Progress 04/01/21 to 03/31/22
Outputs
Target Audience:At invited university and research institute venues our target audience includes undergraduate, graduate, post-doctoral and professors or other researchers. At the Maize Genetics meetings our audience also includes policy experts in agricultural and funding agencies. Research progress our how acidic sesquiterpenoids are regulated, biosynthesized and control maize resistance to biotic stress caused by pathogenic fungi and insects was communicated in 8 significant venues reaching different target audiences as follows: May 2021, invited talk by Dr. Alisa Huffaker: "Exploiting genetic diversity to probe plant responses to herbivore attack." Genetics of maize-microbe interaction working group, virtual online seminar series organized by Dr. Peter Balint-Kurti, NC State University September 2021, invited talk by Dr. Alisa Huffaker: "Mechanisms of grain crop resistance to insect pests." Seminar, Colorado State University Department of Agricultural Biology October 2021, invited talk by Dr. Alisa Huffaker: "Probing mechanisms of plant resistance to pathogens and pests." Seminar, University of Hawaii Department of Tropical Plan and Soil Sciences November 2021, invited talk by Dr. Eric Schmelz "Discovery and disentanglement of maize antibiotic synthesis" Genetics of maize-microbe interaction working group, virtual online seminar series organized by Dr. Peter Balint-Kurti, NC State University February 2022, invited talk by Dr. Alisa Huffaker: "Decoding layered mechanisms regulating innate immunity." Seminar, Max Planck Institute of Chemical Ecology, Jena Germany March 2022, invited talk by Dr. Alisa Huffaker: "Multi-omic approaches for comparative study of innate immune signaling and metabolism." Seminar, Danforth Foundation, St. Louis, MO University of California, San Diego. Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" was run jointly run by Dr. Huffaker and Schmelz during the Spring Quarter 2018, 2019, 2020, 2021 (April 1 to June 8th) and collectively trained more than 100 students for 10 hours per week during the course. In specific laboratory modules the students learned about maize sesquiterpenoid defenses, conducted elicitation experiments, tested for biological activity in highly enriched extracts, analyzed GC/MS data for precursors and products, and in new research conducted forward genetics using association mapping experiments to identify new biosynthetic genes in maize. UCSD Biology Research Opportunity and Orientation for Transfer Students (ROOTS) enrichment program coordinated by Dr. Huffaker and targeted to incoming first generation and/or underrepresented transfer students. Students learned about sesquiterpenoid root defenses and effects on plant microbiome communities. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student in the Huffaker laboratory, Mr. Elly Poretsky has been trained through this project in maize defense, sesquiterpenoid metabolism and the interaction of maize with herbivores and microbes. Mr. Poretsky graduated in June of 2021 and has continued as a postdoctoral researcher on the project. In FY 2021-2022, Dr. Poretsky has focused on (1) proteomics experiments to understand differences underlying variation in terpenoid production between high terpenoid-producing maize roots and low terpenoid producing roots, (2) understanding genetic pathways that upregulate sesquiterpenoid production upon insect attack, (3) delineating the coregulation of sesquiterpenoid defenses with phenylpropanoid defenses and (4) examining microbiome community differences in tps21 knockout plants devoid of selenine-based sesquiterpene defenses and testing the effects of synthetic metabolites on microbial constituents of the microbiome. As a professional development activity, Elly Poretsky developed a module relating to his microbiome work for the ROOTS workshop and was first author on two papers describing his findings. In a classroom setting, the UCSD Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" run jointly run by Dr. Huffaker and Dr. Schmelz trained students for 10 hours per week in plant metabolomics, genetics and disease resistance, students analyzed GC/MS data for terpenoid precursors and products and conducted forward genetics using association mapping experiments to identify a candidate gene transcription factor regulating zealexin biosynthesis. Students were also trained in a classroom setting through the UCSD Biology Research Opportunity and Orientation for Transfer Students (ROOTS) enrichment program coordinated by Dr. Huffaker. ROOTS is an annual summer research experience for incoming first generation and/or underrepresented transfer students prior to their first full quarter at UCSD. Students were trained in basic laboratory techniques, and in the final workshop module assisted with analyzing root metabolite and phenotype data for maize plants knocked out in beta-selinine-derived molecules as compared to wild type. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through four mechanisms. First, as peer-reviewed publications; in the 2021-2022 FY, four manuscripts were published: Murphy KM*, Poretsky E*, Liu H, Mici N, Annika Nyhuis, Bohlmann J, Schmelz EA, Zerbe P, Huffaker A, Bjarnholt N (2022) "Shielding the oil reserves: the scutellum as a source of chemical defenses." Plant Physiology Feb 9:kiac038. doi: 10.1093/plphys/kiac038. Poretsky E, Ruiz M, Ahmadian N, Dressano K, Steinbrenner AD, Schmelz EA, Huffaker A (2021) "Comparative analyses of responses to exogenous and endogenous antiherbivore elicitors enable a forward genetics approach to identify maize gene candidates mediating sensitivity to herbivore-associated molecular patterns." Plant J., 108(5):1295-1316. doi: 10.1111/tpj.15510. Förster C, Handrick V, Ding Y, Nakamura Y, Paetz C, Schneider B, Hughes CC, Castro G, Luck K, Poosapati S, Kunert G, Huffaker A, Gershenzon J, Schmelz EA, Köllner TG (2021) "Biosynthesis and Antifungal Activity of O-methylated Flavonoid Defenses in Maize." Plant Physiology, 188(1):167-190. doi: 10.1093/plphys/kiab496. Ding Y, Northen TR, Khalil A, Huffaker A, Schmelz EA (2021) "Getting back to the grass roots: harnessing specialized metabolites for improved crop stress resilience." Current Opinion in Biotechnology 70, 174-186, https://doi.org/10.1016/j.copbio.2021.05.010 Second, the results were disseminated through six invited talks in the review period: Invited Talks: May 2021, invited talk by Dr. Alisa Huffaker: "Exploiting genetic diversity to probe plant responses to herbivore attack." Genetics of maize-microbe interaction working group, virtual online seminar series organized by Dr. Peter Balint-Kurti, NC State University September 2021, invited talk by Dr. Alisa Huffaker: "Mechanisms of grain crop resistance to insect pests." Seminar, Colorado State University Department of Agricultural Biology October 2021, invited talk by Dr. Alisa Huffaker: "Probing mechanisms of plant resistance to pathogens and pests." Seminar, University of Hawaii Department of Tropical Plan and Soil Sciences November 2021, invited talk by Dr. Eric Schmelz "Discovery and disentanglement of maize antibiotic synthesis" Genetics of maize-microbe interaction working group, virtual online seminar series organized by Dr. Peter Balint-Kurti, NC State University February 2022, invited talk by Dr. Alisa Huffaker: "Decoding layered mechanisms regulating innate immunity." Seminar, Max Planck Institute of Chemical Ecology, Jena Germany March 2022, invited talk by Dr. Alisa Huffaker: "Multi-omic approaches for comparative study of innate immune signaling and metabolism." Seminar, Danforth Foundation, St. Louis, MO Third, results were disseminated through the UCSD Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" run jointly run by Drs. Huffaker and Schmelz (Spring Quarter 2018, 2019, 2020, 2021; April 1 to June 8th) collectively trained more than 100 students for 10 hours per week during the course. In specific laboratory modules the students learned about maize sesquiterpenoid defenses, conduced elicitation experiments, tested for biological activity in highly enriched extracts, analyzed GC/MS data for precursors and products, and in new research conducted forward genetics using association mapping experiments to identify candidate genes underlying variation in metabolite production. Fourth, results were disseminated through the new UCSD Biology Research Opportunity and Orientation for Transfer Students (ROOTS) enrichment program coordinated by Dr. Huffaker and targeted to incoming first generation and/or underrepresented transfer students. Students learned about sesquiterpenoid root defenses and effects on plant microbiome communities. What do you plan to do during the next reporting period to accomplish the goals?This period our microbiome analyses confirmed a dominant role for TPS21-derived metabolites in shaping the soil community, as there were large differences in rhizosphere community composition and diversity in TPS21 knockouts as compared to W22 wild type, including significant differences in the fungal genera Fusarium and Trichoderma. To build on these findings, we will continue extracting large quantities of beta-selenine-derived defenses from maize tissues and will use these for testing the direct effects on maize-associated microbes in these genera on Diabrotica, and on other root-associated organisms. We will examine the effects of the synthetic zealexin B1 we obtained this period on Fusarium, Diabrotica and other root-associated organisms. We are preparing a manuscript describing the new family of beta-selenine derived metabolites that will incorporate the microbiome and bioassay data to be performed and to be submitted prior to the end of the next period.
Impacts
What was accomplished under these goals?
1) Comprehensive metabolic profiling and genetic mapping to identify unstudied root defenses and relevant biosynthetic genes for characterization We previously determined that defense metabolite production for all maize lines is increased when plants are grown in field grown soil rather than potting mix, indicating an environmental factor that is likely due to microbes present in the soil. Moreover, there is a high degree of variation in the extent of metabolites produced among lines, and the variety MoG was selected as a line that consistently produces very large quantities of defense metabolites in field soils. In this reporting period, we examined the molecular underpinnings of high levels of terpenoid production in field-grown roots by using proteomic profiling of roots grown in field soil for approximately 8 weeks, comparing high-and low-terpenoid (MoG versus B73) producing lines. As we are analyzing the data resulting from this experiment through network analysis of proteins associated with high levels of terpenoid production, we have obtained insights into regulation that improve our understanding of defense metabolism and are likely to aid future efforts to engineer increased metabolite production. (1) Importantly, although sesquiterpenoids are generally associated with precursor produced through the MVA pathway, our data indicates that MEP activation is required for sesquiterpenoid defense chemical production. When attempting to engineer plants with increased sesquiterpenoid defense metabolites in the future, increasing substrate availability through the MEP pathway rather than the MVA pathway may be critical. (2) Our proteomics analysis also confirmed a tight linkage between production of sesquiterpenoid defenses and flavonoid defenses, with proteins for both pathways increased in high-terpenoid producing lines, and the FOMT2 and FOMT4 genes tightly linked in network analyses with sesquiterpenoid biosynthetic genes. This corresponded with collaborative work we completed and published in this period, namely the identification of of xilonenins as fungal-inducible flavonoid phytoalexins that inhibit growth of both Fusarium graminearum and Fusarium verticilioides. In addition to demonstrating the anti-Fusarium activity of xilonenins, we identified the O-methyl transferases FOMT2 and FOMT4 as key biosynthetic enzymes for their production (Forster et al.). To better understand maize underground defense metabolism, especially in the early seedling phase when the plant is most vulnerable to Fusarium-mediated damping off, we analyzed the network of maize defense pathways in scutella. As the oil reserve that provides needed energy to the seedling, the scutellum is vulnerable to invasion by pathogens given that it is a rich food source. We found that essentially all known maize chemical defenses that are fungal-inducible in other tissues are highly enriched in the scutellum of developing seedlings. In addition to providing a tool for further metabolic discovery, this finding is likely to help uncover regulatory commonalities between metabolic families. Dr. Elly Poretsy, funded through this project, was co-first author on this work. Dr. Poretsky's also completed a project aimed at understanding regulation of maize sesquiterpenoid defenses against herbivores through publication of a manuscript describing a receptor protein that is specifically required for foliar volatile sesquiterpene production after herbivory, but not for sesquiterpenoid phytoalexins (Portesky et al.). 2) Bioassays of natural and or engineered mutants lacking these specialized metabolites to assess the growth and survival of all three interacting organisms in controlled additive experiments. In the previous period we found that the TPS21-produced β-selenine-derived family of metabolites is much larger than we had previously realized, and is a dominant component of the terpenoids produced by field-grown maize plants. To better understand the role of this metabolite family in shaping soil communities, we expanded our analysis of the soil and root-associated microbiome to TPS21 knock-out lines. Our microbiome analyses confirmed a dominant role for TPS21-derived metabolites in shaping the soil community, as there were large differences in rhizosphere community composition and diversity in TPS21 knockouts as compared to W22 wild type. Fungal genera which differed in relative composition included Fusarium and Trichoderma, whereas the bacterial genus Bradyrhizobium was strongly affected by the presence of beta-selenine metabolites. To build on these findings, we are extracting large quantities of beta-selinine-derived defenses from maize tissues to use for testing direct effects on maize-associated microbes in these genera on Diabrotica, and on other root-associated organisms. In the last period we found that zealexin B1 is one of the predominant metabolites produced by roots in field soil and that plants knocked out in zealexin production have differences in both soil- and root-associated microbial communities. In this period we have been working with a collaborator to synthesize zealexin B1 so that the direct effects of this metabolite on Fusarium, Diabrotica and other root-associated organisms can be studied. We just received the synthetic zealexin B1 and will be testing it in the upcoming period. 3) Creation of novel maize metabolic networks using transgenic 'new-to-maize' genes to enhance resistance to pest complexes (western corn rootworm-Fusarium). As detailed last period, the metabolic complexity revealed by metabolite profiling and forward genetics via association mapping demonstrates that we are not yet able to predict metabolite products that will be produced if given genes are heterologously expressed in maize. For these reasons our predominant focus for Specific Objective 3 continue to focus on defining conditions that consistently drive high levels of sesquiterpenoid production in asymptomatic maize roots. Our studies of genome by environment interactions in this period and network analysis of coexpressed proteins in high terpene producing vs. low producing lines are providing a needed starting point for better understanding factors affecting terpene production.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Murphy KM*, Poretsky E*, Liu H, Mici N, Annika Nyhuis, Bohlmann J, Schmelz EA, Zerbe P, Huffaker A, Bjarnholt N (2022) Shielding the oil reserves: the scutellum as a source of chemical defenses. Plant Physiology Feb 9:kiac038. doi: 10.1093/plphys/kiac038.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Poretsky E, Ruiz M, Ahmadian N, Dressano K, Steinbrenner AD, Schmelz EA, Huffaker A (2021) Comparative analyses of responses to exogenous and endogenous antiherbivore elicitors enable a forward genetics approach to identify maize gene candidates mediating sensitivity to herbivore-associated molecular patterns. Plant J., 108(5):1295-1316. doi: 10.1111/tpj.15510.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
F�rster C, Handrick V, Ding Y, Nakamura Y, Paetz C, Schneider B, Hughes CC, Castro G, Luck K, Poosapati S, Kunert G, Huffaker A, Gershenzon J, Schmelz EA, K�llner TG (2021) Biosynthesis and Antifungal Activity of O-methylated Flavonoid Defenses in Maize. Plant Physiology, 188(1):167-190. doi: 10.1093/plphys/kiab496.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Ding Y, Northen TR, Khalil A, Huffaker A, Schmelz EA (2021) Getting back to the grass roots: harnessing specialized metabolites for improved crop stress resilience. Current Opinion in Biotechnology 70, 174-186, https://doi.org/10.1016/j.copbio.2021.05.010
|
Progress 04/01/20 to 03/31/21
Outputs
Target Audience:At invited university and research institute venues our target audience includes undergraduate, graduate, post-doctoral and professors or other researchers. The Cold Spring Harbor conference has a similar target audience. At the American Society of Plant Biologists and the Maize Genetics meetings our audience also includes policy experts in agricultural and funding agencies. Research progress our how acidic sesquiterpenoids are regulated, biosynthesized and control maize resistance to biotic stress caused by pathogenic fungi and insects was communicated in 7 significant venues reaching different target audiences as follows: August 2020, invited talk by Dr. Alisa Huffaker "Genetic and biochemical deconvolution of pathways controlling maize immunity." Specialized Metabolites Minisymposium, Annual Conference of the American Society of Plant Biologists October 2020, invited talk by Dr. Alisa Huffaker "Decoding layered mechanisms regulating plant innate immunity." Seminar, University of Massachusetts, Amherst Department of Biology February 2021, invited talk by Dr. Eric Schmelz "Untangling plant defense strategies against herbivore attackers." Seminar, Max Planck Institute for Chemical Ecology (Jena, Germany) March 2021, invited talk by Mr. Elly Poretsky " Uncovering the Genetic Basis of Maize Sensitivity to June 2020 Poster presented by Mr. Elly Poretsky: Poretsky E, Huffaker A. "mutRank: An R Shiny web-application for Mutual Rank-based coexpression analysis combined with tools for gene candidate prioritization" Maize Genetics Conference. December 2020, Poretsky E, Schmelz E, Huffaker A. "syntenyZ: BLAST-based, annotation-independent, targeted comparative genomic analyses of syntenic regions in the maize NAM parents" Plant Genomes, Systems Biology and Engineering. University of California, San Diego. Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" was run jointly run by Dr. Huffaker and Schmelz during the Spring Quarter 2018, 2019, 2020 (April 1 to June 8th) and collectively trained more than 100 students for 10 hours per week during the course. In specific laboratory modules the students learned about maize sesquiterpenoid defenses, conducted elicitation experiments, tested for biological activity in highly enriched extracts, analyzed GC/MS data for precursors and products, and in new research conducted forward genetics using association mapping experiments to identify new biosynthetic genes in maize. Changes/Problems:Because our laboratories were closed for approximately 6 months between March 2020 and August 2020, and both PIs were home as full-time primary care providers for preschool-aged offspring, research progress was hampered. Additionally, insect bioassays requireing collaborators were impossible in this time. What opportunities for training and professional development has the project provided?PhD student Mr. Elly Poretsky has been trained through this project in maize defense, sesquiterpenoid metabolism and the interaction of maize with herbivores and microbes. With training and mentorship from Drs. Huffaker and Schmelz, Mr. Poretsky has focused on 1) Continued development of peer-reviewed public bioinformatic tools to help researchers connect genes to phenotypes and application to demonstrate and untangle biosynthetic and regulatory pathways in maize that control biotic stress resistance, 2) expanding our understanding of the beta selinene family of terpenoid root defenses through isolation of new metabolites and mapping to candidate biosynthetic enzymes, 3) examining the effects of endogenous field microbes on production of sesquiterpenoid defenses by roots for all maize NAM parent lines. Together these activities have laid the foundation for better understanding of maize innate immunity contributing to resistance against Diabrotica larvae and pathogens in field soil. Mr. Poretsky presented posters about his work developing bioinformatic tools and how they were leveraged to better understand maize responses to biotic stress at the Cold Spring Harbor Plant Genomes, Systems Biology and Engineering Conference (syntenyZ: BLAST-based, annotation-independent, targeted comparative genomic analyses of syntenic regions in the maize NAM parents) and the Maize Genetics Conference in 2020 (mutRank: An R Shiny web-application for Mutual Rank-based coexpression analysis combined with tools for gene candidate prioritization). As a professional development activity, undergraduate researcher Mr. Miguel Ruiz presented a poster at the SACNAS national conference about his work assisting Mr. Poretsky in characterizing regulatory mechanisms of maize anti-herbivore sesquiterpenoid defense responses. In a classroom setting, the UCSD Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" run jointly run by Dr. Huffaker and Dr. Schmelz trained students for 10 hours per week in plant metabolomics, genetics and disease resistance, students analyzed GC/MS data for terpenoid precursors and products and conducted forward genetics using association mapping experiments to identify candidate genes underlying variation in metabolite production. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through three mechanisms. First, as peer-reviewed publications; in the 2020-2021 FY, four manuscripts were published: Ding Y, Weckwerth PR, Poretsky E, Murphy KM, Sims J, Saldivar E, Christensen SA, Char SN, Yang B, Tong AD, Shen Z, Kremling KA, Buckler ES, Kono T, Nelson DR, Bohlmann J, Bakker MG, Vaughan MM, Khalil AS, Betsiashvili M, Dressano K, Kollner TG, Briggs SP, Zerbe P, Schmelz EA, Huffaker A (2020) "Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity." Nature Plants 6, 1375-1388, doi:10.1038/s41477-020-00787-9 Poretsky E, Dressano K, Weckwerth PR, Ruiz M, Char SN, Shi D, Abagyan R, Yang B Huffaker A (2020) "Differential activities of maize Plant Elicitor Peptides as mediators of immune signaling and herbivore resistance" Plant J. 104, 1582-1602, doi:10.1111/tpj.15022. Poretsky E, Huffaker A (2020) "MutRank: an R shiny web-application for exploratory targeted mutual rank-based coexpression analyses integrated with user-provided supporting information. PeerJ 8, 16, doi:10.7717/peerj.10264. Yajima A, Shimura M, Saito T, Katsuta R, Ishigami K, Huffaker A, & Schmelz EA (2021) "Chemoenzymatic synthesis and absolute configuration of zealexin A1, a sesquiterpenoid phytoalexin from Zea mays." European Journal of Organic Chemistry (7), 1174-1178, https://doi.org/10.1002/ejoc.202001596 Second, the results were disseminated through four invited talks and two poster presentations in the review period: Invited Talks: August 2020, invited talk by Dr. Alisa Huffaker "Genetic and biochemical deconvolution of pathways controlling maize immunity." Specialized Metabolites Minisymposium, Annual Conference of the American Society of Plant Biologists October 2020, invited talk by Dr. Alisa Huffaker "Decoding layered mechanisms regulating plant innate immunity." Seminar, University of Massachusetts, Amherst Department of Biology February 2021, invited talk by Dr. Eric Schmelz "Untangling plant defense strategies against herbivore attackers." Seminar, Max Planck Institute for Chemical Ecology (Jena, Germany) March 2021, invited talk by Mr. Elly Poretsky " Uncovering the Genetic Basis of Maize Sensitivity to Herbivore-Associated Fatty-acid Amino-acid Conjugates." Maize Genetic Conference Poster Presentations: June 2020 Poster presented by Mr. Elly Poretsky: Poretsky E, Huffaker A. "mutRank: An R Shiny web-application for Mutual Rank-based coexpression analysis combined with tools for gene candidate prioritization" Maize Genetics Conference. December 2020, Poretsky E, Schmelz E, Huffaker A. "syntenyZ: BLAST-based, annotation-independent, targeted comparative genomic analyses of syntenic regions in the maize NAM parents" Plant Genomes, Systems Biology and Engineering. Third, the UCSD Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" run jointly run by Dr. Huffaker and Schmelz (Spring Quarter 2018, 2019, 2020, 2021; April 1 to June 8th) collectively trained more than 100 students for 10 hours per week during the course. In specific laboratory modules the students learned about maize sesquiterpenoid defenses, conduced elicitation experiments, tested for biological activity in highly enriched extracts, analyzed GC/MS data for precursors and products, and in new research conducted forward genetics using association mapping experiments to identify candidate genes underlying variation in metabolite production. What do you plan to do during the next reporting period to accomplish the goals?As described, we've determined that defense metabolite production for all maize lines is increased when plants are grown in field grown soil rather than potting mix, indicating an environmental factor that is likely due to microbes present in the soil. Moreover, some lines produce much more of these metabolites than others. In the next reporting period, we will examine the molecular underpinnings of high levels of terpenoid production in field-grown roots. To better understand this process, we will use proteomic profiling of roots grown in field soil for approximately 8 weeks, comparing high-and low-terpenoid producing lines. This information will be used for network analysis of proteins associated with high levels of terpenoid production and generate candidates for future efforts to engineer increased metabolite production. In this period we also found that zealexin B1 is one of the predominant metabolites produced by roots in field soil and that plants knocked out in zealexin production have differences in both soil- and root-associated microbial communities. We will be following up on these findings by working with a collaborator to synthesize zealexin B1, so that the direct effects of this metabolite on Fusarium, Diabrotica and other root-associated organisms can be studied. We also found in this period that the TPS21-produced β-selinine-derived family of metabolites is much larger than we had previously realized and is also a dominant component of the terpenoids produced by field-grown maize plants. To better understand the role of this metabolite family in shaping soil communities, we will expand our analysis of the soil and root-associated microbiome to TPS21 knock-out lines.
Impacts
What was accomplished under these goals?
Objective 1) Analyses of maize roots grown in field soils reveals numerous sesquiterpenoids not previously known in maize. Maize terpene synthase 21 (ZmTPS21) encodes a product specific β-selinene synthase that also yields smaller amounts of α-selinene. Functional ZmTPS21 enzyme is required for the presence of α/β-selinene, α/β -costol and α/β -costic acid. Using heterologous enzyme expression assay in tobacco (Nicotiana benthamiana) we demonstrated that the maize cytochrome P450 (CYP) enzyme ZmCYP71Z19 is sufficient to oxidize β-selinene to β-costic acid. In contrast, related enzymes ZmCYP71Z18 and ZmCYP71Z16 did not display activity on β-selinene (Ding et al., 2020). Our unpublished research on roots of diverse maize germplasm revealed greater complexity and diversity of a/b-selinene derived phytoalexins (termed SX) than previously appreciated. The current SX family is now known to contain more than a dozen family members, including diols and additionally modified acids. Root metabolite association studies in the Goodman diversity panel and the Intermated B73 x Mo17 population (IBM) support SX family genetic connections to ZmTPS21 but also show some SX members as being additionally linked to the zealexin (Zx) biosynthetic genes ZmCYP81A37 (Zx8), ZmCYP81A38 (Zx9) and ZmCYP81A39 (Zx10). These findings expand upon knowledge of ZX biosynthesis where enzyme substrate promiscuity uses diverse substrates to yield complex antibiotic blend: beyond established enzyme promiscuity in the ZmCYP71Z family, we find further genetic evidence for highly-specific selinene pathway precursors interacting with late pathway Zx biosynthetic enzymes, thus identifying a distinct new node of sesquiterpenoid pathway interconnections. Association mapping is powerful for connecting genes to metabolites and ultimately phenotypic traits, but requires measurable genetic variation in the examined populations. An independent approach that does not require genetic variation is co-expression analysis of genes, proteins, metabolites or other combinations of traits. To streamline analyses of transcriptome co-expression analyses, we created a public R shiny web-application for exploratory targeted Mutual Rank-based coexpression analyses termed MutRank (Poretsky and Huffaker, 2020). In this paper, we used a large terpenoid antibiotic pathway and a public transcriptomic dataset to demonstrate that the entire maize biosynthetic pathway could be correctly predicted using an unbiased and non-targeted approach simply requiring knowledge of a single early pathway gene. The same tool powerfully aggregates metabolite families. Simple bioinformatic tools for biologists are needed to make rapid use of large omic datasets to provide candidates for efficient hypothesis testing. Objective 2) The successful design of complex biotic interaction experiment requires in depth knowledge of parameters that can be routinely controlled. To better understand Genotype (G) x Environment (E) interactions that result in significant maize terpenoid levels in asymptomatic roots we grew all Nested Association Mapping (NAM) parent inbred lines in peat-moss based potting soil for 21 days and then added 1 cup of either new potting soil or of Field Soil (F.S.) to the bottom of the pot. 20 days later (day 41), replicated roots (n=3) of each treatment where photographed and harvested for metabolite analyses. On average across all inbreds, ZX levels increased 10.5-fold following the addition of F.S. In nearly all inbred root samples ZX levels increased with F.S. in the absence of any visible disease symptoms. ZX levels following F.S. interactions in P39 and Ki11 roots were approximately 50 mg/g FW, and separate experiments with 7-week old inbred lines using 50% F.S. mixtures resulted in ZX levels of over 300 mg/g FW. Thus, significant progress was made in defining specific germplasm and field relevant growth conditions (G x E interactions) to access maize root biochemistry interactions with WCR predicted to have a significant role in the outcome. Diabrotica spp bioassays with maize defense metabolites require considerable pure material for highly replicated studies at different examined doses. While abundant in the roots of 40-60 day old field grown maize roots, non-volatile maize terpenoids remain challenging to isolate and purify in high quantities. To overcome these research obstacles, we partnered with natural product synthetic chemist. Dr. Arata Yajima (Tokyo University of Agriculture; Department of Chemistry for Life Sciences and Agriculture) to devise a synthetic strategy to efficiently produce ZA1 which is a significant root metabolite (Yajima et al., 2021). This advance now makes it possible to examine ZA1 in WCR bioassays and critically assess insect feeding deterrence and growth rates. To consider sesquiterpenoid regulation and interactions with herbivores, we worked to fully define and give context to maize Plant Elicitor Peptides (ZmPeps) and the cognate receptors (ZmPEPR1 and 2). ZmPeps are present as a 7 member gene family and display specific activities defined by the relative magnitude of their immune response outputs. Requirements for functional ZmPEPR1 receptors were demonstrated with the creation of targeted CRISPR/Cas9 maize mutants. ZmPep3 fails to elicit sesquiterpenes in Zmpepr1 mutants but does not fully abolish the response in Zmpepr2 mutants. In contrast both Zmpepr1 and Zmpepr2 are significantly impaired in ZmPep3 elicited defenses against beet armyworm larvae (Spodoptera exigua) as determined by insect growth bioassays (Poretsky et al., 2020) Objective 3) To complete the initial core ZX biosynthetic pathway (Ding et al., 2020) we redesigned our metabolite extraction and analytical protocols to entirely avoid the use of plastics, and to remove excess derivatization agent by volatilization instead of chemical neutralization. With these changes, we now routinely see not only all known ZX, but additional putative zealexins, a much larger family of ZmTps21 selinene pathway products, and additional products from separate pathways such as terpene synthase (TPS) 8, Tps9, Tps17, kaurene synthase like (KSL) 2, Ksl4 and Ksl1. In nearly all cases Zx5 (ZmCYP71Z19), Zx6 (ZmCYP71Z18) and Zx7 (ZmCYP71Z16) are highly implicated with overlapping/redundant roles. However, mapping efforts in field roots also implicates Zx7 (ZmCYP71Z16) as having specific highly specialized roles in the cleavage of carbons to yield modified nor- and dinorterpenoids (preliminary data). As noted in Objective 1, the expanded maize selinene pathway contains members that map to both ZmTPS21 and a chromosome 1 gene cluster that contains the CYP81A family members Zx8, Zx9, Zx10. This data strongly implicates distinct pathways diverging and reconverging on different nodes which now exceeds the complexity initially described in our accepted hourglass shaped biosynthetic pathway model (Ding et al. 2020) Collectively our maize root metabolite profiling efforts now track over 100 known and candidate terpenoid analytes, which in some germplasm span 6-7 distinct core pathways with more than 10 different hydrocarbon precursors. This complexity demonstrates that we are not yet able to predict metabolite products that would be produced if given genes are heterologously expressed in maize. Thus, our predominant focus for Objective 3 is defining conditions that consistently drive high levels of sesquiterpenoid production in asymptomatic maize roots. These data will be essential to ensure sufficient pathway precursor supply to engineered pathway enzymes for meaningful pathway production levels. A second aspect of success in Objective 3 is understanding pathway enzymes, substrate promiscuity, and interaction between pathways. Real progress is being made, but expensive and higher risk transgenic engineering approaches are not yet feasible.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Ding Y, Weckwerth PR, Poretsky E, Murphy KM, Sims J, Saldivar E, Christensen SA, Char SN, Yang B, Tong AD, Shen Z, Kremling KA, Buckler ES, Kono T, Nelson DR, Bohlmann J, Bakker MG, Vaughan MM, Khalil AS, Betsiashvili M, Dressano K, Kollner TG, Briggs SP, Zerbe P, Schmelz EA, Huffaker A (2020) Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity. Nature Plants 6, 1375-1388, doi:10.1038/s41477-020-00787-9
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Poretsky E, Dressano K, Weckwerth PR, Ruiz M, Char SN, Shi D, Abagyan R, Yang B Huffaker A (2020) Differential activities of maize Plant Elicitor Peptides as mediators of immune signaling and herbivore resistance Plant J. 104, 1582-1602, doi:10.1111/tpj.15022.
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Poretsky E, Huffaker A (2020) MutRank: an R shiny web-application for exploratory targeted mutual rank-based coexpression analyses integrated with user-provided supporting information. PeerJ 8, 16, doi:10.7717/peerj.10264.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Yajima A, Shimura M, Saito T, Katsuta R, Ishigami K, Huffaker A, & Schmelz EA (2021) Chemoenzymatic synthesis and absolute configuration of zealexin A1, a sesquiterpenoid phytoalexin from Zea mays. European Journal of Organic Chemistry (7), 1174-1178, https://doi.org/10.1002/ejoc.202001596
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Progress 04/01/19 to 03/31/20
Outputs
Target Audience:At invited university venues our target audience includes, undergraduate, graduate, post-doctorial and scientist level students and researchers. At the Maize Genetics meeting and the National Corn Growers Association meetings, our audience includes farmers and policy experts in agricultural and funding agencies. At the National Association of Biology Teachers meeting our audience is high school level educators and teachers who seek in incorporate new materials and ideas into their curriculum. Research progress our how acidic sesquiterpenoids are regulated, biosynthesized and control maize resistance to biotic stress caused by pathogenic fungi and root feeding insects was communicated in 6 significant venues reaching different target audiences as follows: Invited talk Dr. Yezhang Ding, Evan Saldivar, Elly Poretsky, Dr. Alisa Huffaker, Dr. Eric Schmelz talk " Convergent evolution on terpenoid metabolic pathways contributes to the protection of diverse crop genera" Maize Genetics Meeting (March 12-15) Kailua-Kona, Hawaii (cancelled at the last minute due to COVID-19 shutdown) February 2020 invited talk by Dr. Alisa Huffaker "Combining association mapping and multi-omic approaches to identify core maize defense pathways." Seminar, Oregon State University Department of Botany and Plant Pathology, Corvallis, OR February 2020 invited talk by Dr. Alisa Huffaker "Identifying components of Panicoid defense responses using association mapping." Seminar, Texas A&M University Department of Entomology, College Station, TX January 2020 invited talk by Dr. Alisa Huffaker "Integrated approaches for defining plant defense pathways." Clayton-Person Lecture, University of British Columbia Department of Botany, Vancouver, Canada August 2019 invited talk by Dr. Alisa Huffaker "Regulation of innate immune responses through post-translational modification of nucleic acid-binding proteins." Biotic Stress Minisymposium, Annual Conference of the American Society of Plant Biologists, San Jose, CA January 2020 invited talk by Dr. Schmelz "Multiple genes recruited from hormone pathways partition maize terpenoid defences" Clayton Person Honorary Lecture, Department of Botany, University of British Columbia, Canada University of California, San Diego. Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" was run jointly run by Dr. Huffaker and Schmelz during the Spring Quarter 2018, 2019, 2020 (April 1 to June 8th) and collectively trained 80 students for 10 hours per week during the course. In specific laboratory modules the students learned about maize sesquiterpenoid defenses, conducted elicitation experiments, tested for biological activity in highly enriched extracts, analyzed GC/MS data for precursors and products, and in new research conducted forward genetics using association mapping experiments to identify new biosynthetic genes in maize. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project has enabled material support for the UCSD Masters student Ahmed Khalil co-supervised by Dr. Schmelz and Dr. Huffaker. Ahmed focused on understanding defense metabolism in the roots of mature (> 30 days after planting) field grown maize plants. Ahmed discovered that unlike the commonly studied B73 inbred, other maize inbreds (such as Mo17 and MoG) could have extremely high levels of root terpenoids (approaching 1% wet weight). In pursuit of identities of highly abundant yet unknown maize root metabolites, Ahmed first purified a metabolite with a molecular weight of 278 Daltons that was predictably related to the methyl ester of ZA9. Surprisingly it was a dolabradiene-derived dinor-diterpenoid (18 carbons). Related purifications identified further related novel diterpenoids and a selinene-diol derivative not previously witnessed in maize. The title of Ahmed Khalil's masters thesis defense (June 1st, 2020) was "Discovery of novel root specific maize dolabralexin defenses: an expanding family of antibiotics", and included genetic mapping efforts to characterize sesquiterpenoid and diterpenoid defense genes present in maize roots. Ahmed routinely participated in joint Huffaker and Schmelz lab meetings and joint meetings with collaborators on maize terpenoids. Ahmed Khalil gave an undergraduate honors thesis talk in 2019 at the UCSD Undergraduate Research Showcase with a poster and oral presentation entitled "Cross leveraging maize and sorghum to understand biochemical layers of immunity". Unfortunately the 2020 final master thesis Research Showcase was cancelled due to COVID-19. In August 2019 the primary funds for the project were allocated to the PhD student Elly Poretsky who has been involved in maize defense and sesquiterpenoid questions since starting in the Huffaker Lab. With training and mentorship from Dr. Huffaker, Elly has focused on 1) the development of peer-reviewed public bioinformatic tools to help researchers connect genes to phenotypes (a public Mutual Rank coexpression analysis tool is near submission), 2) applying developed tools to demonstrate and untangle biosynthetic pathways in maize that control biotic stress resistance and 3) advance maize germplasm (zx1 zx2 zx3 zx4 quadruple mutant) to unambiguously prove that zealexins are large component of maize innate immunity enabling plants to withstand the combined attack of Diabrotica larvae and pathogens in field soil. As a professional development activity, Elly Poretsky attended and presented a poster at the 2019 UCSD Division of Biological Sciences-Salk Institute Annual Retreat (Lake Arrowhead, CA Sept 2019) and at the 2020 Maize Genetics annual meeting (virtually). Elly Poretsky also participated in the First Virtual Maize Annotation Jamboree (March 10-12th, 2020) lead by Dr. Doreen Ware, is featured on the website (https://news.gramene.org/2020_VMAJ) and maintains active membership in the ongoing effort. As a professional development activity, undergraduate researcher Evan Saldivar presented a poster at ASPB (August 2019) entitled "Leveraging cross-species defense pathways to increase metabolic diversity and crop stress resilience (0500-069)" to describe the progress on maize and the pursuit of zingiberene acids. To further his professional development in the plant sciences and mechanisms of biotic stress resistance, Evan Saldivar has joined the PhD program at Stanford (July 2020). How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through 4 mechanisms. First is the free and openly available submission of our discoveries to BioRxiv such that any individual who might lack access to a specific journal can still obtain the information provided an internet connection is available.https://www.biorxiv.org/content/10.1101/2020.03.04.977355v1 To date the material has been examined 1,083 times. Second, the results have been examined by 4 peer reviewers and the revision is now nearly complete at the journal Nature Plants. This process results in the critical evaluation of results. Third, the following invited talks are listed of the review period: Invited talk Dr. Yezhang Ding, Evan Saldivar, Elly Poretsky, Dr. Alisa Huffaker, Dr. Eric Schmelz talk " Convergent evolution on terpenoid metabolic pathways contributes to the protection of diverse crop genera" Maize Genetics Meeting (March 12-15) Kailua-Kona, Hawaii (cancelled at the last minute due to COVID-19 shutdown) February 2020 invited talk by Dr. Alisa Huffaker "Combining association mapping and multi-omic approaches to identify core maize defense pathways." Seminar, Oregon State University Department of Botany and Plant Pathology, Corvallis, OR February 2020 invited talk by Dr. Alisa Huffaker "Identifying components of Panicoid defense responses using association mapping." Seminar, Texas A&M University Department of Entomology, College Station, TX January 2020 invited talk by Dr. Alisa Huffaker "Integrated approaches for defining plant defense pathways." Clayton-Person Lecture, University of British Columbia Department of Botany, Vancouver, Canada August 2019 invited talk by Dr. Alisa Huffaker "Regulation of innate immune responses through post-translational modification of nucleic acid-binding proteins." Biotic Stress Minisymposium, Annual Conference of the American Society of Plant Biologists, San Jose, CA January 2020 invited talk by Dr. Schmelz "Multiple genes recruited from hormone pathways partition maize terpenoid defences" Clayton Person Honorary Lecture, Department of Botany, University of British Columbia, Canada Fourth, the UCSD Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" run jointly run by Dr. Huffaker and Schmelz (Spring Quarter 2018, 2019, 2020; April 1 to June 8th) collectively trained 80 students for 10 hours per week during the course. In specific laboratory modules the students learned about maize sesquiterpenoid defenses, conduced elicitation experiments, tested for biological activity in highly enriched extracts, analyzed GC/MS data for precursors and products, and in new research conducted forward genetics using association mapping experiments to identify new biosynthetic genes in maize. What do you plan to do during the next reporting period to accomplish the goals?Our stated goal is to discover maize biochemical pathways that regulate complex herbivore-root-fungus (Diabrotica-maize-Fusarium) interactions and use defined mutants to empirically demonstrate ecological roles and target the restoration of resistance by engineering novel biochemical networks that suppress pest pressures. In the next reporting period: We will 1) finalize the generation of sufficient seeds for zx1 zx2 zx3 triple mutants, zx1 zx2 zx3 zx4 quadruple mutants and respective wild type siblings. This will allow us to 2) prove the biological importance of zealexins in protecting plants against the stresses of soil microbes such as Fusarium and root feeding beetle larvae (Diabrotica spp). Early experiments already prove that zx1 zx2 zx3 zx4 mutants have altered bacterial microbiomes and increased susceptibility to Fusarium (https://doi.org/10.1101/2020.03.04.977355). 3) Relating to enzyme promiscuity and biochemical layers of innate immunity, we have greatly improved insights that influence the successful path forward on controlling maize antibiotic production and resistance. In published and unpublished we now know the following: in select maize inbreds there can be the following functional terpene synthases (TPS) namely sesquiterpene synthases [a-copaene(1), a-santalene(1), b-selinene(1), b-bisabolene/ b-macrocarpene (most commonly 4 but 3-6 possible)] and diterpene synthases [ent-copalyl-PP (1), ent-isokaurene(1), dolabradiene(1), and complex kaurene synthase-like-1 derived products(1)] that contribute to acidic terpenoid antibiotics. Of the 11 TPS genes listed above, all hydrocarbon olefins appear to rely on the use the chromosome 5 gene cluster encoding closely related enzymes ZmCYP71Z19(Zx5), ZmCYP71Z18(Zx6) and ZmCYP71Z16(Zx7) to generate approximately 100 antibiotics after reconnecting with subsequent pathway specific enzymes. Of nearly 300 inbred lines examined, B73 consistently displays the lowest root production of these products and is thus an imperfect model for demonstrating biological roles. Given the existing published and unpublished complexity in maize, an essential path forward is to establish resources in a specific inbred line that has ALL functional pathways present and displays appreciable antibiotic production in field soil. A collaborator, Dr. Bing Yang (Danforth Center) now has the ability to create CRISPR/Cas9 targeted mutant collections in diverse inbreds. The massive interactive pathway complexity enabled by 11 TPS enzymes funneled through a gene cluster of encoding 3 related CYP71Z enzymes (Zx5/Zx6/Zx7) as a mid pathway initial oxidation step conceptually compels the hourglass biosynthetic pathway to be empirically proven at a functional genetic level using combinatorial single, double and triple mutants in a select inbred line. This work will the enable endogenous origin of diverse terpenoid antibiotic complexities to unambiguously assigned, understood and predictably leveraged in maize and other plants. Partial proof of concept experiments have been performed, for example maize genes such as Zx6 (ZmCYP71Z18) enhance biotic stress resistance in rice (doi: 10.1007/s11103-019-00881-3) by generating novel enzyme pairs and antibiotic products. The phenotypic characterization of maize zx1 zx2 zx3 zx4 mutants with soil microbes and Diabrotica is an essential aspect requiring completion and will compel the concept that terpenoid antibiotics matter for Diabrotica resistance and that the protective network can be further improved. Given that the existing genetic and biochemical complexity greatly exceeds our original vision, a comprehensive final proof of endogenous pathways working through Zx5 to Zx7 is important to achieve prior to confident creation of novel biochemical networks via genetic modification.
Impacts
What was accomplished under these goals?
Description Of Impact: Maize production in the U.S. is enabled by stress resistance traits that provide essential biotic stress protection. Discovery of the genetic and biochemical mechanisms underlying resistance are key to ensuring that systematic improvements can be made through classical breeding and targeted molecular approaches. As a persistent problem, the quality and quantity of maize harvested is negatively impacted by pathogens by the combination of complex biotic pressures. We isolated and identified eight new maize antibiotics, termed zealexins, as part of a biosynthetic pathway of a least 17 related metabolites. Using metabolomics, transcriptomics, proteomics, genetic mapping, heterologous enzyme co-expression assays and defined mutants we discovered 10 zealexin pathway genes as part of a larger and highly interconnected network of terpenoid antibiotics, produced by an unusual biosynthetic hourglass pathway enabling surprising combinatorial complexity. In the biosynthetic hourglass model, structurally diverse precursors from multiple protective pathways are oxidized by a co-regulated gene cluster of related and enzymatically promiscuous cytochrome P450 enzymes to produce the first stage of antibiotics. Subsequently, pathway specific enzymes reconnect with the bioactive substrates to create further biochemical elaborations. Simply stated, maize terpenoid pathways are intimately connected driving extremely diverse antibiotic production. Genetic diversity across maize demonstrates examples of gene family expansion suggestive of selection for antibiotic diversity. As hypothesized, defined maize mutants lacking zealexin biosynthesis display increased susceptibility to pathogens and display significant root necrosis in field soil. Our findings elucidate genes, enzymes and small molecule antibiotics that help maize plants survive field conditions. Analyses of additional interactive pathways informs further engineering approaches to control protective molecules produced through novel associations. Efforts to improve crop protection in rice using zealexin pathway genes have already been achieved (doi: 10.1007/s11103-019-00881-3). The broader impact of our work is the comprehensive elucidation of maize protective antibiotic pathways at the genetic, chemical and functional levels. Our discoveries define opportunities for crop breeding and transgenic applications to leverage protective pathways important for stress resistance. Objective 1: Establish a foundational knowledge of maize biochemicals and genetic pathways that mediate Diabrotica and Fusarium interactions. We largely completed the identification of zealexins present in maize following fungal and insect attack. We found 8 additional β-bisabolene/β-macrocarpene synthase derived antibiotics from 4 maize terpene synthases (TPS) initiating the zealexin pathway namely TPS6 (Zx1), TPS12 (Zx2), TPS11 (Zx3), TPS13(Zx4)] and describe zealexins(Z) ZD1, ZD2, ZA5, ZA6, ZA7, ZA8, ZA9 and ZB3. Significant antifungal activity of the zealexin (Zx) pathway was demonstrated in zx1 zx2 zx3 zx4 mutants displaying increased susceptibility to diverse pathogens (doi: 10.1007/s11103-019-00881-3). Additional experiments conducted included the forward genetic mapping of maize the genes underlying a-santalene and a-copaene. Data collected includes samples of field root samples from association panels, NAM subpopulations, other populations to specifically target zealexins, a-santalene, a-copaene and derived products. Candidate genes identified in mapping intervals, include both an a-copaene synthase and a-santalene synthase that have been cloned, heterologously expressed in tobacco using Agrobacterium and paired experiments with maize cytochrome (CYP)P450 genes encoding CYP71Z19 (Zx5), CYP71Z18 (Zx6) and CYP71Z18 (Zx7). Thus far only CYP71Z19 has demonstrated activity in the formation of acidic sesquiterpenoids form a-copaene synthase and a-santalene. A gene cluster of 5 CYP81A genes responsible was identified using genetic mapping and 3 genes ZmCYP81A37 (Zx8), ZmCYP81A38 (Zx9) and ZmCYP81A39 (Zx10) have been proven to oxidize b-macrocarpene in the production of advanced zealexins. Prior to our research, CYP81 family P450s were unknown to act on terpenoids. Enzyme co-expression efforts in tobacco confirmed the existence of 10 separate functional zealexin biosynthetic pathway genes (Zx1 to Zx10) in maize. Analyses of multiple maize genomes reveals that select maize inbreds, can contain 3 to 6 copies of b-bisabolene/b-macrocarpene synthases (Zx1 to Zx4), up to 6 copies of CYP71Z genes (Zx5 to Zx7), and 5 copies of ZmCYP81A (Zx8 to Zx10). As completed key outcomes and accomplishments, we have established 17 precursors and products in the zealexin pathway and demonstrated roles for 10 Zx pathway genes/enzymes. We found genetic evidence for up to 17 Zx pathway genes across specific inbreds. Using large scale multi-omic analyses we highlighted and placed the surprising complexity of maize terpenoid antibiotic biosynthesis in the context of established defense pathways. Objective 2: Demonstrate the role of biochemical networks on Diabrotica growth, survival, pathogen spread and plant resistance. In an experiment with field soil we examined specific wild type inbred lines paired zx1 zx2 zx3 zx4 mutants. Naturally occurring soil arthropods and microbes resulted in dramatic increases in root necrosis specifically in the zx1 zx2 zx3 zx4 quadruple mutant plants. Furthermore the bacterial root microbiome of zx1 zx2 zx3 zx4 was altered (doi: 10.1007/s11103-019-00881-3). Maize zx1 zx2 zx3 zx4 mutants displayed enhanced spread of Fusarium graminearum. Given the origin of CRISPR/CAS9 derived quadruple mutants from HiII, we are pursing further backcrosses with B73, genotyping, selecting homozygous mutants and segregating wild type siblings, and propagation of sufficient seeds such that Diabrotica assays are not limited. To prepare, we have been growing plants comparing commercial potting soil to potting soil amended with 50% field soil. From these efforts we observed that inbred lines grown in field soil can display healthy white roots yet have vastly different chemical profiles from conspecific plants grown in potting soil. In visually asymptomatic roots, levels of pre-existing root terpenoid antibiotics can be vastly different. Collectively we have now identified a series of select inbreds, mutants, growth conditions and plant/tissue ages to run optimized bioassays. Objective 3: Create novel maize biochemical networks that restore resistance to pest complexes (Diabrotica-Fusarium). As defined in the previous reporting period, we previously made significant progress in using the sorghum SbTPS1 zingiberene synthase and production of a candidate acid using heterologously expressed ZmCYP71Z19 in tobacco. We improved our analytical protocols and have greatly expanded detection maize root terpenoids from field. The terpenoid complexity witnessed exceeds anything we were previously aware of. We now believe that minimally 11 active diverse TPS genes can exist in maize and result in vast array of acidic terpenoid antibiotics. In roots, zealexins, dolabralexins, kauralexins and costic acids can predominate but at least 3 additional families also exist all relying on combinations of the ZmCYP71Z gene cluster (Zx5, Zx6 and Zx7). Given this published (and unpublished) complexity and large quantitative differences between inbreds, it now appears less certain about the value of planned substitution studies of zealexins with a different sorghum TPS. A better approach would be to understand the biology the entire complex network provides. To a measurable extent, significant novel biochemical networks mediated by Zx5, Zx6 and Zx7 continue to be uncovered in maize and are among the most complex known in any significant crop system.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2020
Citation:
Manuscript: Ding Y, Weckwerth PR, Poretsky E, Murphy KM, Sims J, Saldivar E, Christensen SA, Char SN, Yang B, Tong AD, Shen Z,Kremling KA, Buckler ES, Kono T, Nelson DR, Bohlmann J, Bakker MG, Baughan MM, Khalil AS, Betsiashvili M, Briggs SP, Zerbe P, Schmelz EA, Huffaker A (2020) Genetic elucidation of complex biochemical traits mediating maize innate immunity. Accepted with revisions at Nature Plants. Available on BioRXiv: https://doi.org/10.1101/2020.03.04.977355
- Type:
Journal Articles
Status:
Under Review
Year Published:
2020
Citation:
Manuscript: Poretsky E, Dressano K, Weckwerth PR, Ruiz M, Char SN, Shi D, Abagyan R, Yang B Huffaker A (2020) Differential activities of maize Plant Elicitor Peptides as mediators of immune signaling and herbivore resistance In review at The Plant Journal.
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Progress 04/01/18 to 03/31/19
Outputs
Target Audience:At university venues our target audience includes, undergraduate, graduate, post-doctorial and scientist level students and researchers. At the Maize Genetics meeting and the National Corn Growers Association meetings, our audience includes farmers and policy experts in agricultural and funding agencies. At the National Association of Biology Teachers meeting our audience is high school level educators and teachers who seek in incorporate new materials and ideas into their curriculum. Research progress our how acidic sesquiterpenoids are regulated, biosynthesized and control maize resistance to biotic stress caused by pathogenic fungi and root feeding insects was communicated in 5 significant venues reaching different target audiences as follows: May 2018 invited talk by Dr. Schmelz "Leveraging multi-omic approaches to uncover maize specialized metabolic pathways mediating defense". Plant Group Symposium on Plant Signaling in Biotic and Abiotic Stress. University of Missouri (Columbia, Missouri) June 2018 invited talk by Dr. Schmelz "Elucidation of broad-spectrum antifungal maize defense pathways and metabolites underlying pathogen resistance " National Corn Growers Association: Corn Utilization & Technology Conference (St. Louis, Missouri) Dr. Huffaker and Dr. Schmelz served as volunteer staff at the American Society of Plant Biology (ASPB) Education Committee outreach booth at the 2018 National Association of Biology Teachers (NABT) annual meeting to present teaching materials relating to maize sesquiterpene defenses (11/8-9/2018; Sheraton San Diego Hotel and Marina, San Diego) March 14 -17, 2019 invited talk by Dr. Huffaker " Genetic and biochemical delineation of the zealexin biosynthetic pathway reveals coordinated activity of multiple gene clusters to ensure production of a core maize defense" 61st Annual Maize Genetics Conference, Session 6 - INTERACTIONS WITH THE ENVIRONMENT, Union Station St. Louis, Missouri, USA University of California, San Diego. Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" was run jointly run by Dr. Huffaker and Schmelz during the Spring Quarter 2018 and 2019 (April 1 to June 8th) and collectively trained 40 students for 10 hours per week during the course. In specific laboratory modules the students learned about maize sesquiterpenoid defenses, conduced elicitation experiments, tested for biological activity in highly enriched extracts, analyzed GC/MS data for precursors and products, and in new research conducted forward genetics using association mapping experiments to identify new biosynthetic genes in maize. Changes/Problems:As noted above in the "Opportunities for training and professional development" section efforts to identify and mentor a graduate student to focus on this project have reverted back to bringing on a postdoctoral scientist now identified as Dr. Aarthy Thiagarayaselvam in the late summer of 2019. The attempt to focus use of funds for graduate student training and research has slightly delayed the rate of expenditures; however, progress by the PIs and undergraduate students have maintained significant research progress on core objectives. Bringing on the best people for specific projects is rarely an instantaneous process. We do not view this delay as major nor as having a significant impact on the success of the effort. What opportunities for training and professional development has the project provided?The project has enabled material and partial funding support for the undergraduate student worker and Honors Thesis student Evan Saldivar co-supervised by Dr. Schmelz and Dr. Huffaker. Evan Saldivar June 2019 honors thesis was entitled "The elucidation and untangling of maize biosynthetic pathway underlying defense" and focused in part on the role of cytochrome P450 promiscuity in the production of maize terpenoid phytoalexins. Outstanding academic and research accomplishments merited Evan Saldivar being named as a recipient of the Selma and Robert Silagi Award for Undergraduate Excellence carrying monetary gift of $5,000 (3/1/19). Professional development components for Evan Saldivar included UCSD 2019 Student Research Showcase (6/5/19) poster presentation "Complementary adaptations to bioinformatics approaches facilitate the untangling of maize defense biochemistry", UCSD Inter-Sustainability Council Research Symposium (5/30/19) poster presentation "A bioinformatics approach to the untangling of maize defense biochemistry", UCSD Undergraduate Research Conference (5/18/19) oral presentation "A bioinformatic approach to the untangling of maize isoprenoid defense pathways", and the UCSD Summer Research Conference (8/10/18) oral presentation " Elucidating antimicrobial defense pathways in maize mediated by Zealexin biosynthesis". Evan Saldivar has also been award an ASPB travel grant to attend the Plant Biology (2019) meeting in San Jose, CA. as an opportunity to further network for graduate school opportunities in the Plant Sciences. The primary funds for the project were held available for Ms. Devon Birdseye to work as a graduate student starting in the Fall of 2018. With mentorship from Dr. Huffaker and to ensure ample funding for the entire PhD process, Ms. Birdseye applied for additional research support through the NSF Graduate Research Fellowship Proposal program was awarded this honor in the Spring of 2019. With the flexible funds awarded directly to the applicant, Ms. Birdseye decided to change laboratories and research directions. Training in the topics of scientific writing and logic flow considerably helped in the professional development of Ms. Birdseye. The downside of these training elements has been a delay in bringing on board the postdoctoral scientist who will be focused on the core of the research late summer 2019. How have the results been disseminated to communities of interest?Research progress our how acidic sesquiterpenoids are regulated, biosynthesized and control maize resistance to biotic stress caused by pathogenic fungi and root feeding insects was communicated in 5 significant venues reaching different target audiences as follows: May 2018 invited talk by Dr. Schmelz "Leveraging multi-omic approaches to uncover maize specialized metabolic pathways mediating defense". Plant Group Symposium on Plant Signaling in Biotic and Abiotic Stress. University of Missouri (Columbia, Missouri) June 2018 invited talk by Dr. Schmelz "Elucidation of broad-spectrum antifungal maize defense pathways and metabolites underlying pathogen resistance " National Corn Growers Association: Corn Utilization & Technology Conference (St. Louis, Missouri) Dr. Huffaker and Dr. Schmelz served as volunteer staff at the American Society of Plant Biology (ASPB) Education Committee outreach booth at the 2018 National Association of Biology Teachers (NABT) annual meeting to present teaching materials relating to maize sesquiterpene defenses (11/8-9/2018; Sheraton San Diego Hotel and Marina, San Diego) March 14 -17, 2019 invited talk by Dr. Huffaker " Genetic and biochemical delineation of the zealexin biosynthetic pathway reveals coordinated activity of multiple gene clusters to ensure production of a core maize defense" 61st Annual Maize Genetics Conference, Session 6 - INTERACTIONS WITH THE ENVIRONMENT, Union Station St. Louis, Missouri, USA University of California, San Diego. Undergraduate laboratory class BIBC151 "Chemistry of Biological Interactions" was run jointly run by Dr. Huffaker and Schmelz during the Spring Quarter 2018 and 2019 (April 1 to June 8th) and collectively trained 40 students for 10 hours per week during the course. In specific laboratory modules the students learned about maize sesquiterpenoid defenses, conduced elicitation experiments, tested for biological activity in highly enriched extracts, analyzed GC/MS data for precursors and products, and in new research conducted forward genetics using association mapping experiments to identify new biosynthetic genes in maize. What do you plan to do during the next reporting period to accomplish the goals?Our stated goal is to discover maize biochemical pathways that regulate complex herbivore-root-fungus (Diabrotica-maize-Fusarium) interactions and use defined mutants to empirically demonstrate ecological roles and target the restoration of resistance by engineering novel biochemical networks that suppress pest pressures. In the next reporting period: 1) We will finalize our publication on the first 10 genes that encode zealexin (Zx) biosynthetic enzymes and demonstrate the catalytic activity of each. 2) Using a zx1-4 quadruple mutant that lacks the synthesis of all zealexins, we will confirm the biological importance of zealexins in protecting plants against the stresses of soil microbes such as Fusarium and root feeding beetle larvae (Diabrotica spp). 3) We will finish the purification of the sorghum TPS1 plus maize ZmCYP71Z9 product, namely a-zingerbene acid, confirm structure by NMR, and perform bioassays that demonstrate antifungal and anti-Diabrotica activity. The promoter for B73 Zx3 will be cloned and fused to SbTPS1 for transformation directly into B73 to increase the metabolic diversity. Plants generated will be propagated, and crossed into different backgrounds (zx1-4) to prepare the range of materials needed to demonstrate and create novel create novel maize biochemical networks to target the Diabrotica-Fusarium complex.
Impacts
What was accomplished under these goals?
(1) Objective 1: Establish a foundational knowledge of maize biochemicals and genetic pathways that mediate Diabrotica and Fusarium interactions. A major activity completed was the expanded identification of zealexins present in maize following fungal and insect attack in maize. We described the keto acid derivative of b-macrocarpene, termed zealexin A4 in the journal Planta (April 2018, 247 (4) pp 863-873). Additional experiments conducted included the forward genetic mapping of maize the genes underlying a-santalene and a-copaene. Data collected includes 600 samples of field root samples from the Goodman association panel, B73 X M162W Nested Association Panel subpopulation, and the B73 x Mo17 IBM population to specifically target insect and and fungal elicited production of zealexins, a-santalene, a-copaene and derived products. Samples have been examined by GC/MS and integrated for peak areas. Statistics analyzed include significant P values calculated from the general linear model (GLM) and compressed mixed linear model (MLM) identifying genetic intervals where terpenoid synthase and downstream biosynthetic genes occur. From the candidate genes with identified mapping intervals, a-santalene synthase has been cloned, heterologously expressed in Nicotiana benthamiana using Agrobacterium and paired experiments with maize cytochrome (CYP) P450 genes encoding CYP71Z9, CYP71Z16 and CYP71Z18. While strong transcription co-expression exists for CYP71Z18, thus far only CYP71Z9 has demonstrated activity in the formation of α-santalenoic acid. Cloning efforts for the a-copaene synthase are currently underway. Furthermore a gene cluster of 3 CYP81 genes responsible for desaturations and further oxygenations has been mapped using the IBM populations. Large-scale cloning and enzyme co-expression efforts have now confirmed the existence of 10 separate zealexin biosynthetic pathway genes in maize. Importantly Tps6/11 are not 2 genes but instead 4 genes, now termed Zx1-4, that exist in variably functional states in all maize lines. The maize line W22 has 3 functional b-macrocarpene synthases (Zx1,3,4) underlying zealexin production and only lacks a functional Zx2 enzyme due to a single amino acid mutation. These results are being detailed in a manuscript soon to be submitted. The correct identification of the maize a-santalene synthase and the CYP71 enzyme capable of making α-santalenoic acid is an additional key outcome, but efforts to prove the role of genetic knockouts in maize are on-going and not yet finalized for a completed publication. As completed key outcomes and accomplishments, we have proven the identity of zealexin A4, demonstrated regulation by insect and pathogen attack and demonstrated antifungal activity of ZA4. . (2) Demonstrate the role of biochemical networks on Diabrotica growth, survival, pathogen spread and plant resistance. In an experiment with field soil collected from an area with many years of continuous maize production, we examined specific wild type inbred lines paired zx1-4 quadruple mutants that lack all zealexins and Zmtps21 lacking b-costic acid. Naturally occurring soil arthropods and microbes resulted in dramatic increases in root necrosis specifically in the zx1-4 quadruple mutant plants. Experiments are on-going to detail the fungal microbiome communities that differ in these root systems. A large scale Western corn rootworm (Diabrotica virgifera virgifera) experiments were conducted with wildtype and Zmtps21 mutants but growth chamber conditions lead to low vigor of all maize lines examined and yielded unclear results. While not yet published, a key outcome of experiments with zx1-4 mutants is the large-scale role of maize zealexins in preventing root disease in plants exposed to natural field conditions. (3) Create novel maize biochemical networks that restore resistance to pest complexes (Diabrotica-Fusarium). As proposed, we have confirmed the catalytic activity of cloned Sorghum bicolor (Sb) TPS1 as a predominant a-zingerbene synthase using Agrobacterium-mediated heterologous expression in Nicotiana benthamiana. As a component of Objective 1, it was essential that we identify endogenous maize CYP P450s responsible for the conversion of b-macrocarpene to zealexin A1. The maize enzymes CYP71Z9, CYP71Z16 and CYP71Z18 encoded by a chromosome 5 gene cluster all perform this reaction. In related paired experiments with SbTPS1 we found that CYP71Z9 had improved activity on making the respective a-zingerbene acid candidate. These details matter based on the expression levels of ZmCYP71Z9 that exist in the maize lines(s) chosen for transformation. Analyses of diverse maize lines using transcriptomics demonstrates not all maize lines rely on ZmCYP71Z9 equally. As a novel natural product not yet described in nature, the electron ionization spectra lacks a clear parent ion (m/z 248) but otherwise contains meaningful fragment ions. The result is consistent with a labile and more reactive cyclodiene molecule. Before creating the maize transgenics, efforts are underway to purify 0.5 mg from the N. benthamiana expression system, confirm the correct structure and establish activity at least against Fusarium species and WCR if quantities allow. Production of sesquiterpene acids in N. benthamiana has been optimized by co-expression with 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR) to increase pathway flux followed by tissue extraction using b-glucosidase enzymes to release sugar conjugates produced by tobacco. A key outcome is that an identified maize enzyme ZmCYP71Z9 has sufficient promiscuity to produce the candidate a-zingerbene acid as proposed.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Christensen, S. A., Huffaker, A., Sims, J., Hunter, C. T., Block, A., Vaughan, M. M., Willett, D., Romero, M., Mylroie, J. E., Williams, W. P. & Schmelz, E. A. Fungal and herbivore elicitation of the novel maize sesquiterpenoid, zealexin A4, is attenuated by elevated CO2. Planta 247, 863-873, doi:10.1007/s00425-017-2830-5, April 2018, Volume 247, Issue 4, pp 863873
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Block, A. K., Vaughan, M. M., Schmelz, E. A. & Christensen, S. A. Biosynthesis and function of terpenoid defense compounds in maize (Zea mays). January 2019, Planta Volume 249, Issue 1, pp 2130, doi:10.1007/s00425-018-2999-2
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