Submitted to: National Fusarium Head Blight Forum Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 12/10/2002
Publication Date: N/A
Interpretive Summary: Fusarium graminearum continues to destroy much of the U.S. barley and wheat crops. The production of mycotoxins (e.g., DON) by Fusarium makes the harvest unsuitable for food, feed or malting. Genetic transformation with antifungal protein genes may be a viable method for introducing biochemical resistance. Low concentrations of a barley seed protein (thionin) completely suppress germination and growth of F. graminearum spores. However, this thionin is found only in the starchy endosperm. If it can be placed in external tissues such as the lemma/palea and pericarp, it may provide a barrier to F.graminearum, as it attempts to colonize the developing seeds. We have cloned the gene for producing this protein and have placed it into the barley genome so that it is expressed in all tissues. These plants will be tested for Fusarium resistance.
Technical Abstract: A previously unknown gene, D5, has been cloned and found to be expressed only in the lemma/palea and embryo. The promoter was used to drive the expression of a gfp reporter gene in a D5/gfp construct. In transient assays, expression was found in lemmas and not in leaves. As an internal control, the Ubi/GUS vector was simultaneously bombarded. GUS was expressed well in both leaves and lemmas. Several other lemma/palea- and pericarp-specific genes have been cloned. We have successfully transformed Golden Promise with a thionin gene and have so far found five transformants that produce thionin mRNA in seedling leaves, on northern blots. We have also tested subcellular targeting constructs by transient expression assays. All secreted GUS activity (MUG assay) into the apoplast, whereas no MDH activity was secreted (cell leakage control). The unaltered GUS control did not secrete, and had only localized GUS staining spots. Thionin antibodies were developed and tested on western blots of developing seed proteins and purified thionin protein. They did not cross-react with mature thionin, although they did react with two seed proteins of the proper size to represent the unprocessed thionin.