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Post-Transcriptional Gene Silencing is Induced in Plants by Exogenous miRNAs

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1. What were the most important discoveries in our reviewed paper, Betti et al.?

According to Betti et al. (2021), plant-to-plant communication is critical in developing

interspecies relationships. Chemical signals from plants' neighbors may influence a plant's

survival and function. This has ramifications for the peaceful cohabitation of plants and the

assembling of a community. According to several studies, root exudates have played an essential

role in plant-to-plant communication. Inter- and intra-specific assistance in cropping systems,

including mixed-species plantations, is driven by root exudates. Root exudates include signaling

components that may alter relationships. Root ethylene, strigolactones, jasmonic acid, (-)-collide,

and allantoin is signaling molecules that carry information about local circumstances in the

plant-plant interactions. Plants produce an abundance of root-secreted signaling molecules that

set off a cascade of below-ground reactions, including molecular processes in biosynthesis,

secretion, and action, all of which are inter-and intraspecifically coordinated. Several exciting

plant-plant interactions may be discovered via root-secreted chemical signals and associated

molecular processes. However, methodological constraints and root-soil interactions mean many

roots remain undiscovered, especially species-specific signals and their underlying processes.

Studying root-secreted molecular movements and their functions will have many ecological and

agricultural ramifications.

According to Betti et al. (2021), as the minor non-coding RNAs in plants and animals,

microRNAs are the minimal transmitters of gene-encoded, post-transcriptional regulatory

information. Plant and animal miRNAs have a great deal of structural and functional similarity.

Most of the transcriptomes of different cell types have their structure and function determined by

miRNAs from both kingdoms. mRNA stability and translational inhibition seem to be the

primary mechanisms by which plant and animal miRNAs exercise their genetic and
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transcriptomic effects on gene expression. A common origin and functional selection of specific

microRNAs over vast periods of evolution (for example, Arabidopsis thaliana-Homo sapiens

divergence of 1.5 billion years) may be suggested by the finding that plants and animals express

certain miRNA species such as miRNA-155, miRNA-168, and miRNA-854 family members.

When it comes to human health and illness, there is considerable debate about whether plant-

enriched miRNAs, which are involved in cross-kingdom miRNA communication, may have

physiological and pathophysiological effects. The intriguing possibility that dietary miRNAs and

sncRNAs may have the potential to contribute to both intra- and inter-kingdom signaling and, in

doing so, modulate molecular-genetic mechanisms associated with human health and disease, is

highlighted in this research paper, which highlights some recent, controversial, and remarkable

findings in plant- and animal-based miRNA signaling research.

According to Strzyz (2021), RNA silencing has evolved in higher eukaryotes. Non-cell-

autonomous RNA silencing occurs in plants and certain animals, including the worm

Caenorhabditis elegans. As a result, RNA-mediated suppression of homologous regions in

nearby cells occurs due to silencing initiation in one or a few cells. A short-range silencing

signal, most likely 21-nt siRNAs (short interfering RNAs) generated by one of the plant Dicer

enzymes, is responsible for spreading silence in plants. In addition, the phloem system of plants,

which transports compounds from the source to the sink tissues, transmits silencing systemically.

In contrast to the short-range silencing signal, systemic silencing's messengers are primarily

unknown. Some of the genetic components of the short-range silencing spread route have been

uncovered in recent research, shedding light on multiple aspects of the mechanisms involved.

This review aims to identify the similarities and differences between the various RNA silencing

spread mechanisms in plants.

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According to Betti et al. (2021), plants and the worm C. Elegans use a kind of mRNA sequence

known as transitive RNA silencing. Secondary siRNAs are thought to be generated as a

consequence of RDR6 activity. It's been proved time and time again that, unlike transgenes in C.

elegans, endogenes in plants are resistant to RNAi-mediated silencing of sequences other than

the one targeted initially. According to Strzy (2021), endogenes are shielded from RDR action

since the same region is susceptible to transgene transitivity when produced as a transgene.

Considering that more powerful promoters than endogene expression usually control transgenic

transcription, what are the targeted sequences' RNA steady-state levels of the targeted sequences.

Indeed, a high-expression endogene is transitive. In Arabidopsis rdr6-null mutants, on the other

hand, silencing is restricted to 10–15 cells in the vicinity of the silencing source cells.

2. Outline one weakness of the Betti et al. paper.

When faced with environmental stressors such as herbivore attacks and competition with

neighboring plants, plants can alter their phenotypic. Plant defense is mediated by volatile

chemicals generated by the plant itself. Volatile emissions have both advantages and

disadvantages. There has been a lot of research on how specific plants respond to volatiles in

defense. On the other hand, plants are seldom seen independently but in groups where they fight

for resources and share knowledge. Our focus here is on the impact of neighboring plants on

plant volatile-mediated defenses. Volatile molecules have a wide range of functions in the

interactions between plants and other species. We will explain how explosive signaling and

neighbor recognition are intertwined. Here are the most pressing issues that must be addressed in

the future.

According to Betti et al. (2021), exogenous RNA that has been purposefully tailored to target

specific genes seems to be taken up by plants and activated by the RNA interference (RNAi)
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machinery; according to Betti et al. (2021), there is, however, evidence that parasitic plants

exchange short RNAs with their hosts, which suggests that RNAs may be used as signaling

molecules in plant-to-plant communication. RNAi can be induced by exogenous RNAs taken up

by some living organisms. According to Strzyz (2021), exosome-like extracellular vesicles

secreted by host Arabidopsis cells are used to transfer short plant RNAs into Botrytis cinerea.

This phenomenon has been reported in worms and insects. Plants create micro-RNAs (miRNAs)

that operate as signaling molecules that influence gene expression in other nearby plants. To

activate RNAi, both AGO1 and RDR6 must be present for exogenous miRNAs like miR156 and

miR399. Secondary small interfering RNA synthesis is needed, as shown by this finding. This

research indicates that miRNAs are signaling molecules that allow plants to communicate with

one other.

3. Betti et al. show that extracellular miRNAs enter plant cells and silence target genes.

What is one possible pathway(s) that plants use to uptake extracellular miRNAs (or small

RNAs)?

The MiRNA canonical Biogenesis Pathway

According to Treiber, Treiber, & Meister, (2019), the canonical biogenesis route is the central

miRNA processing mechanism. Pre-miRNAs are synthesized by the microprocessor complex,

including the RNA binding protein DiGeorge Syndrome Critical Region 8 (DGCR8) and the

ribonuclease III enzyme Drosha. Drosha cleaves the pri-miRNA duplex at the base of the

distinctive hairpin structure of pri-miRNA. This causes a two nt 3′ overhang on pre-miRNA (29).

Pre-miRNAs are synthesized in the nucleus and exported to the cytoplasm by an exportin 5

(XPO5)/RanGTP complex. The terminal loop is removed, resulting in a mature miRNA duplex.
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According to Salim et al. (2022), the mature miRNA form is named after the miRNA strand's

directionality. From the 5′ end of the pre-miRNA hairpin, the 5p strand emerges, whereas the 3p

strand emerges. ATP-dependent loading of both strands of the mature miRNA duplex into the

Argonaute (AGO) family of proteins (AGO1-4 in humans). The fraction of AGO-loaded 5p or

3p strands changes substantially depending on the cell type or cellular environment. The 5′

extremities of the miRNA duplex or a 5′ U at nucleotide position one are selected as the 5p or 3p

strand. The guide strand has the lowest 5′ stability or 5′ uracil. According to the degree of

complementarity, the unloaded passenger strand will be unwound from the guiding strand.

AGO2 cleaves and degrades miRNA passenger strands with no mismatches, resulting in a

significant strand bias. AGO2-unloaded miRNA duplexes are passively unraveled and destroyed.

Notable Uncanonical miRNA Biogenesis Pathways

According to Treiber, Treiber, & Meister (2019), uncanonical miRNA biogenesis pathways have

been identified. These routes employ alternative combinations of Drosha, Dicer, exportin 5, and

AGO2 proteins from the conventional path. Non-canonical miRNA biogenesis may be classified

as Drosha/DGCR8-dependent or Dicer-dependent. Drosha/DGCR8-independent pre-miRNAs

resemble Dicer substrates. Mirtrons are pre-miRNAs generated from mRNA introns during

splicing. Another example is m7G-capped pre-miRNA. These nascent RNAs are exported

straight into the cytoplasm through exportin one without Drosha cleavage. According to Salim et

al. (2022), the m7G cap prohibiting 5p strand loading into Argonaute causes a substantial 3p

strand bias. On the other hand, Drosha processes Dicer-independent miRNAs from endogenous

shRNA transcripts. Because they are too short to be Dicer-substrates, these pre-miRNAs need

AGO2 to mature in the cytoplasm (38). This enhances pre-miRNA loading into AGO2 and

AGO2-dependent 3p strand slicing. Trimming the 5p strand 3′-5′ completes maturation.

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4. Briefly design an experiment to test your answer to question 2.

In N. benthamiana, the experiment we can find similar findings after RNAi suppression of the

RDR6 gene. To a certain extent, this silencing spread phenotypic resembled the endogene

silencing phenotype in that the suppression of GFP-transgene was limited to 15 cells in a range

in systemic organs. Silencing of rdr mutants by endogene silencing or transgene silencing has

been shown to have this short-range distribution without amplification. Transgenic sequences

may be silenced within a 15-cell radius after being bombarded with DNA or siRNA or even after

a viral vector induces a silencing response. In several GFP transgenic lines, it has been shown

that silencing without systemic dissemination occurs spontaneously. There was not enough

silencing signal generated in this instance to activate the relay mechanism required for further

silencing spread, as we recommended in the preceding situation. An overreaction to a bit of

danger would be prevented by setting a threshold for widespread RNA silencing, which is seen

as a molecular immune system.

5. Describe one possible application for extracellular miRNAs in agriculture or medicine.

According to Han et al. (2021), microRNAs (miRNAs) are short non-coding RNAs that have

emerged as critical regulators of gene expression, mainly by cleavage and translation inhibition

of the target mRNAs during or after transcription. Maintaining the integrity of the plant genome,

development, metabolism, and adaptive responses to environmental challenges are only a few of

the functions of miRNAs. According to Feng et al. (2022), the growing population of the globe

and the need for food necessitates a focus on agricultural plant enhancement to assure long-term

food security.
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According to Feng et al. (2022), different plant gene expressions may be modulated via miRNA

control to manipulate mRNA transcript abundance. MiRNAs are emerging as the next

generation's target for genetic engineering to enhance the agronomic qualities of crops.

Understanding the possibilities and processes involved will help create appropriate tactics for

obtaining desired features with minimal trade-offs in transgenic crops. According to Han et al.

(2021), there are numerous roles for miRNAs in food and industrial crops, both conserved and

newly discovered, and recent advances in the use of miRNAs to improve plants of agronomic

importance to enhance crop yields significantly and increase tolerance of various environmental

factors stress agents from biotic—or abiogenic origin.

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References

Betti, F., Ladera-Carmona, M. J., Weits, D. A., Ferri, G., Iacopino, S., Novi, G., ... & Perata, P.

(2021). Exogenous miRNAs induce post-transcriptional gene silencing in plants. Nature

Plants, 7(10), 1379-1388.

Feng, Y., Liu, Q., Zhao, X., Chen, M., Sun, X., Li, H., & Chen, X. (2022). Framework Nucleic

Acid-Based Spatial-Confinement Amplifier for miRNA Imaging in Living Cells.

Analytical Chemistry.

Han, Y., Jones, T. W., Dutta, S., Zhu, Y., Wang, X., Narayanan, S. P., ... & Zhang, D. (2021).

Overview and update on methods for cargo loading into extracellular

vesicles. Processes, 9(2), 356.

Salim, U., Kumar, A., Kulshreshtha, R., & Vivekanandan, P. (2022). Biogenesis,

characterization, and functions of mirtrons. Wiley Interdisciplinary Reviews: RNA, 13(1),

e1680.
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Strzyz, P. (2021). microRNA communication in plants. Nature Reviews Molecular Cell

Biology, 22(12), 775-775.

Treiber, T., Treiber, N., & Meister, G. (2019). Regulation of microRNA biogenesis and its

crosstalk with other cellular pathways. Nature reviews Molecular cell biology, 20(1), 5-

20.