Our project utilizes two engineered Bacillus strains for nematode control and plant immunity enhancement:

B.velezensis: Colonizes roots, kills nematodes via proteins (Cry6Aa2), RNA interference (shRNA targeting 16D10 gene), and metabolites (TAA from B. thuringiensis).

B.subtilis: Colonizes leaves, boosts plant immunity with proteins (VDAL, Nlp20) secreted via the Splip tag.

Both strains use the MS2-VLPs Delivery System for effector transport, featuring a biosensor module activated by salicylic acid.

A Suicide Module with KillerRed is included for biocontainment.

To sense the salicylic acid signal produced by plants due to disease, we designd salicylic acid sensors. The LysR-type regulator NahR senses salicylic acid signals in the environment.[1] When the salicylic acid concentration reaches a threshold, the Psal promoter is activated to express the HrpR as well as some shRNAs. Figure 1. Salicylic acid biosensor circuit.

HrpR is one of the components of the heterodimer and is involved in the regulation of downstream circuits in a cascade.[2] shRNAs were designed to target key genes in the nematode growth cycle and were packaged in the interior of the VLP.

We plan to use different concentrations of salicylic acid standards to induce engineered bacteria, and verify the NahR and promoter activity with fluorescent proteins as reporter genes.

As a well-studied delivery platform, virus-like particles (VLPs) can be modified to build the expected comprehensive nematode control delivery system. In order to display our functional proteins on the surface of VLP particles, we selected the SpyTag-SpyCatcher system as a scaffold for displaying functional proteins on the surface of VLP through literature review.

In the literature, we found the sequence of the dimeric tandem MS2 coat protein fused with SpyTag peptides obtained from previous studies[3]. The modified protein was able to spontaneously assemble into 22-29 nm VLP[4].

Surface-exposed SpyTag sites can bind to and display functional proteins fused with SpyCatcher on the surface. The MS2 coat protein also binds specifically to RNA with a 19 bp stem-loop motif, encapsuling the RNA with this motif. The result is a multifunctional delivery particle with its surface that displays functional proteins and an inner package of designed specific RNA.

Figure 2. Virus-like particles delivery system circuit.

HrpS is one of the components of the heterodimer and is involved in the downstream of the circuits. When combined with HrpR, it activates the lysis circuit we designed to split the bacteria and release the contained VLP.

In order to improve the disease resistance of plants and to inhibit nematode infestation to a certain extent by enhancing their autoimmunity, we designed several components to activate the plant's immunity. Plant autoimmunity consists of PTI and ETI, so we designed two elements to activate the plant's PTI and ETI responses, respectively.

Considering the VLP releasing is lightly behind the initial time of plant infestation by nematodes, in order to better enhance the effect of our engineered bacteria on controlling nematodes, the project transferred two kinds of plant immune activators carrying exogenic labels of Bacillus proteins. We fused NLP20, a 20-amino acid plant immune-activating peptide from Bacillus subtilis, and Aspf2-like protein (VDAL) from Verticillium dahliae, with the exocytosis signal peptide Splip from Bacillus subtilis[5][6][7][8][10]. Thus, the effectors can be secreted extracellularly from Bacillus subtilis and bind to the surface receptors of the plant cells and activate the PTI response. The expression of these enhancers is regulated by the P_sal. The circuit is shown in Figure 3.

Based on the MS2-Spytag vector, we combined the VDAL protein with the SpyCatcher tag so that it can be carried on the surface of virus-like particles (VLPs) through the specific binding of Spytag-SpyCatcher to improve its transport and binding efficiency. In order to achieve better performance,SR9, the cell-penetrating peptides(CPPs), is fused with SpyCatcher-VDAL.[9] In that case, not only the surface receptors are activated, but also the intracellular receptor is also accessible for VDAL. The principle is based on the electrostatic interaction between the positively charged amino acids of SR9 and the negatively charged membrane phospholipid bilayer on the cell surface.[15] CPPs transform their structure so that their hydrophobic amino acids interact with the hydrophobic core of the cell membrane, making the lipid bilayer sparse. It causes transient or long-term stable imbalance of the membrane at the binding site of the two and enters the plant cell.To avoid the potential influence of SpyCathcer-SpyTag-MS2 particles with VDAL-CPPs on the particle structure of VLP during assembly, SpyCather-CPPs was also expressed to form dodecahedral VLP in normal conformation together with SpyCathcer-VDAL-CPPs. The composite protein is regulated by P_sal as well.

To investigate the possible effect of the 2 circuits, we plan to verify the basic components separately.

Due to the experiment plan, VDAL and Nlp20 is expressed in E.coli BL21(DE3) and B. subtilis 168. The function of VDAL in enhancing plant immunity is demonstrated by co-incubating Arabidopsis thaliana leaves with VDAL and VDAL with CPP, followed by quantification of leaf reactive oxygen species (ROS) content using DAB staining and control with water and salicylic acid (SA) treatments.

Cell-penetrating peptides (CPPs), SR9, ligated with cyan fluorescent proteins are expressed in E. coli BL21 and incubated with Arabidopsis thaliana root tip cells, and fluorescent signals were observed in the protoplasts of the root tip cells by fluorescence confocal microscopy.

1. Cry6Aa2:A crystal protein that kills nematodes

In our project, Cry6Aa2, a nematocidal crystal protein from Bacillus thuringiensis YBT-1518[11], was used with SpyCatcher and cell-penetrating peptides (CPPs) to form a fusion protein linked by a flexible linker. Fusion proteins and spycatchers with CPPs can be loaded onto VLP by bonding with SpyTag on the surface of VLP particles in the presence of isopeptide bonds. CPPs can help VLP particles to enter the nematode body and help the insecticidal protein to play a role in order to reach the purpose of contact.[12]

2. Production and transfer of TAA

The existing literature shows thats trans-aconitonic acid(TAA) has a good killing effect on plant parasitic nematodes.[13] In our project, the aconite isomerase(TbrA) and the TAA transporter(TbrB) were used to isomerize the CAA in the engineered bacteria into TAA and transfer it to the extracellular. TAA spreads through the soil environment near the plant's roots, acting like a protective ring to kill nematodes and protect the plant.

3. shRNA

16D10 is a key gene in plant infestation by root knot nematodes. It encodes a parasitic peptide that interacts with transcription factors in plants and affects root growth.[14] We designed shRNA targeting this gene and used its mediated RNAi to inhibit the expression of parasitic peptides, thereby reducing the ability of nematodes to infect plants.

However, due to time, we only completed the shRNA design of 16D10 gene, but did not synthesize the shRNA and do related experimental verification.

According to reports in the literature, Bacillus mainly relies on the assembly of TasA into amyloid fibrillary structures to form biofilms, while also promoting biofilm formation by producing major and minor wall teichoic acid (WTA).[15]

TapA is a protein of the genus Bacillus that promotes the formation of its biofilm by promoting the assembly of TasA into amyloid fibrillary structures.[16]GgaA is a minor wall teichoic acid (WTA) synthase contained in the genus Bacillus that promotes the formation of Bacillus biofilm by synthesizing minor WTA.[17] Therefore, increasing the expression levels of GgaA and TapA may improve the colonization ability of engineered bacteria.

We designed to combine GgaA and TapA genes with the strong constitutive promoters p43 and SpoVG RBS , in order to increase the expression of these two proteins in Bacillus and enhance the colonization ability of Bacillus.

Since the direct use of Bacillus was not conducive to our verification of successful plasmid construction, we selected E. coli DH5α and Bacillus subtilis B.subtilis 168 as carrier validation chassis and functional validation chassis, respectively.We implemented this idea by inserting this gene circuit into the pHT254 shuttle plasmid.

we expect to verify the module function by staining the biofilm produced by the engineered bacteria with crystal violet solution and then measuring the A₅₉₀ value of dissolved crystal violet.

To release the VLP out of our engineering bacteria,we design a lysis module.we use a promoter activated by Heterodimer protein,with two suicide protein followed.once the proteins in the former circuit,HrpR and HrpS,are expressed,they will form a heteromeric complex,and activate the promoter P_hrpL,express downstream genes,and generate two kinds of toxic proteins:holin and PGHs. The former is a perforin,while the latter is a phage-encoded peptidoglycan hydrolases.Together, they will trigger the cleavage of the bacteria.[18]The VLP is then released into the plant. Figure 9. Lysis circuit.

Since HrpR and HrpS are parts that have already been verified by other teams and their effect were obvious,while lysis-associated proteins are new parts, our verification work focuses on constructing and characterizing the lysis-circuit.

We plan to construct the lysis circuit on the pHT315 plasmid because it contains a xylose operon that we can induce the gene to express with D-xylose. We hope to verify the lysis function by measuring the OD₆₀₀ value of the bacterial solution.

In order to avoid the potential biosafety hazards caused by the leakage of engineered bacteria into the environment, we designed a suicide scheme combining the characteristics of engineered bacteria and the environment of the project application.

Both Bacillus subtilis and Bacillus velezensis colonize crops and are endophytic bacteria of plants, and our object of use is in agriculture, which means that we need to choose a simple and portable method of suicide in order to facilitate the use of our users. Therefore, we selected KillerRed (BBa_K1184000), a phototoxic protein that produces reactive oxygen species (ROS) under yellow-green light (540-585 nm)[19][20]. We combined with the p₄₃ to constitute a constitutive expression,so it can commit suicude under natural light to prevent the leakage of engineered bacteria and ensure the safety of our project. Figure 11. Light-sensitive suicide circuit.

We plan to verify this part on the pHT315 plasmid containing a xylose operon, first inducing the expression of KillerRed with xylose, and then evaluating the suicide effect by measuring the OD₆₀₀ value of the bacterial fluid under different light conditions.