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United States Department of Agriculture

Agricultural Research Service

Title: Transient Expression of Anthocyanin Genes in Barley Epidermal Cells: Potential for Use in Evaluation of Disease Response Genes

Authors
item Nelson, Amy
item Bushnell, William

Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 13, 1996
Publication Date: N/A

Interpretive Summary: Plants respond to fungus diseases by producing antifungal substances that delay or stop development of the fungus pathogens. The plants do this by activating "defense" genes that set in motion metabolic processes that lead to production of the antifungal substances. However, the exact role or importance of individual defense genes is not understood. We have devised a unique way to test for the possible action of individual defense genes by introducing them into epidermal tissues of barley. This is done by coating tiny gold particles with genes (DNA) and blasting them with high pressure helium into cells. We are using genes for anthocyanins, the red-colored pigment seen in fall foliage, to tell if the DNA has entered. In the assay, we inoculate red cells to see if defense genes (introduced along with the anthocyanin genes) have an effect on fungal pathogens. We will use this technique to assay for the importance of the 30 or more defense genes that have been isolated from plants. The results will be important for the scientists around the world who are trying to improve the effectiveness of defense responses in plants.

Technical Abstract: A transient assay is described that should allow evaluation of the role of host genes in disease response by enhancing or disrupting expression of those genes in specific cells and looking for effects on disease development. The assay also has the potential for assessing utility of host and non-host genes in enhancing resistance to disease in transgenic plants. Particle bombardment with a helium discharge particle gun was utilized to transiently express genes in epidermal cells of coleoptiles of barley, Hordeum vulgare. An anthocyanin reporter gene construct provided a means of identifying those cells that were transiently expressing introduced DNA. Optimal transient expression rates were achieved two days following bombardment with 1800 psi helium pressure, 1.0 um diameter gold particles, and coleoptile pre- and posttreatment in 0.30-0.35 M mannitol/sorbitol. Under optimal conditions, at least 35 cells expressed anthocyanin per bombardment. Transiently expressing cells were inoculated with the fungal pathogen, Erysiphe graminis, f.sp. hordei, and fungal development observed. Neither the bombardment procedures, the presence of nearby dead cells, nor accumulation of anthocyanin within living cells affected rates of fungus infection or development in living cells. Therefore, incorporation of disease-related genes onto the same plasmid as the reporter genes will allow evaluation of the role of those genes in disease development or suppression. Since particle bombardment is possible with a great range of different plant tissues, the described methodology should exhibit wide applicability for evaluating genes in diverse plant- pathogen interactions.

Last Modified: 4/16/2014
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