When it comes to dealing with viruses, often no single countermeasure will do. The scientific community has long since found that, in many cases, any one solution will quickly end up useless once the target virus mutates to get around the blockade, no matter what form it initially takes. That’s why in the medical community some of the most sought after viral treatments are those that involve a multi-target cocktail that goes after several parts of the virus or uses multiple combined defenses, as it is close to impossible for any one virus to develop resistance to all of the treatments at the same time. And having resistance against just one or even two won’t prevent the virus from being killed by the other treatments, so long as they are administered simultaneously.

The Viral Specificity Problem

This sort of action is true across the board and also applies when dealing with plant pathogenic viruses. Plants themselves have their own variety of defenses they can bring into play when attacked by viruses, with one of the most prominent being their RNA interference (RNAi) capabilities that targets and chops up any RNA that is on their genetic no-go list. While this system is also used to silence genes in their own genome that they might not want to express all the time, it serves just as well as a line of cellular inhibition against RNA-based pathogens and as a way to prevent viruses from hijacking any of the plant’s own genes. This specific form of usage is referred to as virus-induced gene silencing (VIGS). 

But it’s not perfect. Because of the wide population of viruses out there, that meant that plants would only be able to defend themselves if their virus targeting systems were broad and general. They have a limited amount of targeting ability, so natural selection favored those that could more or less deal with the greater number of viruses. But this had the problem of being too broad at times and even inhibiting the genes of the plants themselves. So, since specific virus systems weren’t all that functional, this became the first avenue of correction when scientists became involved and wanted to engineer pathogen resistant plants. 

Getting around this generality problem was done with the use of artificial microRNAs (amiRNAs) and specific sequence complementarity with known viruses. Also, the portion of the RNAi sequences that had also been shown to have a greater mismatch complication were not used as the portion for the virus targeting, instantly preventing one of the main reasons for RNA silencing to fail in plant defenses. But the main problem remained, in that if the virus developed enough mutations in its own complementary sequence to the amiRNA, then it could avoid detection. This was harder for the viruses to do with these sequences, but it still did happen. 

Thus, researchers brought in the third generation of RNAi technologies, synthetic trans‐acting small interfering RNAs (syn‐tasiRNAs). Arising largely within the past 5 or 6 years, these artificially engineered RNAs can be easily cloned for production and designed with precise desired sequences and, most unique of all, they can be used to target multiple different invading viral sequences all at the same time. They’ve already been used in several plants to confer viral resistance, proving their effectiveness. So how much better do they work in a direct matchup against amiRNAs?

An RNAi Face-off

Researchers at the Valencia Polytechnic University in Spain, alongside the Spanish National Research Council, wanted to find that out, along with developing a cultivar of tomato plants resistant to the tomato spotted wilt virus (TSWV). Their plan was to make independent amiRNA-inserted and syn-tasiRNA-inserted tomato lines and conduct an infectivity assay to not only see how they do against each other, but also what the limit is of their anti-viral capabilities. 

Beforehand, they used the model organism Nicotiana benthamiana, a tobacco relative, to identify the particular small RNA sequences that would have the highest anti-viral activity against TSWV. Then a gene cassette was made for each method to be inserted into the candidate tomato plants. Their results were fairly straightforward and stark in their differences. The amiRNA construct was only able to express a single target sequence, while the comparative syn-tasiRNA constructs could do four sequences expressed simultaneously as developed from the proper single precursor.

Almost all of the syn-tasiRNA tomato plants were resistant to TSWV, while almost all of the amiRNA tomato plants were susceptible and became infected after enough time. As guessed, the viral progenies that developed in the latter were sequenced and found to have developed mutations in the single target sequence the amiRNA was aimed at, allowing the virus to slip by. There were some that were resistant, however, but only the two lines that had been able to accumulate the highest amount of amiRNAs. 

Conversely, the syn-tasiRNA plants did not require a concentration based effect, they functioned as expected in general. Their multi-target site methodology allowed them to keep up the plant resistance and cut up the viral RNA sequences, whereas the TSWV was unable to develop the working mutations in all four of the target sites. Though it was seen that if the syn-tasiRNA did not accumulate to a certain minimum level, that would leave the plants open to infection, as did happen with two of those lines. But any concentration above that level saw no infectivity whatsoever. 

Multiplexing CRISPR and RNAi

The researchers hope to be able to combine the multiplexing action of syn-tasiRNAs with CRISPR complexes in the future. Since one of the big drawbacks in using CRISPR for anti-viral response is that it is possible for virus variants to form that can resist cleavage by the Cas structure. And if the viruses that are cleaved aren’t done so properly due to a mutation, there is the potential to have even more virulent pathogenic forms emerge. But syn-tasiRNAs can get around that by acting as guide RNA across a multi-targeting system to prevent any of the target viruses from escaping detection. 

This multiple targeting and the continuing high specificity of said targeting will likely make syn-tasiRNAs a major candidate for future anti-viral therapies. Regardless of if they are combined with CRISPR or with other delivery methods that can take out the viral sequences once identified. The previously mentioned easy cloning and development of these artificially generated RNAs is just the frosting on the cake of their usage as a tool in biology as a whole. Hopefully we’ll be seeing greater use of them in the coming years in medicine, agriculture, and more. 

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Photo CCs: Münster, Wochenmarkt — 2017 — 2328 from Wikimedia Commons

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