Lanzhou

RNAi Mechanism

RNA interference first discovered as an ancient antiviral system in plants, when long dsRNA come into cells, they will be rapidly cut by dicer or dicer-like enzyme (double-stranded RNA specific endonucleases of Rnase III family), forming 21 to 28-nt RNA duplexes with the characterize of 2-nucleotide 3’overhangs and 5’phosphates, which are called siRNAs (small interfering RNA). SiRNAs conform a ribonucleoprotein complex RISC (RNA-induced silencing complex).

RISC includes slicer and Argonaute protein with Rnase H-like domain, which first mediates the unwinding of siRNA duplex. The passenger strand is degraded by Argonaute, then the guide RNA binds to the target mRNA in a sequence specific manner. This binding mediates target mRNA cleaved by slicer, meanwhile the fragments of mRNA will be recognized as the aberrant and destroyed by cells, inducing gene silence in a post transcriptional level ( PTGS ,Post Transcriptinal Gene Silence).

RNAi mechanism is the basis of our project.

Figure1. In Arabidopsis, four Dicer-like (DCL) proteins (DCL1 to DCL4) have been identified. Three of which are involved in processing dsRNAs that come from different sources. DCL1 is required for the production of siRNAs derived from long dsRNAs, including viruses and transgenic hairpins used in experimental RNA interference.

Tandem RNAi

We noticed that many weeds are perennial plants, and they always chosen by pests as natural shelters to survive the brutal winter. This phenomenon inspired us to wipe out weeds and pests together by a method—tandem RNAi, which means triggering RNA interference in at least two organisms at the same time. In our project, we magnified RNAi effect by selecting vital genes of target organisms, whose silence will result in phenotype change or death. And we used dsRNA irrigation to realize the tandem RNA interference.

In the latest research, Hunter et al. confirmed that citrus trees can absorb dsRNA through roots and phloem injections (Hunter et al. 2012). Besides, It has been proved when Arabidopsis roots were soaked in dsMob1A (Mob1A gene is required for organ growth and reproduction), the root lengths and numbers were significantly suppressed and plants could not bolt or flower. These results directed a possible way to deliver dsRNA into plants through dsRNA irrigation .

Meanwhile, we noticed dsRNA spraying has been verified successfully in disease and mandibulate insects control, but it can’t affect the piercing-sucking pests which live on phloem sap, plant stems or root system. For these sap-sucking insects, dsRNA needs to be delivered through the phloem sap. And this delivery could be realized by dsRNA irrigation. Demonstrated by Araujo that the dsRNA flowing through the plant vascular system could be taken up by psyllids that feed on the phloem. As a result, the insects’ mortality increased when the dsRNA of an arginine kinase was absorbed by the host tree. (Araujo et al. 2006; Hunter et al. 2012).

Taken together, we made an assumption: when designed dsRNA contains both vital genes information of target plant and insect, dsRNA irrigation-mediated RNAi are able to affect insects and their host plants together.

Based on this deduction, we constructed our project—Bio-pesticides

Maybe you would still doubt the power and efficiency of our tandem-RNAi-based bio-pesticides. Here we give u a cardiotonic.

•Amplification of the RNAi system

It was estimated that only two molecules of dsRNA per cell were able to induce RNAi In C. elegans. In another report, injection of dsRNA into the intestine of a C. elegans hermaphrodite generated RNAi, which could be stably inherited to the F2 generation. These two findings led to the proposal that RNAi signals could be systemic and amplifiable in nature.

The amplification effect should be possible with the following mechanisms

1. RISC reuse

RISC can be reused in the last step of RNA silencing pathway and It has been reported that RISC can degrade more than 30 mRNA substrates. Which amplifies and prolongs the silencing effect.
2. Generation of siRNA

A single starting dsRNA molecule are able to generate multiple siRNA molecules (about 45 siRNAs/Kb), it’s a kind of amount magnification, and even without the supply of new dsRNA, the silencing effect could still be maintained for a long time.
3. RDRP mechanism

In addition, RNAi amplification rely on an RNA-dependent RNA polymerase (RdRP) system, the role of RDRP can be explained by"random degradation PCR model": RdRP could use target mRNA as templates to synthesize new dsRNA in the guide of single stand siRNA (as primer), then dicer cut new dsRNAs and generate amount of secondary siRNAs and into the next round of cycle reaction. After multiple cycles, the signal has been maginified, meanwhile there has been enough RISC complexes to finish silencing. In addition, cleaved mRNA fragments can be regarded as aberrant RNA, contributing to dsRNA denovo synthesis( without primer)mediated.by RDRP.

RDRP were demonstrated in nematodes (EGO1, RRF1, RRF3), chondromyces (QDE1) and fission yeast, but there is no direct evidence that RdRP enzyme was found to be active in other organisms. Otherwise, the work of Lipardi demonstrated that RdRP activity was found in the embryo pyrolysis of drosophila, but no RdRP homologous sequence was found in fruit flies and human genomes.

On the whole, the amplification system guarantee the basic strength and duration of RNAi.

•Movement of RNA silencing signal in plant

In plants, RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances through vascular system, leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant.

Molecular transport in plants can occur either symplastically through the channels connecting the adjacent cells termed plasmodesmata, or apoplastically through a transfer process acrossing the intercellular spaces, cell membrane and the cell wall.

In short range silencing spread, the silencing molecules (21nt siRNAs, miRNAs, tasiRNAs or other type) most probably follow the symplastic route. The silencing spread is limited to 10–15 cells beyond the production site and with no need of an amplification mechanism. Silencing molecules are then transported with high velocity through the phloem pathway from photosynthetic sources (i.e. leaves) to sucrose sinks (i.e. roots and growing points).

The long distance or systemic movement of the silencing signal takes place over days. This spread is mediated by PTGS (post transcriptional gene silencing )or TGS (transcriptional gene silencing), which conforms to the mobile 21 nt siRNAs that stem from exogenous dsRNA.

Figure 4. RNA silencing signal movement mechanism

The mechanism ofs silencing signal movement in plants make sure that when dsRNA is absorbed by roots, it will trigger systemic RNAi and silence target gene throughout the entire plant.

Final performance

Our RNAi-based bio-pesticides, unlike chemical pesticides and transgenic crops containing Bacillus thuringiensis (Bt) toxins, are able to target a specific organism, thereby offering pests and weeds species-specific management.

In experiments, designed dsRNA contains both target plant and pest key vital genes information and via dsRNA irrigation plants, realize the “Two birds, one stone” . See more details in construction.

DsRNA irrigation strategies can be improved by mixing the RNAs with chemical reagents or nano materials (like bioclay) to stabilize the RNAs and thus increase the strength and duration of plant protection. Overall, this new generation of tandem-RNAi-based pesticides looks promising to meet our world's increasing demands for increasing safety and quality of crop yields to feed the growing population.

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