Once upon a time, scientists thought genetics and genomes were fairly straightforward. A gene encodes a protein and that protein carries out the actions that cause physical, metabolic effects and even phenotypic effects visible to others. It was a direct and simple system, a functional way for something formed through natural selection to be built. Looking back on their thoughts now, it all appears quite quaint. They had no idea what they were in for.

The discovery of RNA only built in an extra step to the equation, an understandable path to have the proteins actually be encoded without them crowding up in the nucleus. So, of course copying the genes in their reverse and sending those instructions out into the main part of the cell made the most sense. Still simple, right?

No.

RNA was ultimately the key. It opened the door in the scientific community to the wondrous complexity that makes up life itself and all its varying forms. The farther we delved into RNA, the greater the number of sub-types we found, which eventually led to findings involving reverse transcription, transposons, epigenetics, and more. We began to unlock the fact that there was a far greater backdrop to how life functioned than we had ever considered. The regulation of the genome’s expression was an interplay between regulators of varying forms, between wound up spools of genetic material, and enzymatic activity.

In recent decades, the use of RNAi technologies has opened doors into the direct function of genes and how to decipher what each one does in a genome. Because knowing and sequencing a genome is one thing, but having a complete grasp of each gene’s purpose was another level entirely. This has been especially useful in eukaryotic organisms that were resistant to many of our gene knockout tools. Even today, RNAi remains a first-hand alternative when we run across species where CRISPR and other gene modifications just don’t work as we would like.

There are a large number of styles that RNAi can take, whether speaking about human usage of the ability or the inherent capabilities found in nature. Overall, the primary purpose almost exclusively is in the process of gene silencing. This can be to the benefit or detriment of an organism, depending on the silenced gene. We’ll begin our discussion of these modes of operation below.

The Range of RNAi

To find the very beginning of RNA interference, you also have to go back to the early days of genetic modification. From those initial tests, it was found that inserting a reversed, called antisense, version of a gene acts as a way to silence the expression of the normal gene, without having to actively remove said gene from the genome. This was tested in petunias and the anthocyanin production in their petals, resulting in cisgenic petunias with altered color schemes. Two separate groups managed this find in 1990 and things just took off from there.

Tinkering with antisense RNA suppression continued for most of the decade, along with other genetic modification like actual gene insertion. But it wasn’t until 1998 that the next step in the RNAi story began. At that point, another research lab revealed that transgenes capable of forming duplex shapes created a far stronger suppression effect than any other combination of sense or antisense genes. The group hypothesized that the entire observed phenomenon of antisense gene suppression is actually just a related symptom of double-stranded RNA (dsRNA) being produced from the inverted sequences.

Almost simultaneously, another lab put out another dsRNA mechanism that involved silencing of RNA production in the model organism C. elegans. They decided the most appropriate name to use for this system was RNA interference.

Expanding this procedure beyond the species they were originally encountered in was a harder step. Plants especially required a unique construction called a hairpin loop construct. When combined with both the sense and antisense forms of a gene and with a strong promoter of plant-inserted genes, like from cauliflower mosaic virus, the resulting suppression allows for major phenotypic changes. And the suppression success rate was close to 100% efficacy.

Getting back to the briefly mentioned dsRNA, these have to be explained in order to understand the function of RNAi. The common process utilizing them involves degradation of the strands and chopping them into 21 to 24 nucleotides-sized pieces. These are one of many types of small RNAs, with the ones used here being small interfering RNA (siRNA). The enzymes that enact this slicing and dicing are known, appropriately, as the Dicer family of proteins. Plants generally have four or more versions of these unique and useful enzymes.

After cleavage results in siRNAs, their double-strandedness is also cut leaving them to combine with a protein amalgam named the RNA-induced silencing complex (RISC), which then uses the sequence of the siRNA to target transcribed RNAs from the gene in question and destroy them. This effectively silences the gene, while not directly stopping its production of RNA. This silencing can also take place at more than just the post-transcriptional level. Transcriptional silencing can include epigenetic-related changes, such as DNA methylation or histone methylation, or even modification of chromatin binding and folding. Targeted methylation of promoter sequences is a highly effective method to silence a gene’s expression.

The downside of this inherent system is that pathogens can take advantage and hijack it to cause silencing of important genes protecting plants that then, in turn, makes the plants more susceptible to infection. But the opposite is also a possibility, plants using their own RNAi systems to silence virulence genes in the cells of their attackers. We will be discussing all of this and more in the research below. Let us get right into it.

The Mobility of Small RNAs

A key factor in determining the effectiveness of RNAi is to see how mobile the small RNAs that enact it are within the organism in question. The paths that are taken differ significantly between plants and animals and when it comes to transfer of them between organisms, such as pathogen to host or vice versa, the time and manner in which that transfer takes place is crucial for understanding the overall impact. For plants, this comes through the form of the plasmodesmata, a narrow strand of cytoplasm that connects different plant cells, allowing them to essentially communicate with each other through molecular transmission.

This cell to cell structure is the close range system and then the overall plant vascular system facilitates long range movement, like from the stem to the leaves of the plant. This all raises the final big question though: how would these small RNAs travel into or out of the plant in regards to a pathogen? The plasmodesmata would presumably not be an option in that regard and unless the pathogen is living within the vascular walls, it is unlikely for that to be of use.

Generally, from the pathogen side of things, the small RNAs are excreted and then injected in some manner into the host plant, through a physical mechanism. That method is becoming more and more understandable as time goes on, but the opposite manner is still largely a mystery. A group of researchers hailing from UC Riverside in California, Nanjing Agricultural University in China, and National Chiao Tung University in Taiwan decided to work together to take a deeper look into this process. Their research was then published in the journal Science just last month in June.

They decided to go with the basic option, using the model organism Arabidopsis thaliana as their plant host. Then they picked a fungal pathogen by the name Botrytis cinerea, a necrotizing fungus that causes rot in the plants it infects. These two species together are known to be a cross-kingdom bi-directional RNAi system, meaning that they both use small RNAs against each other.

After managing to isolate pure fungal cells, the scientists ran a sRNA profile of the cells and discovered 42 of them that were from Arabidopsis.A similar scan was done on the leaves of the plant to see what kinds of sRNAs were abundant there and whether certain ones were more prone to being transferred. They found that there does appear to be a selective bias in which sRNAs are involved in fungal defense. Specifically, that 25 of the 42 found in the fungus were in low general abundance in the plant leaf tissues, meaning they aren’t generally used for regular activity by the plant.

This information also revealed that the sRNAs were being moved into fungal cells via some method other than concentration-dependent diffusion, as that would favor the more highly concentrated ones rather than the opposite. Since extracellular vesicles, essentially fluid or air filled containers, have been perceived to be involved in such transfers in animals, the researchers sought to see if the same was true for plants. Thus, vesicles isolated from uninfected places on the plant were taken through a similar sRNA screening and, indeed, the majority of the found sRNAs in the fungal cells were also found in the plant vesicles. So the idea that vesicles are used as the transportation mechanism for sRNAs into attacking pathogens is supported and the plants must secrete these into extracellular spaces when under assault.

The exact classifications and way of function of plant extracellular vesicles however has yet to be defined by the scientific community. Animals are known to use exosomes, bodies that secrete microvesicles and cell-damaging molecules, as a defense, so this was something else to look into for plants. Using transgenic Arabidopsis plants, the scientists had them express one of the homologous genes to the animal versions that act as markers for exosome production and only one of the two in total that were found to activate under fungal infection, named TET8. This already existing gene in the plant genome was fused with green fluorescent protein (GFP) to track its activity.

They found that TET8 accumulates at the infection sites and co-localizes with markers for multivesicular bodies (MVBs), allowing them to see that it is these MVBs that fuse with the plasma membrane of the cells in order to release their internal vesicles containing sRNAs. This confirmed that the TET8-related vesicles were indeed exosomes and work in a manner similar to in animals.

Further experimentation on the fungal cells that received these vesicles found that the sRNAs activate the innate systems in the fungi to then cleave mRNA in connected genes, silencing them and their pathogenicity targeting. This makes the fungi less capable at infecting the host plant, along with reducing their overall virulence. The scientists believe that this knowledge may help in the creation of artificial sRNAs in the future to protect plants from the fungal infections in the first place.

Direct Application of Artificial sRNAs

Rather than just investigating the existing capabilities of RNAi as used by plants to defend themselves, other research goes into active usage in order to deal with fungal pathogens that infect crops. A study back in March by USDA scientists desired to find a way to use the technology to deal with the fungus Fusarium verticillioides, a common infector of corn crops that produces mycotoxins dangerous to both livestock and humans that consume the infected corn. The maize seedling blight it creates concentrates high amounts of the toxins in the seed kernels during their development.

The best way to use a plant as a treatment against such a fungus is to use a transgenic overexpression gene for the particular siRNA that counters the fungal activity. The dsRNA that is to be turned into siRNA must be complementary to the gene sequence of interest one wants to silence. This was tested in the past against this very species by using transgenic tobacco plants. But, for corn, there’s only been two cases thus far where anti-fungal RNAi attempts have been made. They did show that host-induced gene silencing functions properly in the plant and can be made to control the production levels of the mycotoxin fumonisin in the pathogen.

For this experiment, the researchers chose to try and downregulate the first two genes in the biosynthesis pathway for fumonisin, FUM1 and FUM8. The complementary dsDNA gene constructs were inserted into the fungal cells. Then a PCR test was done to see if the gene were producing their RNAs in significant quantities still and it was confirmed that silencing those two genes had indeed shut off the entire production pathway.

This was a simple proof of concept for conducting such controlled transformations in fungal pathogens, which have been notoriously difficult to control or even alter with genetic modification techniques.

Targeting Pathogenic Insects

A semi-static fungal infection is not the only thing RNAi defenses can be used for. They work just as well in dealing with members of the kingdom Animalia. For example, insect pests, like the green rice leafhopper species called Nephotettix cincticeps. This pest is normally found in Asian countries and, as the name suggests, feeds on rice plants by sucking juices from the stem, causing leaf wilting and often death to the plant. They are also a vector for viruses that infect rice plants, causing even more damage to fields of rice in the process.

Due to their lifestyle, insecticides have long been the only method to control the spread of the insects. Other studies with different planthopper species to this one have seen RNAi down-regulation of key genes be successful at reducing the impact the insects have on annual rice yields. In February, scientists from the Minnan Normal University in China wanted to finally see if the same held true for leafhopper species as well.

Their goal was to target the gene Troponin C (TnC) that regulates large parts of the insect’s behavior, including its feeding mannerisms. This is done by being involved in muscle contraction and relaxation within the physical body. When this gene was knocked out in the model organisms Drosophila melanogaster, it caused defects in both movement and egg-laying, meaning it could serve as a population control of insects in general. But, whether that same gene silencing would have a similar large impact on leafhoppers had yet to be seen.

Based on the transcriptome sequence of the TnC gene, the researchers cloned a complementary sequence. They also measured that expression of the gene occurred at different stages and rates of development of the insect, especially during formation of specialized tissues. Knocking out or silencing the gene using RNAi had the effect they wanted to come about, with feeding capacity, body weight, and reproductive fecundity being negatively impacted, thereby reducing overall survival rate. They therefore confirmed that gene as a target for future large scale efforts to protect rice crops.

A Two-In-One CRISPR/RNAi System

There exists some amount of overlap between different gene manipulation technologies. Like CRISPR itself, due to having so many variants that can be used for so many things. It is nearly inevitable that some combined usages will become an option once better tools are constructed. And that is just precisely the case when it comes to CRISPR Cas13 and recent items made using RNA control. We’ve discussed its abilities before.

Because RNA is more than just an interim protein-coding device, as non-coding RNAs are oft a part of regulation gene expression, being able to alter the activity of certain RNAs at various times can be crucial to achieving larger effects. It is exactly this that Cas13 can jump in on, as the protein complex is made up of two RNase domains that can cleave RNAs in desired ways. Since crRNAs can be designed to act as targeting systems for any RNA scientists desire, Cas13 can act as both viral and bacterial defense and a regulator of phenotypic expression.

This allows for it to be a combined RNA re-writer and splicer while also enacting silencing mechanisms on desired genes, due to it being able to itself be a Dicer enzyme that destroys any offending RNAs complementary to the strands given to it. Because small RNAs can be complicated to develop, substituting in targeted crRNAs is an easy alternative with plants. There are still the limitations of what species will allow the CRISPR complex to conduct these changes, but that still leaves a wide field of opportunity.

A simpler skill is to use Cas13 as a diagnostic tool, to look for and identify the existence of certain RNAs in a plant, thus telling researchers and farmers what type of pathogen has infected the plant. This is especially useful in cases that have multiple pathogens attacking a field of crops at the same time.

As an additional downside, there is also always the possibility of off-target effects that must be controlled and minimized. New methods let us continue to improve on the technology to make this less and less of a concern, but, for now, it remains one that must have an eye kept on it. Lastly, there are, currently unfounded, concerns that CRISPR usage in this manner could provide a natural selection pressure for viruses and other organisms to develop defenses against the CRISPR tool itself, which would present an entirely new problem to deal with. So far, there is no indication this is possible, but the scientific community remains vigilant.

Cooking Up An RNAi Transgenic Banana

There is another combinative technique that is arguably even superior to what CRISPR Cas13 can be used for. That is, developing crops that are transgenically expressing dsRNA gene products that target pathogens. With such a setup, you could protect plants against the pests that seek to harm them, while not harming any other species outside of those targeted by specific gene silencers.

Researchers at the University of Leeds in the UK have been looking into doing this very thing with the abundant cooking banana. This variant of the banana, often known as plantains, are high in starch and make good sources of nutrients and vitamins, making up 25% of the total daily carbohydrate intake for populations across Africa and South America. The production and farming of the crop is done almost entirely by smallholder or even subsistence farmers that only sell excess within local market regions, meaning that the price of the crop on the larger national and global markets is incredibly stable and not affected by local incidents as significantly.

That doesn’t mean those incidents aren’t devastating for the population in question though. Whenever an annual crop yield is destroyed by pathogen, malnutrition is bound to follow for some time. In addition, because bananas as a whole make up a clonal planting practice, there is little genetic variation within the general group, leaving all the plants wide open to attack. It is not uncommon for parasitic nematodes, one of the worst parasites that affect bananas, to wipe out 50% of total yields. There just exists no real resistance genetics against them, leaving the job up to scientists to find a way to fix that.

The first step the authors of the study took was to study the genomes of the four nematode species they were targeting and find the homologous and unique genes between them, isolating nine genes with 80% similarity across the four species. The genome of the model organism C. elegans was used as a comparator group. Of those nine genes, two were shown to have enough conserved regions to have dsRNA replication be effective against them, as any major variance among a target gene sequence dramatically lowers the impact of RNA interference. All possible off-target effects in other species were identified and truncated from the dsRNA sequence.

The dsRNAs were tested in a petri dish using infected discs of carrot. A 24 hour dosage for a week with the dsRNAs resulted in massive silencing of the targeted genes and that in turn caused a significant loss in locomotion and the ability of the nematodes to spread. This would greatly reduce the amount of plants that could be infected by the pathogens, isolating them to small regions of a field, potentially.

Finally, the hairpin loop forms of the dsRNA were transgenically inserted into carrot hairy roots to test if plant production would succeed in limiting nematode gene expression. And, indeed, they did, with not just a reduction in locomotion, but also replication by 90+%, with differing levels of efficiency depending on the specific variant of the gene sequence they used. The fact that they had a greater effect as compared to the carrot disc sprays implies that continuous exposure via plant generation is far more impactful than just spraying dsRNAs on the nematodes.

The researchers hope that forthcoming efforts in actually applying this dsRNA production to the bananas in question will not only be fruitful, but that this approach guarantees a far higher level of biosafety and prevention of environmental changes with other species. All in all, a combination technique of RNAi with transgenic plants may be the next frontier of genetic engineering for pest management.

The Future of RNAi

While the mechanics of RNA interference have been known for several decades, actual application has been stunted due to the lack of tools involved in more recent genetic modification methods to accordingly apply to RNAi itself. That has begun to slowly change over the past ten years and we are finally seeing some of the labors of scientists vindicated through their experiments.

There are standalone projects being worked on with interference by itself, but it is the doubled up tests that will likely prove to be the most interesting to keep tabs on. No reason exists for items like CRISPR to stay separate and opposed to RNAi. Indeed, many of their traits correlate and complement each other, meaning that a dual system in the near future may prove to be better than the sum of its parts.

For now, all of this remains quite new and novel and there is still ample amounts to learn. As we all strive for a better future, whatever form that may take.

References

1. Doran, T. & Helliwell, C. RNA Interference: Methods for Plants and Animals. 10, (CABI, 2009).
2. Cai, Q. et al. (Jun 2018) Plants send small RNAs in extracellular vesicles to fungal pathogen to silence virulence genes. Science 360 (6393), 1126–1129. doi: 10.1126/science.aar4142
3. Johnson, E. T., Proctor, R. H., Dunlap, C. A. & Busman, M. (Mar 2018) Reducing production of fumonisin mycotoxins in Fusarium verticillioides by RNA interference. Mycotoxin Research 34 (1), 29–37. doi: 10.1007/s12550-017-0296-8
4. Banerjee, S. et al. (May 2017) RNA Interference: A Novel Source of Resistance to Combat Plant Parasitic Nematodes. Frontiers in Plant Science 8 (834). doi: 10.3389/fpls.2017.00834
5. Zotti, M. et al. (Jun 2018) RNA interference technology in crop protection against arthropod pests, pathogens and nematodes. Pest Management Science 74 (6), 1239–1250.
6. Niehl, A., Soininen, M., Poranen, M. M. & Heinlein, M. (Feb 2018) Synthetic biology approach for plant protection using dsRNA. Plant Biotechnology Journal. doi: 10.1111/pbi.12904
7. Lan, H. et al. (Feb 2018) RNA interference-mediated knockdown and virus-induced suppression of Troponin C gene adversely affect the behavior or fitness of the green rice leafhopper, Nephotettix cincticeps. Archives of Insect Biochemistry and Physiology 97 (2). doi: 10.1002/arch.21438
8. Ali, Z., Mahas, A. & Mahfouz, M. (May 2018) CRISPR/Cas13 as a Tool for RNA Interference. Trends in Plant Science 23, 374–378. doi: 10.1016/j.tplants.2018.03.003
9. Roderick, H., Urwin, P. E. & Atkinson, H. J. (Feb 2018) Rational design of biosafe crop resistance to a range of nematodes using RNA interference. Plant Biotechnology Journal 16 (2), 520–529. doi: 10.1111/pbi.12792

Photo CCs: Intercropping maize and beans from Wikimedia Commons

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