Pathogens are a broad field of research. To many, different species and even kingdoms of pathogens must be treated with separate hands, while others feel they are similar enough as to be dealt with together. Bacteria and viruses make up the bulk of this argument and, as with many things, the true answer appears to be somewhere in the middle. Many plant defense systems are evolutionarily constructed against bacterial aggressors, often the primary form of pathogens plants must deal with.

What this results in is often less effective resistance against an onslaught of viruses, which had led many to believe that they must be kept as a separate class of pathogens to be dealt with. This is somewhat true, both the methods of attack and the existing defenses work differently within plants depending on if they are dealing with bacterial or viral attacks. But further, more in-depth research, has also shown some universal exceptions.

The Way Plants Fight Back

The general defense mechanism of plants is the non-host resistance system that has said plant responding to genetic, recognized factors. The pathogen-associated molecular patterns (PAMPs) process is those specific factors and are how the plant is set to work against them. When it comes to the bacteria, several of these factor relate to proteins involving flagellin, the structural molecule that helps to make bacterial flagella.

Perception of this bacterial protein is connected to the FLS2 surface protein on plant cells, which is in turn controlled by its partner activator kinase called BAK1. It turns out that, even though viruses work intracellularly and thus not on the exterior of cells like bacteria do, some of them are still impacted by PAMP-triggered immunity controlled by BAK1. This was then found to be because some viruses, like tobacco mosaic virus, have coat proteins that resemble the molecules included in the PAMP system. Thereby managing to trigger the immune response.

In a distinct manner, double-stranded DNA from viruses has also been found to activate this immunity on occasion, in a manner completely separate from the virus immune system of RNA silencing, the more specific mechanism plants use against viral combatants. But, this non-host resistance immunity isn’t reliable and, as noted, only happens with some viruses. The rest would have free rein against the plant if this was the only protection they had.

Among these other systems, many are not yet well understood. Those best characterized thus far are ones that are controlled by a single dominant (R) gene or by a single recessive (r) gene. These then come in two different flavors of defense. The first creates factors that are capable of inhibiting viral development and infection for a specific stage of that cycle, so are very specialized in nature. The second type involves genes that encode for NLR proteins, molecules that have both Nucleotide-Binding domains and Leucine-Rich Repeat domains.

Some of the strongest forms of the first work to prevent the virus from entering or leaving certain portions of the plant body, such as the vascular system. There are other factors that can inhibit replication by binding to and starting RNA silencing proteins. The latter system is, in contrast, much more complicated.

The NLR proteins act as sensors and detectors to then turn on other immune responses, such as the hypersensitive response, or plant-wide options like the systemic acquired resistance response. The latter requires obtaining part of the virus to recognize it, however, similar to animal immune systems. And while the parts they activate can be broad in scope in what they target, most NLR proteins are nonetheless specific in the kind of pathogen they will activate in reaction to. This focus is based around effector proteins that the pathogens use in attacks on the host plant. That is why NLR proteins as a whole are often called effector-triggered immunity.

For the R genes that have been studied, those that are best described by science are the ones that produce the eukaryotic translation initiation factors (eIFs) exploited primarily by potyviruses and similar families. The viruses use them for not only gene expression, but also actual movement. They produce a special protein with a unique cap that is able to interact with the eIFs and are essential to proper infection activity by the noted viruses. Genetic modification of these eIFs has been found to protect plants against the particular species that target them.

While many, many other types of R genes are known and that they confer resistance against this or that virus is also known, how they accomplish this and the precise nature of their functions has yet to be uncovered. But it can be easily seen that, with systems like this, plants actually have the potential for robust defense systems against viruses, but that it is a multi-layered defense that is hard to define in general terms. There is still so much about it that we have yet to learn.

With that in mind, let’s get into some discussions of recent science that are entirely about explaining newly discovered characteristics and experiments into finding out about and improving the immune defenses of plants against viral pathogens.

Altered Genes, Same Functions

Rather than whole gene insertion or deletion, the advent of synthetic biology and the capability to understand the interconnectedness of hormonal and effector pathways has opened up the option for more subtle displays of genetic engineering. Using nonsynonymous mutations, that is changes that do actually alter an amino acid in a protein, in a way that can allow for resistance but not change the outcome of the pathway is one example of using these methods.

Allowing for normal functions of genes to still be active is paramount, as many of the proteins targeted by viruses are still essential for plant growth and development, so simply shutting them down is not a possibility. This is especially so for the eIFs just discussed. Especially the factor eIF4E, which has shown to negatively confer susceptibility to several major viruses of note across multiple plant species. But, this gene is needed for the initiation of translation and for recruiting the other factors involved in said process. How to get around fixing this open hole in the defenses of so many plants?

The first option was to look across the natural diversity of the plant population to see if any mutations had managed to accomplish what the scientists were looking for. And, indeed, modified eIF4E were found and already deployed into usage across several crops like tomatoes. But not all plant species have had this successful mutation emerge within their population in order to spread it to the rest. Reproductive incompatibility in some populations also lower the capability to allow the rest to obtain this desired alternative gene.

If using a different eIF4E is out, then other options must come forward. For some plants, gene knockout is a possibility due to having redundant duplicates of the gene in their genome, meaning one can be taken out without affecting the cellular system of the plants. But a single knockout has been found to only give limited resistance against the specific viruses and often for only a couple of generations. The viruses usually found a way to get around to go after the other working copy or copies of the gene.

A collaboration of French researchers decided that their best bet was to keep functional alleles of the eIF4E gene intact, but to introduce point mutations that would throw off the ability of the viruses to bind with it. Essentially turning them into special resistance alleles. They chose to run the test on the eIF4E gene from wild type Arabidopsis, due to it being a model organism and this part of its genome having a higher level of accuracy and understanding. Additionally, they specifically chose the variant eIF4E1, which is targeted by the potyvirus Clover yellow vein virus (ClYVV).

It is already known that inactivation of this gene does well enough to provide resistance, but the scientists wanted to make a functioning, yet still resistant option. Since such a variant is not known to naturally occur in any Arabidopsis plant, they looked for equivalent data in other species and found what they wanted in the common pea. That existing natural altered version also gives the pea plant resistance to this exact virus, among others, so the researchers knew it was effective. The homology in the genetic sequences between the eIF4E1 gene in the pea and in Arabidopsis is also over 80%, so they can be presumed more or less similar.

Further investigation of the prior studies showed that there were 5 amino acid differences between the resistant and non-resistant variants in the pea plant, along with a single deletion. Thus, all they had to do was to swap out the existing amino acids in the Arabidopsis gene for the ones discovered in the pea gene, along with using an alanine to take the place of the deletion. Predictive modeling of the structure of the resulting protein showed no drastic changes in structure, implying that it should still be capable of fulfilling its original role.

The method used to change the DNA codons to then alter the amino acids was one called PCR-based site-directed mutagenesis. It was chosen because they were making a synthetic construct and the cheapness of the technique in modern times makes it the best option. Three transgenic lines were made with the altered gene in Arabidopsis plants that originally had the gene knocked out, along with a control given the wild type version.

Their test of exposure to ClYVV showed that the wild type control group accumulated with the virus, while the group with just the knockout obviously had no viral infection, but the groups with the inserted and altered, but working, eIF4E1 gene also did not have any viral replication. This suggests that they were successful at designing a synthetic gene variant that cannot be recruited by the virus for its own duplication.

Further experiments proved that the synthetic variant did not lose any overall yield and also saved the variant with both that and the eIFiso4E genes knocked out from death, as losing the function of both those genes usually prevents the plant from living. But adding in this synthetically constructed gene to the double knockout keeps it alive. This reveal also showed them to be resistant to other strains of ClYVV that rely on both genes to develop. Several other virus species were found as well to not be able to infect the altered plants.

Thus, the researchers were able to show that synthetic alleles can match up to natural resistance alleles variants and can be developed for species where no such variants can be found in the wild population. These transgenic plants also feature no change in behavior or gene functionality, making them barely even transgenic in the first place. They hope that this sort of introduced genetic diversity can allow for creation of many plant cultivars resistant to all the current major viruses that plague farmers and botanists around the world.

Targeting Viral Weaponry Directly

While RNA silencing remains the main tool at the disposal of plants to deal with viral attacks by destroying the genetic material the viruses try to insert into plant cells to take over the cellular machinery, it has its limits and its downsides. An obvious counter is how viruses themselves utilize RNA silencing as well in order to further their assault. Their purposefully inserted double-stranded RNA (dsRNA) is processed by the plant cells into what is referred to as virus-derived small interfering RNAs (vsiRNAs).

These are then used accidentally by the plant cells to silence their own resistance genes, opening themselves up to infection by their aggressor. But even for the viruses themselves, this is a double-edged sword, as plants can develop genes that turn these vsiRNAs against them once more. For Arabidopsis, the genes DCL2, DCL3, and DCL4 confer antiviral defenses by forming vsiRNAs that not only attack viral sequences, but also change the target of vsiRNAs on the host plant to instead focus on transposons and repeat sections, essentially blunting the attack by redirection.

Among recent virus developments, it is the Criniviruses that have begun to emerge as major agricultural pests that cause severe and fatal diseases in crops. And the worst part is that it sometimes feels like a new one is appearing purposefully to target a specific region of the world and the staple foods grown there. In the Middle East, cucurbit yellow stunting disorder virus has been found to feed on the melon and cucumber family and has grown out of that region to become a worldwide threat. Africa has seen the reveal of sweet potato chlorotic stunt virus to be a huge concern for food stocks, due to sweet potatoes being a prime source of nutrition across sub-Saharan Africa.

Dealing with this group of viruses has proven problematic, to say the least. Trying to target vector species that help spread the viruses, such as whiteflies, has seen only limited success. And it is nearly impossible to get local communities to switch to other forms of crops, along with resistant cultivars not lasting long before the viruses find a way around the resistance. While RNA silencing techniques have been tried against the sweet potato virus, they did not end up conferring immunity to it, just limited and partial resistance traits that reduced overall viral replication, but not much more than that.

A collaborative study between American, Polish, and Italian researchers sought to find a more permanent solution. Their focus has been on only a single member of that viral family, lettuce infectious yellow virus. They first attempted to make transgenic species of lettuce and of the tobacco-related model organism Nicotiana benthamiana by using several sequences from the virus itself, to help enact RNA silencing against those sequences in the plant cells. But this first try failed to give immunity to the virus.

It was after that that a more extensive approach was called for. Four transgenic lines of N. benthamiana were made using fragments from a variety of locations along the virus replicase region. Agrobacterium was used to inoculate the sequences into the plant cells. Only one, the RdRp gene fragment responsible for viral replication, appeared to gift the plant with immunity to viral infection, even from a wide number of tests. The MTR fragment, meanwhile, actually proved lethal for the plants to express, while the other two gene fragments just resulted in the plant becoming infected, though one infection admittedly happened far later than the other.

To better understand why this range of responses occurred, the scientists decided to run a deep sequencing of small RNAs in particular from both the transgenic lines and the control non-transgenic lines. These sequencings were done before administering the virus and then again three weeks after. The distribution of sizes of the sRNAs was similar in all the different replicates they sequenced, with 24-nucleotide fragments making up over 50% of the total number. All of them were then aligned with the virus genome and with the RdRp gene sequence in order to see what matched and, therefore, what kinds of sRNAs were being produced in the separate plants.

The non-transgenic and infected plants showed over 500,000 vsiRNAs, making up nearly 6% of the total. In comparison, the control non-transgenic, but non-infected, plants had only 40 in total that matched the viral genome. Thus showing the obvious, that the replicating virus was having the plant cells produce vsiRNAs in order to make more of itself.

For the transgenic groups, the before and after results were highly interesting. Practically all of the sRNAs after inoculation that matched to the virus genome were made from the RdRp gene, though the amount of change was only from 19 to 41 in the before and after. In short, the after managed to match what was expected for the healthy, non-infected control. The most intriguing part was that, looking at the previous non-transgenic infected group, the total percentage of matches coming from the RdRp gene were actually lower, only 0.14% of the total amount as compared to 0.47% in the transgenic lines.

The proportional difference was also focused almost entirely around those 24 nucleotide fragments. The researchers suggest that the accumulation of these particular fragments may play an, as yet unknown, role in conferring immunity to Criniviruses. This was further backed by doing the same test using a transgenic melon and the melon-related virus noted prior. 24 nucleotide fragments also accumulated in those lines that had immunity to the virus. Strangely, no sort of accumulation appears to occur in transgenic lines that are only partially resistant. One would expect there to be some amount of increase due to the fact that they are at least somewhat resistant, but perhaps the buildup is an all or nothing phenomenon that gives full immunity or nothing at all.

Continued research and experimentation on this is clearly a necessity to decipher what role 24 nucleotide small RNA fragments have on giving immunity to Criniviruses, but just this finding is important enough that, with more work, it may shake the agricultural community itself, in a good way. This is, however, only the beginning of that path.

The Power of Artificial MicroRNAs

Jumping over to something completely different, let’s discuss orchids and their pathogens. While this family of flowering plants are among the most prized in floriculture for both their unique structure, colors, and fragrances, they are not without their predators. The annoying part about the two primary viral pathogens of these species is that both have no vectors to speak of and are instead transmitted to new orchid plants via mistakes and mishandling by human horticulturists.

These viruses, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV), cause color breaking, necrosis, and streaky, ringspot symptoms. Prior experimentation has found that co-infection with both of them causes even more severe outcomes, with viral RNA production skyrocketing even higher. Though there has been success in the past at developing resistant transgenic cultivars of orchids, researchers at the National University of Singapore wanted to go with a more targeted approach.

They desired to use artificial microRNAs (amiRNAs) that can be precisely designed to deal with the viral sequences of the two pathogens in question. They are also based off of a precursor miRNA sequence in the orchid with high specificity, meaning the change is passed on to future generations, contributing a permanent weapon against viral aggression. The small size of amiRNAs means several can be included in one transgenic cassette, allowing for resistance sequences aimed at multiple viruses to be inserted. Lastly, they appear to have a stronger resistance effect than other methods and won’t complement non-target sequences, meaning the chance to affect non-target gene sequences is essentially zero.

Since the use of amiRNAs against orchid pathogens has yet to be reported in the literature, this team of scientists decided to take it upon themselves to run just such an experiment. They chose, much like our earlier discussed researchers, to work with the viral Rdrp gene as it appears to give stronger resistance traits. The amiRNAs were constructed to target this sequence and used the commonly employed and strong Caulifower mosaic virus promoter sequence. Due to N. benthamiana being able to be infected by these two viruses, it was chosen as the model for the tests. After three lines of second generation transgenic plants were produced, they were placed up against the two pathogens.

The scientists found very discrete and disparate results from the experiment. For CymMV, all of the control plants began showing significant symptoms after 21 days, while the amiRNA transgenic plants showed none whatsoever, indicating a strengthened resistance response. Leaf extracts that were checked confirmed the absence of the virus at any concentration in the tissues. But, the problem was its counterpart.

The ORSV infected plants indeed still showed symptoms, though they were slightly reduced as compared to the control group. The researchers measured that about 16% of the experimental group showed a resistance response, and it was powerful enough to at least allow them to grow healthily, though some mottling of the leaves and discoloration did occur. They hypothesized that either not enough small RNAs were being produced specifically with ORSV as the target or that the transgene had segregated elsewhere on the genome and was not being expressed fully.

The final part of the study was to dual-infect the plants and see how they did in that situation. What the researchers found in the mixed infection is that some small amount of the transgenic plants exhibited no symptoms at all, but the majority ended up being susceptible to the co-infection and showcased ORSV symptoms in particular. A check showed that ORSV was indeed replicating within the plant tissues.

So, the overall results were complicated. They clearly developed transgenic lines that could resist the impact of CymMV, but not its counterpart virus. There is the possibility that the developed amiRNA was not the correct sequence choice and that the specific Rdrp fragment chosen near the beginning of the gene may not have the same efficiency as fragments taken from near the end of it. Further testing will need to figure out if that is true or not.

Another option is that the ORSV RNA structure for its amiRNA appears to take a higher amount of free energy to form into the necessary stem-loop structure for binding to its complementary sequence in the virus’ genome. This larger requirement may have resulted in less of the amiRNAs being produced overall against this virus, lowering the resistance capabilities of the transgenic plants. So they only gave weak resistance rather than strong.

As a first test of using amiRNAs in orchid defense, the scientists were happy with their data. They had achieved true and complete transgenic resistance against CymMV, a major pathogen of orchids, and they had several ideas left to improve the resistance against ORSV. They knew they were far from done in their efforts and that this initial step had been a huge one with great progress and they were eager for not only their own future research in this area, but for what others will take from their results and produce on their own.

CRISPR-Cas9 and Induced Mutations

For the final course on our trip across plant pathogenic virus research, let us return to the topic of translation initiation factors (eIF) genes from back at the beginning of our trek. Rather than retrace those step though, we’ll take a look at a unique little piece that combines another eIF gene, CRISPR-Cas9, and induced mutations rather than transgenic insertions.

The task of feeding everyone in the world once seemed like a daunting task, especially as the rapid expansion of the global population loomed. But astonishing advances in food production and yields almost seemed like they could reverse that. In the past several decades alone, food insecurity has plummeted and we are well on our way toward having everyone be fed in a nutritious manner.

The threats to the primary world crops, however, continue to be a concern and viruses are no exception. If anything, they remain a dominant force in agriculture and medicine, both. Rice tungro disease (RTD) affects hundreds of thousands of acres of rice farms across Asia, causing stunted growth, yellowed leaves, and outright death to infected plants. The intriguing part of RTD is that it is not caused by a single pathogen, but by the combined and joint interaction of two viruses, rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (RTBV). The former is a single-stranded RNA virus and the latter is a double-stranded DNA virus.

Together, they are transmitted to rice plants via vectors like the green leafhopper. RTBV causes the negative health outcomes for the plant, while RTSV helps by improving the infectivity of its partner and worsening the symptoms that its counterpart has started. Meaning that resistance to RTBV essentially deals with both viruses at once, as the other can’t do much damage at all without any existing symptoms to amplify. However, since RTSV can enhance infection capabilities, it also has the potential to blast through resistances, making it its own kind of unique threat.

The International Rice Research Institute (IRRI) has long been looking around the world to catalog cultivars showing any sign of resistance to the disease. Once discovered, studies began on the most promising candidate to figure out just how its resistance trait functions. It appears that even those exhibiting dual resistances do so via independent traits for each, rather than a combined gene. Specifically, RTSV resistance is a recessive gene tied to translation initiation factor 4 gamma (eIF4G). Looking between the cultivars that were more susceptible to RTSV and the resistant cultivar, they identified the particular mutations that caused the latter to form. Similarly to the first study we discussed, these mutations cause structural changes to the RNAs for the gene, making it impossible for the virus to hijack them for production purposes.

The researchers at IRRI and in collaboration with scientists from the University of Minnesota sought to introduce those same mutations to eIF4G, but in a broadly used rice cultivar. That cultivar, mind you, is also known to already have the susceptible to RTSV form of the gene. They decided CRISPR-Cas9 was one of the better options for targeted mutations. Their guide RNAs for the CRISPR complex were made to fit the previously found nucleotide locations that create resistance and the overall system was optimized to match the nucleotide frequency found in plant genomes. Agrobacterium was utilized as the mediator vector for transporting the CRISPR-Cas9 into the plant cells.

Once put into action, the CRISPR cutters returned with a highly efficient and accurate success story. As expected, depending on the guide RNA targets, and several were used in the different experimental groups, the amount of accurate cuts could vary a fair bit. But, overall, the complex worked as desired and the changes were also confirmed to be inheritable by the subsequent generations. While not all of the members of the experimental groups developed resistance, several did and analysis of their particular alterations suggested that the amino acids asparagine (N) and leucine (L) were correlated to RTSV resistance as well. Lastly, they showed that the second generation plants kept the new and improved genes and no longer had Cas9 residues in their cells, along with no off-target effects being found.

In a simple manner, the researchers were able to prove that CRISPR can be made to induce mutations in rice cultivars that gives them resistance to one of the most devastating viral diseases in a crucial staple food crop grown around the world, especially within its native region of southern and southeastern Asia. Since they have shown that this method works, it can likely be exported to any and all other rice cultivars and may even give insights into other, similar viral diseases of other global crops. Every little piece of knowledge helps add to the whole.

Science’s Fighting Power Grows

We have covered a lot of ground today and yet only covered so little. There is a vast array of research being done out there and it is impossible for anyone to keep up with all of it. But I hope these highlights of some of the more interesting specimens of experimentation in the past year have piqued your curiosity to go and learn more.

Pathogenic viruses that prey on the plants of the world are simultaneously more conniving and more controllable than other forms of pathogens. Their high mutation rate and ability to survive adverse conditions makes them difficult for medicine, as one example, to treat. However, with the advent of genetic modification technologies, viruses are quickly finding themselves adrift without protection, as the very genetic mechanisms they use against their hosts can be so easily turned against them once a human mind focuses in on the project. It may very well be that viruses become ancient terrors of the past sooner than bacteria, fungi, or any other kind of disease-causing pathogen. And in turn, our plants and our persons will be all the better for it.

References

1. Bastet, A. et al. (Mar 2018) Trans-species synthetic gene design allows resistance pyramiding and broad-spectrum engineering of virus resistance in plants. Plant Biotechnology Journal. doi: 10.1111/pbi.12896
2. Carr, J. P., Murphy, A. M., Tungadi, T. & Yoon, J.-Y. (Apr 2018) Plant defense signals: Players and pawns in plant-virus-vector interactions. Plant Science. doi: 10.1016/j.plantsci.2018.04.011
3. Qiao, W., Zarzyńska-Nowak, A., Nerva, L., Kuo, Y.-W. & Falk, B. W. (Apr 2018) Accumulation of 24 nucleotide transgene-derived siRNAs is associated with crinivirus immunity in transgenic plants. Molecular Plant Pathology. doi: 10.1111/mpp.12695
4. Senthilraja, C., Reddy, M. G., Rajeswaran, J., Kokiladevi, E. & Velazhahan, R. (Mar 2018) RNA interference-mediated resistance to Tobacco streak virus in transgenic peanut. Australasian Plant Pathology 47 (2), 227–230. doi: 10.1007/s13313-018-0549-9
5. Petchthai, U., Yee, C. S. L. & Wong, S.-M. (Jul 2018) Resistance to CymMV and ORSV in artificial microRNA transgenic Nicotiana benthamiana plants. Scientific Reports 8. doi: 10.1038/s41598-018-28388-9
6. Macovei, A. et al. (Mar 2018) Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to Rice tungro spherical virus. Plant Biotechnology Journal. doi: 10.1111/pbi.12927

Photo CCs: Potatovirusy from Wikimedia Commons

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