2010 Annual Report 1a.Objectives (from AD-416) Develop transformation expression vectors to target transgene expression to tissues initially infected by Fusarium graminearum. Identify genes upregulated during infection of seed lemma and epicarp; clone and test their promoters for inducibility and tissue-specificity. Develop antifungal genes to use in targeted expression system to develop Fusarium-resistant barley. Identify components of the GA response to predictors of malting quality. Determine whether the gene for a barley aleurone GA receptor can be used as an indicator of malting quality. Analyze gene responses to GA in malting barley and GA response mutants under malting conditions. 1b.Approach (from AD-416) Produce gene macroarrays from our lemma-specific gene library and a new epicarp-specific 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. 3.Progress Report This is the final report for project 3655-21430-008-00D, to be succeeded by project 3655-21430-009-00D. An antifungal thionin gene was cloned and introduced into barley to produce resistance to the fungal pathogen Fusarium graminearum. High levels of thionin protein were produced. Stable transformants were advanced to the T2 generation. Tests for resistance to Fusarium were conducted with leaves. These showed considerably more resistance than controls. These will now undergo field testing. Lines were screened at a collaborating lab for resistance to Ug99 stem rust and are undergoing further testing in Africa. Gene expression analysis is supposed to reflect changes in the biochemical environment during various fungal pathogen infections. Five highly replicated Fusarium graminearum infections were conducted. Uniform lemma and epicarp samples were collected during infections and readied for metabolic profiling using GC-mass spectrometry. Test samples are undergoing analysis to optimize tissue extraction procedures prior to GC-MS analysis. Our initial study showed that numerous metabolyte levels are altered during infection. Most noticeable was a large increase in ribitol in lemmas and epicarps. These studies will help to form a picture of how F. graminearum is able to infect barley. Microarray studies that analyze changes in gene expression use total tissue RNA and assume that amounts of various mRNAs predict corresponding protein levels. It is more logical to analyze polysomal mRNAs, since these are actually translated into protein. In germinating barley, half of the expressed genes differed in expression level by more than two-fold between the total and polysomal RNA populations. A collaborative project verified these results. Polysomes and total RNA were prepared from barley infected with powdery mildew and from Arabidopsis infected with turnip mosaic virus. We found that 15% of gene expressions differed by at least 4-fold, depending on the source of the mRNA. These studies showed that microarray studies around the world could be using the wrong RNA. Gibberellin (GA) hormone activates hydrolytic enzyme genes critical to malting. GA binding protein and an inhibitor of GA-induced transcription were cloned. Antibodies to the GA binding protein and the inhibitor were produced and used in western blots to correlate these proteins with malting quality. We found that the inhibitor is smaller in Steptoe feed barley than Morex malting barley and is more abundant in Steptoe. We cloned the gene promoter for the barley GA hormone receptor and identified sequences that suggest how the promoter works; the sequence of the nuclear gene was released on GenBank. The aleurone’s GA hormone is still unknown. We found that one of the GA hormones, GA3, spikes to high levels at 2 days after imbibition and disappears by day 3. We are following up this study with a larger collaborative project to measure levels of various GAs. 4.Accomplishments 1. Hormone responses in the barley seed control malting and can potentially predict malting quality. Malting quality depends on enzymes produced during the malting process. Most of the genes that direct the production of these enzymes are actived in seed's outer tissue (the aleurone) by gibberellin (GA) hormones. Significant resources are being devoted by American and foreign companies to develop a small set of gene probes that can be used as malting quality predictors; this would save considerable financial resources devoted to breeding programs to produce malting barleys. It has long been believed that, during the malting process, GAs are produced by the embryo and travel to the aleurone, where they activate enzyme production. Surprisingly, GA levels in the aleurone have never been examined. A study was completed showing that GAs may be synthesized in the aleurone (during the first 3 days of germination) from inactive GAs residing in the aleurone and the starchy middle of the seed, the endosperm. Further studies are needed to clarify this point. The appearance of GAs in the aleurone should relate directly to the amounts (over time) of a specific inhibitory protein (DELLA) and a protein that binds to GA. GA causes the amounts of certain malting enzymes to increase, but the inhibitor proteins prevent this. The inhibitor protein is destroyed soon after GA binds to the GA binding protein. Analysis of this system could reveal an important characteristic of superior malting barleys. The impact of these studies would be the establishment of genetic markers for malting quality. Our recent studies showed that a superior malting barley has a larger inhibitor protein than an inferior barley. We have cloned and made public a portion of the genetic code that regulates the production of the GA binding protein. 2. Certain genes are turned on during the the infection of barley by Fusarium graminearum infection, while others are turned off. Many large-scale studies have been conducted to understand changes in gene expression in the developing seed spike during infection by F. graminearum, a fungal pathogen that causes millions of dollars in damage annually to barley (and wheat) crops. It is thought that these studies will show how the barley attempts to defend against infection. All of these studies analyzed the same 22,000 gene products. This has led to little insight into the infection process. It is more logical to use the subset of gene products that lead to protein production (messenger RNA bound to polysomes). Our previous studies showed that the information obtained using this subset is quite different from that obtained with the total population. This study will have an impact in changing the methodology used in this field. The result will be more accurate and, therefore, more useful information. We obtained polysomes from barley infected with powdery mildew and from Arabidopsis infected with turnip mosaic virus. The total gene product population and polysomal subset populations were studied and found to be significantly different. These gene expression patterns are supposed to reflect biochemical changes in the tissues undergoing infection. A much more direct approach is to use metabolic profiling to actually see these biochemical changes. We found previously that the lemma (developing hull) and the epicarp (directly beneath the hull) are the main tissues infected by F. graminearum. In both tissues, the simple sugar derivative ribitol is produced in large amounts. These studies will generate insights into how F. graminearum enters the host in the early stages of infection, whether the host attempts a defense, and how the fungus overcomes these attempts. This will lead to better strategies to develop resistant barley. Review Publications Abebe, T., Wise, R.P., Skadsen, R.W. 2009. Comparative Transcriptional Profiling Established the Awn as the Major Photosynthetic Organ of the Barley Spike while the Lemma and the Palea Primarily Protect the Seed. The Plant Genome. 2(3):247-259.