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shRNA (PG)-1

In order to contain the infection of R.solani, we have identified PG, which is critical to the infectivity of Rhizoctonia solani, as the taget of our first batch of RNAi targets. We have designed siRNA(PG), shRNA(PG) and shRNA(PG) incubated with CNT products successively, see more about siRNA(PG) and shRNA-CNT in our Engineering success.

Sequencing

Sequence and Features

Biology

Polygalacturonase (PG) is a major pectin degrading enzyme of Rhizoctonia solani which hydrolyzes the α-1, 4-glycosidic linkage of D-galacturonic acid in pectin. Pathogenic fungi synthesize PG enzyme for initiation and its establishment during host infection, that is, PG genes were strongly induced during the initial infection process. What is more, pectinases including PG are the most important factor for the pathogenesis of plant fungi which help in decomposition of pectin in the plant cell wall. The degradation of pectin is important not only to weaken the cell wall to facilitate penetration and colonization of the host cell but also is a source of carbon during pathogen proliferation. Thus, we designed this shRNA molecule for the silencing of R.solani-encoded PG gene and suppression of rice sheath blight development.

Design

The PG sequences found was 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. However, it may be due to the lack of relevant research and literature support, and the corresponding mRNA has no variant. Also, the total nucleic acid database blast was carried out on the target CDS to query the similarity of homologous genes in adjacent species, and shRNA was designed in non-conserved regions to improve the species specificity of our shRNA and ensure biological safety.

And then these gene fragments were analyzed by siRNA Design websites. The program scans the DNA sequence of a gene fragment and calculates the binding energy of sense and antisense siRNAs based on the sequence pattern. The higher the score, the stronger the binding ability of the siRNA to the target sequence. Based on the principle of shRNA design, we selected the fragments with high potential siRNA activity from a series of sequences.

For biosafety, the candidate RNAi fragments were submitted to the total mRNA database for blast, and the sequence similarity was compared. Focus on species with more than 90% similarity and their nucleic acid fragments to ensure that there is no matching of common species (human, rice, dog, wheat, etc.) to ensure the specificity of the sequence.

Finally, the chosen RNAi fragment is assembled in the order of sense RNAi fragment — loop — antisense RNAi fragment.

Plasmid construction

We have confirmed that this siRNA sequence has a good binding ability to Rhizoctonia solani. The intrinsic order of this sequence is sense RNAi fragment — loop — antisense RNAi fragment. This sequence was assembled in the pET28a (+) plasmid containing the IPTG-inducible phage T7 promoter and subsequently transferred into RNase-deficient E. coli HT115 (DE3). In our project, shRNA will be industrially produced by E. coli on a large scale, and shRNA will be purified and sprayed on rice fields to inhibit Rhizoctonia solani.

Usage

The shRNA(PG) will be used in crop protection through two ways: host induced gene silencing (HIGS) and spray induced gene silencing (SIGS). Some studies have shown that carbon nanotubes (CNTs), which is non-toxic and efficient in delivering biomolecules, can effectively deliver biological molecules in plant cells and have realized internalization into mature plant cells after binding DNA. Therefore, we will bind our shRNA molecules to CNT in the hope that it can make shRNA more stable in the environment and better absorbed by R.solani cells to start the RNAi process.

Characterization

shRNA production induced by IPTG

We transformed the constructed shRNA expression vector into E. coli HT115 (DE3), and PCR after extracting the plasmid. Our specific primer can amplify a 260 bp band on the plasmid, and the results verify the successful transformation of the plasmid (Fig. 1).

It can be seen from the figure that there is a brighter band between 50-100 bp in the induced sample lane than in the uninduced sample. The size of our shRNA is 59 bp, which proves that our shRNA extraction is successful and the shRNA produced meets the expected size.

shRNA inhibits mycelium growth on leaves

To prove that our RNAi product can inhibit the infection of Rhizoctonia solani, we observed the growth of hyphae on leaves after spraying shRNA under a microscope (Figure 1.). At the same time, we also observed the leaves treated with shRNA after CNT binding (Figure 2.).

After measuring the mycelium extension length in leaves under different treatments, the following statistical results are obtained (Figure 2.).

For spraying shRNA without CNT binding, the growth of mycelia on leaves was inhibited to some extent, but the inhibition effect was not ideal. For shRNA that inhibits the key genes in the infection process of R.solani, only the PG gene is inhibited; However, shRNA of key genes inhibiting the growth of Rhizoctonia solani had no significant effect.

After the shRNA was bound with CNT, it was sprayed. Compared with the control group's 21.5 mm mycelium length, the shRNA targeting the key genes in the infection process of Rhizoctonia solani could inhibit the extension of the mycelium to about 12.1 mm. For shRNA targeting the key genes of R.solani growth, the highest inhibition rate of hyphal extension was 40%.

Inhibition effect detected by qRT-PCR

For shRNA, we also verified its inhibition by the same method as for siRNA . At the same time, after binding CNTs, we also tested their effects (Figure 4.c, d).

The results showed that when CNT was not bound, the average silencing efficiency of shRNAs targeting key genes in the infection process was about 0.33 compared with the control group, while the average silencing efficiency of shRNAs targeting key genes in the survival of Rhizoctonia solani was about 0.37; However, after the same amount of shRNA was bundled with CNT and sprayed, the expression of target genes was significantly inhibited compared with the control group and the group without CNT, and the average inhibition efficiency of the two types of shRNA was 0.59 and 0.66, respectively. To sum up, our shRNA inhibition effect is good, and binding CNT promotes the inhibition of shRNA against R.solani.

Sustained inhibition

The essence of RNAi treatment is that sRNA molecules specifically recognize target genes in R.solani and then conduct post transcriptional silencing. Therefore, in order to verify the therapeutic effect of RNAi, we must measure the changes in the expression level of target genes in R.solani after spraying sRNA. We sprayed siRNA, shRNA and CNT shRNA respectively, and tested the effect of RNAi products through qRT-PCR for 5 consecutive days.

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 5.).

For siRNA and shRNA treatment, the relative expression of target genes in Rhizoctonia solani decreased to the lowest at the third day, 0.144 and 0.103, respectively. But after that, the expression of target genes in both groups increased rapidly. Under the treatment of CNT shRNA, the expression of target gene decreased to 0.019 on the fourth day, and the effect lasted longer.

To sum up, CNT shRNA has more obvious advantages in comparison with the size of the colony of Rhizoctonia solani growing on PDA medium, the growth degree of the mycelia on the leaves, the size of the diseased spot, the expression inhibition level of the target gene and the duration of the inhibition effect. Therefore, we look forward to its outstanding performance as our RNAi product.

Containing leaf lesion

As one of the important indicators of leaf disease, the area of disease spots on leaves can also be used to evaluate the effect of RNAi products. On the fifth day of spraying, we observed the difference of leaf spot under different treatments(Figure 6.), and quantified the area of the spot through image analysis(Figure 7.).

We can find that compared with the control, spraying siRNA will reduce the area of the diseased spot to 45.53%, and shRNA will reduce the area of the diseased spot to 25.91%. The spraying effect of shRNA-CNT was the best, and the area of disease spot was only 8.53% of the control. It can be concluded that shRNA CNT has obvious advantages over siRNA and shRNA.

References

[1]Rao, T.B., Chopperla, R., Methre, R. et al. Pectin induced transcriptome of a Rhizoctonia solani strain causing sheath blight disease in rice reveals insights on key genes and RNAi machinery for development of pathogen derived resistance. Plant Mol Biol 100, 59–71 (2019). https://doi.org/10.1007/s11103-019-00843-9

[2]Chen, X., Lili, L., Zhang, Y. et al. Functional analysis of polygalacturonase gene RsPG2 from Rhizoctonia solani, the pathogen of rice sheath blight. Eur J Plant Pathol 149, 491–502 (2017). https://doi.org/10.1007/s10658-017-1198-5