Plum Pox Virus Coat Protein Gene Intron-hairpin-RNA (ihpRNA) Constructs Provide Resistance to Plum Pox Virus in Nicotiana benthamiana and Prunus domestica

Abstract

<jats:p>Constructs with self-complementary sequences separated by an intron produce “hairpin” RNA [intron-hairpin-RNA (ihpRNA)] structures that efficiently elicit posttranscriptional gene silencing (PTGS). In the current study, the authors use this technology to confer resistance to plum pox virus (PPV) in herbaceous and woody perennial plants by silencing the PPV–coat protein (<jats:italic>CP</jats:italic>) gene. The authors confirmed the high capacity of ihpRNA constructs for inducing RNA silencing in <jats:italic>Nicotiana benthamiana</jats:italic> Domin., as more than 75% of the transformants displayed PTGS as evaluated by specific small interfering RNA (siRNA) production. The authors demonstrated that ihpRNA constructs provided PPV resistance, and they found a correlation between the length of the <jats:italic>PPV</jats:italic> sequence introduced in the ihpRNA constructs and the frequency of transgenic-resistant plants. Plants transformed with the full-length sequence produced a higher percentage of resistant lines. The authors further demonstrated for the first time that ihpRNA technology is applicable to a woody perennial species. A transgenic plum (<jats:italic>Prunus domestica</jats:italic> L.) <jats:italic>PPV-CP</jats:italic> ihpRNA line showed gene silencing characteristics (hypermethylation of the transgene sequence and specific siRNA production) and resistance to PPV infection 16 months after inoculation.</jats:p>

Improvement of resistance to maize dwarf mosaic virus mediated by transgenic RNA interference

“…A longer hpRNA sequence is more efficient than a shorter one. Similar results were presented in tobacco (Chen et al, 2004), tomato (Praveen et al, 2009) and some herbaceous and woody perennial plants (Hily et al, 2007). Further study is necessary to clarify whether the length of the hpRNA sequences or their different target regions in the same gene brought about the different interference efficiency to virus gene expression.…”

“…This is usually attributed to the influence of the length and integration patterns of the transgenic inverted-repeat sequences to the efficiency of RNAi (Yin and Wang, 2000;Wesley et al, 2001;Di Nicola-Negri et al, 2005;Zhu et al, 2005;Rai et al, 2007;Clarke et al, 2008), and RNAi suppress protein (Mallory et al, 2001;Savenkov and Valkonen, 2002;Kubota et al, 2003;Love et al, 2007). A longer hpRNA expressed by the transgenic inverted-repeat sequence was found to be more effective to silence virus gene than a shorter one (Chen et al, 2004;Hily et al, 2007;Praveen et al, 2009), although the short limit of the transgenic inverted-repeat sequence for RNAi was found to be ∼20 bp (Qu et al, 2007). On the other hand, the integration of the exotic expression constructs themselves, especially transformed by Agrobacterium mediation, can induce chromosomal rearrangement including translocation, inversion, deletion and insertion (Azhakanandam et al, 2000;Yin and Wang, 2000;Rai et al, 2007;Zeng et al, 2010;Zhu et al, 2010).…”

Resistance to Citrus psorosis virus in transgenic sweet orange plants is triggered by coat protein–RNA silencing

“…On transcription, the resulting hairpin RNA acts as a strong inducer of post-transcriptional gene silencing (PTGS) (Smith et al, 2000). This strategy has been used to produce virus-resistant transgenic plants (Helliwell and Waterhouse, 2003;Hily et al, 2007;Kreuze et al, 2008;Prins et al, 2008;Shimizu et al, 2009;Fahim et al, 2010), with the accumulation of transgene-derived siRNAs being regarded as a predictor of resistance to virus infection (Kalantidis et al, 2002).…”

Genetically engineered resistance to Plum pox virus infection in herbaceous and stone fruit hosts

“…1) showed that gene constructs encoding intron-spliced hairpin RNAs (ihpRNAs) can induce RNA silencing with high efficiency (>90%) opening the way on how to effectively apply RNA silencing technology to obtain virus-resistant plants. 117 In order to develop robust tools for inducing PPV resistance, that are not highly dependent on a particular and uncontrolled event of transformation, PPV-derived constructs based on the ihpRNA technology 117 were developed using isolates of PPV-D [118][119][120][121] or -M strain. 122 Pandolfini et al utilized a short (Fig.…”

“…Hily et al 120 reported that four ihpRNA constructs, harboring the full length or the second half of the CP gene (Fig. 1), under the transcriptional control of either CaMV 35S or peach Cab promoters, were introduced in N. benthamiana plants.…”