During the brainstorming phase, we learned that crop disease outbreaks would greatly reduce production. Knowing the potential hazards of existing chemical pesticides, we set out to design a novel biopesticide designed by synthetic biology could provide inspiration for solving plant disease problems.

Unlike previous conventional chemical pesticides or some biological pesticides, our Flora Sentinel does not act directly on the pathogen, but uses plants as one of our target gene expression chassis. (You could click on the Design for more details.)

After experimental verification, our modified immune protein designed was successfully expressed in the plant chassis, and our RNA vaccine scaffold we designed has been proven to have excellent abilities for migration and self-amplification. These experimental results demonstrate the feasibility and potential of our design. (For details, please click on the Results.)

On this page, we have compiled the plant-related parts, tools, and protocols used in this project for reference by plant synthetic biology researchers. We sincerely hope that our work this year will provide inspiration to more teams and contribute to the advancement of plant synthetic biology together.

In the face of pests and diseases, the plant's own immunity often fails to meet the needs of agricultural control. Therefore, through synthetic biology, we hope to develop a more efficient and scalable engineered disease control program that utilizes the plant's own immune system. Based on previous studies, we focused on an immune protein pikm in rice, whose ID sequence responsible for specific recognition of pathogen effector is thought to have the potential to be replaced and modified.

Pikm, a member of the Nucleotide-binding domain and Leucine-rich Repeat (NLR) family of immune proteins in rice, can specifically recognize the effector of blast bacteria and play a disease-resistant role. Due to its extensive homology in plants, it has been shown to function in a variety of common crops and model plants[1]. Although there have been many studies on the transformation and replacement of ID sequences in pikm, the relevant transformation has been limited because the binding mechanism between ID sequence and effector has not been clearly revealed. Further, when we want to transform the ID sequence into a more universal recognition structure to meet the effects of engineering applications, the study of Kourelis J et al., which replaced it with nanobodies, provided us with theoretical support[2].

Drawing on the design of mRNA in human, we can inject RNA that can move throughout the plant and enter cells to express modified immune protein (in our project, it is pikm1-enhancer) into plants through transient transfection of Agrobacterium. In this way, pikm1-enhancer can be expressed in cells. So when there is an effector into the plant, that is, when the pathogen invades the plant, the plant can carry out more specific, faster, and strInonger immune response. In addition, what pikm1-enhancer activates is the plant's own immune pathway and does not introduce foreign substances, so our method is also safer.

In addition, this RNA-enabled expression system is non-GMO and very engineered. We can replace the expression of various nanobodies in plant cells to make plants obtain various different resistances; It may even be possible to replace antibodies with sgRNA, or RNA sequences that can express other proteins (such as proteins that can increase the production), and play a more diverse role in plant cells!

Problems with plant transformation

According to the transfection medium, plant transformation technologies mainly include physical/chemical transformation technology, Agrobacterium transient transfection technology and plant virus vector transfection technology. Agrobacterium-facilitated transformation is the much-deployed method for gene transfer in explants such as cotyledonary leaves, vegetative leaves, hypocotyl, and stem explants. It is a more natural manner of transferring DNA and is more acceptable to those who feel that natural is superior. It can infect plant cells, tissues, and organs that are otherwise healthy. As a result, tissue culture restrictions are much less of a worry[3].

We need a new delivery method, overcoming the drawback of traditional transient transfection where expression is limited to the localization of the plant body, while circumventing the ethical dilemmas posed by transgenesis.

Our design

At the beginning of the project, we envisioned delivering proteins throughout the body of the plant through a non-transgenic method. Since plants do not have a human-like blood circulatory system, delivery of substances is a challenge for us. We checked how biomolecules are transported in plants from the perspective of proteins and RNAs, and found that t-RNA-like sequences can help mRNAs to be transported over long distances in plant vascular bundles. Considering the relatively short half-life and short existence of RNA, RNA may be degraded as soon as it moves throughout the body. This problem reminded us of human RNA vaccines, and the emergence of self-replicating RNA vaccines provided us with new inspiration to try to add an RNA self-replicating enzyme to our RNA to increase the mRNA existence time and protein expression time.

At this point, we came up with a completely new model of exogenous protein expression in plants:Agrobacterium delivers vectors containing the target fragments into local tissues of the plant body through transient transfection. mRNAs with TLS and RdRP are produced by transcription and transported throughout the body of the plant, where they are continuously replicated to prolong the half-life, ultimately leading to stable systemic expression within a short period of time.

Our advantages

Compared to conventional transgenic or transient transfection strategies, our expression has the following advantages:

In our wetlab work, we have validated the mobility of 4 TLSs and the replication function of RdRP, and we will explore the conditions under which other TLSs drive mRNA movement in the future to optimize our expression system. We have the ambition to establish a "blood circulation" system in plants.

Utilizing plants' own immune pathways

We hope to address some of the challenges that plants encounter when exposed to pathogenic bacteria, such as insufficient immune responses and suppressed immune pathways. By enhancing plant immunity and overcoming bacterial interference, we hope to improve plant health and productivity.

Plants have evolved two major types of innate immunity - PTI and ETI. PTI is mediated by plant cell surface receptors called PRRs that detect conserved microbial signatures, while ETI is mediated by intracellular proteins(such as NLRs) that recognize specific pathogen effectors. Interestingly, Many pathogens can suppress PTI by secreting effectors that interfere with PRR signaling. To counteract this, plants have evolved ETI that targets these effectors. Moreover, ETI can also modulate PTI, resulting in a coordinated and effective immune response against pathogen invasion.

Utilizing specific sequences in plant NLRs

One of the major challenges for plant immunity against bacterial pathogens is the lack of specificity in their recognition and response mechanisms, which leads to delayed and inefficient defense activation.

Studies of the NLR family and the ETI immune pathway have shown that, some NLRs have a sequence(integrated decoy, ID) that specifically recognizes a pathogenic bacterial effector. The ID sequence triggers a mutation in the NLR protein upon effector recognition and activates downstream signaling for plant immunity. However, the ID-effector interaction is weak and not very specific.

This mechanism of recognition and activation through protein-protein interactions can be naturally associated with the mode of antigenic-antibodies in animals, that supports our proposal of substituting ID sequences with nano-antibodies that have higher specificity.

The specificity and independence of the ID sequence provides us the possibility to replace it with nanobodies. Our experiments also demonstrate that modification of the NLR with nanobodies retains its function of recruiting signals and activating downstream immune responses, and further enables more specific binding to effectors.

Then we hypothesized that by simply fusing our RNA with different tRNA sequences and inserting them into Agrobacterium T-DNA, we would be able to deliver and express them whole after transient transfection of plants by Agrobacterium. We tried different TLS sequences and successfully detected TLS-driven mobility, and we will further explore the conditions and specific mechanisms of TLS-driven transcript transport in future experiments.

Therefore, with the help of the vascular system and tls sequences of plants, we succeeded in obtaining the ability of RNA to be transported in vascular bundles and to be expressed in plant cells, which accomplished non-transgenic, whole-plant delivery and expression after in situ transfection, and developed a completely new delivery mode.

Utilizing RNA molecular transport in plants

In order to achieve systemic delivery of exogenous proteins in plants, we extensively explored different mechanisms of biomolecule transport in plants. For example, plants selectively transport transcription factors through plasmodesmata, and plant extracellular vesicles can also transport cell-derived nucleic acids, proteins, and other bioactive molecules, mediating intercellular communication [4]. In plant vascular tissues, researchers have found a significant accumulation of non-coding RNAs in the phloem, and a RNA structure called TLS plays a crucial role in mediating long-distance RNA transport in vascular tissues.

Among these different molecular transport mechanisms, TLS caught our attention. We envisioned that by fusing our RNA with TLS sequences, we could enable the movement of our RNA throughout the plant and achieve expression of exogenous proteins in the whole plant. This design features simplicity, ease of operation, and modularity, aligning well with the concepts of iGEM and synthetic biology.

We have tried different TLS sequences and successfully detected TLS-driven mobility. In future experiments, we will further explore the conditions and specific mechanisms of TLS-driven transcript transport.

Experimental validation on the model plant——Nicotiana benthamiana

Excellent traits of Nicotiana benthamiana:

(1) It can be easily cultivated in large quantities under laboratory conditions and provides ample plant material for analysis.

(2) It can regulate the expression and post-translational modification of heterologous genes better than E. coli or yeast, making it more suitable for functional verification of foreign genes;

(3) It is susceptible to many pathogenic microorganisms, which makes it a common model for studying plant-microbe interactions;

(4) It allows transient, rapid, and high-level expression of exogenous proteins using Agrobacterium-mediated gene transfer and visual reporter genes (such as fluorescent protein genes), which facilitates the functional study of the proteins of interest;

Parts

Our project hopes to use the plant's own immune system to enhance its resistance to plant pathogenic fungi without altering genes. By reviewing the data, we focused on the NLR family, We uploaded the resistance allele Pikm-1 ( BBa_K4628007 ) and Pikm-2 ( BBa_K4628009 ) sequences from Oryza sativa in our parts library, and also modified the resistance allele Pikm-1. Its ID sequence was replaced by a separate designed eGFP nanoantibody ( BBa_K4628008 ), which showed good immune effect after eGFP stimulation. We also designed a series of nanAbs to verify the immune function of the existing proteins ( BBa_K4628003, BBa_K4628004, BBa_K4628005, BBa_K4628006 ). In the future, specific antibodies could be modularly used to target different fungal effector proteins.

Since Plant cells communicate with each other via called plasmodesmata and Vascular bundle, to allow molecules to pass between cells, we also uploaded a series of tRNA-like sequence (TLS) sequences( BBa_K4628012, BBa_K4628013, BBa_K4628014, BBa_K4628015, BBa_K4628016 ) to the parts library. They are from Arabidopsis thaliana, Nicotiana benthamiana, and have the ability to enhance RNA mobility. It can guide RNA to move in plants. In experiments, we modified their constructs by using reporter genes eGFP( BBa_K4628010 ), GUS( BBa_K4628011 ), etc. They were shown to enter cells and undergo migration. This module has a great space for modification, such as the design of RNA secondary structure and the concatenation of modules( BBa_K4628017 ), which can be carried out on the basis of our design.

To solve the problem of rapid RNA degradation and enhance the effect of our immune module, we also introduced RNA self-amplifying sequences( BBa_K4628021 ) from Tobacco Mosaic virus (TMV) into the design. In order to reduce the plasmid load and reduce the impact of the virus itself on the plant, we deleted the capsid protein and movement protein of the virus( BBa_K4628041, BBa_K4628042 ). By combining with the first two modules, we overcome the disadvantages of low transfection efficiency and small viral load in agrobacterium. We take advantage of the modularity of the plant autoimmune system to create a complete set of modes to enhancing plant immune capabilities.

In addition, we also engineered Agrobacterium tumefaciens commonly used in plant experiments. Taking advantage of the fact that light is required in plant experiments, we introduced photosuicide module( BBa_K4628047, BBa_K4628048 ) to enhance the control ability of engineered bacteria. Meanwhile, in this module, we validated the constitutive and inducible promoters( BBa_K4628022, BBa_K4628023 ) used for expression of protein. These designs not only provide a safety guarantee for the subsequent work of plant related teams, but also facilitate the engineering operation of agrobacterium.

Tools

Hardware

Software

Protocols

Preparation of engineering bacteria and experimental plants

Extraction of Plant RNA and Protein

Measurement of physiological and biochemical indicators of plants