Team:Stony Brook/Poster

Poster: Stony_Brook



LightSwitch

Team Members
Laiba Ahmed, Adit Anand, Yashasvi Bajaj, Justin Beutel, Abhishek Cherath, Alexis Choi, Simranjeet Kaur, Aaliyah Kaushal, Maheen Khan, Melissa King, Chiu Yin Lee, Aneeqa Majid, Julia Petreczky, and Andrew Sillato

Project Advisors
Dr. John Peter Gergen, Dr. Gábor Balázsi, Dr. Steven Glynn, Dr. Joshua Rest

Abstract
Genetically modified (GM) crops have seen widespread adoption in large-scale agriculture, given their potential to improve commercial farming yields and mitigate crop losses from pests and pathogens. With widespread adoption, they could also increase the incidence of gene flow—the transfer of genetic variation across populations—from transgenic to wild crops, threatening biodiversity. Hence, a solution is proposed wherein an optogenetic killswitch, introduced in Nicotiana benthamiana, preventing plant development upon exposure to UV-B light (~311 nm). Through the optogenetic pair comprised of ULTRAVIOLET RESPONSE LOCUS 8 (UVR8) with attached tetracycline repressor domain (TetR) and CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) with attached VP16 transactivation domain, the transcription of synthetic trans-acting small interfering RNAs (syn-tasiRNAs) will be controlled. These syn-tasiRNAs will disrupt the CLAVATA-WUSCHEL signaling pathway through the knockdown of the WUSCHEL (WUS) gene. Stem cells in the shoot apical meristem (SAM) will differentiate, causing stem cell depletion and prevention of further plant growth.
Introduction

Genetically modified (GM) crops such as corn, cotton, and soybeans have been utilized by farmers for decades as a means of improving crop yields and mitigating crop losses due to pests and pathogens[1]. Yet, controversy surrounding the use of GM crops continues to exist today. On one hand, scientists claim that the benefits of GM crops including increased nutrient uptake and reduced dependence on pesticides significantly outweigh their detriments. On the other hand, part of the general public claims that GM crops are “unnatural” and should not be used. We believe this discrepancy can be partially mitigated if there was a way to introduce more control over GM crops. If there was a system that prevented GM crops from inflicting harm outside the controlled environment that they are grown in, both consumers and farmers would be less hesitant to grow them. This is why we created LightSwitch!
Design
CLAVATA-WUSCHEL pathway

The shoot apical meristem (SAM) forms in the plant embryo and contains stem cells that will eventually give rise to all above-ground plant structures. The SAM serves to maintain the active stem cell population in a reservoir at the apex that replenishes the differentiated cells. The organizing center (OC) at the core of the SAM sustains this stem cell population[2]. Additionally, it generates new organs as primordia on meristem flanks. The fate of each SAM cell is determined by positional information based on the domain they are in. These layers are necessary for cells to assess their relative positions in the meristem and behave coordinately with their neighbors.

The CLAVATA-WUSCHEL pathway helps to convey intercellular signals that are critical for shoot and floral meristem maintenance in higher plants. It helps to repress and activate gene transcription in the shoot apical meristem. This is a negative feedback loop that has a CLV3 signal received by the CLV1 receptor or the CLV2 to ultimately limit stem cell production by repressing WUS expression in the OC. CLV3 encodes a signaling peptide that interacts with plasma-membrane localized receptor-like kinases (RLKs) such as CLV1 and CLV2. This triggers a signaling cascade that ultimately down-regulates WUS transcription by restricting the WUS expression domain to the OC. WUS protein moves between cells via plasmodesmata into the apical stem cell domain, where it maintains stem cell fate and and induces the expression of CLV3 genes in a dosage-dependent fashion.



A generalized schematic of the CLAVATA-WUSCHEL pathway

Syntasi-RNA biogenesis

Synthetic trans-acting small interfering RNAs (syntasi-RNAs) will be used to target and degrade WUSCHEL (WUS) mRNAs. The WUSCHEL gene, in the organizing center of the shoot apical meristem (SAM), promotes the maintenance of stem cell populations. Syn-tasiRNA biogenesis begins with the transcription of a syn-tasiRNA precursor with RNA Polymerase II. This single-stranded precursor undergoes Argonaute 1 (AGO1)-mediated cleavage guided by a co-expressed microRNA, miR173. The cleaved syntasi-RNA is then acted upon by RNA-dependent RNA polymerase 6 (RDR6), forming a double-stranded RNA which is cleaved by Dicer-like 4 (DCL4)[3]. This results in the formation of a mature, double stranded long syntasi-RNA, and it is loaded into Argonaute 2 (AGO2), which cleaves the syntasi-RNA passenger strand. The complex of the guide strand and AGO2 then forms the RNA-Induced Silencing Complex (RISC) with the guide strand and mRNA[4]. This silencing would not be isolated to the cells which produce interfering RNAs; syntasi-RNAs can actually move symplastically through the plasmodesmata from N. benthamiana leaves to the SAM to act on WUS mRNAs.


UV-B light inducible gene expression system

Parts

In order to achieve this goal of minimizing gene flow, the Stony Brook iGEM 2020 team has designed an optogenetic kill switch that aims to halt plant development upon exposure of the plant to a specific frequency of light. It incorporates a UV-B light sensitive protein, namely UV-B RESISTANCE LOCUS 8 (UVR8), and its respective binding partner CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1). We take advantage of the ability of UVR8 to monomerize upon exposure to UV-B light (311nm) to induce transcription of a syn-tasiRNA that is designed to bind to WUSCHEL transcript. This will prevent stem cells within the meristem from proliferating.

UVR8 protein is fused to a TetR DNA-binding domain and COP1 is fused to a VP16 activation domain. A flexible Gly4Ser3 linker is utilized for the fusion of these proteins. Furthermore, a self-cleaving T2A peptide is flanked in between these two inserts to produce a polycistronic transcript. These inserts are under the control of a 35S CaMV promoter and terminator. The light responsive promoter consists of a 4x tetO repeat that is bound by the TetR domain. The binding of UVR8-COP1 to the light responsive promoter initiates transcription of the tasi-RNA.




COP1, UVR8, and tasi-RNA inserts

Modeling
I-TASSER

Homology modeling gave us a set of probable structures of the full-length UVR8 monomer, each of which were assigned a confidence score, allowing us to choose the most likely full-length structure of the UVR8 monomer.


Predicted UVR8 structure

COP1 Mutagenesis

We learned that COP1 contained a number of residues which could be altered to abolish or weaken interactions of COP1 with UVR8. Since COP1 sits at a crossroads between many different signaling and gene regulatory pathways, diminishing its interactions with other transcription factors is hypothesized to mitigate any off target effects caused by implementing the designed optogenetic switch in plants. Residues W467 and R465 were identified as adequate targets for mutation as W467 is responsible for many hydrophobic interactions and R465 facilitates charge-charge interactions in the C-terminal tails of COP1’s binding partners[5].

It was hypothesized that a mutation of W467 to phenylalanine might weaken general binding of partners to COP1 though weakening or alteration of hydrophobic interactions. Additionally, it was hypothesized that a mutation of R465 to glutamate would not significantly affect UVR8-COP1 interaction. Unfortunately, these hypotheses could not be examined in great detail. However, the simulations showed that, despite mutation of COP1, COP1-UVR8 interaction is preserved. A total of three mutants were produced: COP1-W467F, COP1-R465E and COP1-R465E/W467F.


COP1-W467 (WT) (top left), COP1-W467F (top right), COP1-R465 (WT) (bottom left), COP1-R465E (bottom right)

Simulating UVR8 and COP1 & COP1 Mutants Together

Both proteins were opened into PyMOL and oriented such that both COP1’s binding interface and the C-terminal extension of UVR8 were close to one another in space.


COP1 (red) and UVR8 (blue)

Human Practices
Indoor Farmers
HeartBeet Farms, Square Roots Farms, Babylon Micro-farms, and the Stony Brook University Life Sciences Greenhouse

We spoke with both indoor farmers, outdoor farmers, and a greenhouse curator in order to gain a better idea of how our research would impact the field. The consensus was that our project would be helpful, but there is not a demand for GMOs in the indoor or vertical farming industry yet, as consumers prefer eating "organic" produce over their GM counterparts.


Dr. Kristen Shorette
Environmental Sociology professor at Stony Brook University

Dr. Kristen Shorette helped us learn that the term “GMO” has a very negative connotation due to its frequent association with certain big corporations. These corporations, which are known for their unethical business practices to produce GM food, cause people to avoid them. We were advised to focus our efforts on teaching the general public about the benefits of GMOs before we attempted to implement our killswitch in a real-life setting.


Dr. Vladislav Verkhusha
Anatomy and Structural Biology professor at the Albert Einstein College of Medicine

One of our previous design idea was to upregulate bax with the BphP1-QPAS1 system to promote apoptosis in plant tissue. Our meeting with Dr. Verkhusha highlighted important issues, such as how introducing the BphP1 system into plants cells would be more challenging than it appears to be. This is due to the significant difference between apoptotic mechanisms found in mammalian cells and plant cells.


Dr. Robert M. Hughes
Assistant professor at East Carolina University

We spoke with Dr. Hughes to improve our understanding of how Bax recruitment to the outer mitochondrial membrane works and how to initiate subsequent oligomerization through an optogenetic system. Although we did not settle on this design idea, we gained further appreciation for how the optogenetic apoptosis mechanism works.


Dr. Kate Creasey
President and Founder of the Grow More Foundation

Our meeting with Dr. Creasey advised us to focus on a more direct approach with our light-inducible system. She mentioned how targeting a pathway that is responsible for making new plant material would guarantee suppressing plant development and helped us move away from our previous design idea involving the BphP1-QPAS1 system.

Education and Engagement

Because of the pandemic, our research, many high schools, programs, and clubs were being administered online, so our team conducted our education initiatives through presentations on Zoom. We presented to pre-freshman groups, freshman groups, and an alumni group. The pre-freshman groups included Shoreham Wading River High School and the Lang Science program, which is organized by the American Museum of Natural History. The freshman groups included the Collegiate Science and Technology Entry Program (CSTEP), and two SBU undergraduate colleges, SSO and ITS. We discussed our project, potential ways to get started on undergraduate research, and how iGEM is not just about biology and research, but modeling, web development, and outreach as well. Additionally, we presented to Stony Brook Alumni, giving them a glimpse into the problem of GMOs potentially giving rise to increased gene flow, how we hope to solve it, and how we hope to implement our kill switch in the real world.

Conclusions

Implementation

Indoor farming allows farmers to grow their products in a controlled environment. With the indoor farming industry projected to reach upwards of 22 billion USD by 2026[6], this growth scenario is becoming more common every year. Conveniently, UV-B light is absent in these environments, allowing us to utilize indoor farms as part of the solution to gene flow. Our UV-B light-activated killswitch could be implemented in GM crops cultivated in these controlled environments. If these plants do somehow find a way to escape to the natural environment, our killswitch could be able to prevent further growth by exploiting RNAi to silence the WUSCHEL gene, a key player in plant development. Combining the controlled environment of indoor farms with our optogenetic killswitch, the net risk of gene flow could be decreased. Indoor farmers would be at greater liberty to modify their crops without worrying about the deleterious ecological effects of gene flow.


Limitations
No laboratory access

Our team was one of the few iGEM teams around the world that were unable to conduct any wet lab experiments. Because of this, we were unable to determine the true effectiveness of our system in our model organism. To demonstrate the efficiency of this optogenetic system, we hope for future research to establish a proof of concept model in N. benthamiana through transient expression of UVR8-COP1 induced transcription of our syn-tasiRNA.

Public fear of GMOs

The current negative stigma around the use of GMOs has unfortunately been increasing, as there has been less transparency between the scientific community and the general public. Talking to farmers helped us realize that the implementation of our system can only occur if the public is fully aware of the potential harms of gene flow and how it can be contained with the issuance of our kill switch. When people hear “genetically modified,” they automatically correlate that to being the opposite of what is “natural” and thus, deem it bad. There is a need for scientists to effectively communicate their work to the general public. We hope that our team, and many others in iGEM, are one of the few that do.

Acknowledgements
Attributions
Professors

Dr. John Peter Gergen, Dr. Gábor Balázsi, Dr. Steven Glynn, Dr. Joshua Rest, Dr. Kristen Shorette, Dr. Robert M. Hughes, Dr. Vlad Verkhusha, Dr. Kate Creasey

Farmers

Jennifer Ross, David Lopez, Maxwell Carmack, Mike Axelrod

iGEM Teaching Assistants

SBU iGEM 2018 and SBU iGEM 2019


References

[1] Digital, G. (2020, January 25). Which genetically engineered crops and animals are approved in the US? Genetic Literacy Project. https://geneticliteracyproject.org/gmo-faq/which-genetically-engineered-crops-and-animals-are-approved-in-the-us/

[2] Somssich, M., Je, B. I., Simon, R., &; Jackson, D. (2016). CLAVATA-WUSCHEL signaling in the shoot meristem. Development, 143(18), 3238-3248. doi:10.1242/dev.133645

[3] Allen, E., & Howell, M. D. (2010). MiRNAs in the biogenesis of trans-acting siRNAs in higher plants. Seminars in Cell & Developmental Biology, 21(8), 798-804. doi:10.1016/j.semcdb.2010.03.008

[4] Carthew, R. W., & Sontheimer, E. J. (2009). Origins and Mechanisms of miRNAs and siRNAs. Cell, 136(4), 642-655. doi:10.1016/j.cell.2009.01.035

[5] Wu, Min & Eriksson, Leif & Strid, Åke. (2013). Theoretical prediction of the protein–protein interaction between Arabidopsis thaliana COP1 and UVR8. Theoretical Chemistry Accounts. 132. 1371. 10.1007/s00214-013-1371-7

[6] Pulidindi, K., & Chakraborty, S. (2018). Vertical Farming Market Trends: Growth Potential 2019-2026. Retrieved from https://www.gminsights.com/industry-analysis/vertical-Farming-market