Submitted to: In Vitro Cellular and Developmental Biology - Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2006
Publication Date: 7/1/2006
Citation: Carlson, A., Skadsen, R.W., Kaeppler, H.F. 2006. Barley hordothionin accumulates in transgenic oat seeds and purified protein retains anti-fungal properties. In Vitro Cellular and Developmental Biology - Plants. 42:318-323. Interpretive Summary: Barley seed hordothionin (HTH) is an innocuous protein that is part of the everyday diet. A very similar protein occurs in wheat seeds and in many other crops. This protein is highly toxic to many fungi which cause diseases of cereal seeds and deposit highly toxic mycotoxins. In an effort to protect seeds from such diseases, we cloned a barley gene which encodes the synthesis of this protein. We introduced this gene into oat plants and tested the effects on the fungal pathogen Fusarium graminearum. The HTH that we extracted from seeds of these transformed oats completely killed the fungus, even when it was used at very low concentrations. It was just as effective as the HTH purified directly from normal barley seeds. These studies will be of value to farmers, millers and feed companies, since they could lead to methods for producing safe disease-free seed.
Technical Abstract: The barley storage protein, hordothionin (HTH), is highly toxic to bacterial and fungal pathogens. In an attempt to increase oat's resistance to pathogens, a hordothionin cDNA (Hth1) was cloned from barley endosperm mRNA, subcloned into a constitutive expression vector, and transformed in oat. Native oat has no seed-specific thionin. Five transgenic oat lines were regenerated and all expressed Hth1 mRNA in the leaves, however the mature peptide only accumulated in the seed. The thionin line with the highest level of HTH accumulation was chosen for in vitro transgenic protein studies. Total protein extraction revealed that barley accumulated 500 micrograms of HTH/g of seed; the transgenic oat line produced 94 micrograms HTH/g of seed, 19% of levels in untransformed barley. The thionin extract was partially purified using ion exchange chromatography. The antifungal activities of HTH fractions from barley cultivar Morex and transgenic and control oat were tested using in vitro spore germination and hyphal growth assays with a seed pathogen, Fusarium graminearum. The control oat did not inhibit fungal growth, while HPLC-purified HTH and the barley and transgenic oat extracts inhibited growth to similar levels over a range of concentrations. Complete inhibition of Fusarium growth was observable at 16 micrograms/mL using HTH from all three sources. These results indicate that hordothionin can be expressed at significant levels in a heterologous cereal species and can maintain its antifungal properties.