Research Project: Fungal Host-Pathogen Interactions and Disease Resistance in Cereal Crops

Location: Crop Production and Pest Control Research

2023 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
For Objective 1.A, sequencing for an improved genome of the tar spot pathogen, Phyllachora maydis, has been completed and analyses are in progress. Due to a similar genome published recently by a group at Michigan State, we are now adding an additional round of sequencing to provide a better annotation to ensure that ours is an improvement over others that are available. For Objective 1.B, proteases and other effectors have been identified from the genome of the wheat pathogen, Zymoseptoria tritici. Expression levels of these effectors were analyzed over time on susceptible and resistant wheat and on the non-host barley over time using data generated from a previous ARS research project. Sequences for 30 of the effectors were generated and transformed into Agrobacterium for expression in the tobacco Nicotiana benthamiana. Microscopic analyses of the expressed proteins identified where they are active within tobacco cells, and thus also should act in cereals. Most localized to the cytosol but several had action in the nuclei or mitochondria. A manuscript describing these results is in preparation for submission to a peer-reviewed journal later this fiscal year. We have made significant progress on identifying virulence (effector) proteins from the genome of Fusarium graminearum. In collaboration with Dr. Kim Hammond-Kosack at the Rothamsted Research Institute, we identified 95 F. graminearum genes potentially coding for secreted effector proteases. This candidate gene list was narrowed further by selecting effector proteases that were upregulated during early stages of infection. These stringent selection criteria identified seven candidate proteases. Sequences for all seven of the effector proteases were generated and transformed into Agrobacterium for expression in the tobacco Nicotiana benthamiana. Microscopic analyses of the expressed proteins identified one effector protease, which we have named TPP1, that localized to chloroplasts and suppressed host immune responses. Importantly, we show that deleting the TPP1 gene from F. graminearum results in reduced fungal colonization of wheat. Furthermore, we recently identified several proteins from wheat that interacted with TPP1. Two of the wheat proteins are localized to chloroplasts. One wheat protein is known to have a role in activating immunity in response to plant pathogens. We are currently performing additional experiments to confirm these observations. A manuscript describing these results is in preparation for submission to a peer-reviewed journal. Effector proteins have been identified from the recently published genome sequence of Phyllachora maydis. In collaboration with researchers at Michigan State University, we selected the top 18 P. maydis effector proteins that are expressed during early disease development. Sequences for all 18 of the effectors were generated and transformed into Agrobacterium for expression in the tobacco Nicotiana benthamiana. Microscopic analyses of the expressed proteins showed that most localize to the nucleus and cytosol. Importantly, of the 18 effectors we investigated, 3 consistently suppressed multiple host immune responses. Hence, these effector proteins contribute to inhibition of immune responses. These results provided valuable insights into the functions of effectors from P. maydis and will stimulate new research aimed at elucidating the molecular mechanisms potentially manipulated by this fungal pathogen. A manuscript on identifying and functionally investigating effector proteins from P. maydis is being prepared for submission to a peer-reviewed journal during Q1 of FY24. For Objective 1.C, genes have been identified and their function is being tested initially using the N. benthamiana system described in the paragraph above. Knocking out genes in Z. tritici is taking longer than anticipated and the grad student hired by our collaborator through a cooperative agreement to help with part of this project is doing an internship with industry over the summer so progress on that part has been slowed. For Objective 2, analyses of the initial microbiome experiments on resistant versus susceptible lines of corn are complete. The results identified large differences between microbiomes with many species of bacteria and fungi differing in their presence and/or abundance on resistant versus susceptible lines. Several species of bacteria and fungi were significantly correlated with the numbers of Phyllachora maydis sequence reads, indicating that they may affect infection levels of this pathogen on corn. Species with a significant positive correlation may be functioning as mycoparasites. Our hypothesis is that as their food source (P. maydis) increases, then the mycoparasites do as well. This hypothesis was supported in our data where corn lines infected with common rust (caused by Puccinia sorghi) had the highest frequencies of sequences of the known rust mycoparasite, Sphaerellopsis filum. Our hypothesis for species that are negatively associated with P. maydis sequence reads is that they may be functioning as antagonists of the tar spot pathogen; with more of the possible antagonist there is less of P. maydis. To test these hypotheses we are sequencing additional samples from a time course from before tar spot symptoms develop, then at early, middle and late times in the disease cycle at the same location in Indiana. We also are trying to obtain samples from Mexico, Central and South America to test whether the microbiomes in regions where tar spot is endemic will have similar microbiomes to what we observe in Indiana. We also are attempting to procure or generate isolates of the major fungi that were positively or negatively correlated with the level of tar spot resistance in corn to test their effects on P. maydis directly. Doing that effectively will require being able to generate tar spot symptoms artificially in a greenhouse by inoculating corn with P. maydis. For that project we teamed up with a collaborator through an agreement with Purdue University. Preliminary work showed that the proposed approach can generate symptoms and a paper on the initial experiments was published in BMC Research Notes (see Publications below). A manuscript analyzing our first microbiome experiments has been written and will be submitted for publication in a peer-reviewed journal later this fiscal year. For Objective 3, we finished analyzing two segregating populations for tar spot resistance in corn. The first population showed a highly significant, very broad quantitative resistance on corn chromosome 9. Further analyses showed that this peak almost certainly represents three closely linked resistance genes. Analyses of the second population identified a highly significant quantitative trait locus for tar spot resistance on corn chromosome 1. This one was more precisely defined with a very narrow peak on the statistical plots. The resistance genes on chromosomes 1 and 9 all appear to be new, adding to the arsenal of resistance that can be deployed against tar spot in corn. By looking at corn genome sequences for the regions involving these resistances we identified numerous candidate genes that could be involved, including several that look like resistance genes cloned against other pathogens in different crops. Analyses of both segregating populations have been completed and manuscripts are being prepared for submission to peer-reviewed journals most likely during Q1 of FY24. For Objective 4.A, PBS1-like (PBL) proteins from barley that activate PBR1 have been successfully identified. To identify which PBS1-like proteins in barley can activate PBR1, we engineered several barley PBS1-like proteins that could be cleaved by an effector protease, with the expectation that cleavage of the modified barley PBL proteins by an effector protease would activate PBR1. Strikingly, our results revealed that proteolytic cleavage of multiple barley PBL proteins activated PBR1. We also show that cleavage of barley PBS1-like orthologs from wheat, rice, maize, and sorghum also activate PBR1, revealing PBR1 can detect cleavage of PBL proteins from other cereal grains. Given the importance and novelty of these results, we have submitted an Invention Disclosure detailing the implications for engineering resistance against fungal pathogens of cereal grains. A manuscript is being prepared for submission to a peer-reviewed journal. For Objective 4.B, our hypothesis is that fungal effectors target and interfere with the function of host proteins that have critical roles in defense. The F. graminearum effector TPP1 described in Objective 1.B. has already been shown to contribute to fungal virulence by suppressing defense. To identify the wheat proteins that TPP1 targets, a yeast-2-hybrid screen was performed using TPP1 as the bait to screen a wheat cDNA library. The candidate interacting wheat proteins will be analyzed by bioinformatics to prioritize those to be tested in VIGS assay. VIGS will be used to knock down expression of the wheat interactors. We will test if the silenced plants become more susceptible to F. graminearum, which would demonstrate that these wheat proteins have significant functions in fungal defense.

Accomplishments
1. Identification of fungi and bacteria that were positively and negatively associated with tar spot in corn. Tar spot, caused by the fungal pathogen Phyllachora maydis, is becoming one of the major constraints to production of corn in many parts of the United States. Because resistance is generally not available, having biological controls that attack tar spot could help maintain yields. However, almost nothing is known about other leaf microorganisms that could serve as biological controls. To address this deficiency, ARS researchers at West Lafayette, Indiana, analyzed the leaf microbiomes of 16 lines of corn that differed for levels of resistance to tar spot. These analyses identified a great diversity of fungi and bacteria in corn. Analyses of the three most resistant versus the three most susceptible lines showed many species of fungi and bacteria with highly significant positive or negative associations with tar spot. Those with positive associations may be either symbionts, or parasites that feed off the tar spot causal fungus. while those that are negatively associated could be antagonists. Discovery of parasitic and antagonistic microbes may identify biocontrol organisms that could be used to mitigate losses from tar spot.

2. Proteins from Phyllachora maydis that suppress corn immune responses for use as bioengineering targets to reduce tar spot. To successfully colonize a host, plant pathogens must reduce host immune responses. To do this, plant pathogens often use proteins known as effectors. Though tar spot is one of the most devastating fungal diseases of corn, very little is known about the effector proteins that suppress corn immune responses. To address this research deficiency, ARS researchers at West Lafayette, Indiana, identified effector proteins that are used by P. maydis to suppress immune responses in corn and may thus have a vital role in causing tar spot disease. Eighteen candidate effector proteins were identified using an advanced P. maydis genome sequence. The candidates were tested in two functional assays to determine if they interfere with well understood plant immune responses. Five of the candidates clearly showed ability to reduce plant defense mechanisms. This work provides the first insights into how this fungal pathogen may induce disease in corn and may be used to bioengineer resistance against this devastating fungal pathogen.

Review Publications
Zhang, C., Lane, B., Fernandez-Campos, M., Cruz-Sancan, A., Lee, D.-Y., Gongora-Canul, C., Ross, T.J., Da Silva, C.R., Telenko, D.E.P., Goodwin, S.B., Scofield, S.R., Oh, S, Jung, J., Cruz, C.D. 2023. Monitoring tar spot disease in corn at different canopy and temporal levels using aerial multispectral imaging and machine learning. Frontiers in Plant Science. 13. Article 1077403. https://doi.org/10.3389/fpls.2022.1077403.
Helm, M.D., Singh, R., Hiles, R., Jaiswal, N., Myers, A., Iyer-Pascuzzi, A., Goodwin, S.B. 2023. Candidate effector proteins from the maize tar spot pathogen Phyllachora maydis localize to diverse plant cell compartments. Phytopathology. 112(12):2538-2548. https://doi.org/10.1094/PHYTO-05-22-0181-R.
Mewa, D.B., Lee, S., Liao, C., Souza, A.M., Adeyanju, A., Helm, M.D., Lisch, D., Mengiste, T. 2022. ANTHRACNOSE RESISTANCE GENE 2 confers fungal resistance in sorghum. Plant Journal. 113(2):308-326. https://doi.org/10.1111/tpj.16048.
Feau, N., Dhillon, B.D., Sakalidis, M., Dale, A.L., Søndreli, K.L., Goodwin, S.B., LeBoldus, J.M., Hamelin, R.C. 2023. Forest health in the Anthropocene: The emergence of a novel tree disease is associated with poplar cultivation. Philosophical Transactions of the Royal Society B. 378: 20220008. https://doi.org/10.1098/rstb.2022.0008.
Singh, R., Shim, S., Telenko, D., Goodwin, S.B. 2023. Parental inbred lines of the Nested Association Mapping (NAM) population of corn show sources of resistance to tar spot in northern Indiana. Plant Disease. 107(2):262-266. https://doi.org/10.1094/PDIS-02-22-0314-SC.
Feurtey, A., Lorrain, C., McDonald, M.C., Milgate, A., Solomon, P.S., Warren, R., Puccetti, G., Scalliet, G., Torriani, S.F.F., Gout, L., Marcel, T.C., Suffert, F., Alassimone, J., Lipzen, A., Yoshinaga, Y., Daum, C., Barry, K., Grigoriev, I.V., Goodwin, S.B., Genissel, A., Seidel, M.F., Stukenbrock, E.H., Lebrun, M.-H., Kema, G.H.J., McDonald, B.A., Croll, D. 2023. A thousand-genome panel retraces the global spread and adaptation of a major fungal crop pathogen. Nature Communications. 14. Article 1059. https://doi.org/10.1038/s41467-023-36674-y.
Gongora-Canul, C., Jimenez-Beitia, F.E., Puerto-Hernandez, C., Avellaneda, M.C., Kleczewski, N.M., Telenko, D.E., Shim, S., Solorzano, J.E., Goodwin, S.B., Scofield, S.R., Cruz, C.D. 2023. Assessment of symptom induction via artificial inoculation of the obligate biotrophic fungus Phyllachora maydis (Maubl.) on corn leaves. BMC Research Notes. 16. Article 69. https://doi.org/10.1186/s13104-023-06341-y.
Gomez-Gutierriez, S.V., Goodwin, S.B. 2022. Loop-mediated isothermal amplification for detection of plant pathogens in wheat (Triticum aestivum). Frontiers in Plant Science. 13. Article 857673. https://doi.org/10.3389/fpls.2022.857673.
Singh, R., Goodwin, S.B. 2022. Exploring the corn microbiome: A detailed review on current knowledge, techniques, and future direction. Phytofrontiers. 2:158-175. https://doi.org/10.1094/PHYTOFR-04-21-0026-RVW.