|Ding, Xin Shun|
Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/19/2006
Publication Date: 11/15/2006
Citation: Ding, X., Schneider, W.B., Chaluvadi, M.A., Mian, R.M., Nelson, R.S. 2006. Characterization of a brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts. Molecular Plant-Microbe Interactions. 19:1229-1239. Interpretive Summary: Virus induced gene silencing (VIGS) is a powerful functional genomics tool for high-throughput assessment of gene functions. The application of this powerful technology in monocotyledonous plants, including rice has been limited due to the limited number of virus expression vectors for monocotyledons. ARS Molecular Geneticist, Rouf Mian, while working at the Samuel Roberts Noble Foundation in Oklahoma, discovered a new strain of Brome mosaic virus (F-BMV) in tall fescue that has been found to be useful for VIGS in a number of grass species. Here we report the cloning and modification of F-MBV for VIGS in rice, barley and a specific cultivar of maize (Va35) under greenhouse conditions. VIGS with this modified virus in maize, barley and rice resulted in visual silencing-like phenotypes that were correlated with decreased targeted host transcript levels in systemic leaves of these plants. The vectorized F-BMV will be useful for analysis of gene function in rice and maize for which no VIGS system is reported. The information reported here expands the number of virus vectors available for functional genomic studies in barley beyond Barley stripe mosaic virus and provides a unique resource for studies with rice and maize.
Technical Abstract: Virus-induced gene silencing (VIGS) is a useful tool for analyzing gene function in dicotyledonous plants. The procedure, however, has not been fully utilized due to the limited number of virus expression vectors for monocotyledonous plants, especially rice. Here we report the cloning and modification for VIGS of a virus from Festuca arundinacea Schreb (tall fescue) that infected rice, barley and a specific cultivar of maize (Va35) under greenhouse conditions. Through capsid analysis and sequencing the virus was determined to be a strain of Brome mosaic virus (BMV). The virus was named F-BMV (F for Festuca) and determinants necessary for systemic infection of rice were genetically mapped to RNAs 1 and 2 of the tripartite genome. cDNA from RNA 3 of the Russian strain of BMV (R-BMV) was modified to accept inserts from foreign genes. Co-inoculation of RNAs 1 and 2 from F-BMV and a modified RNA 3 from R-BMV (infectious virus designated H-BMV; H for hybrid) containing portions of host genes to leaves of maize, barley and rice resulted in visual silencing-like phenotypes that were correlated with decreased targeted host transcript levels in systemic leaves of these plants. The VIGS visual phenotype for targeted host genes varied from stable for actin to transient for phytoene desaturase, lasting through two nodes of growth with incomplete penetration through the leaf lamina. F-BMV RNA 3 also was modified to allow greater accumulation of virus while minimizing virus pathogenicity. The modified vector, C-BMVA/G (C for chimeric), was shown to be useful for VIGS. Vectorized BMV will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.