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

• Main Authors: Spena Angelo, Avesani Linda, Molesini Barbara, Pandolfini Tiziana, Polverari Annalisa
• Format: Article
• Language: English
• Published: BMC 2003-06-01
• Series: BMC Biotechnology
• Online Access: http://www.biomedcentral.com/1472-6750/3/7

<p>Abstract</p> <p>Background</p> <p>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 <it>Drupaceae</it>, although it can also be transmitted to herbaceous species (e.g. <it>Nicotiana benthamiana</it>). Once inside the plant, PPV is transmitted via plasmodesmata from cell to cell, and at longer distances, via phloem. The <it>rolC </it>promoter drives expression in phloem cells. <it>RolC </it>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.</p> <p>Results</p> <p>In the <it>ihprolC-PP197 </it>gene (<it>i</it>ntron <it>h</it>air <it>p</it>in <it>rolC PP</it>V <it>197</it>), 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 <it>rolA </it>intron. This hairpin construct is under the control of the <it>rolC </it>promoter.<it>N. benthamiana </it>plants transgenic for the <it>ihprolC-PP197 </it>gene contain siRNAs homologous to the 197 bp sequence. The transgenic progeny of <it>ihprolC-PP197 </it>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 <it>ihprolC-PP197 </it>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 <it>ihprolC-PP197 </it>construct were detected in transgenic plants before and after inoculation. Transitivity of siRNAs was observed in transgenic plants 6 weeks after viral inoculation.</p> <p>Conclusions</p> <p>The <it>ihprolC-PP197 </it>transgene confers systemic resistance to PPV disease in <it>N. benthamiana</it>. 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.</p>