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

Location: Natural Products Utilization Research

2022 Annual Report

Objectives
1. Discover and develop natural product-based bioherbicides with novel modes of action that are safe and effective tools for weed management. [C2, PS2A] 1.1. Discover uses of new and existing natural products for potential use as herbicides and bioherbicides for weed management. 1.2. Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues, physiological evaluations, and molecular genetics approaches. 2. Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals and determine the role of allelopathy in the success of invasive weeds. 2.1. Identification of transporters required for the extracellular secretion of sorgoleone in Sorghum bicolor root hair cells. 2.2. Manipulation of sorgoleone levels in vivo to generate enhanced S. bicolor germplasm. 2.3. Generation of transgenic maize, wheat and soybean plants containing the complete sorgoleone biosynthetic pathway.

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 Fiscal Year (FY) 2022 and is continuing in FY2023: In 2022, we tested about 150 fungal and plant extracts and pure compounds for phytotoxicity using bioassays with bentgrass (Agrostis stolonifera L.), lettuce (Lactuca sativa L.) and Arabidopsis as model species that served as representatives of monocotyledonous and dicotyledonous plants. These extracts and pure compounds were provided by chemists in our research unit and scientists at the National Center for Natural Products Research. Some compounds were provided by international collaborators. A set of analogs based on the natural products khellin and visnagin were also evaluated for herbicidal activity. The analogs/compounds that demonstrated very satisfactory herbicidal activity are subjected to further investigation. Greenhouse studies are in progress to determine the efficacy of the most active analog as both a post- and a pre-emergent herbicide. Forty bipartite synthetic conjugates of plant growth regulators tested against the model plant Arabidopsis did not exhibit noteworthy post-emerge herbicidal activity. While ten compounds from another pool of chemicals (fifty putative PPO inhibitors) displayed strong phytotoxicity in the primary bioassays and have been selected for further assessment. Fusaricidins A and B, secondary metabolites isolated from bacterium Paenibacillus polymyxa, possess strong antifungal (plant pathogens) and antibacterial (gram-positive bacteria) properties. We tested these compounds for phytotoxic activity. Bioassays showed that they significantly inhibit the growth of Arabidopsis thaliana and Lemna paucicosata with IC50 values of 2.3 µM (fusaricidin A) and 1 µM fusaricidin AB mixture (Pure fusaricidin B was not available for testing since fusaricidin A was associated B during isolation due to the high degree of structural similarity). The investigation of the mode of action of these compounds suggests that the membrane disintegration may be the result of the interaction of fusaricidins with the plasma membrane. The measurements of chlorophyll fluorescence indicated that these compounds do not directly impact photosystems I or II. In collaboration with the National Center for Natural Products Research, we tested a series of synthesized triketone derivatives for putative herbicidal activity. These synthetic compounds exhibit promising herbicidal activity revealed by phytotoxicity assays against Arabidopsis thaliana and Lemna paucicostata. The mechanism of action of these compounds is to inhibit hydroxyphenylpyruvate dioxygenase (HPPD). Hence they have the potential to be developed as herbicides against herbicide-resistant weeds. An invention disclosure was submitted and approved for the preparation of a patent application. Momilactones A and B are diterpenoid phytoalexins with antimicrobial, allelopathic activity and strong phytotoxicity. Despite the fact that most of the genes for momilactone biosynthesis found in gene clusters in rice genome have been reported, the candidates for the missing steps are yet to be characterized. We identified a homolog of momilactone synthase (designated OsMAS2, also known as short-chain alcohol dehydrogenase/reductase) which may be involved in the synthesis of momilactone A. We made five constructs for investigating the function of this gene, which include promoter-driven reporter gene beta-glucuronidase gene (GUS), CRISPR knockout constructs, and overexpression construct for rice transformation. The generation of transgenic rice is in progress. We tested several phytoalexins (momilactones, juglone, aminophenoxazin, protocatechualdehyde, khellin, visnagin) using FluorCam, an instrument for chlorophyll fluorescence imaging of plants, plant tissues and algae. Our results suggested that photosystems I and II are not the primary targets of these phytoalexins. This method was established for detecting an early event in plant response to natural compounds for mode of action studies. Towards achieving Objective 2, the following research was performed in FY 2022 and is continuing in FY2023: 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. Our goals for this work have included transferring the ability to synthesize and secrete sorgoleone to other crops, as well as the development of enhanced sorghum germplasm with increased sorgoleone content. Previously, our research unit successfully completed the isolation and characterization of all genes required for the biosynthesis of sorgoleone from the ubiquitous precursor palmitoleoyl-CoA. Elucidation of the cellular apparatus involved in the secretion of sorgoleone is also critical, as the efflux pumps associated with this process likely provide a mechanism for autotoxicity avoidance to the host plant. Our group has employed a strategy involving the analysis of genes differentially expressed in sorgoleone-deficient transgenic sorghum, relative to wild-type sorghum and null segregant cohorts, for the identification of genes associated with sorgoleone rhizosecretion. The rationale for this approach is the likelihood that within a deficient background, genes directly involved in the biosynthesis and transport of sorgoleone will exhibit expression profiles which differ from those of wild-type plants, thus facilitating their identification. We have now completed these analyses and have identified a candidate list of sequences for follow up tests in transgenic sorghum plants. These candidate sequences will be subjected to CRISPR/Cas-mediated gene editing, and the design of the required guide sequences have also been completed by our group. We will also employ a recently-developed technological approach involving the addition of a morphogenetic marker (Wuschel2 from A. thaliana), which has been demonstrated to dramatically improve the efficiency of both S. bicolor transformation and CRISPR/Cas-mediated gene editing. To facilitate this work, a Non-Assistance Cooperative Agreement is being established with the director of the Donald Danforth Plant Science Center Transformation Laboratory, who has extensive experience performing CRISPR/Cas-mediated gene editing in sorghum. This collaboration will also provide the ARS with access to the proprietary Wuschel2-enhanced CRISPR/Cas technology developed by Corteva Agriscience (Indianapolis, IN). Additionally, the Collaborator's lab has developed a rapid in vitro assay using isolated S. bicolor embryos, which we will use to evaluate the efficiency of various guide RNA sequences prior to their use in stable transformation experiments. Ideally, these experiments will lead to the identification of key cellular components required for the transport of sorgoleone from its site of synthesis in root hairs to the root system-soil interface.

Accomplishments
1. Determination of the mode of action of spliceostatin C.. Spliceostatin C (spC), one of the bioactive components produced from the soil bacterium Burkholderia rinojensis, displayed a high phytotoxic activity at low doses against several dicot weeds. The spliceosome is generally thought to be the target of this compound. However, due to the complexity of the spliceosome which contains a large number of proteins/subunits, the precise mechanism of action of spC is yet to be elucidated. ARS researchers in Oxford, Mississippi, analyzed the genomic sequences retrieved from the genome database for 13 spC susceptible and resistant weeds revealed that there could be mutations in the nucleotide sequence encoding two spliceosome-specific proteins (SF3b14b and SF3b155). Molecular docking showed that spC formed H-bonding with two amino acids which are critical residues for herbicidal activity. These findings provide important information for further investigation into the possibility of generating spC-resistant crops through genetic engineering and breeding and develop spC as a bioherbicide for weed management targeting herbicide-resistant weeds.

Review Publications
Da Cruz-Silva, C.T., Cantrell, C.L., Nobrega, L.P., Ali, A., Duke, S. 2021. Bioassay-guided isolation of phytotoxins from three salvia species. Allelopathy Journal. 54(1):13-24. https://doi.org/10.26651/allelo.j/2021-54-1-1344.
Cárdenas, D.M., Bajsa Hirschel, J.N., Cantrell, C.L., Rial, C., Varela, R.M., Molinillo, J.G., Macías, F.A. 2022. Evaluation of the phytotoxic and antifungal activity of C17-sesquiterpenoids as potential biopesticides. Pest Management Science. https://doi.org/10.1002/ps.7042.
Min, L., Wang, H., Bajsa Hirschel, J.N., Yu, C., Wang, B., Yao, M., Han, L., Cantrell, C.L., Duke, S.O., Sun, N., Liu, X. 2022. Novel dioxolane ring compounds for the management of phytopathogen diseases as ergosterol biosynthesis inhibitors: synthesis, biological activities and molecular docking. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.2c00541.
Rajvi, D., Rodriguez, A., Bajsa Hirschel, J.N., Pan, Z., Machingura, M.C. 2021. Differential gene expression patterns in Sorghum bicolor genotypes in response to high vapor pressure deficit. Journal of Crop Improvement. https://doi.org/10.1080/15427528.2021.2009077.
Duke, S.O., Pan, Z., Bajsa Hirschel, J.N., Boyette, C.D. 2022. The potential future roles of natural compounds and microbial bioherbicides in weed management in crops. Advances in Weed Science. 40(spe1):e020210054. https://doi.org/10.51694/AdvWeedSci/2022;40:seventy-five003.
Ding, C., Lin, X., Zuo, Y., Yu, Z., Lu, Y., Chen, X., Baerson, S.R., Pan, Z., Zeng, R., Song, Y. 2021. Transcription factor OsbZIP49 controls tiller angle and plant architecture through the activation of indole-3-acetic acid-amido synthetases in rice. The Plant Journal. https://doi.org/10.1111/tpj.15515.
Belz, R.G., Duke, S.0. 2022. Modelling biphasic hormetic dose responses to predict sub-NOAEL effects using plant biology as an example. Current Opinion in Toxicology. https://doi.org/10.1016/j.cotox.2022.01.003.
Belz, R.G., Carbonari, C.A., Duke, S.O. 2022. The potential influence of hormesis on evolution of resistance to herbicides. Current Opinion in Environmental Science & Health. https://doi.org/10.1016/j.coesh.2022.100360.
Duke, S.O., Belz, R.G. 2022. Stepping beyond hormesis modelling and sub-NOAEL predictions in plant biology. Current Opinion in Environmental Science & Health. https://doi.org/10.1016/j.coesh.2022.100366.
Nazish, T., Huang, Y., Zhang, J., Xia, J., Alfatih, A., Chao, L., Cai, X., Xi, J., Xu, P., Xiang, C. 2022. Understanding paraquat resistance mechanisms in Arabidopsis thaliana to facilitate developing paraquat-resistant crops. Plant Communications. https://doi.org/10.1016/j.xplc.2022.100321.
Bajracharya, A., Xi, J., Karlie, G., Bayer, E., Grant, C., Clutton, C., Baerson, S.R., Agarwal, A.K., Qiu, Y. 2022. PIF4/HEMERA-mediated daytime thermosensory growth requires the Mediator subunit MED14. bioRxiv. https://doi.org/10.1101/2022.03.02.482654.
Roireau, J.H., Rosano, R.J., Chen, T., Bajsa Hirschel, J.N., Schrader, K.K., Duke, S.O., Wykoff, D., Giuliano, R.M. 2020. Synthesis of pyranopyrans related to diplopyrone and evaluation as antibacterials and herbicides. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.0c02564.