Location: Crop Production and Pest Control Research
2021 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
Objective 1. Analysis of RNA sequencing projects comparing susceptible, incompatible resistance (R)-gene and incompatible non-host resistance responses of the wheat pathogen Zymoseptoria tritici inoculated onto plants of wheat and barley was completed. A draft of a manuscript detailing the non-host versus R-gene responses was begun, but writing was delayed when the ARS postdoctoral associate working on the project accepted a job in private industry. Analysis of pathogen sequences during the infection process showed that there was sufficient signal for thorough analyses, even on the incompatible interactions on wheat plants with resistance genes or on the non-host barley. Therefore, a second manuscript on the pathogen responses is being drafted. This work showed that pathogen gene expression patterns on the two wheat cultivars containing resistance genes were similar to each other but different from those on the susceptible and non-host plants. The highest numbers of differentially expressed genes occurred when the pathogen was growing on the susceptible cultivar at ten days after inoculation, the time of a transition from feeding on living to dead tissue. These analyses identified many interesting candidate genes for additional follow-up work.
Final revisions to a manuscript about light responses of the pathogen were submitted, accepted, and published in the journal BMC Genomics. Follow-up work on a knockout mutant of the VIVID gene, which is involved in light responses in other species, showed that it is required for pathogenicity of Zymoseptoria tritici to wheat. Work to complement the mutant and obtain the final data needed for publication was delayed due to Covid restrictions on laboratory work. Those plus other experiments to try to obtain additional knockout mutants will be resumed as soon as we can enter the lab again safely. From our analyses, we generated a list of 15 genes that are likely to be involved in light responses of Z. tritici and will test in future experiments.
Since we were not able to work in the laboratory during the full year due to Covid, we redirected our efforts for this part of the project to a review on the available literature on Loop-Mediated Isothermal Amplification (LAMP) for identifying wheat pathogens in infected leaves. This analysis shows that the technique has much potential but has not been applied much to wheat diseases. A review summarizing those findings was drafted and will be submitted for publication following internal revisions.
Objective 2. Currently 24 genes have been screened by Virus-Indiced Gene Silencing to assay if they make critical contributions to resistance to Fusarium crown rot. So far none of these genes have shown strong effect. This work was delayed by the COVID closure, but the goal of testing 30 genes should be completed before the end of FY21.
Objective 3. Several virulence proteins were identified from the tar spot pathogen, Phyllachora maydis, that likely have a functional role in plant pathogenesis. Using publicly available software, we identified 59 candidate effectors from among the predicted secreted proteins expressed by P. maydis, 15 of which are predicted with high confidence. We next identified potential proteins that could span cell membranes and those with possible nuclear localization signals, or that could transit membranes of the chloroplasts or mitochondria. Among the 15 candidate effectors predicted with high confidence, four had an identifiable transmembrane capacity, three have putative nuclear localization signals, and one has an identifiable chloroplast transit peptide, suggesting chloroplast localization. Future work will assess the subcellular localizations of the candidate effectors as well as test whether these putative effectors have a functional role in suppressing host defense responses. A manuscript on the identification and functional characterization of candidate-secreted effector proteins is being drafted and we anticipate that it will be finished during the first quarter of FY22. An abstract of work on P. maydis effectors was also submitted for presentation at the annual meetings of the American Phytopathological Society during August of 2021. The work presented at this meeting received a considerable amount of attention from industry scientists and, as a result, will be featured on an upcoming episode of the podcast “Focus on Agriculture.”
Objective 4. Our previous analyses plus results from two other labs in Australia and the UK 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 was not possible to explore alternative approaches in the lab.
Objective 5. Sequencing of bulk populations made between crosses of a resistant and susceptible line chosen from our advanced backcross progeny and that segregates for the Stb2 resistance gene was completed. Analysis of the results identified numerous genes that should be located near the resistance gene, plus two promising candidate genes. Primers for polymerase chain reaction analysis of 100 likely linked genes were designed and are in process of being tested. Preliminary results indicate that many will be useful and will be tested on the full 700-member mapping population that we developed previously. This work has been slowed considerably due to limited access to the laboratory as a protection from Covid but will be pursued vigorously once we are back in the lab full time.
Analysis of a genome-wide association study (GWAS) of Septoria tritici blotch resistance on a large collection of Purdue University wheat cultivars with different origins was completed through collaborative research. A manuscript was prepared and we anticipate that it will be submitted to a journal for publication following additional internal revisions during the last quarter of FY21. A former visitor to the lab from Ethiopia completed a different GWAS analysis of Septoria tritici blotch resistance in a population from Ethiopia and a manuscript on that work was submitted for publication. Revisions based on reviewer comments were prepared and galleys of the accepted manuscript have been returned for publication during the last quarter of FY21.
Corn lines that were parents of publicly available mapping populations plus 200 and 100 progeny from crosses between different parents were planted in the field and scored for resistance to tar spot and other foliar diseases under natural inoculum. Analysis of the results showed very large differences between the parents of various mapping populations and among the progeny. A manuscript on the methods used for phenotyping and on variation among the parents is being prepared for submission to a journal during the last quarter of FY21 or the first quarter of FY22. Sequencing for part of a microbiome analysis of corn was completed and analyses are in progress. Initial analyses showed large differences between the fungal and bacterial microbiomes on a line that was resistant to tar spot versus one that was susceptible. Analysis of the remaining lines (two more that were susceptible to tar spot and two that were resistant) is continuing. We expect to complete those analyses during the last quarter of FY21 and to submit a manuscript on the microbiome results during the first quarter of FY22. In collaborative research, a manuscript on a computer algorithm for phenotyping corn lines for tar spot resistance was developed and has now been accepted for publication in a refereed journal. The galleys have been returned and the paper should be published during September 2021. Other planned labwork on the microbiome project was put on hold pending a return to full-time access to the lab.
Accomplishments
1. The Septoria tritici blotch pathogen of wheat senses and responds to light. Production of toxins and spores of plant-pathogenic fungi can be influenced by light, yet despite its great economic importance, nothing was known about the photobiology of the Septoria tritici blotch pathogen of wheat. ARS scientists in West Lafayette, Indiana, tested whether this fungus can sense and respond to light, gene expression of cultures grown under white, blue or red light were compared to each other and to that seen when grown in the dark. The results showed clearly that this fungus can sense and respond to different wavelengths of light, including some genes that are involved in pathogenicity. Although the largest differences were between cultures grown under any light treatment and the dark, many differences were apparent between the different wavelengths of light, particularly between red and white. These results will be interesting to plant pathologists trying to manage Septoria tritici blotch on wheat and could help fungicide companies design better control strategies by targeting essential genes for sensing and responding to light. A paper describing these results was published less than a year ago but has already been cited by others indicating the international interest and potential high impact of this research.
2. Engineering a novel, new-to-nature disease resistance trait to a viral pathogen of soybean. Genetic-based control using plant disease resistance genes is the most economical and sustainable containment strategy for controlling crop plant pathogens. Though they are indeed effective, resistance genes with desired recognition specificities are not always available. There is thus a need to expand the recognition specificity of pre-existing plant disease resistance proteins such that they recognize multiple plant pathogens. A team of researchers including ARS scientists in West Lafayette, Indiana. In our work, we successfully introduced a novel disease resistance trait in soybean against a viral pathogen. These Our results clearly showed that soybeans expressing the modified resistance protein confer robust resistance against multiple pathogens, including a bacterial and viral pathogen. This work will be of significant interest to plant pathologists as it clearly demonstrates the potential to engineer tailored resistances in a crop plant to pathogens for which effective resistance genes have yet to be identified. This recent work has already cited by others and generated international interest for this approach to engineering new disease resistance specificities in crop plants.
Review Publications
Mccorrison, C.B., Goodwin, S.B. 2020. The wheat pathogen Zymoseptoria tritici senses and responds to different wavelengths of light. BMC Genomics. 21:513 https://doi.org/10.1186/s12864-020-06899-y.
Mekonnen, T., Haileselassie, T., Goodwin, S.B., Tesfayea, K. 2020. Genetic Diversity and Population Structure of Zymoseptoria tritici in Ethiopia as Revealed by Microsatellite Markers. Fungal Genetics and Biology (2020). 141:103413. https://doi.org/10.1016/j.fgb.2020.103413.
