W. Allen Miller, Gennadiy Koev, and B. R. Mohan
Iowa State University, Ames
!RE 4HERE 2ISKS
!SSOCIATED
WITH 4RANSGENIC
2ESISTANCE TO
,UTEOVIRUSES
Engineering crops for disease resistance
is one of the first and most successful examples of the application of plant genetic
engineering for crop improvement. Numerous, diverse approaches have been used
to genetically engineer virus resistance in
plants. These will not be reviewed in this
article; instead the reader is advised to read
other reviews (4,25,49,78). By far the most
common and successful general strategy
has been pathogen-derived resistance, in
which plants are transformed with a gene
or sequence from the virus. These plants
show varying levels of resistance, by a
variety of mechanisms, to the virus from
which the gene was derived. The two most
common viral genes used are those coding
for the coat protein and the RNA-dependent RNA polymerase. The polymerase, also
known as replicase, is the enzyme that
copies the viral genome. (Although the two
terms differ somewhat in meaning, for the
purposes of this review they will be used
interchangeably.) The coat protein gene
Dr. Miller’s address is: Plant Pathology Department, 351 Bessey Hall, Iowa State University,
Ames 50011; 515/294-2436, Fax: 515/294-9420,
E-mail: wamiller@iastate.edu, W3: http://www.
public.iastate.edu/~wamiller/
Publication no. D-1997-0523-04F
© 1997 The American Phytopathological Society
700
Plant Disease / Vol. 81 No. 7
tends to confer resistance to a broader
range of related viruses, but resistance is
often incomplete, with the plant showing
delayed and milder symptoms than the
untransformed plant. Resistance can be
overcome by extremely high levels of inoculum or by inoculation with naked viral
RNA (31). In contrast, polymerase
(replicase) gene-mediated resistance can
confer complete immunity, but only to
virus strains with very high sequence homology to the one from which the transgene was derived (28). Because transgenic
resistance using these two viral genes is the
most widely used, including against
luteoviruses, most of the discussion of risk
will deal with these two approaches.
Like many other major technological
advances, transgenic resistance to pathogens offers not only advantages but also
potential risks (71). These potential risks
have aroused controversy (18,35). We use
a simple definition of risk paraphrased
from Goy and Duesing (29): (probability
of an event occurring) × (potential damage
or loss resulting from the event) = risk.
Throughout the paper, we will distinguish
between probability of an event happening
and the potential damage, because they are
separate aspects of any interaction. This
review is predicated on the notion that (i)
comparison of luteoviral genome sequences, (ii) understanding the replication
mechanisms, (iii) observation of interac-
tions of luteoviruses with each other and
with their hosts, and (iv) observation of
cross-hybridization between luteovirus
host plants and weedy relatives allow prediction of potential risks in virus-derived
transgenic resistance strategies. We discuss
three categories involving risk. The first
comprises interactions that do not involve
any genomic rearrangements between the
transgene product and an invading virus.
The second category is recombination between the transgene and the invading virus
that can create new strains or viruses. The
third category is the potential escape of the
transgene via pollen to weedy relatives of
the transgenic host. Discussion of the first
two points is particularly relevant because
we will provide evidence that the probability of these events occurring may be
greater for luteoviruses than for most other
plant viruses.
Luteoviruses. Luteoviruses represent
one of the most economically significant
groups of plant viruses. The most-studied
members include barley yellow dwarf viruses (BYDVs), beet western yellows virus
(BWYV), and potato leafroll virus
(PLRV), all of which cause serious losses
on their hosts and are worldwide in distribution (44,47). Luteoviruses (62) are
phloem-limited, spherical viruses that often
cause yellowing, reddening, and/or stunting of their hosts (47) (Fig. 1). They are
not mechanically transmissible. Instead,
they are transmitted by aphids (Fig. 2) in a
circulative, nonpropagative manner only
by certain aphid species. Intimate interactions between luteoviruses and their vectors have co-evolved (59). The viral
genome consists of a single RNA molecule
that is about 5.5 kb long and codes for 5 to
6 proteins (46,48). Efforts are under way to
genetically engineer resistance to these
three viruses and to other luteoviruses such
as beet mild yellowing virus, cucurbit
aphid-borne yellows virus, and groundnut
rosette assistor virus.
Based on cytopathology, serology, and
genome sequences, luteoviruses have been
divided into two distinct subgroups. This
distinction is revealed when the genome
organizations of the subgroups are compared (Fig. 3). Two essential genes, those
coding for the RNA-dependent RNA polymerase and the coat protein, have very
different origins, depending on the subgroup (26,48). The polymerase genes of
subgroup I luteoviruses are more closely
related to those of the dianthovirus (e.g.,
red clover necrotic mosaic virus), umbravirus (e.g., carrot mottle virus), and carmovirus (e.g., carnation mottle virus) groups
than they are to genes of the subgroup II
luteovirus polymerases. The polymerases
of subgroup II luteoviruses are most
closely related to those of the sobemoviruses (e.g., southern bean mosaic virus).
Phylogenetic analyses reveal that the
RNA-dependent RNA polymerase genes of
subgroup I and subgroup II luteoviruses are
as different as such genes can get (40,80).
No other virus group in any kingdom has
such an extreme dichotomy in polymerase
gene origins (40,80). In contrast, the coat
protein genes of all luteoviruses are much
more closely related to each other than to
the coat protein genes of viruses in any
other group. The blue shading in Figure 3
demarcates the region of similarity between the subgroups. The other luteovirus
genes that share intragroup homology include an extension to the coat protein that
is probably required for aphid transmission
(AT, Fig. 3) (6,9) and possibly cell-to-cell
movement (MP?) (6), and an overlapping
gene (MP?, Fig. 3) that is required for
systemic infection by BYDV (9) but not by
BWYV (82). It is likely that these genes in
this common region confer on luteoviruses
their distinctive properties, including
icosahedral particle shape, circulative,
nonpropagative transmission by aphids,
serological cross-reactivity, and confinement to the phloem.
Interactions Between
Transgene Products and Virus
Heterologous encapsidation. Coat
protein–mediated resistance is probably the
most widely used form of virus-derived
transgenic resistance (4). This strategy has
been applied successfully against viruses
of many groups, including the luteoviruses
(38). The first virus-resistant transgenic
plant to be marketed was squash expressing coat protein of zucchini yellow mosaic
virus (2), and it is likely that coat protein–
mediated resistance will be used widely in
the near future. In addition to its obvious
roles in encapsidating and protecting the
viral genome, plant virus coat proteins can
Fig. 1. Luteovirus disease symptoms and synergistic interactions. H indicates uninoculated plants. (A) Natural field infection of
oats (Avena sativa) by unidentified strain(s) of barley yellow dwarf virus (BYDV). Severely infected plant on left; moderate
symptoms on right. Note the sterile florets (white, wispy “dead heads”) that produce no grain and the reddening or yellowing of
leaves. (B and C) Oats infected with different strains of RPV (R), PAV (P), or both (R+P) BYDVs. In panel B, the host is GAF/Park oats
on which the RPV-NY isolate is mild, and the PAV-IL isolate is so severe that the mixed infection is only slightly more severe than
PAV alone. In panel C, on an Australian cultivar, stunting is the most obvious symptom caused by Australian isolates, and the
mixed infection causes more stunting than either isolate alone (photo by P. M. Waterhouse). (D and E) Carrot motley dwarf disease
caused by mixed infection of carrot red leaf luteovirus and carrot mottle umbravirus (photos by B. W. Falk). (F) Shepherd’s purse
(Capsella bursa-pastoris) plants infected with a mild strain of beet western yellows virus (BWYV) (B) or the ST9 strain which
contains mild BWYV RNA plus ST9-associated RNA (B+ST9a) (photo by B. W. Falk).
Plant Disease / July 1997
701
affect important biological properties. The
coat protein plays a role in virus movement
within the plant, in manifestation of disease symptoms, and in determining aphid
transmission properties. Does expression
of a viral coat protein in transgenic plants
pose a risk? One possibility is heterologous
encapsidation (Fig. 4). If a transgenic plant
expressing a coat protein of virus A becomes infected with virus B, there is a
chance that genomic RNA of virus B can
get encapsidated in the transgenically expressed virus A coat protein and thereby
acquire characteristics determined by virus
A coat protein, including the ability to
move in the plant or altered vector specificity. Examples that support this scenario
are known. When coat protein–defective
mutants of tobacco mosaic virus (TMV)
were complemented by the coat protein
produced in transgenic tobacco plants, the
mutant acquired the ability to spread systemically in the plant, which the nonencapsidated mutant virus was unable to do
alone (55). Also, an aphid-nontransmissible mutant of zucchini yellow mosaic virus
acquired aphid transmissibility due to heterologous encapsidation upon inoculation
to a plum pox virus coat protein–expressing plant (42). Heterologous encapsidation
Fig. 2. Rhopalosiphum padi (oat bird-cherry aphid), the vector for PAV and RPV barley
yellow dwarf viruses.
Fig. 3. Genome organizations of luteovirus subgroups. Representative members of
each subgroup are listed below each map. Solid black lines represent the viral
genomic RNA. Boxes indicate genes. Blue shading, genes with sequence similarities
between subgroups; yellow, sequence similarity to umbra-, diantho-, and carmoviruses; green, sequence similarity to sobemoviruses. POL, RNA-dependent RNA polymerase; PRO, putative protease; CP, coat protein; MP?, putative movement protein;
AT, read-through domain of the coat protein gene required for aphid transmission.
BYDV, barley yellow dwarf virus; SDV, soybean dwarf virus; SCRLV, subterranean
clover red leaf virus; BWYV, beet western yellows virus; BMYV, beet mild yellowing
virus; CABYV, cucurbit aphid-borne virus; PLRV, potato leafroll virus; GRAV, groundnut rosette assistor virus; CRLV, carrot red leaf virus.
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Plant Disease / Vol. 81 No. 7
was also observed in transgenic potato
plants expressing the coat protein gene of
the O strain of potato virus Y (PVY O) upon
infection with the N strain of the same
virus (PVYN) (20). In natural and laboratory settings, heterologous encapsidation
has been observed in mixed luteovirus
infections (barley yellow dwarf luteoviruses) which resulted in altered vector
specificities (64,77). The numerous and
widespread examples of heterologous encapsidation interactions among luteoviruses suggest that this may be a natural
determinant of luteovirus epidemiology
(65). Thus, it has been speculated that
transgenic heterologous encapsidation
interactions might occur between a genome
of an infecting luteovirus (or other virus)
and the transgenically expressed coat protein of another luteovirus or serotype (Fig.
4).
The risk of transgenic heterologous encapsidation is extremely low, in both the
probability and potential damage variables
of the risk equation. Regarding probability,
in all reported cases of transgenic expression of luteovirus coat protein, the level of
coat protein is so low as to be undetectable
or nearly so (3,38,39). The coat protein
produced by the invading virus would be
orders of magnitude greater in concentration and would thus overwhelmingly favor
encapsidation in the invading virus’s own
coat protein. Secondly, it is possible that
the coat protein does not confer all of the
aphid specificity determinants. A lowabundance form of the coat protein that
contains a long extension at one end, produced by read-through of the stop codon
during translation, is probably required for
aphid transmission (6,10,21,36,75) (Fig.
1). The extended region may also confer
vector specificity. This extended form has
not been used in constructs employing coat
protein–mediated resistance. Thus, any
heterologously encapsidated RNA may not
acquire the vector specificity of the virus
from which the transgene is derived. Even
if the invading RNA did acquire new vector specificity from the transgenic coat
protein, the potential damage is low; the
heterologously encapsidated virus could be
transmitted only once by the new aphid
vector because the genetic material (RNA)
in the heterologously encapsidated virus
still encodes its own vector specificity. The
spread would be limited to a rare transmission event (which could happen anyway
because aphid vector specificity is not
absolute) within the field of transgenic
plants and adjacent plants. Additional research is forthcoming to determine whether
vector specificity is determined by the coat
protein or by the fused read-through protein. Future research strategies could then
further minimize risk by deleting or altering the vector transmission determinant(s)
in the transgenic coat protein gene.
An intriguing new perceived risk has
come to light with the observation that
mixed infections of potato spindle tuber
viroid (PSTVd, an infectious RNA with no
coat protein) and PLRV can result in encapsidation of PSTVd RNA in PLRV virions and subsequent aphid transmission of
PSTVd (61). This could potentially increase the spread of PSTVd, which has no
natural vector and is transmitted only by
mechanical means. However, the encapsidation is highly inefficient, with one
PSTVd molecule encapsidated for every
3,000 to 5,000 PLRV genomes (61). Furthermore, due to the nearly undetectable
levels of transgenically expressed coat
protein and to the absence of the coat protein read-through domain, such an event
need not be considered a significant risk.
Synergistic interactions. Of greater
concern should be the possibility for synergistic interactions between the virusderived transgene product and a challenging virus. In certain combinations, mixed
infections of two viruses produce symptoms much more severe than those caused
by either of the viruses alone. Generally,
these viruses are unrelated or distantly
related, as closely related viruses tend to
cross-protect against one another (24).
Such synergisms are quite common among
luteoviruses and their relatives.
As previously described, luteoviruses
can be divided into two subgroups based
on the homologies of the polymerase genes
(Fig. 1). This dichotomy can be extended
to related luteo-like viruses and RNAs.
These include the umbraviruses, the enamoviruses, and the ST9-associated (ST9a)
RNA that is associated with the ST9 isolate
of BWYV. ST9a RNA enhances BWYV
replication and greatly exacerbates disease
symptoms (Fig. 1F) (19). This RNA codes
for a subgroup I–like polymerase (11) and
can replicate autonomously in laboratory
experiments (56), but it lacks genes for
many luteoviral functions, including a coat
protein. The coat-proteinless umbraviruses
also have subgroup I–like polymerase
genes, are capable of autonomous replication, and are invariably found associated
with subgroup II luteoviruses, upon which
they depend for aphid transmission (54).
Carrot motley dwarf disease results from a
mixed infection of carrot mottle umbravirus and carrot red leaf luteovirus (Fig. 1D
and E). The only known enamovirus is pea
enation mosaic virus (PEMV). PEMV
contains two RNAs, each of which can
replicate autonomously in plant cells, but
which depend on each other for cell-to-cell
movement and encapsidation functions
(13). All of these viruses and RNAs have
been found in various pairs in which an
RNA coding for a subgroup I–like RNA
polymerase enhances replication of an
RNA coding for a subgroup II–like polymerase which, in turn, benefits the subgroup I–like RNA (Table 1). These viruses
and RNAs seem to represent an evolutionary continuum ranging from (i) luteoviruses that replicate on their own but repli-
cate even better when combined with a
synergistic partner of the other subgroup,
to (ii) symbiotic RNAs that rely on a
luteovirus for a function such as encapsidation and in exchange somehow enhance
the luteovirus’s accumulation (ST9a RNA
and umbraviruses), to (iii) two luteoviruslike RNAs that have become so interdependent that they have become a single
bipartite virus (PEMV).
Can individual viral transgenes that confer resistance to one virus act synergistically with unrelated infecting viruses to
exacerbate symptoms? This is not yet
known for luteoviruses, but the work of
Vance et al. (74) demonstrates clearly that
this can occur for a different set of synergistically interacting viruses. The mixed
infection of two unrelated viruses, potato
virus X (PVX, a potexvirus) and a potyvirus such as potato virus Y (PVY), tobacco
vein mottling virus (TVMV), or tobacco
etch virus (TEV), is more severe in to-
bacco plants than infection by either virus
alone (74). Transgenic plants expressing a
specific portion of either the TEV or the
TVMV genome showed the severe symptoms characteristic of the synergistic mixed
infection upon inoculation with PVX
alone. Thus, plants expressing a transgene
from one virus actually were more severely
affected when infected by an unrelated virus.
This type of risk could be controlled by
simply removing the transgenic crop variety from production. However, the consequences are by no means trivial. The widespread use of crop plants that contained a
susceptibility gene analogous to the described synergy events led to one of the
worst epiphytotics in U.S. history. The
1970 southern corn leaf blight epidemic
resulted from millions of acres being
planted with corn containing the T-cytoplasm, which confers cytoplasmic male
sterility (cms). Unfortunately, the same
gene that confers cms (t-urf13) also con-
Fig. 4. Potential heterologous encapsidation of an invading virus by a transgenically
expressed coat protein. Colored lines represent RNA of invading virus (green) or
transgenically expressed coat protein message (red). Ribosomes (gray spheres)
translate coat protein mRNA (colored lines) to produce coat protein subunits (colored
spheres), which assemble on viral RNA to form virions shown at the bottom. As an
example, subgroup II barley yellow dwarf virus (BYDV)-RPV (green) could probably
infect a plant transformed with subgroup I BYDV-PAV coat protein gene (red), because
coat protein amino acid sequences of these viruses are only about 50% identical. If an
RPV RNA was encapsidated in enough PAV coat protein, it could acquire the ability to
be transmitted by the English grain aphid, Sitobion avenae, which is a vector for PAV
but not for RPV. Drawing indicates how coat protein expressed from abundant replicating viral mRNA would be predicted to accumulate at much higher levels than
transgenically expressed coat protein. Thus, phenotypic mixing and transcapsidation
with significant levels of transgenic coat protein would be rare (indicated by <).
Plant Disease / July 1997
703
fers high susceptibility to the T-toxin of
Cochliobolus heterostrophus (60). Favorable weather conditions allowed the pathogen to spread across the Midwest, devastating crops. The problem was “solved” by
discontinuing the use of T-cytoplasm
maize. Theoretically, a similar type of
event could occur if millions of acres were
planted with any crop expressing a viral
gene that confers extra susceptibility to an
unanticipated virus or other pathogen. It
should be noted that the southern corn leaf
blight epidemic actually demonstrates the
risks that can arise even with nontransgenic crop plants when a single genotype is
planted too widely.
What about luteoviruses? The region of
the potyviral genome that conferred synergy did not include the polymerase gene
(74). In contrast, the one feature common
to the luteoviral synergistic interactions is
the paired polymerases of divergent origin
(Table 1). Thus, the most likely candidate
gene for the synergy is the polymerase
gene. Because the most extreme transgenic
resistance to plant viruses, including
luteoviruses (72), is in transgenic plants
encoding the polymerase (28), synergistic
interactions between a polymerase transgene derived from a luteovirus of one subgroup and an invading virus from the other
subgroup may be possible. However, as
described below, these risks can be minimized.
New pathogenic RNAs? Transgenic
plants expressing the viral replicase present
a host of fascinating possibilities in addition to the synergism described above. For
example, many otherwise nonviable deletion mutants of the viral genome could
now be replicated and thus be pathogenic.
The replicase (expressed as a fusion of
open reading frames [ORFs] 1 and 2, Fig.
1) is the only viral gene product needed for
luteoviral RNA replication in plant cells
(51,63). The termini of the viral genome
are also needed, presumably because they
contain the replication origins. Owing to
recombination and the high mutation rate
in RNA replication, many RNA molecules
in a given virus infection are likely noninfectious, but just “go along for the ride”
and get copied if they contain appropriate
replication origins (14). Such defective
interfering (DI) RNAs have been found in
the tombusvirus group (34), which is related to luteovirus subgroup I. They
usually attenuate, but can exacerbate (43)
disease symptoms. Transgenic plants expressing the replicase in every plant cell
could replicate these defective RNAs and
greatly increase the effective population of
viable RNAs. This has been demonstrated
in transgenic plants expressing a tombusvirus replicase (66). Taking this one step
further, it is conceivable that a functional
replicase in an uninfected transgenic plant
may copy host RNAs with some very low
efficiency in the absence of the much more
competitive natural viral template. Given
enough time, these host-derived RNAs
could evolve into efficient templates. This
resembles the proposed origin of variant
subviral bacteriophage RNAs (73) and of
plant satellite RNAs (23). These RNAs
could be new pathogens specific for the
transgenic plants, but could also be acquired by an infecting virus. Acquisition of
new, replicating RNAs by a virus could
effectively generate new virus strains.
Steps to avoid synergistic and related
risks. The possibility that a luteovirus-
derived transgene that confers resistance to
one virus would confer enhanced susceptibility to its synergistic partner virus is real,
but it may depend on the gene used to confer resistance. It is unlikely that the coat
protein is involved directly in synergy
because several of the subgroup I–like
RNAs (e.g., ST9a RNA) that enhance subgroup II RNA (e.g., BWYV RNA) replication lack coat proteins. In contrast, polymerase-mediated resistance could pose a
risk if the above hypothesis proposing
involvement of this gene product in synergy is correct or if the scenario of RNA
amplification occurs. Unfortunately from
this viewpoint, in the case of PLRV, transgenic plants expressing the full polymerase
gene showed far more complete resistance
than did those transformed with the coat
protein gene (72). On the bright side, resistance in transgenic plants transformed
with the coat protein or polymerase genes
often is not due to the gene product, but
rather is due to an unsolved mechanism in
which presence of the transgenic RNA
somehow suppresses both its own accumulation and that of the invading virus
(52,69). These plants express very little
viral transgene product and thus would not
be likely to interact synergistically with an
invading virus. If it turns out that this
mechanism applies to luteoviruses, the
possibility of polymerase-mediated synergistic interaction would be very remote.
Another safety valve would be to mutate or
truncate the polymerase transgene so that
the encoded protein is inactive in replication but the gene still confers resistance.
This has been achieved for several viruses
(7,28,45).
Recombination
Table 1. Continuum of synergistically interacting pairs of viruses and RNAa
Polymerase homology
Subgroup II
(-like)
Subgroup I
(-like)
Host
BYDV-RPV
BYDV-PAV
Cereals
BWYV
ST9a RNA
CRLV
CMoV
(umbra)
Beet, lettuce,
Shepherd’s purse,
others
Carrot
GRAV
GRV
(umbra)
Groundnut
(peanut)
PEMV RNA1
(enamo)
PEMV RNA2
(enamo)
Legumes
a
704
Effect
More severe stunting, yellowing,
reddening, sterility (Fig. 1). Higher
virus titer
More severe stunting, necrosis in
Shepherd’s purse (Fig. 1). Higher
virus titer (19)
Carrot motley dwarf disease (Fig. 1).
More severe symptoms, aphid
transmission of CMoV (76)
GRAV alone is symptomless.
Presence of GRV and its satellite
RNA causes rosette symptoms (53).
GRAV allows aphid transmission of
these RNAs
Ability to infect plants. These are two
components of a bipartite virus, but
each RNA is capable of independent
replication in plant cells (13)
Virus group assigned to the nonluteovirus or RNA is in parentheses. BYDV, barley yellow
dwarf virus; BWYV, beet western yellows virus; CRLV, carrot red leaf virus; CMoV, carrot
mottle virus; GRAV, groundnut rosette assistor virus; GRV, groundnut rosette virus; PEMV,
pea enation mosaic virus.
Plant Disease / Vol. 81 No. 7
Recombination is the joining or splicing
together of two separate RNA molecules to
create a molecule with a new sequence. In
theory, recombination between a transgenically expressed mRNA with a luteoviral
sequence and the genomic RNA of an infecting virus could create a new viral RNA
with new properties. Such events have
been observed for other viruses. Transgenic Nicotiana benthamiana plants
expressing a portion of the coat protein
gene of cowpea chlorotic mottle virus were
inoculated with a mutant form of the virus
in which a part of the coat protein gene
corresponding to the transgene was deleted
(30). Progeny virus was recovered in
which the coat protein gene defect was
repaired by recombination with the
transgenic RNA. Some of the progeny had
mutations that allowed them to cause more
severe chlorosis in indicator plants than did
the parental virus (1). In a second example,
transgenic tobacco (Nicotiana bigelovii)
plants expressing gene VI from a strain of
cauliflower mosaic virus (CaMV) capable
of infecting N. bigelovii were inoculated
with a different strain of CaMV that was
unable to infect nontransgenic N. bigelovii
(67). Two weeks after inoculation, the
originally nonpathogenic strain acquired
the ability to accumulate in the transgenic
N. bigelovii plants because it acquired the
transgenic gene VI by recombination. The
recombinant progeny virus could now
infect nontransgenic N. bigelovii plants,
and it caused new, including more severe,
symptoms in some hosts (67). The ability
of this virus to acquire a new host range
and symptomatology may be a cause for
concern. However, the investigators think
that the probability of recombination was
much greater for the cauliflower mosaic
virus group (caulimoviruses) than for other
groups because the replication enzymes
must perform a recombination-like event
during normal caulimoviral genome
replication.
Falk and Bruening (18) argued that recombination between viruses has occurred
throughout evolutionary history and that
natural selection has generated the most fit
viruses. All the recombination events described occurred under strong artificial
selective pressure favoring the recombinants. In the field, this is less likely to be
the case. Thus, any new viruses generated
by recombination with transgenes would
be selected against, unable to compete with
“natural” viruses that have been honed by
millions of years of evolution. However,
agricultural practices generally are not at
evolutionary equilibrium. Thus, the
chances of new viruses being generated
and spreading, at least transiently, are
worth considering.
How do luteoviruses measure up in
terms of the probability of recombination
occurring? We predict that it is more likely
than for most viruses, based on the clear
evidence that recombination has occurred
more recently in the evolution of luteoviruses than in other plant viruses (26,48).
The probable sites of recombination are the
regions of the genome sequence at which
the similarity between subgroups begins
and ends (Fig. 5). It turns out that these
boundaries, one located between the polymerase (POL) and coat protein (CP)
genes and another located after the aphid
transmission gene (AT), coincide with the
start sites for synthesis of subgenomic
mRNAs (48). A half-genome-length subgenomic mRNA is the message for translation of the coat protein and neighboring
genes (Fig. 5, in blue) of all luteoviruses,
and a smaller one may serve for translation
of the smaller gene near the end of the
genome of BYDV-PAV. During replication,
the viral polymerase not only copies the
whole genome, but also synthesizes these
subgenomic mRNAs. Based on work on
other viruses (15), we propose that it
recognizes a specific RNA sequence, binds
the RNA there, and then begins copying to
make new RNA. Such recognition sites are
likely to be located at the end of the
genomic RNA for full-length genome rep-
lication and also internally at the subgenomic mRNA start sites. Indeed, a conserved nucleotide sequence is present at
these locations (48).
We propose that, occasionally during
evolution, mixed infections occurred in
which the replicase fell off in the process
of copying a template RNA. Then, without
releasing the nascent strand, it bound the
RNA of another virus at the subgenomic
mRNA promoter sequence for which it has
affinity. After this strand switch, it would
resume copying until it completed the new
template, resulting in a hybrid virus (Fig
5). The subgenomic mRNA start sites of
subgroup I–like dianthoviruses and subgroup II luteoviruses are known to have
homology (48,81), so the model in Figure
5A is particularly feasible. This would
create a hybrid virus with a polymerase
gene of a dianthovirus and the coat protein
and neighboring genes of a subgroup II
luteovirus. After a subsequent recombination occurring downstream of the aphid
transmission gene at the next subgenomic
RNA promoter, the resulting progeny
Fig. 5. Model for origin of luteovirus subgroups. Solid black lines represent viral
genomic RNA. Dashed lines indicate subgenomic RNAs. Boxes indicate genes. Blue
shading, genes with sequence similarities between subgroups; yellow, sequence
similarity to umbra-, diantho-, and carmoviruses; green, sequence similarity to sobemoviruses. Gray boxes represent putative origins of replication and subgenomic
mRNA promoters. POL, RNA-dependent RNA polymerase; PRO, putative protease; CP,
coat protein; MP?, putative movement protein; AT, read-through domain of the coat
protein gene required for aphid transmission. Pink line shows the proposed path of
the replicase as it switched strands during copying of viral RNAs in a mixed infection.
Plant Disease / July 1997
705
would be a subgroup I luteovirus. A reciprocal recombination between a sobemovirus similar to cocksfoot mottle virus
(CfMV) and a subgroup I luteovirus would
require only a single crossing over event
followed by premature termination to generate a subgroup II luteovirus.
Recombination at the predicted sequences has not been observed directly in
luteoviruses but has occurred between an
invading virus and a transgene of a closely
related virus. Tobacco plants were constructed to express RNA2 of red clover
necrotic mosaic dianthovirus (RCNMV)
lacking its 5′ terminus, including the proposed replication origin that is shared with
subgroup II luteoviruses. After inoculation
of RNA1, which is not infectious on its
own, infectious progeny viruses were obtained in which recombination allowed
transgenic RNA2 to acquire the putative
origin of replication sequence either from
the end of RNA1 or from a sequence in the
middle of the RNA (79), both of which
have sequence homology to the predicted
origin (48,81). The original transgenic
RNAs had the 3′ replication origin, which
may have allowed replicase access to facilitate the recombination event. Thus, the
risk could be greatly reduced by ensuring
that no replication origins are included in
the transgene. To aid in this design, our lab
is in the process of identifying replicase
recognition sites.
Transgene Escape
Many cultivated plants have relatives
that are weeds. Some of these crop species
can cross-pollinate with their weedy relatives and thereby exchange genetic information. Thus, there is a possibility that
viral genes that confer resistance on transgenic crops might escape with the pollen
and end up in weeds. The transgene transfer probability is determined by the biological characteristics of the species,
whereas the consequences are a subject for
speculation. Although pollen escape is not
an environmental risk peculiar to transgenic plants (37) or to luteovirus hosts, the
fact that some plants from the luteovirus
host range readily cross-pollinate with their
wild relatives should be considered. For
instance, important hosts of BWYV, lettuce
(Lactuca sativa), and cultivated beets (Beta
vulgaris) have weedy relatives with which
they can cross-pollinate (5,37). On the
other hand, all weedy relatives with which
cultivated beets can hybridize are from the
same species, Beta vulgaris, which does
not seem to serve as a virus reservoir (17).
There is also a possibility of transgene
escape from transformed, PLRV-resistant
potatoes to some wild species of the family
Solanaceae. Twenty-four species have been
identified that can successfully cross with
seven potato cultivars (32). However, the
closest wild potato relatives in Europe
would be highly unlikely to be fertilized
with potato pollen (16). Regarding hosts of
BYDVs, related weeds also exist. For cultivated oat (Avena sativa), the outcrossing
rate with its wild relative A. sterilis has
been shown to vary from 1.8 to 8.7% (68).
We transferred transgenes from A. sativa to
the weed A. fatua by manual pollination of
emasculated A. fatua plants. Fertilization
and germination rates were very low, but
the A. sativa × A. fatua hybrid plants that
were recovered were transgenic and fertile
(G. Koev, unpublished). Whether such an
outcrossing event is likely to occur in the
field in this self-pollinating species remains to be determined. Another BYDV
host, cultivated rice, was found to hybridize with the weed red rice at rates between
1 and 52%, depending on the cultivar, under fairly natural conditions (41).
With luteoviruses, there are two potential scenarios. First, having acquired genes
for resistance, the weeds could become
more of a problem if the virus had been a
natural limiting factor for their growth.
There is no evidence to support this. On
the other hand, resistant weeds could reduce the natural reservoir of the virus and
lower the infection pressure on the crop.
BYDVs can infect a wide range of host
plants in the Poaceae. However, the importance of wild relatives as an inoculum
resource has not been clearly determined
for BYDVs. Some scientists do not con-
An advertisement appears in the printed journal in this space.
706
Plant Disease / Vol. 81 No. 7
sider weedy Poaceae species an important
reservoir of the virus, at least not in North
America (33). However, adjacent reservoirs have been reported to be important
sources for infection by BYDV (8). Recent
field surveys suggest that wild grasses
have a very high incidence of BYDVs, but
infection is often symptomless (58). Thus,
wild species are at least tolerant of BYDV.
In fact, some wild Poaceae such as Thinopyron (Agropyron) and others have been
used as resistance sources for cereal
breeding (12). Because virus infection can
positively or negatively affect seed production of wild grasses (58), the escape of
a transgene that confers high resistance or
immunity to BYDV could affect the ecology of native grasses and weeds if it were
to spread through the population. This
could also reduce the reservoir of BYDV
that could serve as an inoculum source.
Conclusions
Most of this article is speculative because more knowledge is needed. Areas of
research that would be particularly relevant
include the following:
1. Identification of the sequences in the
coat protein and aphid transmission
genes that are required for aphid transmission and that confer vector specificity would be valuable in predicting the
likelihood of heterologous encapsidation
conferring new vector specificity. If
such regions could be omitted while still
conferring resistance, the probability
would be reduced.
2. Identification of the viral genes that
confer the ability to synergistically enhance accumulation of other virus(es)
would be important to avoid or predict
the possibility of particular invading viruses causing more severe symptoms.
3. In the established and soon-to-be constructed transgenic, luteovirus-resistant
plants, it would be valuable to determine
whether the resistance is conferred by
the “homology-dependent” suppression
identified by Mueller et al. (52). This
would be indicated if resistant lines
show less transgene RNA accumulation
than do some more susceptible transgenic lines, and if low RNA accumulation and resistance co-segregate in a
dominant fashion. If so, one of the most
exciting fundamental questions in plant
molecular biology should be asked:
what is the mechanism of this homology-dependent, RNA-based suppression? This is an example of a plant
pathology study that may lead to the
discovery of new mechanisms of gene
expression regulation in general.
4. A better understanding of the mechanism of replication is essential to allow
prediction of recombination probability.
Mapping the origins of replication and
the subgenomic mRNA promoters
would be a good start. Such sequences
5.
6.
7.
8.
should be avoided in transgenic plants.
A more empirical approach of seeking
recombination hot spots is to simply sequence more luteovirus isolates. This
has allowed identification of recombination sites not predicted by our subgenomic promoter model (27). This information is also useful to get an idea of
how much variation exists in the field.
This would allow more informed predictions of the effectiveness of a particular transgene and of the probability
of a more distantly related luteovirus
being available to act synergistically or
recombine with the transgene.
Actual observation of recombination
between virus and transgene, such as the
work begun by Xiong et al. (79) with
the related dianthoviruses, is obviously
valuable. An intriguing extension of this
work would be to look for recombination between viruses of different groups
to test the models in Figure 5.
Outcrossing rates of luteovirus hosts
and weedy relatives under field conditions must be determined. Currently,
only anecdotes of such events exist for
most luteovirus hosts. Data that would
allow estimates of probability of hybridization occurring are insufficient.
Ecological effects of resistant weeds
need to be tested. If the probability of
transgene escape is determined, then the
potential consequences of such an event
must also be determined in a reasonably
quantitative way.
Finally, despite all possible predictions
and knowledge, we will never fully
know the risks until transgenic crops are
planted widely. Events too rare to detect
in lab or field trials may show up only
after many years of planting millions of
acres. Thus, we must continue to be
aware of the possibility that unexpected
diseases or weeds that arise from time to
time may be a consequence of transgenic, pathogen-derived resistance. If
the suggested areas of research have
been pursued in advance, an explanation
for and thus a means of prevention of
such a transgene-induced outbreak could
be applied quickly.
In addition to the measures we described
to avoid risks of pathogen-derived resistance, other novel strategies could be tried
to induce transgenic resistance, for example, expression of antiviral antibodies in
plants (70) or the use of human antiviral
pathways such as the interferon-induced
RNase L system, which has been adapted
to plants (50). Engineering aphid resistance
would effectively provide resistance to
luteoviruses. However, these approaches
may pose their own new risks and, being
less developed, are likely to be available
only in the next generation of transgenic
resistant plants.
Finally, although this review is on the
risks and not the benefits of transgenic
resistance, we would like to finish with a
brief but very important mention of the
latter. We think that virtually all of the
above scenarios have a very low probability and would cause less damage than if no
transgenic resistance is pursued. Few natural resistance genes to luteoviruses exist.
Current means of controlling luteoviruses
involve eliminating reservoir species and
aphicidal spraying, which is expensive and
more harmful to the environment than the
risk scenarios. Incidents of waterfowl kills
owing to insecticide spraying in order to
control aphids in wheat have been documented (22). The more common alternative to pesticides is simply living with
occasional luteovirus outbreaks. Thus, in
the present situation, the probability of
either insecticide use or luteovirus infection is high. The damage of either is evident. The worst consequences of transgenic
resistance discussed in this article would
be less damaging than the luteovirus outbreaks we currently live with. Thus, the
risks would certainly be outweighed by the
benefits and, in most cases, readily controlled. This will be tested soon. NatureMark’s NewLeaf potatoes (developed by
Monsanto Company) with combined replicase-mediated resistance to PLRV (72)
and Bt-resistance to Colorado potato beetle
are being marketed with the promise of
greatly reduced pesticide inputs (57).
Acknowledgments
We thank Bryce Falk for valuable comments on
the manuscript, Peter Waterhouse and Bryce Falk
for providing photographs, and Zhongguo Xiong
for providing unpublished data. This work was
funded by USDA Risk Assessment Research
Grants Program, grant no. 94392100531. This is
paper J-17141 of the Iowa State University Agricultural and Home Economics Experiment Station
Project 3270 and is supported by Hatch Act and
State of Iowa funds.
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translation mechanisms, and transgenic resistance in oats.
Mr. Koev is a graduate student in the
Plant Pathology Department of Iowa
State University. In 1994, he received
his Specialist degree at the National
Agricultural University, Kiev, Ukraine,
majoring in plant protection. The same
year he started his M.S. program at
Iowa State in the laboratory of W. Allen
Miller. His research focused on
characterizing transgenic oats for
resistance to BYDV and assessing
potential environmental risks, including
transgene
escape
and
possible
synergistic interactions. Mr. Koev
completed his M.S. studies in 1996 and
began working toward his Ph.D. in
plant pathology with Dr. Miller, studying
the replication of BYDV RNA.
Mr. Mohan is a graduate student
working toward a Ph.D. degree in
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of Agricultural Sciences, Bangalore,
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