Post-Transcriptional Gene Silencing of the p23 Silencing Suppressor of Citrus tristeza virus Confers Resistance to the Virus in Transgenic Mexican Lime

Abstract

Previously, we have shown that most Mexican limes (Citrus aurantifolia (Christ.) Swing.) expressing the p23 gene of Citrus tristeza virus (CTV) exhibit aberrations resembling viral leaf symptoms. Here we report that five independent transgenic lines having normal phenotype displayed characteristics typical of post-transcriptional gene silencing (PTGS): multiple copies of the transgene, low levels of the corresponding mRNA, methylation of the silenced transgene, and accumulation of p23-specific small interfering RNAs (siRNAs). When graft- or aphid-inoculated with CTV, some propagations of these silenced lines were immune: they neither expressed symptoms nor accumulated virions and viral RNA as estimated by DAS-ELISA and Northern blot hybridization, respectively. Other propagations were moderately resistant because they became infected later and showed attenuated symptoms compared to controls. The susceptible propagations, in addition to symptom expression and elevated virus titer, accumulated p23-specific siRNAs at levels significantly higher than immune or non-inoculated propagations, and showed transgene demethylation. This variable response among clonal transformants indicates that factors other than the genetic background of the transgenic plants play a key role in PTGS-mediated resistance.

Citrus tristeza virus infection induces the accumulation of viral small RNAs (21–24-nt) mapping preferentially at the 3′-terminal region of the genomic RNA and affects the host small RNA profile

“…is resistant to CTV at the w hole plant level, w ith the resistance being conferred by the single d om inant locus Ctv, w hich m ost likely blocks virus m ovem ent because CTV replicates and form s virions in protoplasts of this species (Albiach -Martí et al 2004). In add ition to the gRN A and sgRN As, CTV propagation occurs w ith the accum ulation of vsRN As (Fagoaga et al 2006), but there is no d etailed inform ation about their physical properties, relative abund ance and d istribution along the viral genom e, as w ell as w hether CTV infection has any effect on plant sRN As. Moreover, it is unclear not only for CTV but in general, w hich are the substrates for antiviral DCLs because (+) ssRN A viruses m ay prod uce d sRN As d uring replication of the gRN A as well as d uring transcription of the sgRN As (Aliyari et al 2008), and highly -structured regions of the ss (+) gRN A and sgRN As m ay also be targeted by DCLs (Molnar et al 2005;Szittya et al 2010).…”

The Complex Genetics of Citrus tristeza virus

“…However, the symptomatology pattern developed in these transgenic plants is different than those induced by natural virus infection. Additionally, the grade of symptom severity observed in these p23 transgenic plants is directly proportional to the p23 production level, and independent of the viral source or sequence of the p23 gene [67,68]. Nevertheless, the symptom intensity in wild virus-infected limes or sour oranges is radically different between severe and mild isolates of virus.…”

Molecular Virology and Pathogenicity of Citrus tristeza virus

“…Advances in genetically engineered protection against viruses by the generation of transgenic plants have lately been remarkable. However, incorporation of pathogen-derived resistance by plant transformation of CP and p23 or the 3´NTR has yielded variable results Dominguez et al, 2002;Fagoaga et al, 2006;López et al, 2010). Another biotechnological approach to control the virus, and eventually turn it from a pathogen into a molecular tool for citrus improvement, is the custom engineering of a recombinant mild strain cross-protection (Albiach-Martí et al 2010).…”