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United States Department of Agriculture

Agricultural Research Service

Title: Engineering Resistance to Multiple Stone Fruit Viruses Through Expression of Chimeric Hairpins

Authors
item Liu, Zongrang
item Hily, Jean Michel
item Scorza, Ralph
item Scott, Simon - CLEMSON UNIVERSITY
item James, Delano - CFIA, SIDNEY, CANADA

Submitted to: Journal of the American Society for Horticultural Science
Publication Type: Proceedings
Publication Acceptance Date: February 10, 2006
Publication Date: May 1, 2007
Citation: Liu, Z., Hily, J., Scorza, R., Scott, S., James, D. 2007. Engineering resistance to multiple stone fruit viruses through expression of chimeric hairpins. American Society of Plant Biologists Annual Meeting. 132:283-436.

Technical Abstract: Stone fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. Here, we report a post-transcriptional gene silencing-based approach for engineering multi-virus resistance in plants. A single chimeric transgene PTRAP6 was created by the fusion of 400 -500 bp gene fragments from six major Prunus fruit viruses including american plum line pattern virus (APLPV), peach mosaic virus (PMV), plum pox virus (PPV), prunus dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an approximately 2.5 kb transcript in plants without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV, ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific siRNA species were detected along with the disappearance of transgene transcript in the resistant lines, indicating that post-transcriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus viruses.

Last Modified: 12/20/2014