Engineering Virus-Induced African Cassava Mosaic Virus Resistance by Mimicking a Hypersensitive Reaction in Transgenic Cassava

Abstract

A new African cassava mosaic virus (ACMV) resistance strategy was developed by mimicking a hypersensitive reaction using barnase (the ribunuclease produced by Bacillus amyloliquefaciens) and barstar (its specific inhibitor) genes in planta. The barnase gene was cloned under the control of AV promoter (AVp), which is trans-activated by the TrAP protein of ACMV. Synchronously, the barstar gene was driven by the AC promoter to counteract the basal expression of barnase. Upon viral infection, the ratio of barnase/barstar would be expected to shift in favour of the barnase due to the upregulation of AVp by TrAP, resulting in local cell death before the virus can spread to adjacent cells. 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 transformation. Among 24 transgenic lines, which have a normal phenotype, only 3 confirmed the presence of ACMV DNA-A promoter, and barnase and barstar genes. Southern analysis of the 3 lines showed the integration of the barnase and bastar genes, but transgene rearrangements were also observed. The basic expression level of barnase and barstar was detected at RNA level by RT-PCR in 2 plant lines. In vitro viral replication assay using leaves of transgenic plants compared with wildtype plants showed the reduction of viral replication in transgenic leaves by 86-99%.