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[SANET-MG] USDA transgenic plum
There are still 5 days left to get your comment into USDA.
ISIS Press Release 11/07/06
USDA Proposes to Deregulate Its Own Transgenic Plum
Should not be approved, major gaps in risk assessment Prof. Joe Cummins
and Dr. Mae-Wan Ho
This report was submitted to the USDA on behalf of the Independent
Science Panel
Transgenic plum for plum poxvirus resistance
The United Sates Department of Agriculture (USDA) has announced that its
Animal and Plant Health Inspection Service (APHIS) has received a
petition from its Agricultural Research Service (ARS) seeking
non-regulated status for a transgenic plum designated transformation
event C5, genetically engineered to resist infection by plum poxvirus
(PPV), and is soliciting public comments on whether this plum presents a
plant pest risk. The closing date for making public comment is 17 July
2006 (http://www.regulations.gov/fdmspublic/component/main)
It is worth mentioning that the transgenic plum petition is the first
temperate transgenic tree to be petitioned for non-regulated status.
Petitions for a number of transgenic trees are certain to follow in
short order including transgenic forest trees, which would be really
disastrous for the world?s forests [1] (GM forest trees, the ultimate
threat, SiS 26). Those petitions will be of low quality unless
sufficient public participation is encountered in this first petition.
So please enter your objections now. Use this article freely.
A version of the same petition was first submitted to the USDA in 2004,
and the current petition open for public comment is a revised version
submitted in March 2006 [2], together with an updated environment
assessment [3]. The most salient feature of the revised petition and
assessment is that the gene for the viral coat protein was found not to
produce a viral protein but to initiate a process called
post-transcriptional gene silencing associated with a small inhibitory
RNA, a short sequence of RNA which can be used to silence gene expression.
The proposed commercial release is the patented plum variety ?Honey
Sweet? plum developed jointly by USDA, Institut National de la Recherche
Agronomique, Paris, France and Cornell University. The plum tree has the
plum poxvirus (PPV) coat protein gene incorporated to provide resistance
to the major plum pest PPV. The female parent of the plum is ?Bluebyrd?
(named for Senator Robert Byrd), while the pollen parent is ?unknown?.
The plant is not self fertile, a pollinator is required. The variety is
propagated by bud grafting to standard rootstocks [3]. The plum fruit is
a typical drupe in which the skin and flesh of the fruit contain only
maternal genes; the seed embryo and endosperm contain both paternal and
maternal genes. The seeds of the transgenic plum are viable and could
produce viable plants. A non-transgenic plum tree pollinated by the
transgenic plum will give fruits that will not contain the PPV gene in
their flesh, only the seed would. All the plums produced on transgenic
tree, however, fruit and seed, would be transgenic, regardless of the
status of the pollinator.
The transgenic plum contains the PPV coat protein gene along with the
selectable markers NPTII (Kanamycin resistance) and GUS
(?-Glucuronidase). There are multiple copies of the PPV coat protein
gene linked at the insertion site. The genetic modification of the plums
was done using a gene cassette containing the NPTII gene driven by the
relatively weak nos promoter from Agrobacterium and terminated by the
nos terminator. The PPV-CP was driven by the cauliflower mosaic virus
(CaMV) promoter and transcription was terminated by the nos terminator
from Agrobacterium. The GUS gene was also driven by the CaMV promoter
and transcription terminated with nos. Analysis of the genes inserted
into the plum clone C5 showed that there was a second PPV gene insert
unlinked to the primary NPII, GUS, PPV-CV gene insert. Fragments of the
NPII gene and the GUS gene were also detected in the transgenic plum.
The multi-copy PPV inserts appeared to behave like a single gene in
crosses, indicating that they are relatively close together on a
chromosome.
There were no attempts to characterize the inserts with fluorescent in
situ hybridization of chromosomes and to identify genome sequences
flanking the insert(s), both of which are now standard.
Sequencing was done, and results were ?inconclusive? on account of
?transgene duplications, rearrangements, and ?an inverted repeat of the
PPV-CP gene...? [2, p. 29] Sequences from the plasmid vector pBR322 were
also present in the insert(s).
PPV-CV insert was apparently methylated and its CaMV promoter more
lightly so, unlike the promoter or the GUS gene [2]. Nevertheless, the
PPV-CV is actively transcribed, and then presumably degraded in
post-transcriptional gene silencing, so only low levels of mRNA accumulated.
Post-transcriptional gene silencing is a sequence-specific
post-transcriptional RNA degrading system that is programmed by the
transgene-encoded RNA sequence [5].
The insertion of the PPV-CP gene cassette into the plum is necessary but
not sufficient to produce strong stable resistance to PPV. For example
plum transformation events C2, C3 and C4 accumulated high levels of
PPV-CP messenger RNA and coat protein but were not resistant to PPV. In
contrast, event C5 produced little PPV-CP messenger RNA and barely
detectable coat protein [6], but its PPV resistance appeared stable in
open field trials together with controls either without transgenes or
with other transformation events that were not virus resistant such as
event C3 [2, 7]. The stable viral resistance of C5 was associated with
the duplication and methylation of the PPV-CP gene, and also with a
small RNA species present in high concentrations (see later).
Horizontal spread of antibiotic resistance gene
One potential problem with the C5 event released into the environment is
the transfer of the NPTII kanamycin resistance gene to soil bacteria and
in turn, to animal pathogens. The NPTII gene was extensively transferred
from transgenic sugar beet to a soil bacterium, Actinobacter, in an
experimental situation [8]. Even though the rootstock for the C5 plum is
not transgenic and not able to transfer the NPII gene, the autumn
leaves, shed bark and flowers of the plum would certainly deliver a good
quantity of the antibiotic resistance gene to the soil. Sequence
homologies to bacterial sequences, including GUS gene and nos terminator
and pBR322 plasmid, are expected to greatly increase the frequency of
horizontal gene transfer, up to a billion-fold [9]. Furthermore, the
horizontal transfer of non-homologous DNA occurs at relatively high
frequencies when a homologous DNA ?anchor sequence? is present, which
can be as short as 99bp. There are at least 87 species of naturally
transformable bacteria in the soil [10]. As trees are long-lived, there
is every opportunity for horizontal gene transfer to take place from
transgenic trees.
Scientific Advisory Panel Report inadequate
In 2004, the United States EPA published a Scientific Advisory Panel
(SAP) Report on Plant Incorporated Protectant, specifically those based
on viral coat proteins (PVCP-PIPS) [11]. The report provided extensive
discussion of concerns such as the spread of virus resistance to weedy
relatives, but did not deal with the implications of
post-transcriptional gene silencing, horizontal gene transfer, or viral
interactions in the wild.
Transgene instability and viral recombination
We have drawn attention to the recombination between viral transgenes
and invading viruses in connection with the hazards of the cauliflower
mosaic virus 35S promoter [12] (Hazards of transgenic plants containing
the cauliflower mosaic viral promoter) that is in practically every
transgenic plant commercially grown, and is present in the transgenic
plum, driving both the PPV-CP gene and the GUS gene. We pointed out that
as the CaMV 35S promoter contains a recombination hotspot, it is more
likely to take part in horizontal gene transfer recombination and is a
major cause of transgene instability. This prediction has been confirmed
since [13-15] (Transgenic lines proven unstable; SiS 20, Unstable
transgenic lines illegal, SiS 21). Five out of five transgenic lines
commercially approved had rearrangements of the transgenic insert, and
the CaMV 35S promoter was a frequent breakpoint. The issue of transgenic
instability remains unresolved to this day, and we are not convinced
that the transgenic plum petitioned for non-regulated status is stable
in the absence of the appropriate molecular genetic data. In fact, there
are signs that it too is unstable (see later).
There are other ways in which viruses interact: heterologous
encapsidation (the transgenic coat protein adding to the capsid of an
unrelated invading virus and therefore helping it escape inactivation by
the host, and synergy, in which invading viruses supply suppressors of
post-transcriptional gene silencing mounted by the host, therefore
cancelling out the viral resistance. The SAP believed that heterologous
encapsidation and synergy were relatively unimportant in PVPCP-PIPS [11]
and felt that recombination could be prevented by removing the
untranslated tail end of the gene construct, even though there was
limited support for that supposition. The panel concluded that eating
transgenic viral coat protein should be considered safe (without
experimental verification) because people have been eating virus
infected plant material for a long time. We have argued in detail why
that assumption is invalid in the case of viral DNA such as the coat
protein gene or the CaMV 35S promoter [12], basically because a viral
gene isolated and placed in a foreign genetic and evolutionary context
can never be equated with the gene in the natural virus (Hazards of
transgenic plants containing the cauliflower mosaic virus promoter). As
viral coat protein is not produced, there is little concern over that
impact, though the viral coat protein DNA is present, and can take part
in recombination.
Safety of novel small RNA in transgenic plum not considered
Furthermore, the transgenic plum was found to produce a novel small RNA
molecule [2] associated with post-transcriptional gene silencing and
virus resistance, and its safety to consumers has not been considered.
Animals and humans may be exposed not just through consuming the plum,
but also through breathing pollen or to the fruit juice through skin
abrasions. This is particularly remiss, as interfering RNA species
(RNAi) are now known to be ubiquitous, with many effects on biological
functions [16, 17] (Life after the Central Dogma series; Subverting the
genetic text, SiS 24). A small bacterial RNA was found to elicit RNA
interference in mammals [18].
RNAi gene therapy, the injection of small RNAs to silence genes, touted
as a ?breakthrough? in precision in 2002, was found to have significant
off target effects in 2005. In May 2006, RNAi gene therapy was reported
killing dozens upon dozens of mice [19, 20] (Gene therapy nightmare for
mice, could humans be next? this issue). The effects were not
sequence-specific. Out of 49 different sequences of RNA tested, 23 were
lethal, killing the animals within 2 months. Another 13 sequences were
?severely toxic? to the liver. Against this background, it is reasonable
to ask if the small RNA in the transgenic plum is safe for humans and
animals.
The USDA documents on the plums also do not appear to include any report
on the impact of the transgenic plums on the mortality and behaviour of
bees, which are the pollinators for plums. We should also ask whether
the small resistance RNA produced in the transgenic plum is safe for bees.
Instability of viral resistance
The SAP report on PVCP-PIPS [11] provides poor guidance for risk
assessment of the PPV-CP plum in yet another respect. There are well
known post-transcriptional gene-silencing suppressors in the poty
viruses related to PPV and in PPV itself; and the extent of homologous
recombination between PPV and the PPV-CP transgene has not been
adequately investigated. Post-transcriptional gene silencing and the
stability of resistance of presumably the same transgenic plum had been
studied earlier [21], and high levels of transgene mRNA were detected in
the nucleus and low levels of transgene RNA in the cytoplasm. But the
later reports [2, 5] stated that the inserted viral coat protein genes
are methylated and show low levels of mRNA, indicating that transgene
expression was unstable.
Transgenic C5 trees inoculated with virus appeared to show no infection
during several years of virus exposure. However, the stability of PTGS
has been questioned in studies showing that plum poxvirus silencing can
easily be reversed through mutations in the small RNA targeting
sequence, or by mutations that activate the virus? suppressor of the
host?s silencing [22, 23].
There is a clear need for fuller testing of the small silencing RNA from
the transgenic plum for its effects on both plants and animals including
bees and humans, and to consider fully the consequences of horizontal
gene transfer and recombination and transgene instability.
USDA deems virus transgene-contaminated plums to be organic
Finally, there is the issue of transgene contamination. As bees are the
pollinators for plums, it would not be surprising if the transgenic plum
pollinates and contaminates non-transgenic varieties long distances
away. The actual fruit of the plum will not be transgenic, but the pits
of the fruit will be. USDA has deemed that accidentally GM-pollinated
organic fruit is still organic, and has not commented on transgene
pollution or on the patent-infringement issue, should the patent holders
detect the transgene in non-transgenic plums, organic or otherwise. US
has about 80 percent of the world prune export market and countries
which look for transgenes in prune flesh will not find transgenes, but
if whole prunes are measured the seed transgenes will show up.
The comment on organic plums and transgenic plums from the USDA petition
is as follows:
?The presence of a detectable residue of a product of excluded methods
alone does not necessarily constitute a violation of the National
Organic Standards. The unintentional
presence of the products of excluded methods will not affect the status
of an organic
product or operation when the operation has not used excluded methods
and has taken
reasonable steps to avoid contact with the products of excluded methods
as detailed in
their approved organic system plan. Organic certification of a
production or handling
operation is a process claim, not a product claim.
?It is not likely that organic farmers, or other farmers who choose not
to plant transgenic varieties or sell transgenic plum, will be
significantly impacted by the expected
commercial use of this product since: (a) nontransgenic plum will likely
still be sold and
will be readily available to those who wish to plant it; (b) plum trees
propagated by
grafting and growers purchasing bud wood or grafted plants will know
that this product is
transgenic because it will be marketed as plum pox virus resistant plum.
Additionally,
decreasing the overall incidence of plum pox in conventional orchards
may lower the
likelihood of an organic orchard becoming infected.?
We would be very surprised if growers purchasing plants marketed as
?plum pox virus resistant plum? would automatically know that the plants
are transgenic. It is a blatant attempt on the part of the regulator to
avoid labelling and hence mislead the public.
US organic plum producers may feel protected by the USDA position. But
the export market may look at whole plums, not just the flesh of the plums.
References
1.Ho MW and Cummins J. GM forest trees the ultimate threat Science in
Society 2005, 26, 14-16. http://www.i-sis.org.uk/isisnews.php
2.Scorza,R. Application for determination of non-regulatory status for
C5 (honey sweet) plum resistant to plum pox virus Revised petition 2006
ARS-PLMC5-6
3.USDAIAPHIS Environmental Assessment In response to USDA-ARS Petition
04-264-01P seeking a Determination of Non-regulated Status for C5 Plum
Resistant to Plum Pox Virus OECD Unique Identifier ARS-PLMC5-6 2006
4.Scorza R, Ravelonandro M. and Gonsaloves D. Plum tree named ?Honey
Sweet? United States Patent PP15,154 2004.
5.Lindbo J and Dougherty W. Plant pathology and RNAi: A brief history
Ann. Rev. Phytopathol. 2005, 43 (7) 1- 14.
6.Ravelonandro M, Scorza R, Bachelier JC, Labonne G, Levy L, Damsteegt
V, Callahan AM and Dunez J. Resistance of transgenic Prunus domestica to
plum pox virus infection. Plant Dis.1997, 81,1231-5.
7.Hily JM, Scorza R, Malinowski T, Zawadzka B. and Ravelonandro M.
Stability of gene silencing-based resistance to Plum pox virus in
transgenic plum (Prunus domestica L.) under field conditions Transgenic
Res. 2004,13, 427-36.
8.Nielsen K, van Elsas J.and Smalla, K. Transformation of Acinetobacter
sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil
microcosms and effects of kanamycin on selection of transformants. Appl
Environ Microbiol. 2000, 66,1237-42.
9.de Vries J, Herzfeld T and Wackernagel W. Transfer of plastid DNA from
tobacco to the soil bacterium Acinetobacter sp. By natural
transformation. Molecular Microbiology 2004, 53, 323-34.
10. de Vries J, Herzfeld T and Wackernagel W. Transfer of plastid DNA
from tobacco to the soil bacterium Acinetobacter sp. By natural
transformation. Molecular Microbiology 2004, 53, 323-34.
11. Agency Regarding: ISSUES ASSOCIATED WITH DEPLOYMENT OF A TYPE OF
PLANT-INCORPORATED PROTECTANT (PIP), SPECIFICALLY THOSE BASED ON PLANT
VIRAL COAT PROTEINS (PVCP-PIPS) SAP Report No. 2004-09 FIFRA Scientific
Advisory Panel Meeting, October 13-15, 2004.
12. Ho MW, Ryan A and Cummins J. Hazards of transgenic plants containing
the cauliflower mosaic viral promoter. Microbial Ecology in Health and
Disease 2000, 12, 6-11.
13. Collonier C, Berthier G, Boyer F, Duplan M-N, Fernandez S, Kebdani
N, Kobilinsky A, Romanuk M, Bertheau Y. Characterization of commercial
GMO inserts: a source of useful material to study genome fluidity.
Poster presented at ICPMB: International Congress for Plant Molecular
Biology (n°VII), Barcelona, 23-28th June 2003. Poster courtesy of Pr.
Gilles-Eric Seralini, Président du Conseil Scientifique du CRII-GEN,
www.crii-gen.org
14. Ho MW. Transgenic lines proven unstable. Science in Society 2003,
20, 35, http://www.i-sis.org.uk/isisnews.php
15. Ho MW. Unstable transgenic lines illegal. Science in Society 2004,
21, 23, http://www.i-sis.org.uk/isisnews.php
16. Ho MW. Life after the Central Dogma series, Science in Society 2004,
24, 4-13, http://www.i-sis.org.uk/isisnews.php
17. Ho MW. Subverting the genetic text. Science in Society 2004, 24,
6-8, http://www.i-sis.org.uk/isisnews.php
18. Xiang S, Fruehauf J and Li CJ. Short hairpin RNA-expressing bacteria
elicit RNA interference in mammals. Nat Biotechnol. 2006, 24(6), 697-702.
19. Ho MW. Gene therapy nightmare for mice, could humans be next?
Science in Society 2006, 31 (in press), http://www.i-sis.org.uk/isisnews.php
20. Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR,
Marion P, Salazar F and Kay MA. Fatality in mice due to oversaturation
of cellular microRNA/short hairpin RNA pathways. Nature 2006, 441(7092),
537-41.
21. Scorza R, Callahan A, Levy L, Damsteegt V, Webb K. and Ravelonandro
M. Post-transcriptional gene silencing in plum pox virus resistant
transgenic European plum containing the plum pox potyvirus coat protein
gene. Transgenic Res. 2001,10, 291-9.
22. Simon-Mateo C and Garcia JA. MicroRNA-guided processing impairs Plum
pox virus replication, but the virus readily evolves to escape this
silencing mechanism. J Virol. 2006, 80(5), 2429-36.
23. Goldbach R, Bucher E and Prins M. Resistance mechanisms to plant
viruses: an overview. Virus Res. 2003, 92(2), 207-12.
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