Part:BBa_K4653907
T7 promoter (lacⅠ repressible) - shRNA (Pme1)-2 - T7 Terminator
In order to inhibit the infection of B. cinerea, the pathogen of grey mold and control the disease in tomato, we designed two pieces of shRNAs targeting the Bcpme1 gene of the pathogen, which is helping B. cinerea produce pectin methylesterase (PME), based on RNAi technology. shRNA can be actively absorbed by B. cinerea, then enter into its cells to be processed into siRNA, and further specifically target mRNA to achieve degradation. Or, entering plant cells, the related proteins in the cell will process and deliver shRNAs to B. cinerea, which can also achieve the effect of silencing mRNA, reducing the level of its specific protein, and finally forming the inhibition of the pathogen. We have designed naked shRNA(Pme1)-2, CPP-shRNA and CPP+bi-shRNA, learn more in our engineering.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 107
Biology
The cell wall is the main interface for plant and microbial interaction, limiting the invasion of pathogens and the spread of infections. When B. cinerea invades the plant host, it synthesizes exogenous enzymes that degrade pectin, a major component of the plant cell wall. Pectin methylesterase (PME) is a hydrolase that catalyzes the hydrolysis of α ester bonds on pectin molecules in plant cell walls. By silencing Bcpme1, an important gene for the expression of PME by B. cinerea, it is capable to prevent B. cinerea from harming the plant cell wall and blocking its invasion from the early stage of infection.
Design of shRNA
After confirming the selection of targets Bcpme1, we searched the cDNA library of B. cinerea according to the sequences or primes provided in the literature, and found the homologous cDNA sequence of B. cinerea. Then, the sequence was input into the National Center for Biotechnology Information (NCBI) website for analysis and prediction, and the CDS sequence of the target gene was input into the total nucleic acid database BLAST to query the homologous similarity of neighboring species. siRNA sequences were designed in non-conserved regions to ensure species-specific and biosafety of our shRNAs.
Next, we used a professional siRNA designed website (https://www.genscript.com/tools/sirna-target-finder) to predict the siRNA sequences that would effectively target the mRNA, and then screened out siRNA fragments with high potential activity in a series of predictions based on shRNA design principles. By making structural predictions of the mRNA (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi), we ensured that the selected siRNA sequences targeted relatively loose positions in the mRNA structure. For biosafety reasons, we BLAST the candidate siRNA fragments into the total mRNA database to ensure that it does not target any genes of common species (such as human, tomato, dog, rice, wheat, etc.), ensuring sequence specificity.
Finally, we assembled the selected siRNA sequence into our shRNA in the sequence of siRNA sense strand - loop - reversed siRNA antisense strand.
Plasmid construction
We have assembled our shRNA in the sequence of siRNA sense strand - loop - reversed siRNA antisense strand, then sequence was assembled in the pET28a (+) plasmid. The recombinant vector was transferred into RNase-deficient E. coli HT115(DE3), and the large-scale fermentation production of shRNA in E. coli could be achieved by induction of IPTG. In our experiment, the results of treatment of different shRNAs at both phenotypic and molecular levels were analyzed to screen out the effective shRNAs.
Usage
The shRNA(Pme1) will be used in crop protection through the way of spraying induced gene silencing (SIGS), which is an emerging, non transgenic RNAi strategy. After screening out an effective shRNA against B. cinerea, the shRNA will be wrapped in KH9-BP100, which belongs to cell-penetrating peptides in a spherical shape and can be used to deliver biological molecules, to forming a CPP-shRNA complex. Then, spraying the complex on the tomato infected by B. cinerea, we hope that it can play a more effective and more steady role in controlling grey mold.
Characterization
shRNA production induced by IPTG
After plasmid extraction, we transformed the constructed shRNA expression vector into E.coli HT115 (DE3) and performed PCR. Our specific primers successfully amplified a 260 bp band from the plasmid, confirming the successful transformation of the plasmid. This indicates that the shRNA expression vector has been successfully introduced into E.coli and can be detected and confirmed by PCR. This is an important milestone that lays the foundation for further experiments.
1-5: plasmids control; 5-10: Plasmids extracted from E.coli.
Compared to the non-induced sample, there is a brighter band between 50-100 bp and 100-150 bp in the induced sample lane. Our shRNA has a size of 69 bp, while the Box-Survival shRNAs, being a concatenation of two shRNAs, have a size of 124 bp. This confirms the successful extraction of our shRNA, and the generated shRNA is of the expected size.
CPP-shRNA under SEM
However, the instability of shRNA in the field environment hinders the optimal performance of our product. Understanding our expectations, our PI suggested that we could try using cell-penetrating peptides (CPP) in combination with shRNA for spray application, and provided us with KH9-BP100 as our CPP material. KH9-BP100 is a carrier peptide-based gene delivery system that enhances the endocytic uptake and cytoplasmic transfer of shRNA in plants, allowing for more efficient transfection of plant callus cells with shRNA. To understand the morphology of the shRNA and KH9-BP100 complex, we used scanning electron microscopy (SEM) to observe the morphology of shRNA, KH9-BP100, and shRNA+KH9-BP100 separately. As shown in the figure, we observed that CPP-shRNA complex form spherical aggregates under electron microscopy. These small spherical aggregates further tend to aggregate with each other. We speculate that this stacking aggregation is due to electrostatic forces.
Distribution of disease spots
We designed shRNA to target and silence genes necessary for the survival of B. cinerea and virulence genes of infected tomatoes. Therefore, we wanted to test whether spraying shRNA can really reduce the attack of B. cinerea on tomato fruits. We used a black marker to draw a circle with a diameter of about 3 mm on the surface of the tomato fruit, and poked five small holes within the circle with a sterilized thin needle.
For the naked shRNA treatment, we added 10 μL solution containing 10 μg shRNA in and around the circle of the fruit surface. After the liquid dried, we drilled holes on the edge of the B. cinerea plate with a 10 μL transparent suction head, and then covered the surface of the fruit in the dotted line area with the mycelium side of the cake. In the control group, we selected non-specific shRNA GFP. The treated tomato fruits were placed in a humid environment at 21 ℃. After three days, ImageJ software was used to conduct a quantitative analysis of the lesion area, which was determined by the area covered by mycelia. Error bars represent standard deviations (SD) obtained from 11-15 biological replicates, the data are F-tested and T-tested, the level of significant difference is passed by a single-tail test, and shown above the bar chart (ns P > 0.05；* P < 0.05；** P < 0.01；*** P < 0.001). For the treatment coated with transmembrane peptide (CPP), the treatment was consistent with the naked shRNA treatment, except that the solution of dripping per sample was changed to 12 μL containing 10 μg shRNA and 8.2 μL 1mg/mL CPP (Figure 6).
At the phenotypic level, the shRNA(Pme1)-2 treatment was effective in reducing the relative plaque area by 27.2% compared with the control group. When combined with CPP, the relative plaque area treated by shRNA(Pme1)-2 was reduced by 31.2% compared with control, proving that CPP can help shRNA more effective.
Detection of inhibition effect by qRT-PCR
On the third day of the experiment, after sampling the lesions of tomato fruits, the sample RNA was extracted, reverse-transcribed, and qRT-PCR was performed to detect the inhibition effect of shRNA on mycelium target mRNA in infected fruits (Figure 7).
From the results of molecular experiments, the silencing rates of the experiment that spraying the naked shRNA(Pme1)-2 is 58.4%, after binding with CPP, the silencing rates could reach 59.0%. This result have showed that CPP was really poly a good role in helping shRNA to silencing target mRNA.
Sustained inhibition
To investigate the duration of action and the optimal timing of our RNAi products, we conducted stability testing on the produced shRNA. Due to time constraints, we selected only the shRNA(Pme1)-2 combined with CPP for validation in this experiment. After droplet application of 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 shRNA-CPP. The results are shown in the figure below.
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 70%. Subsequently, the expression levels of the target genes show an increasing trend.
Delayed Differential Equation Model of shRNA
To further characterize the process of RNAi interference, we introduced biological delays on the basis of the conventional system of ordinary differential equations, taking into account the time for mRNA to be transported out of the nucleus and the time required for mRNA to be broken down. This model was used to predict the silencing effect of shRNA in cells, and the mean square error was only about 10% compared with experimental data.
Furthermore, we used optimal control theory and optimization theory to optimize the cost function and developed a mathematical model to predict the optimal application rate of RNAi pesticides. Feeding the result into our previously developed DDE model concerning RNAi, it was found that RNA pesticides performed significantly better under an acceptable cost after optimization.
References
[1] Niu D, Hamby R, Sanchez JN, Cai Q, Yan Q, Jin H. RNAs - a new frontier in crop protection. Curr Opin Biotechnol. 2021 Aug;70:204-212. doi: 10.1016/j.copbio.2021.06.005. Epub 2021 Jul 1.
[2] Sarkar A, Roy-Barman S. Spray-Induced Silencing of Pathogenicity Gene MoDES1 via Exogenous Double-Stranded RNA Can Confer Partial Resistance Against Fungal Blast in Rice. Front Plant Sci. 2021 Nov 26;12:733129. doi: 10.3389/fpls.2021.733129.
[3] Qiao L, Lan C, Capriotti L, Ah-Fong A, Nino Sanchez J, Hamby R, Heller J, Zhao H, Glass NL, Judelson HS, Mezzetti B, Niu D, Jin H. Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake. Plant Biotechnol J. 2021 Sep;19(9):1756-1768. doi: 10.1111/pbi.13589. Epub 2021 May 4.
[4] Bofill-De Ros X, Gu S. Guidelines for the optimal design of miRNA-based shRNAs. Methods. 2016 Jul 1;103:157-66. doi: 10.1016/j.ymeth.2016.04.003. Epub 2016 Apr 12.
[5] Thagun C, Horii Y, Mori M, Fujita S, Ohtani M, Tsuchiya K, Kodama Y, Odahara M, Numata K. Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts. ACS Nano. 2022 Mar 22;16(3):3506-3521. doi: 10.1021/acsnano.1c07723. Epub 2022 Feb 23.
[6] Rao DD, Senzer N, Wang Z, Kumar P, Jay CM, Nemunaitis J. Bifunctional short hairpin RNA (bi-shRNA): design and pathway to clinical application. Methods Mol Biol. 2013;942:259-78. doi: 10.1007/978-1-62703-119-6_14.
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