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

Location: Chemistry Research

2021 Annual Report

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.

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
Progress has been made on the research project 6036-11210-001-00D by Gainesville, Florida, ARS scientists. Objective 1A, several genome wide association studies (GWAS) were performed by ARS researchers in Gainesville, Florida, using the Goodman Diversity Panel (~300 diverse maize lines). These collective analyses identified more than 1000 single nucleotide polymorphisms (SNPs) highly associated with variation in the expression of known and unknown molecular features. Among the analyses performed was GWAS on maize lines with a stem infection of Southern leaf blight (SLB) using the metabolite 10-oxo-11-phytoenoic acid (10-OPEA) as a molecular phenotype. This analysis was used to map to a novel cyclase gene in the fatty acid/oxylipin biosynthesis pathway. Biochemical analyses confirmed that the novel cyclase can produce 10-OPEA and CRISPR/Cas9 targeted mutagenesis genetically affirmed the cyclase’s role in 10-OPEA biosynthesis. Functional characterization of this gene demonstrated a role for this cyclase in conferring resistance to the ear-, stalk-, and root-rot pathogen Fusarium graminearum. In FY2020 ARS researchers in Gainesville, Florida, performed GWAS on Goodman Diversity Panel maize stems infected with Fusarium verticillioides. This 440-sample data set (n=4) yielded 52,000+ unknown molecular features. GWAS, co-expression, and statistical analyses were performed on this data set and more than 100 genetic loci hypothesized to be involved in disease resistance were identified. In FY2021 to narrow down targets related to pathogen resistance, ARS researchers in Gainesville, Florida, performed association analyses and selected the 100 top features significantly correlated to maize lines with low levels of fungal growth. GWAS results from these features led to 12 loci with a total of 36 candidate genes associated with resistance to F. verticillioides. Of particular interest was a highly significant SNP on chromosome 3 near to the fatty-acid biosynthesis gene, 12-oxo-phytodienoic acid reductase 1 (OPR1). To confirm that OPR1 produces the unknown compound associated with variation at this locus and to investigate the significance of this putative fatty acid in maize resistance, CRISPR/Cas9 targeted mutagenesis was performed and the initial characterization of the resultant OPR1 mutants is underway. ARS researchers in Gainesville, Florida, also investigated the impact of heat stress on maize defenses against SLB. Targeted and untargeted metabolomics on maize leaf tissue under heat stress, SLB infection, and the combination of heat and SLB were performed using ultra-high-performance liquid chromatography-high-resolution mass spectrometry. This study revealed significant metabolic responses to both SLB infection and heat stress. Furthermore, combinatorial experiments with heat and fungal inoculation demonstrated that heat stress prior to infection can compromise important disease resistant mechanisms. Specifically, plants exposed to heat prior to inoculation were deficient in the antimicrobial hydroxycinnamic acid, p-coumaric acid. Collectively, these findings demonstrate that heat stress can predispose crops to more severe disease symptoms, underlining the increasing need to investigate defense chemistry in plants under combinatorial stress. Objective 1B, ARS researchers in Gainesville, Florida, used Arabidopsis as a homologous system with which to test the function of a maize gene hypothesized to produce green leaf volatiles. Transgenic Arabidopsis plants constitutively expressing a recombinant version of the maize candidate gene accumulated the green leaf volatile (Z)-3-hexanal, thus confirming the candidate gene’s biochemical function. ARS researchers in Gainesville, Florida, previously isolated six CRISPR/Cas-9 generated loss of function mutants in three farnesyl diphosphate synthase mutants in maize. These genes make farnesyl diphosphate, a precursor to many herbivore-induced sesquiterpene volatiles. The production of these volatiles was measured in the loss of function mutants in response to fall armyworm infestation and no difference was observed compared to wildtype plants. ARS researchers in Gainesville, Florida, therefore crossed the mutant lines to obtain farnesyl diphosphate double mutants with which to test for gene redundancy and continue functional analyses. The impact of flooding on insect induced chemical defenses was completed ahead of schedule in previous years. The impact of other abiotic stresses on insect induced chemical defenses has not yet been started. Objective 2, forward genetic screens using high-copy Mutator transposon lines have been used to successfully identify over 30 mutants with phenotypes indicative of hormone signaling disruptions. Phenotypes include differences in plant growth, leaf pigmentations, leaf development, and spontaneous lesion formation. These mutants have been verified to be heritable and show normal Mendelian segregation, indicating they are being caused by single mutations. Transposon mapping experiments are in progress to identify transposon insertions that co-segregate with mutant phenotypes and thus determine which gene mutations may be responsible for the phenotypes. Isolation of CRISPR/Cas9-generated mutations in more than 50 high-priority target genes involved in hormone signaling continues. Mutations in maize genes responsible for jasmonic acid biosynthesis, including two allene oxide cyclases, three OPDA reductases, and four lipoxygenases, have been isolated as homozygous mutants and full characterization of the mutants is ongoing. Mutations have also been isolated in five abscisic acid hydroxylases responsible for the degradation of the hormone abscisic acid (ABA). These mutant lines are disrupted in normal hormone signaling and are slated for use in biotic, abiotic, and combinatorial stress assays to help understand the roles of the hormones in stress responses. Results from stress assays thus far have shown significant alterations in hormone levels but little effect on stress responses, though tests are ongoing. Progress has also been made in the characterization of maize albescent1 (al1) mutants, showing that the mutants are disrupted in a gene encoding a plastidial alternative oxidase. These mutants, in combination with the previously characterized white seedling 3 (w3) mutants, are being used to help understand the biosynthetic pathways of carotenoids and ABA. Double mutants between al1 and w3 show exaggerated ABA deficient phenotypes that would not be expected if the current understanding of their roles is complete. ARS researchers in Gainesville, Florida, have also uncovered a novel pathway by which Mutator transposons swap internal and external components with one another, potentially allowing them to escape host silencing pathways. These transposon dynamics help explain the strong tendency of Mutator transposons to target the 5’ regions of genes in maize, a well-documented phenomenon that has long perplexed transposon researchers and which has greatly facilitated gene and genome evolution in maize.

1. Identification of corn aroma compounds that alter fall armyworm behavior. Fall armyworm is a major global threat for the production of corn and other crops. It is currently having a devastating impact in sub-Saharan Africa, where small-holder farmers often lose their entire crop to these voracious pests. The fast spread and nocturnal habits of these long-distance flyers mean that rapid detection of these moths is vitally important for their control. ARS scientists from Gainesville, Florida, have identified several odors produced by corn that alter fall armyworm behavior. A number of these identified odors attract fall armyworm and therefore could be used to trap and monitor fall armyworm populations. As part of early warning detection system, they would allow farmers to apply pesticides at the start of a fall armyworm infestation, potentially reducing damage to their crops. Other compounds were identified that can repel fall armyworm. As these compounds are naturally produced by corn, long-term guided breeding strategies could be used to increase their levels, making corn plants less attractive to fall armyworm.

2. Understanding how heat waves can affect crop disease resistance. The joint impact of pests and extreme weather events is a major global agricultural problem. For example, billions of bushels of corn are lost to problems such as fungal infections and extreme temperatures. While scientists moderately understand the effects of individual pests or single types of extreme weather, very little is known about how the combination of these events impact crops. To investigate this, ARS scientists from Gainesville, Florida, exposed corn plants to a simulated heat wave and then infected them with the common corn pathogen, southern leaf blight. The scientists observed that heat wave-exposed corn plants had more disease than plants kept at normal temperatures, and p-coumaric acid, an antibiotic compound produced by corn is involved in this altered disease resistance. Selection by breeders and farmers of corn varieties that maintain high levels of this endogenous antibiotic is therefore important for retaining disease resistance in heat wave prone regions.

Review Publications
Block, A.K.; Mendoza, J.S.; Rowley, A.L.; Stuhl, C.J.; Meagher Jr, R.L. 2021. Approaches for assessing the Impact of Zea mays on the behavior of Spodoptera frugiperda and its parasitoid Cotesia marginiventris. Florida Entomologist. 103:505-513.
Christensen, S.A.; Santana, E.; Alborn, H.T.; Block, A.K.; Chamberlain, C.A. 2021. Metabolomics by UHPLC-HRMS reveals the impact of heat stress on pathogen-elicited immunity in Maize. Metabolomics. 17:6.
Soubeyrand, E., Latimer, S., Bermert, A.C., Keene, S.A., Johnson, T.S., Shin, D., Block, A.K., Colquhoun, T.A., Shaefnner, A.R., Kim, J., Basset, G.J. 2021. 3-O-glycosylation of kaempferol restricts the supply of the benzenoid precursor of ubiquinone (Coenzyme Q) in Arabidopsis thaliana. Phytochemistry. 186,112738.
Plant, S.R.; Irigoyen, S.; Liu, J.; Bedre, R.H.; Christensen, S.A.; Schmelz, E.A.; Sedbrook, J.; Scholthof, K.G.; Mandadi, K.K. 2021. Brachypodium phenylalanine ammonia lyase (PAL) promotes antiviral defenses against Panicum mosaic virus and its satellites. mBio. 12:e03518-20. https//
Marcon, C.; Altrogge, L.; Win Y.N.; Stöcker, T.; Gardiner, J.M.; Portwood, J.L. 2nd; Opitz, N.; Kortz, A.; Baldauf, J.A.; Hunter, C.T.; McCarty, D.R.; Koch, K.E.*; Schoof, H.; Hochholdinger, F. 2020 BonnMu: A sequence-indexed resource of transposon-Induced maize mutations for functional genomics studies. Plant Physiology 184(2):620-631.
Y-S. Park; E.J. Borrego; X. Gao, S.A. Christensen; E. Schmelz; A. Lanubile; D.A. Drab; W. Cody; H. Yan; W-B. Shim; M.V. Kolomiets. 2021. Fusarium verticillioides induces maize-derived ethylene to promote virulence by engaging fungal G-protein signaling. Molecular Plant Microbe Interactions https//
Hunter, C.T. 2021. Considerations for using CRISPR/Cas9 in targeted mutagenesis for functional genetics in plants. Plants. 10(4): 723.
Tahjib-Ul-Arif, M.; Zahan, M.; Karim, M.; Imran, S.; Hunter, C.T.; Islam, M.; Mia, M.; Hannan, M.; Rhaman, M.S.; Hossain, M.; Brestic, M.; Skalicky, M.; Murata, Y. 2021. Citric acid-mediated abiotic stress tolerance in plants. International Journal of Molecular Sciences. 22(13): 7235.
Yactayo Chang, J.P.; Tang, H.V.; Mendoza, J.S.; Christensen, S.A.; Block, A.K. 2020. Plant defense chemicals against insect pests. Agronomy. 10:1156. 2020
Block, A.K.; Xin, Z; and Christensen, S.A. 2020 The 13-lipoxygenase MSD2 and the w-3 fatty acid desaturase MSD3 impact Spodoptera frugiperda resistance in Sorghum. Planta 252:62 https://doi:10.1007/s00425-020-03475-2
Kim, S., Karre, S., Choi, H., Christensen, S.A., Wang, G., Jo, Y., Cho, W., Balint Kurti, P.J. 2021. Analysis of the transcriptomic, metabolomic, and gene regulatory responses to Puccinia sorghi in maize. Molecular Plant Pathology.
Ding, Y.; Murphy, K.M.; Poretsky, E.; Mafu, S.; Yang, B.; Char, S.; Christensen, S.A.; Saldivar, E.; Wu, M.; Wang, Q.; Ji, L.; Schmitz, R.J.; Kremling, K.A.; Buckler, E.S.; Shen, Z.; Briggs, S.P.; Bohlmann, J.; Sher, A.; Castro-Falcon, G.; Hughes, C.C.; Huffaker, A.; Zerbe, P.; Schmelz, E.A. 2020. Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity. Nature Plants. 6(11):1375-1388.
Perez, V.E.; Dai, R.; Bing Bai, B.; Tomiczek, B.; Askey, B.; Zhang, Y.; Ding, Y.; Grenning, A.; Block, A.K.; and Kim, J. 2021 Aldoxime-derived auxin biosynthesis occurs in both Arabidopsis and maize. New Phytologist. https://doi: 10.1111/nph.17447