Page
1
of
20
MEETING
DOCUMENT:
FIFRA
SAP
MEETING
CONCERNING
PLANTINCORPORATED
PROTECTANTS
BASED
ON
PLANT
VIRAL
COAT
PROTEIN
GENES
(
PVCP­
PIPS)

DECEMBER
6­
8,
2005
HOLIDAY
INN­
NATIONAL
AIRPORT,
ARLINGTON,
VIRGINIA
Purpose
The
purpose
of
this
meeting
is
to
seek
the
advice
of
the
Federal
Insecticide,
Fungicide
and
Rodenticide
Act
(
FIFRA)
Scientific
Advisory
Panel
(
SAP)
on
a
number
of
technical
issues
associated
with
a
type
of
pesticide,
plant­
incorporated
protectants
based
on
plant
virus
coat
protein
genes
(
PVCP­
PIPs).
To
this
end,
the
Environmental
Protection
Agency
(
EPA)
is
providing
to
the
FIFRA
SAP
this
cover
document
which
poses
a
series
of
questions
to
the
SAP,
and
four
attached
documents,
which
provide
additional
information
on
the
topics
before
the
Panel.
The
four
attachments
are:

Attachment
I:
Draft
Approach
to
Exempting
Certain
PVCP­
PIPs
from
Regulation
under
FIFRA
Attachment
II:
Draft
Approach
to
Exempting
Certain
PVC­
Proteins
from
the
Requirement
of
a
Tolerance
under
FFDCA
Attachment
III:
Environmental
Risk
Assessment
of
Plant
Incorporated
Protectant
(
PIP)
Inert
Ingredients
Attachment
IV:
Minutes
of
the
October
13­
15,
2004
FIFRA
Scientific
Advisory
Panel
Meeting
on
Issues
Associated
with
Deployment
of
a
Type
of
Plant­
Incorporated
Protectant
(
PIP),
Specifically
those
Based
on
Plant
Viral
Coat
Proteins
(
PVCPPIPs

The
material
introducing
each
question
articulated
in
this
cover
document
gives
a
general
cursory
overview
of
the
issue
on
which
EPA
requests
technical
advice
from
the
SAP.
For
additional
information
on
these
questions,
SAP
members
are
directed
to
the
relevant
sections
of
first
three
attachments
which
provide
more
detailed
descriptions
of
the
Agency
analysis
of
the
technical
issue.
In
some
questions,
the
Agency
asks
the
SAP
whether
Agency
interpretations
of
available
information
could
support
the
technical
rationales
advanced
by
the
EPA
in
these
attachments.

As
this
meeting
builds
on
a
previous
meeting
of
the
SAP
on
PVCP­
PIPs,
EPA
includes
in
this
package
the
minutes
of
the
October
13­
15,
2004
meeting
as
the
fourth
attachment
to
this
cover
document.
Page
2
of
20
Introduction
A
plant­
incorporated
protectant
(
PIP)
is
defined
as
a
pesticidal
substance
that
is
intended
to
be
produced
and
used
in
a
living
plant,
or
in
the
produce
thereof1,
and
the
genetic
material
necessary
for
production
of
such
a
pesticidal
substance.
The
definition
includes
both
active
and
inert
ingredients2.

PIPs
may
be
genetically
engineered
into
plants3.
Some
specific
genetic
sequences,
when
incorporated
into
a
plant's
genome,
can
endow
the
plant
with
the
ability
to
resist
damage
from
certain
pests.
EPA
considers
plant
virus
coat
protein
PIPs
(
PVCP­
PIPs)
to
be
those
PIPs
based
on
one
or
more
genes
that
encode
a
coat
protein
of
a
virus
that
naturally
infects
plants.
This
includes
PVCP­
PIPs
that
produce
no
protein.
Incorporation
of
plant
viral
coat
protein
gene
sequences
into
plant
genomes
has
been
found
to
confer
resistance
to
the
virus
from
which
it
was
derived,
and
often
to
related
viruses
(
OECD
Environment
Directorate
1996).

PVCP­
PIPs
are
regulated
as
pesticides
by
EPA
under
the
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA)
because
they
meet
the
FIFRA
definition
of
a
pesticide,
being
intended
for
preventing,
destroying,
repelling,
or
mitigating
a
pest.
Residues
of
PVCP­
PIPs
in
food
are
regulated
by
EPA
under
the
Federal
Food,
Drug,
and
Cosmetic
Act
(
FFDCA).

Background
 
FIFRA
Regulation
In
1994,
EPA
proposed
two
options
to
exempt
PVCP­
PIPs
under
FIFRA.
The
first
option
was
a
full
categorical
exemption
based
on
the
rationale
that
PVCP­
PIPs
generally
pose
a
low
probability
of
risk
to
human
health
and
the
environment.
However,
recognizing
that
other
plants
could
acquire
the
virus
resistance
through
hybridization
with
a
transgenic
plant,
an
alternative
to
a
full
categorical
exemption
was
also
proposed.
Under
this
alternative
exemption
option,
the
Agency
defined
a
set
of
criteria
to
identify
those
PVCP­
PIP/
plant
combinations
with
the
lowest
potential
to
confer
selective
advantage
on
wild
or
weedy
plant
relatives.
Only
those
PVCP­
PIPs
that
met
the
criteria
would
have
been
exempt
from
regulation.

EPA
has
yet
to
finalize
a
FIFRA
exemption
for
PVCP­
PIPs,
in
part
because
more
recent
information
has
raised
questions
about
whether
all
PVCP­
PIPs
pose
low
risks,
and
EPA
must
be
able
to
make
such
a
finding
in
order
to
exempt
all
PVCP­
PIPs
under
FIFRA.
For
example,
the
2000
National
Research
Council
(
NRC)
report,
Genetically
Modified
Pest­
Protected
Plants,
recommended
that
1
The
phrase
"
or
produce
thereof"
is
included
in
the
definition
of
a
PIP
to
make
it
clear
that
pesticidal
substances
active
in
the
fruit
or
other
plant
product
for
pesticidal
purposes
are
also
considered
to
be
PIPs.
2
"
Inert
ingredient"
means
any
substance,
such
as
a
selectable
marker,
other
than
the
active
ingredient,
where
the
substance
is
used
to
confirm
or
ensure
the
presence
of
the
active
ingredient,
and
includes
the
genetic
material
necessary
for
the
production
of
the
substance,
provided
that
genetic
material
is
intentionally
introduced
into
a
living
plant
in
addition
to
the
active
ingredient.
3
PIPs
may
also
be
found
naturally
occurring
in
plants
or
may
be
introduced
through
conventional
breeding.
However,
the
focus
here
is
on
PIPs
based
on
plant
viral
coat
protein
genes
that
are
introduced
into
plants
through
genetic
engineering.
Page
3
of
20
"
EPA
should
not
categorically
exempt
viral
coat
proteins
from
regulation
under
FIFRA.
Rather,
EPA
should
adopt
an
approach,
such
as
the
agency's
alternative
proposal,
that
allows
the
agency
to
consider
the
gene
transfer
risks
associated
with
the
introduction
of
viral
coat
proteins
to
plants."

In
addition
to
the
risks
associated
with
gene
transfer,
the
2000
NRC
report
considered
other
risk
issues
in
the
context
of
PVCP­
PIPs,
including
the
potential
for
adverse
effects
following
recombination,
heterologous
encapsidation,
and
synergy.
Although
the
report
concluded
that,
"[
m]
ost
virus­
derived
resistance
genes
are
unlikely
to
present
unusual
or
unmanageable
problems
that
differ
from
those
associated
with
traditional
breeding
for
virus
resistance,"
it
also
suggested
strategies
to
reduce
or
eliminate
such
concerns.
Neither
formulation
of
the
exemption
EPA
proposed
in
1994
contained
provisions
that
would
have
enabled
EPA
to
ensure
that
the
risk
management
strategies
suggested
by
the
NRC
would
be
implemented,
e.
g.,
elimination
of
specific
sequences
to
limit
the
potential
for
recombination.

Background
 
FFDCA
Regulation
In
1994
EPA
proposed
exempting
the
plant
virus
coat
protein
portion
of
a
PVCP­
PIP
(
PVCprotein
from
the
requirement
of
a
food
tolerance
under
FFDCA
based
on
the
rationale
that
(
1)
virus
infected
plants
have
always
been
a
part
of
the
human
and
domestic
animal
food
supply
and
(
2)
plant
viruses
have
never
been
shown
to
be
infectious
to
humans
or
other
mammals.
The
safety
of
consuming
plant
virus
genes
has
since
been
supported
by
experimental
investigations
(
Chen
et
al.
2003;
Rogan
et
al.
2000;
Shinmoto
et
al.
1995)
and
expert
consultations
including
the
2000
NRC
report
which
concluded
that,
"
viral
coat
proteins
in
transgenic
pest­
protected
plants
are
not
expected
to
jeopardize
human
health
because
consumers
already
ingest
these
compounds
in
nontransgenic
food"
(
National
Research
Council
2000).

EPA
has
not
finalized
the
proposed
tolerance
exemption
for
PVC­
proteins4,
in
part
because
it
has
been
unclear
how
to
describe
the
PVC­
proteins
that
have
a
history
of
safe
human
dietary
consumption
and
that
would
therefore
fall
within
the
base
of
information
supporting
the
1994
proposal.
EPA
recognizes
that
PVCP­
PIP
developers
may
need
to
modify
the
coat
protein
gene
for
appropriate
gene
expression
or
may
wish
to
modify
the
coat
protein
gene
to
achieve
other
goals,
e.
g.,
to
reduce
the
frequency
of
recombination.
Such
modifications
might
result
in
changes
to
the
protein
produced
such
that
the
rationale
used
to
support
exemption
(
i.
e.,
a
history
of
exposure)
might
not
apply.

EPA's
Current
Approach
In
October
2004,
EPA
consulted
the
FIFRA
SAP
on
a
number
of
scientific
issues
identified
for
PVCP­
PIPs.
After
carefully
considering
this
advice
and
other
available
scientific
and
regulatory
4
Although
a
general
tolerance
exemption
covering
a
category
of
PVC­
proteins
has
not
been
finalized,
tolerance
exemptions
for
specific
PVC­
proteins
have
been
issued
(
i.
e.,
coat
protein
of
Potato
Virus
Y,
coat
protein
of
Watermelon
Mosaic
Virus­
2
and
Zucchini
Yellow
Mosaic
Virus,
coat
protein
of
Papaya
Ringspot
Virus,
and
coat
protein
of
Cucumber
Mosaic
Virus).
Nucleic
acids
are
also
currently
exempt
from
FFDCA
tolerance
requirements
(
40
CFR
174.475).
Page
4
of
20
information,
the
Agency
is
now
developing
proposals
for
an
exemption
under
FIFRA
and
an
exemption
under
FFDCA.

Under
FIFRA,
one
approach
that
EPA
is
considering
would
involve
criteria
that
would
clearly
identify
and
exempt
only
those
PVCP­
PIPs
that
fall
within
the
base
of
experience
used
to
support
the
exemption
and
pose
low
risk
to
human
health
and
the
environment.
Under
this
approach,
EPA
has
identified
three
possible
criteria,
each
of
which
could
be
satisfied
in
one
of
two
ways.
The
first
method,
articulated
in
paragraph
(
1),
describes
an
objective,
well­
defined
characteristic
of
the
PVCP­
PIP
and
could
therefore
be
self­
evaluated
by
a
product
developer.
The
second
method,
described
in
paragraph
(
2),
involves
consideration
of
several
types
of
information,
and
an
Agency
review
would
therefore
be
required
to
determine
qualification.
PVCP­
PIPs
that
do
not
qualify
for
any
proposed
FIFRA
exemption
could
be
submitted
for
a
case­
by­
case
review
for
registration
as
is
required
for
non­
exempted
PIPs
distributed
or
sold
in
commerce.

Under
FFDCA,
EPA
is
considering
two
possible
tolerance
exemptions
for
PVC­
protein
residues:
a
categorical
exemption
for
a
subset
of
PVC­
proteins
based
on
objective,
well­
defined
characteristics
of
the
PVC­
protein,
and
an
exemption
conditional
on
an
Agency
determination
after
review
that
certain
other
criteria
are
met.
The
potential
criteria
in
both
exemptions
are
intended
to
identify
clearly
only
those
residues
for
which
a
long
history
of
safe
exposure
and
consumption
can
support
exemption.
PVC­
proteins
that
qualify
for
neither
proposed
tolerance
exemption
could
be
submitted
for
a
case­
by­
case
review
for
an
individual
tolerance
exemption
as
is
required
for
all
non­
exempted
PIP
residues
that
may
be
present
in
food
or
feed.

The
FIFRA
SAP
is
being
asked
to
advise
the
Agency
on
a
number
of
scientific
issues
pertinent
to
determining
the
potential
impact
of
these
approaches
on
human
health
and
the
environment.
Four
relevant
documents
are
attached.

Attachment
I,
"
Draft
Approach
to
Exempting
Certain
PVCP­
PIPs
from
regulation
under
FIFRA,"
outlines
the
scientific
issues
EPA
has
considered
in
evaluating
PVCP­
PIPs
for
possible
exemption.

Attachment
II,
"
Draft
Approach
to
Exempting
Certain
PVC­
Proteins
from
the
Requirement
of
a
Tolerance
under
FFDCA,"
outlines
the
scientific
issues
EPA
has
considered
in
evaluating
PVCproteins
for
a
possible
tolerance
exemption.

Attachment
III,
"
Environmental
Risk
Assessment
of
Plant
Incorporated
Protectant
(
PIP)
Inert
Ingredients,"
covers
EPA's
environmental
risk
assessment
of
six
selectable
markers.

Attachment
IV,
"
Minutes
of
the
October
13­
15,
2004
FIFRA
Scientific
Advisory
Panel
Meeting
on
Issues
Associated
with
Deployment
of
a
Type
of
Plant­
Incorporated
Protectant
(
PIP),
Specifically
those
Based
on
Plant
Viral
Coat
Proteins
(
PVCP­
PIPs),"
is
the
minutes
of
the
most
recent
SAP
meeting
convened
to
address
issues
associated
with
PVCP­
PIPs.
Page
5
of
20
Charge
Questions
to
the
Panel
Gene
Flow
Issues
The
first
criterion
EPA
is
considering
under
any
proposed
FIFRA
exemption
concerns
gene
flow/
transfer.
While
gene
flow
from
plants
containing
PVCP­
PIPs
does
not
necessarily
constitute
an
environmental
risk,
there
are
concerns
that
wild
or
weedy
relatives
that
could
acquire
the
PVCP­
PIP
might
acquire
the
potential
to
escape
any
significant
growth
and/
or
reproduction
constraints
imposed
on
the
plant
by
natural
virus
infection,
and
such
events
could
in
turn
impact
the
ecosystem
to
a
degree
that
is
not
yet
predictable.

Although
many
events
must
occur
before
the
transfer
of
a
PVCP­
PIP
from
crop
plants
to
wild
or
weedy
relatives
would
significantly
change
plant
population
dynamics,
such
a
series
of
events
could
reasonably
be
expected
to
occur
for
a
few
PVCP­
PIP/
plant
combinations.
Research
showing
that
plants
infected
with
viruses
often
have
decreased
growth,
survivorship,
and/
or
reproduction
(
Yahara
&
Oyama
1993;
Friess
&
Maillet
1996;
Funayama
et
al.
1997;
Maskell
et
al.
1999)
suggests
that
introgression
of
a
virus
resistance
gene
into
some
plant
populations
might
allow
a
population
to
outcompete
other
plant
species
(
Power
2002)
or
have
other
effects
on
ecosystem
relationships.
In
addition,
a
few
studies
confirm
that
virus
infection
can
in
some
cases
affect
plant
population
dynamics
(
Jones
&
Nicholas
1998;
Funayama
et
al.
2001).
Acquisition
of
virus
resistance
has
also
been
found
in
some
cases
to
decrease
plant
fitness
(
e.
g.,
Remold
2002),
due
in
one
case
to
plants
becoming
more
attractive
to
herbivores
when
not
infected
by
viruses
(
Gibbs
1980).
Such
considerations
may
be
important
in
evaluating
effects
on
endangered/
threatened
species.
Whether
these
types
of
changes
could
result
in
adverse
environmental
effects
is
still
unresolved.

In
order
to
develop
a
proposed
exemption,
EPA
seeks
a
straightforward,
easy­
to­
understand
criterion
to
identify
those
crop
plants
containing
a
PVCP­
PIP
that
pose
low
probability
of
risk
with
respect
to
the
potential
of
gene
transfer
to
lead
to
significant
changes
in
plant
population
dynamics.
The
inability
of
the
plant
to
form
viable
hybrids
with
wild
or
weedy
plants
in
the
United
States
provides
straightforward
assurance
in
a
clearly
articulated
criterion
that
adverse
effects
due
to
gene
transfer
are
unlikely
to
occur
for
a
particular
PVCP­
PIP/
plant
combination.

With
the
assistance
of
the
October
2004
SAP,
EPA
has
identified
the
following
plants
as
not
having
wild
or
weedy
relatives
in
the
United
States,
its
possessions,
or
territories5
with
which
they
can
produce
viable
hybrids
in
nature:
almond
(
Prunus
communis),
apricot
(
Prunus
armeniaca),
asparagus
(
Asparagus
officinale),
avocado
(
Persea
americana),
banana
(
Musa
acuminata),
barley
(
Hordeum
vulgare),
bean
(
Phaseolus
vulgaris),
black­
eyed
pea
(
Vigna
unguiculata),
cacao
(
Theobroma
cacao),
celery
(
Apium
graveolens),
chickpea
(
Cicer
arietinum),
citrus
(
Citrus
spp.),
coffee
(
Coffea
arabicua),
corn
(
Zea
maize),
cucumber
(
Cucumis
sativus),
eggplant
(
Solanum
melongena),
guava
(
Psidium
guajava),
kiwi
(
Actinidia
spp.),
mango
(
Mangifera
indica),
nectarine
(
Prunus
persica),
okra
(
Abelmoschus
esculentus),
olive
(
Olea
europaea),
papaya
(
Carica
papaya),
parsley
(
Petroselinum
crispum),
pea
(
Pisum
sativum),
peach
(
Prunus
persica),
peanut
(
Arachis
hypogaea),
pineapple
(
Ananas
comosus),
pistachio
(
Pistacia
vera),
plum
(
Prunus
domestica),
potato
(
Solanum
tuberosum),
soybean
(
Glycine
max),
spinach
5
Includes
the
Commonwealth
of
Puerto
Rico,
the
Virgin
Islands,
Guam,
the
Trust
Territory
of
the
Pacific
Islands,
and
American
Samoa.
Page
6
of
20
(
Spinacia
oleracea),
starfruit
(
Averrhoa
carambola),
taro
(
Colocasia
esculenta),
tomato
(
Lycopersicon
lycopersicum),
or
watermelon
(
Citrullus
lanatus).

1(
a).
Does
this
list
identify
plant
species
that
would
present
low
risk
of
conferring
any
selective
advantage
on
a
wild
or
weedy
relative
in
the
United
States,
its
possessions,
or
territories5
were
they
to
contain
a
PVCP­
PIP?
Please
explain
the
basis
for
your
answer,
providing
documentation
to
support
your
decision.

The
October
2004
SAP
noted
that
some
of
the
plants
on
this
list
(
i.
e.,
asparagus
and
celery)
were
able
to
escape
cultivation
and
form
occasional
volunteer
populations.
EPA
notes
that
in
addition
to
these
two
species,
many
other
species
from
this
list
have
naturalized
populations
in
the
United
States
(
i.
e.,
plants
occurring
in
natural
areas
outside
of
agricultural
fields
and
not
simply
volunteer
plants
within
other
fields).
For
example,
the
USDA
PLANTS
database
(
accessible
at
http://
plants.
usda.
gov/)
indicates
that
almond,
apricot,
avocado,
banana,
barley,
bean,
black­
eyed
pea,
cacao,
chickpea,
citrus,
coffee,
corn,
cucumber,
eggplant,
guava,
mango,
okra,
olive,
papaya,
parsley,
pea,
peach,
peanut,
pineapple,
plum,
potato,
soybean,
spinach,
taro,
tomato,
and
watermelon
have
"
native
or
naturalized"
6
populations
in
the
United
States.

Based
on
this
information,
it
appears
that
the
ability
to
naturalize
is
common
for
crops.
However,
it
can
be
hypothesized
that
naturalized
populations
of
these
particular
crop
plants
possess
a
suite
of
traits
that
facilitate
cultivation
in
a
managed
habitat
and
likely
confer
a
selective
disadvantage
on
plants
in
the
wild.
Acquisition
of
a
single
trait,
i.
e.,
virus
resistance,
would
therefore
not
be
expected
to
provide
sufficient
competitive
advantage
to
make
naturalized
populations
of
these
plants
significant
weed
problems
outside
of
agricultural
fields.

1(
b).
What
data
supports
or
refutes
the
rationale
above
that
naturalized
populations
of
plants
on
the
list
in
question
1(
a)
would
not
be
expected
to
become
weedy
or
invasive
outside
of
agricultural
fields
if
they
were
to
acquire
virus
resistance
from
a
PVCP­
PIP
(
assuming
that
the
cultivated
crop
is
negatively
affected
by
virus
infection
and
a
PVCP­
PIP
targeted
at
that
virus
is
developed)?
If
the
rationale
does
not
apply
to
all
the
crops
on
this
list,
is
there
an
alternative
rationale
that
would
apply
to
particular
plant
species?

1(
c).
Please
list
any
additional
plants
(
including
genus
and
species)
that
both
(
1)
have
no
wild
or
weedy
relatives
in
the
United
States,
its
possessions,
or
territories
with
which
they
can
form
viable
hybrids
in
nature
and
(
2)
have
low
potential
to
naturalize
and
become
weedy
or
invasive
outside
of
agricultural
fields
with
the
acquisition
of
any
PVCP­
PIP.
For
each
identified
plant
please
explain
why
(
2)
is
likely
the
case.

6
In
the
PLANTS
database,
Native
means
naturally
occurring
in
North
America
and
the
U.
S.
and
its
territories
at
the
time
of
Columbus;
Introduced
means
arrival
from
some
other
part
of
the
world
since
Columbus's
time.
Naturalized
plants
are
introduced
plants
that
now
exist
in
the
wild
without
assistance
from
humankind;
in
PLANTS
these
are
called
Introduced
since
this
term
is
more
familiar
to
most
people
than
naturalized.
In
PLANTS,
"
Native
and
Introduced"
applies
to
species
with
both
native
and
introduced
varieties
or
subspecies.
And
if
a
plant
is
native
to
one
part
of
the
U.
S.
and
introduced
in
another,
it
is
coded
as
Native.
Page
7
of
20
A
list
of
plants
such
as
those
in
question
1
offers
a
well­
defined
criterion
for
identifying
crop
plants
that
pose
low
probability
of
risk
with
respect
to
concerns
associated
with
gene
transfer
when
they
contain
a
PVCP­
PIP.
However,
EPA
recognizes
that
other,
perhaps
less
well­
defined
criteria
could
also
identify
PVCP­
PIPs
that
are
low
risk.
EPA
is
requesting
the
SAP's
advice
on
whether
a
PVCP­
PIP
would
pose
low
risk
with
respect
to
concerns
associated
with
gene
flow
if
the
Agency
determines
that
the
plant
containing
the
PVCP­
PIP
(
i)
is
itself
not
a
weedy
or
invasive
species
outside
of
agricultural
fields
in
the
United
States,
its
possessions,
or
territories,
and
(
ii)
does
not
have
relatives
outside
of
agricultural
fields
in
the
United
States,
its
possessions,
or
territories
that
are
weedy
or
invasive
species
or
endangered/
threatened
species
with
which
it
can
produce
viable
hybrids
in
nature.

The
rationale
for
such
a
criterion
would
be
that
there
is
a
low
probability
that
acquisition
of
a
virus­
resistance
trait
would
confer
on
plants
that
are
not
already
weedy
or
invasive
sufficient
additional
competitive
advantage
to
lead
to
adverse
environmental
outcomes.
With
regard
to
consideration
of
whether
a
plant
population
is
under
viral
disease
selection
pressure
and
whether
this
pressure
is
the
only
condition
restraining
the
population,
conventional
agriculture
offers
some
insight.
Virus­
resistant
varieties
of
certain
crops
have
been
bred
and
grown
in
the
past,
generally
using
a
wild
relative
of
the
crop
plant
as
the
source
of
resistance.
There
is
no
indication
that
growing
such
crop
plants
near
wild
or
weedy
relatives
results
in
these
relatives
becoming
any
more
of
a
weed
problem
due
to
acquiring
virus
resistance
from
the
crop
(
National
Research
Council
1989).
It
is
unlikely
that
use
of
PVCP­
PIPs
would
affect
wild
or
weedy
relatives
differently
than
virus
resistant
varieties
developed
through
traditional
breeding
have
affected
wild
or
weedy
relatives
in
the
past.
In
addition,
outbreeding
depression
between
crop
plants
and
their
wild
relatives
appears
to
be
more
common
than
hybrid
vigor
(
Hails
&
Morley
2005).
In
outbreeding
depression,
mating
between
individuals
from
two
different
environments
can
disrupt
gene
combinations
that
are
favored
by
natural
selection
in
each
environment.
Resulting
offspring
may
have
phenotypes
that
are
poorly
adapted
to
the
habitat
of
either
parent.
Thus,
hybrid
offspring
acquiring
a
PVCP­
PIP
are
often
likely
to
be
less
competitive
than
their
wild
parent
in
nature.
When
EPA
asked
the
FIFRA
SAP
in
2004
about
the
likelihood
that
plant
populations
freed
from
viral
pressure
could
have
increased
competitive
ability
leading
to
changes
in
plant
population
dynamics,
the
FIFRA
SAP
offered
the
following
opinion:
"[
b]
ased
on
knowledge
obtained
from
observation
of
cultivated
crops
in
the
agroecosystem,
the
majority
of
the
[
2004]
Panel
concluded
that
it
would
be
unlikely
that
a
plant
population
freed
from
viral
pressure
would
give
a
plant
species
a
competitive
advantage"
(
Ref.
U.
S.
Environmental
Protection
Agency
2004).

2(
a).
Please
comment
on
whether
the
following
criteria
would
allow
the
Agency
to
identify
correctly
those
PVCP­
PIPs
that
present
low
risk
with
respect
to
environmental
concerns
associated
with
gene
flow
of
a
PVCP­
PIP.
What
data
supports
or
refutes
the
Agency's
rationale
for
developing
these
criteria?

(
i).
the
plant
containing
the
PVCP­
PIP
is
itself
not
a
weedy
or
invasive
species
outside
of
agricultural
fields
in
the
United
States,
its
possessions,
or
territories,
and
Page
8
of
20
(
ii).
the
plant
containing
the
PVCP­
PIP
does
not
have
relatives
outside
of
agricultural
fields
in
the
United
States,
its
possessions,
or
territories
that
are
weedy
or
invasive
species
or
endangered/
threatened
species
with
which
it
can
produce
viable
hybrids
in
nature.

2(
b).
Are
there
other
factors
besides
a
plant's
weediness,
invasiveness,
and/
or
endangered/
threatened
status
that
should
be
taken
into
consideration
when
evaluating
whether
a
PVCP­
PIP
poses
low
risk
with
respect
to
environmental
concerns
associated
with
gene
flow
of
a
PVCP­
PIP?

In
its
evaluation
of
(
i)
and
(
ii)
above,
EPA
would
consider
the
most
recent
scientific
information
about
the
plant
species
containing
the
PVCP­
PIP
and
its
wild
or
weedy
relatives
to
evaluate
the
potential
for
weedy
or
invasive
behavior,
including
whether
any
of
these
species
are
extending
their
range.
The
Agency
would
evaluate
a
number
of
sources
including
existing
lists
of
invasive
weeds,
e.
g.,
the
Federal
Noxious
Weed
List.
Inclusion
on
any
given
list
would
be
informative,
but
not
determinative
for
the
Agency's
evaluation.
Examination
of
existing
lists
has
shown
that
different
organizations
use
different
criteria
for
listing
species
depending
on
the
goals
and
missions
of
those
organizations.
Thus,
the
Agency
is
considering
using
existing
lists
as
a
resource
much
as
it
would
use
published
literature,
rather
than
as
determinative
sources.
For
example,
plants
that
may
form
volunteer
populations
in
agricultural
fields
are
considered
weeds
by
some
organizations
and
may
appear
on
those
organizations'
weed
lists,
but
EPA
would
not
consider
propensity
to
volunteer,
i.
e.,
to
grow
in
a
field
from
seeds
dropped
from
the
previous
crop
rotation,
to
be
indicative
of
general
weediness
potential
for
a
plant.

2(
c).
Please
describe
any
additional
factors
beyond
those
listed
above
that
the
Agency
could
use
to
evaluate
whether
a
PVCP­
PIP
meets
(
i)
or
(
ii).

Viral
Interactions
Issues
The
second
criterion
EPA
is
considering
as
part
of
a
FIFRA
exemption
concerns
viral
interactions.
The
Ecological
Society
of
America
noted
that
such
interactions
could
lead
to
the
creation
of
viruses
with
enhanced
disease
characteristics
or
new
transmission
properties
(
Snow
et
al.
2005).
However,
mixed
viral
infections
are
extremely
common
in
crops
and
other
plants
(
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
(
e.
g.,
through
recombination,
heterologous
encapsidation,
or
synergy).
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,
numerous
recombination
events
among
tomato­
infecting
begomoviruses
around
the
Nile
and
Mediterranean
Basins
are
thought
to
be
at
least
partially
responsible
for
numerous
whiteflytransmitted
tomato
diseases
that
have
emerged
in
the
last
20
years
(
Fauquet
et
al.
2005).
In
Page
9
of
20
addition,
as
the
2004
SAP
pointed
out,
"[
i]
n
contrast
to
heterologous
encapsidation
and
synergy,
at
least
in
theory,
the
impact
of
recombination
could
be
much
greater,
since
there
is
no
abundant
bioinformatics
evidence
that
recombination
has
indeed,
as
had
been
long
suspected,
played
a
key
role
in
the
emergence
of
new
viruses
over
evolutionary
time"
(
U.
S.
Environmental
Protection
Agency
2004).
Interactions
between
viral
transgenes
and
an
infecting
virus
may
be
a
concern
to
the
extent
that
such
events
are
novel,
i.
e.,
involve
viruses
that
would
otherwise
not
be
expected
to
interact
in
a
mixed
infection
found
in
nature.

The
Agency
asked
the
FIFRA
SAP
during
the
October
2004
meeting
to
what
extent
PVCP­
PIPs
in
plants
might
present
a
potential
concern
should
interactions
with
infecting
viruses
occur.
The
Panel
expressed
concern
only
"
about
certain
limited
situations"
and
stated
that
"
except
perhaps
for
a
very
few
cases,
neither
heterologous
encapsidation
nor
synergy
should
be
considered
to
be
of
serious
concern"
and
"
in
most
cases
there
is
little
a
priori
reason
to
believe
that
recombinants
between
viruses
and
transgenes
will
be
more
of
a
problem
than
recombinants
between
two
viruses
infecting
the
same
plant,
unless
transgenes
are
derived
from
severe
or
exotic
isolates.
The
general
recommendation
to
use
mild,
endemic
isolates
as
the
source
of
the
transgene
(
e.
g.
Hammond
et
al.
1999)
should
minimize
any
potential
for
creation
of
novel
isolates
that
would
not
equally
easily
arise
in
natural
mixed
infections."

Based
on
the
advice
from
the
October
2004
SAP
meeting,
EPA
has
developed
the
following
language
to
identify
PVCP­
PIPs
that
present
low
risk
with
respect
to
concerns
associated
with
viral
interactions
(
i.
e.,
recombination):
the
viral
pathotype7
used
to
create
the
PVCP­
PIP
has
naturally
infected8
plants
in
the
United
States,
its
possessions,
or
territories
and
naturally
infects
plants
of
the
same
species
as
those
containing
the
PVCP­
PIP.

The
rationale
for
such
a
criterion
would
be
that
if
the
viral
pathotype
meets
these
conditions,
the
recombinants
that
could
be
produced
in
that
plant
would,
in
principle,
be
no
different
than
what
could
occur
in
a
natural
mixed
infection
in
the
United
States
involving
that
virus.
Mixed
virus
infections
occur
frequently
in
nature
(
Hammond
et
al.
1999)
and
thus
provide
numerous
opportunities
for
viruses
in
the
United
States
that
infect
the
same
plant
species
to
interact.
EPA
seeks
to
identify
those
situations
that
clearly
pose
low
risk
with
respect
to
viral
interactions,
i.
e.,
those
situations
in
which
recombination
in
a
transgenic
plant
would
involve
segments
of
viruses
that
already
have
the
opportunity
to
recombine
in
a
natural,
mixed
infection.

7
EPA
uses
the
term
"
viral
pathotype"
rather
than
the
more
generic
term
"
virus"
in
response
to
the
FIFRA
SAP
comment
in
October
2004,
that
"[
n]
ot
all
isolates
of
a
virus
infect
and
cause
disease
in
all
plant
genotypes
and,
as
a
consequence,
the
unqualified
use
of
the
term
"
virus"
when
setting
a
condition
for
applicants
to
the
Agency
[
is]
not
adequate
in
this
context.
It
is
therefore
appropriate
in
the
context
of
biosafety
as
well
as
virus
epidemiology
to
recognize
the
value
of
defining
specific
viral
pathotypes
or
host
range
variants."
8
EPA
means
by
the
term
"
naturally
infect"
to
infect
by
transmission
to
a
plant
through
direct
plant­
to­
plant
contact
(
e.
g.,
pollen
or
seed),
an
inanimate
object
(
e.
g.,
farm
machinery),
or
vector
(
e.
g.,
arthropod,
nematode,
or
fungus).
It
does
not
include
infection
by
transmission
that
occurs
only
through
intentional
human
intervention.
The
Agency
wants
specifically
to
exclude
transmission
that
occurs
only
through
intentional
human
intervention,
e.
g.,
manual
infection
in
a
laboratory
or
greenhouse
setting,
because
such
transmission
would
have
little
relevance
to
normal
human
dietary
exposure.
EPA
intends
to
include
viruses
that
are
likely
to
have
been
part
of
the
human
diet
due
to
their
ability
to
spread
without
intentional
human
intervention.
EPA
recognizes
that
humans
may
play
an
inadvertent
role
in
infection
(
e.
g.,
by
transmitting
the
virus
on
farm
machinery).
Such
unintentional
(
and
often
unavoidable)
transmission
can
be
an
important
means
of
virus
transmission,
and
this
mode
of
transmission
would
be
included
under
"
naturally
infects."
Page
10
of
20
3.
Please
comment
on
the
usefulness
of
the
following
criteria
(
i)
and
(
ii)
in
correctly
identifying
PVCP­
PIPs
that
present
low
risk
with
respect
to
environmental
concerns
associated
with
novel
viral
interactions9.
Please
explain
the
basis
for
your
answer,
including
whether
the
limitations
imposed
by
the
use
of
"
viral
pathotype,"
7
"
naturally
infect,"
8
"
species,"
and
"
United
States,
its
possessions,
or
territories"
are
necessary
and/
or
sufficient.
For
example,
could
other
parts
of
North
America
be
included
as
part
of
criterion
(
i)?

(
i)
the
viral
pathotype
used
to
create
the
PVCP­
PIP
has
naturally
infected
plants
in
the
United
States,
its
possessions,
or
territories
and
(
ii)
the
viral
pathotype
used
to
create
the
PVCP­
PIP
naturally
infects
plants
of
the
same
species
as
that
containing
the
PVCP­
PIP.

Other
characteristics
of
the
PVCP­
PIP
may
also
indicate
low
risk,
even
though
criteria
describing
such
characteristics
cannot
be
as
clearly
articulated
as
those
described
by
the
language
in
question
3.
EPA
is
requesting
the
SAP's
advice
on
whether
a
PVCP­
PIP
would
pose
low
risk
with
respect
to
viral
interactions
if
the
Agency
determines
that
(
i)
the
properties
of
the
viral
pathotype
that
are
determined
by
the
coat
protein
gene
used
to
create
the
PVCP­
PIP
are
substantially
similar
to
the
properties
of
a
viral
pathotype
that
naturally
infects
plants
in
the
United
States,
its
possessions,
or
territories,
and
the
viral
pathotype
used
to
create
the
PVCP­
PIP
naturally
infects
plants
of
the
same
species
as
that
containing
the
PVCP­
PIP,
or
(
ii)
viruses
that
naturally
infect
the
plant
containing
the
PVCP­
PIP
are
unlikely
to
acquire
the
coat
protein
sequence
through
recombination
and
produce
a
viable
virus
with
significantly
different
properties
than
either
parent
virus.

The
rationale
supporting
criterion
(
i)
is
that
if
the
properties
of
the
viral
pathotype
that
are
determined
by
the
coat
protein
gene
are
substantially
similar
to
the
properties
of
a
viral
pathotype
that
naturally
infects
plants
in
the
United
States
and
the
viral
pathotype
used
to
create
the
PVCPPIP
naturally
infects
plants
of
the
same
species
as
those
containing
the
PVCP­
PIP,
the
viral
interactions
that
could
occur
in
a
plant
containing
such
a
PVCP­
PIP
would
be
no
different
than
what
could
occur
in
a
natural
mixed
infection
in
the
United
States
involving
that
virus.
To
evaluate
this
criterion,
EPA
would
consider
first
the
sequence
similarity
in
the
coat
protein
gene
of
the
pathotype
used
and
pathotypes
found
in
the
United
States.
Then,
when
information
is
available,
EPA
would
evaluate
the
extent
to
which
any
significant
deviations
from
pathotypes
in
the
United
States
are
likely
to
influence
phenotypic
properties
of
the
virus.
The
rationale
supporting
criterion
(
ii)
is
that
even
if
criterion
(
i)
is
not
met,
it
may
be
possible
to
reduce
the
frequency
of
recombination
to
such
an
extent
that
few
viral
interactions,
novel
or
not,
are
expected
to
occur.
Recombination
is
the
sole
type
of
viral
interaction
focused
upon
based
in
part
on
conclusions
of
the
2004
SAP
that
"
except
perhaps
for
a
very
few
cases,
neither
heterologous
encapsidation
nor
synergy
should
be
considered
to
be
of
serious
concern"
(
U.
S.
Environmental
Protection
Agency
2004).

9
A
"
novel
viral
interaction"
is
an
interaction
(
i.
e.,
recombination,
heterologous
encapsidation,
or
synergy)
between
viral
transgenes
and
an
infecting
virus
involving
viruses
that
would
otherwise
not
be
expected
to
interact
in
a
mixed
infection
found
in
nature.
Page
11
of
20
4(
a).
Please
comment
on
the
usefulness
of
the
following
criteria
(
i)
and
(
ii)
in
allowing
the
Agency
in
its
review
of
the
product
to
identify
correctly
whether
the
PVCP­
PIP
presents
low
risk
with
respect
to
environmental
concerns
associated
with
novel
viral
interactions9.
Please
explain
the
basis
for
your
answer.

(
i)
the
properties
of
the
viral
pathotype
that
are
determined
by
the
coat
protein
gene
used
to
create
the
PVCP­
PIP
are
substantially
similar
to
the
properties
of
a
viral
pathotype
that
naturally
infects
plants
in
the
United
States,
its
possessions,
or
territories,
and
the
viral
pathotype
used
to
create
the
PVCP­
PIP
naturally
infects
plants
of
the
same
species
as
that
containing
the
PVCP­
PIP,
or
(
ii)
viruses
that
naturally
infect
the
plant
containing
the
PVCP­
PIP
are
unlikely
to
acquire
the
coat
protein
sequence
through
recombination
and
produce
a
viable
virus
with
significantly
different
properties
than
either
parent
virus.

The
Agency
review
of
(
i)
could
involve
a
consideration
of
data
from
a
number
of
different
sources
including
virus
coat
protein
sequence
data
from
public
repositories
and
developergenerated
data
on
the
natural
range
of
variation
of
coat
protein
genes
for
particular
viral
pathotypes.
In
review
of
(
ii),
the
Agency
might
consider
(
a)
if
the
PVCP­
PIP
confers
virus
resistance
through
post­
transcriptional
gene
silencing
thereby
greatly
reducing
the
amount
of
RNA
available
for
recombination,
(
b)
if
the
PVCP­
PIP
construct
is
designed
to
reduce
the
frequency
of
recombination
(
e.
g.,
Miller
2000;
Nagy
&
Bujarski
1996;
Nagy
&
Bujarski
1998;
Nagy
et
al.
1999;
Teycheney
et
al.
2000),
or
(
c)
if
the
inserted
coat
protein
sequence
is
only
a
relatively
small
portion
of
the
naturally
occurring
sequence
suggesting
that
viruses
acquiring
the
region
are
unlikely
to
acquire
a
novel
phenotype.
EPA
recognizes
the
comments
of
the
2004
SAP
that
"
methods
for
minimizing
recombination
are
only
partially
effective.
For
this
reason,
the
question
remains
whether
novel
recombinants
would
be
created
in
transgenic
plants,
and
simply
reducing
the
frequency
of
these
events
is
not
an
answer
to
the
question"
(
U.
S.
Environmental
Protection
Agency
2004).
However,
a
combination
of
two
or
more
methods,
or
even
perhaps
a
single
method
in
some
cases,
might
reduce
the
expected
frequency
of
recombination
such
that
a
PVCP­
PIP
would
pose
low
risk
with
respect
to
viral
interactions.
Such
a
determination
would
probably
best
be
made
on
a
case­
by­
case
basis.

4(
b).
Please
comment
on
the
usefulness
of
the
analyses
described
above
for
evaluating
whether
a
PVCP­
PIP
meets
(
i)
or
(
ii).
Please
describe
any
additional
factors
that
the
Agency
could
use
in
this
evaluation
(
e.
g.,
consideration
of
whether
the
plant
virus
species
has
an
inherently
low
natural
recombination
frequency
with
respect
to
the
coat
protein
gene).

PVC­
Protein
Production
Issues
When
evaluating
PVCP­
PIPs
for
possible
exemption
under
FIFRA,
EPA
must
consider
nontarget
and
human
non­
dietary
risks
from
exposure
to
any
potentially
expressed
PVC­
proteins.
Based
on
Page
12
of
20
the
information
currently
available
to
the
Agency
to
support
an
exemption,
EPA
is
considering
a
criterion
that
describes
PVC­
proteins
that
are
within
the
range
of
natural
variation
of
the
virus
and
therefore
have
a
long
history
of
safe
nontarget
and
human
exposure.

EPA
consulted
the
2004
SAP
about
possible
nontarget
effects
of
PVC­
proteins.
The
panel
confirmed
that
PVC­
proteins
within
the
range
of
natural
variation
of
the
virus
would
not
be
anticipated
to
present
risks
to
nontarget
organisms,
concluding
that,
"[
l]
ethal
effects
in
animal
life
after
feeding
on
PVCP­
PIP
plants
are
highly
unlikely
because
plant
viruses
are
not
known
to
have
deleterious
effects
on
animal
life.
Additionally,
animals
routinely
feed
on
non­
engineered
virus­
infected
plants
and
do
not
die .
[
S]
ublethal
effects
are
not
expected
to
be
manifested
in
animal
life,
again
because
wildlife
and
insects
regularly
feed
on
non­
engineered
virus­
infected
plants
with
no
apparent
sublethal
damage"
(
U.
S.
Environmental
Protection
Agency
2004).

Based
on
the
above
advice,
PVCP­
PIPs
that
present
low
risk
with
respect
to
nontarget
and
human
non­
dietary
exposures
to
PVC­
proteins
could
be
described
by
the
following
language:
the
genetic
material
encodes
only
a
single
contiguous
portion
of
each
unmodified
viral
coat
protein.
This
would
include
multiple
proteins
expressed
from
a
single
PVCP­
PIP
construct,
but
not
chimeric
proteins.
Under
these
conditions,
such
PVC­
proteins
would
be
identical
to
plant
viral
coat
proteins
that
are
widespread
in
plants
and
are
not
known
to
have
any
toxic
effects
on
nontarget
organisms
or
to
have
any
toxic
or
allergenic
effects
in
humans.
Therefore,
no
nontarget
or
human
non­
dietary
safety
issues
would
be
raised
by
PVC­
proteins
meeting
this
criterion.

The
Agency
recognizes
that
PVCP­
PIP
developers
may
wish
to
modify
PVCP­
PIP
constructs
to
achieve
certain
product
development
goals
such
as
greater
efficacy,
and
such
modifications
might
result
in
changes
to
the
protein(
s)
produced.
Many
modifications
to
the
genetic
material
may
be
so
minor
that
they
are
unlikely
to
cause
changes
to
the
protein
that
would
be
significant
from
a
human
or
nontarget
organism
perspective.
Many
of
the
modifications
are
likely
to
produce
proteins
that
fall
within
the
range
of
natural
variation
of
the
virus.
However,
it
is
not
currently
possible
to
a
priori
define
a
regulatory
standard
describing
the
range
of
variation
of
plant
virus
coat
proteins
in
general
or
even
of
any
particular
virus.
(
See
discussion
in
Unit
II.
B
of
Attachment
II.)
Given
the
large
number
and
wide
variety
of
changes
that
could
be
made
to
a
genetic
construct
containing
a
plant
viral
coat
protein
gene
and
the
differences
among
virus
species
in
the
amount
of
natural
variation
they
exhibit,
it
would
not
be
possible
to
decide
a
priori
that
any
particular
predetermined
type
or
number
of
modifications
would
consistently
produce
a
protein
that
falls
within
the
range
of
natural
variation.
However,
such
a
determination
could
be
made
on
a
case­
by­
case
basis.

Therefore,
PVCP­
PIPs
that
present
low
risk
with
respect
to
nontarget
and
human
non­
dietary
exposures
to
PVC­
proteins
could
be
described
by
the
following
language:
the
genetic
material
(
i)
encodes
a
protein
that
is
minimally
modified
from
a
coat
protein
from
a
virus
that
naturally
infects
plants
or
(
ii)
produces
no
protein.
In
determining
whether
a
PVC­
protein
is
"
minimally
modified"
from
a
natural
viral
coat
protein,
EPA
would
consider
first
whether
the
protein
is
substantially
similar
to
a
natural
viral
coat
protein
by
evaluating
information
on
the
genetic
construct,
amino
acid
sequence,
and
molecular
weight
of
the
PVC­
protein.
EPA
might
also
evaluate
information
developed
by
the
submitter
from
public
sequence
databases
on
where
the
PVC­
protein
sequence
falls
relative
to
the
range
of
natural
variation.
Those
PVC­
proteins
that
are
determined
to
be
substantially
similar
would
be
further
evaluated
to
determine
whether
the
Page
13
of
20
modified
PVC­
protein
is
as
safe
as
an
unmodified
protein
by
considering
information
on
the
expression
level
of
the
PVC­
protein
relative
to
levels
generally
found
in
plants
and
information
from
amino
acid
sequence
comparisons
with
known
toxins
and
allergens.
The
type
and
extent
of
information
that
would
need
to
be
provided
with
an
exemption
request
in
order
for
EPA
to
determine
whether
a
PVC­
protein
is
"
minimally
modified"
and
therefore
qualifies
for
the
exemption
would
be
determined
on
a
case­
by­
case
basis.

5.
Please
comment
on
the
usefulness
of
the
above
factors
in
allowing
the
Agency
to
identify
correctly
those
PVCP­
PIPs
that
present
low
risk
with
respect
to
nontarget
and
human
non­
dietary
exposure
to
PVC­
proteins.

Post­
Transcriptional
Gene
Silencing
(
PTGS)
Issues
PVCP­
PIPs
may
confer
resistance
in
at
least
two
different
ways.
In
protein­
mediated
resistance,
the
coat
protein
is
thought
to
impede
the
infection
cycle
by
interfering
with
the
disassembly
of
infecting
viruses.
In
such
cases,
the
PVC­
protein
appears
to
be
the
active
ingredient
directly
effecting
the
pesticidal
action.
In
other
cases,
prevention
or
mitigation
of
viral
disease
is
not
correlated
with
the
level
of
coat
protein
expression,
and
RNA
fragments
appear
to
be
the
active
ingredient,
e.
g.,
through
post­
transcriptional
gene
silencing
(
PTGS;
Goldbach,
2003).
Regardless
of
the
mechanism
of
resistance,
plants
containing
plant
viral
coat
protein
genes
for
the
purpose
of
preventing
or
mitigating
viral
disease
contain
a
pesticide
subject
to
regulation
by
EPA
under
both
FIFRA
and
FFDCA.
EPA
is
seeking
guidance
from
the
SAP
as
to
how
the
mechanism
of
resistance
affects
the
evaluation
of
risk
associated
with
PVCP­
PIPs
in
order
that
the
Agency
can
appropriately
address
all
types
of
resistance
mechanisms.

EPA
is
considering
whether
any
proposed
exemption
under
FIFRA
should
include
criteria
to
address
concerns
associated
with
(
1)
gene
flow,
(
2)
viral
interactions,
and
(
3)
exposure
to
PVCproteins
sufficiently
different
from
those
with
which
organisms
generally
interact
when
associating
with
the
plant.
Concerns
associated
with
gene
flow
are
not
influenced
by
the
mechanism
of
resistance
based
on
the
assumption
that
virus
resistance
could
be
expressed
in
a
plant
that
acquired
the
genetic
material
regardless
of
whether
resistance
is
protein­
or
RNAmediated
However,
concerns
associated
with
recombination
and
PVC­
proteins
may
be
significantly
reduced
when
resistance
is
RNA­
mediated.
For
example,
when
a
plant
employs
PTGS,
RNA
with
sequence
similarity
to
the
transgene
will
be
broken
down,
including
that
produced
by
the
transgene
(
Goldbach
et
al.
2003).
Thus,
the
potential
for
recombination
with
expressed
transcripts
from
the
transgene
will
be
likewise
reduced.
The
decrease
in
RNA
also
leads
to
a
significant
decrease
in
protein
production
(
and
therefore
PVC­
protein
exposures).
In
some
cases,
protein
production
may
be
reduced
to
levels
below
the
detection
limit
or
even
to
zero.

Under
FFDCA,
any
PVC­
protein
residues
in
food
must
be
covered
by
a
tolerance
or
tolerance
exemption.
Even
when
a
PVCP­
PIP
prevents
or
mitigates
viral
disease
through
PTGS
in
which
the
pesticidal
substance
appears
to
be
RNA,
if
any
PVC­
protein
were
to
be
produced
in
small
Page
14
of
20
quantities,
in
certain
tissues,
at
certain
life
stages,
under
certain
environmental
conditions,
or
in
the
case
of
suppression
of
gene
silencing,
this
PVC­
protein
is
part
of
the
PVCP­
PIP.
(
See
Béclin
et
al.
1998;
Mitter
et
al.
2001;
Pang
et
al.
1996;
Savenkov
&
Valkonen
2001;
Szittya
et
al.
2003;
and
discussion
in
Unit
II.
D
of
Attachment
II.)

6(
a).
Please
identify
any
characteristics
of
a
PVCP­
PIP
construct
that
would
indicate
it
is
unlikely
to
produce
PVC­
protein.
Please
discuss
the
likelihood
that
protein
production
could
nevertheless
occur
from
constructs
with
these
characteristics
(
i)
in
some
tissues,
(
ii)
at
some
life
stages,
(
iii)
under
some
environmental
conditions,
(
iv)
in
the
case
of
suppression
of
gene
silencing,
or
(
v)
under
any
other
circumstances.
For
example,
how
likely
is
PVC­
protein
production
from
a
construct
containing
an
inverted
repeat
of
the
coat
protein
gene
(
e.
g.,
Mitter
et
al.
2003)
or
from
a
construct
lacking
a
start
codon
(
AUG
sequence)
and/
or
a
ribosome
binding
site
on
the
expressed
RNA?

6(
b).
Assuming
a
PVCP­
PIP
construct
does
not
possess
any
characteristics
that
would
indicate
a
low
likelihood
of
protein
expression
but
PVC­
protein
is
not
detected
in
plants
containing
the
PVCP­
PIP,
presumably
because
virus
resistance
is
conferred
through
RNA,
please
comment
on
the
likelihood
and
expected
quantity
of
both
RNA
and
protein
that
would
be
present
(
i)
only
transiently,
(
ii)
only
in
certain
tissues,
(
iii)
only
at
certain
life
stages,
(
iv)
only
under
certain
environmental
conditions,
or
(
v)
in
the
case
of
suppression
of
gene
silencing?
How
likely
is
suppression
of
gene
silencing
to
occur
in
the
environment
over
time?

6(
c).
Please
identify
conditions
under
which
protein
detection
methods
should
be
conducted
to
determine
whether
PVC­
protein
is
produced
from
the
PVCP­
PIP.
For
example,
how
many
replicates
and
what
particular
tissues,
life
stages,
and/
or
environmental
conditions
should
be
tested?

6(
d).
Compared
with
protein­
mediated
virus
resistance,
how
does
RNA­
mediated
virus
resistance
(
e.
g.,
during
PTGS)
affect
the
likelihood
and
possible
environmental
impact
of
(
i)
gene
flow
of
a
PVCP­
PIP
transgene
and
(
ii)
recombination
of
an
infecting
virus
with
a
PVCP­
PIP
transgene
or
RNA
transcript.

Food
Safety
Issues
EPA's
base
of
experience
with
viruses
infecting
food
plants
has
led
the
Agency
to
draw
three
conclusions
on
which
it
would
rely
to
support
any
tolerance
exemption
for
residues
of
PVCproteins
in
food.
First,
virus­
infected
plants
have
always
been
a
part
of
the
human
and
domestic
animal
food
supply.
Most
crops
are
frequently
infected
with
plant
viruses,
and
food
from
these
crops
has
been
and
is
being
consumed
without
adverse
human
or
animal
health
effects.
Second,
plant
viruses
are
not
infectious
to
humans,
including
children
and
infants,
or
to
other
mammals.
Third,
plant
virus
coat
proteins,
while
widespread
in
food,
have
not
been
associated
with
toxic
effects
to
animals
or
humans.
EPA
believes
these
conclusions
are
derived
from
a
sufficient
Page
15
of
20
experience
and
information
base
to
support
a
proposed
exemption
from
the
requirement
of
a
food
tolerance
under
FFDCA.

EPA
is
attempting
to
determine
whether
there
would
be
any
safety
issues
raised
from
exposure
to
PVC­
proteins
if
the
virus
used
to
create
the
PVCP­
PIP
does
not
naturally
infect
the
particular
plant
species
into
which
the
PVCP­
PIP
is
inserted.
A
PVC­
protein
may
be
expressed
in
a
food
plant
that
the
virus
does
not
naturally
infect
when
heterologous
resistance
to
a
particular
virus
is
conferred
through
a
different
virus'
coat
protein
gene
(
e.
g.,
Dinant
et
al.
1993).
Such
situations
may
also
arise
when
a
small
segment
of
a
plant
virus
coat
protein
gene
is
used
to
achieve
expression
of
a
coat
protein
gene
from
a
different
virus
(
e.
g.,
Gonsalves
1998).
The
Agency
is
attempting
to
determine
whether
such
PVC­
proteins
present
low
dietary
risk
based
on
the
rationale
that
these
proteins
are
reasonably
expected
to
be
part
of
the
current
diet.
Based
on
their
broad
host
range,
plant
viruses
are
known
generally
to
infect
a
wide
variety
of
plants
that
humans
consume.
People
generally
eat
a
broad
range
of
food
plants
through
which
they
would
reasonably
be
expected
to
be
exposed
to
a
wide
variety
of
plant
virus
coat
proteins.
In
addition,
EPA
is
not
aware
that
any
plant
viral
coat
proteins
have
been
identified
as
allergens,
so
it
is
unlikely
that
a
person
with
food
allergies
avoids
a
particular
food
plant
because
of
an
allergic
reaction
to
a
viral
coat
protein.
Based
on
this
rationale
and
in
the
absence
of
contravening
evidence,
EPA
believes
that
a
PVC­
protein
expressed
in
a
plant
that
is
not
normally
infected
by
the
corresponding
virus
would
raise
no
safety
issues.

7(
a).
What
is
the
potential
for
novel
human
exposure
to
a
PVC­
protein
when
it
is
expressed
in
food
from
a
plant
species
that
the
virus
used
to
create
the
PVCP­
PIP
does
not
naturally
infect
(
assuming
that
the
virus
naturally
infects
another
food
plant
species)?
What
is
the
potential
for
allergenicity
to
be
associated
with
such
PVC­
proteins?
How
would
use
of
a
small
segment
of
such
a
protein
(
e.
g.,
to
achieve
gene
expression)
affect
relative
concern
for
allergenicity?

The
rationale
for
exempting
truncated
PVC­
proteins
from
food
tolerance
requirements
would
be
that
segments
of
coat
proteins
exist
in
nature
due
to
processes
such
as
incomplete
translation
of
transcripts
and
partial
degradation
of
proteins.
Incomplete
translation
may
occur
due
to
routine
replication
errors
causing
a
ribosome
to
dissociate
from
an
RNA
transcript
or
if
mutation
introduces
a
premature
stop
codon,
i.
e.,
a
nonsense
mutation.
Truncated
plant
virus
coat
proteins
are
indeed
known
to
occur
in
nature
(
Sacher
&
Ahlquist
1989).
Thus,
PVC­
proteins
that
are
truncated
forms
of
naturally
occurring
plant
virus
coat
proteins
would
not
significantly
increase
the
likelihood
of
exposure
to
a
toxic
or
allergenic
protein
since
humans
are
currently
exposed
to
them
in
the
diet
along
with
complete
plant
virus
coat
proteins.

7(
b).
Please
comment
on
the
likelihood
that
PVC­
proteins
containing
terminal
deletions
are
within
the
range
of
natural
variation
of
plant
virus
coat
proteins.
What
is
the
likelihood
such
truncated
proteins
would
have
increased
toxicity
or
allergenicity
relative
to
the
corresponding
full­
length
plant
virus
coat
protein?
What
relevance
does
the
size
of
the
deletion
have
to
this
issue?
What
relevance
does
deletion
at
the
Cterminus
versus
N­
terminus
have
to
this
issue?
Page
16
of
20
The
AUG
codon
for
methionine
initiates
translation
in
eukaryotes
(
Berg
et
al.
2002).
Among
certain
viruses
such
as
the
Potyviridae,
the
coat
protein
is
produced
as
part
of
a
polyprotein,
so
the
coding
region
for
the
coat
protein
is
excised
from
the
genetic
material
encoding
the
polyprotein
to
create
a
PVCP­
PIP
and
thus
normally
lacks
a
start
codon.
Insertion
of
an
AUG
codon
allows
for
PVC­
protein
expression,
which
may
be
needed
to
confer
virus
resistance.
EPA
believes
the
addition
of
a
single,
N­
terminal
methionine
residue
would
be
unlikely
to
affect
a
PVC­
protein's
toxicity
or
allergenicity
relative
to
a
naturally
occurring
plant
virus
coat
protein.

7(
c).
Please
comment
on
the
likelihood
that
a
PVC­
protein
modified
by
an
additional
methionine
at
the
N­
or
C­
terminus
would
have
increased
toxicity
or
allergenicity
relative
to
the
corresponding
unmodified
plant
virus
coat
protein.
What
relevance
does
the
terminus
at
which
the
amino
acid
is
added
have
to
this
issue?
Of
what
relevance
is
the
particular
amino
acid
added?
Of
what
relevance
is
the
number
of
additional
amino
acids?

Viruses
have
a
wide
range
of
natural
variation
and
it
is
likely
that
many
modifications
in
addition
to
truncations
and
the
addition
of
an
AUG
codon
could
be
introduced
into
the
genetic
material
encoding
the
PVC­
protein
and
result
in
exposure
to
PVC­
proteins
similar
to
plant
viral
coat
proteins
currently
in
the
diet.
However,
protein
modifications
have
been
recognized
as
having
the
potential
to
significantly
alter
a
protein's
properties.

7(
d).
Please
identify
type(
s)
of
protein
modification(
s)
(
e.
g.,
internal
deletions,
amino
acid
substitutions,
addition
of
certain
amino
acid
residues)
that
could
be
introduced
without
resulting
in
a
PVC­
protein
that
would
have
increased
toxicity
or
allergenicity
relative
to
the
corresponding
unmodified
plant
virus
coat
protein,
e.
g.,
because
the
changes
are
expected
to
be
within
the
range
of
natural
variation
for
all
virus
families.

Under
EPA's
current
approach,
in
determining
whether
a
PVC­
protein
is
"
minimally
modified"
from
a
natural
viral
coat
protein,
the
Agency
would
consider
first
whether
the
protein
is
substantially
similar
to
a
natural
viral
coat
protein
by
evaluating
information
on
the
genetic
construct,
amino
acid
sequence,
and
molecular
weight
of
the
PVC­
protein.
EPA
might
also
evaluate
information
developed
by
the
submitter
from
public
sequence
databases
on
where
the
PVC­
protein
sequence
falls
relative
to
the
range
of
natural
variation.
Those
PVC­
proteins
that
are
determined
to
be
substantially
similar
would
be
further
evaluated
to
determine
whether
the
modified
PVC­
protein
is
as
safe
as
an
unmodified
protein
by
considering
information
on
the
expression
level
of
the
PVC­
protein
relative
to
levels
generally
found
in
plants
humans
consume
and
information
from
an
amino
acid
sequence
comparison
with
known
toxins
and
allergens.
The
type
and
extent
of
information
that
would
need
to
be
provided
in
order
for
EPA
to
determine
whether
a
PVC­
protein
is
"
minimally
modified"
would
be
determined
on
a
case­
by­
case
basis.

8.
Please
comment
on
the
usefulness
of
the
factors
described
above
for
evaluating
food
safety
of
the
encoded
PVC­
protein.
How
important
is
it
to
characterize
the
expressed
Page
17
of
20
protein,
e.
g.,
to
determine
whether
any
post­
translational
modifications
have
occurred?

Some
PVC­
proteins
may
be
chimeric
proteins
that
are
encoded
by
a
sequence
constructed
from
portions
of
two
or
more
different
plant
virus
coat
protein
genes.
Such
constructs
may
be
made
to
enable
appropriate
expression
of
the
desired
coat
protein
gene
and
confer
resistance
in
the
plant
(
Gonsalves
1998;
Ravelonandro
et
al.
1992)
or
to
expand
the
range
of
viruses
to
which
the
plant
is
resistant
(
Lindbo
et
al.
1993).

9(
a).
What
is
the
likelihood
that
a
chimeric
PVC­
protein
would
have
increased
toxicity
or
allergenicity
relative
to
the
corresponding
non­
chimeric
plant
virus
coat
proteins?
Can
you
describe
any
objective
criteria
to
identify
those
chimeric
PVC­
proteins
with
novel
toxic
or
allergenic
properties?

9(
b).
Please
address
the
relevance
of
the
following
factors
to
the
potential
toxicity
or
allergenicity
of
a
chimeric
PVC­
protein:

(
i)
the
size
of
the
various
segments
comprising
the
chimeric
PVC­
protein,

(
ii)
the
viral
source(
s)
of
the
various
segments,
and/
or
(
iii)
the
location
on
the
protein
where
fusions
occur.

9(
c).
Are
the
factors
specified
in
question
8
applicable
to
evaluating
the
safety
of
chimeric
PVC­
proteins?
Are
there
any
additional
factors
specific
to
chimeric
proteins
that
should
be
considered?

Other
Issues
Under
the
regulatory
structure
established
for
PIPs,
selectable
markers
(
i.
e.,
inert
ingredients
by
definition
under
FIFRA)
are
considered
to
be
part
of
the
PIP.
EPA
has
identified
three
selectable
markers
that
it
believes
present
a
low
probability
of
risk
to
human
health
and
the
environment
when
used
in
any
plant
on
the
list
in
question
1(
a):
CP4
enolpyruvylshikimate­
3­
phosphate
(
CP4
EPSPS),
glyphosate
oxidoreductase
(
GOX
or
GOXv247),
and
phosphinothricin
acetyltransferase
(
PAT).
In
addition,
EPA
has
identified
three
selectable
markers
that
it
believes
pose
low
risk
to
human
health
and
the
environment
when
used
in
any
plant
as
part
of
a
PIP:
beta­
D­
glucuronidase
(
from
E.
coli),
neomycin
phosphotransferase
II
(
NPTII),
and
phosphomannose
isomerase
(
PMI).
Each
of
these
proteins
already
has
a
tolerance
exemption
under
FFDCA
section
408.
The
Agency
is
now
considering
exempting
these
selectable
markers
from
regulation
under
FIFRA.

10.
Please
comment
on
the
Agency's
environmental
risk
assessment
of
each
of
the
six
selectable
markers
(
found
in
attachment
III).
Does
the
SAP
concur
that
CP4
EPSPS,
GOX/
GOXv247,
PAT
each
pose
a
low
probability
of
risk
to
the
environment
when
Page
18
of
20
used
in
one
of
the
plants
listed
in
question
1(
a)?
Does
the
SAP
concur
that
beta­
Dglucuronidase
NPTII,
and
PMI
each
pose
a
low
probability
of
risk
to
the
environment
when
used
in
any
plant?

In
examining
approaches
to
identifying
and
describing
PVCP­
PIPs
likely
to
present
low
risk,
EPA
has
developed
criteria
to
address
the
risk
issues
most
commonly
identified
as
potentially
associated
with
PVCP­
PIPs.
The
SAP
has
been
asked
to
comment
on
aspects
of
these
criteria
in
this
meeting.
The
Agency
recognizes
that
other
viral
components
have
been
or
are
being
used
to
develop
PIPs
intended
for
use
in
protecting
against
viral
disease,
e.
g.,
PIPs
based
on
viral
replicase
genes.

The
Agency
asked
the
SAP
in
1992
to
what
extent
the
rationale
used
in
the
proposed
exemption
for
PVCP­
PIPs
could
be
applied
to
other
viral
components.
The
SAP
responded:

"
Other
viral
gene
products
usually
are
expressed
at
lower
levels
in
the
infected
plants
than
viral
coat
protein.
Also,
their
turnover
rate
is
much
higher.
Thus,
in
some
cases,
transgenic
plants
expressing
other
viral­
encoded
proteins
may
express
these
proteins
at
levels
higher
than
in
a
naturally
infected
plant.
Hence,
a
generalization
cannot
be
made
for
such
proteins
as
can
be
made
for
coat
protein.
Although
at
present
this
approach
seems
reasonable,
additional
research
will
be
necessary
before
any
generalizations
can
be
made"
(
U.
S.
Environmental
Protection
Agency
1992).

11.
Please
comment
on
whether
the
criteria
discussed
above
that
EPA
is
considering
for
PVCP­
PIPs
(
i.
e.,
relating
to
gene
flow,
viral
interactions,
and
protein
production)
would
be
applicable
for
other
PIPs
conferring
virus
resistance,
e.
g.,
those
based
on
virus
replicase
genes
(
Ehrenfeld
et
al.
2004)
or
defective
interfering
RNA
(
Kollar
et
al.
1993).
Please
indicate
the
scientific
rationale
for
including
any
additional
PIPs
under
such
an
exemption
and
whether
any
additional
(
or
fewer)
qualifications
would
be
needed.

References
Béclin,
C.,
Berthomé,
R.,
Palauqui,
J.
C.,
Tepfer,
M.,
and
Vaucheret,
H.
1998.
Infection
of
tobacco
or
Arabidopsis
plants
by
CMV
counteracts
systemic
post­
transcriptional
silencing
of
nonviral
(
trans)
genes.
Virology
252:
313­
317.
Berg,
J.,
Tymoczko,
J.,
Stryer,
L.,
and
Clarke,
N.
2002.
Biochemistry.
W.
H.
Freeman
and
Company,
New
York.
Chen,
Z.
L.,
Gu,
H.,
Li,
Y.,
Su,
Y.,
Wu,
P.,
Jiang,
Z.,
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