Location: Corn Insects and Crop Genetics Research
2020 Annual Report
Objectives
Objective 1. Discover diverse fungal disease resistance mechanisms in cereal (barley and maize) crops.
Sub-Objective 1A. Use expression quantitative trait locus (eQTL) analysis in combination with genome-wide promoter-motif enrichment strategies to discover master regulators of immunity.
Sub-Objective 1B: Identify host targets of pathogen effectors by next generation yeast-two-hybrid interaction screens.
Sub-Objective 1C: Identify and characterize the genetic and molecular pathological modes of action for isolate-specific and non-specific Quantitative Disease Resistance (QDR) mechanisms that protect corn plants against northern leaf blight.
Objective 2: Generate novel sets of disease defense alleles for mechanistic dissection and application to crop protection.
Sub-Objective 2A: Functional confirmation via integrated reverse genetic analysis.
Sub-Objective 2B: Evaluate yield and northern leaf blight resistance properties of QDR alleles in hybrid genetic contexts.
Approach
Large-scale sequencing of plant and pathogen genomes has provided unprecedented access to the genes and gene networks that underlie diverse outcomes in host-pathogen interactions. Determination of regulatory focal points critical to these interactions will provide the molecular foundation necessary to dissect important disease resistance pathways. This knowledge can be used to guide modern plant breeding efforts in response to pathogens that present diverse challenges to the host.
Progress Report
Fungal pathogens are the greatest threats to cereal grain production worldwide. Effector proteins secreted by these pathogens manipulate host processes in order to create an ideal environment for colonization. To defend themselves, plants have evolved a battery of receptors that activate immune responses. Using plant-pathogen interaction systems of barley and corn, this project aims to identify both host disease defense components and pathogen signaling molecules that suppress them. By understanding how plants and pathogens manipulate each other during complex interactions, geneticists and breeders can tip the scales in favor of the crop plants to promote more stable and more efficient production.
Significant new insights into disease resistance signaling were obtained in FY2020. ARS scientists in Ames, Iowa, in collaboration with colleagues at the Danforth Center, St. Louis, Missouri, and funded by the National Science Foundation-Plant Genome Research Program, performed a genome-wide investigation of regulatory small RNAs (sRNAs), both in the barley host and its powdery mildew pathogen. sRNAs regulate a wide range of biological processes by degrading the RNA transcripts and the resulting proteins in control of these processes.
From these sRNAs, an important subclass of microRNAs (miRNAs), expressed during barley powdery mildew infection, were identified with their corresponding RNA transcript targets. These candidates were validated with parallel analysis of RNA ends (PARE), resulting in identification of novel miRNA candidates predicted to target transcriptional regulation and disease resistance signaling. In addition, another novel subclass of sRNAs, termed phased siRNAs (phasiRNAs), were identified which require a trigger miRNA to initiate their biogenesis. PhasiRNA loci were identified in powdery mildew infected barley leaves that overlap with protein-encoding transcripts encoding a mix of functional categories including signaling, metabolism, transcriptional regulation, and defense. Hence, miRNAs and phasiRNAs carefully control the expression of disease defense components to promote full growth potential during non-infection conditions, while allowing a switch to defense during pathogen challenge.
This research demonstrates for the first time that small RNAs are integral to gene regulation during infection by this destructive fungal pathogen. Knowledge from this research will impact how plant breeders select for disease resistance, one of the most important traits that affect crop yield, and thus food security.
In contrast to barley, whose disease defense relies heavily on classical disease resistance genes that initiate signaling cascades resulting in qualitative resistance, maize relies more on quantitative disease resistance (QDR) mechanisms. This is consequential to breeding because it dictates the types of approaches that should be used to prevent and mitigate disease outbreaks. Significant progress was made by establishing genetic dosage effects of the QDR alleles for Northern corn leaf blight (NLB). Specifically, the first year of a replicated inbred-hybrid genetic experiment was executed, allowing comparative performance assessment of zero, one and two copies of the resistance allele at each QDR locus in question. This was accomplished by growing, inoculating and evaluating the inbred lines (N=215) together with their respective Parent 1 (N=215) and Parent 2 (N=215) iso-hybrid backcross progenies in randomized, replicated blocks. In all, more than 20,000 plants were manually inoculated and evaluated in order to capture the phenotypic responses to NLB infection. Genetic analyses remain underway, but preliminary results show that several QDR alleles appear to be effective in single copy, which can dramatically simplify the breeding work necessary to leverage the allele for commercial breeding. An important additional finding of the work has been that prior genetic results were recapitulated to a reasonably high degree, indicating environmental stability for several of the QDR alleles, which is an important attribute for commercial performance.
While breeding is quite effective for crop protection, it is also vital to understand the mechanisms of disease defense and the magnitude and stability of their effects using genetic-based research approaches. To this end, the genetics of quantitative disease resistance against Setosphaerica turcica, the causal agent of NLB, was dissected using inbred line populations. In the past year, a myriad of breeding tasks were accomplished to enable follow-on genetic experiments that will allow yield and northern corn leaf blight resistance effects of transgenic and natural QDR alleles at varying dosages to be assessed in replicated multi-environment iso-hybrid trials. In May 2019, much of the corn planted for these efforts experienced unseasonably cold and wet conditions, resulting in poor germination, flood damage, and washout losses. Variation in outcomes arose from planting date, planting location, and type of material planted. Fortunately, the team’s staggered and distributed investment allowed for partial and full success of many breeding and evaluation efforts.
During this period of several years that elapse while the introgression breeding efforts occur, new approaches have been initiated to expand the experimental framework to include multiple genomic data sets acquired from NLB-inoculated and mock-inoculated corn lines. Using season one biological materials in the NLB experiment described above, a collaboration with Iowa State University scientists was initiated to conduct transcriptomic and proteomic evaluations for which data collection and analysis are already underway. The execution of this work is foundational to future success in identifying the genetic mechanisms that underpin the QDR alleles we have characterized.
Accomplishments
1. Small RNA transcripts during infection by barley powdery mildew control transcriptional regulation and disease resistance signaling. Fungal pathogens are the greatest threats to cereal grain production worldwide. Among the wide array of nucleic acids in eukaryotic cells, small RNAs (sRNAs) are important regulatory molecules for diverse physiological processes. ARS scientists in Ames, Iowa, in collaboration with colleagues at the Danforth Center, St. Louis, Missouri, and funded by the National Science Foundation-Plant Genome Research Program, implemented a genome-wide investigation of sRNAs in the cereal grain crop barley, and its powdery mildew pathogen, and identified multiple roles in disease resistance and pathogen virulence. Using a sophisticated informatics approach, they demonstrated for the first time, that small RNAs are integral to gene regulation during infection by this destructive fungal pathogen. Knowledge from this research will impact how plant breeders select for disease resistance, one of the most important traits that affect crop yield, and thus food security.
2. Elucidation of the genomic basis for rapid environmental adaptation in maize. Environmental maladaptation is the foremost barrier to capitalizing on intraspecific variation in plant breeding. ARS scientists in Raleigh, North Carolina; Ames, Iowa; and Columbia, Missouri, collaborated with university colleagues to implement a novel study design to measure the adaptability of a tropical maize landrace under selection for performance in a temperate environment. The basis for rapid adaptation while maintaining ample genetic diversity was shown to involve a multitude of genes responding in successive phases. The findings of this study provide an important guide for mining tropical germplasm resources of corn that will be crucial for continued gains in crop protection and utilization.
3. Science, Technology, Engineering, and Math (STEM) program on plant genetics. Most young people are influenced to pursue STEM careers in secondary school rather than college. Thus, there is a need to enable these students, and their teachers, to understand the important impact and satisfaction that comes from ‘doing good science’. Over the past ten years, ARS scientists and staff in Ames, Iowa, have mentored forty teachers from Iowa and Tuskegee, Alabama, to implement the iTAG (inheritance of Traits and Genes) outreach curriculum in 195 classrooms, impacting 4,660 students, 1/3 of which are underrepresented from urban and rural communities. In FY 2019, this program impacted 144 new students from nine classrooms in six schools. Notably, one teacher then had 10 students that chose STEM career paths. Providing scientific opportunity to a diverse population will ensure the long-term development of a globally competitive STEM workforce.
Review Publications
Dennison, T., Qin, W., Loneman, D.M., Condon, S.G., Lauter, N.C., Nikolau, B.J., Yandeau-Nelson, M.D. 2019. Genetic and environmental variation impact the cuticular hydrocarbon metabolome on the stigmatic surfaces of maize. Biomed Central (BMC) Plant Biology. 19. https://doi.org/10.1186/s12870-019-2040-3.
Do Vale Martins, L., Yu, F., Zhao, H., Dennison, T., Lauter, N.C., Wang, H., Deng, Z., Thompson, A., Semrau, K., Rouillard, J., Birchler, J.A., Jiang, J. 2019. Meiotic crossovers characterized by haplotype-specific chromosome painting in maize. Nature Communications. 10:4604. https://doi.org/10.1038/s41467-019-12646-z.
Falcon, C.M., Kaeppler, S.M., Spalding, E.P., Miller, N.D., Haase, N., Alkhalifah, N., Bohn, M., Buckler IV, E.S., Campbell, D.A., Ciampitti, I., Coffey, L., Edwards, J.W., Ertl, D., Flint Garcia, S.A., Gore, M.A., Graham, C., Hirsch, C.N., Holland, J.B., Jarquin, D., Knoll, J.E., Lauter, N.C., Lawrence-Dill, C.J., Lee, E.C., Lorenz, A., Lynch, J.P., Murray, S.C., Nelson, R., Romay, M., Rocheford, T., Schnable, P., Scully, B.T., Smith, M.C., Springer, N., Tuinstra, M., Walton, R., Weldekidan, T., Wisser, R.J., Xu, W., De Leon, N. Relative utility of agronomic, phenological, and morphological traits for assessing genotype-by-environment interaction in maize inbreds. Crop Science. 2020; 60:62-81. https://doi.org/10.1002/csc2.20035
Hunt, M., Banerjee, S., Surana, P., Liu, M., Fuerst, G.S., Mathioni, S., Meyers, B.C., Nettleton, D., Wise, R.P. 2019. Small RNA discovery in the interaction between barley and the powdery mildew pathogen. Biomed Central(BMC)Genomics. 20. https://doi.org/10.1186/s12864-019-5947-z.
Mcfarland, B.A., Alkhalifah, N., Bohn, M., Bubert, J., Buckler IV, E.S., Ciampitti, I., Edwards, J.W., Ertl, D., Gage, J.L., Falcon, C.M., Flint Garcia, S.A., Gore, M., Graham, C., Hirsch, C., Holland, J.B., Hood, E., Hooker, D., Jarquin, D., Kaeppler, S., Knoll, J.E., Kruger, G., Lauter, N.C., Lee, E.C., Lima, D.C., Lorenz, A., Lynch, J.P., Mckay, J., Miller, N.D., Moose, S.P., Murray, S.C., Nelson, R., Poudyal, C., Rocheford, T., Rodriguez, O., Romay, M., Schnable, J.C., Schnable, P.S., Scully, B.T., Sekhon, R., Silverstein, K., Singh, M., Smith, M., Spalding, E.P., Springer, N., Thelen, K., Thomison, P., Tuinstra, M., Wallace, J., Walls, R., Wills, D., Wisser, R.J., Xu, W., Yeh, C., De Leon, N. Maize genomes to fields (G2F): 2014 –2017 field seasons: genotype, phenotype, climatic, soil and inbred ear image datasets. BMC Research Notes. 13,71 (2020). https://doi.org/10.1186/s13104-020-4922-8.
Wisser, R.J., Fang, Z., Holland, J.B., Teixeira, J.E., Dougherty, J., Weldekidan, T., De Leon, N., Flint-Garcia, S.A., Lauter, N.C., Murray, S.C., Xu, W., Hallauer, A. 2019. The genomic basis for short-term evolution of environmental adaptation in maize. Genetics. 213(4):1479-1494. https://doi.org/10.1534/genetics.119.302780.
Nino-Liu, D.O., Bancroft, T., Rajaram, M.L., Moscou, M., Caldo, R., Schmidt, C., Dorman, K., Yang, B., Patil, P., Szurek, B., Koebnik, R., Van Sluys, M., Leach, J.E., Salzberg, S.L., White, F.F., Nettleton, D., Wise, R.P., Bogdanove, A.J. 2017. Targeting of the rice transcriptome by TAL effectors of Xanthomonas oryzae. In: Wolpert, T., Shiraishi T., Collmer A., Akimitsu K., and Glazebrook J., editors. Genome-Enabled Analysis of Plant-Pathogen Interactions. St. Paul, Minnesota: American Phytopathological Society Press. p.193-206. https://doi.org/10.1094/9780890544983.021.
