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[SANET-MG] Honey Sweet plum poised
May 7, 2005
Prof. Joe Cummins
“Transgenic virus resistant plums poised for release”
On 9 September 2004 the Agricultural Research Service of the United
States Department of Agriculture (USDA) petitioned that virus resistant
transgenic plums be given non-regulated status. That petition is still
under consideration but should be available for public comment soon. The
proposed commercial release is, I believe, 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 (1). 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(1).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. There
was a high level of PPV-CP .gene transcription in the plum cell nucleus
, there was a low level of PPV-CP messenger RNA in the cytoplasm and
very little detectable coat protein produced. Attempts to infect the
transgenic plum with large innocula of PPV showed that little virus
was produced in the plum. The resistance to PPV in the transgenic plum
was based on a process called post transcriptional gene silencing (2).
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 (3,4).
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(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). This SAP
report like many others tends to long on speculation and discourages
full and adequate experimentation in areas where safety experiments are
few and inadequate.
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. The
extent of homologous recombination between PPV and the PPV-CP transgene
has not been adequately investigated . Furthermore, even the small
amount of PPV coat protein produced in the transgenic plants should be
evaluated for human consumption because the PPV-CP gene has been
manipulated and is the product of a nuclear gene not a virus. The SAP
report, essentially ignored phenomena such as the read through
effects associated with the nos transcription terminator in roundup
ready soy (9) The read through produced transcription variants and
potentially unsuspected fusion proteins.
In conclusion, transgenic plum pox resistant Honey Sweet Plum is
based on posttranscriptional gene silencing. It evaluation prior to the
application for non-regulated status does not appear to be fully
adequate. USDA may have acted peremptorily in seeking release of a
novel but poorly tested variety of a fruit tree. Given the outlook of
USDA the PPV resistant fruit tree may be imposed on all plum producers
in the near future to contain the spread of PPV. The period of public
consultation may near at hand and it seems wise to comment on the
petition for un-regulated status.
References
1.Scorza,R, Ravelonandro,M. and Gonsaloves,D. Plum tree named ‘Honey
Sweet’ United States Patent PP15,154 2004
2. 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,:201-9
3.Lindbo,J. and Dougherty,W. Plant pathology and RNAi: A brief history
Annu.Rev. Phytopathol. 2005,43,7.1-7.14
4.Ho.M. Subverting the genetic text ISIS Press Release 2004
http://www.i-sis.org.uk/
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. A Set of Scientific Issues Being Considered by the Environmental
Protection 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.Rang,A,Linke,B. and Jansen,B. Detection of RNA variants transcribed
from the transgenes in roundup ready soy Eur.Food Res.Technol.
2005,220,438-43
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