Location: Crop Production and Pest Control Research
2025 Annual Report
Objectives
Objective 1: Investigate the mechanisms of fungal pathogenicity and other important biological traits in cereal crops.
Sub-objective 1.A: Develop an improved genome sequence for the tar spot pathogen of maize, Phyllachora maydis.
Sub-objective 1.B: Identify proteases and other potential effectors expressed by pathogens of wheat, barley and maize that are involved in pathogenicity.
Sub-objective 1.C: Identify and test the function of genes expressed by fungal pathogens of wheat that are involved in survival and pathogenicity.
Objective 2: Analyze microbiomes associated with resistance and susceptibility to identify vulnerabilities in fungal pathogens of cereal crops.
Objective 3: Identify, genetically map and functionally characterize host resistance against fungal pathogens of cereal crops.
Objective 4: Exploit knowledge of host-pathogen interactions and pathogen vulnerabilities to develop novel methods for increasing resistance in cereal crops.
Sub-objective 4.A: Engineer gene-for-gene resistance to Fusarium Head Blight in wheat and barley.
Sub-objective 4.B: Functional identification of wheat genes able to confer resistance to Fusarium head blight and crown rot (FCR) when their expression is induced by ethylene treatment.
Approach
Diseases caused by fungal pathogens pose significant economic threats to grain crop production. Currently, little is known about the molecular and genetic mechanisms that govern host resistance and fungal virulence in wheat. Research objectives and approaches in this project focus on identifying genes expressed by the host and the fungal pathogens during infection. The primary subjects of research will be septoria tritici blotch (STB) and Fusarium head blight (FCHB) and crown rot (FCR) of wheat. We will utilize RNA sequencing to identify wheat genes expressed during different types of resistance responses and fungal genes involved in pathogenicity and other important biological processes. Some of the host materials will include recently developed isogenic lines for resistance genes against STB. These genes are on different wheat chromosomes and the isogenic lines will allow us to test the hypothesis that they use different mechanisms for resistance. We will analyze nonhost resistance responses in interactions between barley and wheat inoculated with Mycosphaerella graminicola and Septoria passerinii, respectively. Gene function in the pathogens will be confirmed by generating knockout mutants and testing for phenotype and in the host by Virus-Induced Gene Silencing (VIGS). We also will use comparative genomics of resequenced isolates to identify essential genes in M. graminicola and will use these plus others identified from the RNA-seq experiments for both pathogens to identify genes that can be targeted for Host-Induced Gene Silencing (HIGS) to increase the level of resistance in wheat. Additional objectives are to develop a CRISPR/Cas9 system for M. graminicola and to do finescale genetic mapping for developing additional molecular markers linked to the resistance genes. Successful completion of the objectives will contribute to the basic understanding of diseases caused by plant-pathogenic fungi and will provide clues about potential targets for genetic modification of the crop to prevent or circumvent damage resulting from fungal pathogens.
Progress Report
Sub-objective 1.A. In FY25, publishing the improved genome sequence for the tar spot pathogen of corn was delayed because the sequencing company sent incorrect data that took time to identify, and decreased the overall coverage of the genome. The improved genome is important because it will serve as a reference for future research done by ARS, industry and academia. Due to this mistake, additional DNA samples were sent and we are awaiting the results. We arranged with the Joint Genome Institute to analyze the final assembly, help with annotation, and host the genome on their Mycocosm portal so that it will be immediately accessible to the worldwide research community. Preliminary analyses of our improved genome identified genes for interacting with its corn host that were not identified in the currently available genome. This finding suggests that these genes may change rapidly, which could allow for enhanced ability to infect its host and rapid adaptation to overcome host defenses.
Sub-objective 1.B. In FY25 significant advances in the identification of putative maize host targets of Phyllachora maydis effector proteins have been made. During FY2025, research focused on a candidate effector gene that can be secreted by the fungus to attack proteins produced by corn and appeared to be expressed during host colonization. However, subsequent analysis of its sequence and structural analysis of the protein revealed a mistake in how the gene was described—this gene does not function as believed. This situation indicates why it is extremely important for us to generate an improved reference genome for this fungus to avoid these kinds of errors and wasted effort in the future.
As a result, the scope of the investigation was broadened to include the characterization of more candidate genes expressed by P. maydis that could help it to infect its host. Several of these proteins were found to interfere with specific aspects of plant immunity. To systematically identify maize host proteins that interact with those produced by the pathogen, a high-throughput biotechnology approach was outsourced using expressed genes from maize and the pathogen protein coding sequences. Results from this approach are expected in Q1 of FY2026 and will provide critical insights into the molecular mechanisms by which proteins produced by the tar spot pathogen manipulate host proteins to cause disease.
Sub-objective 1.C. In FY25 a new biotechnology tool (CRISPR/Cas9-based system) was developed for knocking out genes in the wheat pathogen Zymoseptoria tritici and used to knock out several protein genes that previous research showed were highly expressed at different stages of infection and may be involved in attacking host defenses. Phenotypic analysis of the knockout mutants during host inoculations showed that their pathogenicity was reduced but not eliminated, most likely indicating redundant functions of the proteins expressed by different genes. Experiments to knock out additional effector genes plus an unrelated gene that may affect pathogenicity are in process. A detailed protocol for the CRISPR/Cas9 knockout system is being prepared for publication so that this major advance can be shared with the global research community.
Objective 2. In FY25, microbiomes on corn leaves that were infected with tar spot in Ecuador and Guatemala were sequenced and compared to those from samples collected in Indiana. The analyses identified a higher diversity of fungi on corn leaves in the tropics compared to Indiana. Also identified, were high frequencies of a Microdochium species on corn leaves from some locations in Ecuador and Guatemala but not in Indiana. This is significant because Microdochium was initially believed to form part of a complex of organisms causing tar spot but the exact species has not been identified and it has not been reported in the U.S. or Canada. Some investigators have recently suggested that reports of this species were due to misidentification of fungi in the genus Fusarium. However, our analyses showed clearly that Microdochium occurs at very high frequencies in some locations. The high-Microdochium samples often had lesions that looked different from typical tar spot and could indicate that there is a different, but similar, disease to tar spot on corn that could pose a threat to U.S. agriculture. Attempts to isolate the Microdochium in pure culture and to assess genetic variation of tar spot samples in other countries are continuing.
Sub-objective 3.A. In the past year, our research made important progress in understanding how the fungus Fusarium graminearum—the cause of head blight in wheat and other grains—overcomes plant defenses. This fungus produces a protein, called FgTPP1, that plays a key role in helping the pathogen infect crops. We discovered that FgTPP1 interacts with specific plant proteins in ways that weaken the plant’s natural immune system. Normally, plants use their chloroplasts—the same parts of the cell that capture sunlight for photosynthesis—to generate protective molecules called reactive oxygen species (ROS) that help fight off infection. Our work showed two surprising things about FgTPP1: 1) The protein can change its form once inside plant cells, which affects how strongly it can suppress the plant’s defenses and 2) before it even reaches the chloroplasts, the protein can trap an important wheat protein (called PetC) outside of the chloroplasts. By keeping PetC from entering chloroplasts, the fungus prevents the plant from producing the protective ROS molecules it needs. These results reveal a completely new way that Fusarium disarms plant defenses. Understanding this mechanism is an important step toward developing crops that are better able to resist infection. In the long term, this knowledge will help agricultural producers by guiding the development of wheat varieties with stronger, more durable resistance to head blight, ultimately reducing crop losses and protecting the global grain supply.
Sub-objective 3.D.In FY25 a report on mapping of tar spot resistance genes in corn was prepared and was submitted to a peer-reviewed journal for publication. This work identified new quantitative resistance on corn chromosome 9 using the B73xCML52 genetic population. Importantly, candidate genes were identified for follow-up work to identify which function in the resistance mechanism. Plans for FY26 are to screen additional genetic materials generated from the B73xCML52 population to further refine the list of candidate genes to these that are most likely to have an effect. Analyses were completed for another project on resistance to tar spot in a different genetic population that identified a significant quantitative resistance on corn chromosome 1. All of these quantitative resistances appear to be different from those that have been mapped previously so should provide additional tools for the arsenal plant breeders can use to increase the level of resistance to tar spot in U.S. corn cultivars.
Sub-objective 4. In FY25 substantial progress was made in refining a bacterial-based screening assay designed to identify the cleavage sites of proteins produced by fungal pathogens. This system relies on the co- expression of fungal proteins with a synthetic, randomized substrate library within bacterial cells. Cellular growth depends on successful cleavage of a protein, thereby identifying the most important sites for protein-substrate interactions. Development of this assay necessitated the construction of a high-diversity library and rigorous evaluation. During FY25, research focused on optimization and troubleshooting of the system. Although early results were promising, subsequent modifications required for efficient library expression—particularly changes to the time and level of expression of relevant genes—were identified as difficulties to be overcome. Detailed analysis revealed that inconsistent numbers of protein and substrate constructs were the main problems. Efforts are ongoing to tweak the system and restore functionality. Identification of candidate cleavage sequences for target proteins is anticipated by Q2 of FY26 and will be essential for designing more effective resistance against fungal pathogens of crop plants in the future.
Accomplishments
1. New biotechnological method for studying an important wheat pathogen. The pathogenic fungus Zymoseptoria tritici causes Septoria tritici blotch disease of wheat. This disease occurs throughout the world and is usually one of the top three foliar disease problems that can only be managed by increasing host resistance or by expensive fungicide sprays, which usually are not cost effective for U.S. growers. Progress in understanding pathogenicity of this fungus has been hindered by an inability to easily introduce genetic changes, such as deleting potential pathogenicity genes. To address this problem, ARS scientists at West Lafayette, Indiana, collaborated with scientists at Purdue University and at Wageningen University in the Netherlands to develop a method that uses CRISPR/Cas9 gene editing to create fungal mutations. The researchers then used this new technique to confirm the importance of a pathogenicity gene by showing reduced infections on wheat leaves when gene functionality was eliminated. Development of a CRISPR/Cas9 knockout system for this pathogen is a major advance that is already being used globally by other scientists. This technology will facilitate the development of improved methods for fighting this pathogen and help agricultural producers meet growing demands for wheat.
2. How Maize Fights Back: New Clues to Combat Tar Spot Disease. Tar spot is a fungal disease that has quickly become a major threat to maize (corn) crops across the U.S. and Canada, causing significant yield losses. Most commercial corn hybrids are highly susceptible, which allowed tar spot to become the number one foliar pathogen of corn within a few years after its introduction to the U.S. in 2015. Because the disease had not been present previously, no work on breeding for resistance had been done in the U.S. and breeders have very little to work with. Therefore, both naturally existing and engineered forms of resistance are being explored to combat this devastating, emerging disease. ARS scientists at West Lafayette, Indiana, collaborated with scientists at Michigan State University to track how maize genes respond to tar spot exposure over time. By analyzing which maize genes were turned on or off during infection, the researchers discovered thousands that are involved in plant defense, including those that strengthen cell walls, produce natural anti-fungal chemicals, and signal immune responses. Some of these genes had already been linked to disease resistance, while others were newly identified as potential regulators of defense responses. This is the first in-depth analysis of how maize biologically responds to tar spot in a real-world setting, offering important new leads for breeding stronger, more resistant maize varieties.
3. Disarming a Deadly Wheat Fungus: How Scientists Are Uncovering a New Way to Fight Fusarium Head Blight. Fusarium head blight is a devastating disease that attacks wheat and barley; reducing yields and contaminating grain with toxins. ARS scientists at West Lafayette, Indiana identified a specific fungal protein, called FgTPP1, that helps the fungus weaken a plant's immune defenses and allows infection to occur more easily. They found that this protein is secreted by the fungus and enters plant cells, where it travels to the plant’s chloroplasts, the part of the cell responsible for energy and some defense responses. There, FgTPP1 interferes with the plant’s natural immune signals, making it easier for the fungus to spread. When researchers removed the FgTPP1 gene from the fungus, the disease was much less severe, proving the protein’s importance. Due to this protein also being found in many related fungi, it could be a promising universal target for developing new, long-lasting forms of resistance in crops. This discovery opens the door to bioengineering wheat and barley that can recognize and fight off fungi before they cause serious harm.
Review Publications
Jaiswal, N., Liao, C., Hewavidana, A., Mengiste, T. 2025. GCN5-related histone acetyltransferase HOOKLESS2 regulates fungal resistance and growth in tomato. New Phytologist. https://doi.org/10.1111/nph.70025.
Gomez-Gutierrez, S.V., Sic-Hernandez, W., Haridas, S., Labutti, K., Eichenburger, J., Kaur, N., Lipzen, A., Barry, K., Goodwin, S.B., Gribskov, M., Grigoriev, I.V. 2024. Comparative genomics of the extremophile Cryomyces antarcticus and other psychrophilic Dothideomycetes. Frontiers in Fungal Biology. https://doi.org/10.3389/ffunb.2024.1418145.
Lee, D., Na, D., Gongora-Canul, C., Jimenez-Beita, F.E., Goodwin, S.B., Cruz, A., Delp, E.J., Acosta, A.G., Lee, J., Falconi, C.E., Cruz, C.D. 2024. Optimizing corn tar spot measurement: a deep learning approach using red-green-blue imaging and Stromata Contour Detection Algorithm for leaf-level disease severity analysis. Plant Disease. https://doi.org/10.1094/pdis-12-23-2702-re.
Velasquez-Zapata, V., Smith, S., Priyanka, S., Chapman, A.V., Jaiswal, N., Helm, M.D., Wise, R.P. 2024. Diverse epistatic effects in barley-powdery mildew interactions localize to host chromosome hotspots. iScience. https://doi.org/10.1016/j.isci.2024.111013.
Hiles, R., Rogers, A., Jaiswal, N., Zhang, W., Butchacas, J., Merfa, M.V., Klass, T., Barua, P., Thirumalaikumar, V.P., Jacobs, J.M., Staiger, C.J., Helm, M.D., Iyer-Pascuzzi, A.S. 2024. A Ralstonia solanacearum type III effector alters the actin and microtubule cytoskeleton to promote bacterial virulence in plants. PLoS Pathogens. https://doi.org/10.1371/journal.ppat.1012814.
Darino, M., Jaiswal, N., Darma, R., Kroll, E., Urban, M., Xiang, Y., Srivastava, M., Kim, H., Myers, A., Scofield, S.R., Innes, R., Hammond-Kosack, K., Helm, M.D. 2025. The Fusarium graminearum effector protease FgTPP1 suppresses immune responses and facilitates Fusarium Head Blight disease. Molecular Plant-Microbe Interactions. https://doi.org/10.1094/MPMI-08-24-0103-FI.
Roggencamp, E.M., Jaiswal, N., Helm, M.D., Thompson, A., Chilvers, M.I. 2025. Survey of maize differential gene expression upon environmental exposure to the tar spot pathogen, Phyllachora maydis. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-12-24-0136-R.