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.
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.
Objective 1: Investigate the mechanisms of fungal pathogenicity and other important biological traits in cereal crops. We have continued working on improved methods for transformation of fungal strains since the new project was initiated five months ago. We have made significant research progress with regards to identifying proteases from several fungal pathogens, including Fusarium graminearum, Zymoseptoria tritici, and Phyllachora maydis. Analysis of the draft genome sequence of Phyllachora maydis and the finished reference genome of Zymoseptoria tritici revealed that both fungal pathogens encode proteases with predicted signal peptide sequences. It is thus likely these proteases are secreted during infection and may have a functional role during plant pathogenesis. Future work will aim to address whether these proteases modulate host immune responses in wheat, barley, and maize. Objective 2. Analyze microbiomes associated with resistance and susceptibility to identify vulnerabilities in fungal pathogens of cereal crops. Analyses of microbiome sequences from corn lines that showed varying levels of resistance to tar spot during the 2019 growing season were completed. These results showed differences in both bacterial and fungal species present on resistant versus susceptible corn lines and a clear gradation in the number of reads from Phyllachora maydis, the causal fungus of tar spot. Some corn inbreds clearly were susceptible to other fungal pathogens as revealed by high numbers of sequence reads from the causal organisms. A manuscript on these results is being prepared for submission during the last quarter of fiscal year 2022. Objective 3. Identify, genetically map and functionally characterize host resistance against fungal pathogens of cereal crops. During the field season of 2022, we have selected a progeny of the corn Nested Association Mapping population to screen for tar spot resistance based on our phenotyping of the parental inbred lines during the 2019- 2021 field seasons. The resistant parent for this population derives from North Carolina so we anticipate it may contain different resistance gene(s) from what has been reported previously. The parental and progeny lines have been planted and germination was good but we have to rely on natural infection so do not yet know whether there will be enough disease on the susceptible lines for identification of tar spot resistance in the field. Based on mapping efforts from our previous five-year project, we have developed primers for molecular markers that should be linked to resistance genes to Septoria tritici blotch of wheat and tar spot of corn, which will be tested beginning the fourth quarter of this fiscal year. Objective 4: Exploit knowledge of host-pathogen interactions and pathogen vulnerabilities to develop novel methods for increasing resistance in cereal crops. The first step in developing our decoy engineering-based disease resistance system is the identification of pathogen-encoded effector proteases that are secreted in the plant cell and contribute to initiating the infection process. We have made substantial research progress in identifying F. graminearum (Fg) effector proteases. We identified 95 F. graminearum genes potentially coding for secreted proteases. This candidate gene list was narrowed further by selecting only endopeptidases and genes that were upregulated during early stages of infection. These stringent selection criteria identified seven candidate proteases. A manuscript on these results is being prepared for submission.
1. Identification of candidate effector proteins from the maize tar spot pathogen Phyllachora maydis and determination of their cellular locations. Plants have a sophisticated immune system capable of detecting and conferring resistance to a variety of plant pathogens. During the infection process, pathogens inject proteins known as ‘effectors’ into the plant host cell to “shut-off” the plant immune system, thereby promoting pathogen growth and disease and consequently reducing crop yields. Therefore, studying how plant pathogens use these proteins during infection is important as it often provides insight into how pathogens reduce crop yields. One such fungal pathogen of corn known as Phyllachora maydis injects ‘effector’ proteins into corn leaves resulting in a disease called tar spot. Importantly, tar spot disease can significantly reduce overall corn yields and, in turn, induce severe financial penalties to farmers. Therefore, there is an urgent need to investigate how this fungal pathogen uses its effector proteins to infect corn leaves. Here, we show that this fungal pathogen injects ‘effector’ proteins into host cells, where they specifically target plant organelles such as the nucleus and chloroplast. ARS researchers in West Lafayette, Indiana, therefore, hypothesized this fungal pathogen interferes with and manipulate host immune responses by targeting the plant organelles. Our work presented here provides valuable insights into the host processes potentially manipulated by this fungal pathogen and lays the foundation for generating testable hypotheses for future work. A manuscript describing these results was recently published in Phytopathology.