 1
v
,I,
i
0-4-0"6 ~
1-2-1.4
,..
m
ii
,
iv
,
v
0"6-0-8
Fig. 1. Incidence of different PSbMV concentration (measured as A405
from the indirect ELISA) in populations of embryos of different
developmental stages (i to v) taken from infected plants of cvs Vedette
(a) and Progreta (b). Vertical blocks: percentage of infected embryos at
each PSbMV concentration. Only the percentages of infected embryos
are shown.
Northern blot hybridization analysis of PSbMV in
embryos of early developmental stages
Immunological assays indicated that there was an
increasing incidence and concentration of PSbMV in
Positive/total*
Stage ii
15/24
Stage iii
19/24
Fig. 2. Northern blot hybridization of RNA extracted in a single experiment from individual embryos at stage ii (40 mg fresh weight)
and stage iii (80 mg fresh weight) in development, using a cDNA clone specific for PSbMV RNA. Frequency of detection (*) of the 9.5
kb genomic RNA (arrowed) in embryos of Vedette is listed on the right; no positive hybridization was obtained with any RNA samples
from Progreta embryos.
T a b l e 3. Seed-borne infection from seeds of cvs Progreta and Vedette harvested at three
stages of dessication
Progreta
Dehydration
stage*
1
2
3
Expt.
Expt.
Expt.
Expt.
Expt.
Expt.
1
2
1
2
1
2
Vedette
No. infected/
no. tested
Infection (%)t
No. infected/
no. tested
Infection (%)
0/24
1/24
0/24
0/24
0/24
0/24
0
4
0
0
0
0
14/24
-:1:
12/24
17/24
13/24
19/24
58
50
71
54
79
* Stage l, pods and seeds were green and soft; stage 2, pods were yellow and soft, and seeds were grey and
soft; stage 3, pods were yellow and hard and seeds were grey and hard.
t Seed-borne infection assessed as symptoms on young seedlings, confirmed by ELISA.
:~ (-) Not tested.
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P S b M V seed transmission in pea
Vedette embryos with time (Table 2, Fig. 1). The
possibility that the apparent low frequency of infection
in early stage embryos reflected the sensitivity of the
ELISA was examined by extracting total RNA from
individual stage ii and stage iii embryos and testing for
PSbMV RNA by Northern blot hybridization. It was
found that in Vedette a high incidence of viral RNA was
already present in the embryos at stage ii and increased
only slightly at stage iii (Fig. 2), whereas in Progreta no
viral RNA was detected at either stage in any embryos.
The frequency of viral RNA detection by hybridization
at stage ii corresponded with the final seed transmission
efficiency for Vedette and Progreta although the concentration of viral RNA in Vedette varied significantly
between embryos and increased overall from stage ii to
stage iii.
Discussion
Pollen transmission of PSbMV was shown not to occur in
five susceptible cultivars of pea which varied widely in
their efficiency of virus seed transmission. Hence, it was
not surprising that pollen grains from infected plants
showed no PSbMV infection when tested by ELISA or
electron microscopy. Previously, a low level ( < 1~) of
pollen transmission of PSbMV has been detected in a
single pea cultivar with 6 ~o seed-borne infection (Stevenson & Hagedorn, 1973); this cultivar and the PSbMV
isolate were not available for our studies. Pollen
transmission occurs for the potyvirus BCMV in P.
vulgaris and its extent is cultivar-specific (Nelson &
Down, 1933; Medina & Grogan, 1961). The importance
of pollen transmission in either of these cases is
questionable since both plants are predominantly selffertilized.
Invasion of the female gamete prior to fertilization is
difficult to assess without extensive electron microscopy.
However, the absence of detectable (using ELISA and
electron microscopy) virus in pea ovules prior to
fertilization makes the female gamete an unlikely source
of virus for the embryo infection observed later in
development. BCMV was detected in 80~ of ovules
prior to fertilization but the extent to which this led to
egg cell infection and finally to 15~ seed-borne
infection, is not clear (Schippers, 1963). In fact, the
isolation of the female megaspore or the egg cell from the
surrounding maternal tissues make the former unlikely
targets for virus invasion. Infection of the female
gametophyte is believed to be critical for the invasion of
barley embryos by barley stripe mosaic virus (hordeivirus) and probably occurs prior to the completion of the
meiotic divisions (Carroll, 1981).
Cultivars Vedette and Progreta are equally susceptible
1619
to PSbMV infection in the vegetative tissues of the plant
(Wang et al., 1992b) and hence, the maternal tissues of
the flower, including the seed coat, were infected in both
cases. Differences which correlated with different
efficiency of seed transmission between these cultivars
appeared in only the embryo sac fluid and the embryo. A
low incidence and small amounts of virus were detected
in samples of each from Progreta but the absence of
embryo infection when assessed using the sensitive
method of Northern blot hybridization probably indicates that the positive detection was derived from
contamination from infected testa tissues. Progreta,
therefore, is probably blocked in seed transmission at the
stage of virus invasion of the early embryo, although the
possibility that the embryos themselves may be resistant
to infection by PSbMV has not been addressed in these
experiments. Studies of SMV in soybean showed a
different mechanism regulating seed transmission. SMV
multiplied in the embryonic tissues of both transmitting
and non-transmitting cultivars but the incidence of virus
in the latter decreased during embryo development by a
process of virus inactivation (Bowers & Goodman, 1979;
Irwin & Goodman, 1981).
These experiments have highlighted the importance of
the early embryo in the establishment of a seed-borne
infection of PSbMV in pea. Northern blot hybridization
detected PSbMV RNA in a proportion of stage ii
embryos corresponding to the final proportion of seed
transmission assessed as infected seedlings. Quantitative
ELISA showed that the virus then multiplied to a
maximum in mature fresh embryos and did not decrease
during seed dessication. It is acknowledged, however,
that the titre of infectious virus in these samples was not
measured. The source of the virus for embryo infection
and the precise stage at which early embryo invasion
occurs is the subject of further study, but the detection of
virus in the embryo sac fluid might indicate a possible
route. The variability in the concentration of viral RNA
in stage ii and, to a lesser extent, stage iii embryos
indicates that either the virus might invade individual
embryos at different stages or that the rate of virus
accumulation may differ between embryos. However,
consistency of the incidence of embryo invasion between
stages ii and iii might suggest that that there is only a
limited 'window' in the developmental process during
which virus can enter. Such a 'window' might easily
differ with the physiological status of the host plant and
could account in part for the commonly observed
environmental influence on seed transmission.
From these studies it would appear that although
differences in detail exist between the behaviour of
different potyviruses in relation to seed transmission,
early embryo invasion may be a common feature of the
process. Embryo invasion probably occurs for BCMV in
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D. Wang and A. J. Maule
P. vulgaris, was shown to be important for PStV of
peanut (Xu et al., 1991), and occurred for SMV in
soybean although in this case the determinant of
resistance to seed transmission was different. A further
detailed study will be required to determine the genetic
control of embryo invasion and the biology of the
'invasion process'.
The authors thank Drs A. J. Cockbain and R. D. Woods for advice,
and Professor J. W. Davies and Dr M. I. Boulton for critical reading of
the manuscript. For part of this work, D. W. was jointly funded by
Imperial Chemical Industries plc., Plant Breeding International
Cambridge Ltd, Sharpes International Seeds Ltd, Nickersons SA and
the Processors and Growers Research Organization. The virus was
held under MAFF licence No. PHF 1185A/68(21).
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