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Journal of General Virology (1992), 73, 1615-1620. 1615 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 16:11:21 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 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 16:11:21 PSbMV seed transmission in pea 1617 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 16:11:21 1618 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. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 16:11:21 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 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 16:11:21 1620 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). References BE~a~mx-r,C. W. (1969). 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(Received 5 February 1992; Accepted 31 March 1992) Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Wed, 14 Jun 2017 16:11:21