Expression of self-complementary hairpin RNA under the control of the rolC promoter confers systemic disease resistance to plum pox virus without preventing local infection.
BACKGROUND
Homology-dependent selective degradation of RNA, or post-transcriptional gene silencing (PTGS), is involved in several biological phenomena, including adaptative defense mechanisms against plant viruses. Small interfering RNAs mediate the selective degradation of target RNA by guiding a multicomponent RNAse. Expression of self-complementary hairpin RNAs within two complementary regions separated by an intron elicits PTGS with high efficiency. Plum pox virus (PPV) is the etiological agent of sharka disease in Drupaceae, although it can also be transmitted to herbaceous species (e.g. Nicotiana benthamiana). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The rolC promoter drives expression in phloem cells. RolC expression is absent in both epidermal and mesophyll cells. The aim of the present study was to confer systemic disease resistance without preventing local viral infection.


RESULTS
In the ihprolC-PP197 gene (intron hair pin rolC PPV 197), a 197 bp sequence homologous to the PPV RNA genome (from base 134 to 330) was placed as two inverted repeats separated by the DNA sequence of the rolA intron. This hairpin construct is under the control of the rolC promoter.N. benthamiana plants transgenic for the ihprolC-PP197 gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of ihprolC-PP197 plants are resistant to PPV systemic infection. Local infection is unaffected. Most (80%) transgenic plants are virus free and symptomless. Some plants (20%) contain virus in uninoculated apical leaves; however they show only mild symptoms of leaf mottling. PPV systemic resistance cosegregates with the ihprolC-PP197 transgene and was observed in progeny plants of all independent transgenic lines analyzed. SiRNAs of 23-25 nt homologous to the PPV sequence used in the ihprolC-PP197 construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenic plants 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 used to confer PPV resistance to fruit trees where systemic disease causes economic damage.
Pandolfini
Tiziana
T
Dipartimento Scientifico-Tecnologico, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy. tiziana.pandolfini@univr.it
Molesini
Barbara
B
Avesani
Linda
L
Spena
Angelo
A
Polverari
Annalisa
A
D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods; D007113:Immunity, Innate / Q000235:genetics*; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000821:virology; D017801:Plum Pox Virus / Q000187:drug effects* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034622:RNA Interference / Q000187:drug effects; D018075:RNA, Complementary / Q000235:genetics / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D014026:Nicotiana / Q000187:drug effects / Q000235:genetics / Q000821:virology; D019076:Transgenes; D001617:beta-Glucosidase / Q000235:genetics
D018075:RNA, Complementary; D034741:RNA, Small Interfering; C071257:cytokinin-beta-glucosidase; D001617:beta-Glucosidase