Proceedings of the XLVII Italian
Society of Agricultural Genetics - SIGA Annual Congress
Verona,
Italy - 24/27 September, 2003
ISBN 88-900622-4-X
Poster
Abstract - 1.25
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
B. MOLESINI, T.
PANDOLFINI, L. AVESANI, A. SPENA, A. POLVERARI
Dipartimento
Scientifico-Tecnologico, University of Verona, Strada Le Grazie 15, 37134
Verona, Italy
PTGS, virus
resistance, sharka, hairpin construct
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.
In
the ihprolC-PP197
gene (intron hairpin 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 is 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.
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.