[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]

[SANET-MG] transgenic virus resistant plums



June 11, 2006

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 (?-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.

********************************************************
To unsubscribe from SANET-MG:
1- Visit http://lists.sare.org/archives/sanet-mg.html to unsubscribe or;
2- Send a message to <listserv@sare.org> from the address subscribed to the list. Type "unsubscribe sanet-mg" in the body of the message.

Visit the SANET-MG archives at: http://lists.sare.org/archives/sanet-mg.html.
Questions? Visit http://www.sare.org/about/sanetFAQ.htm.
For more information on grants and other resources available through the SARE program, please visit http://www.sare.org.