Part:BBa_K4653015
shRNA(Box-survival)
In order to kill B. cinerea, the pathogen of grey mold and control the disease in tomato, we designed two pieces of shRNAs targeting the genes BcchsIIIa and Bccyp51 of the pathogen, which is essential for its cell wall formation and cell membrane maintaining, based on RNAi technology. Then, we stringed two pieces of shRNAs to form bi-shRNA, which can be actively absorbed by B. cinerea, then enter into its cells to be processed into different siRNAs, and further specifically target mRNA of genes BcchsIIIa and Bccyp51 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 mRNAs, reducing the level of its specific protein, and finally killing the pathogen. We have designed naked shRNA, CPP-shRNA and CPP+bi-shRNA, learn more in our engineering.
Sequencing
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Biology
Chitin synthase (CHS) catalyzes the synthesis of chitin, a major structural component of fungal cell walls consisting of β-1, 4-linked N-acetylglucosamine polymers. CHS can be divided into seven classes, and ascomycetes usually have representatives of all seven classes, with each enzyme having a specific role. CHS (III, V, and VI) are specific to molds, suggesting that they may play an important role in hyphal growth. Like other ascomycetes, B. cinerea contains two CHSIII genes. It has been shown that the BcchsIIIa gene is most expressed in the CHSIII genes, whereas the BcchsIIIb gene is not expressed in any of the growth conditions used. Ergosterol (ERG) is a C28 sterol that is particularly present in fungal cell membranes and plays a crucial role in the structure and function of fungal cell membranes. Ergosterol is responsible for maintaining membrane fluidity, regulating membrane permeability, affecting membrane-related enzyme activity, and influencing fungal cell growth. Silencing Bccyp51, a key gene in the ergosterol biosynthesis pathway, can destroy the cell membrane of B. cinerea and produce killing effect on it. By silencing the important chitin synthase gene BcchsIIIa and ergosterol synthase gene Bccyp51, the formation of the cell wall and cell membrane of B. cinerea could be affected, thus killing pathogenic fungi.
To reduce the cost of RNAi bio-pesticides, and to strengthen the effect of a single RNAi molecule, we have used a novel vector-driven bifunctional short hairpin RNA (bi-shRNA) technology that harnesses both cleavage-dependent and cleavage-independent RISC loading pathways to enhance knockdown potency. Consequent advantages provided by the bi-shRNA include a lower effective systemic dose than comparator siRNA/shRNA to minimize the potential for off-target side effects, due to its ability to induce both a rapid (inhibition of protein translation) and delayed (mRNA cleavage and degradation) targeting effect depending on protein and mRNA kinetics, and it has been proved a longer duration of effectiveness for clinical applications.
Design of shRNA
After confirming the selection of targets BcchsIIIa and Bccyp51, 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.
Then, we assembled the selected siRNA sequence into our shRNA in the sequence of siRNA sense strand - loop - reversed siRNA antisense strand. After we got the shRNAs targeting BcchsIIIa and Bccyp51, using a specific sequence AGGCAT to connect two different shRNA sequences. Finally, we finished the design our bi-shRNA and named it shRNA(Box-survival).
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(Box-survival) 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(Box-survival) treatment was effective in reducing the relative plaque area by 19.3% compared with the control group, while the session sizes treating by shRNA(CHSIIIa) and shRNA(cyp51) were 25.6% and 16.8% respectively, showing little different effects among them. However, after binding CPP, the relative plaque area of the shRNA(Box-survival) treatment group was reduced by 42.0%, which was more effective than other two group treating by CPP-shRNA(CHSIIIa) and CPP-shRNA(cyp51) obviously. The results not only showed that CPP can help shRNA play a better role, also confirmed that our shRNA(Box-survival) could work well.
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 CPP-shRNA(Box-survival) is 69.6%, while the rates of CPP-shRNA(CHSIIIa) and CPP-shRNA(cyp51) were 50.5% and 65.2% respectably, further confirmed that the design of bi-shRNA can help shRNAs be more effective.
References
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