Research Project: New Weed Management Tools from Natural Product-Based Discoveries

Location: Natural Products Utilization Research

2019 Annual Report

Objectives
The overall goal of this project is to discover and develop natural product-based weed management solutions. The research is divided into discovery and development of biochemical bioherbicides and creation of crops that are resistant to weeds by transgenically imparting or improving plant/plant allelopathy. This research should provide new avenues for the development of affordable and effective, yet sustainable, weed control strategies. 1. Discover and develop natural product-based bioherbicides that provide environmentally safe and toxicologically benign tools for weed management, with novel modes of action, to address current problems associated with herbicide resistant weeds. 1.1. Discover new and existing natural products for potential use as herbicides and bioherbicides. 1.2. Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues and physiological evaluations. 1.3. Develop natural products as new weed management tools. 2. Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals. 2.1. Complete the characterization of the gene products of putative genes for enzymes of the sorgoleone biosynthetic pathway. 2.2. The use of sorghum transformants possessing altered sorgoleone levels to investigate the ecophysiological role of sorgoleone. 2.3. Identification of plant promoters to facilitate root hair-specific metabolic engineering of sorgoleone biosynthesis. 2.4. Engineering de novo sorgoleone biosynthesis in non-producing host plants.

Approach
Bioassay-directed isolation of phytotoxin will be followed by their evaluation of their potential as bioherbicides and determination of their modes of action. Genes of the sorgoleone synthesis pathway with root hair-specific promoters will be inserted into plants with the intent to impart or improve allelopathic capacity for enhanced weed management.

Progress Report
Towards achieving Objective 1, the following research was performed in FY 2019 and milestones have been met: Determination of the mode of action of spliceostatin C. Spliceostatin C (sp C) is an active component of bioherbicide MBI-014 isolated from soil bacterium Burkholderia rinojensis. The chemical structure of this natural product is related to spliceostatin A (sp A) which was characterized as an anticancer agent and splicing inhibitor. The comprehensive study included phytotoxic activity and mode of action analysis of this natural product. Spliceostatin C also significantly inhibited the growth of Arabidopsis thaliana seedlings and triggered leaf bleaching, with growth IC50 of 2.2 µM. To elucidate the inhibitory functions of sp C in planta, 7-day-old Arabidopsis seedlings were treated with sp C at the concentration of IC50, and RNAs were extracted for semiquantitative RT-PCR (RT-sqPCR) analysis. Among 20 genes that were selected for the assays, five transcripts (tubulin alpha-5, mRNA splicing factor, SF3b14b, flowering focus M and circadian clock associated 1) underwent intron rearrangements such as intron retention and alternative 5’ or 3’ splicing side upon exposure to sp C. The results obtained from RT-sqPCR confirmed that spliceostatin C inhibits plant spliceosome catalysis. To investigate the impact of sp C on gene expression further, global proteome profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed. After exposure to sp C for 6 h, 145 protein isoforms were identified with fold changes greater than 1.5 (p = 0.05), and, among them, 134 were decreased and 11 increased. KEGG pathway analysis revealed that these proteins are associated with metabolic pathways, carbon metabolism, the ribosome, and secondary metabolite pathways. Further analysis of these proteins could provide insight into the mechanisms of phytotoxicy of sp C to the plant cell. Identification of a Natural Product-Based Potentiator of Echinocandin Class Fungicides. Cell wall-targeting echinocandin fungicides such as caspofungin are highly potent, yet lack efficacy against some fungal species due to their propensity to induce pathogen resistance. One potential strategy to address this limitation is to combine echinocandins with a potentiating compound that improves their activity by disrupting cellular adaptation pathways. In collaboration with University of Mississippi investigators, we identified the marine sponge-derived sesquiterpene quinone puupehenone (PUUP) as a potentiator of the echinocandin fungicide caspofungin (CAS) in CAS-resistant fungal pathogens. Using RNA-Seq analysis, coupled with genetic and molecular studies in the model organism S. cerevisiae, we determined that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2, the terminal MAP kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its co-chaperone Cdc37. Through these experiments we’ve demonstrated that PUUP likely potentiates echinocandin fungicide activity via the disruption of Hsp90 activity and the CWI pathway. The identification of a novel natural product-based potentiator of echinocandin fungicides has important implications for the development of novel approaches for the control of pathogenic fungi. Bioassay-guided isolation of phytotoxic compounds was accomplished from several sources. For example, confertin and salsolol A and B from Ambrosia salsola had IC50 values of 261, 275, and 251 µM, respectively, against duckweed. New sesquiterpenoids were isolated from the fungus Stereum complicatum and some of these compounds were moderately toxic to duckweed. The microbial broth of Burkholderia rinojensis strain A396 is herbicidal to a number of weed species with greater observed efficacy against broadleaf than grass weeds. A portion of this activity is attributed to romidepsin, a 16-membered cyclic depsipeptide bridged by a 15-membered macrocyclic disulfide. Romidepsin, which is present in small amounts in the microbial broth, was isolated and purified using standard chromatographic techniques. We established that pure romidepsin is a natural proherbicide that targets the activity of plant histone deacetylases (HDAC). Molecular dynamic simulation of the binding of romidepsin to atHDAC19 indicated the reduced form of the compound could reach deep inside the catalytic domain and interact with an associated zinc atom required for enzyme activity. For Objective 2, milestones have been met and significant progress has been made. Manipulation of Sorgoleone Biosynthesis in Planta - The allelochemical sorgoleone likely plays a major role in the sorghum plant’s natural ability to fend off weed infestations, and also represents a promising natural product-based alternative to synthetic herbicides. Previously, our research unit successfully completed a long term effort to clone all of the genes required for the biosynthesis of sorgoleone from the ubiquitous precursor palmitoleoyl-CoA, thus providing us with the necessary tools for manipulating sorgoleone production in planta. Our goals for this work have included transferring the entire biosynthetic pathway to other crops, as well as the development of enhanced sorghum germplasm with increased sorgoleone content. We reported on the generation of transgenic rice plants containing the complete sorgoleone biosynthetic pathway in our 2018 annual report, and have now developed and utilized high-throughput methods to screen the transgenic rice events using qRT-PCR and GC-MS. It was also necessary for this screening work to devise an in vitro culture approach for increasing root hair production, which was accomplished by 1-aminocyclopropane-1-carboxylic acid (ACC) addition to the plant growth media, and the design of polypropylene mesh supports in the culture vessels to increase root biomass. We are currently nearing completion of our transgenic rice seedling screening efforts, and we will also be initiating similar experiments in other crops this FY, including corn, wheat, and soybean. We have also initiated experiments for generating transgenic sorghum with increased sorgoleone content this FY by using root hair-specific desaturase and glutathione-s-transferase gene promoters driving the expression of ARS1, which encodes an enzyme required for the production of the (rate limiting) 5-pentadecatrienyl resorcinol sorgoleone pathway intermediate. The root hair-specific desaturase and glutathione-s-transferase gene promoters we used were also isolated by our research unit and were mentioned in a previous annual report. We anticipate that positive outcomes from these efforts will represent major breakthroughs in the plant-produced biopesticide field, resulting in the generation of novel germplasm possessing enhanced resistance to weed infestations, and potentially other agronomic pests. NP / Component Coding: 304 2 B 2008

Accomplishments

Review Publications
Duke, S.O., Owens, D.K., Dayan, F.E. 2019. Natural product-based chemical herbicides. In: Weed Control: Sustainability, Hazards and Risks in Cropping Systems Worldwide. Korres, N.E, Burgos, N.R and Duke, S.O. Taylor and Francis. pp. 153-165.
Duke, S.O., Reddy, K.N. 2018. Is mineral nutrition of Glyphosate-resistant crops altered by Glyphosate treatment? Outlooks on Pest Management. 29(5):206-208. https://doi.org/10.1564/v29oct05.
Duke, S.O., Dayan, F.E. 2018. Herbicides. In eLS. John Wiley & Sons, Ltd: Chichester. https://doi:10.1002/9780470015902.a002526.
Mu, J., Zhai, Z., Tan, C., Weng, J., Wu, H., Duke, S.O., Zhang, Y., Liu, X. 2019. Synthesis and herbicidal activity of 1,2,4-triazole derivatives containing a pyrazole moiety. Journal of Heterocyclic Chemistry. http://doi.org/10.1002/jhet.3476.
Korres, N., Burgos, N., Travlos, I., Vurro, M., Gitsopoulos, T., Varanasi, V., Duke, S.O., Kudsk, P., Brabham, C., Rouse, C., Salas-Perez, R. 2019. New directions for integrated weed management: Modern technologies, tools and knowledge discovery. Advances in Agronomy. https://doi.org/10.1016/bs.agron.2019.01.006.
Duke, S.O., Powles, S.B., Sammons, R. 2018. Glyphosate – how it became a once in a hundred year herbicide and its future. Outlooks on Pest Management. 29:247-251. https://doi.org/10.1564/v29_dec_03.
Tan, L., Wang, M., Kang, Y., Azeem, F., Zhou, Z., Tuo, D., Preciado-Rojo, L., Khan, I.A., Pan, Z. 2018. Biochemical and functional characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L. Molecules. 23(11):2876-2896. https://doi.org/10.3390/molecules23112876.
Kumarihamy, M., Ferreira, D., Croom, E.M., Sahu, R., Tekwani, B.L., Duke, S.O., Khan, S., Techen, N., Nanayakkara, N.D. 2019. Antiplasmodial and cytotoxic cytochalasins from an endophytic fungus, Nemania sp. UM10M, isolated from a diseased torreya taxifolia leaf. Molecules. 24(4):777. https://doi.org/10.3390/molecules24040777.
Duke, S.O., Stidham, M.A., Dayan, F.E. 2018. A novel approach to herbicide and herbicide mode of action discovery. Pest Management Science. https://doi.org/10.1002/ps.5228.
Favaretto, A., Cantrell, C.L., Fronczek, F.R., Duke, S.O., Wedge, D.E., Abbas, A., Scheffer-Basso, S.M. 2019. New phytotoxic Cassane-like diterpenoids from Eragrostis plana (Nees). Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.8b06832.
Maroli, A.S., Gaines, T.A., Foley, M.E., Duke, S.O., Dogramaci, M., Anderson, J.V., Horvath, D.P., Chao, W.S., Tharayil, N. 2018. OMICS in weed science research: A perspective from genomics, transcriptomics and metabolomics approaches. Weed Science. https://doi.org/10.1017/wsc.2018.33.
Nandula, V.K., Riechers, D.E., Ferhatoglu, Y., Barrett, M., Duke, S.O., Dayan, F.E., Goldberg-Cavalleri, A., Tetard-Jones, C., Wortley, D.J., Onkokesugn, N., Brazier-Hicks, M., Edwards, R., Gaines, T., Iwakami, S., Jugulam, M., Ma, R. 2019. Herbicide metabolism: Crop selectivity, bioactivation, weed resistance mechanisms, and regulation. Weed Science. 67:149-175.
Rosa, L.H., Zani, C.L., Cantrell, C.L., Duke, S.O., Dijck, P.V., Desideri, A., Rosa, C.A. 2019. Fungi in Antarctica: diversity, ecology, effect of climate changes, and bioprospection for bioactive compounds. Book Chapter. https://doi.org/10.1007/978-3-030-18367-7_1.
Costa, F.R., Rech, R., Duke, S.O., Carvalho, L.B. 2018. Lack of effects of glyphosate and glufosinate on growth, mineral content, and yield of glyphosate- and glufosinate-resistant maize. GM Crops & Food. https://doi.org/10.1080/21645698.2018.1511204.
Diaz-Tielas, C., Grana, E., Sanchez-Moreiras, A.M., Reigosa, M.J., Vaughn, J.N., Pan, Z., Bajsa Hirschel, J.N., Duke, S.O. 2019. Transcriptome responses to the phytotoxin t-Chalcone in Arabidopsis thaliana L. Pest Management Science. https://doi.org/10.1002/ps.5405.
Reddy, K.N., Cizdziel, J.V., Williams, M., Maul, J.E., Rimando, A.M., Duke, S.O. 2018. Glyphosate resistance technology has minimal or no effect on maize mineral content and yield. Journal of Agricultural and Food Chemistry. 66:10139-10146. https://doi.org/10.1021/acs/jafc.8b01655.
Lazzara, N.C., Rosano, R.J., Vagadia, P.P., Giovine, M.T., Bezpalko, M.W., Piro, N.A., Kassel, W.S., Boyko, W.J., Zubris, D.L., Schrader, K.K., Wedge, D.E., Duke, S.O., and Giuliano, R.M. Synthesis and biological evaluation of 6-[(1R)-1-hydroxyethyl]-2,4a(R),6(S),8a(R)-tetrahydropyrano-[3,2-b]-pyran-2-one and structural analogues of the putative structure of Diplopyrone. The Journal of Organic Chemistry. 2019;84:666-678. https://doi.org/10.1021/acs.joc.8b02490.
Perera, W.H., Meepagala, K.M., Fronczek, F.R., Cook, D., Wedge, D.E., Duke, S.O. 2019. Bioassay-guided isolation and structure elucidation of fungicidal and herbicidal compounds from Ambrosia salsola (Asteraceae). Molecules. 24:1-12. https://doi.org/10.3390/molecules24050835.
Wu, X., Lin, X., Baerson, S.R., Ding, C., Zhang, L., Wu, C., Song, Y., Zeng, R. 2018. The roles of jasmonate (JA) signaling in nitrogen uptake and allocation in rice (Oryza sativa L.). New Phytologist. https://doi.org/10.1111/pce.13451.