Poster: Cell Walls

Abs # 1249: Viral induced gene silencing of cinnamate 4-hydroxylase in barley using a Barley Stripe Mosaic Virus vector

Presenter:	Tobias, Christian M., ctobias@pw.usda.gov
Authors	Tobias, Christian M. (A)   Chow, Elaine  (A)
Affiliations:	(A): USDA, ARS Western Regional Research Center

Reduction of lignin levels in forage grasses and energy crops such as switchgrass is desirable for improved forage digestibility, and improved process economics for ethanol production. We are exploiting the Barley Stripe Mosaic Virus (BSMV) vector system developed by Large Scale Biology Inc. to induce VIGS in barley tissue. The goal is to rapidly assess the impact on cell wall structure of silencing candidate genes identified through public EST sequencing projects that may be involved in lignin biosynthesis. As an initial test of the system we chose to downregulate independently two isoforms of cinnamate 4-hydroxylase(C4H). This enzyme is necessary for the formation of p-coumaric acid from cinnamic acid. Based on previous research, one of these isoforms is believed to be a rate limiting step for monolignol synthesis. Clones encoding the two isoforms of barley C4H were obtained from the Arizona Genomics Institute. These sequences were cloned distal to the gb coding sequence of BSMV, and Barley seedlings of the cultivar Black Hull-less were inoculated with infective BSMV transcripts to induce silencing. At 15-19 days post inoculation lignin levels were analyzed in upper, non-inoculated leaves undergoing silencing using acetyl bromide lignin determinations. Histological staining for lignin was also performed on fresh tissue sections. The effects on lignin levels and cell wall structure of downregulating C4H in barley using BSMV will be presented. Further enhancements to the BSMV VIGS system are anticipated for achieving more efficient silencing and higher throughput.