Location: Crop Production and Pest Control Research
2024 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, we made significant research progress investigating the functional role of effector proteins from Phyllachora maydis. In fiscal year (FY) 2023, we reported the identification of eighteen effector proteins from P. maydis that are upregulated during the early stages of disease development. In FY 2024, we showed that of the eighteen effector proteins, three consistently attenuate production of reactive oxygen species (ROS) in plant cells. Using AlphaFold2, we showed that one effector adopts a predicted structure similar to that of a previously characterized effector from Verticillium dahliae, suggesting this P. maydis protein is a bona fide effector. A manuscript detailing this work was submitted and accepted during FY 2024.
We also initiated a project to discover P. maydis effector proteins that specifically localize to and interfere with chloroplast-derived immune responses. We discovered one effector protein that localizes to host chloroplasts and showed that it suppresses plant defense responses, suggesting this effector contributes to the attenuation of plant immunity. A manuscript detailing these observations is currently being prepared and will likely be submitted in FY 2025.
To annotate our improved P. maydis genome sequence, we have provided all data to our collaborators at the Joint Genome Institute. They will provide improved annotations with addition of our isoSeq data which has already identified many corrections to a previously published genome sequence. They also will make the data public with an easily accessible genome browser that will facilitate future research by the global community. A manuscript on the new genome and its improvements will be prepared during Quarter (Q) 4 of FY 2024 or Q1 of FY 2025.
Sub-objective 1.B, we made substantial progress on identifying candidate effector proteases from the genome of Fusarium graminearum. In FY 2023, we reported the identification of a trypsin precursor protein, designated FGSG_11164 as Fusarium graminearum Trypsin precursor protein 1 (FgTPP1). In FY 2024, we showed the FgTPP1 protein encodes a predicted 17-amino acid signal peptide sequence and is secreted from fungal cells. Using AlphaFold2, we showed FgTPP1 has three predicted catalytic active sites, histidine-67 (His67), aspartic acid-112 (Asp112), and serine-208 (Ser208) as well as a three putative substrate binding sites, aspartic acid-202 (Asp202), serine-224 (Ser224), and glycine-226 (Gly226), suggesting this protein is a bona fide protease. Furthermore, we show that FgTPP1 localizes to host chloroplasts and suppresses numerous plant defense, revealing FgTPP1 is a functional effector. A manuscript on the discovery and functional characterization of this effector protease from F. graminearum is being prepared for submission to a peer-reviewed journal during Q4 of FY 2024.
Sub-objective 1.C, our initial work is complete but transformation with this pathogen has been very difficult. To solve that problem, a student funded by a Non-Assistance Cooperative Agreement is now trying to develop a CRISPR-based transformation system with colleagues in the Netherlands who have it working with a related pathogen. The student is currently in the Netherlands working out the technique. Having a CRISPR-based system for Zymoseptoria tritici would be a major breakthrough that would alleviate a major research bottleneck. We should know whether it is working by Q4 of FY 2024.
Objective 2.A, a manuscript on our first microbiome experiment was submitted. Data from the second microbiome experiment have been analyzed and turned out to be even more interesting. Tar spot in Latin America has been considered a disease complex comprised of two pathogens and a mycoparasite. The primary pathogen is the obligately biotrophic fungus Phyllachora maydis. The second pathogen, Monographella (aka Microdochium) maydis, is associated with fisheye symptoms, while Coniothyrium is a possible mycoparasite of one or both pathogens. Previous analyses had not identified Monographella in the United States, however, in Mexico it has been attributed to a species of Fusarium (which is related to Microdochium). Our analyses of microbiomes from corn in Guatemala and Ecuador compared to Indiana identified Monographella in samples from parts of Ecuador and Guatemala but not in Indiana. In some samples from Ecuador Monographella was the most common and Phyllachora (the usual cause of tar spot) was only the fifth-most commonly detected fungus. These results explain the difference between species identified in the United States versus Latin America and raise the surprising possibility that Monographella can cause tar spot on its own, a hypothesis that needs to be tested by future research. A manuscript describing this work is in preparation and hopefully will be submitted during Q4 of FY 2024.
Objective 2.B, numerous fungal cultures were isolated and identified by sequencing the ITS region of ribosomal DNA. Many of these appear to be Paraphaeoshaeria, another fungus that could function as a mycoparasite and thus could be of interest as a potential biocontrol organism. Based on our microbiome results from Guatemala and Ecuador, we are now increasing the scope of this objective to include samples from Latin America with the hope of isolating Monographella maydis for future research. Unfortunately, Phyllachora maydis is an obligate pathogen and cannot be grown in culture. However, we are developing a protocol for greenhouse inoculations using pathogen spores grown on the host. A preliminary report was published in FY 2023 and a manuscript on an improved protocol is in preparation.
Sub-objective 3.A, we made significant research progress on identifying host proteins that interact with a candidate effector protease from F. graminearum, FgTPP1. We have now identified proteins from wheat that interacted with FgTPP1 using a yeast two-hybrid (Y2H) assay. We specifically identified six wheat proteins that interact strongly with the FgTPP1. Using co-immunoprecipitation and bimolecular fluorescence complementation assays, we confirmed that FgTPP1 interacts with these six proteins. We also show that two of the proteins from wheat specifically co-localize with FgTPP1 in host chloroplasts. We thus predict that FgTPP1 is interacting with the wheat proteins at the chloroplast to interfere with photosynthesis or defense responses initiating in the chloroplast. A manuscript detailing these discoveries is currently being prepared and will likely be submitted in Q1 of FY 2025.
Sub-objective 3.B, we are in the process of developing and testing markers. Our bioinformatics specialist working on this project retired during August of 2023 and a laboratory technician will complete this part of the project,
Sub-objective 3.C, we have developed an efficient, genotype-independent transformation system for wheat. We are currently trying to develop a CRISPR-based gene knockout system for functional analysis of wheat that, if successful, will be much better than the VIGS approach we proposed several years ago and will allow us to test genes much more thoroughly and faster (see 4.B.).
Sub-objective 3.D, the RNAseq analyses were completed and a number of candidate genes were identified. A postdoc recently went for training in CRISPR-based functional analysis of corn and we hope to use that technology to test resistance gene candidates that are identified from the mapping project. Field testing of a third population for tar spot resistance was completed during Q1.
Field research with P. maydis has many complications, so we have developed a protocol for greenhouse inoculations. This is difficult with an obligate pathogen that cannot be cultured in vitro but persistence is paying off and reproducibility is almost good enough to be used for testing segregating populations. We should be able to use the greenhouse assay starting in Q1 of FY 2025. Being able to test in a greenhouse would bring more consistency to the results and allow us to test year-round.
Objective 4.A, we made significant progress towards developing a novel, E. coli-based assay to determine the target cleavage sequences proteases from fungal pathogens, such as P. maydis, Z. tritici, and F. graminearum. The assay works by co-expressing a protease of interest with a randomized substrate library. The only way a bacterial cell can grow is if the fungal protease cleaves the peptide sequence. This process requires the generation of a randomized substrate library as well as verification of selection for protease-substrate matches. Efforts during FY 2024 has primarily focused on troubleshooting and refining the assay. While initial results have been encouraging, generating a randomized library required significant alterations to how these genes were expressed. Cells carrying these changes failed to reproduce our initial positive results. We determined that the problem rests in the relative copy numbers (i.e., how many copies of our genes each cell carries) between substrate and protease. We are now troubleshooting these issues and expect to have initial results and potential peptide sequences that are cleaved by fungal proteases in Q2 of FY 2025.
Sub-objective 4.B., we found that the BSMV-VIGS system has too much variability to reliably detect genes that make contributions to Fusarium head blight resistance. The gene candidates we tested are likely to contribute to resistance, not be determinative. Given the variation with the greenhouse Fusarium Head Blight (FHB ) assay and VIGS, no genes were found making clear and reproducible contributions to resistance. Fortunately, we have made a major breakthrough in wheat transformation. In collaborative work we have developed a simple and efficient genotype- independent transformation system. A manuscript describing this discovery has been submitted. This system will allow us to use CRISPR-Cas to knockout candidate genes, thereby avoiding the variation of VIGS.
Accomplishments
1. Investigated the infection process used by the maize tar spot pathogen Phyllachora maydis to colonize maize leaf tissue. Tar spot, caused by the fungal pathogen Phyllachora maydis, is an emerging disease since 2015 that now is responsible for significant yield loss in most maize-growing regions within the continental United States. Importantly, genetic-based host resistance against this fungal pathogen is generally not available. Hence, there is an urgent need to investigate how P. maydis colonizes host tissue during infection as such knowledge will likely lead to an enhanced understanding of host-microbe interactions as well as potentially lead to the development of effective disease control strategies. To further our understanding of how P. maydis colonizes host cells, ARS researchers at West Lafayette, Indiana, in collaboration with researchers from Purdue University, analyzed the fungal colonization patterns of infected maize leaves. Using advanced microscopy methods, we showed this fungal pathogen produces both sexual and asexual fungal structures during infection, suggesting P. maydis may have greater genetic diversity than previously reported. We also showed this fungal pathogen is unable to colonize the vasculature, demonstrating that spread within a maize leaf likely does not require the vasculature. This study, to the best of our knowledge, is the first to provide foundational knowledge and insights into how this devastating fungal pathogen colonizes and spreads within maize leaves. Our study addresses a significant knowledge gap in our understanding of how this fungal pathogen causes disease in maize. The results presented in our study will likely be used to mitigate the economic losses caused by this disease as well as decrease the environmental impacts of fungicide application. Importantly, the knowledge gained from our work will likely lead to more sustainable and enhanced disease management strategies to control this disease.
2. Initial steps towards developing resistance against tar spot disease of corn. Phyllachora maydis is a significant fungal pathogen of maize that causes tar spot disease and is responsible for significant economic losses to U.S. farmers. Despite tremendous efforts to identify durable, genetic-based disease resistance against this disease, no such host resistance has been reported. Moreover, there is very limited information with regards to how this fungal pathogen uses virulence molecules, known as effectors, to cause tar spot disease in maize. To address this deficiency, ARS researchers at West Lafayette, Indiana, developed an assay to analyze and characterize the activities of effector molecules from P. maydis. Significantly, the ARS researchers were the first to identify several effector molecules that suppress host defense responses. This study lays the foundation for investigating the molecular interactions between maize and P. maydis. Our work is the first to investigate how this fungal pathogen uses effector molecules to “shut-off” the plant immune system. Studying how plant pathogens use effector molecules during the infection process is quite informative as their study often provides insight into how pathogens cause disease. Hence, our results are, therefore, expected to significantly advance our ability to develop resistance against this fungal pathogen.
3. Developed a simple and efficient genotype-independent wheat transformation system. The application of biotechnology for wheat improvement and basic research is greatly impeded by the inability to introduce DNA constructs into the plant. A solution to this problem would open the door for dramatic improvements for wheat; making it possible to introduce DNA constructs that edit genes to improve disease resistance, remove glutens from seed proteins, or creating dwarfing phenotypes that were so critical for the green revolution. ARS scientists at West Lafayette, Indiana, in collaboration with Purdue University researchers developed a simple and efficient system for wheat transformation. This breakthrough is a gateway for applying biotechnology for wheat improvement. Because the system is genotype independent unlike any existing methods, transformation and gene editing can be done directly in the elite wheat varieties currently grown or that seed companies are developing for future sale, thereby cutting years from developing new lines that farmers can grow.
Review Publications
Jaiswal, N., Myers, A., Cameron, T.L., Carter, M.E., Scofield, S.R., Helm, M.D. 2023. Analysis of cell death induction by the barley NLR immune receptor PBR1. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-01-23-0005-R.
Caldwell, D., Da Silva, C., McCoy, A., Avila, H., Bonkowski, J., Chilvers, M., Helm, M.D., Telenko, D., Iyer-Pascuzzi, A. 2024. Uncovering the Infection Strategy of Phyllachora maydis during Maize Colonization: A Comprehensive Analysis. Phytopathology. https://doi.org/10.1094/PHYTO-08-23-0298-KC.
Scofield, S.R. 2023. Loss of foreign dna inserts from barley stripe mosaic virus vectors- potential consequences for use in functional genomics studies. International Journal of Plant Biology. https://doi.org/10.3390/ijpb14040080.
Garg, A., Brandt, A.S., Scofield, S.R. 2024. Analysis of the time course of the establishment of systemic gene silencing by barley stripe mosaic virus-induced gene silencing constructs in wheat. International Journal of Plant Biology. https://doi.org/10.3390/ijpb15010011.
Rogers, A., Jaiswal, N., Roggenkamp, E., Kim, H., MacCready, J.S., Chilvers, M.I., Scofield, S.R., Iyer-Pascuzzi, A.S., Helm, M.D. 2024. Genome-informed trophic classification and functional characterization of virulence proteins from the maize tar spot pathogen Phyllachora maydis. Phytopathology. https://doi.org/10.1094/PHYTO-01-24-0037-R.
Jaiswal, N., Helm, M.D. 2024. Pseudomonas syringae pv. tomato DC3000 induces defense responses in diverse maize inbred lines. PhytoFrontiers. https://doi.org/10.1094/PHYTOFR-11-23-0149-SC.
Gonhora-Canul, C., Puerto, C., Jimenez-Beitia, F.E., Telenko, D.E., Kleczewski, N.M., Rosas, J., Avellaneda, C., Sanders, A., Rodriguez, I.Y., Goodwin, S.B., Fernandez-Campos, M., Lee, D., Cruz, A.P., Cruz, C.D. 2024. Comparing tar spot epidemics in high-risk areas in the U.S. and Honduras. Canadian Journal of Plant Pathology. https://doi.org/10.1080/07060661.2023.2300077.