Team:Tec-Chihuahua/Description

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Inspiration
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Description

Inspiration

When we first heard about iGEM, we also heard about the wonders of synthetic biology, and were more than inspired by the projects we read about. Learning about all the things that had been done only reaffirmed the notion that not even the sky was our limit. So what a better idea than to use all these incredible tools to solve a real world issue in such a way that we could notice an impact in our own community?

Our team is comprised of people who have lived most of their lives in the state of Chihuahua. Knowing our home and our people for so many years and in so many different aspects, we wanted to do something that would truly influence our community. We wanted to create a meaningful change in our surroundings.

Before being part of iGEM Tec-Chihuahua 2019, some of our team members had the chance to meet and work with people immersed in the rural area, while others had developed small research projects in our school regarding agronomic issues. Somehow all of us had some sort of experience with agriculture and its importance for Mexican people.

For our country, agriculture fulfills the nutritional needs of millions of citizens, preserves natural environments, stimulates progress by improving the quality of life in rural areas, and has also been ingrained in our culture and traditions since the very beginning.1

A certain concern seemed to stand out among the people our team members knew and had worked with. Both agronomic engineers and crop producers involved in the cotton agricultural business mentioned Verticillium wilt. This is a disease caused by phytopathogenic fungus Verticillium dahliae that wilts their crops and has only worsened with every new season. This particular disease has been one of their major problems for more than 7 years, and still, they haven’t received any help whatsoever. This year the overall production is expected to lower considerably compared to previous seasons. One of the principal measures agricultors use as an attempt to control this situation is applying great amounts of agrochemicals; representing a hazard regarding soil, water, and air pollution.

Unfortunately, so far there is no product or efficient treatment to deal with this fungal infection. Besides, the lack of proper methods to detect this disease in its earliest stages represent another crucial concern.

Once we learned about this and heard just how much impact it represents to our community, we decided we would do something about it. We looked forward to using synthetic biology to reach the best possible solution to this issue. One that aids in Verticillium dahliae’s inhibition, while preserving our environment.

The fact that we found a problem people were concerned about, hasn’t been effectively solved, and affected a relevant cause for our population that could also escalate to a worldwide issue, was more than enough to fire up our motivation and led us to develop what today we call V-TION.

Description

Verticillium dahliae has a wide range of hosts, overall affecting more than 300 species, including cotton, strawberries, raspberries, blueberries, potatoes, lettuce, herbaceous plants, perennial woods, among many others. But more importantly, its effects are worldwide.2

Figure 1. Regions affected by Verticillium wilt

China is the world’s second biggest cotton producer and Verticillium wilt’s rates have been increasing in the past decade. V. dahliae, and another fungal wilting disease, whose incidence is decreasing, have had a great impact in the country’s cotton production, losing more than 150,00 tons per year.3

Meanwhile, in the US cotton wilt causes annual losses of around 0.5% and 3.5% of the crops at a national level. In certain states such as New Mexico, these rates have reached 5% and in Alabama cropping fields have decreased 1.5% of their extension. These losses translate into roughly 3 million dollars.4

In Mexico, cotton production is highly important. Over 80% of our county's cotton demand is met by national production. In 2018, cotton production represented a profit of over $10,343,005,600(MXN) for the country.5 Chihuahua, our state, is the biggest cotton producer in Mexico with its yield being 70.2% of all national production.1

Currently, given that there is not a Verticillium dahliae aimed product, cotton producers turn to the conventional approach. They add agrochemicals to their crops without any proper instructions and in unawareness of the potential consequences. This elicits unwanted effects, such as eliminating non-target organisms and affecting the food chain, while posing high risks to the environment and human health. Over 3 million agricultors have suffered from serious poisoning, while 25 million from mild poisoning.6

Even trophic chains have been affected by this. A given example is the bee population reducing a 36% due to the excessive use of triazoles.7

Another method employed to reduce Verticillium dahliae caused infections is crop rotation, which consists of alternating plants with different nutritional needs in one place during different cycles; but ,unfortunately, it can result in disease propagation. Another frequently used technique is solarization. It refers to soil disinfestation through the heat generated from captured solar energy, but despite its positive outcomes, it demands a great amount of time.8

What about this fungus?

Verticillium dahliae is stimulated by humid soils and temperatures between 21°C and 27°C (70°F-80°C). This makes tropical and subtropical regions a suitable environment for its growth. The fungus can survive winters in its mycelial form into perennial hosts, plant debris, and parts of vegetative propagation.9

Life cycle and action mechanism

It can be divided into three phases:

Latent phase: as microsclerotia, made from melanin, Verticillium dahliae can survive in the soil from a single year to over two decades. Once roots exudates are found in the surroundings, the microsclerotia starts to germinate and spreads inside the plant through its mycelial form or as spores called microconidia. Once this happens, it travels into the plant’s vascular system, worsening the infection.10

Parasitic phase: The fungus colonizes the plant’s structure, especially its roots and stems,producing in turn more microsclerotia. Infected plant tissue turns necrotic since the vascular system is obstructed with mycelia, conidias, and fungal metabolism byproducts. The pathogen itself produces a glue-like substance that also obstructs the plant’s vessels, provoking water blockage and tissue wilting.11

Saprophytic phase:the plant dies, therefore the microsclerotia gets into the soil and starts with the cycle all over again.12

Usually, Verticillium wilt symptoms vary among hosts, but certain common symptoms can be pinpointed: premature leaf chlorosis, necrosis and vascular discoloration in stems and roots.13

Methodology

Looking forward to helping our environment and the cotton industry, to develop a systemic, preventive, and treating biofungicide using recombinant antifungal peptides as active ingredients is the goal. So, in order to fulfil our objective, the following methodology was designed to turn our project from an idea to reality.

BioBrickTM Design: a total of five DNA sequences were designed to express recombinantly the peptides in E. coli. The constructs count with the essential parts needed for their correct translation into proteins, purification, and proper folding. Recombinant production of disulfide bonded peptides, like PsDEF1 and WAMP1b, can be complicated in prokaryotic systems, thus we have as well created DNA sequences for the co-expression of these peptides with Erv1p, a disulfide bond forming protein.14

E. coli Cells Transformation: chemically competent E. coli cells are prepared and transformed with our vectors. E. coli BL21 (DE3) was chosen as a chassis for the expression of AtPFN1 for being ideal for protein expression. In the case of PsDef1 and WAMP1b, E. coli SHuffle T7 Express, a specially designed strain to aid in the proper formation disulfide bonds, was chosen as an expression chassis.

IPTG induced protein expression: after the cells are correctly transformed, expression of the peptides can be induced and controlled by Isopropyl β-D-1-thiogalactopyranoside (IPTG); both BL21 (DE3) and SHuffle are T7 expression strains. Once IPTG is added to these strains, T7 RNA polymerase is produced and capable of transcribing downstream of promoters like those found in our DNA constructs.15

Soluble Protein Extraction: after induction and a period of incubation, cells are lysed through a series of freeze/thaw cycles with the help of a lysis solution and sonication. The lysate is then centrifuged to obtain the cells' soluble proteins in the supernatant.

Protein Purification: our peptides are designed to be expressed as fusion proteins with a 6x His-Tag. This allows the peptides to be purified through immobilized metal affinity chromatography, a purification procedure based on the interaction of metal ions with polyhistidine sequences in tagged proteins.16

Antifungal Assay: next, the antifungal activity of the peptides can be tested against V. dahliae. For this, a spore suspension is treated with the purified peptides individually, as well as a mixture of all three of them to prove their individual and collaborative effect against the fungus.

Delivery: nanoencapsulation has been shown to be a promising alternative to deliver active ingredients more efficiently and accurately. This is the main reason to get our recombinant peptides nanoencapsulated by creating a chitosan-poly(lactice) copolymer nanocapsule. 17

Our peptides

WAMP1b


WAMP1b is part of the WAMPs antimicrobial peptides, a type of chitinase-like peptides from Triticum kiharae. These antimicrobial peptides are also called hevein-like proteins due to their similarity to hevein and for having a chitin binding site similar to chitinases.18 The peptide’s action mechanism consists in the inhibition of fungal defense metalloproteases called fungalysins. This metalloproteases cleave chitinases, plant proteins that degrade chitin found in the fungal cell wall.19 Since V. dahliae produces fungalysin, WAMP1b can be used to inhibit the fungus’ defense system, thus decreasing its pathogenicity.20

AtPFN1


AtPFN1 is a profilin from Arabidopsis thaliana. Profilins are proteins that bind to chitin, an important mechanism for their antifungal action. It degrades spores and hyphae cell wall, subsequently accumulating in the cytoplasm where it generates reactive oxygen species and induces apoptosis. AtPFN1 has been shown to break down fungal cell walls, even in the fungus dormant, spore phase, ideally preventing the spread of V. dahliae and other fungi.21

PsDef1


PsDef1 stands for Pinus sylvestris Defensin 1, a plant defensin that has been shown to produce morphological changes to fungal mycelium. It presents significant similarities to RsAFP2 ( Raphanus sativus antifungal peptide). RsAFP2, upon binding to GlcCer, produces reactive oxygen species inside the fungal cell, effectively killing it from the inside.22 Additionally, PsDef1 contains four disulfide bonds which grant it resistance against environmental changes.23


The peptides’ nature and the delivery properties of the nanocapsule will allow the biofungicide to act both as a preventive and treating product.

Once the nanoencapsulation is achieved, the product could be lyophilized and turned into wettable powder (WP). In this presentation it can be applied to the crops through the irrigation system. After completing a field research, we realized people involved in agriculture learned about new agricultural products through their technical assessors, who in turn learned about them through distributors. Considering this informational chain, our aim is that the final product reaches the agrochemical products development company.

Finally, bibliographic and field research were carried out, seeking for the final product to assure the greatest benefit for all parties involved (cotton producers, textile industry, cotton consumers, distributors, the environment, among many others). Through interviews the producer’s wishes were validated and through detailed consultation of NOMs like NOM-232-SSA1-2009 and NOM-045-SSA1-1993, the proposal of a law-abiding project was aimed for. Likewise, a thorough risk analysis was developed, considering any unfavorable situation our project might pose.

References

  1. SAGARPA. (2017). Planeación Agrícola Nacional. Retrieved from: https://www.gob.mx/cms/uploads/attachment/file/255627/Planeaci_n_Agr_cola_Nacional_2017-2030-_parte_uno.pdf
  2. Farr, D., & Rossman. (2014). Fungal Databases, Systemic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from: http://nt.ars-grin.gov/fungaldatabase
  3. Li, X., Zhang, Y., Ding, C., Xu, W., & Wang, X. (2017). Temporal patterns of cotton Fusarium and Verticillium wilt in Jiangsu coastal areas of China. Scientific Reports, 7(1). doi:10.1038/s41598-017-12985-1
  4. Land, C., Lawrence, K., & Newman, M. (2016). First Report of Verticillium dahliae on cotton in Alabama. APS Publications. https://doi.org/10.1094/PDIS-10-15-1143-PDN
  5. SIAP. (2018). Estadísticas de Producción Agrícola. Retrieved from: http://infosiap.siap.gob.mx/gobmx/datosAbiertos.php
  6. Özkara, A., Akyil, D., & Konuk, M. (2016). Pesticides, Environmental Pollution, and Health. Environmental Health Risk - Hazardous Factors to Living Species. doi:10.5772/63094
  7. Mahmood, I., Imadi, S. R., Shazadi, K., Gul, A., & Hakeem, K. R. (2016). Effects of Pesticides on Environment. Plant, Soil and Microbes, 253–269. doi:10.1007/978-3-319-27455-3_13
  8. FAO. (2004). Manejo de malezas para países en desarrollo. Retrieved from: http://www.fao.org/3/y5031s/y5031s00.htm#Contents
  9. Miroux, B., & Walker, J. E. (1996). Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels. Journal of Molecular Biology, 260(3), 289– 98. doi:10.1006/jmbi.1996.0399
  10. Berlanger, I., & Powelson, M. (2005). Verticillium wilt. The Plant Health Instructor. doi: 10.1094/PHI-I-2000-0801-01
  11. Fradin, E., & Thomma, B. (2006). Physiology and molecular aspects of Verticillium wilt diseases caused by V. dahliae and V.albo-atrum. Molecular Plant Phatology, 7(2), 71-86. doi: 10.1111/j.1364-3703.2006.00323.x
  12. Goud, J.-K. C., Termorshuizen, A. J., & Gams, W. (2003). Morphology of Verticillium dahliae and V. tricorpus on semi-selective media used for the detection of V. dahliae in soil. Mycological Research, 107(7), 822–830. doi:10.1017/s09537562030080
  13. Missouri Botanical Garden. (2003). Verticillium wilt. Retrieved from: http://www.missouribotanicalgarden.org/gardens-gardening/your-garden/help-for-the-home-gardener/advice-tips-resources/pests-and-problems/diseases/cankers/verticillium-wilt.aspx
  14. Lee, J.-E., Hofhaus, G., & Lisowsky, T. (2000). Erv1pfromSaccharomyces cerevisiaeis a FAD-linked sulfhydryl oxidase. FEBS Letters, 477(1-2), 62–66. doi:10.1016/s0014-5793(00)01767-1
  15. Miroux, B., & Walker, J. E. (1996). Over-production of Proteins inEscherichia coli: Mutant Hosts that Allow Synthesis of some Membrane Proteins and Globular Proteins at High Levels. Journal of Molecular Biology, 260(3), 289– 98. doi:10.1006/jmbi.1996.0399
  16. Gaberc-Porekar, V., & Menart, V. (2001). Perspectives of immobilized-metal affinity chromatography. Journal of biochemical and biophysical methods, 49(1-3), 335-360.
  17. Xu, L., Cao, L.-D., Li, F.-M., Wang, X.-J., & Huang, Q.-L. (2014). Utilization of Chitosan-Lactide Copolymer Nanoparticles as Controlled Release Pesticide Carrier for Pyraclostrobin AgainstColletotrichum gossypiiSouthw. Journal of Dispersion Science and Technology, 35(4), 544–550. doi:10.1080/01932691.2013.800455
  18. Nawrot, R., Barylski, J., Nowicki, G., Broniarczyk, J., Buchwald, W., & Goździcka-Józefiak, A. (2013). Plant antimicrobial peptides. Folia Microbiologica, 59(3), 181–196. doi:10.1007/s12223-013-0280-4
  19. Slavokhotova, A. A., Naumann, T. A., Price, N. P., Rogozhin, E. A., Andreev, Y. A., Vassilevski, A. A., & Odintsova, T. I. (2014). Novel mode of action of plant defense peptides–hevein‐like antimicrobial peptides from wheat inhibit fungal metalloproteases. The FEBS journal, 281(20), 4754-4764. doi: 10.1111/febs.13015
  20. Naumann, T. A., & Wicklow, D. T. (2013). Chitinase modifying proteins from phylogenetically distinct lineages of Brassica pathogens. Physiological and molecular plant pathology, 82, 1-9. doi: https://doi.org/10.1016/j.pmpp.2012.12.001
  21. Park, S.-C., Kim, I. R., Kim, J.-Y., Lee, Y., Kim, E.-J., Jung, J. H., … Lee, J. R. (2018). Molecular mechanism of Arabidopsis thaliana profilins as antifungal proteins. Biochimica et Biophysica Acta (BBA) - General Subjects, 1862(12), 2545–2554. doi:10.1016/j.bbagen.2018.07.028
  22. Khairutdinov, B. I., Ermakova, E. A., Yusypovych, Y. M., Bessolicina, E. K., Tarasova, N. B., Toporkova, Y. Y., … Nesmelova, I. V. (2017). NMR structure, conformational dynamics, and biological activity of Ps Def1 defensin from Pinus sylvestris. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1865(8), 1085–1094. doi:10.1016/j.bbapap.2017.05.012
  23. Kovalyova, V. A., Gout, I. T., Kiyamova, R. G., Filonenko, V. V., & Gout, R. T. (2007). Cloning and analysis of defensin 1 cDNA from Scots pine. Biopolymers and Cell, 23(5), 398-404. doi: http://dx.doi.org/10.7124/bc.000779

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