We are working to develop a safer and more efficient biopesticide. We designed an RNA strand as a functional part of the vaccine and a biosafety module for engineered bacteria to ensure good biosafety.

The plant RNA vaccine we constructed can be divided into three parts: tRNA-liked sequences, RNA dependent RNA polymerase, and the module of plant immunity. The biosafety module will be introduced into the engineered bacteria as an independent plasmid.

Part 1 tRNA-like sequences(TLS)

Part2 RNA-dependent RNA polymerase(RdRp)

Part 3 The module of plant immunity

Part4 The module of biosafety

tRNA-like structures (TLSs) are RNA structures that resemble transfer RNA (tRNA) molecules in their secondary and tertiary structures. TLS has been found to pose transport capabilities for RNA molecules, and according to existing research, this ability can help transport RNA molecules in plant vascular bundles.

In order to allow our RNA to diffuse from the Agrobacterium infestation site to the whole plant, then expressing target proteins in the whole plant, we added a tRNA-like structure (TLS) module to the RNA. RNAs with TLS can be recognized by specific proteins and trigger long-distance transport in the phloem[1].

We tried TLS of different amino acids from different species, and also modified their sequences to find several sequences that help the most with RNA mobility. RNAs with our optimized TLS can move through plant vascular bundles and express target proteins in non-transformed plant cells.

One prominent limitation of employing RNA in vaccines is its inherent instability, leading to a reduced half-life. The delicate structure and susceptibility of RNA molecules pose significant hurdles in terms of storage, transportation, and longevity of efficacy. Sustaining a durable protective response necessitates multiple administrations or booster doses due to the transient presence of RNA vaccines. Therefore, we take inspiration from RNA viruses and human RNA vaccines.

We assemble the replicase from Tobacco mosaic virus (TMV) to our RNA [2]. TMV is a kind of positive-sense single-stranded RNA virus. The replicase from TMV can transcribe genomic (+)RNA into genomic (-)RNA. In a later phase, genomic (-)RNA can serves as a template for the production of new copies of the original genomic RNA but also for the production of subgenomic RNA which express protein of interest. As a result, RdRp module can help extend the half-life of RNA, also increasing the time to express the target protein.

We selected two genes of NLR proteins, pikm1 and pikm2. Pikm1 is the receiver NLR responsible for recognizing effects, while pikm2 is the signaling NLR triggers immediate responses (please click on the Description for theoretical details).

Our design is to replace the ID sequence with a nanobody that specifically binds to effects by directly replacing the ID sequence fragment (base 509-730) of pickm-1 with enhancer, and trying to retain its ability to repeat signaling NLR and activate downstream immediate responses after modification.

Due to the need for both sensors and helper proteins to activate plant immunity, we designed two functional paths, located in two different types of engineered bacteria, to reduce the impact of artificially synthesized genes on the growth of engineered bacteria

In this module, we introduced a constitutive promoter expressed in Agrobacterium and achieved regulation of cell death by coordinating with phototoxic proteins, ensuring the safety of our project.

Light induced kill switch

Promoter