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Zongrang Liu, Ralph Scorza, Jean-Michel Hily, Simon W. Scott, and Delano James

Prunus L. 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. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta 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 Domin. 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 small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.

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Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu, and Ralph Scorza

Constructs with self-complementary sequences separated by an intron produce “hairpin” RNA [intron-hairpin-RNA (ihpRNA)] structures that efficiently elicit posttranscriptional gene silencing (PTGS). In the current study, the authors use this technology to confer resistance to plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV–coat protein (CP) gene. The authors confirmed the high capacity of ihpRNA constructs for inducing RNA silencing in Nicotiana benthamiana Domin., as more than 75% of the transformants displayed PTGS as evaluated by specific small interfering RNA (siRNA) production. The authors demonstrated that ihpRNA constructs provided PPV resistance, and they 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 full-length sequence produced a higher percentage of resistant lines. The authors further demonstrated for the first time that ihpRNA technology is applicable to a woody perennial species. A transgenic plum (Prunus domestica L.) PPV-CP ihpRNA line showed gene silencing characteristics (hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection 16 months after inoculation.