RNAi-Mediated Protection Against Citrus Tristeza Virus in Transgenic Citrus Plants

RNA silencing is a sequence-specific mechanism of inhibition of gene expression evolutionarily conserved in most eukaryotes. RNA interference (RNAi), a technology based on the use of double-stranded RNA (dsRNA) to trigger RNA silencing, can be achieved in plants by genetic transformation with sense and antisense cDNAs derived from target viral sequences separated by an intron (intron-hairpin constructs). Upon transcription, the resulting hairpin RNA transcript usually acts as a strong inducer of RNA silencing. This strategy has been widely used to produce virus-resistant transgenic plants. Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) is the causal agent of the most devastating viral diseases of citrus trees in the world. It only infects phloem-associated tissues of Citrus species and relatives within the family Rutaceae. CTV is one of the largest and most complex plant RNA viruses, with a single-stranded, plus-sense RNA genome of 19.3 kb, organized in 12 open reading frames (ORFs), potentially coding for at least 17 polypeptides, and two 5′ and 3′ unstranslated regions (UTRs). Replication and expression of the genomic RNA results in more than 30 different plus and minus RNA species, as well as their corresponding dsRNA forms. Concomitantly, citrus hosts have developed a strong antiviral response through RNA silencing, as inferred from the high level of virus-derived siRNAs observed in infected tissues. As a counterdefense, CTV encodes at least three silencing suppressor proteins acting intracellularly and/or intercellularly to overcome antiviral defense. Under these circumstances, searching for RNAi-induced resistance against CTV in transgenic citrus plants becomes a real challenge. We have used intron-hairpin constructs targeting several viral regions, with our present interest focusing on one or the three CTV genes encoding silencing suppressors, or on conserved domains important for viral replication and encapsidation.