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shRNA(Box-infect)

In order to inhibit the infection of B. cinerea, the pathogen of grey mold and control the disease in tomato, we designed four pieces of shRNAs targeting the genes Bcpme1, BcOAH, Bcdcl1 and Bcdcl2 of the pathogen, which can help B. cinerea produce virulence factors, based on RNAi technology. Then, we stringed four 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 Bcpme1, BcOAH, Bcdcl1 and Bcdcl2 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.

Sequencing

Sequence and Features

Biology

During infection, B. cinerea will express oxaloacetate hydrolase, which catalyzes the hydrolysis of oxaloacetate in B. cinerea to oxalic acid. After oxalic acid enters the host plant, the free calcium ions in the host body can form calcium oxalate crystals, which is easy to cause the symptoms of plant blockage. The release of oxalic acid can also inhibit the outbreak of reactive oxygen species (ROS) in plants, which is not conducive to the plant to defense the infection of B. cinerea. In addition, the cell wall degrading enzymes of pathogenic bacteria have high activity in the acidic environment provided by oxalic acid, which is conducive to the penetration of pathogenic fungi. Silencing the gene BcOAH of B. cinerea can reduce the production of oxalic acid and prevent further infection and spread of pathogens after invasion.

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.

Dicer-like proteins DCL1 and DCL2 of B. cinerea participate in the synthesis of siRNA. Then siRNAs will be delivered to host plant cells to participate in the formation of RISC complexes in the host RNAi mechanism, and subsequently silence and inhibit host immune-related genes, coding mitogen activated protein kinase (MAPK) and cell wall-associated kinases (WAK), making plant cells more susceptible to infection. The shRNA was designed to silence B. cinerea genes Bcdcl1 and Bcdcl2, preventing them from synthesizing siRNA that interferes with the plant cell's immune system, thus allowing the plant to effectively defend itself against B. cinerea.

To reduce the cost of RNAi bio-pesticides, and to reduce the off-target rate of shRNA molecules, 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 Bcpme1, BcOAH, Bcdcl1 and Bcdcl2, 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 Bcpme1, BcOAH, Bcdcl1 and Bcdcl2, we made a bold attempt to connect two different shRNA sequences with a specific sequence AGGCAT . Finally, we finished the design of our tetra-shRNA and named it shRNA(Box-infect).

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

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 21.2% compared with the control group, with no significant difference compared with other groups. However, after binding CPP, the relative plaque area of the shRNA(Box-infect) treatment group was reduced by 21.8%, showed no significant difference compared with shRNA treatment without CPP, also seen almost to be same as the groups treated by CPP-shRNA. According to the results, we hypothesized that tandem RNA can improve the efficiency of RNAi, but when the tandem shRNA reaches a specific number, the binding of Dicer to the molecules will be blocked due to the structure of the tandem molecules in the reaction of processing into different siRNAs, which will lead to the limitation of siRNAs production and ultimately weaken the effect of RNAi.

References

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