Prof. Joe Cummins

Transgenic virus resistant plums

The United Sates Department of Agriculture (USDA) announced "We are
advising the public that the Animal and Plant Health Inspection Service
has received a petition from the U.S. Department of Agriculture's
Agricultural Research Service seeking a determination of nonregulated
status for plum designated as transformation event C5, which has been
genetically engineered to resist infection by plum pox virus (PPV). The
petition has been submitted in accordance with our regulations
concerning the introduction of certain genetically engineered organisms
and products. In accordance with those regulations, we are soliciting
public comments on whether this plum presents a plant pest risk. We are
also making available for public comment an environmental assessment for
the proposed determination of nonregulated status." The closing date for
making public comment is July 17,2006.

To make comments on line visit Regulation.gov :
<http://www.regulations.gov/fdmspublic/component/main> scroll down to
Animal and Plant Health Inspection Service. On that page find U.S.
Department of Agriculture, Agricultural Research Service; Availability
of Petition and Environmental Assessment for Determination of
Nonregulated Status for Plum Genetically Engineered for Resistance to
Plum Pox. There you may comment directly or attach a file in pdf format.
It worth mentioning that the transgenic plum petition is the first
transgenic tree to be put forward in a petition for nonregulated status.
Petitions for a number of transgenic trees are certain to follow in
short order. Those petitions will be of low quality unless there is
public participation is encountered in this first petition.

In 2004 a petition for nonregulated status for C5 Honey Sweet Pox
Potyvirus Resistant plum was submitted to USDA , a revised petition was
submitted during March 2006 and that was the petition opened for public
comment(1) An updated environment assessment of the resistant plum was
submitted with the revised petition (2). The most salient feature of the
revised petition and assessment was 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 related to 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, the Institut National de la
Recherche Agronomique ,Paris France and Cornell University. The plum
tree has the plum pox virus (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. In the event
that the pollen was produced on the PPV transgenic plum the flesh of the
fruit produced when a normal plum is pollinated would not contain the
PPV gene but the seed would

The transgenic plum contained the PPV coat protein gene ,along with the
selectable markers NPTII (Kanamycin resistance ) and GUS
(β-Glucuronidase**).** There were 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 gene from Agrobacterium finally the GUS
gene was driven by the CaMV promoter and transcription was terminated
with the nos gene.. Analysis of the genes inserted into the plum clone
C5 showed that there was a second insertion of the PPV gene insert that
was "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 behaved like single genes in crosses
indicating that hey were relatively close together on a chromosome. The
PPV-CV inserts were found to be methylated unlike the promoters or the
GUS gene(1).

Post transcriptional gene silencing is a cellular based sequence
specific post transcriptional RNA degrading system that is programmed by
the transgene encoded RNA sequence. RNA viruses produce double stranded
RNA during replication and these replicating virus is rapidly degraded
by the enzyme complex programmed by the transgene RNA (4).

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 (5). Event C5 PPV resistance was stable in open
field trials including controls either without transgenes or the
transformation events that were not virus resistant such as event
C3(1,6). The reason that event C5 was stably resistant to PPV appears to
be the duplication and methylation of the PPV-CP gene in event C5.

One problem with the C5 event in the environment may be the transfer of
the NPTII gene to soil bacteria and in turn horizontal transfer of the
gene to animal pathogens. The NPTII gene was shown to be extensively
transferred to a soil bacterium, Actinobacter, from transgenic sugar
beet (7). Even though the root stock for the C5 plum was 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.

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). The report provided extensive
discussion of concerns such as the spread of virus resistance to weedy
relatives but did not deal with the special consideration of the
implications of post transcriptional gene silencing. Viral interaction
including recombination between a viral transgene and an invading virus
(either the same or a different virus from which the transgene was
recovered), ,heterologous encapsidation ( adding the transgenic coat
protein to the capsid of an unrelated invading virus) and synergy
(synergy is illustrated by viral suppressors of post transcriptional
gene silencing). The panel believed that heterologous encapsidation and
synergy were relatively unimportant in PVPCP-PIPS and felt that the
recombination could be prevented by removal of the three prime (tail end
of the gene construct) un-translated end of the gene construct even
though there was limited support for that conclusion. 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 (8). Since viral coat
protein is not produced there is little concern over that impact but in
the transgenic plums a novel small RNA molecule is produced and the
safety of that RNA to consumers of transgenic plums should be
considered. The SAP report on PVCP-PIPS provides poor guidance for the
PPV-CP plum. There are well known post transcriptional gene silencing
suppressors in the poty viruses related to PPV and within PPV. Along
with The extent of homologous recombination between PPV and the PPV-CP
transgene has not been adequately investigated .

Post transcriptional gene silencing (PTGS) and the stability of
resistance has been studied . High levels of transgene mRNA was observed
in the nucleus accompanied by low levels of transgene RNA in the
cytoplasm (9) but later reports indicated that the inserted viral coat
protein genes were hypermethylated and showed no detectable expression,
transgenic trees inoculated with virus repeatedly showed no infection
during several years of virus exposure (1,4). The stability of PTGS has
been questions in studies showing that plum pox virus silencing can
easily be reversed through mutations in the small RNA targeting sequence
or by silencing suppressor mutations (10).

A small RNA related to PTGS was identified in the transgenic plum (1).
That unique RNA was not studied for its impact on animals including
humans that consume the plum or are exposed to it through breathing
pollen or exposure juices through skin abrasions. A small bacterial RNA
was found to elicit RNA interference in mammals (11) thus it is not
unreasonable to suggest that a unique interfering plum RNA may be active
in humans and animals. Regulators may giggle at such comments but it has
been found that overexposing mice to regulatory RNA may cause
fatality(12) Common sense requires adequate safety experiments.

The revised petition for nonregulated status for the virus resistant
plum is a major improvement over the previous petition. The previous
petition more or less ignored the small RNA virus gene silencing that
was the key to antiviral capacity of the transgenic plum. However, a
growing body of evidence indicated that the small silencing RNAs can
effect other plants and animals. There is a need for fuller testing of
the small silencing RNA from the transgenic plum for their effect on
both plants and animals including humans. The fact that people may have
eaten virus infected plums does not really indicate that the transgenic
plum that resists virus infection in a novel way is safe for people and
animals to consume.

# References

1.  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

2.  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

3.Scorza,R, Ravelonandro,M. and Gonsaloves,D. Plum tree named 'Honey
Sweet' United States Patent PP15,154 2004

4.Lindbo,J. and Dougherty,W. Plant pathology and RNAi: A brief history
Annu.Rev. Phytopathol. 2005,43,7.1-7.14

5.Ravelonandro, M., Scorza, R., Bachelier, J. C., Labonne, G., Levy, L.,
Damsteegt, V. Callahan,A. M., and Dunez, J. Resistance of transgenic
Prunus domestica to plum pox virus infection Plant Dis.1997, 81,1231-5

6\. 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

7\. 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

8.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

9\. 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

10\. 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 Mar;80(5):2429-36.

11\. Xiang S, Fruehauf J and Li CJ. Short hairpin RNA-expressing
bacteria elicit RNA interference in mammals. Nat Biotechnol. 2006
un;24(6):697-702

12\. 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 May
25;441(7092):537-41.