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United States Department of Agriculture

Agricultural Research Service

Research Project: CONTROL OF FUSARIUM MYCOTOXINS IN CORN, WHEAT, AND BARLEY

Location: Bacterial Foodborne Pathogens & Mycology Research Unit

Title: Activation tag screening to identify novel genes for trichothecene resistance

Authors
item Mcclaughlin, John -
item Salmon-Denikos, Emily -
item Umer, Anwar Bin -
item Basu, Debaleena -
item McCormick, Susan
item Gregory, Brian -
item Tumer, Nilgun -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 9, 2010
Publication Date: December 9, 2010
Citation: Mcclaughlin, J., Salmon-Denikos, E., Umer, A., Basu, D., Mccormick, S.P., Gregory, B., Tumer, N. 2010. Activation tag screening to identify novel genes for trichothecene resistance. Meeting Abstract.

Technical Abstract: The goal of our research is to identify plant genes which enhance trichothecene resistance and, ultimately, Fusarium Head Blight resistance in wheat and barley. We are taking a two pronged approach using Arabidopsis to identify plant genes which confer resistance to trichothecenes. The first approach identified Arabidopsis orthologs of previously identified yeast knockouts from a viability screen using a trichothecene mycotoxin, trichothecin. An alternative method to identify trichothecene targets in plants is to perform mutagenesis followed by selection. T-DNA mutagenesis coupled with phenotypic selection has proven to be an extremely successful strategy to identify and isolate genes. One drawback from screening traditional T-DNA mutants is the problem of gene redundancy whereby knockouts do not present identifiable phenotypes. An alternative version of T-DNA mutatgenesis, termed activation tagging, provides an effective approach to overcome this limitation. 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 >45,000 activation tagged Arabidopsis seeds for resistance to trichothecin and identified 15 lines that showed a very high level of 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. We will present the preliminary characterization of two of these mutants. Sequence analysis of the resistant lines by TAIL-PCR demonstrated T-DNA insertions in two novel genes, termed Arabidopsis thaliana resistant root formation1 and 5 (AtTRRF1 and AtTRRF5). Arabidopsis plants with independently generated knockouts (T-DNA) in these two genes are currently being tested for resistance. In addition, we are testing expression of neighboring genes by qPCR for upregulation due to the enhancer sequences. We propose that screening a large activation tagged Arabidopsis collection on media containing trichothecene mycotoxins provides an extremely flexible and efficient method to identify novel genes for trichothecene resistance in plants.

Last Modified: 9/10/2014
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