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The siRNA silencing Polygalacturonase (PG) of Rhizoctonia solani AG1-IA.

Usage and 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

Next, the PG 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. 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.

Sequencing

Sequence and Features

Assembly

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.

Characterization

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(Fig. 1), and quantified the area of the spot through image analysis(Fig. 2).

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.

Sustained inhibition of siRNA

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(Fig. 3).

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