Effectiveness of RNA interference in transgenic plants

Kerschen, Arthur; Napoli, Carolyn A.; Jorgensen, Richard A.; Müller, Andreas E.

doi:10.1016/j.febslet.2004.04.043

Cited by 194 publications

(119 citation statements)

References 9 publications

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“…The resulting 339 bp PCR product was cloned into a plasmid vector and sequenced. Subsequently, the fragment was directionally cloned at Bam HI and Xba I sites into the expression vector pFGC5941 [9]. This is a dsRNA (hairpin) vector which in this case was used to express only the AV2 gene in the antisense orientation.…”

Section: Resultsmentioning

confidence: 99%

Silencing of the AV2 gene by antisense RNA protects transgenic plants against a bipartite begomovirus

Mubin

Mansoor

Hussain

et al. 2007

Virol J

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Whitefly-transmitted geminiviruses (genus Begomovirus) are phytopathogens that cause heavy losses to crops worldwide. Efforts to engineer resistance against these viruses are focused mainly on silencing of complementary-sense virus genes involved in virus replication. Here we have targeted a virion-sense gene (AV2) to develop resistance against Tomato leaf curl New Delhi virus, a bipartite begomovirus prevalent throughout the Indian subcontinent. We show that tobacco plants transformed with an antisense construct targeting this gene are resistant to the virus. Following challenged with the virus, transgenic plants remained symptomless, although viral DNA could be detected in some plants by PCR. This is the first report of transgenic resistance against a bipartite begomovirus obtained by targeting a virion-sense gene. The relatively conserved nature of the gene suggests that the technology may be useful to develop broad-spectrum resistance which is required because of the fact that plants are often infected with multiple begomoviruses in the field.

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Section: Resultsmentioning

confidence: 99%

Silencing of the AV2 gene by antisense RNA protects transgenic plants against a bipartite begomovirus

Mubin

Mansoor

Hussain

et al. 2007

Virol J

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“…The GFP ORF was ampliWed from U70495 (Davis and Vierstra 1998) using the primers GFPorfFXba and GFPorfnsKpn. The RAP2.4 deletion fragments were ligated to the 3Ј end of the GFP coding region and the resulting fragments cloned between the CaMV35S promoter and Tnos in the vector pFGC5941 (Kerschen et al 2004). The resulting plasmids were introduced into Agrobacterium tumefaciens strain GV3101.…”

Section: Subcellular Localisation Of Gfp-rap24 Fusion Proteinsmentioning

confidence: 99%

“…Restriction sites included in the forward and reverse primers, respectively, were used to clone the genes between the CauliXower Mosaic Virus (CaMV) 35S promoter and the nopaline synthase terminator (Tnos). The resulting expression cassettes [CaMV35S-RAP2.4B (or RAP2.4)-Tnos] were excised from pROK2 and recloned into pFGC5941 (Kerschen et al 2004), which contains the bar gene, conferring tolerance to the herbicide phosphinothricin.…”

Section: Construction Of Promoter-gus Fusions and Overexpression Plasmentioning

confidence: 99%

Regulation of multiple aquaporin genes in Arabidopsis by a pair of recently duplicated DREB transcription factors

Rae

Lao

Kavanagh

2011

Planta

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Identifying the transcription factors that mediate responses to abiotic stress is of fundamental importance in plant biology, not least because of their potential utility in crop improvement. The recently duplicated genes RAP2.4B and RAP2.4 encode transcription factors belonging to the abiotic stress-associated DREB A-6 clade in Arabidopsis thaliana. Both proteins localise exclusively to nuclei and show similar DRE-element-binding characteristics. Expression analysis of stressed and non-stressed plants revealed partially overlapping expression patterns. Both genes were highly expressed in stems and roots and were differentially induced in response to cold, dehydration and osmotic stress. RAP2.4B, however, was uniquely expressed at a high level in dry seeds and was induced by heat stress, while RAP2.4 was uniquely induced at a high level by salt stress. Microarray-based transcriptional profiling of double knockout and overexpression lines revealed altered expression of genes associated with adaptation to drought stress. Most strikingly, six aquaporin genes, five of which are members of a recently identified co-expression network, were downregulated in the double knockout line and correspondingly upregulated in the overexpression line, suggesting that these DREBs play a role in the regulation of water homeostasis.

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“…An RNAi-based silencing construct was produced using the first 200 nucleotides of the GFP gene [38]. The gene fragment was first cloned in the RNAi vector pFGC5941 [39,40] and then the expression cassette was sub-cloned into pGreen0029 at EcoRI and HindIII restriction sites. All binary vector constructs were finally transformed into Agrobacterium tumefaciens strain GV3101.…”

Section: Production Of Gene Expression Constructsmentioning

confidence: 99%

Identification of a major pathogenicity determinant and suppressors of RNA silencing encoded by a South Pacific isolate of Banana bunchy top virus originating from Pakistan

et al. 2010

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Five genes encoded by Banana bunchy top virus (BBTV) originating from Pakistan were expressed in Nicotiana benthamiana using a Potato virus X (PVX) vector. Expression of the master replication-associated protein (mRep) and movement protein (MP) resulted in necrotic cell death of inoculated tissues, as well as leaf curling and necrosis along the veins in newly emerging leaves. The systemic necrosis induced by the expression of MP was discolored (dark) in comparison to that induced by mRep. Expression of the cell-cycle link protein (Clink), the coat protein (CP), and the nuclear shuttle protein from the PVX vector induced somewhat milder symptoms, consisting of mild leaf curling and mosaic, although expression of the CP caused a necrotic response in inoculated leaf. The accumulation of viral RNA was enhanced by MP, Clink, and CP. Of the five BBTV-encoded gene products two, the MP and Clink, stabilized GFP-specific mRNA and reduced GFP-specific small interfering RNA in N. benthamiana line 16c when expressed under the control of the 35S promoter and co-inoculated with a construct for the expression of GFP hairpin RNA construct. These results identified MP and Clink as suppressors of RNA silencing. Taken together the ability of MP to induce severe symptoms in plants and suppress RNA silencing implicates this product as a major pathogenicity determinant of BBTV.

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Effectiveness of RNA interference in transgenic plants

Cited by 194 publications

References 9 publications

Silencing of the AV2 gene by antisense RNA protects transgenic plants against a bipartite begomovirus

Silencing of the AV2 gene by antisense RNA protects transgenic plants against a bipartite begomovirus

Regulation of multiple aquaporin genes in Arabidopsis by a pair of recently duplicated DREB transcription factors

Identification of a major pathogenicity determinant and suppressors of RNA silencing encoded by a South Pacific isolate of Banana bunchy top virus originating from Pakistan

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