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United States Department of Agriculture

Agricultural Research Service

Title: Transformation of Barley with Antifungal Genes and Strategies for Their Targeted Expression

Authors
item Skadsen, Ronald
item Nuutila, Anna - VTT, FINLAND
item Sathish, Puthigae - UNIVERSITY OF WISCONSIN
item Kaeppler, Heidi - UNIVERSITY OF WISCONSIN

Submitted to: Proceedings of National Scab Forum
Publication Type: Proceedings
Publication Acceptance Date: October 1, 1998
Publication Date: N/A

Interpretive Summary: Our objective is to make barley and other cereals resistant to the fungal pathogens that destroy grain quality. A specific Fusarium fungus causes a disease of barley and wheat known as scab or Fusarium head blight. Fusarium has caused devastating losses to barley and wheat crops each year, since 1993. Barley and wheat have little resistance to Fusarium, which begins its attack on the seed by colonizing the leaf-like organs that surround the seed. Barley seeds produce proteins that can destroy fungi. However, these proteins are inside the seed and cannot provide a defense against Fusarium. We have cloned the genes that specify the production of these proteins and put the genes back into barley in such a way that they will potentially produce the antifungal proteins in the organs surrounding the seed. We are now determining whether the antifungal proteins are produced in these organs, as predicted. If successful, this may intercept and neutralize the Fusarium. In order to visualize the invasion route taken by Fusarium, the fungus was genetically modified so that it glows green. The Fusarium-resistant barleys and the knowledge of Fusarium that will be generated will benefit barley growers, processors and the food and beverage industries. The reliance on imported grains will be greatly reduced. This technology will also benefit consumers by preventing contamination of cereals with mycotoxins. Our methods can also be directly applied to wheat.

Technical Abstract: Antifungal permatin proteins occur in many cereal seeds, but little is known of their regulation and roles. Permatin cDNA clones, pBARPERM1 and 2, were produced from developing barley seeds, and pOATPERM1 was produced from oat. Developing seeds accumulate permatin mRNA bimodally, peaking very early, disappearing, and reaching a second peak in the doughy stage. Barperm1 mRNA is largely confined to tissues around the starchy endosperm, while Oatperm1 mRNA is distributed between the endosperm and surrounding tissues. Small amounts of permatin mRNAs also occur in roots and epicotyls. Developing seeds of a cultivar moderately resistant to Fusarium graminearum, had more Barperm1 mRNA than a highly susceptible cultivar. For genetic transformation of barley by particle bombardment, expression vectors were constructed from the pAHC25 ubi/GUS/BAR expression vector, after replacing the GUS gene with a permatin gene. Only one regenerated plant contained the Oatperm1 gene. Some of its progeny are growing and have the Oatperm1 gene present, as judged by PCR and Southern blots. Transformation with Barperm1 in the antisense orientation was undertaken to knock out expression of the native gene. This produced a number of regenerants with a grassy phenotype, apparently from altering a basic process. Transformation with hordothionin resulted in 100 healthy regenerants. These also have a distinctive phenotype, including curly flag leaves and wrinkling of other leaves. We are using the differential display technique to detect genes expressed mainly in spike tissues. The products will be used to location tissue-specific promoter sequences. These will serve in the future to direct the synthesis of antifungal proteins in lemma and palea tissues surrounding the seed, thus intercepting Fusarium.

Last Modified: 4/16/2014
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