Page
1
of
17
VIRAL
INTERACTIONS
IN
VIRAL
COAT
PROTEIN
TRANSGENIC
PLANTS:
A
LITERATURE
REVIEW
Virus
infection
is
a
serious
problem
in
agricultural
production.
Virtually
every
plant
species
is
susceptible
to
infection
by
at
least
one
of
more
than
500
known
plant
viruses
(
Waterhouse
et
al.
2001).
Plant
viruses
create
economic
losses
for
a
vast
variety
of
crops
by
reducing
yields
and
negatively
affecting
the
quality
of
the
crop
(
Tolin
1991).
Growers
may
need
to
use
several
control
methods
during
a
crop
season
in
an
attempt
to
prevent
viral
infection
and
dissemination,
primarily
by
planting
virus­
free
material
for
mechanically
transmitted
viruses.
For
vectortransmitted
viruses,
control
measures
have
often
focused
on
chemical
insecticides,
fungicides,
and
nematicides
to
reduce
the
population
of
vectors
that
transmit
viruses
from
plant
to
plant.
However,
control
of
vectors
is
not
always
feasible
or
effective
as
a
way
to
control
virus
transmission
(
OECD
Environment
Directorate
1996).
In
another
common
control
strategy,
plants
are
infected
with
a
mild
form
of
a
virus
to
confer
resistance
to
a
more
virulent
form.
This
method
has
serious
limitations
as
well.
In
some
cases,
the
development
of
resistant
cultivars
can
be
the
only
viable
means
of
virus
control.
Plants
developed
through
conventional
breeding
techniques
offer
some
degree
of
virus
resistance.
However,
breeding
for
resistance
has
not
been
successful
for
the
majority
of
field
crops
that
are
severely
affected
by
viruses
(
Tolin
1991).
More
recent
transgenic
methods
for
creating
resistant
varieties
incorporate
viral
DNA
into
the
plant's
genome
to
confer
effective
resistance
to
infection
by
that
virus
as
well
as
others
of
similar
sequence.

Many
different
types
of
transgenes
have
been
used
to
confer
resistance
to
viral
infection
including
viral
replicase,
movement
protein,
and
nuclear
inclusion
genes
as
well
as
nonviral
sequences
from
a
variety
of
species
(
Tepfer
2002).
However,
the
most
common
type
of
transgenes
used
to
confer
virus
resistance
are
viral
coat
protein
(
VCP)
genes
(
White
2000).
VCPs
encapsidate
the
viral
nucleic
acid
and
are
thought
to
be
important
in
nearly
every
stage
of
viral
infection
including
replication,
movement
throughout
an
infected
plant,
and
transport
from
plant
to
plant
(
Callaway
et
al.
2001).
Given
their
central
role
in
viral
infection,
the
interaction
of
a
VCP
transgene
with
the
genome
of
an
invading
virus
could
potentially
exacerbate
viral
disease
through
a
change
in
the
disease
characteristics
or
transmission
properties
of
that
virus.
Continued
development
and
testing
of
new
VCP­
transgenic
plants
has
facilitated
evaluation
of
such
potential
impacts
in
the
18
years
since
the
first
one
was
created
(
Abel
et
al.
1986).
This
review
evaluates
the
current
scientific
understanding
of
the
potential
risks
associated
with
viral
interactions
in
VCP­
transgenic
plants.

Viral
Interactions
Mixed
viral
infections
can
be
extremely
common
in
crops
and
other
plants
(
for
review
see
Hammond
et
al.
1999).
In
natural,
mixed
infections,
viral
genomes
from
different
strains
and/
or
different
species
simultaneously
infect
the
same
plant
and
thus
have
opportunities
to
interact.
In
spite
of
many
opportunities
for
interaction
in
nature,
such
events
rarely
lead
to
any
detectable
adverse
outcome
(
Falk
&
Bruening
1994).
However,
such
in
planta
interactions
do
have
the
potential
to
result
in
a
virus
that
causes
increased
agricultural
or
other
environmental
damage.
For
example,
the
epidemic
of
severe
cassava
mosaic
disease
in
Uganda
is
thought
to
be
due
to
the
combination
and/
or
sequential
occurrence
of
several
phenomena
including
recombination,
Page
2
of
17
psuedorecombination,
and/
or
synergy
among
cassava
geminiviruses
(
Pita
et
al.
2001).
VCPtransgenic
plants
potentially
raise
concerns
because
every
virus
infection
is
essentially
a
mixed
infection
with
respect
to
the
CP
gene
(
de
Zoeton
1991).
The
pertinent
question
is
whether
the
risks
associated
with
VCP­
transgenic
plants
may
be
either
greater
in
degree
or
different
in
kind
than
are
presented
by
natural,
mixed
infections
(
Tepfer
2002).

Recombination,
heterologous
encapsidation,
and
synergy
are
discussed
individually
to
evaluate
each
viral
interaction
as
it
occurs
under
natural
conditions,
its
potential
to
occur
in
VCPtransgenic
plants,
and
ways
that
the
frequency
of
its
occurrence
could
be
reduced
if
warranted.
Then
the
limited
number
of
field
evaluations
of
viral
interactions
in
VCP­
transgenic
plants
are
discussed.
Finally,
the
ecological
significance
of
these
studies
is
put
into
context
by
considering
whether
viral
interactions
in
transgenic
plants
could
occur
at
an
increased
frequency
or
be
unlike
those
that
occur
in
natural,
mixed
infections.

Recombination
Recombination
under
natural
conditions
Recombination
plays
a
significant
role
in
virus
evolution.
Evidence
of
past
recombination
having
led
to
the
creation
of
new
DNA
and
RNA
viruses
has
been
found
in
a
number
of
different
groups
including
bromoviruses
(
Allison
et
al.
1989),
caulimoviruses
(
Chenault
&
Melcher
1994),
luteoviruses
(
Gibbs
&
Cooper
1995),
nepoviruses
(
Le
Gall
et
al.
1995),
cucumoviruses
(
Masuta
et
al.
1998),
and
geminiviruses
(
Pita
et
al.
2001;
Zhou
et
al.
1997).
Sequence
analysis
of
viruses
from
the
family
Luteoviridae
indicated
that
this
family
has
evolved
via
both
intra­
and
interfamilial
recombination
(
Moonan
et
al.
2000).

The
propensity
to
recombine
and
thus
the
significance
of
recombination
varies
within
and
across
virus
groups
(
Worobey
&
Holmes
1999),
possibly
due
to
the
degree
of
dissimilarity
the
viral
replicase
can
tolerate
(
Hammond
et
al.
1999).
Sequence
analysis
of
the
CP
and
3'
untranslated
region
(
UTR)
of
109
potyvirus
isolates
revealed
intraspecies
recombination
led
to
viable
recombinants
in
four
out
of
the
eight
species
represented
(
Revers
et
al.
1996).

Recombination
is
more
likely
to
occur
and
thus
more
likely
to
result
in
viable
recombinants
that
have
the
appropriate
transcription
recognition
signals
the
more
closely
related
the
viruses
are.
However,
experiments
suggest
that
recombination
between
even
unrelated
viruses
may
prove
significant.
A
pseudorecombinant
strain
created
by
experimentally
combining
regions
of
the
cucumber
mosaic
virus
(
CMV)
and
tomato
aspermy
cucumovirus
(
TAV)
genomes
was
found
to
have
more
severe
symptoms
than
either
of
the
parentals,
although
the
recombinant
wasn't
able
to
move
beyond
infection
of
the
initially
infected
cells
(
Salánki
et
al.
1997).
Experiments
have
also
shown
interspecific
recombination
between
CMV
and
TAV
under
conditions
in
which
recombinants
would
not
be
expected
to
have
any
particular
fitness
advantage
(
Aaziz
&
Tepfer
1999).
In
another
example,
alteration
of
the
host
range
of
tobacco
mosaic
virus
(
TMV)
occurred
when
a
chimeric
virus
expressed
the
CP
from
alfalfa
mosaic
virus
(
AMV)
instead
of
its
own
(
Spitsin
et
al.
1999).
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3
of
17
In
addition
to
virus­
virus
recombination,
recombination
has
also
been
found
to
occur
between
virus
and
plant
host
RNA.
Sequence
analysis
of
the
5'
terminal
sequence
of
potato
leafroll
virus
(
PLRV)
suggests
that
it
arose
via
recombination
with
host
mRNA
(
Mayo
&
Jolly
1991).
Evidence
suggests
that
such
recombination
events
can
affect
virus
virulence
(
for
review
see
Rubio
et
al.
1999).

Recombination
only
rarely
leads
to
the
persistence
of
a
new,
viable
virus.
Whether
a
recombinant
virus
survives
in
the
field
depends
on
how
well
it
is
able
to
compete
with
other
viruses
at
all
stages
of
the
infective
cycle:
transmission,
gene
expression,
replication,
and
assembly
of
new
virions
(
Falk
&
Bruening
1994).
An
analysis
of
CMV
isolates
in
natural
populations
showed
that
while
mixed
infections
are
common,
viable
recombinants
were
very
rarely
recovered.
Such
results
suggest
that
recombinants
are
infrequent
or
are
at
a
selective
disadvantage
relative
to
parental
viruses
(
Fraile
et
al.
1997).
However,
a
virus
that
has
altered
transmission
patterns
from
parental
varieties
might
be
able
to
invade
a
new
niche
and
thus
escape
such
intense
competition
(
Gibbs
1994).
Laboratory
experiments
have
demonstrated
that
some
RNA­
RNA
recombinant
viruses
are
potentially
able
to
have
increased
fitness,
possibly
due
to
increased
replication
efficiency
or
increased
stability
of
the
viral
RNA
(
Fernández­
Cuartero
et
al.
1994).

Laboratory
experiments
of
recombination
in
transgenic
plants
with
viral
transgenes
Like
a
plant
host
genome,
viral
transgenes
would
be
available
for
recombination
with
infecting
viruses,
and
portions
of
the
transgene
could
thus
be
incorporated
into
the
replicating
virus.
Laboratory
experiments
with
pseudorecombinant
transcripts
of
papaya
ringspot
virus
(
PRSV)
have
shown
that
recombinant
viruses
that
theoretically
could
be
produced
in
field­
grown
transgenic
papaya
would
be
able
to
affect
the
virulence
of
the
infecting
strains
(
Chiang
et
al.
2001).
Viruses
that
are
pathogens
of
the
host
plant
would
be
available
for
recombination
with
transgenes.
In
addition,
other
viruses
that
are
generally
not
pathogens
may
also
be
available
if
the
plant
resists
infection
by
that
virus
by
restricting
its
movement
while
still
allowing
its
replication
(
Greene
&
Allison
1994).
The
theoretical
possibility
exists
that
recombination
between
a
replication­
competent
but
nonpathogenic
virus
and
a
transgene
could
convert
the
virus
to
a
pathogen
(
Allison
et
al.
1996).

Several
laboratory
experiments
have
investigated
the
potential
for
recombination
between
the
RNA
of
viral
genes
in
a
transgenic
plant
and
an
infecting
RNA
virus
of
the
same
type:

 
Transgenic
Nicotiana
benthamiana
containing
the
red
clover
necrotic
mosaic
virus
cell­
tocell
movement
protein
transgene
were
inoculated
with
infectious
transcripts
of
the
virus.
Sequence
analysis
of
virus
found
in
noninoculated,
systemically
infected
leaves
showed
that
the
infecting
virus
had
recombined
with
the
cell­
to­
cell
movement
protein
transgene
to
yield
a
virus
capable
of
systemic
infection
(
Lommel
&
Xiong
1991).

 
Transgenic
Nicotiana
benthamiana
containing
the
3'
2/
3
of
the
cowpea
chlorotic
mottle
virus
(
CCMV)
CP
gene
were
inoculated
with
a
CCMV
deletion
mutant
lacking
the
3'
1/
3
of
the
gene
such
that
recombination
within
the
central
1/
3
region
of
the
gene
would
allow
restoration
of
a
virus
capable
of
systemic
infection.
Three
percent
of
inoculated
plants
produced
viable
recombinants,
each
from
a
separate
aberrant
homologous
recombination
Page
4
of
17
event,
in
spite
of
experimental
conditions
designed
to
favor
precise
homologous
recombination
(
Greene
&
Allison
1994).

 
Transgenic
Nicotiana
benthamiana
containing
the
CP
coding
sequence
from
tomato
bushy
stunt
virus
(
TBSV)
were
inoculated
with
CP
mutants
of
TBSV.
Double
recombination
events
restored
wild­
type
virus
phenotype
in
up
to
20%
of
the
plants
(
Borja
et
al.
1999).

 
Transgenic
Nicotiana
benthamiana
containing
the
CP
coding
region
of
TMV
was
inoculated
with
a
CP
deletion
mutant.
Recombinant
RNA
was
detected
in
32%
of
the
infected
plants,
but
encapsidated
recombinant
virions
were
not
detected.
No
recombinant
RNA
was
detected
when
the
region
investigated
was
a
gene
nonessential
for
infection
rather
than
the
CP
gene
(
Adair
&
Kearney
2000).

 
Transgenic
Nicotiana
benthamiana
containing
the
CP
coding
region
of
plum
pox
virus
(
PPV)
with
either
a
complete
or
a
partially
deleted
3'
UTR
were
inoculated
with
CP
mutants
of
an
infectious
full­
length
clone
of
PPV.
Recombination
produced
viable
PPV,
but
only
when
the
transgenic
plants
contained
the
complete
3'
UTR
(
Varrelmann
et
al.
2000).

Similar
experiments
show
that
recombination
between
the
RNA
of
viral
transgenes
and
infecting
DNA
viruses
can
also
occur.
The
transgenic
plants
used
in
these
experiments
(
detailed
below)
actually
show
no
resistance
to
the
DNA
virus
whose
coat
protein
they
contain,
and
attempts
to
develop
transgenic
DNA
virus­
resistant
plants
in
general
have
had
little
success
(
Hammond
et
al.
1999).
Thus,
the
relevance
of
the
recombination
events
observed
in
these
plants
as
an
indicator
of
environmental
concern
might
be
questioned.

 
Transgenic
Brassica
were
infected
with
a
defective
CaMV
genome.
A
gene
that
complements
the
defective
region
was
inserted
into
the
plants
such
that
recombination
between
virus
and
transgene
could
produce
a
functional
copy
of
the
virus
genome
that
would
enable
virus
replication
and
spread.
Although
CaMV
is
a
DNA
virus,
it
replicates
through
an
RNA
intermediate.
Both
RNA
and
DNA
recombination
events
appeared
to
be
responsible
for
generation
of
viable
recombinants
in
four
out
of
12
inoculated
plants
(
Gal
et
al.
1992).

 
Transgenic
Nicotiana
bigelovii
containing
a
transgene
from
an
infective
strain
of
CaMV
were
inoculated
with
isolates
of
a
CaMV
strain
that
do
not
infect
N.
bigelovii
systemically.
Complementation
but
not
recombination
occurred
with
one
isolate,
and
recombination
but
not
complementation
occurred
in
eight
out
of
ten
plants
of
another
isolate.
Recombination
between
the
CaMV
transgene
and
infecting
viruses
was
able
to
alter
the
symptomatology
and
expand
the
host
range
of
the
infecting
virus
strain
(
Schoelz
&
Wintermantel
1993).

 
Transgenic
Nicotiana
bigelovii
containing
a
transgene
from
CaMV
were
inoculated
with
CaMV
strains
that
could
infect
N.
bigelovii
systemically,
so
recombinant
viruses
would
be
expected
to
have
little
selective
advantage
over
the
parentals.
Recombinant
viruses
were
recovered
in
three
out
of
23
transgenic
plants
inoculated
with
one
isolate
and
three
out
of
32
plants
inoculated
with
another.
Under
strong
selection
pressure
for
recombination,
recombinants
were
recovered
from
all
24
transgenic
plants
tested
(
Wintermantel
&
Schoelz
1996).

 
Several
transgenic
Nicotiana
benthamiana
lines
containing
the
CP
coding
sequence
from
African
cassava
mosaic
virus
(
ACMV;
a
geminivirus)
were
inoculated
with
a
CP
deletion
mutant
of
ACMV.
Recombinant
progeny
were
recovered
from
transgenic
lines
only
when
Page
5
of
17
they
contained
geminiviral
noncoding
sequence
on
both
sides
of
the
CP
transgene.
The
frequency
of
recombinants
varied
from
<
1%
to
67%
of
plants
in
a
given
line
(
Frischmuth
&
Stanley
1998).

Experimental
evidence
thus
demonstrates
that
recombination
can
occur
between
viral
transgenes
and
invading
viruses.
However,
to
facilitate
the
detection
of
recombinants,
most
of
these
experiments
were
conducted
under
conditions
of
high
selective
pressure,
i.
e.,
the
virus
was
not
viable
unless
a
recombination
event
occurred.
The
selective
pressure
is
usually
likely
to
be
much
weaker
under
normal
field
conditions.
Parental
viruses
will
outnumber
the
new
recombinant
and
will
be
competent
in
all
of
the
functions
needed
for
propagation.
The
relevance
of
experiments
demonstrating
recombination
under
conditions
of
strong
selection
is
thus
unclear
beyond
documenting
that
recombination
between
viral
genomes
and
transgenes
can
occur.

Although
the
laboratory
experiments
outlined
above
artificially
establish
a
high
selection
pressure
for
recombinants
that
is
unlikely
to
occur
in
nature,
such
selection
might
arise
in
transgenic
plants
when
viruses
partially
overcome
the
engineered
resistance,
as
they
have
been
known
occasionally
to
do
(
Carrington
et
al.
2001).
If
the
VCP
transgene
only
partially
eliminated
an
infecting
virus,
recombinant
virions
that
acquired
a
CP
gene
from
a
different
infecting
virus
would
be
able
completely
to
evade
the
resistance
conferred
by
the
VCP
transgene.
Such
recombination
events
among
different
infecting
viruses
could
occur
in
the
absence
of
the
transgene.
However,
the
transgene
could
create
a
selective
environment
that
would
favor
persistence
of
recombinants
and
thus
increase
their
potential
to
be
epidemiologically
significant
(
Jakab
et
al.
1997).

The
potential
environmental
impact
of
any
recombinant
viruses
that
might
arise
in
VCPtransgenic
plants
and
the
likelihood
of
their
arising
need
to
be
evaluated
in
the
context
of
the
events
that
occur
in
non­
transgenic
plants.
The
recombinants
that
arise
in
transgenic
plants
are
theoretically
unlikely
to
be
qualitatively
different
from
those
that
could
arise
in
natural,
mixed
infections
if
the
transgene
is
from
a
virus
that
normally
infects
the
plant
and
is
expressed
in
the
same
cells
that
normally
are
infected.
Under
such
circumstances
the
potential
new
viruses
that
could
be
created
through
recombination
are
expected
in
theory
to
be
the
same
in
transgenic
and
nontransgenic
plants
(
Roossinck
1997).

Reducing
the
frequency
of
recombination
in
transgenic
plants
All
evidence
suggests
that
recombination
among
RNA
viruses
occurs
via
template
switching
by
the
viral
replicase
during
replication
such
that
a
hybrid
molecule
is
formed
(
AIBS
1995).
Modifications
of
CP
transgenes
may
reduce
the
possibility
of
their
participation
in
recombination.
A
number
of
ways
have
been
suggested,
including:

 
Remove
the
3'
untranslated
region
(
UTR)
in
the
CP
mRNA
transcript
(
Teycheney
et
al.
2000).
Inclusion
of
this
region
may
enable
replication
to
begin
on
the
mRNA
transcript
and
then
switch
to
the
RNA
of
the
invading
virus.
Removal
of
this
region
would
necessitate
two
separate
template­
switching
events
to
form
a
successful
recombinant
and
thus
reduce
its
likelihood
of
occurrence
(
Greene
&
Allison
1994).
Experiments
with
CCMV
demonstrated
that
deletions
in
the
3'
UTR
did
indeed
reduce
the
recovery
of
recombinant
viruses
(
Greene
Page
6
of
17
&
Allison
1996).
Since
functional
resistance
is
still
conferred
by
constructs
containing
a
CP
lacking
the
3'
UTR,
this
region
may
not
be
necessary.

 
Reduce
the
extent
of
shared
sequence
similarity
between
the
infecting
virus
and
the
transgene
to
reduce
the
opportunities
for
homologous
recombination
(
Nagy
et
al.
1999).

 
Exclude
any
sequences
containing
replicase
recognition
sites
that
are
potential
sites
of
recombination
and
any
sequences
known
or
thought
to
be
recombination
hotspots,
e.
g.,
promoters
for
genomic
and
subgenomic
RNA
synthesis
(
Miller
et
al.
2000).

 
Avoid
potential
hairpin
structures
in
the
transgene
that
might
function
as
acceptor
structures
for
the
replicase
complex
(
Nagy
et
al.
1998).

 
Insert
GC­
rich
sequences
downstream
of
any
AU­
rich
region
thought
to
be
important
for
conferring
resistance
(
Hammond
et
al.
1999).
AU­
rich
regions
were
found
to
be
associated
with
imprecise
homologous
recombination
in
brome
mosaic
virus
(
BMV;
Nagy
&
Bujarski
1996),
and
insertion
of
GC­
rich
sequences
in
BMV
was
found
to
reduce
the
incidence
of
recombination
in
spite
of
increasing
the
total
amount
of
sequence
identity
(
Nagy
&
Bujarski
1998).
The
applicability
of
these
results
to
other
viruses
has
yet
to
be
demonstrated.

 
Use
the
smallest
viral
fragment
that
confers
effective
resistance,
as
longer
fragments
provide
larger
recombination
targets
(
Nagy
et
al.
1999).
In
addition,
longer
segments
are
more
likely
to
encode
functional
modules
that
could
be
incorporated
into
another
virus
as
a
single
unit,
thus
increasing
the
chance
that
recombinants
would
be
viable.

Heterologous
encapsidation
Heterologous
encapsidation
under
natural
conditions
For
many
viruses,
transmission
from
plant
to
plant
occurs
by
insect
vectors
and
each
virus
tends
to
be
transmitted
by
only
one
type
of
insect
(
Callaway
et
al.
2001).
The
CP,
possibly
in
conjunction
with
other
viral
factors,
is
essential
for
transmission
and
responsible
for
conferring
the
high
degree
of
specificity.
Heterologous
encapsidation
occurs
when
the
capsid
protein
subunits
of
one
virus
surround
the
nucleic
acid
of
a
different
virus,
thus
potentially
changing
its
vector
specificity.

Most
evidence
of
heterologous
encapsidation
is
derived
from
laboratory
or
greenhouse
studies.
The
high
frequency
of
mixed
infections
suggests
the
potential
for
heterologous
encapsidation
to
occur
in
nature
is
great,
but
most
mixed
infections
do
not
lead
to
heterologous
encapsidation,
and
those
virus
interactions
that
do
occur
are
very
specific
(
Falk
et
al.
1995).
Heterologous
encapsidation
is
however
known
to
be
a
regular
occurrence
among
some
plant
viruses.
Its
frequency
depends
on
the
viruses
involved
and
is
more
likely
to
occur
among
close
relatives
(
Tepfer
1993).
An
expansion
of
aphid
vector
specificity
due
to
heterologous
encapsidation
was
first
observed
in
plants
infected
with
two
different
isolates
of
barley
yellow
dwarf
luteovirus
(
BYDV;
Rochow
1970)
and
was
later
shown
to
be
a
general
phenomenon
among
these
viruses
in
natural
populations
of
several
plant
species
(
Creamer
&
Falk
1990).
Heterologous
encapsidation
was
also
shown
to
occur
in
potyviruses.
An
isolate
of
zucchini
yellow
mosaic
virus
(
ZYMV)
that
is
normally
non­
aphid
transmissible
due
to
a
transmission­
deficient
CP
was
found
to
be
aphid
Page
7
of
17
transmissible
due
to
heterologous
encapsidation
when
in
a
mixed
infection
with
another
potyvirus,
papaya
ringspot
virus
(
Bourdin
&
Lecoq
1991).
Heterologous
encapsidation
may
be
an
important
route
of
disease
transmission
for
viruses
that
have
no
CP.
For
example,
potato
spindle
tuber
viroid
is
transmitted
only
by
foliar
contact
or
botanical
seed
unless
encapsidated
by
the
coat
protein
of
PLRV
which
renders
it
aphid
transmissible
(
Querci
et
al.
1997).

Heterologous
encapsidation
is
considered
a
possible
environmental
concern
because
if
the
new
vector
has
a
host
range
different
from
the
original
vector,
the
virus
may
be
spread
to
new
plant
varieties
that
can
support
its
replication
but
that
it
would
not
have
infected
if
not
for
the
heterologous
encapsidation
(
de
Zoeton
1991).
Such
concerns
are
largely
mitigated
by
several
factors.
If
replication
is
possible
in
the
new
host,
it
would
cause
the
virus
to
be
encapsidated
by
its
own
CP,
thus
limiting
the
epidemiological
consequences
of
heterologous
encapsidation.
Secondly,
the
vector
transmission
of
the
encapsidating
virus
may
require
regions
of
the
genome
other
than
the
CP
for
effective
transmission,
so
heterologous
encapsidation
could
not
lead
to
a
change
in
vector
specificity
(
Robinson
1996).
Thirdly,
the
vector
may
have
a
limited
host
range
in
the
area
where
the
crop
is
to
be
grown
such
that
it
would
be
unlikely
to
transmit
a
heterologous
virus
to
a
novel
host.
Rather,
the
vector
would
transmit
virus
only
to
the
same
plant
that
the
virus
is
already
able
to
infect
(
Robinson
1996).

However,
under
certain
limited
circumstances
heterologous
encapsidation
theoretically
might
still
have
environmental
or
agricultural
impacts.
For
one,
a
virus
may
become
available
for
transmission
by
new
potential
vectors
that
feed
on
the
new
host
but
not
the
virus'
original
host
(
Hammond
et
al.
1999).
Through
such
a
mechanism,
theoretically
both
the
host
and
vector
range
of
a
virus
may
be
expanded,
and
the
change
of
both
plant
host
and
vector
could
make
detection
of
heterologous
encapsidation
difficult.
In
addition,
with
a
high
enough
frequency
of
vector
transmission
to
a
new
host
due
to
heterologous
encapsidation,
secondary
spread
among
new
plant
hosts
might
not
be
required
for
the
phenomenon
to
affect
them.
Due
to
the
lack
of
a
proofreading
mechanism
during
replication
of
RNA
viruses,
they
are
thought
to
exist
as
"
quasispecies"
in
which
each
viral
genome
differs
by
a
few
nucleotides
from
a
consensus
sequence.
After
expansion
to
a
new
host,
rapid
selection
of
variants
best
adapted
to
the
new
environment
might
lead
to
the
evolution
of
a
new
virus
(
Hammond
et
al.
1999).
No
direct
evidence
of
such
events
exists,
but
their
occurrence
is
suggested
by
the
appearance
of
"
new"
viruses
in
areas
that
have
had
recent
agricultural
expansions.
When
previously
wild
areas
are
cultivated,
novel
interactions
may
occur
among
potential
vectors,
viruses
in
the
local
natural
vegetation,
and
crops
previously
unexposed
to
these
viruses
(
Hammond
et
al.
1999).

Laboratory
experiments
of
heterologous
encapsidation
in
transgenic
plants
with
viral
transgenes
Experimental
studies
have
shown
that
the
protein
from
VCP
genes
in
transgenic
plants
has
the
ability
to
encapsidate
even
unrelated
infecting
viruses:

 
Transgenic
tobacco
plants
engineered
with
the
CP
of
TMV
were
infected
with
strains
of
TMV
defective
for
the
CP
gene.
The
transgenic
CP
was
able
to
complement
the
defective
TMV
and
enable
long­
distance
transport
(
Osbourn
et
al.
1990).
Page
8
of
17
 
Transgenic
tobacco
plants
engineered
with
the
CP
gene
of
AMV
were
infected
with
CMV.
Heterologous
encapsidation
was
observed
to
occur
between
these
two
unrelated
viruses
in
one
third
of
plants
at
a
frequency
of
about
5
x
10­
7
(
Candelier­
Harvey
&
Hull
1993).

 
Transgenic
tobacco
plants
engineered
with
the
CP
of
PPV
were
infected
with
a
strain
of
ZYMV
that
is
non­
aphid
transmissible
due
to
the
lack
of
a
functional
CP
gene.
Aphid
transmission
of
ZYMV
was
found
to
occur,
most
likely
by
heterologous
encapsidation
in
the
PPV
CP
(
Lecoq
et
al.
1993).

 
Transgenic
tobacco
plants
engineered
with
the
CP
gene
of
PPV
were
infected
with
various
potyviruses
and
with
viruses
from
other
groups.
Newly
formed
potyvirus
particles
were
found
to
contain
PPV
CP,
but
no
other
viral
particles
did
(
Maiss
et
al.
1994).

Heterologous
encapsidation
can
only
occur
in
transgenic
plants
if
the
coat
protein
is
expressed.
Therefore,
in
transgenic
VCP
plants
that
express
very
little
CP
(
i.
e.,
those
relying
on
posttranscriptional
gene
silencing
to
effect
resistance),
any
effects
associated
with
heterologous
encapsidation
would
be
minor
except
in
cases
of
resistance
breakdown.
In
addition,
as
with
recombination,
as
long
as
the
VCP
inserted
in
the
transgenic
plant
is
from
a
virus
that
normally
infects
the
plant
in
the
area
where
it
is
planted,
the
outcome
of
any
heterologous
encapsidation
that
may
occur
is
expected
to
be
qualitatively
the
same
in
transgenic
plants
as
in
natural,
mixed
infections.

Reducing
the
frequency
and/
or
impact
of
heterologous
encapsidation
in
transgenic
plants
Steps
can
be
taken
to
reduce
the
likelihood
of
heterologous
encapsidation
and/
or
vector
transmission
occurring.
Specific
locations
within
the
CP
gene
of
several
viruses
have
been
shown
to
affect
aphid
transmission:

 
Amino
acid
changes
in
the
CP
of
CMV
differentially
reduce
the
efficiency
of
transmission
by
two
different
aphid
species
(
Perry
et
al.
1998).

 
The
readthrough
domain
(
RTD)
of
the
CP
plays
a
key
role
in
determining
aphid
transmission
specificity
in
PLRV
(
Jolly
&
Mayo
1994),
BYDV
(
Chay
et
al.
1996),
and
beet
western
yellows
luteovirus
(
BWYV;
Brault
et
al.
1995;
Reinbold
et
al.
2001;
Bruyère
et
al.
1997;
Brault
et
al.
2000).

 
Point
mutations
in
the
major
capsid
protein
(
P3)
of
BWYV
affect
aphid
transmission
(
Brault
et
al.
2003).

 
Amino
acid
changes
in
a
conserved
loop
structure
of
the
CP
of
CMV
affect
aphid
transmission
(
Liu
et
al.
2002).

 
A
three
amino
acid
sequence,
asp­
ala­
gly
(
DAG)
is
conserved
in
aphid­
transmissible
strains
of
potyviruses
including
tobacco
vein
mottling
virus
(
Atreya
et
al.
1991)
and
ZYMV
(
Gal­
On
et
al.
1992).
Mutations
in
this
region
in
either
virus
render
it
non­
aphid­
transmissible.
The
particular
context
in
which
this
amino
acid
triplet
is
found
also
appears
to
be
important
in
determining
aphid
transmissibility
in
TVMV
(
López­
Moya
et
al.
1999).
Page
9
of
17
Experiments
suggest
that
VCP
gene
modifications
could
reduce
the
frequency
of
heterologous
encapsidation
and/
or
vector
transmission.
Mutations
in
the
two
assembly
motifs
(
RQ
and
D)
of
the
CP
of
PPV
were
found
to
suppress
heterologous
encapsidation,
particle
assembly,
and
complementation
without
affecting
resistance
to
viral
infection
(
Varrelmann
&
Maiss
2000).
Likewise,
the
CP
gene
of
PPV
was
modified
or
truncated
in
order
to
reduce
any
potential
impacts
of
heterologous
encapsidation:
either
the
DAG
triplet
was
deleted
or
the
first
420
nucleotides
of
the
PPV
CP
gene
were
removed
(
Jacquet
et
al.
1998b).
Both
transgenic
lines
were
resistant
to
PPV
infection,
indicating
that
the
full­
length
CP
region
is
not
needed
for
resistance.
Further
experiments
confirmed
that
such
changes
in
the
CP
did
effectively
mitigate
the
potential
for
heterologous
encapsidation
to
have
any
effect
(
Jacquet
et
al.
1998a).
However,
investigation
of
the
DAG
motif
in
begomoviruses
revealed
that
it
is
not
the
precise
determinant
of
whitefly
transmission
of
this
virus.
Rather,
amino
acids
123
to
149
of
the
CP
are
minimally
required
for
transmission,
and
amino
acids
149
to
174
contribute
to
efficient
transmission
(
Höhnle
et
al.
2001).

If
changes
that
resulted
in
loss
of
aphid
transmissibility
were
to
be
incorporated
into
the
VCP
genes
used
in
transgenic
plants,
then
heterologous
encapsidation
would
be
extremely
unlikely
to
change
vector
specificity,
since
the
CP
would
no
longer
confer
any
specificity
at
all.
Experiments
with
various
potyviruses
showed
that
the
degree
of
heterologous
encapsidation
failed
to
correlate
with
the
degree
of
resistance
conferred
by
different
CP
constructs
(
Hammond
&
Dienelt
1997),
suggesting
that
such
strategies
could
be
deployed
without
compromising
product
efficacy.
However,
while
particular
changes
in
the
CP
are
effective
at
reducing
any
potential
impacts
associated
with
heterologous
encapsidation,
variation
among
viruses
may
preclude
a
single
generic
solution
that
could
be
used
for
all.

Synergy
Synergy
under
natural
conditions
In
addition
to
the
creation
of
a
novel
virus
through
recombination
and
the
alteration
of
transmission
properties
of
an
existing
virus
through
heterologous
encapsidation,
viral
interactions
may
cause
increased
disease
severity
through
alteration
of
the
replication
and/
or
movement
efficiency
of
a
virus.
In
a
synergistic
disease
the
severity
of
two
viruses
together
is
greater
than
expected
based
on
the
severity
of
each
alone.
When
potato
virus
X
(
PVX)
is
coinfected
with
a
number
of
potyviruses
including
TVMV,
TEV,
and
pepper
mottle
virus,
the
disease
symptoms
are
considerably
worsened
and
PVX
accumulates
to
a
greater
concentration
(
Vance
et
al.
1995).
CMV
and
TAV
cause
no
synergistic
disease
in
double
infections,
but
an
interspecies
hybrid
was
significantly
more
virulent
than
either
parent
in
all
plant
species
tested
(
Ding
et
al.
1996).
The
increased
severity
was
most
likely
due
to
a
synergistic
interaction
between
TAV
and
the
protein
produced
from
the
CMV
portion
of
the
hybrid.
A
listing
of
reported
viral
synergisms
has
been
compiled
(
OECD
Environment
Directorate
1996).

Synergistic
interactions
are
extremely
common
among
the
luteoviruses,
but
the
CP
is
considered
much
less
likely
to
be
responsible
for
synergism
than
other
regions
of
the
viral
genome,
for
example
the
polymerase
gene
(
Miller
et
al.
1997).
Within
potyviruses,
the
5'
proximal
1/
3
of
the
Page
10
of
17
viral
genome
is
thought
to
contain
the
factors
that
mediate
synergism,
including
the
5'
UTR
and
the
coding
region
for
the
5'
N­
terminal
portion
of
the
viral
polyprotein
(
Pruss
et
al.
1997).

Synergy
in
transgenic
plants
with
viral
transgenes
Synergy
in
transgenic
plants
is
generally
an
agroeconomic
rather
than
environmental
concern.
Any
negative
effects
are
expected
primarily
to
affect
the
transgenic
crop
itself
which
would
be
quickly
abandoned
once
such
effects
were
detected.
Temporarily
increased
viral
loads
caused
by
synergistic
disease
could
result
in
limited
damage
to
nearby
conventional
crops
if
the
transgenic
crop
were
already
deployed
in
large
field
acreages
when
synergistic
disease
was
discovered
(
Miller
et
al.
1997).
However,
synergistic
interactions
can
be
evaluated
in
transgenic
plants
before
deployment
by
experimental
inoculation
with
all
of
the
viruses
likely
to
be
encountered
in
the
field
(
Robinson
1996).
Developers
have
a
strong
incentive
to
undertake
such
efforts
to
ensure
the
efficacy
of
their
product
after
deployment.

Reducing
the
frequency
of
synergy
in
transgenic
plants
As
with
heterologous
encapsidation,
constructs
can
be
engineered
to
reduce
the
likelihood
of
synergy.
Particular
transgenes
known
to
participate
in
synergistic
interactions
could
be
avoided
or
defective
copies
of
genes
could
be
used.
Stacking
multiple
resistances
within
the
same
plant
such
that
it
will
have
reduced
viral
loads
for
all
of
its
normal
pathogens
has
also
been
suggested
(
Palukaitis
2000).
The
benefits
of
this
approach
may
however
be
outweighed
by
the
concerns
associated
with
stacking
in
VCP­
transgenic
plants
(
discussed
below).

Field
evaluations
of
viral
interactions
in
transgenic
plants
with
viral
transgenes
The
many
laboratory
experiments
outlined
above
that
investigated
the
potential
viral
interactions
in
VCP­
transgenic
plants
are
only
one
part
of
evaluating
potential
environmental
impacts
of
such
events.
Consideration
of
the
potential
impact
under
natural,
field
conditions
evaluated
in
the
context
of
the
likelihood
of
any
such
event
occurring
is
equally
important
(
García­
Arenal
et
al.
2000).
Relatively
few
field
studies
have
been
conducted
to
address
these
questions,
but
those
that
have
been
done
have
found
no
significant
impact
associated
with
deployment
of
VCP­
transgenic
plants
beyond
natural
background
events.
A
six­
year
experiment
searched
for
and
failed
to
find
evidence
of
interactions
involving
viral
transgenes
in
25,000
transgenic
potato
plants
transformed
with
various
PLRV
CP
constructs.
Both
greenhouse
and
field
tests
failed
to
show
any
change
in
the
type
or
severity
of
disease
symptoms,
and
all
viruses
isolated
were
previously
known
to
infect
the
plants
and
had
the
expected
transmission
characteristics
(
Thomas
et
al.
1998).

An
experiment
with
transgenic
melon
and
squash
expressing
CP
genes
of
an
aphid­
transmissible
strain
of
CMV
failed
to
find
evidence
that
either
recombination
or
heterologous
encapsidation
enabled
spread
of
an
aphid
non­
transmissible
strain
of
CMV
in
the
field
(
Fuchs
et
al.
1998).
A
similar
experiment
with
transgenic
squash
expressing
CP
genes
of
an
aphid­
transmissible
strain
Page
11
of
17
of
watermelon
mosaic
virus
(
WMV)
showed
that
an
aphid
non­
transmissible
strain
of
ZYMV
was
not
detected
in
nontransgenic
fields
but
was
transmitted
to
2%
of
plants
in
transgenic
fields,
likely
due
to
heterologous
encapsidation.
However,
this
rate
of
transmission
failed
to
lead
to
the
development
of
an
epidemic
of
ZYMV
in
transgenic
squash
fields
(
Fuchs
et
al.
1999).

In
an
experiment
to
assess
the
biological
and
genetic
diversity
of
California
CMV
isolates
before
and
after
deployment
of
transgenic
melon
containing
the
CMV
CP
gene
showed
that
the
only
CMV
isolate
to
show
significant
sequence
changes
after
infecting
the
transgenic
squash
was
not
the
result
of
recombination
(
Lin
et
al.
2003).
The
only
field
experiment
to
directly
assess
the
effect
of
recombination
in
a
VCP­
transgenic
plant
found
no
detectable
grapevine
fanleaf
virus
(
GFLV)
recombinants
containing
the
inserted
CP
sequence
over
the
course
of
a
four­
year
study
(
Vigne
et
al.
2004).

The
limited
number
of
field
evaluations
thus
indicate
that
the
likely
environmental
consequences
of
viral
interactions
in
VCP­
transgenic
plants
are
minimal.
However,
large
acreages
of
VCPtransgenic
plants
grown
over
many
years
may
provide
increased
opportunity
for
rare
events
to
occur
that
are
unlikely
to
be
detected
in
experimental
studies
(
Miller
et
al.
1997),
so
consideration
of
theoretical
outcomes
and
their
likelihood
of
occurrence
may
be
necessary
to
facilitate
a
complete
evaluation
of
the
risk
concerns.

Is
the
frequency
of
viral
interactions
in
transgenic
plants
different
than
in
natural,
mixed
infections?

Few
experimental
studies
have
addressed
the
relative
degree
of
risk
directly,
mostly
because
it
is
hard
under
any
circumstances
to
determine
the
frequency
of
viral
interactions
that
lead
to
a
measurable
effect
given
how
rare
these
events
are.
Some
characteristics
of
VCP­
transgenic
plants
suggest
that
the
frequency
of
interactions
may
be
lower
than
in
natural,
mixed
infections.
Other
characteristics
suggest
that
the
frequency
may
be
higher
(
see
below).

One
factor
that
may
decrease
the
frequency
of
interactions
in
transgenic
systems
is
that
the
cellular
concentration
of
viral
RNA
transcripts
expressed
from
transgenes
will
be
orders
of
magnitude
lower
than
the
concentration
of
viral
RNA
commonly
found
in
natural,
mixed
infections
(
Allison
et
al.
1996).
The
concentration
of
infecting
viral
RNA
from
the
target
virus
will
also
be
considerably
reduced,
particularly
when
the
mechanism
of
resistance
relies
on
posttranscriptional
gene
silencing
to
remove
all
viral
RNA
transcripts
with
homology
to
the
transgene
(
Rovere
et
al.
2002).
However,
the
significance
of
these
observations
is
difficult
to
interpret.
While
it
is
known
that
greater
concentrations
of
RNA
will
provide
greater
opportunity
for
interactions,
meaningful
values
for
high
or
low
concentrations
are
unavailable
(
AIBS
1995).

The
frequency
of
interactions
in
transgenic
systems
may
be
increased
because
promoters
currently
used
in
VCP­
transgenic
plants
cause
constitutive
expression
of
transgenes
at
developmental
stages
that
might
otherwise
be
unaffected
by
viral
infection
and
often
in
tissues
that
the
virus
does
not
normally
infect
(
Allison
et
al.
2000).
For
example,
luteoviruses
are
normally
expressed
only
in
phloem
tissue,
but
the
cauliflower
mosaic
virus
(
CaMV)
promoter
drives
expression
of
luteoviral
CP
in
all
plant
cells.
Some
evidence
suggests
that
in
natural
Page
12
of
17
infections
different
viruses
have
different
temporal
or
spatial
expression
patterns
that
would
limit
their
interactions
(
Gibbs
1994;
Hull
1994;
Aaziz
&
Tepfer
1999).
Viruses
must
simultaneously
replicate
in
the
same
cellular
compartment
for
their
RNA
to
be
able
to
interact.
However,
when
a
virus
invades
a
cell,
it
often
replicates
and
then
moves
to
other
cells
within
the
plant.
The
RNA
remaining
in
the
initially
infected
cell
becomes
encapsidated
and
is
no
longer
available
for
interactions
with
another
invading
virus
(
Allison
et
al.
2000).

How
these
competing
factors
balance
out
to
affect
the
relative
frequency
of
viral
interactions
in
transgenic
versus
nontransgenic
plants
is
thus
likely
to
vary
with
the
virus,
the
plant,
and
the
mechanism
of
resistance.
Whether
the
nature
of
the
interactions
that
do
occur
may
be
different
in
VCP­
transgenic
plants
than
expected
in
natural,
mixed
infections
must
also
be
considered.

When
could
viral
interactions
in
transgenic
plants
be
unlike
those
likely
to
occur
in
natural,
mixed
infections?

Another
key
question
for
evaluating
the
significance
of
the
above
studies
is
whether
the
viral
interactions
in
transgenic
plants
may
be
unlike
those
likely
to
occur
in
non­
transgenic
plants.
The
potential
risks
associated
with
viral
interactions
in
VCP
transgenic
plants
are
similar
to
those
that
arise
with
natural,
mixed
infections.
Recombination,
heterologous
encapsidation,
and
synergy
may
occur
in
both
cases,
although
a
comparison
of
frequencies
is
difficult.
Theoretically,
the
hazards
associated
with
these
phenomena
are
expected
to
be
similar
whether
these
events
occur
in
transgenic
or
nontransgenic
plants.
However,
under
certain
circumstances,
transgenic
plants
may
in
theory
provide
opportunities
for
unique
interactions
that
would
not
be
expected
to
occur
in
a
natural,
mixed
infection:

 
Transgenic
multiresistances:
A
plant
may
be
engineered
to
resist
infection
from
multiple
viruses
by
incorporation
of
several
CPs
into
the
same
plant.
While
generally,
a
transgenic
plant
that
is
resistant
to
a
particular
virus
will
be
planted
only
where
the
virus
is
a
problem,
with
stacked
resistances,
the
likelihood
of
doing
otherwise
increases.
For
example,
a
cucurbit
resistant
to
CMV,
WMV2,
and
ZYMV
has
been
created
(
Fuchs
et
al.
1997).
Such
a
plant
would
probably
be
used
in
all
areas
where
ZYMV
is
prevalent,
including
tropical
areas
where
WMV2
never
or
only
rarely
occurs
(
Hammond
et
al.
1999).
WMV2
could
then
interact
with
a
local
strain
that
would
otherwise
have
had
no
opportunity
for
interaction
with
it.

 
Heterologous
resistance:
A
plant
may
be
resistant
to
infection
by
a
particular
virus
in
spite
of
having
the
CP
of
another
virus
incorporated
into
its
genome.
For
example,
VCP
genes
from
LMV
were
used
to
provide
resistance
to
PVY
in
tobacco
which
is
not
infected
by
LMV
(
Dinant
et
al.
1993).
In
such
plants,
LMV
might
have
a
new
opportunity
to
interact
with
viruses
that
infect
tobacco.

 
Plants
may
be
engineered
with
VCP
genes
from
an
exotic
strain
of
a
virus
that
may
be
more
virulent
or
have
other
properties
different
from
endemic
isolates.
Under
certain
circumstances,
the
desire
that
transgenes
be
from
an
endemic
isolate
may
be
balanced
by
other
concerns.
For
example,
when
the
costs
of
an
exotic
viral
invasion
would
be
particularly
devastating,
incorporation
of
a
viral
transgene
intended
to
prevent
such
establishment
might
be
warranted
(
Hammond
et
al.
1999).
In
addition,
identification
of
local
virus
isolates
may
Page
13
of
17
not
always
be
possible,
especially
given
that
the
spectrum
of
isolates
in
a
given
area
will
change
over
time.
Even
the
definition
of
what
an
endemic
isolate
is
can
be
complicated
by
international
trade
in
seed
and
vegetative
propagating
material.
Nevertheless,
thorough
evaluation
of
a
VCP­
transgenic
plant
requires
knowledge
of
the
plant
viruses
that
are
present
in
the
release
environment
and
that
naturally
infect
the
host
(
Robinson
1996).

 
Plants
may
express
VCP
genes
in
cells
and/
or
tissues
that
the
virus
does
not
normally
infect
(
as
discussed
above).

 
Plants
may
be
engineered
with
VCP
genes
that
have
been
altered
such
that
they
do
not
resemble
any
that
exist
in
nature.
Any
interactions
involving
such
VCP
genes
would
thus
be
novel.

Such
situations
may
present
the
opportunity
for
novel
viral
interactions
in
VCP­
transgenic
plants,
i.
e.,
interactions
between
portions
of
two
or
more
different
viruses
not
expected
to
occur
in
a
mixed
infection
found
in
nature.
Such
situations
may
be
avoided
in
designing
and
deploying
VCP­
transgenic
plants.
However,
if
avoidance
is
impossible
or
undesirable,
strategies
such
as
those
discussed
above
to
prevent
the
occurrence
of
viral
interactions
would
greatly
reduce
the
frequency
of
all
interactions,
whether
novel
or
not.

Conclusion
While
the
nature
of
the
potential
risks
posed
by
novel
interactions
among
viral
genomes
in
transgenic
plants
is
relatively
well
understood,
data
to
evaluate
the
actual
impact
and
likelihood
of
such
events
under
a
wide
range
of
natural
conditions
for
different
VCP­
transgenic
plants
are
sparse.
Further
experimental
data
may
help
to
provide
a
more
complete
picture
that
would
allow
a
careful
evaluation
of
the
likelihood
of
adverse
impacts
and
the
subsequent
evaluation
of
the
risks
and
benefits
of
deploying
VCP­
transgenic
plants.
Although
current
evidence
suggests
that
the
risks
are
unlikely
to
be
much
greater
than
those
found
in
natural,
mixed
infections
(
Hammond
et
al.
1999;
Bruening
&
Falk
1994),
steps
may
be
taken
when
designing
products
to
minimize
any
potential
impacts.

References
Aaziz,
R.
and
Tepfer,
M.
1999.
Recombination
between
genomic
RNAs
of
two
cucumoviruses
under
conditions
of
minimal
selection
pressure.
Virology
263:
282­
289.
Abel,
P.
P.,
Nelson,
R.
S.,
Hoffmann,
N.,
Rogers,
S.
G.,
Fraley,
R.
T.,
and
Beachy,
R.
N.
1986.
Delay
of
disease
development
in
transgenic
plants
that
express
the
tobacco
mosaic
virus
coat
protein
gene.
Science
232:
738­
743.
Adair,
T.
L.
and
Kearney,
C.
M.
2000.
Recombination
between
a
3­
kilobase
tobacco
mosaic
virus
transgene
and
a
homologous
viral
construct
in
the
restoration
of
viral
and
nonviral
genes.
Arch.
Virol.
145:
1867­
1883.
AIBS
.
Transgenic
virus­
resistant
plants
and
new
plant
viruses.
http://
www.
aphis.
usda.
gov/
ppq/
biotech/
virus/
95_
virusrept.
pdf
.
1995.
Allison,
R.
F.,
Janda,
M.,
and
Ahlquist,
P.
1989.
Sequence
of
cowpea
chlorotic
mottle
virus
RNAs
2
and
3
and
evidence
of
a
recombination
event
during
bromovirus
evolution.
Virology
172:
321­
330.
Allison,
R.
F.,
Schneider,
W.
L.,
and
Greene,
A.
E.
1996.
Recombination
in
plants
expressing
viral
transgenes.
Semin.
Virol.
7:
417­
422.
Page
14
of
17
Atreya,
P.
L.,
Atreya,
C.
D.,
and
Pirone,
T.
P.
1991.
Amino
acid
substitutions
in
the
coat
protein
result
in
loss
of
insect
transmissibility
of
a
plant
virus.
Proc.
Natl.
Acad.
Sci.
88:
7887­
7891.
Borja,
M.,
Rubio,
T.,
Scholthof,
H.
B.,
and
Jackson,
A.
O.
1999.
Restoration
of
wild­
type
virus
by
double
recombination
of
tombusvirus
mutants
with
a
host
transgene.
Mol.
Plant
Microbe
Interact.
12:
153­
162.
Bourdin,
D.
and
Lecoq,
H.
1991.
Evidence
that
heteroencapsidation
between
two
potyviruses
is
involved
in
aphid
transmission
of
a
non­
aphid­
transmissible
isolate
from
mixed
infections.
Phytopathology
81:
1459­
1464.
Brault,
V.,
Bergdoll,
M.,
Mutterer,
J.,
Prasad,
V.,
Pfeffer,
S.,
Erdinger,
M.,
Richards,
K.
E.,
and
Ziegler­
Graff,
V.
2003.
Effects
of
point
mutations
in
the
major
capsid
protein
of
beet
Western
yellows
virus
on
capsid
formation,
virus
accumulation,
and
aphid
transmission.
J.
Virol.
77:
3247­
3256.
Brault,
V.,
Mutterer,
J.,
Scheidecker,
D.,
Simonis,
M.
T.,
Herrbach,
E.,
Richards,
K.,
and
Ziegler­
Graff,
V.
2000.
Effects
of
point
mutations
in
the
readthrough
domain
of
the
beet
western
yellows
virus
minor
capsid
protein
on
virus
accumulation
in
planta
and
on
transmission
by
aphids.
J.
Virol.
74:
1140­
1148.
Brault,
V.,
van
den
Heuvel,
J.
F.
J.
M.,
Verbeek,
M.,
Ziegler­
Graff,
V.,
Reutenauer,
A.,
Herrbach,
E.,
Garaud,
J.
C.,
Guilley,
H.,
Richards,
K.,
and
Jonard,
G.
1995.
Aphid
transmission
of
beet
western
yellows
luteovirus
requires
the
minor
capsid
read­
through
protein
P74.
EMBO
J.
14:
650­
659.
Bruening,
G.
and
Falk,
B.
W.
1994.
Risks
in
using
transgenic
plants?
­
Author
reply.
Science
264:
1651­
1652.
Bruyère,
A.,
Brault,
V.,
Ziegler­
Graff,
V.,
Simonis,
M.
T.,
van
den
Heuvel,
J.
F.
J.
M.,
Richards,
K.,
Guilley,
H.,
Jonard,
G.,
and
Herrbach,
E.
1997.
Effects
of
mutations
in
the
beet
western
yellows
virus
readthrough
protein
on
its
expression
and
packaging
and
on
virus
accumulation,
symptoms,
and
aphid
transmission.
Virology
230:
323­
334.
Callaway,
A.,
Giesman­
Cookmeyer,
D.,
Gillock,
E.
T.,
Sit,
T.
L.,
and
Lommel,
S.
A.
2001.
The
multifunctional
capsid
proteins
of
plant
RNA
viruses.
Annu.
Rev.
Phytopathol.
39:
419­
460.
Candelier­
Harvey,
P.
and
Hull,
R.
1993.
Cucumber
mosaic
virus
genome
is
encapsidated
in
alfalfa
mosaic
virus
coat
protein
expressed
in
transgenic
tobacco
plants.
Transgenic
Research
2:
277­
285.
Carrington,
J.
C.,
Kasschau,
K.
D.,
and
Johansen,
L.
K.
2001.
Activation
and
suppression
of
RNA
silencing
by
plant
viruses.
Virology
281:
1­
5.
Chay,
C.
A.,
Gunasinge,
U.
B.,
Dinesh­
Kumar,
S.
P.,
Miller,
W.
A.,
and
Gray,
S.
M.
1996.
Aphid
transmission
and
systemic
plant
infection
determinants
of
barley
yellow
dwarf
luteovirus­
PAV
are
contained
in
the
coat
protein
readthrough
domain
and
17­
kDa
protein,
respectively.
Virology
219:
57­
65.
Chenault,
K.
D.
and
Melcher,
U.
1994.
Phylogenetic
relationships
reveal
recombination
among
isolates
of
cauliflower
mosaic
virus.
J.
Mol.
Evol.
39:
496­
505.
Chiang,
C.
H.,
Wang,
J.
J.,
Jan,
F.
J.,
Yeh,
S.
D.,
and
Gonsalves,
D.
2001.
Comparative
reactions
of
recombinant
papaya
ringspot
viruses
with
chimeric
coat
protein
(
CP)
genes
and
wild­
type
viruses
on
CP­
transgenic
papaya.
J.
Gen.
Virol.
82:
2827­
2836.
Creamer,
R.
and
Falk,
B.
W.
1990.
Direct
detection
of
transcapsidated
barley
yellow
dwarf
luteoviruses
in
doubly
infected
plants.
J.
Gen.
Virol.
71:
211­
217.
de
Zoeton,
G.
A.
1991.
Risk
assessment:
Do
we
let
history
repeat
itself?
Phytopathology
81:
585­
586.
Dinant,
S.,
Blaise,
F.,
Kusiak,
C.,
Astier­
Manifacier,
S.,
and
Albouy,
J.
1993.
Heterologous
resistance
to
potato
virus
Y
in
transgenic
tobacco
plants
expressing
the
coat
protein
gene
of
lettuce
mosaic
potyvirus.
Phytopathology
83:
818­
824.
Ding,
S.
W.,
Shi,
B.
J.,
Li,
W.
X.,
and
Symons,
R.
H.
1996.
An
interspecies
hybrid
RNA
virus
is
significantly
more
virulent
than
either
parental
virus.
Proc.
Natl.
Acad.
Sci.
93:
7470­
7474.
Falk,
B.
W.
and
Bruening,
G.
1994.
Will
transgenic
crops
generate
new
viruses
and
new
diseases?
Science
263:
1395­
1396.
Falk,
B.
W.,
Passmore,
B.
K.,
Watson,
M.
T.,
and
Chin,
L.
S.
1995.
The
specificity
and
significance
of
heterologous
encapsidation
of
virus
and
virus­
like
RNAs.
In
Bills,
D.
D.
and
Kung,
S.
D.,
eds.
Biotechnology
and
Plant
Protection:
Viral
pathogenesis
and
disease
resistance.
World
Scientific,
Singapore,
pp
391­
415.
Fernández­
Cuartero,
B.,
Burgyàn,
J.,
Aranda,
M.
A.,
Salánki,
K.,
Moriones,
E.,
and
García­
Arenal,
F.
1994.
Increase
in
the
relative
fitness
of
a
plant
virus
RNA
associated
with
its
recombinant
nature.
Virology
203:
373­
377.
Fraile,
A.,
Alonso­
Prados,
J.
L.,
Aranda,
M.
A.,
Bernal,
J.
J.,
Malpica,
J.
M.,
and
García­
Arenal,
F.
1997.
Genetic
exchange
by
recombination
or
reassortment
is
infrequent
in
natural
populations
of
a
tripartite
RNA
plant
virus.
J.
Virol.
71:
934­
940.
Frischmuth,
T.
and
Stanley,
J.
1998.
Recombination
between
viral
DNA
and
the
transgenic
coat
protein
gene
of
African
cassava
mosaic
geminivirus.
J.
Gen.
Virol.
79:
1265­
1271.
Fuchs,
M.,
Ferreira,
S.,
and
Gonsalves,
D.
Management
of
virus
diseases
by
classical
and
engineered
protection.
Molecular
Plant
Pathology
On­
Line.
http://
www.
bspp.
org.
uk/
mppol/
1997/
0116fuchs
.
1997.
Page
15
of
17
Fuchs,
M.,
Gal­
On,
A.,
Raccah,
B.,
and
Gonsalves,
D.
1999.
Epidemiology
of
an
aphid
nontransmissible
potyvirus
in
fields
of
nontransgenic
and
coat
protein
transgenic
squash.
Transgenic
Research
8:
429­
439.
Fuchs,
M.,
Klas,
F.
E.,
McFerson,
J.
R.,
and
Gonsalves,
D.
1998.
Transgenic
melon
and
squash
expressing
coat
protein
genes
of
aphid­
borne
viruses
do
not
assist
the
spread
of
an
aphid
non­
transmissible
strain
of
cucumber
mosaic
virus
in
the
field.
Transgenic
Research
7:
449­
462.
Gal,
S.,
Pisan,
B.,
Hohn,
T.,
Grimsley,
N.,
and
Hohn,
B.
1992.
Agroinfection
of
transgenic
plants
leads
to
viable
cauliflower
mosaic
virus
by
intermolecular
recombination.
Virology
187:
525­
533.
Gal­
On,
A.,
Antignus,
Y.,
Rosner,
A.,
and
Raccah,
B.
1992.
A
zucchini
yellow
mosaic
virus
coat
protein
gene
mutation
restores
aphid
transmissibility
but
has
no
effect
on
multiplication.
J.
Gen.
Virol.
73:
2183­
2187.
García­
Arenal,
F.,
Malpica,
J.
M.,
and
Fraile,
A.
2000.
Evolution
of
plant
virus
populations:
The
role
of
genetic
exchange.
In
Fairbairn,
C.,
Scoles,
G.,
and
McHughen,
A.,
eds.
Proceedings
of
the
6th
International
Symposium
on
the
Biosafety
of
Genetically
Modified
Organisms.
University
Extension
Press,
University
of
Saskatchewan,
Saskatoon,
Canada,
pp
91­
96.
Gibbs,
M.
1994.
Risks
in
using
transgenic
plants?
Science
264:
1650­
1651.
Gibbs,
M.
J.
and
Cooper,
J.
I.
1995.
A
recombinational
event
in
the
history
of
luteoviruses
probably
induced
by
basepairing
between
the
genomes
of
two
distinct
viruses.
Virology
206:
1129­
1132.
Greene,
A.
E.
and
Allison,
R.
F.
1994.
Recombination
between
viral
RNA
and
transgenic
plant
transcripts.
Science
263:
1423­
1425.
Greene,
A.
E.
and
Allison,
R.
F.
1996.
Deletions
in
the
3'
untranslated
region
of
cowpea
chlorotic
mottle
virus
transgene
reduce
recovery
of
recombinant
viruses
in
transgenic
plants.
Virology
225:
231­
234.
Hammond,
J.
and
Dienelt,
M.
M.
1997.
Encapsidation
of
potyviral
RNA
in
various
forms
of
transgene
coat
protein
is
not
correlated
with
resistance
in
transgenic
plants.
Mol.
Plant
Microbe
Interact.
10:
1023­
1027.
Hammond,
J.,
Lecoq,
H.,
and
Raccah,
B.
1999.
Epidemiological
risks
from
mixed
virus
infections
and
transgenic
plants
expressing
viral
genes.
Adv.
Virus
Res.
54:
189­
314.
Höhnle,
M.,
Höfer,
P.,
Bedford,
I.
D.,
Briddon,
R.
W.,
Markham,
P.
G.,
and
Frischmuth,
T.
2001.
Exchange
of
three
amino
acids
in
the
coat
protein
results
in
efficient
whitefly
transmission
of
a
nontransmissible
Abutilon
mosaic
virus
isolate.
Virology
290:
164­
171.
Jacquet,
C.,
Delecolle,
B.,
Raccah,
B.,
Lecoq,
H.,
Dunez,
J.,
and
Ravelonandro,
M.
1998a.
Use
of
modified
plum
pox
virus
coat
protein
genes
developed
to
limit
heteroencapsidation­
associated
risks
in
transgenic
plants.
J.
Gen.
Virol.
79:
1509­
1517.
Jacquet,
C.,
Ravelonandro,
M.,
Bachelier,
J.
C.,
and
Dunez,
J.
1998b.
High
resistance
to
plum
pox
virus
(
PPV)
in
transgenic
plants
containing
modified
and
truncated
forms
of
PPV
coat
protein
gene.
Transgenic
Research
7:
29­
39.
Jakab,
G.,
Vaistig,
F.
E.,
Droz,
E.,
and
Malnoë,
P.
1997.
Transgenic
plants
expressing
viral
sequences
create
a
favourable
environment
for
recombination
between
viral
sequences.
In
Tepfer,
M.
and
Balázs,
E.,
eds.
Virus­
resistant
Transgenic
Plants:
Potential
Ecological
Impact.
Springer,
Berlin,
pp
45­
51.
Le
Gall,
O.
L.,
Lanneau,
M.,
Candresse,
T.,
and
Dunez,
J.
1995.
The
nucleotide
sequence
of
the
RNA­
2
of
an
isolate
of
the
English
serotype
of
tomato
black
ring
virus:
RNA
recombination
in
the
history
of
nepoviruses.
J.
Gen.
Virol.
76:
1279­
1283.
Lecoq,
H.,
Ravelonandro,
M.,
Wipf­
Scheibel,
C.,
Monsion,
M.,
Raccah,
B.,
and
Dunez,
J.
1993.
Aphid
transmission
of
a
non­
aphid­
transmissible
strain
of
zucchini
yellow
mosaic
potyvirus
from
transgenic
plants
expressing
the
capsid
protein
of
plum
pox
potyvirus.
Mol.
Plant
Microbe
Interact.
6:
403­
406.
Lin,
H.
X.,
Rubio,
L.,
Smythe,
A.,
Jiminez,
M.,
and
Falk,
B.
W.
2003.
Genetic
diversity
and
biological
variation
among
California
isolates
of
Cucumber
mosaic
virus.
J.
Gen.
Virol.
84:
249­
258.
Liu,
S.,
He,
X.,
Park,
G.,
Josefsson,
C.,
and
Perry,
K.
L.
2002.
A
conserved
capsid
protein
surface
domain
of
Cucumber
mosaic
virus
is
essential
for
efficient
aphid
vector
transmission.
J.
Virol.
76:
9756­
9762.
Lommel,
S.
A.
and
Xiong,
Z.
1991.
Reconstitution
of
a
functional
red
clover
necrotic
mosaic
virus
by
recombinational
rescue
of
the
cell­
to­
cell
movement
gene
expressed
in
a
transgenic
plant.
J.
Cell
Biochem.
15A:
151.
López­
Moya,
J.
J.,
Wang,
R.
Y.,
and
Pirone,
T.
P.
1999.
Context
of
the
coat
protein
DAG
motif
affects
potyvirus
transmissibility
by
aphids.
J.
Gen.
Virol.
80:
3281­
3288.
Maiss,
E.,
Koenig,
R.,
and
Lesemann,
D.
E.
1994.
Heterologous
encapsidation
of
viruses
in
transgenic
plants
and
in
mixed
infections.
In
Jones,
D.
D.,
ed.
Biosafety
Results
of
Field
Tests
of
Genetically
Modified
Plants
and
Microorganisms.
University
of
California
,
Division
of
Agriculture
and
Natural
Resources,
Oakland,
pp
129­
139.
Page
16
of
17
Masuta,
C.,
Ueda,
S.,
Suzuki,
M.,
and
Uyeda,
I.
1998.
Evolution
of
a
quadripartite
hybrid
virus
by
interspecific
exchange
and
recombination
between
replicase
components
of
two
related
tripartite
RNA
viruses.
Proc.
Natl.
Acad.
Sci.
95:
10487­
10492.
Mayo,
M.
A.
and
Jolly,
C.
A.
1991.
The
5'­
terminal
sequence
of
potato
leafroll
virus
RNA:
evidence
of
recombination
between
virus
and
host
RNA.
J.
Gen.
Virol.
72:
2591­
2595.
Miller,
W.
A.,
Koev,
G.,
and
Beckett,
R.
2000.
Issues
surrounding
transgenic
resistance
to
the
Luteoviridae.
In
Schiemann,
J.,
ed.
The
Biosafety
Results
of
Field
Tests
of
Genetically
Modified
Plants
and
Microorganisms.
Biologische
Bundesanstalt
für
Land­
und
Forstwirtschaft,
Berlin,
pp
203­
209.
Miller,
W.
A.,
Koev,
G.,
and
Mohan,
B.
R.
1997.
Are
there
risks
associated
with
transgenic
resistance
to
luteoviruses?
Plant
Dis.
81:
700­
710.
Moonan,
F.,
Molina,
J.,
and
Mirkov,
T.
E.
2000.
Sugarcane
yellow
leaf
virus:
an
emerging
virus
that
has
evolved
by
recombination
between
luteoviral
and
poleroviral
ancestors.
Virology
269:
156­
171.
Nagy,
P.
D.
and
Bujarski,
J.
J.
1996.
Homologous
RNA
recombination
in
brome
mosaic
virus:
AU­
rich
sequences
decrease
the
accuracy
of
crossovers.
J.
Virol.
70:
415­
426.
Nagy,
P.
D.
and
Bujarski,
J.
J.
1998.
Silencing
homologous
RNA
recombination
hot
spots
with
GC­
rich
sequences
in
brome
mosaic
virus.
J.
Virol.
72:
1122­
1130.
Nagy,
P.
D.,
Ogiela,
C.,
and
Bujarski,
J.
J.
1999.
Mapping
sequences
active
in
homologous
RNA
recombination
in
brome
mosaic
virus:
prediction
of
recombination
hot
spots.
Virology
254:
92­
104.
Nagy,
P.
D.,
Zhang,
C.,
and
Simon,
A.
E.
1998.
Dissecting
RNA
recombination
in
vitro:
role
of
RNA
sequences
and
the
viral
replicase.
EMBO
J.
17:
2392­
2403.
OECD
Environment
Directorate
.
Consensus
document
on
general
information
concerning
the
biosafety
of
crop
plants
made
virus
resistant
through
coat
protein
gene­
mediated
protection.
http://
www.
olis.
oecd.
org/
olis/
1996doc.
nsf/
62f30f71be4ed8a24125669e003b5f73/
ce3a104b8ada9e8ac1256
3e2003183bb/$
FILE/
11E63213.
ENG
.
1996.
Osbourn,
J.
K.,
Sarkar,
S.,
and
Wilson,
T.
M.
A.
1990.
Complementation
of
coat
protein­
defective
TMV
mutants
in
transgenic
tobacco
plants
expressing
TMV
coat
protein.
Virology
179:
921­
925.
Palukaitis,
P.
2000.
Synergy
of
virus
accumulation
and
pathology
in
transgenic
plants
expressing
viral
sequences.
In
Schiemann,
J.,
ed.
The
Biosafety
Results
of
Field
Tests
of
Genetically
Modified
Plants
and
Microorganisms.
Biologische
Bundesanstalt
für
Land­
und
Forstwirtschaft,
Berlin,
pp
197­
202.
Perry,
K.
L.,
Zhang,
L.,
and
Palukaitis,
P.
1998.
Amino
acid
changes
in
the
coat
protein
of
cucumber
mosaic
virus
differentially
affect
transmission
by
the
aphids
Myzus
persicae
and
Aphis
gossypii.
Virology
242:
204­
210.
Pita,
J.
S.,
Fondong,
V.
N.,
Sangare,
A.,
Otim­
Nape,
G.
W.,
Ogwal,
S.,
and
Fauquet,
C.
M.
2001.
Recombination,
pseudorecombination
and
synergism
of
geminiviruses
are
determinant
keys
to
the
epidemic
of
severe
cassava
mosaic
disease
in
Uganda.
J.
Gen.
Virol.
82:
655­
665.
Pruss,
G.
J.,
Ge,
X.,
Shi,
X.
M.,
Carrington,
J.
C.,
and
Vance,
V.
B.
1997.
Plant
viral
synergism:
the
potyviral
genome
encodes
a
broad­
range
pathogenicity
enhancer
that
transactivates
replication
of
heterologous
viruses.
Plant
Cell
9:
859­
868.
Querci,
M.,
Owens,
R.
A.,
Bartolini,
I.,
Lazarte,
V.,
and
Salazar,
L.
F.
1997.
Evidence
for
heterologous
encapsidation
of
potato
spindle
tuber
viroid
in
particles
of
potato
leafroll
virus.
J.
Gen.
Virol.
78:
1207­
1211.
Reinbold,
C.,
Gildow,
F.
E.,
Herrbach,
E.,
Ziegler­
Graff,
V.,
Gonçalves,
M.
C.,
van
den
Heuvel,
J.
F.
J.
M.,
and
Brault,
V.
2001.
Studies
on
the
role
of
the
minor
capsid
protein
in
transport
of
Beet
western
yellows
virus
through
Myzus
persicae.
J.
Gen.
Virol.
82:
1995­
2007.
Revers,
F.,
Le
Gall,
O.,
Candresse,
T.,
Le
Romancer,
M.,
and
Dunez,
J.
1996.
Frequent
occurrence
of
recombinant
potyvirus
isolates.
J.
Gen.
Virol.
77:
1953­
1965.
Robinson,
D.
J.
1996.
Environmental
risk
assessment
of
releases
of
transgenic
plants
containing
virus­
derived
inserts.
Transgenic
Research
5:
359­
362.
Rochow,
W.
F.
1970.
Barley
yellow
dwarf
virus:
phenotypic
mixing
and
vector
specificity.
Science
167:
875­
878.
Roossinck,
M.
J.
1997.
Mechanisms
of
plant
virus
evolution.
Annu.
Rev.
Phytopathol.
35:
191­
209.
Rubio,
T.,
Borja,
M.,
Scholthof,
H.
B.,
and
Jackson,
A.
O.
1999.
Recombination
with
host
transgenes
and
effects
on
virus
evolution:
an
overview
and
opinion.
Mol.
Plant
Microbe
Interact.
12:
87­
92.
Salánki,
K.,
Carrère,
I.,
Jacquemond,
M.,
Balázs,
E.,
and
Tepfer,
M.
1997.
Biological
properties
of
pseudorecombinant
and
recombinant
strains
created
with
cucumber
mosaic
virus
and
tomato
aspermy
virus.
J.
Virol.
71:
3597­
3602.
Schoelz,
J.
E.
and
Wintermantel,
W.
M.
1993.
Expansion
of
viral
host
range
through
complementation
and
recombination
in
transgenic
plants.
Plant
Cell
5:
1669­
1679.
Page
17
of
17
Spitsin,
S.,
Steplewski,
K.,
Fleysh,
N.,
Belanger,
H.,
Mikheeva,
T.,
Shivprasad,
S.,
Dawson,
W.,
Koprowski,
H.,
and
Yusibov,
V.
1999.
Expression
of
alfalfa
mosaic
virus
coat
protein
in
tobacco
mosaic
virus
(
TMV)
deficient
in
the
production
of
its
native
coat
protein
supports
long­
distance
movement
of
a
chimeric
TMV.
Proc.
Natl.
Acad.
Sci.
96:
2549­
2553.
Tepfer,
M.
1993.
Viral
genes
and
transgenic
plants:
What
are
the
potential
environmental
risks?
Biotechnology
(
N
Y)
11:
1125­
1132.
Tepfer,
M.
2002.
Risk
assessment
of
virus­
resistant
transgenic
plants.
Annu.
Rev.
Phytopathol.
40:
467­
491.
Teycheney,
P.
Y.,
Aaziz,
R.,
Dinant,
S.,
Salánki,
K.,
Tourneur,
C.,
Balázs,
E.,
Jacquemond,
M.,
and
Tepfer,
M.
2000.
Synthesis
of
(­)­
strand
RNA
from
the
3'
untranslated
region
of
plant
viral
genomes
expressed
in
transgenic
plants
upon
infection
with
related
viruses.
J.
Gen.
Virol.
81:
1121­
1126.
Thomas,
P.
E.,
Hassan,
S.,
Kaniewski,
W.
K.,
Lawson,
E.
C.,
and
Zalewski,
J.
C.
1998.
A
search
for
evidence
of
virus/
transgene
interactions
in
potatoes
transformed
with
the
potato
leafroll
virus
replicase
and
coat
protein
genes.
Molecular
Breeding
4:
407­
417.
Tolin,
S.
A.
1991.
Persistence,
establishment,
and
mitigation
of
phytopathogenic
viruses.
In
Levin,
M.
A.
and
Strauss,
H.
S.,
eds.
Risk
Assessment
in
Genetic
Engineering.
McGraw­
Hill,
Inc.,
New
York,
pp
114­
139.
Vance,
V.
B.,
Berger,
P.
H.,
Carrington,
J.
C.,
Hunt,
A.
G.,
and
Shi,
X.
M.
1995.
5'
proximal
potyviral
sequences
mediate
potato
virus
X/
potyviral
synergistic
disease
in
transgenic
tobacco.
Virology
206:
583­
590.
Varrelmann,
M.
and
Maiss,
E.
2000.
Mutations
in
the
coat
protein
gene
of
Plum
pox
virus
suppress
particle
assembly,
heterologous
encapsidation
and
complementation
in
transgenic
plants
of
Nicotiana
benthamiana.
J.
Gen.
Virol.
81:
567­
576.
Varrelmann,
M.,
Palkovics,
L.,
and
Maiss,
E.
2000.
Transgenic
or
plant
expression
vector­
mediated
recombination
of
Plum
pox
virus.
J.
Virol.
74:
7462­
7469.
Vigne,
E.,
Komar,
V.,
and
Fuchs,
M.
2004.
Field
safety
assessment
of
recombination
in
transgenic
grapevines
expressing
the
coat
protein
gene
of
Grapevine
fanleaf
virus.
Transgenic
Research
13:
165­
179.
Waterhouse,
P.
M.,
Wang,
M.
B.,
and
Lough,
T.
2001.
Gene
silencing
as
an
adaptive
defence
against
viruses.
Nature
411:
834­
842.
White,
J.
L.
2000.
An
overview
on
cultivation
of
virus­
resistant
crops
in
the
United
States.
In
Schiemann,
J.,
ed.
The
Biosafety
Results
of
Field
Tests
of
Genetically
Modified
Plants
and
Microorganisms.
Biologische
Bundesanstalt
für
Land­
und
Forstwirtschaft,
Berlin,
pp
177­
181.
Wintermantel,
W.
M.
and
Schoelz,
J.
E.
1996.
Isolation
of
recombinant
viruses
between
cauliflower
mosaic
virus
and
a
viral
gene
in
transgenic
plants
under
conditions
of
moderate
selection
pressure.
Virology
223:
156­
164.
Worobey,
M.
and
Holmes,
E.
C.
1999.
Evolutionary
aspects
of
recombination
in
RNA
viruses.
J.
Gen.
Virol.
80:
2535­
2543.
Zhou,
X.,
Liu,
Y.,
Calvert,
L.,
Munoz,
C.,
Otim­
Nape,
G.
W.,
Robinson,
D.
J.,
and
Harrison,
B.
D.
1997.
Evidence
that
DNA­
A
of
a
geminivirus
associated
with
severe
cassava
mosaic
disease
in
Uganda
has
arisen
by
interspecific
recombination.
J.
Gen.
Virol.
78:
2101­
2111.
