STEM

Our Class

This course focuses on scientific research and engineering. During the first part of the year, we conduct independent research projects that incorporate reviewing literature, making conjectures, developing methodology, designing experiments, and communicating findings. Our final projects are presented at a school-wide science fair, with the possibility for advancement to regional, state, and international fairs. During the second part of the year, we work in small teams in order to engineer assistive technology devices. We meet with clients, conduct patent searches, design and build prototypes, demonstrate our products to expert judges, and deliver the products to our clients. Throughout the course, we practice incorporating purpose, clarity, organization, mechanics, and audience appeal as they communicate about topics in science and technology. Assignments consist of research papers, short essays, technical reports, and presentations.

Application of Muropeptides for the Sustainable Management of Invasive Plant Species

My STEM project focuses on evaluating the feasibility of a biological invasive plant removal technique. In order to conserve biodiversity in ecosystems, the removal of exotic species is of utmost importance as they greatly harm other species through methods such as out competition and alteration of habitat among others. Many of the herbicides used in removing invasive plants have both positive and negative consequences as they harm water systems, human health, agriculture, and native plants and animals. I hypothesized that muropeptides, fragments of bacterial peptidoglycan, could trigger immune responses in plants that had receptors recognizing bacterial peptidoglycan strong enough to kill them. This way, invasive plants could be selectively eliminated from ecosystems as all other organisms that do not have the receptors would have no reaction to the biological agents.

Abstract

Invasive organisms threaten the health of fragile ecosystems and endangered animals. The removal of such invasive species is of great importance to maintain high biodiversity in ecosystems. Invasive plant removal is often achieved through several methods: chemical removal, biological removal, and mechanical removal; however, each method has its own limitations and shortcomings. The pitfalls include damage to water systems, harm to native plants and animals, and the disruption of the soil microbial communities. A potential alternative for the herbicides frequently used in removal processes is the application of muropeptides. Muropeptides, fragments of bacterial peptidoglycan, can induce immune responses in plant species that have pattern recognition receptors recognizing peptidoglycan. After gathering a plant sample and isolating plant RNA, it will be tested for receptors then later submitted to application of muropeptides. Through the implementation of a killing assay, perception of peptidoglycan in plants with and without receptors will be observed. This research will contribute to a better understanding of the various pattern recognition receptors in plants and is expected to find muropeptide application to be a new possible biological removal method for invasives.

Graphical Abstract

Phrase 1

Is there a method capable of effectively eliminating invasive plants while simultaneously preserving the health of surrounding species?

Phrase 2

Muropeptides and their immune-stimulating effects can be used to selectively kill invasive plant species in a sustainable and eco-friendly manner.

Background

Invasive organisms reduce biodiversity across the globe, damaging both ecosystems and species. These exotic animals and plants often replace the native species through various methods such as predation, alteration of habitat, disease transmission, and out competition (Kumar Rai & Singh, 2020). Many invasive plants, like Kudzu, an invasive Chinese arrowroot, possess the ability to overshadow entire forests of native species, inhibiting their access to sunlight, and in turn, deactivating their natural photosynthesizing ability. Exotic species benefit from their evolutional abilities, that are in balance with their natural ecosystem, when relocated into previously uninhabited environments (Meyer et al., 2021). Lacking predators and parasites, these species can uncontrollably thrive, throwing off the biological system of checks and balances, and predators and prey. As climate change accelerates, the spread of invasive species is expected to intensify, further disrupting ecosystems, and threatening biodiversity, making it crucial to address this growing issue now to safeguard the health of our planet's natural systems. The strongest natural defense against climate change is biodiversity, only attained and upheld by the abatement of invasive species (Biodiversity - our strongest natural defense against climate change, n.d.). To limit the invasive plant presence in ecosystems, several removal techniques exist: mechanical, chemical, and biological. Mechanical removal involves cutting or pulling the plants out of the ground and is extremely labor intensive. This method limits environmental impact but is difficult and exhaustive to execute. Conversely, using chemicals to eliminate invasive species is effective and resource-efficient but has the potential to release toxins into the environment, posing risks to surrounding wildlife and water systems. Biological removal requires the use of natural enemies, such as plant diseases or insect predators, that will either outcompete the invasive species or directly target it. However, biological removal can have inadvertent consequences and requires high amounts of research on all possible outcomes as to not introduce control agents that can harm non-target species and disrupt the balance of the ecosystem, potentially resulting in further loss of biodiversity (Pearson et al., 2021). Both chemical and biological removal have the potential to drive hundreds of species to extinction if not used properly and with thorough testing. Many of the herbicides used in killing invasive plant species damage surrounding native plants and have immense negative environmental impacts. There is no established method capable of effectively eliminating invasive plants while simultaneously preserving the health of surrounding species. This research seeks to investigate the escalating expansion of invasive, non-native plant species that have caused widespread degradation of the environment, water systems, agriculture, biodiversity, and essential ecosystem services in regions worldwide. Muropeptides are a main component of bacterial cell wall peptidoglycan, which is a crucial structural component that provides rigidity and strength in bacteria and helps them protect against osmotic pressure. Peptidoglycan receptors have been identified in some plant species, but very little research has been conducted on how they work (Andrea A. Gust, 2015). However, it has been established that different plant immune responses are linked to variations in the carbohydrate or peptide parts of the peptidoglycan (Erbs et al., 2008). Observing the effects of peptidoglycan on plants, this research has the potential to address the substantial environmental damage caused by invasive, exotic species across the globe. By gaining a deeper understanding of the mechanisms behind their spread and impact, this work could lead to more effective strategies for mitigating their disruptive effects on ecosystems. Invasive species are known to threaten biodiversity, alter habitats, and disrupt ecological balance, and thus, efforts to control and prevent their development could have profound benefits for the preservation of natural environments and the health of ecosystems worldwide.

Procedure

This study will begin by assessing pattern recognition of various plants, invasive and native. Arabidopsis thaliana will serve as my positive control, and human peripheral blood mononuclear cells (PMBCs). RNA from each plant species and control will be isolated and tested in a lab setting. To do this, a Qiagen RNeasy RNA isolation kit will be used. Data on the purity and amount of RNA will be collected and subsequently analyzed. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) will be performed to determine which pattern recognition receptors are exhibited by the sample plant species. The first step, reverse transcription, involves RNA extraction, conversion to cDNA, and priming. The next step will be Polymerase Chain Reaction (PCR) which requires amplification, thermal cycling, and lastly exponential amplification. Muropeptides will be purified then applied to new plant samples. The application will involve another test regarding whether the roots or leaves are better for maximizing the absorption. To do this, a well-characterized reporter gene such as β- glucuronidase will be used to express color change after the application of peptidoglycan.

Figure 3

The figure above relates the relative expression of the target pattern recognition receptor, CERK1 (Chitin Elicitor Receptor Kinase 1), in each plant in the sample as well as the negative control, H2O. CERK1 is a pattern recognition receptor (PRR) in plants that detects chitin, a key component of fungal cell walls. Upon recognizing chitin, CERK1 activates immune signaling pathways to trigger defense responses against fungal pathogens.

Figure 4

The figure above relates the relative expression of the target pattern recognition receptor, LYM1, in each plant in the sample as well as the negative control, H2O. LYM1 is a lysin-motif receptor-like protein that functions as a pattern recognition receptor (PRR) in plants, where it helps detect bacterial cell wall components such as peptidoglycan. By recognizing these microbial signatures, LYM1 plays a critical role in activating the plant's innate immune responses against potential pathogens.

Analysis

The RNA isolation data illustrates the amount of RNA and the purity of the RNA in each plant species and the positive control. A260/A280 and A260/A230 are measurements of a sample of RNAs purity.
The results of the qPCR test are as seen in Figures 2, 3, and 4 above. The figures illustrates the expression of three target genes, along with a negative control (H2O), in the selected plant sample. Bittersweet had a relative expression of 7 for the FLS2 pattern recognition receptor and the pea plant had a relative expression of 2 while Arabidopsis had 0.2. Arabadopsis expresses the CERK1 Chitin receptor the most at a relative expression level of 0.4 while Bittersweet was lower at 0.03. The LYM1 pattern recognition receptor recognizing bacterial peptidoglycan was expressed the highest by Arabidopsis at a relative expression level of 1.0 and the pea plant and Bittersweet were both lower at around 0.5.

Discussion/Conclusions

As anticipated, Arabidopsis thaliana successfully expressed all three genes of interest. This confirms that the experiment was conducted properly as the entire genome of this plant has been sequenced and published upon. The data suggests that muropeptides could be a viable method for biological removal because of how the LYM1 pattern recognition receptor was expressed in both the Bittersweet and pea plants. One significant consideration is that the experiment is an n of 1, meaning it was performed only once and for its results to carry more significance and lead to stronger conclusions, the experiment needs to be repeated. For qPCR tests to be conclusive, they must be conducted multiple times. A limitation in this experiment is how the replicants varied from one another. This inconsistency could be due to impurities in the samples that could have potentially affected the accuracy of the results. It is crucial that the results are reproducible across multiple trials to draw definitive conclusions. With the data collected to this point, it can be concluded that pattern recognition receptors recognizing bacterial peptidoglycan, LYM1, are conserved in Bittersweet and pea plants. Testing on the other hypotheses is yet to be completed.
This project conforms with the understanding of LYM1 and peptidoglycan in Arabidopsis Thaliana presented by Erbs et al. 2008. This study explored the innate immune response of plants to peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas in Arabidopsis Thaliana. Along with this study, the results of my project demonstrate the expression of PRRs recognizing bacterial peptidoglycan in Arabidopsis Thaliana.
Future research for this project includes repeating qPCR tests so that the results can carry more significance. The next step is to do functional assays involving the reporter gene beta glutaminase that uniformly models the PR1 gene transcription through visible color change. This way, the ideal concentration of muropeptides can be observed. The beta glutaminase is in a genetically modified recombinant Arabidopsis plant. PR1 gene transcription is associated with the innate immune system’s defense against pathogens or immune response (Erbs et al., 2008).

References

Crystal-Ornelas, R., Hudgins, E. J., Cuthbert, R. N., Haubrock, P. J., Fantle-Lepczyk, J., Angulo, E., Kramer, A. M., Ballesteros-Mejia, L., Leroy, B., Leung, B., López-López, E., Diagne, C., & Courchamp, F. (2021). Economic costs of biological invasions within North America. NeoBiota, 67(67), 485–510. https://doi.org/10.3897/neobiota.67.58038

Erbs, G., Silipo, A., Aslam, S., De Castro, C., Liparoti, V., Flagiello, A., Pucci, P., Lanzetta, R., Parrilli, M., Molinaro, A., Newman, M. A., & Cooper, R. M. (2008). Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity: structure and activity. Chemistry & Biology, 15(5), 438–448. https://doi.org/10.1016/j.chembiol.2008.03.017

Gust, A. A. (2015). Peptidoglycan Perception in Plants. PLoS Pathogens, 11(12). https://doi.org/10.1371/journal.ppat.1005275

Gust, A. A., Biswas, R., Lenz, H. D., Rauhut, T., Ranf, S., Kemmerling, B., Götz, F., Glawischnig, E., Lee, J., Felix, G., & Nürnberger, T. (2007). Bacteria-derived Peptidoglycans Constitute Pathogen-associated Molecular Patterns Triggering Innate Immunity in Arabidopsis. Journal of Biological Chemistry, 282(44), 32338–32348. https://doi.org/10.1074/jbc.m704886200

Kumar Rai, P., & Singh, J. S. (2020). Invasive alien plant species: Their impact on environment, ecosystem services and human health. Ecological Indicators, 111, 106020. https://doi.org/10.1016/j.ecolind.2019.106020

Lamkanfi, M., & Walle, L. (2016). Europe PMC. Europepmc.org. https://europepmc.org/article/med/27404251

Meyer, S. E., Callaham, M. A., Stewart, J. E., & Warren, S. D. (2021). Invasive Species Response to Natural and Anthropogenic Disturbance. In Invasive Species in Forests and Rangelands of the United States, 85–110. https://doi.org/10.1007/978-3-030-45367-1_5

National Invasive Species Information Center. (n.d.). Www.invasivespeciesinfo.gov. https://www.invasivespeciesinfo.gov/subject/climate-change

Patten, K., O’Casey, C., & Metzger, C. (2017). Large-Scale Chemical Control of Smooth Cordgrass (Spartina alterniflora) in Willapa Bay, WA: Towards Eradication and Ecological Restoration. Invasive Plant Science and Management, 10(3), 284–292. doi:10.1017/inp.2017.25

Pearson, D. E., Clark, T. J., & Hahn, P. G. (2021). Evaluating unintended consequences of intentional species introductions and eradications for improved conservation management. Conservation Biology. https://doi.org/10.1111/cobi.13734

Qiao, P., Wang, A., Xie, B., Wang, L., Han, G., Mei, B., & Zhang, X. (2019). Effects of herbicides on invasive Spartina alterniflora in the Yellow River Delta. Acta Ecologica Sinica, 39(15), 5627–5634.

Saur, I. M. L., Panstruga, R., & Schulze-Lefert, P. (2020). NOD-like receptor-mediated plant immunity: from structure to cell death. Nature Reviews Immunology, 21. https://doi.org/10.1038/s41577-020-00473-z

United Nations. (n.d.). Biodiversity - our strongest natural defense against climate change. United Nations. https://www.un.org/en/climatechange/science/climate-issues/biodiversity

USDA. (n.d.). Economic and Social Impacts | National Invasive Species Information Center | USDA. National Invasive Species Information Center (NISIC). https://www.invasivespeciesinfo.gov/subject/economic-and-social-impacts

Use of Herbicides for Invasive Plant Control. (n.d.). Mass Audubon. https://www.massaudubon.org/nature-wildlife/invasive-plants-in-massachusetts/herbicides

Willmann, R., Lajunen, H. M., Erbs, G., Newman, M.-A., Kolb, D., Tsuda, K., Katagiri, F., Fliegmann, J., Bono, J.-J., Cullimore, J. V., Jehle, A. K., Gotz, F., Kulik, A., Molinaro, A., Lipka, V., Gust, A. A., & Nurnberger, T. (2011). Arabidopsis lysin-motif proteins LYM1, LYM3, CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection. Proceedings of the National Academy of Sciences, 108(49), 19824–19829. https://doi.org/10.1073/pnas.1112862108

Xie, B., Han, G., Qiao, P., Mei, B., Wang, Q., Zhou, Y., Zhang, A., Song, W., & Guan, B. (2019). Effects of mechanical and chemical control on invasive Spartina alterniflora in the Yellow River Delta, China. PeerJ, 7, e7655. https://doi.org/10.7717/peerj.7655

Zenni, R. D., Essl, F., García-Berthou, E., & McDermott, S. M. (2021). The economic costs of biological invasions around the world. NeoBiota, 67, 1–9. https://doi.org/10.3897/neobiota.67.69971

Zipfel, C. (2014). Plant pattern-recognition receptors. Trends in Immunology, 35(7), 345–351. https://doi.org/10.1016/j.it.2014.05.004