Proof of Concept


According to our iHP feedback, pesticide abuse is a common problem in the use of traditional packaged pesticides by farmers. The pesticide preparations available to farmers in the market are usually in large volumes, and farmers often add excessive amounts based on their subjective requirements, which aggravates pesticide residues in crops and even spreads to the farmland environment, which is a serious and non-negligible problem. In addition, combined with the easy degradation of our RNAi biopesticides and the characteristics of immune-inducing engineered bacteria, we designed the product Polycobead. Polycobead uses a high alcoholysis degree water-soluble polyvinyl alcohol (PVA) as the outer film to wrap our CPP-shRNAs preparation and the engineered chassis-bacteria B. subtilis together to form a bead. In order to verify our design, in addition to completing basic molecular biology experiments for verification (see Results page), we also conducted a variety of different confirmatory experiments to support our design as below.

PolycoBead.png Figure 1. PolycoBead

Polycobead anti-B. cinerea

We dissolved the product Polycobead in water, and then sprayed it on tomato fruits inoculated with B. cinerea in advance to verify whether our product can effectively block the further infection of B. cinerea to tomato. The contents released by Polycobead were sprayed only once. We recorded the lesion area on the tomato surface in the first three days. After calculation, the RNA quality received by each tomato does not exceed 10 μg. As shown in the figure (Figure 2-1), compared with the control group, the lesion area of the treatment group is not large, and the spread is not obvious, which proves that Polycobead can effectively inhibit the further infection of tomato by B. cinerea.

fruit-d1-d8 Figure 2-1. Polycobead treated tomato fruits inoculated with gray mold fungus after dissolution in water

Release of Polycobead

In our project, we hope that the product Polycobead can be directly applied in the fields after simple treatment by users. Farmers only need to calculate the number of Polycobeads used according to the size of farmland and the planting density of tomatoes, and then put our Polycobeads into a certain volume of water for simple stirring, releasing effective ingredients CPP-shRNA preparation and immune-inducible engineering bacteria, so as to apply them to tomato fields to prevent and control tomato gray mold.

electrophoresis Figure 3-1. Electrophoresis result.
Figure 3-2. Structure of CPP-shRNA complex solution in water under SEM.
Figure 3-3. Structure of CPP-shRNA complex solution in glycerol under SEM.

In order to ensure the activity and stability of Polycobead contents after release, nucleic acid electrophoresis was performed for verification. As shown in Figure 3-1, from left to right are marker, the naked shRNA aqueous solution, the naked shRNA glycerin-aqueous solution (glycerin: water = 9:1), the CPP-shRNA complex aqueous solution, the CPP-shRNA complex glycerin-aqueous solution (glycerin: water = 9:1), and marker. Except lane 0 and lane 5 are markers, lane 1 is a normal RNA electrophoresis band. Lane 2 has a blocking effect on electrophoresis due to the presence of glycerol, which makes the nucleic acid sink in the electric field, but the brightness of the band is the same, so the possibility of RNA molecules being degraded is extremely low. The third lane is the aqueous solution of the CPP-shRNA complex. It can be seen that the CPP-bound RNA does not show any bands during electrophoresis, and the fourth lane does not show bands, indicating that the presence of glycerol will not lead to the separation of CPP and RNA, otherwise, RNA bands will appear.

In summary, we can conclude that the dissolution of Polycobead can completely release the complete CPP-shRNAs complex, and does not cause degration. In addition, scanning electron microscopy was used to observe the structural differences between CPP-shRNA in aqueous solution and glycerol solution under microscopic field of view, as shown in FIG. 3-2 and FIG. 3-3. It can be seen that glycerol does not damage the structure of CPP-shRNA complex.

RNA preparation

1.Distribution of disease spots

The shRNA we designed can target and silence genes that essential for the survival of B. cinerea and virulence genes that involved in infecting tomatoes. Therefore, we want to verify from a phenotypic perspective whether spraying shRNA can indeed reduce the damage of B. cinerea to tomato fruits. Before starting the formal experiment, we poked holes in the surface of the tomato.

For the naked shRNA treatment, we added 10μL system (containing 10μg shRNA) to the surface wounds of each tomato. After the liquid dried, we used a 10μL transparent pipette tip to punch holes at the same radius on a plate full of B. cinerea, and connect the bacterial cake to the center of the shRNA coating area of each tomato. For the control group, we chose non-specific shRNA (gfp). Afterwards, we placed the treated tomato fruits in a moist environment at 21℃. According to relevant literature, we learned that more obvious effects can be observed around the third day. Therefore, on the third day after the inoculation, we used ImageJ software to quantitatively analyze the area of the lesion on the fruit. The area of the lesion is determined by the area covered by the mycelium (Figure 4-1, Figure 4-2). You can refer to Protocol for more data analysis methods.

For the shRNA treatment combined with cell-penetrating peptide (CPP), besides changing the system that added to each sample to 12μL (containing 10μg shRNA and 8.2μL 1mg/mL CPP), the rest of the treatment is the same as the naked shRNA treatment (Figure 4-1, Figure 4-3).

Figure 4-1. Distribution of disease spots on tomato fruits infected with B. cinerea.
relative-lesion-area-naked-shrna.png Figure 4-2. Relative lesion area of tomato fruits after treatment with naked shRNA.

relative-lesion-area-shrna-cpp.png Figure 4-3. Relative lesion area of tomato fruits after treatment with CPP-shRNA.

At the phenotypic level, it can be observed from the above figure that most of the shRNA have shown effectiveness. Compared to the control group, the relative lesion area in the experimental group can be reduced by 10% to 30%. Furthermore, when combined with CPP, all shRNA show effectiveness, and the effects of most shRNA are further enhanced, resulting in a reduction of the relative lesion area by 20% to 40% in the experimental group. Particularly, our concatenated shRNA - bi-shRNA(Box-survival) shows a significant effect, reducing the relative lesion area by 42%. Additionally, due to issues such as low extraction yield during the extraction process of shRNA(cyp51)-1, it was not able to undergo the second stage of iteration (combining CPP with naked shRNA) for experimental validation. You can refer to Engineering for more iteration information.

2.Detection of inhibition effect by qRT-PCR

In addition, we also verified at the molecular level whether the application of shRNA can indeed reduce the damage of B. cinerea on tomato fruits. On the third day after the inoculation, samples were taken from the infected areas of tomato fruits, followed by RNA extraction and reverse transcription and qRT-PCR to detect the inhibitory effects of shRNA on the target genes of fungal mycelium in infected fruits (Figure 4-4, 4-5).

q-naked-shrna.png Figure 4-4. Inhibitory effects of naked shRNA on the target genes.

q-shrna-cpp.png Figure 4-5. Inhibitory effects of CPP-shRNA on the target genes.

From the molecular experimental results, it can be observed that most of the tested shRNA can achieve silencing rates of 30% to 60% before combining with CPP. After combining with CPP, the silencing rates of most shRNA can reach 50% to 70%. Consistent with the phenotypic results, our concatenated shRNA - bi-shRNA(Box-survival) exhibits remarkable performance, with a silencing rate of 70% on the target genes after combining with CPP.

3.Continuous qRT-PCR

To investigate the duration of action and the optimal timing of our RNAi products, we conducted stability testing on the shRNA. Due to time constraints, we selected only the shRNA(Pme1)-2 combined with CPP for validation in this experiment. After adding CPP-shRNA on the surface of tomato fruits, they were infected with B. cinerea. Samples were taken every 12 hours for the following 4 days to measure the expression levels of the target genes and evaluate the duration of silencing activity of CPP-shRNA. The results are shown in the figure below.

szu-poc-rnai-1.jpg Figure 4-6. Variation in silencing efficiency of target gene by CPP-shRNA over 12-96 hours

According to the graph, it is evident that the expression levels of the target genes exhibit a decreasing trend before 60 hours. At 60 hours, the relative expression level of the target genes reaches its lowest point, with a gene silencing rate of approximately 0.7. Subsequently, the expression levels of the target genes show an increasing trend.

This experiment proves that the combination of CPP with shRNA(Pme1)-2 achieves optimal results at 60 hours. If time permits, we would like to further investigate the stability of other shRNAs and determine the maximum duration of RNAi action. This will guide us in determining the application method and frequency of our final product. Additionally, the time variation of the silencing efficiency of CPP-shRNA in the experiment is the same as that of our RNAi model. You can refer to Modeling for more information.

Engineered B. subtilis

In Polycobead, our engineered immune-inducing bacteria B. subtilis are wrapped in calcium alginate beads. We hope that the presence of engineered bacteria in the product will not affect the CPP-shRNAs preparation, and at the same time, when the bead-engineered bacteria are released into the field, they can be planted to the tomato roots and release BvEP to induce plant immunity. When the concentration of sodium alginate was high, the cross-linking between the molecules was enhanced, and the bead structure formed was relatively dense, resulting in a slow release rate of the engineered bacteria. On the contrary, when the concentration of sodium alginate is low, the gel structure is looser and the release rate of engineering bacteria is faster. Therefore, it is imperative that we conduct meticulous testing and refinement of the sodium alginate concentration introduced during the production process, along with optimizing the bead structure for optimal performance, aimed at preventing premature rupture or hindered release of the engineered bacteria. Therefore, we made the following time-release curve of sodium alginate concentration with engineering E. coli.

Figure 5-1. Place A calcium alginate-engineered bacteria
on the plate overnight.

Figure 5-2. Release of engineering bacteria from calcium
alginate bead.

As illustrated in the figure, it is observed that when the concentration of sodium alginate is insufficient, the formation of a stable condensation structure becomes unattainable, thereby hindering the effective encapsulation of the engineered bacteria. Conversely, when the concentration is excessively high, the condensation becomes overly stable, leading to the confinement of the engineered bacteria, impeding their release.

Ultimately, our chosen approach involved blending a 1% sodium alginate solution with a highly concentrated bacterial solution. This mixture was subsequently introduced into a 2% CaCl2 solution to fabricate calcium alginate-engineered bacterial condensation beads. Subsequent experimentation revealed that, following a week-long immersion in a glycerol system, these beads exhibited a mere 5% reduction in volume. Remarkably, they exhibited rapid degradation in soil, releasing the bacterial solution into the soil within a mere 10 hours. These calculations were based on the assumption of condensation beads with a 1cm diameter, glycerol purity at 99%, and the utilization of soil sourced from the tomato cultivation area within the Tissue Culture Room of the College of Life and Oceanography Science at Shenzhen University, characterized by a pH level of approximately 6.3.

The immune-inducing factor BvEP

The BvEP protein is a elicitor protein phosphopentomutase from B. velezensis LJ02, which has been isolated and screened. This enzyme enhances tomato resistance to B. cinerea by upregulating the expression of PTI and ETI related genes, ROS content, and related factors in tomato fruits. It is important to note that BvEP treatment does not impact the weight or nutritional composition of tomato fruits. We expressed and purified BvEP using E. coli and applied it to tomato leaves. The activity of reactive oxygen species (ROS) in tomato leaves was assessed by measuring the formation of brown-red compounds generated through the Diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide reaction, serving as an indicator of plant immune pathways.

We treated the leaves with drip and soak methods and different protein concentrations. Then we analyzed the gray scale of tomato leaves after ethanol decolorization. We counted the relative gray scale values in each group of leaves and compared them. The lower the gray level, the more brown pigment was deposited on the leaves, that is, the more ROS exploded in the leaves. The results shown in FIG. 6-1 and FIG. 6-2 are obtained, which means that the induced effect of BvEP expressed by engineered bacteria on plants is moderate and effective.

To learn more about our experimental results on immune induction, please click Results .

Figure 5-3. Relative grayscale values of purified BvEP-soaked tomato leaves after immunization at different concentrations.
Figure 5-4. Relative grayscale values of purified BvEP-dripped tomato leaves after immunization at different concentrations.

11 Figure 5-5. Third day of B. cinerea infection experiment on tomato fruits treated with induced BvEP protein (at a maximum concentration of 0.530 mg/ml) for 16 hours and water (control).

Epidemiological models of plant mycoses

In addition, we simulated the natural infection of gray mold on tomatoes in a relatively enclosed tent. We placed 500 small tomatoes on the floor. For the 10 tomatoes in the middle, we conducted a pre-infection experiment with B. cinerea. At the same time, we randomly selected about 50 tomatoes for scratch treatment. Inside the tent, we placed a humidifier to maintain the humidity. On the top of the tent, we hung a ceiling fan to create wind for the dispersion of gray mold spores. The experiment lasted for 10 days, and the video below shows the recordings from day 0 to day 5. Finally, we calculated the incidence rate of gray mold disease in tomatoes and constructed an epidemiological model for plant fungal diseases (Figure 7-1). You can refer to modeling for more information.

liuxing.jpg Figure 7-1. Model predictions and experimental results.