Because of the high specificity, safety and sustainability of RNAi, we chose this technology to achieve the treatment of rice sheath blight. After screening, we selected the key gene, PG, which plays a role in the process of rice infection by R.solani as our target gene. For the selection of sRNA molecules that can trigger RNAi process, because siRNA is more widely used and the application system is more mature, we initially decided to use siRNA to achieve our silencing of target genes of R.solani. On the basis of previous studies proving that R.solani can independently absorb sRNA molecules, we hope that through spraying, siRNA molecules from the outside world can enter R.solani and start the RNAi process in its body, so as to achieve the silencing effect of target gene PG, and then inhibit the infection of R.solani on rice.

After determining the target gene, we searched the cDNA database of Rhizoctonia solani AG1-IA according to the sequences provided by the literature, and found the homologous cDNA sequences of AG1-IA strain. Next, the sequences found by this method were analyzed, queried or predicted on the National Center for Biotechnology Information (NCBI) website whether there were multiple spliced versions of mRNA, and if there were, the homologous region was taken as the target region of RNAi interference target. Through BLAST, we checked the biosafety of the target sequence. After confirming that its good specificity, and then by professional siRNA Design websites to analyze these gene fragments. Based on the principle of shRNA design, we selected the fragments with high potential siRNA activity from a series of sequences(Table1). Then we sent the sequence to the biological company and obtained our siRNA molecule by chemical synthesis.

Table 1. siRNA information targeting the PG gene of R.solani

The rice leaves infected with R.solani were sprayed with siRNA, and the expression level of target genes of R.solani on the leaves was detected for 5 consecutive days after spraying. From the results of continuous qRT-PCR, it can be seen that the target gene was silenced after siRNA treatment for 3 days, with a silencing rate of 85.6%, and began to rise rapidly after the third day(Figure 2). (More details can be viewed in Proof of Concept.)

Figure 2 Silencing level of siRNA to target gene

Seen from the silencing effect, siRNA can not effectively silence target genes for a long time, which means that we need to spray siRNA several times in practical applications to achieve our desired effect. However, chemical synthesis of siRNA in vitro is expensive, so it is not practical to achieve large scales of continuous spraying. In addition, the exposed ends of siRNA make it easy to be degraded by RNase in the air. Therefore, we have to consider selecting other sRNA molecules and developing new sRNA production methods.

We further screened out more target genes. Silencing these target genes can achieve the purpose of inhibiting infectivitity and killing pathogens. Considering that shRNA is more stable than siRNA, we decided to use shRNA to realize the RNAi process in Rhizoctonia solani, and designed different target sequences according to the selected target genes. Because a large amount of shRNAs are required during spraying, they need to be produced by a low-cost and high-yield method. So we thought that we could use fermentation engineering technology to obtain a large amount of shRNA molecules. We selected the most common strain, E.coli, for fermentation production, and used nuclease deficient E.coli HT115. The designed target sequence is constructed through the order of sense fragment loop-antisense fragment, and connected to the plasmid pET-28a (+) to obtain our shRNA expression vector.
During the wet experiment, we also decided to build an ODE model of RNAi to predict the effect of shRNA molecules.

In order to verify our successful transformation, we used specific primers to amplify the plasmid extracted from E. coli by PCR, and proved that our shRNA expression vector successfully entered E. coli HT115 by agarose gel electrophoresis. Then, after IPTG induction, we extracted the RNA of Escherichia coli, and confirmed the successful production of shRNA in Escherichia coli according to the band size(Figure 3).

Figure 3 Electrophoresis of RNA extracted from HT115 (DE3)

In order to ensure that our shRNA can enter the cell of Rhizoctonia solani smoothly, we also observed its spraying under the laser confocal microscope (Figure 4).

Figure 4 Adding GFP-shRNA of Cy5 labeled bundled CNT to R.solani on leaves

At the same time, the shRNA produced was sprayed on the leaves infected by R.solani, and the inhibition effect of shRNA on target genes of Rhizoctonia solani was detected by qRT-PCR. The result shows that the target gene was silenced after spraying shRNA for 3 days, with a silencing rate of 89.7%, and began to rise rapidly after the third day(Figure 5).The data showed that shRNA had a better inhibition effect than siRNA, and had a longer effect.（More details can be viewed in Proof of Concept).

Figure 5 Silencing level of shRNA to target gene

We substituted the data in E.coli into the ODA model of RNAi, and obtained the theoretically silent effect and action duration of shRNA(Figure 6).The effect of model simulation is highly similar to the experimental results.

Figure 6 RNAi ODE model results

More details about the RNAi ODE model, please click model.

Our actual application scenario is in the field, and it is difficult to ensure the stability and sustainability of shRNA molecules under complex environmental conditions. In the face of this problem, we have to find a more effective way to deliver our shRNA.

Nanomaterials can contribute to the stability of sRNA molecules, and their applications have been widely verified in delivering RNAi drugs in medicine. However, few people have completed this work in the field of crop protection. We hope that by binding with nanomaterials, we can help our shRNA molecules to be more stable in the air and complete steady release for a period of time, so that they can be applied to spraying in the field.

We obtained our nanomaterial shRNA particles by co incubating the nanomaterial and shRNA in a fixed proportion at room temperature for 30 minutes. For the binding of LDH, we verified its successful packaging by agarose gel electrophoresis (Figure 7). In addition, we also sprayed carbon nanotube(CNT) separately to ensure that they are not toxic to our rice leaves and R.solani (Figure 8).

Figure 7 Research on binding conditions with LDH

Figure 8 Incidence of leaves after CNT treatment

Similarly, in order to ensure that our shRNA can still enter the cells of Rhizoctonia solani smoothly after binding CNT, we also carried out verification (Figure 9).

Figure 9 Adding GFP shRNA of Cy5 labeled bundled CNT to R.solani

Spray shRNA bound with nanomaterials on the leaves infected by R.solani, and detect its inhibition effect on target genes by qRT-PCR (Figure 10). From the results, CNT shRNA has a strong inhibitory effect on target genes. On the fourth day, the inhibition rate reached the maximum of 98.13%, and the inhibition rate was slow, ensuring that CNT shRNA can continue to play its role for a period of time (More details can be viewed in Proof of Concept).

Figure 10 Silencing level of CNT-shRNA to target gene

From the above results, our nanomaterial-shRNA has a excellent inhibitory effect on target genes, and the silencing effect lasts longer. Therefore, we can ensure that the nanomaterial-shRNA can be sprayed in the field as our RNAi product to achieve a precise and continuous attack on pathogenic fungi. Our engineering cycle of RNAi Silence System also ends after the verification of this step.

In order to block the transmission of R.solani from aquatic interface and soil, we hope to design a carrier that can accurately deliver engineered T.atroviride spores to the susceptible parts of rice, such as the rice stem at the aquatic interface. The first material we noticed was starch Graft Acrylate Polymer, which has strong water absorption and retention.

We prepare the TACE1.0 according to the following steps. Firstly, prepare the spore suspension and TACE1.0, which are 40 mesh or 80 mesh starch Graft Acrylate Polymer particles. Then add spore suspension according to the liquid-said ratio of 45:1, and spray after its full imbibition.

To confirm whether it is the best choice for us to load T.atroviride spores, we tested the performance of TACE1.0 delivering spores at the aquatic interface. The experiments include the sustained release capability, adsorption radius, floatability, encapsulation and loading rate.

For sustained release capability, the result shows that spores in TACE1.0 are almost completely released in 100 minutes. We measured the distance from the point where TACE1.0 particles can be adsorbed to the plant, and measured that the adsorption radius of TACE1.0 is 0.824±0.209cm.

Figure 11 TACE1.0 is adsorbed to the rice stem

Then we found that the floating capacity of TACE is unstable, and it is easy to settle in advance or disengage from rice stem. We also calculated the encapsulation and loading rate of the spores after TACE's imbibition. However, we found that even under the optimal liquid-said ratio, the encapsulation efficiency of TACE1.0 was only 17%, and the loading rate was only 13%.

Figure 12 Carry capacity of TACE 1.0 to Trichoderma spores at different liquid-said ratio

In addition, we pay great attention to whether our products are environmental-friendly, so we test whether TACE1.0 will be residues in the environment and come to the conclusion that TACE1.0 will not be completely degraded after its dissemination in paddy fields . There remains 0.2% - 0.3% residues of refractory substances in the environment.

Figure 13 Degradation of TACE 1.0

The data obtained from the experimrnts shows that the duration of spore sustained release failed to meet our expectation of long-term release at aquatic interface. In addition, the firmness of adsorption of TACE1.0 is not ideal and is greatly affected by external forces. This means that once TACE1.0 is blown away, it is difficult to gather at the rice stem again.As for its floatability, TACE1.0 is hard to stay afloat. Moreover, the encapsulation and loading rate are still below our expectations.
Having learned from the shortcomings of TACE1.0 above, we successfully iterated the TACE.

Inspired from pharmaceutics, we designed the second generation of TACE. TACE2.0 is a hydrophilic sustained-release matrix preparation with Hypromellose (HPMC) as the main component and T.atroviride spore as the effective component. We expect TACE2.0 to have more efficient carry capacity, better sustained release capability, more stable floating and adhesion performance.

After investigation and experiment, we came up with a recommended prescription of TACE2.0. Through a modern pharmaceutical process, we mixed 15% Trichoderma spore powder, 15% glucose, 60% HPMC400, 10% HPMC15000 and trace magnesium stearate and tablet , obtaining TACE2.0 of different particle sizes.

Figure 14 TACE2.0 of different particle size

After testing, we obtained the following performance data of TACE2.0:
(1)Constancy:
At the same time, we verify that the 3mm TACE2.0 could release for at least 8h and 6mm for 1~2 days, forming a continuous local high spore concentration environment at the aquatic interface.
In order to simulate the release process of spores in TACE2.0, we used first-order kinetic equation to fit the release profile of TACE (More details can be viewed in model). The release curve should have three typical release phases that TACE2.0 doesn't have. From the anomaly modeling results, we have learned that TACE2.0 is not smooth enough, leading to rapid gelation of surface and entering the intermediate rapid release phase.

Figure 15 Fitted by the first order kinetic equation

(2)Floatage: The dry TACE2.0 has a measured density of about 0.87 g/ml and can suspend on the water surface itself.

Figure 16 Adhesion of TACE2.0 to Rice Stem

(3)Adhesion: Due to the gelation of surface, TACE2.0 has good adhesion performance so that it is not easy to be peeled off.
(4)Encapsulation efficiency and loading rate: TACE2.0's theoretical encapsulation rate reaches 100%, and the loading rate reaches 15%. That is, a single 3mm TACE2.0 tablet can carry approximately 3.27 million T.atroviride spores.
(5)Dissolution: The HPMC matrix is dissoluble and degradable, which means TACE2.0 does not leave any residue in paddy fields.

Our TACE2.0 has marked improvements over TACE1.0 in terms of carry capacity, sustained release capability, stable floating and adhesion performance and so on. What's more, we achieved delayed activation of the conditional triggered switch by adding glucose. Through a series of verification, TACE2.0 has been proved to be a successful Trichoderma's carrier.