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United States Department of Agriculture

Agricultural Research Service

Title: An in Vivo Transient Expression Assay for Antifungal Genes in Barley Coleoptiles Infected with Blumeria Graminis

Authors
item Bucciarelli, Bruna - UNIVERSITY OF MINNESOTA
item Bushnell, William
item Baldridge, Gerald - UNIVERSITY OF MINNESOTA
item Giroux, Randal - GRAIN RES LAB, CANADA
item Szabo, Les

Submitted to: International Congress of Plant Molecular Biology
Publication Type: Abstract Only
Publication Acceptance Date: June 18, 2000
Publication Date: N/A

Technical Abstract: We have utilized an in vivo prescreen method to evaluate the efficacy of genes for antifungal proteins (AFPs) in a transient expression assay. Genes tested were introduced into epidermal cells of barley coleoptiles by particle bombardment. The AFP genes evaluated were: two chitinases (rice and barley), a B-1,3- glucanase (alfalfa), an inactivated B-1,3- glucanase (barley), a ribosome inhibiting protein (barley) and 6 thaumatin-like proteins (3 from oats, 1 from wheat, 1 from arabidopsis and 1 acidic form from barley). Four promoters were also evaluated to determine optimal gene expression: the CaMV 35S promoter, the maize ubiquitin promoter, and two forms of a sugarcane bacilliform virus promoter (GScBv and BScBv). The tissue was challenged with conidia of Blumeria graminis f.sp. hordei and then evaluated microscopically for appressorium and haustorium development. Concomitantly with each AFP gene, the maize C1 and R genes that regulate anthocyanin biosynthesis were also introduced. Cells expressing the C1 and R genes turned red two days after bombardment. The red cells had a high probability of coexpressing the test AFP gene. Genes that were effective in reducing infection rates were: rice and barley chitinase, one oat thaumatin-like protein, and the barley acidic thaumatin-like protein. No promoter was consistently better than others. Some promoter::gene combinations increased infection rates in ways unrelated to a specific gene product. These included the ribosome inhibiting protein gene driven by the CaMV 35S and the inactivated B-1,3- glucanase gene driven by the maize ubiquitin promoter. We are currently developing techniques to confirm gene expression and measure protein content in individual transformed cells.

Last Modified: 8/29/2014
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