Location: Chemistry Research
2023 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
This is the final report for the project 6036-11210-001-000D which terminated in March 2023. ARS researchers in Gainesville, Florida, investigated the impact of heat stress on maize defenses against Southern leaf blight (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.
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 confirmed the cyclase’s role in 10-OPEA biosynthesis. Ongoing functional characterization of this gene indicates a potential role for this cyclase in conferring resistance to the ear-, stalk-, and root-rot pathogen Fusarium graminearum.
Gainesville ARS scientists also identified 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 was identified as a location where high levels of anti-fungal chemicals are produced. Mutants with disrupted aleurone formation were found to be more susceptible to fungal infection during germination, and work is ongoing to confirm that this cell layer is important for fungal resistance.
Fall armyworm is a major global threat for the production of maize 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 maize 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. The ARS scientists also found that the production of several of these compounds including methyl salicylate, which is a fall armyworm oviposition attractant, were increased if maize plants were subjected to combined flooding stress and herbivory.
As these compounds are naturally produced by maize, long-term guided breeding strategies could be used to alter their levels, making maize plants less attractive to fall armyworm. To identify genes involved in producing these volatiles, we ran metabolite GWAS on a sweetcorn diversity panel (~350 lines) screening for fall armyworm induced volatiles. This analysis led to the identification of maize terpene synthase 1 (TPS1) as an enzyme involved in the production of the monoterpene volatiles linalool and beta-myrcene. Maize plants containing a loss-of-function mutant of TPS1 were less attractive to fall armyworm, making it a good potential target for molecular breeding.
The combinatorial stress of flooding and fall armyworm infestation in maize was found to increase the resistance of maize to this insect pest. Transcriptomics using RNAseq was used to identify maize genes that are differentially regulated in response to these combinatorial stresses. 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.
We have isolated numerous other characterized mutants for genes with roles in maize biotic stress responses. More than 30 mutants have been isolated from the UniformMu maize population with Mutator transposon insertions in genes with predicted roles in pathogen defense. Additionally, more than 20 CRISPR-derived gene edited mutants in genes involved in metabolism of stress signaling hormones, including jasmonic acid (JA) and abscisic acid (ABA), have been isolated. Finally, more than 30 uncharacterized maize mutants have been identified in forward genetic screens for phenotypes indicative of disrupted hormone signaling. These lines have been tested for heritability and are undergoing hormone analysis.
Mutant phenotyping and metabolite analysis by Gainesville ARS scientists showed that two of the CRISPR edited genes, allene oxide cyclase (AOC) 1 and 2 are central to the biosynthesis of the JA precursor 12-OPDA. Homozygous double mutants for AOC1 and AOC2 show complete feminization of tassels, producing female florets in place of male florets, similar to other JA-deficient maize mutants. The double mutants are highly susceptible to fall armyworm herbivory and SLB infection due to the failure to upregulate JA and JA-reliant defense metabolites in response to biotic stresses. These mutants are being used to determine which defense-related changes to gene expression and metabolism are under the control of the JA signaling pathway.
Co-expression analysis with publicly available RNAseq/microarray data using known phytoalexin biosynthesis genes was used to identify a receptor-like protein kinase in maize associated with the regulation of phytoalexin production. Transposon tagged mutants of this gene were obtained and shown to be compromised in their ability to produce phytoalexins in response to fungal attack. This gene was also shown to be induced in response to fungal infection and was named fungal induced-receptor like protein kinase (FI-RLPK). Furthermore, the loss-of-function mutants in FI-RLPK displayed enhanced susceptibility to the necrotrophic fungal pathogen Cochliobolus heterostrophus and increased resistance to stem inoculation with the hemibiotrophic fungal pathogen Fusarium graminearum, indicating that FI-RLPK is important for fungal recognition and activation of defenses.
ARS researchers used CRISPR approaches to generate loss-of-function mutants in three farnesyl diphosphate synthase (fps) genes in maize. Mutants in fps3 showed significantly reduced production of fungal induced sesquiterpene phytoalexins. Mutants in fps1 showed significant decreases in developmental related compounds including ubiquinone, a vital redox co-factor of mitochondrial electron transport. These mutants were dwarf, with pale leaves and had impaired chlorophyll production. These data indicate that the different fps isoforms play specific roles in metabolism in maize and may aid in circumventing growth-defense tradeoffs.
ARS researchers in Gainesville, Florida conducted metabolic, phylogenetic, and gene expression analyses of JA-dependent defense responses in Setaria viridis, a proposed model plant for C4 grasses, and maize. By doing so, they showed that many of the core chemical defense signaling pathways are maintained between maize and S. viridis, and thus S. viridis would serve as an effective model species for examining core defense pathways. However, many of the downstream defense chemicals that maize produces are absent in S. viridis, and these chemical pathways must be studied in the species of interest directly.
Accomplishments
1. Analysis of corn transposons provides insight into the evolution of these jumping genes. The Mutator (Mu) transposon family are mobile DNA elements “jumping genes” that promote the genetic variation and genome instability necessary for the evolution of plants and animals. In collaboration with the University of Florida, ARS scientists from Gainesville, Florida, have gained new insights into the evolution and behavior of these transposons. Analysis of sequenced corn genomes revealed that separate lineages of Mu transposons exchange components, including genes responsible for transposon movement and function. This finding improves our ability to use these natural mobile elements to study the function of many different genes in corn and facilitate the molecular breeding of climate resilient corn.
Review Publications
Perez, V.C., Dai, R., Tomiczek, B., Mendoza, J.S., Greening, A., Reed, E.S., Vermerris, W., Block, A.K., Kim, J. 2023. Metabolic link between auxin production and specialized metabolites in Sorghum bicolor. Journal of Experimental Botany. 74:364-376. https://doi.org/10.1093/jxb/erac421.
Saldivar, E.V., Ding, Y., Poretsky, E., Bird, S., Block, A.K., Huffaker, A., Schmelz, E.A. 2023. Maize terpene synthase 8 (ZmTPS8) contributes to a complex blend of fungal-elicited antibiotics. Plants. 12(5):1111. https://doi.org/10.3390/plants12051111.
Yactayo Chang, J.P., Hunter III, C.T., Alborn, H.T., Christensen, S.A., Block, A.K. 2022. Production of the green leaf volatile (Z)-3-hexenal by a zea mays hydroperoxide lyase. Plants. 11(17):2201. https://doi.org/10.3390/plants11172201.
Yactayo Chang, J.P., Boehlein, S., Libertini, G., Beiriger, R., Resende, M., Bruton, R.G., Alborn, H.T., Tracy, W.F., Block, A.K. 2022. The impact of post-harvest storage on sweetcorn aroma. Phytochemistry Letters. 52:33-39. https://doi.org/10.1016/j.phytol.2022.09.001.
Tang, H.V., Berryman, D.L., Mendoza, J.S., Yactayo Chang, J.P., Li, Q., Christensen, S.A., Hunter III, C.T., Best, N.B., Soubeyrand, E., Akhtar, T., Basset, G.J., Block, A.K. 2022. Dedicated farnesyl diphosphate synthases circumvent isoprenoid-derived growth-defense tradeoffs in Zea mays. Plant Journal. https://doi.org/10.1111/tpj.15941.
Abraham-Juarez, M.J., Busche, M., Anderson, A.A., Lunde, C., Winders, J.R., Christensen, S.A., Hunter Iii, C.T., Hake, S.C., Brunkard, J.O. 2022. Liguleless narrow and narrow odd dwarf act in overlapping pathways to regulate maize development & physiology. The Plant Journal. https://doi.org/10.1111/tpj.15988.
