Accumulation of transgene‐derived siRNAs is not sufficient for RNAi‐mediated protection against Citrus tristeza virus in transgenic Mexican lime

Abstract

Mexican lime plants transformed with the 3'-terminal 549 nucleotides of the Citrus tristeza virus (CTV) genome in sense, antisense and intron-hairpin formats were analysed for transgene-derived transcript and short interfering RNA (siRNA) accumulation, and for CTV resistance. Propagations from all sense, antisense and empty-vector transgenic lines were susceptible to CTV, except for a single sense-line plant with a complex transgene integration pattern that showed transgene-derived siRNAs in association with low levels of the transgene-derived transcript. In contrast, nine of 30 intron-hairpin lines showed CTV resistance, with 9%-56% of bud-propagated plants, depending on the line, remaining uninfected on graft inoculation, and the others being susceptible. Although resistance was always associated with the presence of transgene-derived siRNAs, their level in different sense and intron-hairpin transformants was variable irrespective of the response to CTV infection. In intron-hairpin lines with single transgene integration, CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs.

Strategic RNA Silencing for Plant Viral Resistance

“…However, a recent report showed that accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated resistance by transforming Mexican lime plants ( Citrus aurantifolia) with the 3′-terminal 549 nt of the Citrus tristeza virus (CTV) genome in intron-hairpin format. Analysis of intronhairpin lines with single copy transgene insertion, showed that CTV resistance was correlated with low accumulation of the transgene-derived transcript rather than with high accumulation of transgene-derived siRNAs (LÓpez et al 2010). A similar phenomenon occurred in RSV-resistant transgenic rice based on RNA silencing (Shimizu et al 2011).…”

RNA interference: concept to reality in crop improvement

“…There are many other transgenic plants with enhanced defence by RNAi-mediated gene silencing, such as Tomato Yellow Leaf Curl Virus (TYLC)-resistant tomato (Fuentes et al 2006), Rice Tungro Bacilliform Virus (RTBV)-resistant rice (Tyagi et al 2008) and Citrus Tristeza Virus (CTV)-resistant mexican lime (López et al 2010). Schwind et al (2009) have even shown that using hpRNA construct in Solanum lycopersicum (tomato) against Potato Spindle Tuber Viroid (PSTVd) produce PSTVd-resistant tomato plant varieties.…”

The Complex Genetics of Citrus tristeza virus

“…The SY determinant was mapped to the region encompassing the p23 gene and the 3' NTR (nt 18394-19296) ( Figure 6) [20]. The 3'NTR has been used to generate transgenic plants resistant to CTV [69] and it has also been related to symptom development for other virus [70]. Likewise, the p23 is an obvious candidate for SY symptom determinant since it is one of the most highly expressed CTV proteins [54], a RNA-binding protein responsible for asymmetric replication [16,17], and it is a viral suppressor of RNAmediated gene silencing mechanism [18].…”