The small grain cereal crops of wheat, oats, and barley are important agricultural commodities in the United States and many other countries of the world. Unfortunately, they are attacked by rust diseases caused by the fungal pathogens Puccinia graminis f. sp. tritici (Pgt), P. coronata f. sp. avenae (Pca), and Puccinia hordei (Ph). Yield losses due to these rust diseases can exceed 40% during severe epidemics. The deployment of resistant varieties is the most effective means for control of the cereal rust diseases. This strategy is based on exploiting genetic variation for novel resistance (R) genes. Most R genes encode immune receptor proteins that recognize pathogen 'effector' proteins which are delivered into host cells during infection. Effectors recognized by immune receptor proteins are referred to as Avirulence (Avr) factors as their recognition leads to the pathogen's inability to infect lines carrying the appropriate R gene immune receptor. The major challenge in breeding cereal crops for durable rust resistance is the evolution of pathogen populations to overcome resistance conferred by R genes through mutations or other genetic processes in the corresponding Avr genes. This has resulted in many "boom and bust" cycles, whereby newly deployed resistance genes provide protection for only a limited time and are then overcome by newly evolved virulent pathogen isolates. There is a dearth of knowledge concerning Avr genes as only three have been identified in the cereal rust fungi, all from Pgt. Characterization of the broad repertoire of Avr genes in cereal rusts is critical for understanding how interactions with the plant's host immune system influence the evolution of virulence in these important pathogens. This knowledge, in turn, is critical for developing novel resistance breeding strategies for cereal crops, thereby reducing the impact of these devastating diseases for producers and end-users alike.Thus, this research project aims to address these knowledge gaps by 1) generating complete haplotype-phased genome assemblies for diverse sets of Pgt, Pca, and Ph isolates from across the world; 2) identifying genes underlying avirulence phenotypes and their genetic diversity; and 3) understanding the functions of Avr effectors in disease. By studying three different rust pathogens on different cereal hosts, we will be able to conduct cross-species comparisons to identify common mechanisms of virulence in these systems.Our specific objectives are as follows:1) Identify Avr gene candidates through genome sequence comparisons. Some comparative genome sequence analyses have been completed for Pgt and Pca. Here we will expand the sequence resources available for this analysis with additional Pgt and Pca isolates, as well as investigate a large collection of global barley leaf rust (Ph) isolates. Haplotype-phased genome references will be constructed for key isolates and resequencing of additional isolates will allow for further identification of Avr loci through either genome-wide association studies (GWAS) of sexually related populations or mutation screening in clonal lineages.2) Validate Avr gene candidates through functional assays. To assess whether the candidate genes identified in Objective 1 are recognized by the corresponding resistance genes in wheat, oat, or barley, we will generate recombinant viral expression vectors expressing these candidates. The ability of recombinant virus strains to infect wheat, oat, or barley lines containing the corresponding resistance genes will be assessed using infection assays similar to those used for the functional validation of AvrSr50 and AvrSr27 in previous investigations. As a further test, we will verify Avr function using a protoplast transient transformation assay to express candidate genes in cells with or without the corresponding resistance genes. Where the cloned gene sequences of the corresponding resistance genes are available, we will also conduct transient expression assays in Nicotiana benthamiana to test for R-Avr recognition.3) Examine Avr gene diversity and evolution in rust populations. Sequence and structural variation (copy number variation and large insertions/deletions) in Avr genes will be examined in the genome assemblies (Objective 1) to determine the diversity and levels of homo/heterozygosity at these loci. Comparison of fully resolved genome assemblies is absolutely necessary to generate a true picture of both sequence and structural diversity in Avr loci across rust populations. For all currently known Avr genes and those identified from Objectives 1 and 2, we will assess genetic diversity by examining the orthologous genes in each haplotype of all the reference genome sequences. This will identify the nature of genetic variation, such as presence/absence polymorphisms, amino acid sequence diversity, insertions, and expression level variation. Sequence variants of known Avr genes can be tested for functional recognition using assays described above.4) Determine the role of Avr proteins in rust infection. Avr proteins are a subset of secreted effector proteins delivered into host cells during infection that are thought to facilitate disease by suppressing host immunity and/or altering host metabolism and physiology. However, our understanding of the function of rust effectors in supporting the infection processes is very limited. This objective will address this knowledge gap using the following set of experiments:4-1A. As each of the previously cloned effectors (AvrSr27, AvrSr35, and AvrSr50) have been shown to be functional when fused to Yellow Fluorescent Protein (YFP) or Green Fluorescent Protein (GFP), we will produce transgenic wheat lines (cv. Cadenza) overexpressing these effectors as tagged with GFP at the N-terminus.4-1B. The generated transgenic wheat lines will be used in co-immunoprecipitation/mass spectrometry (coIP/MS) analyses to identify the in planta interacting wheat proteins.4-2. coIP/MS may miss some of the important interactors (for example because of the low concentration of the interacting protein in the protein extract), but may also identify proteins that interact indirectly, e.g., as a part of the protein complex. Therefore, in parallel with coIP/MS, yeast two-hybrid (Y2H) screens will be carried out with these same effectors to identify the binary protein-protein interactions.4-3. The two complementary protein-protein interaction screens described in 4-1 and 4-2, will provide us with a list of candidate genes and pathways targeted by the Pgt effectors AvrSr27, AvrSr35, and AvrSr50 for further validation and functional analyses using reverse genetics approaches such as Virus-induced gene silencing (VIGS) and/or Targeting Induced Local Lesions in Genomes (TILLING).4-4. The validated wheat interactors (up to ten) will then be functionally analyzed using VIGS to determine their function in infection. Here we will use Barley Stripe Mosaic Virus (BSMV) VIGS to specifically silence the target wheat genes.