RNA Silencing of the Introduced Coat Protein Gene of Turnip mosaic virus Confers Broad-Spectrum Resistance in Transgenic Arabidopsis

Abstract

The coat protein (CP) gene derived from Turnip mosaic virus (TuMV) isolate JO was introduced into Arabidopsis thaliana and the resulting transgenic progenies were analyzed for resistance to TuMV. Transgenic Arabidopsis plants with no detectable transcripts of the introduced CP gene exhibited complete resistance to TuMV. There was no significant correlation between the resistance and the copy number of the transgene. Instead, small interfering RNAs (siRNAs) were detected in these resistant plants, indicating that the resistance is attributed to RNA silencing. The RNA-mediated resistance was not only inherited over successive generations but also effective against 17 worldwide TuMV isolates with different pathogenicity. Comparative analysis of the CP genes among the 17 TuMV isolates revealed that the 380-nt in the 3' region is highly conserved, suggesting the importance of the 3' conserved region for broad-spectrum resistance. These results indicate that introduction of the TuMV-CP gene into the target Brassicaceae plants followed by selecting transformants that show RNA silencing for the transgenes can be an effective and reliable strategy for developing crucifer crops with a broad spectrum of resistance to TuMV.

Tomato Chlorotic Mottle Virus Is a Target of RNA Silencing but the Presence of Specific Short Interfering RNAs Does Not Guarantee Resistance in Transgenic Plants

“…Indeed, transgenic plants were shown to contain transgene-specific siRNAs. In contrast to experiments with RNA viruses (18,33,42,46) completely immune lines were not observed. Most transgenic lines, however, did show significant delays in symptom development, and two lines displayed highly resistant and even immune plants.…”

“…Noris et al (47) reported similar results for Tomato yellow leaf curl Sardinia virus, where this virus was able to infect tomato plants expressing siRNAs specific to AC1. Both cases contrast with RNA silencing-mediated resistance to RNA viruses, where the presence of virus-specific siRNAs in transgenic plants strongly correlates with virus resistance (18,33,42,46), indicating that RNA viruses are more susceptible to RNA silencing, possibly because both mRNAs and genomic RNAs can be the target of the silencing machinery.…”

Lectin-Mediated Resistance Impairs Plant Virus Infection at the Cellular Level

“…Arabidopsis plants were also inoculated mechanically with an extract of PlAMV-GFP-infected N. benthamiana plants, which was prepared by grinding infected leaf tissues in 0.1 M phosphate buffer, pH 7.0, as described previously . N. benthamiana plants were also inoculated with AV3 (Hashimoto et al, 2008), CMV (Suzuki et al, 1991), PVX (Komatsu et al, 2010), PVY (Hidaka et al, 1992), RaMV (Komatsu et al, 2007), TMV , TRV (Ratcliff et al, 2001), TuMV (Nomura et al, 2004), or WClMV (Nakabayashi et al, 2002) mechanically. These viruses were detected by RT-PCR with total RNA isolated from the upper leaves of virus-inoculated plants at 20 DAI using the primers indicated in Supplemental Table 4 online.…”

Suppression of transgene RNA silencing in transgenic Arabidopsis thaliana by a mild strain of Cucumber mosaic virus

“…Previously, we reported that transgenic Arabidopsis thaliana carrying the full coding region of a Turnip mosaic virus (TuMV)-CP gene showed complete resistance to TuMV isolates (Nomura et al 2004). The purpose of the present study was to determine the RNA silencing suppression ability of a mild strain of CMV isolated from tomato using TuMV-resistant transgenic A. thaliana plants (TuR-At) carrying posttranscriptionally silenced TuMV-CP transgenes.…”

“…Three TuR-At Arabidopsis lines, CP112-R1, CP202-R1, and CP232-R1, carrying TuMV-CP transgenes with posttranscriptionally silenced expression, and a TuMV strain, TuMV-JO, were described previously (Nomura et al 2004). Nontransgenic wild-type Col-0 of Arabidopsis thaliana was used as the control in these experiments.…”