Location: Chemistry Research2020 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 has been made on the research project 6036-11210-001-00D by ARS scientists in Gainesville, Florida. For Objective 1A, ARS scientists, at Gainesville, Florida, performed genome wide association studies (GWAS) on several experiments and identified candidate genes for functional analysis. Among the analyses performed was GWAS on our Southern leaf blight stem infection experiment using the concentrations of the metabolite 10-oxo-11-phytoenoic acid (10-OPEA) as a molecular phenotype and mapped to a novel cyclase gene in the fatty acid/oxylipin biosynthesis pathway. Collaborative efforts in biochemistry have confirmed that this novel cyclase is directly involved the biosynthesis of 10-OPEA. CRISPR/Cas9 targeted mutagenesis of this cyclase gene is near completion at the University of Missouri and functional analysis of this gene in fatty acid biosynthesis and plant-microbe interactions will soon be performed. In addition to targeted mutagenesis, ARS scientists, at Gainesville, Florida, have also initiated the use of viral induced gene silencing (VIGS) to study the signaling function of 10-OPEA in programmed cell death using three metacaspases mapped with GWAS (see FY19 progress report) as our targets. ARS scientists, at Gainesville, Florida, also performed untargeted metabolomics on a large diversity panel experiment comprised of 110 maize inbred lines (55 resistant and 55 susceptible) that were infected with the economically important pathogen Fusarium verticillioides. This 440-sample data set (n=4) yielded 52,000+ unknown molecular features. GWAS was performed on this data set and more than 100 loci hypothesized to be involved in disease resistance were identified. Co-expression and statistical analyses have helped us to identify additional gene targets of interest. Objective 1B. ARS scientists, at Gainesville, Florida, have successfully employed CRISPR-based mutagenesis to cause loss-of-function alleles in 34 high-priority target genes involved in chemical defense production or defense signaling. Homozygous lines carrying loss-of-function alleles have been isolated for 12 of the target genes and are being used in phenotypic analyses, biotic, abiotic, and combinatorial stress assays. The remaining targets are undergoing genetic analyses and crossing to isolate mutants in desired combinations and backgrounds. Homozygous lines were obtained for mutants in four genes related to the defense of maize against insect pests and levels of insect induced volatiles determined for two of the four lines and levels of benzoxazinoids for one of the lines. Mutants in farnesyl diphosphate synthases displayed compromised developmental and fungal resistance phenotypes, so the levels of some fungal and developmental related compounds were also measured in these lines. For the CRISPR/Cas9 terpene synthase mutants additional generations were grown to attempt to isolate the homozygous lines and although homozygous plants were identified, these lines had background mutations in additional terpene synthases making them unsuitable for metabolic evaluation at this time. The plants were therefore selected for backcrossing into the wildtype parent to remove these confounding mutations. In addition to the genetics two new methods were developed for this subobjective. These are bioassays to functionally assess the roles of maize volatiles in impacting the behavior of fall armyworm. The first assay is a gas chromatographic-electroantennographic detection method for assessing the ability of fall armyworm moths to perceive specific volatile compounds or blends of compounds. The second assay is an oviposition choice assay which will enable the comparison of mutant and wild type plants for their ability to attract ovipositing fall armyworm female moths. Objective 2. Homozygous lox10 mutant lines were isolated and initial trials on the effect of this mutant phenotype on combinatorial drought and southern leaf blight infestations were performed. No major phenotypes were observed for the mutant in the initial trials. More detailed metabolic phenotyping is planned to assess less obvious effects. ARS scientists, at Gainesville, Florida, research last year demonstrated the hormone salicylic acid was associated with flooding-induced insect resistance in maize. During this research ARS scientists, at Gainesville, Florida, assessed the impact of a salicylic acid receptor mutant on this combinatorial stress response. This year ARS scientists, at Gainesville, Florida, have identified a second allele of the salicylic acid receptor mutant and are in the process of progressing the generations to obtain a homozygous mutant line for further studies. Furthermore, ARS scientists, at Gainesville, Florida, performed RNAseq analysis on maize plants subjected to flooding, herbivory or a combinatorial treatment and identified several genes that showed differential responses to the combinatorial treatments. The forward genetic screen for maize mutants is ahead of schedule with phenotypes indicative of impaired hormone production or defense signaling was conducted on high-copy Mutator maize lines from the UniformMu population. The initial screen identified 56 lines with phenotypes of interest (altered leave blade development, different growth rate, lesion formation, early necrosis, etc.), and confirmatory grow-outs showed 38 of those to have heritable, segregating phenotypes that will be pursued in genetic analyses. Lines carrying these mutants will be planted in the 2020 Fall field to generate material for cosegregation analyses to identify candidate Mutator insertions associated with phenotypes. Examination of CRISPR-generated mutants in maize allene oxide cyclases (AOC1 and AOC2), enzymes involved in the synthesis of jasmonic acid (JA), revealed developmental defects indicative of JA deficiency. However, metabolic analyses showed that double mutants retained approximately 50% of normal JA levels. This was unexpected as genetic analyses show only two copies of AOC in the maize genome and our AOC double mutants should not have active AOC. Ongoing analyses are testing whether the remaining JA may be coming from a source other than AOCs or whether our mutants are not complete loss-of-function alleles. Cloning and isolation of the albescent1 (al1) maize mutant revealed its association with an IMMUTANS-like plastidial alternative oxidase (PTOX) that functions in recycling of plastoquinone-9 (PQ9). Analyses showed that the al1 mutants are severely carotenoid deficient. These mutants are being combined with previously-discovered w3 alleles to better understand the role of PQ9 and PTOX in phytoene desaturase, an early step in carotenoid and carotenoid-dependent secondary metabolite biosynthesis.
1. Flooding leads to increased insect resistance in corn. To achieve optimum yields in crops it is important for farmers to be able to manage the various pests and weather-related problems that their crops will be exposed to. It is usual for crops to be exposed to multiple such problems at the same time and detailed knowledge of the combined effects of these threats can enable farmers to effectively prioritize their management plans. To expand this knowledge base ARS scientists from Gainesville, Florida, examined the effect of flooding on pest resistance in corn. They discovered that flooding increased the resistance of corn to the insect pest fall armyworm and caused changes in defense related compounds in the plants. These findings mean that farmers now can know that treatment for insect pests may be less of a priority in corn fields subjected to flooding.
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