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United States Department of Agriculture

Agricultural Research Service

Title: Transformation of Barley with Native Antifungal Genes - Targeted Expressionand Monitoring of Fusarium Infection

Authors
item Skadsen, Ronald
item Nuutila, Anna - VTT, FINLAND
item Sathish, Puthigae - UNIVERSITY OF WISCONSIN
item Kaeppler, Heidi - UNIVERSITY OF WISCONSIN
item Hohn, Thomas - NOVARTIS AGRI BIOTECH RES

Submitted to: Barley Improvement Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: January 5, 1999
Publication Date: N/A

Interpretive Summary: Our objective is to redirect the expression of genes encoding antifungal proteins to make barley and wheat resistant to the fungal pathogen Fusarium graminearum. This pathogen causes a mycotoxin-producing disease, known as scab. Losses to scab (Fusarium head blight or FHB) continue to devastate the barley and wheat industries. Although there is little resistance to Fusarium, barley already produces several potential defenses. Antifungal permatin proteins are found in seeds of barley (hordomatin) and oat (avematin). In addition, a storage protein (hordothionin) in seeds of barley has potent antimicrobial properties. However, these proteins are not found in the lemma and palea, the leaf-like organs that surround the seed. It is in these organs that Fusarium infection is established. We have cloned the permatin and hordothion genes and used them to genetically transform barley so that the genes are stably integrated into the barley genome. We are now determining whether the antifungal proteins encoded by these genes are actually produced. If successful, this may intercept and neutralize the pathogen. We are developing tissue-specific gene promoters so that antifungal genes can be expressed exclusively in the lemma and palea tissue. We are also conducting research with a modified form of the Fusarium fungus, which will allow us to determine precisely the subcellular route taken by the fungus during infection. This will allow us to place the proteins directly in path of the fungus. The Fusarium-resistant barleys and the knowledge of Fusarium that will be generated will be of value to barley and wheat growers and processors.

Technical Abstract: Antifungal permatin protein genes were cloned and used for transformation in order to learn their roles in cereal seeds and to produce barley lines with resistance to Fusarium graminearum. Permatin cDNA clones were produced from developing barley (pBARPERM1 and 2)and oat (pOATPERM1) seeds. These were used as northern blot probes and showed that permatins are expressed bimodally - peaking very early, disappearing, and reaching a second peak in the doughy stage. Weak expression occurs in roots and epicotyls. Barley was transformed by particle bombardment, using the pAHC25 ubi/GUS/BAR vector, after replacing GUS with a permatin gene. One Oatperm1 transformant was obtained. Its progeny are growing and contain the Oatperm1 gene, 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 several regenerants with a grassy phenotype, apparently from altering critical processes. Transformation with hordothionin resulted in 100 healthy regenerants. These have a distinctive phenotype, including curly flag leaves and wrinkling of other leaves. Barperm1 antibodies were produced to determine whether this protein accumulates in the lemma/palea of transformants.

Last Modified: 10/1/2014
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