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United States Department of Agriculture

Agricultural Research Service

Title: Plum pox virus coat protein gene intron-hairpin-RNA (ihpRNA) constructs provide resistance to Plum pox virus in Nicotiana benthamiana and Prunus domestica

Authors
item Hily, Jean Michel
item Scorza, Ralph
item Ravelonandro, Michel - INRA, FRANCE
item Damsteegt, Vernon
item Bassett, Carole
item Petri, Cesar - CLEMSON UNIVERSITY
item Liu, Zongrang

Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 21, 2007
Publication Date: November 21, 2007
Citation: Hily, J., Scorza, R., Ravelonandro, M., Damsteegt, V.D., Bassett, C.L., Petri, C., Liu, Z. 2007. Plum pox virus coat protein gene intron-hairpin-RNA (ihpRNA) constructs provide resistance to Plum pox virus in Nicotiana benthamiana and Prunus domestica. Journal of the American Society for Horticultural Science. 132(6):850-858.

Interpretive Summary: Genetic engineering offers the promise of disease control through non-chemical means. This technology could have important uses for tree fruits which are subject to a number of difficult to control disease problems including virus diseases. One of the most devastating virus diseases is plum pox virus which is an exotic disease organism that has recently invaded the U.S. We have developed plum plants that can "silence" the virus infection process. These plum plants were inoculated with plum pox virus and shown to be resistant to the virus. This work shows that we can now produce new plum pox virus resistance lines for breeding and variety release. Our technology can protect the U.S. plum industry should plum pox virus establish itself in the U.S. This is especially important because there are no chemical or other methods of protecting trees from this disease.

Technical Abstract: The efficiency and strength of RNA silencing appears to depend on the formation of dsRNA. Constructs with self-complementary sequences separated by an intron produce "hairpin" RNA (ihpRNA) structures that efficiently elicit post-transcriptional gene silencing (PTGS). In the present study, we used this technology to confer resistance to Plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV-CP (coat protein) gene. We confirm the high capacity of ihpRNA constructs for inducing RNA silencing in Nicotiana benthamiana, with more than 75% of transformants displaying PTGS as evaluated by specific DNA methylation and small interfering RNA (siRNA) production. We show that ihpRNA constructs provide PPV resistance, and we found a correlation between the length of the PPV sequence introduced in the ihpRNA constructs and the frequency of transgenic resistant plants. Plants transformed with the longer sequence produced a higher percentage of resistant lines. We further demonstrate for the first time that this technology is applicable to a woody perennial species. Two transgenic Prunus domestica PPV-CP ihpRNA lines show gene silencing characteristics (low level of PPV-CP mRNA, hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection sixteen months post inoculation.

Last Modified: 9/22/2014