2012 Annual Report
1a.Objectives (from AD-416):
Objective 1: Develop transformation expression vectors to target transgene expression specifically to tissues that are initially infected by Fusarium graminearum.
Objective 2: Develop antifungal candidate genes and gene constructs that can be used in targeted expression system to develop Fusarium-resistant barley.
Objective 3: Identify components of the GA response that can be used as predictors of malting.
1b.Approach (from AD-416):
Produce gene macroarrays from our lemma-specific gene library and a new epicarpspecific library. Probe libraries with cDNA from Fusarium-infected lemma and epicarp. Clone and identify the upregulated genes, and confirm tissue-specificity with RNA blots. A modified inverse PCR will be used to clone their promoters from barley. Promoter (upstream) regions will be ligated upstream of the green fluorescent protein gene in an expression vector and functionally confirmed in transient bombardment assays where tissues will be examined for fluorescence before and after infection with Fusarium. If successful, barley will be stably transformed with antifungal protein genes driven by these promoters using the Agrobacterium vector pRSHyg. These genes (cloned in this lab) include lemma thionin, Ltp, and germin. Transformants will be tested for Fusarium resistance. The gene for the barley gibberellin (GA) hormone receptor will be cloned using homologies to the rice receptor. The gene will be compared in GA response mutants. Receptor sequence and mRNA levels will be analyzed in barleys of varying malting qualities. The Barley1 GeneChip will be used to examine transcripts in 7 malting barleys and GA response mutants. The differences in transcript profiles will provide insights into the relationship of the GA signal transduction pathway and malting quality.
An “embryo factor”, released from the germinating barley embryo at a specific time interval, is thought to cause the alpha-amylase enzyme production in the aleurone. We have refined this observation and showed that the factor comes from the shoot axis (most likely from the apical meristem). Studies just completed raise doubts as to whether this factor is a gibberellin (GA) hormone. (See companion project – Specific Cooperative Agreement with a GA expert at the University of Calgary, Canada). The final analysis of all detectable GAs in all dissected seedling organs (aleurone, sub-aleurone, scutellum and root/shoot axis) has been completed. GA levels in the aleurone did not support the model that GAs from the embryo turn on hydrolytic enzyme genes in the aleurone. Metabolic profiling studies of the seed organs most susceptible to infection by the fungal pathogen, Fusarium graminearum, have been completed, except for final univariate statistical analysis. This study showed that perhaps the main biochemical change accompanying infection is the conversion of 7 sugars to sugar alcohols in both the lemma and the epicarp. This does not show up in microarray studies conducted to date, perhaps because the enzymes involved have not been fully identified and cloned from barley.
Fusarium graminearum, a fungal pathogen that causes Fusarium head blight, may hijack the seed’s metabolism, providing metabolites that the fungus needs for a successful infection. The infecting threads (hyphae) attack the lemma and palea (which form the hull) and epicarp (soft tissue layer under the lemma). A detailed metabolic profiling analysis of infection in these tissues revealed strong changes in their biochemical makeup. We found that the infected tissues produced sugar alcohols between 3 and 5 days after inoculation. These compounds are known to benefit fungi as nutrients, as protection from free radicals, and as protection from osmotic stress. This study provides insight into how this pathogen infects and could impact strategies to make barley resistant to Fusarium.
Factors governing the production of the great variety of enzymes produced in the germinating barley seed. There are substantial gaps in our knowledge of how the production of the great variety of enzymes produced in the germinating barley seed is regulated. We are trying to determine if a postulated “embryo factor”, released from the embryo, dictates the enzyme profile that will be produced in the outer aleurone layer. We found that the factor comes from the shoot axis at about 36 hours after imbibition begins. This factor is still thought by others to be a gibberellin (GA) hormone. Analysis of all detectable GAs in all dissected seedling tissues showed that the bioactive GA1 did not accumulate in the aleurone in the right amounts and at the right timing for it to be the embryo factor. All repetitions had different GA patterns, yet the alpha-amylase genes were turned on exactly at the same time in all. The impact will be an improved method for identifying superior malting barleys.
Moeller, J.R., Moscou, M.J., Bancroft, T., Skadsen, R.W., Wise, R.P., Whitham, S.A. 2012. Differential regulation of host mRNA translation during obligate pathogen-plant interactions. Molecular Biosystems. 8:2153-2165.