Research Project: Molecular and Biochemical Characterization of Biotic and Abiotic Stress on Plant Defense Responses in Maize

Location: Chemistry Research

2019 Annual Report

Objectives
The overall goal of this project is to provide stakeholders with increased knowledge of the innate immune responses of maize to insect and fungal attack and determine how these defense mechanisms are affected by abiotic stress factors. Objective 1. Molecularly characterize the production and function of chemical defense responses to biotic and abiotic stress of maize to evaluate and elucidate the cumulative effect of multiple stressors. Sub-objective 1A. Molecularly characterize defense metabolites (i.e., fatty acids) and their mediated plant responses in fungal infected tissues, and determine the impact of abiotic stress on these responses. Sub-objective 1B. Identify maize genes involved in the production of chemical defenses against insect pests, use mutants in these genes to elucidate the production and function of chemical defenses in insect resistance, and assess the effect of abiotic stress on these defenses. Objective 2. Identify and functionally characterize genetic components that mediate the defense response of maize to biotic stress, and determine the impact of abiotic stress on these mechanisms to mitigate yield loss.

Approach
The production of select chemical defenses in maize in response to specific biotic stressors will be analyzed by profiling novel free fatty acids, hormones, inducible volatiles, and flavones in maize in response to fungal or insect attack. Candidate genes responsible for the biosynthesis and regulation of these metabolites will be identified using co-expression analysis and forward genetic approaches. Once the genes have been selected they will be prioritized for further characterization and mutant resources such as the UniformMu maize population mined for mutations in those genes and the presence of mutant alleles confirmed by gene-by –gene PCR genotyping. Mutants will also be generated in genes of interest using CRISPR/Cas9 technology. Loss-of-function mutants, coupled with metabolic profiling and bioassays will then be used to assess the function of select high priority candidate genes and their products in biotic stress resistance. Furthermore, defense responses will be characterized under abiotic stress conditions (heat, drought, elevated carbon dioxide) to determine the integrity of defense pathways under situations of combinatorial stress.

Progress Report
Good progress has been made on the research project 6036-11210-001-00D by Gainesville ARS scientists. For Objective 1A, the following experiments were conducted using mapping populations. First, plants from Goodman diversity panel were treated with our recently characterized fatty acid cell death-inducing signal (10-oxo-11-phytoenoic acid). Lesion areas from this treatment were quantified and used in Genome Wide Association Study (GWAS) mapping to identify three candidate metacaspase genes involved in programmed cell death processes. Second, young inner whorl and stem tissues from the Goodman diversity panel were inoculated with Cochliobolus heterostrophus (the causal agent of Southern corn leaf blight) and fatty acid metabolites were extracted and utilized as traits for GWAS mapping to identify two novel fatty acid biosynthesis genes involved in plant defense against fungal pathogens. Other mapping experiments that have been conducted and are being processed include Fusarium verticillioides stem inoculations on the Goodman diversity panel, drought stress on the Goodman diversity panel, and F. verticillioides stalk inoculations on the intermated B73xMo17 (IBM) population. For Objective 1B, co-expression analysis, literature searches and qRT-PCR were used to identify several genes potentially involved in the chemical defenses of maize against insect pests. Potential loss-of function alleles were identified in 3 of these genes in the UniformMu population. These lines are currently planted in the spring field for genetic evaluation. A CRISPR/Cas9 construct was made to target 10 terpene synthase genes potentially involved in the production of herbivore induced volatiles. Screening of the first generation of transgenic plants reveled effective loss-of-function alleles for at least seven of the targeted genes. These lines are being progressed to obtain homozygous single mutants for the appropriate bioassays and analysis. For Objective 2, more than 50 mutant lines are currently being introgressed into W22, B104 or B73 inbred lines. Maize lines altered in hormone production or perception are being used in combinatorial stress bioassays. The combinatorial stress of flooding and fall armyworm infestation in maize was found to increase the resistance of maize to this insect pest. Metabolite profiling revealed that the production of the plant hormone salicylic acid was strongly increased, specifically in response to this combined stress. A loss-of-function mutant in the salicylic acid receptor NPR1 was then used to show that salicylic acid production/perception was important for flooding induced fall armyworm resistance in maize. Maize lines with lower jasmonic acid (allene oxide cyclase mutants, lipoxygenase 10 mutants), higher jasmonic acid (hairy sheath frayed-1 mutants), and lower abscisic acid (viviparous14 mutants) were used in fungal infection and insect infestation bioassays. Results from these initial bioassays show little effect on biotic resistance to date, but these mutants and others continue to be examined under a series of biotic, abiotic, and combinatorial stresses. Another important advancement made by Gainesville ARS scientists was in identifying an important role of the maize aleurone in protecting germinating kernels against fungal infection. The aleurone is a specialized cell layer surrounding the maize kernel and Gainesville ARS scientists identified it as a location where high levels of anti-fungal chemicals are produced. Using mutants with disrupted aleurone formation, it was shown that aleurone-defective mutants were susceptible to fungal infection during germination, demonstrating that the cell layer is important for protecting against infection.

Accomplishments

Review Publications
Jones, A.C., Seidl-Adams, I., Engelberth, J., Hunter III, C.T., Alborn, H.T., Tumlinson, J.H. 2019. Herbivorous caterpillars can utilize three mechanisms to alter green leaf volatile emission. Environmental Entomology. 48(2):419–425. https://doi.org/10.1093/ee/nvy191.
Soubeyrand, E., Johnson, T.S., Latimer, S., Block, A.K., Kim, J., Colquhoun, T.A., Butelli, E., Martin, C., Chapple, C., Basset, G.J. 2018. The peroxidative cleavage of kaempferol contributes to the biosynthesis of the benzenoid moiety of ubiquinone in plants. The Plant Cell. 30:2910-2921. https://doi.org/10.1105/tpc.18.00688.
Wang, M., Toda, K., Block, A.K., Maeda, H.A. 2019. TAT1 and TAT2 tyrosine aminotransferases have both distinct and shared functions in tyrosine metabolism and degradation in Arabidopsis thaliana. Journal of Biological Chemistry. 294(10):3563-3576. https://doi.org/10.1074/jbc.RA118.006539.
Block, A.K., Yakubova, E., Widhalm, J.R. 2019. Specialized naphthoquinones present in Impatiens glandulifera nectaries inhibit the growth of fungal nectar microbes. Plant Direct. 3(5):1-7. https://doi.org/10.1002/pld3.132.
Block, A.K., Vaughan, M.M., Schmelz, E.A., Christensen, S.A. 2018. Production and function of terpenoid defense compounds in maize (Zea mays). Planta. https://doi.org/10.1007/s00425-018-2999-2.
Christensen, S.A., Hunter III, C.T., Block, A.K. 2018. Pesticides on the inside: Exploiting the natural chemical defenses of maize against insect and microbial pests. ACS Symposium Series. https://doi.org/10.1021/bk-2018-1294.ch006.
Springer, N., Anderson, S., Andorf, C.M., Ahern, K., Bai, F., Barad, O., Barbazuk, W., Bass, H.W., Baruch, K., Gen-Zvi, G., Buckler IV, E.S., Bukowski, R., Campbell, M.S., Cannon, E.K., Chomet, P., Dawe, R., Davenport, R., Dooner, H.K., He Du, L., Du, C., Easterling, K., Gault, C., Guan, J., Jander, G., Hunter III, C.T., Jiao, Y., Koch, K.E., Kol, G., Kudo, T., Li, Q., Lu, F., Mayfield-Jones, D., Mei, W., McCarty, D.R., Noshay, J., Portwood II, J.L., Ronen, G., Settles, M.A., Shem-Tov, D., Shi, J., Soifer, I., Stein, J.C., Suzuki, M., Vera, D.L., Vollbrecht, E., Vrebalov, J.T., Ware, D., Wei, X., Wimalanathan, K., Woodhouse, M.R., Xiong, W., Brutnell, T.P. 2018. The maize W22 genome provides a foundation for functional genomics and transposon biology. Nature Genetics. 50:1282-1288. https://doi.org/10.1038/s41588-018-0158-0.
Chandrasekhar, K., Shavit, R., Distefeld, A., Christensen, S.A., Tzin, V. 2018. Exploring the metabolic variation between domesticated and wild tetraploid wheat genotypes in response to corn leaf aphid infestation. Plant Signaling and Behavior. 13(6):1-5. https://doi.org/10.1080/15592324.2018.1486148.