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Journal of General Virology (1992), 73, 1615-1620.

Printed in Great Britain 1615

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 tration in pea seeds of different developmental stages
mosaic virus (PSbMV) in several pea genotypes has demonstrated that in the cultivar with a high incidence
been studied. Cross-pollination experiments showed of seed transmission, P S b M V directly invaded imma-
that pollen transmission of P S b M V did not occur and ture embryos, multiplied in the embryonic tissues and
accordingly, virus was not detected in pollen grains by persisted during seed maturation. In contrast, the
ELISA or electron microscopy. Comparative studies cultivar without seed transmission did not show
between two pea cultivars, one with a high incidence of invasion of immature embryos by the virus; there was
seed transmission and one with none, showed that no evidence for virus multiplication or persistence
PSbMV infected the floral tissues (sepals, petals, during embryo development and seed maturation.
anther and carpel) of both cultivars, but was not Hence seed transmission of PSbMV resulted from
detected in ovules prior to fertilization. Virus was direct invasion of immature pea embryos by the virus
detected equally well in seed coats of the progeny in and the block to seed transmission in the non-
both cultivars. Analysis of virus incidence and concen- permissive cultivar probably occurred at this step.

Introduction which are compatible with all the aforenoted host


changes are seed-transmissible, and (ii) the complexity of
Since the identification (Reddick & Steward, 1919) of the process provides many stages at which seed
seed transmission of viruses evidence has accumulated transmission may be regulated.
that indicates that the phenomenon is the end result of a There are many examples which illustrate the influ-
complex interaction between the host and the virus ence of host and viral genotypes on seed transmission
which may be influenced by a variety of environmental and the regulation of seed transmission at various
factors (for reviews, see Bennett, 1969; Shepherd, 1972; developmental steps but there are few examples of
Bos, 1977; Carroll, 1981). This is illustrated by the fact systematic analyses of the seed transmission process in a
that a seed-transmitted virus often has variants that are well-defined genetic system. General mechanisms which
not seed-transmitted, that different genotypes of the regulate the efficiency of seed transmission in many
same host species can differ in their efficiencies of host-virus interactions have not emerged from these
transmission of a single isolate and that the transmission studies.
efficiency can be affected by temperature and daylength. We have chosen to study seed transmission of the
To accomplish the process of seed transmission, the potyvirus pea seed-borne mosaic virus (PSbMV) in pea
virus must initiate an infection during the vegetative as it is a well-defined genetic and developmental system,
growth of its host, establish itself in the developing and constitutes a problem of major importance in plant
embryos, remain stable during seed dessication and pathology (Khetarpel & Maury, 1988). Pea cultivars
storage and eventually be reactivated during, or after, have been identified which vary from a high frequency of
seed germination. While the genetic structure of the virus seed transmission to zero (Stevenson & Hagedorn, 1969,
remains the same throughout, the host changes genetical- 1973; Wang et al., 1992b) and there is a substantial
ly from diploid to haploid and back to diploid, and understanding of the molecular and physiological aspects
physiologically from vegetative growth to reproductive of pea embryology (Casey, 1990). The complete sequence
growth and back to vegetative growth. This developmen- of PSbMV has been published (Johansen et al., 1991).
tal progression reveals two important aspects of plant Furthermore, seeds of many other major food legume
virus seed transmission: (i) only those viral genotypes species such as bean, cowpea, lentil, peanut and soybean

00014)900 © 1992 SGM


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1616 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 - derived from the embryo sac fluid might be a problem, embryos were
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 washed several times in distilledwater before grinding. The embryo sac
fluid from each immature seed was drawn off in a capillary tube and
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 &
diluted in 300 I11 extraction buffer prior to assay. Samples from all
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 stages were scored for the presence or absence of PSbMV and for the
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 concentration of virus present [absorbance at 405 nm (-4405)obtained
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 - from the indirect ELISA (Cockbain et al., 1988).
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 Northern blot hybridization analyses of PSbMV in immature embryos.
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 Genomic RNA of PSbMV-28 was prepared and reverse-transcribed as
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 described in Wang et al. (1992a). The eDNA was cloned into EcoRI-
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. digested 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
Methods hybridization analysis of PSbMV in immature embryos, total RNA
was extracted from individual embryos according to Casey et al. (1985)
PSbMV isolate andpea genatypes. An isolate of PSbMV, PSbMV-28,
and electrophoresed through 1% agarose containing formaldehyde
originally derived from a PSbMV-infected seed of Pisum sativum cv.
Waverex (Wang et al., 1992b) was maintained on, and purified (Wang (Sambrook et al., 1989). Separated RNAs were vacuum-transferred
et al., 1992a) from P. sativum cv. Dark Skinned Perfection. Combining onto nylon membrane (Hybond-N, Amersham) and cross-linked by
pea cvs. Bunting and Progreta 0ear-type, marrowfat type), Helka u.v. illumination. The filter was hybridized, washed and exposed to X-
(semi-leafless [afila]-type, non-marrowfat type), Birte and Vedette ray film as described in Sambrook et al. (1989).
(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. Results
Cross-pollination experiments. Healthy plants of cvs. Bunting, Helka, Pollen transmission o f P S b M V
Birte and Vedette were cross-pollinated with pollen from infected
plants of the homologous genotype and grown to give mature seed. In 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
the case of cv. Progreta which was shown to lack seed transmission in a to include p e a s o f the leaf- a n d afila-types, o f t h e
previous study (Wang et aL, 1992b), pollen grains from the infected
marrowfat and non-marrowfat type and of variable
plants were used to pollinate healthy plants of cv. Vedette in order to
resolve whether there was any PSbMV in Progreta pollen grains. Seeds 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 ,
from cross-pollinated plants and infected pollen-donor plants were 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
harvested and germinated to assess pollen transmission and embryo 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
transmission rates. Seed-borne infection was determined from the 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).
appearance of symptoms 3 weeks after germination and by indirect
ELISA (Cockbain et al., 1988) using polyclonal antibodies raised to 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
PSbMV-28. 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 virus-
i 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
Detection of PSbMV in inflorescence samples. Sepal, petal, pollen,
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
anther and carpel samples were collected from infected plants of
Progreta and Vedette and assayed for PSbMV using indirect ELISA 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
(Cockbain et al., 1988) or processed for electron microscopy (Wang et 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
al., 1991). Pollen grains from single flowers were washed several times et al., 1992b).
by mild sonication in extraction buffer (20 raM-sodium phosphate-
buffered saline, pH 7.4, containing 0.05% v/v Tween 20, 2% polyvinyl
pyrrolidone and 0.1% Triton X-100) and centrifugation, and finally Detection o f P S b M V in f l o r a l tissues
ground by mortar and pestle to be used as individual samples in indirect
ELISA. This washing procedure removed PSbMV attached to the A more thorough investigation of the distribution of
surface of a pollen grain as well as PSbMV-infected tissue debris 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
derived from anther epidermal tissues. Ovules dissected from a single 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
flower were combined and treated as a single sample in indirect 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
ELISA.
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,
Detection of PSbMV in embryos of different developmental stages. 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
Seeds representative of five different developmental stages (20, 40, 80, 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
200 and 280 mg embryo fresh weight) before dessication and three
E L I S A ( T a b l e 1). A l l t h e inflorescence p a r t s were
stages after dessication (24 seeds for each stage) were separately
analysed for the incidence of virus infection. Seeds were dissected into 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
testa, embryo and, for the two earliest stages, embryo sac fluid. When 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
dissecting immature embryos in which contamination by PSbMV 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 1617

Table 1. Virus incidence in the inflorescenceparts of cvs Progreta and Vedette

Progreta Vedette

Flower Incidence of ELISA Incidence of ELISA


part virus invasion* (A40s)i" virus invasion* (A4os)t

Sepal 30]30 0.411 ___ 0.331 30]30 0.613 + 0.312


Petal 30/30 0.651 _ 0-212 30]30 0.532 + 0-165
Anther 30/30 1.358 + 0.240 30/30 1.132 + 0.253
Carpel 30/30 0.204 + 0-047 30/30 0.128 + 0.059
Ovule 0/20 ND~ 0/20 NO
Pollen 0]10 ND 0/10 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 Embryonic sac fluid Embryo


Pea Seed
cultivar* transmission (%) it ii iii iv v i ii iii iv v i ii iii iv v

Progreta 0 100 0 12 -:~ - - 4 12 0 8 4


Vedette 60-80 100 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 possible to avoid cross-contamination of the samples.
sections of sepal, petal, anther-epidermal tissues and The developmental stages chosen were from late embryo
carpel. Virus particles were also observed, most fre- histogenesis to the mature seed stage just before
quently as bundles of particles (Wang et al., 1991). In dessication. The results are summarized in Table 2.
contrast, none of these structures were seen inside pollen Virus antigen was detected by ELISA in the testa
grains or ovules from either cultivar; funicle tissue tissues of all immature seeds from both cultivars
adjacent to the ovule showed some cells containing indicating that the ovule was invaded early in seed
PSbMV. To confirm these observations, pollen grains development. In contrast, virus was never detected in
and ovules from individual flowers were tested by ELISA 100% of the embryo or embryo sac fluid samples for
as described in Methods. Neither pollen grains nor ovule either cultivar. The maximum incidence was in Vedette
samples from infected Progreta orVedette plants gave a (75% of stage v embryos and 75% of stage i embryo sac
positive reaction with antibody to PSbMV (Table 1). 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.
Detection of PSbMV in embryos of different In Vedette, virus was readily detected in the embryo sac
developmental stages
fluid at stage i and appeared to decline in abundance by
If virus invasion of the ovule occurred post-fertilization, stage ii. Very little virus was present in these samples or
it was important to determine whether this happened whole embryos at all five stages from Progreta. PSbMV
early or late in seed development, whether all parts of the was barely detectable in the two earliest stage embryos of
seed became infected at the same time and whether two Vedette but was then detected with increasing frequency
pea cultivars, permissive and non-permissive for seed to a maximum at stage v. The patterns of virus
transmission, behaved similarly. The strategy followed accumulation in embryos of Vedette and Progreta
was to test by ELISA, seeds of different developmental differed not only in the frequency of detection but also in
status for PSbMV incidence after dissection into testa, the concentration of virus (Fig. 1), indicating that
embryo and embryo sac fluid, taking precautions where PSbMV multiplied in embryos of Vedette.

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1618 D. Wang and A. J. Maule

50 I I I I I I I
Stability of the virus during seed maturation and
- (a) ~(b) dehydration has been shown to be a determinant of the
40 efficiency of seed transmission in SMV-infected soybean
-7 (Bowers & Goodman, 1979; Irwin & Goodman, 1981).
30
Mature seeds from PSbMV-infected Progreta and
-~ 20
Vedette plants were harvested during natural seed
dessication, dissected into testa and embryo (cotyledons
10 plus axis) and analysed for the incidence of PSbMV

0 ....
i 11 iii iv v
,I,
i ii
,..
m
,
iv
,
v
(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
A4o5 ~ 0 0"2 ~ 0"2-0,4 ~ 0-4-0"6 ~ 0"6-0-8 incidence of virus infection, or virus concentration (data
~0-8-1-0 1 1"0-1-2 1 1-2-1.4
not shown), with time.

Fig. 1. Incidence of different PSbMV concentration (measured as A405


from the indirect ELISA) in populations of embryos of different Northern blot hybridization analysis of PSbMV in
developmental stages (i to v) taken from infected plants of cvs Vedette embryos of early developmental stages
(a) and Progreta (b). Vertical blocks: percentage of infected embryos at
each PSbMV concentration. Only the percentages of infected embryos Immunological assays indicated that there was an
are shown. 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 Vedette

Dehydration No. infected/ No. infected/


stage* no. tested Infection (%)t no. tested Infection (%)

1 Expt. 1 0/24 0 14/24 58


Expt. 2 1/24 4 -:1: -
2 Expt. 1 0/24 0 12/24 50
Expt. 2 0/24 0 17/24 71
3 Expt. 1 0/24 0 13/24 54
Expt. 2 0/24 0 19/24 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 1619

Vedette embryos with time (Table 2, Fig. 1). The to PSbMV infection in the vegetative tissues of the plant
possibility that the apparent low frequency of infection (Wang et al., 1992b) and hence, the maternal tissues of
in early stage embryos reflected the sensitivity of the the flower, including the seed coat, were infected in both
ELISA was examined by extracting total RNA from cases. Differences which correlated with different
individual stage ii and stage iii embryos and testing for efficiency of seed transmission between these cultivars
PSbMV RNA by Northern blot hybridization. It was appeared in only the embryo sac fluid and the embryo. A
found that in Vedette a high incidence of viral RNA was low incidence and small amounts of virus were detected
already present in the embryos at stage ii and increased in samples of each from Progreta but the absence of
only slightly at stage iii (Fig. 2), whereas in Progreta no embryo infection when assessed using the sensitive
viral RNA was detected at either stage in any embryos. method of Northern blot hybridization probably indi-
The frequency of viral RNA detection by hybridization cates that the positive detection was derived from
at stage ii corresponded with the final seed transmission contamination from infected testa tissues. Progreta,
efficiency for Vedette and Progreta although the concen- therefore, is probably blocked in seed transmission at the
tration of viral RNA in Vedette varied significantly stage of virus invasion of the early embryo, although the
between embryos and increased overall from stage ii to possibility that the embryos themselves may be resistant
stage iii. to infection by PSbMV has not been addressed in these
experiments. Studies of SMV in soybean showed a
different mechanism regulating seed transmission. SMV
Discussion multiplied in the embryonic tissues of both transmitting
and non-transmitting cultivars but the incidence of virus
Pollen transmission of PSbMV was shown not to occur in in the latter decreased during embryo development by a
five susceptible cultivars of pea which varied widely in process of virus inactivation (Bowers & Goodman, 1979;
their efficiency of virus seed transmission. Hence, it was Irwin & Goodman, 1981).
not surprising that pollen grains from infected plants These experiments have highlighted the importance of
showed no PSbMV infection when tested by ELISA or the early embryo in the establishment of a seed-borne
electron microscopy. Previously, a low level ( < 1~) of infection of PSbMV in pea. Northern blot hybridization
pollen transmission of PSbMV has been detected in a detected PSbMV RNA in a proportion of stage ii
single pea cultivar with 6 ~o seed-borne infection (Steven- embryos corresponding to the final proportion of seed
son & Hagedorn, 1973); this cultivar and the PSbMV transmission assessed as infected seedlings. Quantitative
isolate were not available for our studies. Pollen ELISA showed that the virus then multiplied to a
transmission occurs for the potyvirus BCMV in P. maximum in mature fresh embryos and did not decrease
vulgaris and its extent is cultivar-specific (Nelson & during seed dessication. It is acknowledged, however,
Down, 1933; Medina & Grogan, 1961). The importance that the titre of infectious virus in these samples was not
of pollen transmission in either of these cases is measured. The source of the virus for embryo infection
questionable since both plants are predominantly self- and the precise stage at which early embryo invasion
fertilized. occurs is the subject of further study, but the detection of
Invasion of the female gamete prior to fertilization is virus in the embryo sac fluid might indicate a possible
difficult to assess without extensive electron microscopy. route. The variability in the concentration of viral RNA
However, the absence of detectable (using ELISA and in stage ii and, to a lesser extent, stage iii embryos
electron microscopy) virus in pea ovules prior to indicates that either the virus might invade individual
fertilization makes the female gamete an unlikely source embryos at different stages or that the rate of virus
of virus for the embryo infection observed later in accumulation may differ between embryos. However,
development. BCMV was detected in 80~ of ovules consistency of the incidence of embryo invasion between
prior to fertilization but the extent to which this led to stages ii and iii might suggest that that there is only a
egg cell infection and finally to 15~ seed-borne limited 'window' in the developmental process during
infection, is not clear (Schippers, 1963). In fact, the which virus can enter. Such a 'window' might easily
isolation of the female megaspore or the egg cell from the differ with the physiological status of the host plant and
surrounding maternal tissues make the former unlikely could account in part for the commonly observed
targets for virus invasion. Infection of the female environmental influence on seed transmission.
gametophyte is believed to be critical for the invasion of From these studies it would appear that although
barley embryos by barley stripe mosaic virus (hordei- differences in detail exist between the behaviour of
virus) and probably occurs prior to the completion of the different potyviruses in relation to seed transmission,
meiotic divisions (Carroll, 1981). early embryo invasion may be a common feature of the
Cultivars Vedette and Progreta are equally susceptible process. Embryo invasion probably occurs for BCMV in

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1620 D. Wang and A. J. Maule

P. vulgaris, was shown to be important for PStV of Edited by K. Maramorosch & K. F. Harris. New York & London:
Academic Press.
peanut (Xu et al., 1991), and occurred for SMV in JOrt~NSEN, E., RAMUSSEN,O. F., H~IDE, M. & BORKrt~Dr, B. (1991).
soybean although in this case the determinant of The complete nucleotide sequence of pea seed-borne mosaic virus
resistance to seed transmission was different. A further RNA. Journal of General Virology 72, 2625-2632.
KrmT~d~PAL, R. K. & MAURY, Y. (1987). Pea seed-borne mosaic virus:
detailed study will be required to determine the genetic a review. Agronomie 7, 215-224.
control of embryo invasion and the biology of the MEDINA, A. C. & GRO~AN, R. G. (1961) Seed transmission of bean
'invasion process'. mosaic viruses. Phytopathology 51, 452-456.
NELSON, R. & Down, E. E. (1933). Influence of pollen and ovule
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and Professor J. W. Davies and Dr M. I. Boulton for critical reading of 25.
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Imperial Chemical Industries plc., Plant Breeding International diagnosis in seed health testing. Seed Science Technology 2, 3-155.
Cambridge Ltd, Sharpes International Seeds Ltd, Nickersons SA and REDDICK, D. & STEWART, V. B. (1919). Transmission of the virus of
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