Expression of self complementary hairpin RNA under the control of rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection

Background: Homology-dependent selective degradation of RNA, or post-transcriptional genesilencing (PTGS), is involved in several biological phenomena, including adaptative defensemechanisms against plant viruses. Small interfering RNAs mediate the selective degradation oftarget RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAswithin two complementary regions separated by an intron elicits PTGS with high efficiency. Plumpox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also betransmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV istransmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolCpromoter drives expression in phloem cells. RolC expression is absent in both epidermal andmesophyll cells. The aim of the present study was to confer systemic disease resistance withoutpreventing local viral infection.Results: In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologousto the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated bythe DNA sequence of the rolA intron. This hairpin construct is under the control of the rolCpromoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologousto the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPVsystemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free andsymptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they showonly mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAsof 23–25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detectedin transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenicplants 6 weeks after viral inoculation.Conclusions: The ihprolC-PP197 transgene confers systemic resistance to PPV disease in N.benthamiana. Local infection is unaffected. This transgene and/or similar constructs could be usedto confer PPV resistance to fruit trees where systemic disease causes economic damage.