Team:OhioState/Description

Cereal crops, such as maize, are an essential component of Ohio’s agricultural industry. Maize, the largest crop in Ohio, alone was valued to over 2 billion dollars gross revenue in 2018^[1]. Ohio corn does more than just stimulate the economy. Ethanol distilled from corn is combined with gasoline to produce cleaner way to fuel the world, producing less soot and emissions. As much as Americans love corn, cultivating such large quantities of corn has significant environmental impacts.

Close to home, Lake Erie shoulders much of Ohio’s fertilizer runoff. 50% of the fertilizers applied to corn remains unused and 10-20% of this runoff enters water systems leading to eutrophication^[2] . This has encouraged the toxicity of cyanobacterial harmful algal blooms, which presents issues of bacterial toxins entering water supplies of surrounding areas. The algal blooms may also reduce the biodiversity of Lake Erie, creating dead zones where oxygen is unable to dissolve for use of other aquatic animals^[3].

Like other cereal crops, corn (Zea mays) relies on nitrogen as a primary nutrient. Despite the abundance of nitrogen in the atmosphere, this form of nitrogen cannot be of use to cereal crops. Atmospheric nitrogen (N2) must be reduced by to ammonia (NH3) before it can be of use to plants.

This process is accomplished one of two ways:

Rhizobia and a few other types of bacteria can take in nitrogen from the atmosphere and fix it using nitrogenase into a form that plants can use in vital cellular processes. However, these symbioses are highly species specific, and extending this capability to other plants would require massive amounts of genetic engineering^[4].
The Haber-Bosch process has been used since the early 1920s to fix atmospheric nitrogen into chemical fertilizers^[5]. However, most crops can only incorporate about 50% of applied fertilizer and roughly 10-20% of the nitrogen fertilizer is leached into nearby waterways^[2]. This causes serious environmental damage including deterioration of soil conditions, eutrophication, and irreversible damage to nearby ecosystems.

Our project, Maizotroph, introduces the nitrogen-fixing capabilities of the Rhodopseudomonas species into identified bacterial endosymbionts of Zea mays.

Previous research from Oldroyd and Dixon indicates the possibility of supplementing crop plants with nitrogen-fixing bacteria, introducing nitrogen fixation to non-leguminous plants^[6], and work by SCU iGEM 2015 indicated that transformation of nitrogenase genes is possible^[7]. We have identified two bacterial species, Rhodopseudomonas palustris and Pseudomonas stutzeri, which both have well-characterized Nitrogen fixation gene clusters, but are not natural colonizers of corn^[8][9]. We plan to transfer these genes to Pseudomonas strains that do colonize corn^[10][11]. The clusters will be incorporated into the genetic machinery of Pseudomonas protegens PF5, P. putida KT2440, and sp. CT364 (whose root colonization ability was well characterized by Newcastle iGEM 2018)^[12]. We are exploring two methods of cloning these genes: via mini-CTX1 plasmid, and RP4::Mini-mu cloning. Once successful transformants are obtained, their ability to supplement Zea mays and arabidopsis growth in soil will be investigated. Their ability to colonize developing plant roots will be visualized and growth characteristics of colonized plants will be assessed.

References

[1] 2018 STATE AGRICULTURE OVERVIEW. (n.d.). Retrieved October 18, 2019, from https://www.nass.usda.gov/Quick_Stats/Ag_Overview/stateOverview.php?state=OHIO.

[2] Jagdish K. Ladha, Himanshu Pathak, Timothy J. Krupnik, J. Six, Chris van Kessel, Efficiency of Fertilizer Nitrogen in Cereal Production: Retrospects and Prospects, Advances in Agronomy, Academic Press,Volume 87, 2005, Pages 85-156, ISSN 0065-2113, ISBN 9780120007851, https://doi.org/10.1016/S0065-2113(05)87003-8.

[3] Nutrient Pollution - Eutrophication. (n.d.). Retrieved October 18, 2019, from https://oceanservice.noaa.gov/education/kits/estuaries/media/supp_estuar09b_eutro.html.

[4] Mus, F., Crook, M. B., Garcia, K., Costas, A. G., Geddes, B. A., Kouri, E. D., … Peters, J. W. (2016). Symbiotic Nitrogen Fixation and the Challenges to Its Extension to Nonlegumes. Applied and Environmental Microbiology, 82(13), 3698–3710. https://doi.org/10.1128/AEM.01055-16

[5] Lochheim, J. (2016, October 29). Fertilizer History: The Haber-Bosch Process. Retrieved October 18, 2019, from https://www.tfi.org/the-feed/fertilizer-history-haber-bosch-process.

[6] Oldroyd, G. E., & Dixon, R. (2014). Biotechnological solutions to the nitrogen problem. Current Opinion in Biotechnology, 26, 19–24. https://doi.org/10.1016/j.copbio.2013.08.006

[7] Team:SCU China/NitrogenFixation - 2015.igem.org. (n.d.). Retrieved June 13, 2019, from https://2015.igem.org/Team:SCU_China/NitrogenFixation

[8] Venieraki, A., Dimou, M., Vezyri, E., Vamvakas, A., Katinaki, P.-A., Chatzipavlidis, I., … Katinakis, P. (2014). The Nitrogen-Fixation Island Insertion Site Is Conserved in Diazotrophic Pseudomonas stutzeri and Pseudomonas sp. Isolated from Distal and Close Geographical Regions. PLoS ONE, 9(9), e105837. https://doi.org/10.1371/journal.pone.0105837

[9] Oda, Y., Samanta, S. K., Rey, F. E., Wu, L., Liu, X., Yan, T., … Harwood, C. S. (2005). Functional Genomic Analysis of Three Nitrogenase Isozymes in the Photosynthetic Bacterium Rhodopseudomonas palustris. Journal of Bacteriology, 187(22), 7784–7794. https://doi.org/10.1128/JB.187.22.7784-7794.2005

[10] Setten, L., Soto, G., Mozzicafreddo, M., Fox, A. R., Lisi, C., Cuccioloni, M., … Ayub, N. D. (2013). Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions. PloS One, 8(5), e63666. https://doi.org/10.1371/journal.pone.0063666

[11] Planchamp, C., Glauser, G., & Mauch-Mani, B. (2015). Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants. Frontiers in Plant Science, 5. https://doi.org/10.3389/fpls.2014.00719

[12] Newcastle iGEM 2018. (n.d.). Team:Newcastle - 2018.igem.org. Retrieved June 12, 2019, from https://2018.igem.org/Team:Newcastle