Location: Chemistry Research
2022 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
Progress has been made on the research project 6036-11210-001-000D by ARS scientists at Gainesville, Florida. For Objective 1A, CRISPR/Cas-9 approaches were used to target genes involved in defense signaling and/or defense chemical production. New gene edited mutants were isolated in 5 genes for abscisic acid (ABA) hydroxylases (ABA degradation), 4 genes for 9-cis-epoxycarotenoid dioxygenases (ABA biosynthesis), 3 lipoxygenase genes (jasmonic acid biosynthesis), and 2 hydroperoxide lyases (defense-related volatile production). In addition, transposon insertion alleles have been isolated for 10 high priority target genes from the UniformMu reverse genetics resource. All of these mutants are being progressed through a field genetics pipeline to isolate desired alleles in the needed combinations and in the proper genetic backgrounds for phenotypic testing and functional characterization. They will be added to the growing library of maize mutants (50+) with altered defense hormone metabolism or defense chemical production.
Co-expression analysis with 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. These data can be used to guide breeding of maize with optimized resistance to pathogens of concern to specific growers or to a particular region.
For Objective 1B, previously isolated six CRISPR/Cas-9 generated loss of function mutants in three farnesyl diphosphate synthase (fps) genes 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. However, when these lines were assessed for the production of fungal induced sesquiterpene phytoalexins, mutants in one of the genes, fps3, showed significant reductions in phytoalexin production. Mutants in another gene, fps1, showed significant decreases in developmental related compounds including ubiquinone (CoQ10), 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.
The investigation on the impact of loss of perception of the plant hormone salicylic acid on the combined impact of flooding and insect induced chemical defenses using the homozygous npr1 mutant was completed ahead of schedule in previous years. Further studies were undertaken to determine if the altered volatiles produced during this combined stress response impacted fall armyworm oviposition preferences, but no significant impacts were observed.
For Objective 2, CRISPR-derived mutants in the jasmonic acid (JA) biosynthetic genes allene oxide cyclase (AOC) 1 and 2 were tested for developmental defects and changes to insect susceptibility. Homozygous double mutants for AOC1 and AOC2 shows complete feminization of tassels, producing female florets in place of male florets, similar to other JA-deficient maize mutants. The double mutants were also highly susceptible to fall armyworm herbivory, leading to dramatically faster growth of caterpillars feeding on tissue from the double mutant plants, assumedly because of the disruption in JA signaling and the failure to synthesize anti-feeding defenses (metabolic analyses are ongoing). The AOC1 and AOC2 genes appeared to be completely redundant based on single mutants having no detectable differences from wildtype in either plant morphology or insect bioassays. Tissue was collected from these lines for use in RNAseq experiments to identify downstream components of these JA signaling pathways in maize.
Forward genetic screens to identify and isolate mutants impaired in hormone signaling have successfully identified 30 mutants with various developmental defects from high-copy Mutator transposon lines. These mutants have been checked for Mendelian segregation patterns and heritability and are currently being bulked for co-segregation analyses to identify specific transposon insertions associated with the phenotypes. Tissue for this co-segregation analysis has been collected and grow-outs for phenotypic assays can begin once seeds are harvested from the Spring 2022 maize nursery.
Efforts to explain the unexpected ability of phytoene desaturase (PDS) to function in the absence of plastoquinone-9 (PQ9) in the maize w3 mutant (previously published) we have identified the maize albescent1 (al1) mutant as being caused by mutations in a gene for the maize plastidial alternative oxidase (PTOX). Double mutants between w3 and al1 resulted in a strong viviparous (premature germination) phenotype not seen in either of the single mutants, indicative of a complete loss of ABA. This supports the hypothesis that the maize PTOX plays a role in phytoene desaturation by supplementing PQ9 as a cofactor. It also provides another tool for understanding the roles of carotenoids and ABA in plant defense and development.
Accomplishments
1. Factors underlying the natural chemical defenses of corn against fungal diseases. Corn can actively protect itself from pathogens using a variety of natural chemical defenses. Among these defenses are antimicrobial chemicals called zealexins that protect against diseases such as southern leaf blight and stalk rot. ARS scientists from Gainesville, Florida, have identified a gene family in corn in which specific members produce the chemical ingredients needed for zealexin production, while other members produce the same ingredients for growth and development. This separation of function allows the plants to produce high levels of defense chemicals while at the same time maintaining their growth and yield. These findings reveal how crops can effectively utilize defense chemicals and can be used to guide the breeding of optimized natural pest resistance. (NP301, C3, PS3A)
2. Identification of a corn receptor protein that mediates resistance to fungal pathogens. In order to defend itself from diseases, corn, like many plants, needs to be able to recognize and rapidly respond to specific pathogens. ARS scientists from Gainesville, Florida, have identified a receptor protein from corn that allows it to co-ordinate its defense responses to fungal pathogens such as southern leaf blight and stalk rot. Presence of this protein increases the resistance of corn to southern leaf blight while decreasing its resistance to fusarium stalk rot. These findings can guide corn breeding to prioritize resistance to either one of these diseases depending on growers concerns and predicted disease pressures. (NP301, C3, PS3A)
Review Publications
Latimer, S., Keene, S.A., Berger, A., Bernet, A., Soubeyrand, E., Wright, J., Clarke, C.F., Block, A.K., Colquhoun, T.A., Elowsky, C., Christensen, A., Basset, G.J. 2021. A dedicated flavin-dependent monooxygenase catalyzes the hydroxylation of demethoxyubiquinone into ubiquinone (Coenzyme Q) in Arabidopsis. Journal of Biological Chemistry. 297,101283. https://doi.org/10.1016/j.jbc.2021.101283.
Ul Islam, M., Nupur, J.A., Hunter III, C.T., Sohag, A.A., Sagor, A., Hossain, M., Latef, A., Tahjib-Ul-Arif, M. 2022. Crop improvement and abiotic stress tolerance promoted by Moringa leaf extract. Phyton International Journal of Botany. 91, 1557-1583. https://doi.32604/phyton.2022.021556
Berger, A., Latimer, S., Stutts, L.R., Soubeyrand, E., Block, A.K., Basset, G.J. 2022. Kaempferol as a precursor for ubiquinone (coenzyme Q) biosynthesis: An atypical node between specialized metabolism and primary metabolism. Current Opinion in Plant Biology. 66, 102165. https://doi.org/10.1016/j.pbi.2021.102165.
Cowles, K.N., Block, A.K., Barak, J.D. 2022. Xanthomonas hortorum pv. gardneri TAL effector AvrHah1 is necessary and sufficient for increased persistence of Salmonella enterica on tomato leave. Scientific Reports. 12, 7313. https://doi.org/10.1038/s41598-022-11456-6
Perez, V.C., Dai, R., Block, A.K., Jeongim, K. 2021. Metabolite analysis of Arabidopsis CYP79A2 overexpression lines reveals turnover of benzyl glucosinolate and an additive effect of different aldoximes on phenylpropanoid repression. Plant Signaling and Behavior. 16, 1966586. https://doi.org/10.1080/15592324.2021.1966586.
Ingber, D.A., Christensen, S.A., Alborn, H.T., Hiltpold, I. 2021. Detecting the conspecific: herbivory induced olfactory cues in the fall armyworm (Lepidoptera: Noctuidae). Metabolites. 11, 583. https://doi.org/10.3390/metabo11090583.
Yactayo Chang, J.P., Mendoza, J.S., Willms, S.D., Beck, J.J., Rering, C.C., Block, A.K. 2021. Zea mays volatiles that influence oviposition and feeding behaviors of Spodoptera frugiperda. Journal of Chemical Ecology. 47, 799-809. https://doi.org/10.1007/s10886-021-01302-w.
Block, A.K., Tang, H.V., Hopkins, D., Mendoza, J.S., Solemslie, R.K., Christensen, S.A., Du Toit, L.J. 2021. A maize leucine-rich repeat receptor-like protein kinase mediates responses to fungal attack. Planta. 254, 73. https://doi.org/10.1007/s00425-021-03730-0.
Souza, D., Christensen, S., Wu, K., Lyle, B., Kleckner, K., Darrisaw, C., Shirk, P., Siegfried, B. 2022. RNAi-induced knockdown of white gene in the southern green stink bug (Nezara viridula L.). Scientific Reports. 12, 10396. https://doi.org/10.1038/s41598-022-14620-0.
