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Multiple viral determinants affect seed transmission of pea

seedborne mosaic virus in Pisum sativum

I. E. J o h a n s e n , 1 W. G. D o u g h e r t y , 2 K. E. Keller, 3 D. W a n g 4 a n d R. O. H a m p t o n 3

1Biotechnology Group, The Danish Institute of Plant and Soil Science, Lyngby, DK-2800 Denmark
z Department of Microbiology and Center for Gene Research and Biotechnology, Oregon State University, Corvallis 97331-3804, USA
3 Department of Botany and Plant Pathology, Oregon State University, Corvallis 97331-2902, USA
4 Department of Virus Research, John Innes Centre, Norwich NR4 7UH, UK

Two pea seedborne mosaic potyvirus (PSbMV)
isolates, P-1 DPD1 (P-l), which is highly seed-
transmitted, and P-4 NY (P-4), which is rarely seed-
transmitted, and chimeras between P-1 and P-4
were analysed to map the viral genetic determinants
of seed transmission. Infectivity of chimeric viruses
was evaluated by inoculating Pisum sativum with
RNA transcribed in vitro from recombinant full-
length cDNA clones. The chimeric viruses that were
used demonstrated that a genomic segment
encoding the 49 kDa protease and putative RNA
polymerase was responsible for symptom induction.
Attempts to determine transmission of the chimeric
viruses in P. sativum cultivars known to transmit P-
1 at high frequencies showed that seed transmission
is a quantitative character influenced by multiple
viral determinants. Seed transmission frequency did
not correlate with accumulation of virus in vege-
tative tissue. The 5' 2"5 kb of the 10 kb PSbMV
genome had a major influence on the seed trans-
mission frequency and was analysed further. This
showed that, while the helper-component protease
was a major determinant of seed transmission, the
potyviral Pl-protease exerted no measurable
influence.

Introduction inherited as a quantitative character controlled by the action of

Transmission through seed has been described for 108
plant viruses in one or more of their hosts. For all of these,
except tobacco mosaic virus, successful seed transmission
depends on the virus entering and surviving in the embryo
(Mink, 1993). Seed transmission is precluded when the virus is
unable to infect the gametes prior to fertilization, unable to
enter the embryo during development, or when the virus is
inactivated in the embryo during seed maturation and storage
(Maule & Wang, 1996). In virus-host combinations with
potential for seed transmission, the frequency of seed trans-
mission depends on both host and virus genotype and may
range from 0% to almost 100% (Mink, 1993).
The mechanisms of resistance to seed transmission are not
resolved and inheritance of resistance has been investigated in
only a few cases. In Hordeum vulgare cultivar Modjo, resistance
was reported to be conditioned by a single recessive gene
(Carroll et al., 1979), whereas resistance to seed transmission of
pea seedborne mosaic potyvirus (PSbMV) in Pisum sativum is
Author for correspondence: I. E. Johansen.
Fax +45 45 93 22 13. e-mail e.johansen@dips-lyngby.dk
multiple maternal genes (Wang & Maule, 1994). In a single
host cultivar, different virus isolates are seed-transmitted to
different degrees. These differences may reflect differences in
virus replication and movement which determine the frequency
at which the virus successfully enters the gametes or the
embryo (Carroll, 1981).
Pseudorecombination studies with isolated viral RNAs
from strains differing in seed transmissibility showed that the
seed transmission phenotype was linked to RNA 1 of raspberry
ringspot and tomato black,zing nepoviruses (Hanada &
Harrison, I977) and to RNA I of cucumber mosaic cucumo-
virus (Hampton & Francki, 1992). Analyses of barley stripe
mosaic hordeivirus (BSMV) chimeras facilitated mapping of
the major determinants of seed transmission in barley (H.
vulgare) to the 5' untranslated leader of RNAT, a 369 bp repeat
in the 7a gene, and the 7b gene (Edwards, 1995).
The seed transmission frequency of PSbMV is influenced
by the genotype of the virus isolate (Kohnen et al., 1995). The
complete nucleotide sequences of two isolates of PSbMV
which differ widely in seed transmission frequency in several P.
sativum cultivars have been determined (Johansen et al., 1991,
1996). However, a comparison of the primary sequences

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0001-4205 © 1996 SGM
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revealed too m a n y differences to conclude which part of the
g e n o m e determined seed transmission (Johansen et al., I996).
Therefore a study was undertaken to analyse the 10 kb R N A
g e n o m e of P S b M V b y creating chimeras b e t w e e n the two
virus isolates a n d analysing their transmission through seed in
P. sativum cultivars k n o w n to transmit P S b M V at high
frequencies.
Methods
• Virus i s o l a t e s . PSbMV isolate P-1 DPD1 was recovered from a pea
seed sample analysed at the Danish Plant Directorate (Lyngby, Denmark).
The PSbMV P-4 NY isolate was recovered from USDA Pisum P.I.
accession 471128 and kindly provided by R. Provvidenti (New York
State Agriculture Experiment Station, Geneva, NY, USA). For con-
venience P-1 DPD1 and P-4 NY will be referred to hereafter as isolates
P-I and P-4; however, it should be noted that seed transmissibility is not
necessarily linked to the pathotype of PSbMV. Cloning and sequencing
of P-1 and P-4 were reported by Johansen el al. (1991, 1996).
• cDNA modificationsand construction of full-length clones.
A cistron map of PShMV is shown in Fig. 1 (a). Locations of natural and
engineered restriction sites in the cDNA of isolates P-1 and P-4 are
identified by the first nucleotide (nt) of the recognition sequence. Maps
and names of isolates P-I and P-4 intermediary clones are shown in Fig.
I (b).
cDNA of the 5' termini of isolates P-1 and P-4 covering nt 1-Sph[ 965,
and 1-SphI 924, respectively, were amplified by RT-PCR (Kohnen et at.,
(a)
/ / e'
P1Pr HC-ProI Pa II c, IivPgl
I 6kl 6k~
% %
(b)
Dral Sphl BamHI Pstl
4, $ 4, 4,
i i: ] pPe
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1992) with primers that added a DraI site at each 5' end and an XbaI site
at each 3' end. The DraI site allowed precise excision of the viral 5'
termini, and the XbaI site facilitated subsequent assembly of full-length
clones. An XbaI linker was inserted 3' to the cDNA covering the viral
poly(A) tails, thus creating a unique XbaI site for linearization of the full-
length plasmids prior to in vitro transcription. The cDNA sequences were
modified by site-directed in vitro mutagenesis (Sculptor, Amersham). A
HindIII site was introduced at nt 1276 of P-l, and the P-4 sequence was
modified in three positions, introducing a HindIII site at nt I234, a BamHI
site at nt 2259 and a PstI site at nt 5812. All nucleotide substitutions were
translationally silent.
Full-length clones for in vitro transcription were assembled in the
vector pTTE19( + ) (Petty, 1988), which was modified to remove the SphI
and HindIII sites from the polylinker. The virus sequences were cloned
directly behind the T7 promoter by digesting modified pT7E19( + ) with
Sad, treating with T4 DNA polymerase and then digesting with XbaI
before inserting the 5' terminus of either P-1 or P-4 as a DraI-XbaI
fragment. These plasmids were digested with SphI and XbaI, and cloned
cDNAs (covering Sph1965 to BamH12300 of P-1 and SphI 924 to BamHI
2259 of P-4) were inserted as SphI-XbaI fragments creating plasmids pPa
and pPb, respectively. Cloned cDNAs covering HindIII 8647 to the
poly(A) tail of isolate P-1 and HindIII 8585 to the poly(A) tail of isolate
P-4 were inserted as BamHI-XbaI fragments 3' to both BamHI 2300 of
pPa and BamHI 2259 of pPb resulting in pPc, pPd, pPe and pPf.
pPg (containing cDNA covering BamH12300 to HindlII 8647 of P-l)
and pPh (containing cDNA covering BamHI 2259 to HindlII 8585 of P-
4) were digested with BamHI and PstI, and the BamHI-PstI fragment of
pPg was inserted into pPh, creating pPi.
Full-length plasmids pP-1, pP-1114 and pP-4111 were assembled by
I I
I RdRp I CP ~poly(A)
I Fig. I. (o) Cistron map of PSbMV
showing the noncoding regions (solid
line), the open reading frame encoding
the potyvirus polyprotein (open box) and
putative proteolytic cleavage sites
(vertical lines). Relevant restriction sites
in the cDNA are shown above (P-I) and
Hindlll Xbal below (P-4) the cistron map. Restriction
sites marked with an asterisk were
engineered into the sequences. PI Pro,
pPa PI protease; HC-Pro, helper-component
protease; P3, P3 protein; 6ki, 6 kDa
pPb protein I ; CI, cylindrical inclusion protein;
6k2, 6 kDa protein 2; 49k-Pro, 49 kDa
r-----~ pPc protease; VPg, genome linked virus
protein; RdRp, putative RNA dependent
pPd RNA polymerase; CP, coat protein. (b)
Maps and names of intermediary clones
used in the assembly of the full-length
clones pP-1, pP-1114, pP-411 I, pP-
1144 and pP-4144. The P-I isolate
I ]I pP! sequences (light grey) and P-4 isolate
sequences (dark grey) contained in the
PPg clones are shown as boxes, positioned
according to the sequence in the genome
pPh they represent. Areas represented by a
solid line are not present in the clones.
pPi Vector sequences are not shown.
 (a) (b)
Symptoms Virus acc. Inf./Tot. % ST % Germination

P-1 Severe 0.66 c 116/464 25 b 94 b,c

P-4 " Mild 0.37 d 1/505 0.2 d 91 c,d

vP-1 ~4~ ~<~i~@~<~;~ Severe 0.71 b,c 136/366 37 a 96 a,b

H
vP-1114 ~:<~:~-.'~'u~ Severe 0.34 d 93/521 18 b 88 d
B
vP-4111 ~ ; ~ : ~ & ~ ; ~ ; ~ ; ~ ; ~ Severe 0.93 a 28/614 4.6 c 98 a
P
vP-1144 ~t~ I Mild 0.81 a,b 44/626 7.0 c 95 a,b
B P
vP-4144 I Mild 0.34 d 12/729 1.6 d 95 a,b

Fig. 2. (o) Cistron maps of isolates P-I (light grey), P-4 (dark grey), transcript derived vP-1 and chimeras vP-1114, vP-411 I,
vP-1144 and vP-4144. (b) Symptoms and virus accumulation in infected P. sativum '549 ', seed transmission frequency and
percentage germination of seeds from infected plants. The restriction sites B (BamHl), P (Pstl) and H (Hindlll) were used for
assembly of the recombinant full length clones. Severe, P-I -like symptoms; mild, P-4-1ike symptoms. Virus acc., virus
accumulation in the youngest fully expanded leaves of P. sativum 3 weeks after inoculation determined by ELISA (A4o5 values,
mean of eight plants). Inf./Tot., total number of infected progeny seedlings detected/total number of viable progeny seedlings
assayed (pooled from three separate experiments). % ST, percentage seed transmission detected (average of three separate
experiments). Means followed by the same letter are not statistically different at the 95% level.

(a) (b) pPa with the HinclI I02-HindlII 1234 of pPb; and the HindIII 1276--
Inf./Tot. % ST BamHI 2300 of pPa with the HindIII 1234-BamHI 2259 of pPb,
respectively, followed by assembly of the full-length clones as described
vP-1 for pP-I.
169/316 53 a
• In vitro transcription and inoculation of RNA. Full-length
P-4 PSbMV cDNAs were cloned and amplified in Escherichia coli strain SURE
0/105 0.0 d
(Stratagene). Caesium chloride gradient-purified plasmids were linearized
vP-1 (p:4 5'UTR) with XbaI, and 2-3 lag of linearized plasmid was used for in vitro synthesis
54/184 29 b
of capped transcripts (mMESSAGE MACHINE, Ambion). Approximately
fi H vP-1 (P-4 Pl pro) 2 lag of transcript was inoculated mechanically onto two carborundum-
57/103 55 a dusted leaves of each P. sativum plant. Two weeks after inoculation,
infectivity of the in vitro-synthesized transcripts was assayed by double-
H a vP-I(P-4 HCpro N)
18/210 8.6 c antibody sandwich (DAS)-ELISA (Converse & Martin, 1990) using an
antiserum specific for PSbMV coat protein (Hampton & Mink, 1989).
Fig. 3. (a) Cistron maps of vP-1 (P-4 5'UTR), vP-1 (P-4 P/pro) and
Virus derived from plasmid pP-1 is referred to as vP-1, and chimeric
vP-1 (P-4 HCproN). The restriction sites B (BamHl), H (Hindlll) and Hi viruses are referred to as vP-1114, vP-4111 etc.
(Hincll) were used to exchange the 5' UTR, P/pro and HCproN in the Accumulation of viruses in young, fully expanded, systemically
recombinant full-length clones. (b) Seed transmission in P. sativum infected leaves was compared by measuring (by DAS-ELISA) the
'Vedette'. Inf./Tot., total number of infected progeny seedlings accumulation of coat protein in plants infected with native isolates P-1 or
detected/total number of viable progeny seedlings assayed (pooled from P-4, or transcript-derived viruses, respectively. For each virus, eight
two separate experiments). % ST, percentage seed transmission detected
(average of two separate experiments). Means followed by the same letter plants were tested and confidence intervals (95%) were calculated,
are not statistically different at the 95% level. assuming a standard normal distribution of A40~ values.
• Determination of seed transmission frequency. The pea
cultivar '549' was used for the first three seed transmission experiments
inserting the BamHI-Hindi[I fragment of pPg into BamHI/HindIII- under greenhouse conditions described by Kohnen et al. (1995). ' Vedette'
digested pPc, pPe and pPf, respectively, pP-II44 and pP-4144 were was used for the fourth and fifth experiment because this cultivar matures
assembled by inserting the BarnHI-HindIII fragment of pPi into early and transmits PSbMV P-1 through seed at very high frequencies
BamHI/HindlII-digested pPe and pPd. The full-length clones are shown (Wang et aI., 1993). lnoculum was raised in leaves of P. sativum '549' or
in Fig. 2 (a). 'Vedette' infected with in vitro-synthesized transcripts. Infection was
Plasmids pP-I(P-4 5'UTR), pP-I(P-4 Plpro) and pP-I(P-4 HCpro ~¢) confirmed by DAS-ELISA 2 weeks after inoculation. Leaves of infected
(Fig. 3 a) were created by substituting the HincI[ 102-BamH12259 of pPb plants were homogenized in buffer (50 raM-sodium phosphate pH 7"0)
with the HinctI I46--BamHr 2300 of pPa; the HinclI I4&-HindlII 1276 of and used to inoculate 2-3-week-old P. sativum '549" or 'Vedette'.

[
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Infectedplants were grown to maturityand seeds were harvested as the
seeds matured. Seeds from infected plants were sown and seedling
infection was determined by DAS-ELISA 3 weeks after emergence.
Confidence intervals (95 %) were calculated for the probability of the
observed seed transmissionfrequency,assuminga binomial distribution
of the data.
Results and Discussion
Infectivity of RNA transcripts from native and
recombinant full-length cDNA clones
Inoculation of P. sativum with in vitro-synthesized trans-
cripts of pP-1, pP-1114, pP-4111, pP-1144 or pP-4144 (Fig.
2a), and pP-I(P-4 5'UTR), pP-I(P-4 Plpro) or pP-I(P-4
HCpro N) (Fig. 3 a) all resulted in infection of 50 to 100 % of the
inoculated plants. In subsequent mechanical transfers, using
infected plant material as inoculum, the infectivity of vP-1 and
the chimeric viruses was comparable to native isolates P-I and
P-4. In contrast, transcripts of pP-4 and reciprocal chimeras to
those shown in Fig. 2 (a) were not infectious. All these plasmids
contained cDNA covering nts 2259-5812 of P-4, suggesting
that this cDNA segment was defective. However, substitution
of this region with cDNA from a new cDNA synthesis reaction
did not result in the generation of biologically active
transcripts.
Symptom induction and accumulation of transcript-
derived viruses
Symptoms induced by vP-1, vF-1114, vP-4111, vP-I(P-4
5'UTR), vP-I(P-4 Plpro) and vP-I(P-4 HCpro •) were similar
to those of native P-1 virus (transient vein clearing, downward
rolling of leaflets and shortened internodes). Symptoms of vP-
1144 and vP-4144 were mild, causing only a slight growth
reduction similar to plants inoculated with native P-4 virus
(Fig. 2 b).
These observations suggest that the region of the PSbMV
genome between PstI 5874/5812 and HindIII 8647/8585 has
a major influence on symptom severity in P. salivum. This
region contains the potyvira149 kDa protease and the putative
RNA dependent RNA polymerase, both proposed to be
involved in replication of the potyvirus genome (Riechmann et
al., 1992).
To determine whether the differences in symptom in-
duction correlated with differences in virus accumulation, the
relative accumulation of virus in systemically infected leaves
was determined 3 weeks after inoculation (Fig. 2 b).
vP-4111 accumulated to a higher concentration than P-l,
vP-1 and vP-1144, which, in turn, accumulated to higher
concentrations than P-4, vP-1114 and vP-4144. These data
demonstrate that P-l, P-4 and the chimeras accumulated to
different levels in vegetative tissues, but that the accumulation
of virus was not correlated with symptom induction. Therefore,
the symptoms induced were not merely a result of competitive
inhibition of host growth, but rather might have been due to an
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interference with host growth regulator metabolism and gene
regulation (Fraser et al., 1986; Wang & Maule, 1995).
Seed transmission of vP-1114, vP-1144, vP-4144
and vP-4111 in P. sotivum ~549'
The seed transmission frequencies of vP-1114, vP-1144,
vP-4144 and vP-4111 were determined in three experiments.
The absolute seed transmission frequency varied between
experiments, which was probably a result of seasonal changes
affecting greenhouse conditions. However, in all three experi-
ments, vP-1 was transmitted at the highest frequency, followed
by P-l, vP-1114, vP-1144, vP-4111 and vP-4144. P-4 was
transmitted in only one seed. Results of the combined data are
shown in Fig. 2 (b). The seed transmission frequency of vP-1
was higher than P-1 in all three experiments. The difference,
which was significant (P < 0"05), could be a result of the
maintenance of P-1 by repeated mechanical transfers without
selection for seed transmissibility. In contrast, vP-1 was derived
from a cDNA of viral RNA isolated from P-1 shortly after the
virus isolate was first recovered from an infected seed sample.
The seed transmission frequencies of the chimeric viruses
were intermediate between P-1/vP-1 and P-4. Of the chimeras,
vP-4144 was transmitted at the lowest frequency (slightly, but
not significantly, higher than P-4). The increasing contribution
of sequences from P-1 found in vP-4144, vP-1144 and vP-1114
correlated with increasing seed transmission, demonstrating
that seed transmission of PSbMV is influenced by determinants
contained in each of the exchanged genome fragments. The 5'
region (nt 1-2259) of P-4 appeared to have the strongest
influence on the seed transmission frequency, reducing the
seed transmission frequency of vP-4111 to 4"6%, compared
with 37% for vP-I.
The seed transmission frequency was not correlated to the
virus concentration in infected vegetative tissues (Fig. 2 b). This
is in agreement with results obtained by Wang et al. (1993)
who found no obvious relationship between virus content and
the efficiency of seed transmission of PSbMV in different pea
cultivars. Also, Ligat & Randles (1993) observed that repeated
transfers through seed in the cultivar 'Dundale' resulted in a
gradual reduction of PSbMV accumulation in vegetative tissue
to a level where it was not detectable by ELISA, while the seed
transmission frequency exceeded 90 %.
The percentage germination of seeds from infected plants
ranged from 88% (pP-1114) to 98% (pP-4II1). However,
there was no apparent correlation of seed viability with
symptom severity, virus accumulation or seed transmission
frequency.
Seed transmission of vP-1 (P-4 5'UTR), vP-1 (P-4
P1 pro) and vP-I (P-4 HCpro N) in P. sotivum 'Vedette'
As described above, the region of isolate P-4 from
nt 1-2259, which covers the 5'-untranslated leader (UTR), the
P1 protease (Plpro) and the N-terminal two-thirds of the
helper-component protease (HCpro ~¢) appeared to have a
major effect on seed transmission. Three recombinant, full-
length clones were therefore created in which the 5' UTR, the
Plpro and HCpro N of P-1 were exchanged with the cor-
responding region of P-4. Seed transmission of the resulting
chimeric viruses was assayed in 'Vedette', and the combined
data from experiments four and five are shown in Fig. 3 (b).
The seed transmission frequencies of vP-I(P-4 5'UTR) and
vP-I(P-4 HCpro N) were reduced to 50% and 20% of vP-1,
respectively, while vP-I(P-4 Plpro) was seed transmitted at
the same frequency as vP-1.
The 5' UTRs of P-1 and P-4 are 143 and 99 nt in length,
respectively, and there are 32 amino acid differences in the
HCpro N region between the two viruses. The 5' UTR is likely
to affect both translation and replication of the potyvirus
genome (Riechmann et al., 1992). The region of HCpro
contained in HCpro N affects aphid transmission, replication
(Atreya et al., 1992) and long-distance movement (Cronin et al.,
1995).
Wang & Maule (1994) demonstrated that the suspensor can
act as a channel for transmission of PSbMV to the embryo
proper. A prerequisite for embryo infection is, therefore, that
virus reaches the micropylar region of the testa before
disintegration of the suspensor. This suggests that the HCpro ~
region may affect replication and/or long-distance movement
in reproductive tissues and thus influence the number of
embryos which become infected and hence, the final seed
transmission frequency.
The observation that seed transmission is affected by
multiple determinants in the viral genome agrees well with the
results of W a n g & Maule (1994) who demonstrated that
resistance to seed transmission in P. sativum involves several
nuclear genes. This suggests that several virus-host factor
interactions determine the spread and accumulation of PSbMV
in the maternal testa tissue. Whereas PSbMV can enter the
embryo after fertilization (Wang & Maule, 1992), seed
transmission of BSMV depends mainly on the ability of the
virus to reach the reproductive tissues prior to fertilization
(Edwards, 1995). Despite this difference, replication and
movement also play central roles in seed transmission of
BSMV, the major determinants being the RNA~ 5' UTR, a 369
repeat in the 7a gene, and the 7b gene. Therefore, seed
transmission of both PSbMV and BSMV appears to be
determined by the ability to replicate and move in the
reproductive tissues and to reach the suspensor or mega-
gametophyte, respectively, before a certain critical point in
development.
We thank I. T. D. Petty for pT7E19(+) and Kristian Kristensen for
statistical advice and analysis.
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}15~
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Received 13 June 1996; Accepted 22 August 1996

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