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Title: IDENTIFICATION, CHARACTERIZATION AND USE OF A BROME MOSAIC VIRUS STRAIN AS A VECTOR FOR GENE SILENCING IN MONOCOTYLEDONOUS HOSTS

Author
item DING, XIN - NOBLE FOUNDATION
item Schneider, William
item CHALUVADI, SRINIVASA - NOBLE FOUNDATION
item MIAN, M.A. - NOBLE FOUNDATION
item NELSON, RICHARD - NOBLE FOUNDATION

Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2006
Publication Date: 11/1/2006
Citation: Ding, X.S., Schneider, W.L., Chaluvadi, S., Mian, M.R., Nelson, R.S. 2006. Identification, characterization and use of a brome mosaic virus strain as a vector for gene silencing in monocotyledonous hosts. Molecular Plant-Microbe Interactions. MPMI 19:1229-1239

Interpretive Summary: Gene silencing relies on a natural plant pathway to regulate excessive accumulation of messenger RNAs (mRNA). Any mRNA that is transcribed above acceptable levels is targeted and destroyed. Virus induced gene silencing (VIGS) occurs when the host plant uses the same pathway to recognize high levels of viral RNAs and degrade them. This has been a useful tool for studying genetics in dicot plants, as any gene can be silenced by expressing a portion of its sequence in a viral RNA. For monocots (grasses, corn, wheat, rice, etc…) this tool has not been utilized because of a lack of acceptable monocot plant viruses. This paper describes the discovery and characterization of a new strain of the monocot virus Brome mosaic virus (BMV). The virus was named F-BMV, and it was found to be capable of infecting rice, barley and a particular variety of corn. The genes necessary for the expanded monocot host range were identified, and the virus was cloned and modified for use as a VIGS vector. Vectorized BMV will be useful for analysis of gene function in rice and maize for which no VIGS system is reported.

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 pratensis (tall fescue) that infected rice, barley and a specific cultivar of maize (Va35) under our 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 maintained 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.