Journal of General Virology (2009), 90, 1767–1774
DOI 10.1099/vir.0.009647-0
Vertical transmission of Prunus necrotic ringspot
virus: hitch-hiking from gametes to seedling
Khalid Amari, Lorenzo Burgos, Vicente Pallás3
and Maria Amelia Sánchez-Pina
Correspondence
Maria Amelia Sánchez-Pina
Dpto. de Biologı́a del Estrés y Patologı́a Vegetal, CEBAS-CSIC, Campus Universitario de Espinardo,
PO Box 164, 30010 Espinardo-Murcia, Spain
[email protected]
Received 16 December 2008
Accepted 4 March 2009
The aim of this work was to follow Prunus necrotic ringspot virus (PNRSV) infection in apricot
reproductive tissues and transmission of the virus to the next generation. For this, an analysis of
viral distribution in apricot reproductive organs was carried out at different developmental stages.
PNRSV was detected in reproductive tissues during gametogenesis. The virus was always
present in the nucellus and, in some cases, in the embryo sac. Studies within infected seeds at the
embryo globular stage revealed that PNRSV infects all parts of the seed, including embryo,
endosperm and testa. In the torpedo and bent cotyledon developmental stages, high
concentrations of the virus were detected in the testa and endosperm. At seed maturity, PNRSV
accumulated slightly more in the embryo than in the cotyledons. In situ hybridization showed the
presence of PNRSV RNA in embryos obtained following hand-pollination of virus-free pistils with
infected pollen. Interestingly, tissue-printing from fruits obtained from these pistils showed viral
RNA in the periphery of the fruits, whereas crosses between infected pistils and infected pollen
resulted in a total invasion of the fruits. Taken together, these results shed light on the vertical
transmission of PNRSV from gametes to seedlings.
INTRODUCTION
Seed transmission is a common mechanism of virus
perpetuation, as about 20 % of plant viruses are seed
transmitted (Hull, 2002; Mink, 1993). This process may
play an important role in dissemination of a virus,
especially as a result of breeding programmes, the
international exchange of germplasm and the use of
seedlings as rootstocks. The low seed transmission rate of
some viruses is not a good indicator of the potential
importance of a disease. The introduction of a poorly seedtransmitted virus in a new area can be catastrophic if an
efficient vector is available.
In general, viral seed transmission (Mink, 1993; Maule &
Wang, 1996) depends on infection of the embryo, which
can occur by three alternative pathways: the virus can (i)
reach the embryo during fertilization after being transported by the male gametophyte, (ii) invade the ovule and
then the embryo, also during fertilization (both ways are
indirect) and/or (iii) directly invade the embryo during its
development after fertilization. However, little is known
about how viruses can move from sporophytic to
gametophytic and back again into sporophytic tissues, in
3Present address: Instituto de Biologı́a Celular y Molecular de Plantas,
UPV-CSIC, Avda. de los Naranjos s/n, 46022 Valencia, Spain.
A supplementary figure showing dwarfed plants grown from PNRSVinfected seeds is available with the online version of this paper.
009647 G 2009 SGM
a process known as vertical transmission. The best example
of the processes involved in vertical transmission is the
work of Wang & Maule (1997) who demonstrated that pea
early browning virus is transmitted by seed after infection
of both gametes (indirect way) and that pea seed borne
mosaic virus (PSbMV) is seed-transmitted, although it is
not able to infect the gametes (direct way). The model of
PSbMV transmission was elucidated by Roberts et al.
(2003) who showed that the virus was able to first infect the
testa and then invade the endosperm using the existing
symplastic connection. The virus subsequently reached the
embryo through the pore-like structures that are located in
the sheath separating the embryo from the suspensor.
To improve our knowledge of how viruses infect seeds, we
have studied, in this work, the possible routes by which
Prunus necrotic ringspot virus (PNRSV) is vertically
transmitted in Prunus, more specifically, in apricot trees.
Apricot is an important stone fruit tree crop in the
Mediterranean region and there is a relatively high
incidence of PNRSV in orchards of this species (Myrta
et al., 2002).
PNRSV is the causal agent of several diseases that affect
most cultivated stone fruit trees. It causes serious economic
damage (Uyemoto & Scott, 1992), decreasing tree growth
by up to 30 % and their yield by 20–56 %; PNRSV infection
also makes infected trees very susceptible to chilling
damage. PNRSV belongs to the genus Ilarvirus and has
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K. Amari and others
the same genomic organization as, and encodes translation
products functionally similar to, those of the genera
Bromo-, Cucumo-, Olea- and Alfamovirus, which all belong
to the family Bromoviridae (Aparicio et al., 2001, 2003,
2006; Aparicio & Pallás, 2002; Sánchez-Navarro & Pallás,
1997; Sánchez-Navarro et al., 2006; Herranz & Pallás 2004;
Herranz et al., 2005). The virus is pollen- and seedtransmitted (Cole et al., 1982; Hamilton et al., 1984; Kelley
& Cameron, 1986; Aparicio et al., 1999; Amari et al., 2007),
which contributes to its rapid spread in stone fruit trees
(Mink, 1992). The seed transmission rate for PNRSV varies
among the infected species. Previous studies have demonstrated that, in the case of cherry, PNRSV was transmitted
in 88.8–76.9 % (Vértesy, 1976; Kryczynski et al., 1992) of
cases, while in almond it was only 9 % (Barba et al., 1986).
The routes by which the seeds become infected are not
known in detail, mainly due to the difficulties involved in
working with stone fruit trees, which have long generation
cycles and bloom only once a year.
This paper studies the progress of infection of PNRSV in
apricot reproductive organs during their development up
until the seedling stage, in an attempt to explain its mode
of transmission.
METHODS
(Roche). A PNRSV non-radioactive riboprobe was obtained by
transcribing a pPN4-890 clone corresponding to an 890 nt full-length
cDNA of PNRSV subgenomic RNA 4, that codes for the coat protein
(Sánchez-Navarro & Pallás, 1997).
Tissue-printing hybridization. Tissue-printing of fruits was carried
out as described previously (Más & Pallás, 1995). A total of 12 fruits
divided into groups of three (making a total of four membranes) from
different sides of the trees were freshly cut and imprinted directly
onto nylon membranes. The membranes were treated as described for
dot-blot hybridization.
Seed germination. Seeds collected from infected and virus-free trees
were cold-stratified in humid vermiculite for 3 months in a cold room
(4–7 uC). When the seeds began to germinate, they were individually
transplanted to soil and the seedlings were maintained in the
greenhouse to observe any symptoms and detect PNRSV.
In situ hybridization. Ten flower buds, ovaries and seeds were fixed
by immersion in a freshly made mixture of 4 % paraformaldehyde
and 2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), at 4 uC
for 4 h and embedded in paraffin (Paraplast Plus) as described
previously (Garcı́a-Castillo et al., 2001; Amari et al., 2007). The same
probe used for dot-blot hybridization was used for in situ
hybridization. In situ hybridization was performed at 55 uC
overnight. Slide pre-treatments, hybridization and colourigenic
detection were carried out as described previously (Garcı́a-Castillo
et al., 2001; Amari et al., 2007). Sections were examined and
photographed with a Leica DC 500 digital camera using a DMRB
Leitz microscope.
Plant material. Apricot trees (Prunus armeniaca L.) cultivar ‘Búlida’
from an open field at Calasparra (Murcia, Spain) were checked for the
presence of PNRSV during routine virus indexing, using a previously
described RT-PCR method (Sánchez-Navarro et al., 2005) that
permits simultaneous detection and differentiation of eight important
viruses that affect stone fruit trees, among them PNRSV. Flower buds
at different developmental stages were collected from both virus-free
and infected trees and examined for the presence of PNRSV. The
ovaries were separated from pistils prior to fixation and paraffin
embedding. Seeds were obtained from fruits at different ripening
stages.
Total RNA extraction. Several embryos, testa and cotyledons were
excised from ten immature seeds, of both virus-free and infected
trees, and homogenized in liquid nitrogen. Samples of homogenized
material and carefully separated endosperm were extracted with 2
volumes of 1 : 1 ratio of extraction buffer (350 mM glycine, 48 mM
NaOH, 34 mM NaCl, 40 mM EDTA and 4 % SDS) and phenol/
chloroform/isoamylalcohol (25 : 24 : 1). After shaking for 2 min, the
homogenate was centrifuged for 10 min at 21 920 g at 4 uC; the upper
phase was extracted twice with phenol, followed by precipitation with
1/10 volume of 3 M sodium acetate, pH 5.2, and 2.5 volumes of
ethanol. The pellet was resuspended in the appropriate volume of
nuclease-free water and total RNA integrity was checked by agarose
gel electrophoresis before being quantified using a Nanodrop ND1000 Spectrophotometer (Thermo Fisher Scientific) and equalized to
0.2 mg ml21.
Dot-blot hybridization. Five microlitres (1 mg) total RNA extracted
from infected and virus-free immature seeds were applied to nylon
membranes (Roche), either directly or in a serial fivefold dilution.
Membranes were air-dried and the nucleic acids were cross-linked by
exposure to UV irradiation on a transilluminator. Pre-hybridization
and hybridization were carried out as described previously (Pallás et
al., 1998). Chemiluminescent detection with CSPD reagent as
substrate was performed as recommended by the manufacturer
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RESULTS
PNRSV invades apricot reproductive organs from
early stages of development
PNRSV was detected in the early stages of ovule
development (Fig. 1a) in both the ovule and the ovary.
As expected, no viral RNA was detected in virus-free flower
buds at the same developmental stage (Fig. 1b). PNRSV
was detected in ovules in the megaspore stage (Fig. 1c)
when it was present in the nucellus, while the megaspore
itself remained free of the virus. PNRSV was localized, in
later developmental stages, in the funiculus as well as in its
corresponding ovule (Fig. 2a). However, at this developmental stage, distribution of virus was not homogeneous
inside the ovary, since the other funiculus and its
corresponding ovule were both virus-free (Fig. 2a).
The study of later developmental stages showed that
PNRSV invades all parts of the ovule, including inner and
outer integuments surrounding the nucellus, which
encloses the embryo sac (Fig. 2b, c). At this stage, two
different infection patterns were observed in the ovule: in
the first, the whole ovule was infected (Fig. 2b) and in the
second, the nucellus was free of PNRSV except for the cells
located close to and surrounding the embryo sac (Fig. 2c).
However, PNRSV was always observed in the nucellar cells
located around the embryo sac, even in the more advanced
developmental stage studied (Fig. 2d), where viral RNAs
were rarely detected inside the embryo sac and, when
found, they were in the fused polar cells (Fig. 2e).
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Journal of General Virology 90
Vertical transmission of PNRSV in apricot
Fig. 1. In situ hybridization of PNRSV in cross sections of young
winter flowers. (a) Infected young flower bud showing viral RNA
(blue) in the pistil (P) and ovule (Ov) inside the ovary (O), as well as
in anthers (A). (b) No viral RNA was detected in flower buds from
virus-free trees. (c) Ovule at the early developmental stage
showing viral RNA in the nucellus (Nu) surrounding the megaspore
(M), which is not infected.
Infection of apricot embryos and seeds by PNRSV
Dot-blot hybridization of PNRSV RNA obtained from
different parts of the seeds (embryo, endosperm, testa and
cotyledons) showed that in the torpedo (Fig. 3a) and bent
cotyledon (Fig. 3b) stages, the lowest amount of PNRSV
was detected in the embryos, while the greatest amount was
detected in the endosperm and testa (Fig. 3a, b). In the
mature seed stage, endosperm was totally absent and so
embryos were separated from their cotyledons for the
analysis; which showed that the PNRSV signal in the
embryos was strongest at this stage, and even slightly
higher than in the corresponding cotyledons (Fig. 3c).
The PNRSV invasion of the apricot embryo in early
developmental stages was confirmed by in situ hybridization studies of immature infected seeds, where PNRSV was
detected even in embryos at the globular stage; here, it
showed a heterogeneous distribution but was mostly
present in the apical region (Fig. 4a). In addition,
PNRSV RNA was detected in embryos in the globular
developmental stage obtained from the hand-pollination of
virus-free flowers with infected pollen grains (Fig. 4b). No
in situ hybridization signal was observed in virus-free
embryos at this stage (Fig. 4c).
We also observed viral infection of the seed integuments,
nucellus and testa in immature seeds (Fig. 4d–g) using in
http://vir.sgmjournals.org
Fig. 2. Distribution of PNRSV in female reproductive organs. (a)
PNRSV RNA was localized in the funiculus (F) and its corresponding ovule (Ov) tissues (on the right). (b) Ovule showing
infected outer and inner integument (OI and II, respectively), as
well as the totally infected nucellus (Nu) surrounding the embryo
sac (ES). (c) Infected ovule showing the uninfected nucellar tissue,
except for the zone enclosing the embryo sac. (d, e) High
magnification micrographs of the infected nucellus and their
corresponding embryo sacs, including the egg cell (EC) and
central (CC) cell, which is invaded by the virus in (e).
situ hybridization. Infection of the testa (Fig. 4e) mainly
affected areas in and around its vascular tissue (Fig. 4f, g).
Seed transmission of PNRSV in apricot
Tissue-printing hybridization analysis of apricot fruits
resulting from the hand-pollination of virus-free plants
with infected pollen revealed the presence of PNRSV only
in the outer part of these fruits (Fig. 5a). However, in fruits
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K. Amari and others
Fig. 3. Dot-blot hybridization to detect
PNRSV RNAs from different parts of the seed.
(a, b) Serial dilutions of total RNA extracted
from embryo, endosperm and testa at torpedo
(a) and bent cotyledon (b) developmental
stages were blotted and hybridized against
viral RNA. (c) Total RNA from mature embryos
and cotyledons was analysed separately
showing a slightly higher accumulation of
PNRSV in the embryos. Note that no positive
hybridization signal was observed from virusfree controls. The positive control was RNA
extracted from PNRSV-infected cucumber.
resulting from the hand-pollination of infected plants with
infected pollen, the virus was uniformly distributed
(Fig. 5b). No signal was obtained from the tissue-printings
of virus-free fruits (Fig. 5c). Furthermore, when seedlings
obtained by germination of seeds from infected trees were
analysed by dot-blot hybridization using total RNA
extraction from leaves, 10 % of the seedlings were seen to
be infected (data not shown) and the plants that were
obtained showed a severe dwarfing (Supplementary Fig. S1,
available in JGV Online).
DISCUSSION
It has been demonstrated previously that seed transmission
is related to the ability of the virus to infect flower organs,
especially the meristematic reproductive tissue, during
early developmental stages (Mink, 1993; Bennett, 1969;
Vazquez Rovere et al., 2002). We have already demonstrated that PNRSV invades apricot reproductive organs,
including the pollen mother cell (PMC) stage, early in
development (Amari et al., 2007). In that work, we showed
that the virus reduces pollen fitness, causing poor
germination and delayed growth inside the pistil.
However, in the south-eastern Spanish climate (where this
study was carried out), the delay in ovule maturity (Egea &
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Burgos, 1998; Alburquerque et al., 2000) gives the infected
pollen the opportunity to fertilize the ovules (Amari et al.,
2007); this was confirmed in the present study by obtaining
infected embryos after hand-pollinating virus-free pistils
with infected pollen.
In the present study, we investigated whether female
reproductive organs are infected as early in their development as the male gametophyte. Our results show that
PNRSV invades the ovary in early developmental stages,
since viral RNA was detected from the beginning of ovary
formation (Fig. 1). Unlike our earlier work with pollen, we
were unable to detect viral RNA in megaspores from young
ovules by using in situ hybridization (Fig. 1c), which
suggests that PNRSV cannot invade the megaspore,
although the possibility that the amount of viral RNA
present in this tissue was below the detection limit of the in
situ hybridization method cannot be ruled out. The
detection of PNRSV in the funiculus and its corresponding
ovule (Fig. 2a) explains the mechanism of PNRSV invasion
of the ovule, since the funiculus constitutes the last vascular
connection of the ovule with the pericarp (Reiser &
Fischer, 1993). Moreover, infection of the integuments
(Fig. 2b) would be an early indication of testa infection,
since the testa originates from the integuments (Swamy &
Krishnamurthy, 1980).
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Journal of General Virology 90
Vertical transmission of PNRSV in apricot
Fig. 4. In situ hybridization to determine the
localization of PNRSV RNAs in embryos and
seeds. (a) Infected embryo (E) at the globular
stage from an infected tree, where viral RNAs
are concentrated in the apical region. (b)
Immature seed obtained from hand-pollination
of a virus-free flower with infected pollen
grains, showing infection of embryo and
endosperm (En). (c) Virus-free embryo at the
globular stage, showing no positive signal. (d)
Cross-section of infected immature seed
showing PNRSV RNAs in all parts of the seed
(Mi, micropil; II, inner integument; Nu, nucellus;
En, endosperm; E, embryo). (e) Infected
immature seed at a later developmental stage
than those shown before, showing that
PNRSV RNA is infecting the testa (T) where
it is located in and around its vascular tissue, as
can be seen at higher magnification (f and g).
Unlike in the megaspore stage, PNRSV was detected in the
mature embryo sac (Fig. 2e), which may explain its vertical
transmission in apricot. This infection takes place after
invasion of the embryo by virus from the highly infected
Fig. 5. Detection of viral RNAs by tissue-printing hybridization. (a)
Cross-section of three different fruits obtained from hand
pollination of virus-free flowers by PNRSV-infected pollen, where
viral RNA is located only on the periphery of fruits. (b) Fruits
obtained from infected trees showing uniform infection by PNRSV.
(c) Tissue-printing from two different virus-free fruits.
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nucellar tissue (Fig. 2b). This hypothesis is supported by
the fact that the nucellus is responsible for providing
nutrients to the embryo sac during its development (Reiser
& Fischer, 1993; Radchuk et al., 2006), as well as by
previous studies which have demonstrated that the callose
layer separating the megaspore from the maternal tissues is
discontinuous, unlike in the case of PMCs (Johansen et al.,
1994). Moreover, according to Kapil & Bhatnagar (1981),
this callose layer of the female organ is absorbed and
replaced by a pectocellulase layer later in development.
This process could well be exploited by PNRSV to invade
the embryo sac from the nucellus.
The process of embryo development in dicots is often
divided into four stages: globular, heart- and torpedoshaped and cotyledonary. The invasion of apricot seeds by
PNRSV was studied from early (globular in immature
seeds) to mature embryo stages. Our studies on viral
accumulation in infected apricot seeds have shown that
PNRSV is able to infect young embryo tissues during their
development, at least during fertilization, since viral RNA
was detected during the globular developmental stage
(Fig. 4b) in young embryos obtained by the handpollination of virus-free pistils with infected pollen.
Infection of the endosperm (Fig. 3a, b) may occur during
double fertilization by one of the two sperm cells of
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K. Amari and others
infected pollen or by the central cell which, as we have
shown, can be infected by PNRSV (Fig. 2e). Even if PNRSV
cannot move from the gametes to the endosperm, infection
of the latter could originate in the sporophytic maternal
tissue, as has already been shown in the case of PSbMV,
which is not able to infect the gametes but is able to reach
the endosperm through the existing connections between
the testa and the endosperm (Roberts et al., 2003).
Moreover, PNRSV was localized in the testa of infected
apricot seeds (Figs 3a, b and 4e–g). This localization might
explain the seed transmission of the virus but only if PNRSV
can invade the embryo via the endosperm from the testa,
because until now, no virus except tobacco mosaic virus
(TMV) has been shown to be transmitted to seedlings
directly from seeds. However, in the case of TMV, the
stability of the virus allowed transmission to seedlings as a
result of external contamination during seed manipulation
(Broadbent, 1965; Taylor et al., 1961). The increase in
PNRSV accumulation observed in the testa from the
torpedo and bent cotyledon stages (Fig. 3a, b) could be
explained by the existence of viral replication in this tissue or
by continuous viral invasion from the maternal tissue
following the route of plant assimilates, which are directed
towards the fruit during development. In our particular case,
we cannot rule out any of these hypotheses, although it is
most probable that embryo infection takes place during
fertilization, since PNRSV was detected in pollen grains,
particularly in the generative cell (Amari et al., 2007), as well
as in the embryo sac (Fig. 2e), both of which are responsible
for the formation of the zygote and, later on, the embryo.
The slightly higher level of accumulation of PNRSV in the
embryo compared with the cotyledon (Fig. 3c) suggests
that the virus could replicate in the embryo tissue, since
RNA integrity was checked. This is not the case with other
seed-transmitted viruses such as alfalfa mosaic virus, which
only infect the embryo (Bailiss & Offei, 1990). In the case
of PSbMV, the virus invades the embryo and partially
invades the cotyledons (Wang & Maule, 1997). In our
study, PNRSV invaded both the embryo and cotyledons
(Figs 3c and 4a, b). It seems that infection of the embryo is
a prerequisite for seed transmission, given that other
viruses that are able to infect only the cotyledons are not
seed-transmitted, as in the case of plum pox virus
(Pasquini & Barba, 2006).
The seed transmission rate obtained for PNRSV in apricot
trees was similar (10 %) to that obtained for other genera
of Prunus (Mink, 1992; Bassi & Martelli, 2003). It must be
taken into account that the low rate of seed transmission of
a given virus is not a good indicator of its ability to invade
the embryo, since virus inactivation can occur during seed
maturation or storage, which will, in some cases, reduce
virus transmission (Bowers & Goodman 1979; Bailiss &
Offei, 1990; Johansen et al., 1994). Thus, invasion of the
embryo is not sufficient to classify the virus as seedtransmitted, since it must also be able to survive during the
maturation and storage of the seed.
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In our study, we detected viral RNA in immature fruits
obtained by hand-pollinating virus-free pistils with
infected pollen (Fig. 5a), which demonstrates that
PNRSV, like many other seed-transmitted viruses, can be
transmitted into a pollinated virus-free plant or, at least, to
the fruits in apricot. It has been described that some
viruses, including PNRSV in cherry trees, can invade the
pollinated plant (Cameron et al., 1973; Davidson, 1976;
George & Davidson, 1964; Murant et al., 1974). However,
the percentage of this type of transmission, called ‘retroinfection’ by Mandahar (1985), is very low. This mode of
transmission has been questioned, since even if a virus is
able to escape from the ovule during or after fertilization, it
would not be able to rapidly invade the adjacent maternal
tissue to establish a systemic infection in the whole plant.
Phloem transport is mostly directed to the developing
fruits and the cell-to-cell movement would be too slow for
the virus to leave the fruit and invade other parts of the
plant (Bennett, 1969). Our observation of the external
localization of PNRSV in the fruits (Fig. 5a) suggests an
alternative route that this virus can use to escape from
pollen and infect the fruit. This might take place during
pollen tube germination, since the callose layer separating
pollen and pistil is not present in the growing zone of the
pollen tip (Keijzer & Willemse, 1989). This callose layer is
formed by deposition of vesicular components of the
pollen tube cytoplasm (Franklin-Tong, 1999) and we
suggest that PNRSV could be transported within these
vesicles and so leave the pollen tube. This suggestion is
based on our previous work, where we observed a high
concentration of PNRSV in the growing zone of germinating apricot pollen tubes (Amari et al., 2007).
Taking all our data into account, we suggest that PNRSV is
most probably transmitted by apricot seeds through
gametes and not indirectly from the testa.
ACKNOWLEDGEMENTS
We thank Mr P. Thomas for revising the English grammar. This
research was supported by grants BIO2002-04099-C02-01, BIO200507331 and BIO2008-03528 from the Spanish granting agency
DGYCIT and FEDER.
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