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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC CONTROL OF FUSARIUM MYCOTOXINS TO ENHANCE FOOD SAFETY

Location: Bacterial Foodborne Pathogens & Mycology Research Unit

Title: An activation tagging screen to identify novel genes for Fusarium head blight (FHB) resistance

Authors
item Mclaughlin, John -
item Bin Umer, Anwar -
item Basu, Debaleena -
item McCormick, Susan
item Tumer, Nilgun -

Submitted to: National Fusarium Head Blight Forum Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: December 6, 2011
Publication Date: December 6, 2011
Citation: Mclaughlin, J., Bin Umer, A., Basu, D., Mccormick, S.P., Tumer, N.E. 2011. An activation tagging screen to identify novel genes for Fusarium head blight (FHB) resistance. National Fusarium Head Blight Forum Proceedings. Session3,p.90.

Technical Abstract: The goal of this project is to identify plant genes that confer resistance against FHB and reduced DON accumulation. The identification of such genes offers the possibility to more fully understand the mechanisms of Fusarium susceptibility and to design transgenic strategies to increase FHB resistance in barley and wheat. We are using activating tagging to identify genes which confer resistance to trichothecenes. Activation tagging uses a modified T-DNA vector which contains multiple copies of the cauliflower mosaic virus (CaMV) 35S gene enhancer arranged in tandem. In addition to knocking out genes, the modified T-DNA vector can also function as an enhancer when inserted either upstream or downstream of a gene to produce gain-of-function phenotypes. The genomic location of the tag is readily identifiable by thermal asymmetric interlaced (TAIL) PCR. Using this approach, we have screened ~250,000 activation tagged Arabidopsis seeds for resistance to trichothecin and identified 30 lines that showed resistance. These plants were able to form roots on 4 µM Tcin, a concentration which severely inhibits germination and prevents root formation of the Col-0 wild type. Characterization of two of these lines using RT-qPCR identified an activation genotype in one line, termed Arabidopsis thaliana resistant root formation1 or AtRRF1 and a knockout genotype in the other, termed AtRRF5. In AtTRRF1, two novel lipid transfer protein (LTP) genes, designated as LTP4 and LTP5, were overexpressed compared to the wild-type control. LTPs are small cysteine-rich proteins that transfer lipids between membranes in vitro. To verify resistance, both LTP4 and 5 have been cloned into Gateway expression vectors and transformed into Arabidopsis. In AtRRF5, the activation tag was found in the second exon of TBR (Trichome Birefringence-Like), a gene that controls synthesis and deposition of secondary wall cellulose. Expression of AtRRF5 was not detected in the activation tagged line while the wild-type control showed a detectable level of expression, confirming that the insertion created a knockout genotype. Resistance in AtRRF5 was confirmed by testing two independent knockout lines which were obtained from the Arabidopsis Information Resource (TAIR) collection. This research has shown that activation tagging is a useful method to identify plant genes which play a role in trichothecene resistance. The next step will be to determine if the novel genes identified from the screen in Arabidopsis will confer resistance to DON and FHB in transgenic wheat and barley plants. In addition, we are exploring the use of activation tagging in both wheat and barley to directly identify genes for trichothecene resistance in these crop species.

Last Modified: 12/22/2014
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