Location: Crop Production and Pest Control Research
2022 Annual Report
Objectives
Objective 1: Identify candidate genes expressed by the host and fungal pathogens during resistant, susceptible and non-host interactions to elucidate mechanisms of host resistance of wheat, corn and barley.
Sub-objective 1a. Identify genes expressed by hosts containing different resistance genes to identify the mechanisms involved in each resistance response.
Sub-objective 1b. Identify genes expressed by the pathogens during different growth conditions that are involved in survival and pathogenicity.
Objective 2: Determine whether selected genes function in fungal resistance responses by virus-induced gene silencing (VIGS) in wheat, and test if the best candidates confer improved resistance in transgenic plants.
Sub-objective 2a. Utilize BSMV-VIGS to assess function of candidate genes in FCR and Septoria leaf blotch resistance.
Sub-objective 2b. Attempt to engineer FCR, FHB or STB resistance in transgenic wheat utilizing genes confirmed in VIGS analysis.
Objective 3: Analyze multiple fungal genomes to identify effectors and other biologically important genes involved in pathogenicity on wheat and corn.
Objective 4: Determine the effectiveness of Host-Induced Gene Silencing (HIGS) to control Septoria and Fusarium diseases in wheat.
Objective 5: Refine the map locations of genes for resistance to Septoria diseases in wheat and fungal diseases in corn to identify tightly linked molecular markers for marker-assisted selection in cereal improvement programs.
Sub-objective 5a. Identify molecular markers tightly linked to the Stb2 and Stb3 resistance genes in wheat.
Sub-objective 5b. Identify molecular markers linked to resistance genes in corn.
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 non-host 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 fine-scale 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
This is the final report for this project, which terminated in March 2022. See the report for the replacement project, 5020-21220-014-000D, “Fungal Host-Pathogen Interactions and Disease Resistance in Cereal Crops” for additional information.
Objective 1. The overall goal of this objective was to use RNA sequencing (RNAseq) experiments to identify genes from wheat that are involved in resistance responses to the fungal pathogen Zymoseptoria tritici (the cause of Septoria tritici blotch disease) and of genes for important biological processes in the pathogen. Several experiments were completed looking at both host (including major resistance genes) and non-host (against the closely related barley pathogen Septoria passerinii) resistance. The analyses identified sets of differentially expressed genes that may be involved in host or non-host resistance and will be the subject of continuing analyses in our next five-year project. On the pathogen side, we were able to identify genes expressed during growth on a susceptible host as well as the changes involved in resistant hosts (where the pathogen is suppressed but not killed). We also used RNAseq analyses to prove that the pathogen can sense and respond to different wavelengths of light and that work was published during a previous fiscal year. While it was assumed that the pathogen could respond to light, our experiments were the first to prove it and to identify some of the genes involved. A follow-up experiment was conducted to analyze the response of host and pathogen during a diurnal cycle but the RNAs were not extracted because of Covid restrictions that kept us out of the lab for more than a year.
During the most recent fiscal year, RNAseq analyses of host and pathogen responses were completed and two additional manuscripts are in process, one on non-host responses in wheat and a second on pathogen gene expression on a susceptible host during disease development. We hope to have both manuscripts submitted for publication later this fiscal year or at the beginning of the next.
A review paper on Loop-Mediated Isothermal Amplification (LAMP) for identifying wheat pathogens in infected leaves was submitted during January of 2022 and published in March in Frontiers in Plant Science.
Objective 2. The goal of this objective was to identify and functionally test using Virus-Induced Gene Silencing (VIGS) genes that might be involved in resistance to Fusarium crown rot (FCR) in wheat. Work during previous fiscal years identified lists of candidate genes for testing and made progress on analyzing responses to FCR. For the most recent fiscal year, despite silencing 20 new genes, no sequences were found that reproducibly affected Fusarium head blight resistance in wheat. This is almost certainly because the genes tested only make partial contributions to resistance, and so silencing them one at a time did not give significant enough effects to be reproducibly detected in the Fusarium head blight greenhouse assay.
Objective 3. The goal of this objective was to analyze pathogen genomes to identify genes that might be involved in important biological traits for future functional analyses. Comparative genomic analyses with the wheat pathogen Zymoseptoria tritici identified numerous genes on both core and accessory chromosomes that might be important for pathogenicity and are being followed up by work in our continuing projects as well as other laboratories in other countries. However, some of this work was delayed due to our inability to be in the laboratory during most of 2020-2021. Due to the recent emergence and high importance of tar spot disease, caused by the fungal pathogen Phyllachora maydis, to corn production in Indiana and adjacent states, we refocused much of our effort to this extremely important pathogen, which led to the generation and publishing through collaborative research of its first draft genome. During fiscal year 2022, analysis of the P. maydis genome sequence revealed 462 putatively secreted proteins, of which 40 contain characteristics expected of effectors that may assist the pathogen in causing disease. Laser-scanning confocal microscopy of epidermal cells from the tobacco Nicotiana benthamiana revealed that most of the putative P. maydis effectors localized to the nucleus and cytosol. One candidate effector localized to multiple subcellular compartments including the nucleus, nucleolus, and plasma membrane while an additional putative effector preferentially labelled both the nucleus and nucleolus. One of the candidate effectors consistently localized to the stroma of chloroplasts as well as stroma-containing tubules (stromules). Collectively, these data suggest that effector candidate proteins from P. maydis target diverse cellular organelles and may provide valuable insights into their possible functions as well as host processes potentially manipulated by this fungal pathogen. A manuscript on these results was submitted and has been accepted for publication in Phytopathology.
Objective 4. Our previous analyses plus results from two other labs in Australia and the United Kingdom showed that host- induced gene silencing (HIGS) is not possible against our targeted wheat pathogen. Therefore, this project as originally designed was deemed unfeasible and work was stopped. With Covid restrictions it has not been possible to explore alternative approaches in the lab.
Objective 5. The original goal of this objective was to identify molecular markers linked to genes for resistance to Septoria tritici blotch in wheat. Additional populations segregating for the Stb2 and Stb3 genes for resistance were generated and phenotyped by inoculating plants in the greenhouse. Beginning in 2019 most work on this objective was switched to tar spot of corn due to the pressing need for information on how to manage this disease. Analysis of the parents of the Nested Association Mapping (NAM) population in corn previously identified large variation among the inbred lines with those from North America mostly being highly susceptible but with very strong resistance in tropical corn lines plus one from North Carolina. A manuscript on these results was submitted and has been accepted for publication in Plant Disease.
To follow up on that project we phenotyped two segregating populations for tar spot disease. Analysis of one population is complete and identified a very large single or possibly double peak corresponding to a quantitative trait locus (or loci) (QTL) for tar spot resistance on corn chromosome 9. This resistance gene appears to confer a very strong effect and has not been identified previously. It is different from a previously identified quantitative trait locus on chromosome 8 and combining both in a single corn line could lead to greatly increased resistance to tar spot. A manuscript on that work is being prepared that we anticipate submitting during the first quarter of the next fiscal year (FY). Phenotyping on a second population has been completed but the analysis is not finished. Depending on the results, analysis of that population may comprise a second manuscript or it may be combined with the first one. To address problems with inoculating and scoring tar spot symptoms we have initiated a collaboration with Purdue University to better understand the basic biology of the pathogen. Part of that work involved developing a computer algorithm for discriminating tar spot lesions from other markings on corn leaves from analysis of digital images. A manuscript on that work was submitted and published during the first quarter of this fiscal year. A second project was to compare development of tar spot epidemics in different environments in the United States and Honduras. A manuscript on that work has been prepared and will be submitted during the fourth quarter of this FY. A third aim of this collaborative project was to develop a protocol for artificial inoculation of the tar spot pathogen in a greenhouse. This pathogen is an obligate pathogen so cannot be cultured and inability to make artificial inoculations is a severe limiting factor in research progress. Work with fresh inoculum in Honduras gave reasonable results and greenhouse work with various inoculum sources in the United States showed that controlled inoculations are possible. The success rate is not as high as we would like but may be high enough to screen germplasm under controlled conditions in a greenhouse. A manuscript on those results is being prepared and will be submitted by the end of this FY.
Accomplishments
1. Identifying sources of resistance to tar spot in corn. Helped to mitigate future losses to this disease, which were estimated at 231 million bushels during 2021 alone. ARS researchers in West Lafayette, Indiana, analyzed 26 parents of the Nested Association Mapping (NAM) population of corn for resistance to tar spot in replicated field trials during the 2019-2021 growing seasons. All lines were phenotyped for disease scores at three times (early, middle and late) during the epidemic and the results were analyzed for differences. The NAM population contained a great variability with some lines being rated consistently as highly resistant while most were scored as moderately or highly susceptible. Lines from North America were mostly highly susceptible while many of those from tropical areas showed varying levels of resistance with some being almost immune. A line from North Carolina was among those that were the most highly resistant indicating the potential to breed in resistance from adapted germplasm from North America.
Results from these analyses were used to choose populations for phenotyping in the field to identify the numbers and locations of any resistance genes they may carry. A manuscript describing these results has been accepted for publication in Plant Disease. After first arriving in 2015, by 2021 tar spot was the most important disease of corn in the United States, losses estimated at over 231 million bushels. Host resistance is the most economical method for limiting disease losses and if this new resistance gene can be transferred into elite corn inbreds it could save tens of millions of dollars of lost yield every year.
Review Publications
Helm, M.D., Margets, A., Rima, S., Carter, M.E. 2021. Molecular mechanism & structure — zooming in on plant immunity. Molecular Plant-Microbe Interactions. 12:1346-1349. https://doi.org/10.1094/MPMI-08-21-0208-MR.
Lee, D., Na, D., Gongora-Canul, C., Baireddy, S., Lane, B., Cruz, A.P., Fernandez-Campos, M., Kleczewski, N.M., Telenko, D., Goodwin, S.B., Delp, E.J., Cruz, C. 2021. Contour-based detection and quantification of tar spot stromata using RGB imagery. Frontiers in Plant Science. 12:675975. https://doi.org/10.3389/fpls.2021.675975.
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.
Mekonnen, T., Sneller, C.H., Haileselassie, T., Ziyomo, C., Bekele, A.G., Goodwin, S.B., Lule, D., Tesfaye, K. 2021. Genome-Wide Association Study reveals novel genetic loci for quantitative resistance to Septoria tritici blotch in wheat (Triticum aestivum L.). Frontiers in Plant Science. 12:671323. https://doi.org/10.3389/fpls.2021.671323.
Crane, C.E., Nemacheck, J.A., Subramanyam, C.F., Williams, S.N., Goodwin, S.B. 2022. SLAG: A program for seeded local assembly in complex genomes. Molecular Ecology Resources. 22:596–616. https://doi.org/10.1111/1755-0998.13580.
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.
