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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVEMENT OF BARLEY SEED QUALITY THROUGH MOLECULAR AND FUNCTIONAL GENOMIC ANALYSIS OF GENE EXPRESSION

Location: Cereal Crops Research

Title: Thionin antifungal peptide synthesis in transgenic barley

Authors
item Fu, Jianming -
item Patel, Minesh -
item Nuutila, Anna Maria -
item Skadsen, Ronald

Submitted to: Book Chapter
Publication Type: Book / Chapter
Publication Acceptance Date: September 20, 2011
Publication Date: September 25, 2012
Citation: Fu, J., Patel, M., Nuutila, A., Skadsen, R.W. 2012. Thionin antifungal peptide synthesis in transgenic barley. Small Wonders: Peptides for Disease Control. Washington, D.C. American Chemical Society Books. 359-377.

Interpretive Summary: The inner starchy portion of barley seeds contains a variety of proteins that kill invading bacteria and fungi. However, one of the main fungal pathogens of barley and wheat (Fusarium graminearum) attacks developing seed by first colonizing outer tissues - the protruding seed tip and the hull (lemma). We are trying to raise the amount of one anti-fungal protein in these tissues. Only small amounts of this protein, called HTH, kills the fungus. We determined the lethal levels of this protein and other variants of the protein. We found that the protein can only be produced in the starchy endosperm, where it belongs. We worked with another variant of this protein and were able to make these outer tissues and the leaves produce high amounts of the protein. We are now developing stable genetic lines of these new barleys. Once stable, they will be tested for resistance to Fusarium. These studies will lead to improved pathogen resistance in barley and improved seed quality.

Technical Abstract: In seeds and vegetative organs of barley and other cereals, thionins are processed into peptides with pronounced anti-microbial properties. In vitro studies demonstrated the toxicity of a- and ß-hordothionins (HTHs) to the fungal pathogen Fusarium graminearum. Increasing the expression of thionin genes may, therefore, provide resistance against this and other pathogens. Before a transgenic strategy can succeed, obstacles to Hth expression must be overcome. Barley transformed with a seed-specific barley aHth1 gene produced very little Hth1 mRNA in non-endosperm tissues. Removal of the first of two nearby 5’ methionine codons (producing aHth1') increased Hth1 mRNA levels. However, not even the association of Hth1 with membrane-bound polyribosomes led to HTH protein accumulation. Transformation of oat with this Hth1 vector (Carlson et al., 2006) showed that HTH1 protein was produced but only in the endosperm. These findings suggest that cereals have a mechanism (or deficiency) that prevents HTH1 protein accumulation beyond the endosperm. A ß-hordothionin cDNA, Lemthio1, cloned from lemma (hull) mRNA, produced high levels of LEMTHIO1 protein in lemmas of transformants. These will be tested for resistance to F. graminearum. In addition, we found that the population of the large thionin gene family variants was skewed toward cell wall bound forms. This may reflect the fibrous nature of the lemma.

Last Modified: 4/16/2014
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