CBN-V Video Archives - S7-36
Engineering Virus-induced ACMV Resistance by Mimicking a Hypersensitive Reaction in Transgenic Cassava Plants

Zhang P, P. Frey, J. Fütterer, I. Potrykus, J. Puonti-Kaerlas and W. Gruissem

Institute for Plant Sciences, ETH-Zentrum / LFW E 17, CH-8092 Zürich, Switzerland ZHANG.PENG@IPW.BIOL.ETHZ.CH

        To develop a new strategy towards African cassava mosaic virus (ACMV) resistance in cassava, the regulation of bidirectional ACMV DNA A promoter in plant cells and the barnase-barstar based toxin/anti-toxin system were explored. The barnase ORF was cloned under the ACMV virion-sense promoter, which is trans-activated by the TrAP protein of ACMV. Additionally, the barstar was introduced under the control of the ACMV complementary-sense promoter to counteract basal expression of barnase. Upon viral infection the ratio of barnase/barstar would be expected to shift in favor of the barnase due to the up-regulation of virus-sense by viral protein TrAP, resulting in local cell death before the virus can spread to adjacent cells. In order to adjust the expression level of barnase, constructs with different additional short open reading frames in front of the barnase gene were designed and used for transforming cassava via particle bombardment-mediated suspension transformation. Southern analysis of several transgenic cassava plant lines showed the integration of the barnase gene and bastar gene. The basic expression level of barnase and barstar ORFs could also be detected at RNA level by RT-PCR. An in vitro viral replication assay using leaves of transgenic plants compared with wildtype plants showed the reduction of viral replication in transgenic leaves.

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