In our project, after consulting with relevant instructors and conducting a series of preliminary investigations, we decided to utilize a biocontrol bacterium, Pseudomonas chlororaphis B3-3G, identified by the mentor's research group for its insect-resistant properties against the Plagiodera versicolora. We integrated this bacterium with RNAi technology to achieve a safe and effective control of the Plagiodera versicolora.
1. Knockout of RNase III: RNase III, a key enzyme for dsRNA degradation in prokaryotes, was targeted for knockout to enhance the accumulation of dsRNA expression in Pseudomonas chlororaphis B3-3G. Using the pK18mobsacB plasmid, we successfully knocked out RNase III, resulting in the mutant strain KC-P.
2. Construction of dsRNA Expression Vectors and Expression in Pseudomonas chlororaphis B3-3G and KC-P: The actin gene of the Plagiodera versicolorawas selected as the target gene, and we successfully constructed the dsRNA expression vector KC1. Simultaneously, the gfp gene was chosen as a control, and the dsRNA expression vector KC2 was constructed. Subsequently, KC1 and KC2 were separately transformed into the wild-type Pseudomonas chlororaphis B3-3G and the mutant strain KC-P, achieving dsRNA expression. Furthermore, Northern blot analysis revealed higher expression of actin dsRNA in the mutant strain KC-P, contributing to increased accumulation in Pseudomonas chlororaphis B3-3G.
3. Construction and Validation of a Safety Switch: To ensure the biosafety of our engineered bacteria, LacI operators were introduced into the KC1 and KC2 vectors, leading to the successful construction of KC3 and KC4 for regulating dsRNA expression. Additionally, KillerRed protein expression cassettes were further integrated into the KC3 and KC4 vectors, resulting in the construction of KC5 and KC6 vectors to enhance the safety of the engineered bacteria.
4. Validation of Pseudomonas chlororaphis B3-3G Combined with RNAi for Plagiodera versicoloraControl: Through feeding experiments on detached leaves, we measured relevant data such as mortality, weight, pupation rate, and eclosion rate of the Plagiodera versicolorain the experimental and control groups. At the molecular level, qRT-PCR was utilized to detect the expression levels of the actin gene. The results demonstrated that the combination of Pseudomonas chlororaphis B3-3G and RNAi improved the control effectiveness against the Plagiodera versicolora.
In prokaryotes, RNase III is the primary enzyme for dsRNA degradation. After discussions with our principal investigator (PI), we decided to use the pK18mobsacB plasmid to knockout the RNase III gene.
Figure 1 How pK18mobsacB works (Kvitko et al., 2011)
According to the working principle of the pK18mobsacB plasmid (as illustrated in Figure 1), we successfully integrated the upstream and downstream fragments of RNase III from Pseudomonas chlororaphis B3-3G into the pK18mobsacB plasmid, resulting in the knockout plasmid PK18-2.
Figure2
(PK18-2 plasmid, where "Down sequence" represents the downstream fragment of RNase III in Pseudomonas chlororaphis B3-3G, and "Up sequence" represents the upstream fragment of RNase III.)
Within the PK18-2 plasmid, there are kana resistance genes and SacB genes. Through electroporation, we introduced this plasmid into B3-3G, aiming to achieve the knockout of the RNase III gene. The SacB gene expresses levansucrase, catalyzing the hydrolysis of sucrose into levans that are lethal to cells. Simultaneously, the kana resistance gene allows host cells to survive in a kanamycin-resistant environment.
After electroporation, a period of antibiotic-free cultivation was initiated to recover transformed bacteria. Subsequently, the bacteria were spread on plates containing 15% sucrose and 100 mg/mL kanamycin to screen for mutants. The results were as follows:
| Kana Resistance | Survival in Sucrose Environment | Theoretical Result | |
|---|---|---|---|
| B3-3G (No homologous recombination) | No | Yes | Death |
| B3-3G Single exchange (Plasmid integrated into the genome) | No | Yes | Death |
| B3-3G (Homologous recombination, double exchange) | No | Yes | Survival |
On the sucrose-kanamycin plates, six randomly selected single colonies were verified through PCR. Primers were designed with one primer matching the original genomic sequence and the other matching the sequence on the integrated plasmid. PCR testing showed that colonies 1 and 6 exhibited the expected results, confirming them as mutants with double exchange events. To further validate these findings, the PCR products were sequenced, confirming that colonies 1 and 6 were indeed the desired mutants (as shown in Figure 3-a, 3-b, and 3-c).
Figure 3-a shows left-end PCR verification, 3-b is right-end PCR verification, and 3-c is a schematic diagram of sequencing results. M is Marker, ck is Pseudomonas chlororaphis B3-3G wild-type, 1, 2, 3, 4, 5, 6, PCR is performed using the selected 6 single colonies as a template.
With the replacement of the RNase III gene in B3-3G by the resistance gene in the PK18-2 plasmid, the next step involved the removal of the resistance gene integrated into the B3-3G genome.
Figure 4 - pFLP2 plasmid
Through large-scale dot-blotting, we selected colonies on plates with 100 mg/mL kanamycin and 500 mg/mL ampicillin. The high concentration of ampicillin was used because B3-3G inherently possessed some resistance to ampicillin, and after testing, it was determined that a higher concentration was necessary to ensure the death of B3-3G not transformed with the pFLP2 plasmid.
Results from the selection process on kanamycin and ampicillin plates were as follows:
| Kana Resistance (100 mg/mL) | Amp Resistance (500 mg/mL) | Theoretical Result | |
|---|---|---|---|
| B3-3G (No homologous recombination) | Yes | No | Survival on kana plate, death on amp plate |
| B3-3G + pFLP2 plasmid | Yes | Yes | Survival on both kana and amp plates |
| B3-3G (Homologous recombination) | No | No | Death on both kana and amp plates |
| B3-3G + pFLP2 plasmid | No | Yes | Death on kana plate, survival on amp plate |
According to the results in Table 1, colonies that survived on the ampicillin plate but died on the kanamycin plate were selected for further verification.
Figure 5 - Results of dot-blotting on kanamycin and ampicillin plates
Primers were designed with one primer on the Down sequence and the other on the Up sequence. PCR testing revealed that Pseudomonas chlororaphis B3-3G amplified a 2401 bp DNA fragment, non-homologous recombination resulted in a 3226 bp DNA fragment, and homologous recombination produced an 1877 bp DNA fragment. Results from the PCR verification indicated that all four selected colonies exhibited the expected results, confirming that they had undergone homologous recombination. Further sequencing confirmed the successful homologous recombination events.
Figure 6-a, 6-b ,PCR verification and sequencing results
In conclusion, we successfully obtained the mutant strain of Pseudomonas chlororaphis B3-3G, named KC-P, by knocking out the RNase III gene.
We selected the actin gene of the Plagiodera versicoloraas our target gene and proceeded to construct dsRNA expression vectors based on this gene.
As illustrated in Figure 7, we incorporated tac promoters into both segments of the actin gene sequence, leading to the construction of the KC1 plasmid for expressing actin-dsRNA. To mitigate the potential impact of non-target gene dsRNA, we also constructed the KC2 plasmid for expressing gfp-dsRNA.
Figure 7-KC1 and KC2 plasmid plots
By electroporation, we introduced KC1 and KC2 plasmids into Pseudomonas chlororaphis B3-3G and Pseudomonas chlororaphis KC-P, respectively, and then used the actin sequence as a probe for northern blot verification, knot The result is shown in Figure 8.
Figure 8 presents the northern blot detection results from left to right, including Marker, KC-P+KC1, KC-P+KC2, B3-3G, B3-3G+KC1, and B3-3G+KC2.
According to Figure 8, it is evident that the accumulation of actin-dsRNA significantly increased after the knockout of RNase III.
1.Construction of the LacI Operator Switch
Considering that the dsRNA expression in KC1 and KC2 plasmids is constitutive, we introduced a LacI operator at one end of the tac promoter, transforming it into an inducible dsRNA expression plasmid for both KC3 and KC4. The expression of dsRNA was regulated by IPTG.
Figure 9 illustrates the KC3 and KC4 plasmids.
Through electroporation, we introduced KC3 and KC4 plasmids into the Pseudomonas chlororaphis KC-P. Subsequently, using the actin sequence as a probe, we conducted northern blot verification, and the results are shown in Figure 10.
Figure 10 represents the northern blot results from left to right, including Marker, KC-P+KC3+IPTG, KC-P+KC4+IPTG, KC-P+KC3, KC-P+KC3, and KC-P+KC4.
According to Figure 10, the LacI operator significantly inhibits the expression of actin-dsRNA. However, the KC-P+KC3 without IPTG induction still exhibits slight coloration. We believe there are two possible reasons for this result:
The LacI operator is not a completely stringent switch, allowing KC-P to express a small amount of actin-dsRNA.
We added the LacI operator only between one tac promoter and the actin sequence, while the other tac promoter can still transcribe actin-ssRNA in the absence of IPTG induction. Although we introduced RNAase in the Northern blot experiment to degrade actin-ssRNA, there may still be a small amount of actin-ssRNA binding to the probe.
Based on the analysis of Figure 10, we can still observe a certain level of inhibition in the expression of actin-dsRNA without induction.
2. The Second Switch: KillerRed
Our team places particular emphasis on biosafety. After introducing the LacI operator, we decided to add a suicide switch—KillerRed. The KillerRed protein, expressed by the killerRed gene, undergoes a structural change upon light stimulation, generating highly toxic oxygen radicals in cells, leading to cell death.
To achieve this, we further integrated the KillerRed protein expression cassette into the KC3 and KC4 plasmids, successfully constructing the KC5 and KC6 plasmids (Figure 11), further ensuring the safety of our engineered bacteria.
Figure 11 illustrates the KC5 and KC6 plasmids.
Through electroporation, we introduced the KC5 and KC6 plasmids into the Pseudomonas chlororaphis KC-P and validated them.
The results in Figure 12 indicate that on the plate after 1 hour of light exposure, the number of viable bacteria has already decreased by more than 95%, demonstrating the excellent lethality of our plasmids.
Figure 12, from left to right, represents plates after 0h, 0.5h, 1h, and 2h of light exposure.
We also conducted modeling analysis using ordinary differential equations to describe the system, where:
N represents the number of viable bacteria,t represents the light exposure time,k represents the bacterial death rate
We used the ode45 function in MATLAB to solve this differential equation, compared the results with actual data, and calculated the error. Then, we used the fminsearch function to fit the unknown parameter k, minimizing the error. Since there might be multiple local optimal solutions, we performed multiple fittings to find the global optimal solution. Based on the fitting results, we re-solved the differential equation to obtain the final model and plotted the fitting curve, as shown in the figure. The fitted value for the parameter k in the exponential fit was 0.07.
Exponential Fit:
Using the MATLAB Curve Fitting Toolbox to fit the existing data, we found that an exponential fit provided the best results. The adjusted R2 was as high as 97.03%, and the root mean square error (RMSE) was 5.883. The final expression for the fitted curve is:
Figure: represents our data analysis modeling for the KillerRed suicide switch.
This model is similar to the logarithmic residual model of high-temperature sterilization bacteria. From the experimental results and model predictions, it can be observed that after introducing KillerRed, the majority of bacteria can be killed with only a short period of light exposure. However, unlike high-temperature sterilization, it does not completely eliminate almost all bacteria. This also indicates that compared to physical methods of killing, the bacterial death caused by the biological suicide switch is relatively gentle, yet sufficient to meet our biosecurity needs.
We conducted feeding experiments on detached leaves to determine the mortality rate, body weight, pupation rate, and eclosion rate of Plagiodera versicoloraunder different conditions. The experimental groups included: Pseudomonas chlororaphis B3-3G, Pseudomonas chlororaphis B3-3G+KC5, Pseudomonas chlororaphis B3-3G+KC6, Pseudomonas chlororaphis KC-P+KC5, and Pseudomonas chlororaphis KC-P+KC6.(Figure 9)
Figure 13: Data analysis of the mortality rate, average weight, pupation rate, and eclosion rate of Plagiodera versicolora.
After conducting statistical analysis, we concluded that the experimental group with Pseudomonas chlororaphis B3-3G showed some impact on the mortality rate, average weight, pupation rate, and eclosion rate of Plagiodera versicoloracompared to the control group, but it did not show significant differences in statistical analysis.
After combining RNAi, the experimental group with Pseudomonas chlororaphis B3-3G+KC5 showed significant differences in the mortality rate, average weight, pupation rate, and eclosion rate of Plagiodera versicoloracompared to the control group. The pupation rate decreased by approximately 46% compared to the control group, the average weight decreased by over 35% compared to the control group, and the eclosion rate decreased by approximately 48% compared to the control group.
Furthermore, we observed that the Pseudomonas chlororaphis KC-P+KC5, which knocked out RNase III, performed better in controlling Plagiodera versicolora. The experimental group with Pseudomonas chlororaphis KC-P+KC5 showed extremely significant differences in the mortality rate, average weight, pupation rate, and eclosion rate of Plagiodera versicoloracompared to the control group. The pupation rate decreased by approximately 75% compared to the control group, the average weight decreased by over 60% compared to the control group, and the eclosion rate decreased by approximately 67% compared to the control group.
As shown in Figure 14-d, in the statistical analysis of the mortality rate of Plagiodera versicolorawith Pseudomonas chlororaphis B3-3G+KC5, the mortality rate reached over 50% on the 5th day, while in the KC-P+KC5 experimental group after knocking out RNase III, the mortality rate reached over 50% on the 2nd day.
We can see that after knocking out RNase III, the combination of Pseudomonas chlororaphis and RNAi greatly improved the control effect on Plagiodera versicolora.
To further validate the results, we conducted qRT-PCR analysis to detect the expression level of the actin gene in Plagiodera versicoloraon the third day of feeding.
FIG 15: The expression level of actin genePlagiodera versicolora.
As shown in Figure 15, in the experimental group fed with Pseudomonas chlororaphis B3-3G+KC5, the expression level of the actin gene in Plagiodera versicolorashowed significant differences compared to the control group. In the experimental group fed with Pseudomonas chlororaphis KC-P+KC5, the expression level of the actin gene in Plagiodera versicolorashowed extremely significant differences compared to the control group.
By incorporating RNAi technology and optimizing the expression of dsRNA through the knockout of RNase III in Pseudomonas chlororaphis, we have successfully developed the KC-P+kc5 strain with significantly improved insect resistance. With these optimizations, our team has completed the optimization of Plagiodera versicoloracontrol techniques and expression systems.