**[Plant-Incorporated Protectants Based on Plant Viral Coat
Proteins]{.smallcaps}**

**Introduction**

A plant-incorporated protectant (PIP) is a pesticidal substance that is
intended to be produced and used in a living plant, or in the produce
thereof[^1], and the genetic material necessary for production of such a
pesticidal substance. The term includes both active and inert
ingredients. 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.

PIPs may be genetically engineered into plants[^2]. Some specific
genetic sequences, when incorporated into a plant's genome, can endow
the plant with the ability to resist damage from certain pests. Plant
virus coat protein PIPs ([PVCP-PIP]{.smallcaps}s) are PIPs in which the
inserted genetic material is derived from a plant virus sequence that
encodes a coat protein. Plant virus coat proteins encapsidate the viral
nucleic acid and are known to have a role in nearly every stage of viral
infection including replication, movement throughout an infected plant,
and transport from plant to plant. Incorporation of plant viral coat
protein (PVCP) 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).

**Background**

In 1994 EPA published its policy regarding regulation of PIPs (then
called plant-pesticides) under FIFRA. Recognizing the breadth of the
definition of a PIP and that some PIPs present a lower overall risk and
thus may not require regulation, the Agency proposed several exemptions
to regulation. Included were two options to exempt PVCP-PIPs. A full
categorical exemption from regulation under FIFRA was proposed for
PVCP-PIPs based on the rationale that they generally pose a low
probability of risk to human health or 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 to promote full discussion of
this issue. Under this alternative exemption option, the Agency defined
a set of criteria to identify those viral coat protein/plant
combinations with the lowest potential to confer selective advantage on
wild or weedy plant relatives. Only those PVCP-PIPs so identified would
have been exempt from regulation. The 1994 policy statement described
this alternative exemption as follows:

> Coat proteins from plant viruses \[would be exempt\] if the genetic
> material necessary to produce a coat protein is introduced into a
> plant's genome and the plant has at least one of the following
> characteristics:
>
> \(1\) The plant has no wild relatives in the United States with which
> it can successfully exchange genetic material, i.e., corn, tomato,
> potato, soybean, or any other plant species that EPA has determined
> has no sexually compatible wild relatives in the United States.
>
> \(2\) It has been demonstrated to EPA that the plant is incapable of
> successful genetic exchange with any existing wild relatives (e.g.,
> through male sterility, self-pollination).
>
> \(3\) If the plant can successfully exchange genetic material with
> wild relatives, it has been empirically demonstrated to EPA that
> existing wild relatives are resistant or tolerant to the virus from
> which the coat protein is derived or that no selective pressure is
> exerted by the virus in natural populations.

Some proposals in the 1994 policy statement were finalized in 2001.
However, neither the full nor alternative exemption for PVCP-PIPs was
finalized, in part because more recent information had questioned the
idea that PVCP-PIPs would generally pose a low probability of risk and
would not pose unreasonable adverse effects to human health or the
environment in the absence of any regulation under FIFRA. For example,
the 2000 National Research Council (NRC) report, [Genetically Modified
Pest-Protected Plants]{.underline}, recommended that,

> "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 concerns associated with the use 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," neither formulation of the
exemption 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.

In 2001 the Agency reopened the comment period on the PVCP-PIP exemption
proposals and sought advice from the public on how best to proceed. EPA
is in the process of reevaluating how PVCP-PIPs will be treated by the
Agency in light of comments and advice it received. The Agency is
reconsidering the scientific merit and feasibility of exempting a subset
of PVCP-PIPs from regulation under FIFRA as one of several regulatory
options. Potential conditions intended to reduce the risks associated
with the use of PVCP-PIPs are included in the questions to the panel to
provide a structure that may facilitate discussion of environmental
concerns raised with regard to commercial use of PVCP-PIPs.

**Charge to the SAP**

EPA requests that the Scientific Advisory Panel provide scientific
advice to assist EPA in its evaluation of plant-incorporated protectants
(PIPs) based on plant virus coat proteins (PVCP-PIPs). During the public
comment periods for PVCP-PIPs in 1994 and again in 2001, the Agency
received comments from the scientific community that covered viewpoints
ranging from support for a full categorical exemption to opposition to
any exemption procedure for PVCP-PIPs. These comments highlighted
several areas of scientific uncertainty that are addressed in our
questions to the panel. The Agency asks for the panel's scientific
advice in three areas: gene flow, viral interactions, and other issues.
EPA seeks the assistance of the SAP in evaluating whether gene flow and
viral interactions may pose environmental hazards, and if so, whether
these concerns could be mitigated under certain circumstances. EPA seeks
the advice of the SAP to ensure that PVCP-PIPs are appropriately
described and to develop a more complete technical record.

***Charge -- Gene Flow***

Gene flow from plants containing PVCP-PIPs is a potential concern to the
extent that new genes conferring virus resistance may be introduced into
wild or weedy relatives with the potential either to release them from
growth and/or reproduction constraints imposed by naturally occurring
virus(es) or conversely to decrease their fitness through changes in
gene expression. These relatives of transgenic plants may then have the
potential for increased weediness and/or the ability to outcompete other
species. Conversely, plants also may become less competitive.

A broad review of hybridization between plants and their wild relatives,
including the potential for and possible implications of the movement of
transgenes, was recently published (Ellstrand 2003), and the issue of
introgression from genetically modified crops to their wild relatives
was considered in a recent review (Stewart et al. 2003). In addition,
several scientific review panels have recently analyzed the general
issue of gene flow with particular attention to the regulatory issues
facing both U.S. and foreign government agencies (National Research
Council 2000; GM Science Review Panel 2003)[^3]. The potential effects
of gene flow to wild relatives specifically from plants containing
PVCP-PIPs has been addressed in a few experimental studies (Bartsch et
al. 1996; Bartsch et al. 2001; Spencer & Snow 2001; Ilardi & Barba 2001;
Fuchs et al. 2004a; Fuchs et al. 2004b). This issue is considered in
greater depth in several reviews (Bartsch 1997; Power 2002; Tepfer
2002). The theoretical possibility that gene flow from plants containing
PVCP-PIPs to wild or weedy relatives could, in some instances, lead to
environmental impacts is widely accepted. However, empirical or
observational data on the likelihood or magnitude of such impacts are
lacking. Consequently EPA is seeking the assistance of the SAP to
attempt to reach a better understanding of the circumstances in which
the flow of PVCP-PIPs from transgenic plants to wild or weedy relatives
could occur and the potential for adverse impacts from such gene flow.

***Questions -- gene flow***

Concerns about the transfer of virus resistance to wild or weedy
relatives include the assumption that such resistance might confer a
selective advantage to a wild or weedy relative that could increase its
competitive ability and potential to become weedy or invasive. The
Agency would like the panel to consider the evidence supporting this
assumption.

1.  **What scientific evidence supports or refutes the idea that plant
    viruses have significant effects on reproduction, survival, and
    growth of plant populations in natural settings? Is there scientific
    evidence that plant populations freed from viral pressure could have
    increased competitive ability leading to changes in plant population
    dynamics?**

In the 1994 proposal, EPA identified tomato, potato, soybean, and corn
as having no wild relatives in the United States with which they can
successfully exchange genetic material, although the 2002 NRC report ,
[Environmental Effects of Transgenic Plants]{.underline}, suggests that
corn and its wild relative Eastern gamagrass (*Tripsacum dactyloides*)
might produce hybrids with some fertility at a very low frequency in the
United States.

2.  **Please comment on the validity of the Agency list of crops that
    have no wild or weedy relatives in the United States with which they
    can produce viable hybrids in nature (i.e., tomato, potato, soybean,
    and corn).**

3.  **Please identify other crops that have no wild or weedy relatives
    in the United States with which they can produce viable hybrids in
    nature, e.g., papaya, peanut, and/or chickpea.**

The Agency anticipates the need to evaluate data addressing whether
transgenic plant species are capable of genetic exchange with wild or
weedy plant relatives. In general, EPA is focused on the potential for
genetic exchange that can occur in the field. However, evaluations of
the potential for genetic exchange are likely to include laboratory
studies that are not necessarily an accurate indicator of plants'
ability to exchange genetic material outside the lab.

4.  **What laboratory techniques used to achieve genetic exchange
    between species (e.g., embryo rescue, use of intermediate bridging
    crosses, protoplast fusion) are *not* indicative of possible genetic
    exchange between these species in the field? Conversely, what
    techniques, if any, used in laboratory or greenhouse experiments
    provide the most reliable indication of ability to hybridize in the
    field?**

EPA recognizes that it may be possible to genetically engineer a plant
such that concerns about gene flow to wild or weedy relatives are
significantly reduced. However, according to the 2004 NRC report,
[Biological Confinement of Genetically Engineered
Organisms]{.underline}, current techniques for bioconfinement (e.g.,
sterile triploids, male sterility) are imperfect and are not guaranteed
to eliminate entirely gene flow to existing wild relatives. Recent
modeling studies suggest imperfections in bioconfinement could result in
significant levels of gene introgression in compatible plant relatives
over a period of decades (Haygood et al. 2004).

5.  **Given that current bioconfinement techniques are not 100%
    effective, what would the environmental implications be of extremely
    low transfer rates of virus-resistance genes over time?**

EPA recognizes that concerns about gene flow to wild or weedy relatives
may be ameliorated if the introduced virus-resistance trait would give
little or no selective advantage to the recipient plant, as would occur
if the plant were already tolerant or resistant[^4] to the virus to
which resistance is conferred. It is obvious that such resistance does
exist in some populations because traditional breeding for resistance
relies on finding a source of resistance within related cultivated
species, old varieties, or wild species (Khetarpal et al. 1998).

6.  **Please comment on the prevalence of tolerance and/or resistance to
    viruses in wild relatives of crops.**

7.  **Please specify techniques that do or do not provide measures of
    tolerance and/or resistance that are relevant to field conditions.**

8.  **How do environmental or other factors (e.g., temporal variations)
    affect tolerance and/or resistance? Given the expected variability,
    what measures of tolerance and/or resistance would be reliable?**

9.  **What would be the ecological significance if a plant population
    acquired a small increase in viral tolerance and/or resistance above
    a naturally-occurring level?**

Based on the hypothesis that concerns about the consequences of gene
flow to a wild or weedy relative in the United States may be negligible
in certain cases, the Agency is considering whether there are mechanisms
to adequately address concerns associated with gene flow so that certain
types of VCPs would be of such low risk that they would not need to be
regulated by EPA. Below are examples of three conditions (modified from
those proposed in 1994) that are intended to significantly reduce any
potential adverse effects of gene flow with plants containing a
PVCP-PIP.

1.  The plant into which the PVCP-PIP has been inserted has no wild or
    weedy relatives in the United States with which it can produce
    viable hybrids in nature, e.g., corn, tomato, potato, or soybean; or

2.  Genetic exchange between the plant into which the PVCP-PIP has been
    inserted and any existing wild or weedy relatives is substantially
    reduced by modifying the plant with a scientifically documented
    method (e.g., through male sterility); or

3.  It has been empirically demonstrated that all existing wild or weedy
    relatives in the United States with which the plant can produce a
    viable hybrid are tolerant or resistant to the virus from which the
    coat protein is derived.

```{=html}
<!-- -->
```
10. **Please comment on how necessary and/or sufficient these conditions
    are to minimize the potential for the PVCP-PIP to harm the
    environment through gene flow from the plant containing the PVCP-PIP
    to wild or weedy relatives. Would any other conditions work as well
    or better?**

***Charge -- Viral Interactions***

Interactions between introduced plant virus sequences and other invading
viruses in transgenic plants (e.g., during recombination or heterologous
encapsidation) may be a concern to the extent that such events may
increase in frequency or be unlike those expected to occur in nature. It
has been hypothesized that such events could lead to the creation of
viruses with new disease states or transmission properties. The Agency
is evaluating the circumstances that might increase the potential for
such events to occur and the potential environmental consequences of
novel viral interactions[^5] in light of the 2000 NRC report which
stated 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." The report
went on to suggest that risks might be managed by particular ways of
engineering transgenes[^6]. However, under either of the 1994 proposed
exemptions, the Agency would be unable to ensure that such strategies
were implemented. The Agency's literature review, "Viral interactions in
viral coat protein transgenic plants," discusses possible ways of
managing these potential risks in detail.

***Questions -- viral interactions***

Viral interactions may occur in natural, mixed infections which are
common in plants. Hypothetical concerns related to potential adverse
effects resulting from viral interactions between infecting viruses and
PVCP-PIPs in transgenic plants may be attributed to opportunities for
interactions not expected to occur in nature. EPA is interested in
evaluating the significance of *novel* viral interactions involving a
viral transgene[^7].

11. **To what extent are *novel* viral interactions (e.g.,
    recombination, heterologous encapsidation) involving a viral
    transgene an environmental concern?**

Mixed viral infections can be extremely common in crops and other
plants. However, scientific uncertainty exists as to whether
recombination and heterologous encapsidation would occur more or less
frequently in the case of a viral transgene and an infecting virus
interaction as compared to such interactions in mixed infections of a
transgenic plant's non-bioengineered counterpart[^8].

12. **What conclusions can be drawn as to whether the likelihood of
    recombination and/or heterologous encapsidation would be increased
    or decreased in a transgenic plant compared to its non-bioengineered
    counterpart?**

A number of methods for reducing the frequency of recombination and
heterologous encapsidation have been identified. While the effectiveness
of these techniques has been verified for particular cases, their
applicability to all PVCP-PIPs is unclear. Recognizing that it would be
difficult for a product developer to measure rates of recombination,
heterologous encapsidation, or vector transmission under field
conditions, EPA is considering whether it would be necessary to verify
that such methods worked in any particular instance by measuring rates
in modified versus unmodified plants.

13. **How effective is deleting the 3' untranslated region of the PVCP
    gene as a method for reducing the frequency of recombination in the
    region of the PVCP gene? Is this method universally applicable to
    all potential PVCP-PIP constructs? Would any other methods work as
    well or better? Which methods are sufficiently effective and
    reproducible such that actual measurement of rates to verify rate
    reduction would be unnecessary?**

14. **Are any methods for inhibiting heterologous encapsidation or
    transmission by insect vectors universally applicable to all
    PVCP-PIPs? Which methods are sufficiently effective and reproducible
    such that actual measurement of rates to verify rate reduction would
    be unnecessary?**

15. **How technically feasible would it be to measure rates of
    recombination, heterologous encapsidation, and vector transmission
    in PVCP-PIP transgenic plants in order to show that rates are
    reduced?**

EPA recognizes that scientific disagreement exists as to the likelihood
of environmental impacts due to novel viral interactions in transgenic
plants modified with PVCP-PIPs. The Agency is considering whether there
are available mechanisms to adequately address concerns associated with
novel viral interactions so that certain types of PVCP-PIPs would be of
such low risk that they would not need to be regulated by EPA. Below are
examples of conditions that might significantly reduce either the
novelty \[(1) and (2)\] or frequency \[(3) and (4)\] of viral
interactions in PVCP-PIP transgenic plants.

1.  The genetic material of the PVCP-PIP is translated and/or
    transcribed in the same cells, tissues, and developmental stages
    naturally infected[^9] by every virus from which any segment of a
    coat protein gene used in the PVCP-PIP was derived.

2.  The genetic material of the PVCP-PIP contains coat protein genes or
    segments of coat protein genes from viruses established throughout
    the regions where the crop is planted in the United States and that
    naturally infect the crop into which the genes have been inserted.

3.  The PVCP-PIP has been modified by a method scientifically documented
    to minimize recombination, (e.g., deletion of the 3' untranslated
    region of the coat protein gene).

4.  The PVCP-PIP has been modified by a method scientifically documented
    to minimize heterologous encapsidation or vector transmission, or
    there is minimal potential for heterologous encapsidation because no
    protein from the introduced PVCP-PIP is produced in the transgenic
    plant or this virus does not participate in heterologous
    encapsidation in nature.

```{=html}
<!-- -->
```
16. **Please comment on how necessary and/or sufficient each of these
    conditions is to minimize the potential for novel viral
    interactions. Please address specifically what combination would be
    most effective or what conditions could be modified, added, or
    deleted to ensure that potential consequences of novel viral
    interactions in PVCP-PIP transgenic plants are minimized.**

***Other questions***

In 1994 EPA proposed exempting plant viral coat proteins from the
requirement of a food tolerance under the Federal Food, Drug, and
Cosmetic Act[^10] based on 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
mammals. The safety of consuming plant virus genes has been supported by
experimental work (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." However, EPA recognizes
that PVCP-PIP developers may wish to modify the PVCP-PIP construct and
that some methods of mitigating potential risks associated with
recombination and heterologous encapsidation might actually require them
to do so. Such modifications might result in changes to the protein(s)
produced thus creating potential food safety concerns, e.g., inadvertent
production of new toxins or allergens (Day 1996). Modifications of the
construct and alteration of the proteins produced creates the potential
for health impacts on non-target species as well as humans.

17. **To what degree and in what ways might a PVCP gene be modified
    (e.g., through truncations, deletions, insertions, or point
    mutations) while still retaining scientific support for the idea
    that humans have consumed the products of such genes for generations
    and that such products therefore present no new dietary exposures?**

18. **What are the potential adverse effects, if any, of such
    modifications on nontarget species (e.g., wildlife and insects that
    consume the PVCP-PIP)?**

Modifications of the construct may also potentially create the
opportunity for novel viral interactions because the inserted virus
sequences could be unlike any that occur naturally.

19. **To what degree and in what ways might a PVCP gene be modified
    (e.g., through truncations, deletions, insertions, or point
    mutations) before it would be a concern that novel viral
    interactions due to the modifications could occur because the PVCP
    gene would be significantly different from any existing in nature?**

The potential risk issues identified in this paper are specific to
virus-resistant transgenic plants. However, the Agency recognizes that
it may be necessary to evaluate other information related to the
PVCP-PIP.

20. **Would any additional requirements related to PVCP-PIP identity and
    composition (e.g., demonstration that the transgene has been stably
    inserted) be needed for significant reduction of risks associated
    with PVCP-PIPs?**

21. **Are there any considerations beyond gene flow, recombination, and
    heterologous encapsidation as posed in the preceding questions that
    the Agency should consider in evaluating the risk potential of
    PVCP-PIPs (e.g., synergy)?**

**References**

**Appendix: Definitions**

*Resistant*, when referring to PVCP-PIPs only, means the plant is not
infected by or is a non-host of the virus concerned.

*Naturally infects* means a naturally occurring virus is transmitted to
a plant by direct plant-to-plant contact, inanimate objects, or vectors
such as pollen, arthropods, nematodes, or fungi; and the virus
replicates and moves within the recipient plant. It does not include
human intervention, e.g., manual inoculation.

*Novel viral interaction* means interaction between portions of two or
more different viruses (e.g., through recombination or heterologous
encapsidation) not expected to occur in a mixed viral infection found in
nature.

*PVCP-PIP* means a plant-incorporated protectant created from the gene,
or a segment of the gene, that codes for a coat protein of a virus that
naturally infects crop plants.

*Tolerant*, when referring to PVCP-PIPs only, means the plant is able to
sustain the effects of a virus infection with negligible or mild symptom
expression and negligible or mild effects on fitness or growth despite
the presence of the virus within the host.

[^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]: PIPs may also be found naturally-occurring in plants or may be
    introduced through conventional breeding. However, the focus here is
    on PIPs, specifically those based on viral coat proteins, that are
    introduced into plants through genetic engineering.

[^3]: The UK Government, the Scottish Executive, the National Assembly
    for Wales and the administration in Northern Ireland have been
    promoting a national dialogue on genetic modification (GM) issues.
    One part of this was a review of the science of GM, led by Sir David
    King (the Government\'s Chief Scientific Adviser) working with
    Professor Howard Dalton (the Chief Scientific Adviser to the
    Secretary of State for the Environment, Food and Rural Affairs),
    with independent advice from the Food Standards Agency.

[^4]: See Appendix for definitions.

[^5]: See Appendix for definition of novel viral interactions.

[^6]: The report says, "\[c\]an transgenes be engineered to reduce or
    eliminate the risk that recombination will spawn new pathogens?
    Evidence suggests that elimination of genome replication-control
    sequences from transgenes can limit recombination and therefore
    risk\... Furthermore, strategies to produce resistance-mediating
    transgenes that encode nonfunction proteins or no protein can be
    used effectively against viruses. For example, resistant plants that
    express nontranslatable RNA can confer immunity through induction of
    post-transcriptional gene silencing..." (National Research Council
    2000; pg. 94)

[^7]: See EPA literature review "Viral Interactions in Viral Coat
    Protein Transgenic Plants" pp. 15-16.

[^8]: See EPA literature review "Viral Interactions in Viral Coat
    Protein Transgenic Plants" pp. 14-15.

[^9]: See Appendix for definition of naturally infects.

[^10]: EPA determines whether limits (tolerances) should be set on the
    amount of residues of PVCP-PIPs in food derived from the improved
    plant. When there is substantial information indicating safety and
    history of safe use, the developer may request an exemption from the
    requirement of a tolerance. Although a general tolerance exemption
    for all PVCP-PIPs has not been finalized, tolerance exemptions for
    specific PVCP-PIPs have been . They can be found in 40 CFR Part
    180.1182 (potato), 180.1184 (watermelon), 180.1185 (papaya), and
    180.1186 (cucumber).