The diseases out there in the world seem limitless. Every new nook and cranny we uncover shows new lifeforms and new diseases they cause or that are subjected upon them. The Earth is just a melting pot of infection (and parasitism) of every species trying to take advantage of every other. Due to their ubiquity, the focus of medical and animal science largely goes to the topic of bacteria or viruses when it comes to disease-causing agents, especially infectious ones. They are largely the only organisms we know about outside of actual parasitic species that can cause a rapidly spreading pandemic of infection through a species, including us.
But there is at least one more example, and probably many more besides, that gets less attention, but only because they usually stay contained within their hosts and specific actions on the part of others are needed to cause an epidemic. As you might have guessed from the title, today we are talking about prions, the protein-based, infection-causing agents that are so unique in their structure and mechanisms that they have fascinated many a disease researcher.
A Short Discussion On Prions
The history of these bizarre “only self” proteins are accented in their mysteriousness by the fact that it has only been just over 30 years that we’ve known they existed at all. There were suspicions since the 60’s about some sort of self-propagating protein being the cause for brain disease, but the hypothetical didn’t become real until 1982 when a prion was finally successfully purified.
But the confusion only deepened upon that discovery, since it was found that prions could hold multiple different shapes, some strange and nonsensical in relation to other proteins, and that usually only one of these configurations led to the disease in question. When a prion folded into this particular form, it began a process of rewriting the non-active functioning protein form to be the same as itself, as a form of replication similar to the actions of viruses, but without requiring any sort of genetic material other than protein sequences. The non-infectious forms are usually just the “normal” form of the acting protein that can, upon contact with the replicating form, misfold itself into a copy.
Even in the past decade momentous discoveries have been made in prion research, with entirely new proteins being found to be susceptible to misfolding into infectious prions and causing their own new diseases. This process is commonly found in proteins associated with the brain and nervous system, which is why the common disease type is neurodegenerative in origin.
This localization is due to how prions always form from a subset of proteins in the group known as amyloids, protein aggregates that have a tendency to clump together to form what are called fibrils. Due to their structure, these fibrils are commonly a part of neurological and nervous systems and misfolding can lead to diseases like Parkinson’s and Huntington’s. The type of amyloids that misfold and go on to be infectious are what we call prions.
There are quite likely to be several more prion types out there in the amyloids that have not yet been discovered. But ongoing research with them in animals has revealed amazing capabilities of these resilient proteins related to their high stability and resistance to chemical or other forms of environmental degradation. Thanks to this, once an organism infected with prions dies, that doesn’t mean the risk of an infection spreading is abated. Quite the opposite, in fact.
But that is a topic that requires some backstory to unveil and we’ll get to that in the section below.
Uptake of Prions by Plants
The History of Chronic Wasting Disease
For animal researchers involved in wildlife management, a concerning disease that has been spreading across North America has been an area of intense study. That condition is known as chronic wasting disease (CWD) and it has been moving between deer species across the continent. Not just deer even, but also elk and moose have been found to be affected, including those being held in captivity.
It was first identified in 1967 among the mule deer in Colorado, but has spread rapidly in the decades since. It is now subsumed 19 states in the northwest and midwest regions. In one state alone, Wyoming, the amount of deer infected went from 15% in 1997 when first tested to over 40% in 2013. This one disease appears to be responsible for the collapse of deer herd populations across the country.
Prior research was able to determine that CWD is a prion disease that accumulates the misfolded proteins in the central nervous system, causing heavy salivation, excitability, and other behavior changes, before ultimately proving fatal. There is no effective treatment available for the condition other than preventing its spread in the first place. But to do that, the first step is to figure out how and why it is spreading, as incidences of deer consuming other deer meat and becoming infected that way is rare. So, how are the prions spreading?
Direct infection is definitely one noted method of transmission, as the CWD prions are shed in the excretions of the deer in any form and can be spread onto other deer in this manner, including through sexual transmission. But the amount such an infection can spread in this way is limited in speed and doesn’t entirely match up with the recorded emergence throughout North America, especially when it comes to jumping between deer species and also to other cervids.
The first suspicion was that the prions were surviving the decomposition of their hosts and continuing to exist within the soil. Their resilience certainly would make this possible, but it’s also not common for animals to consume soil itself, so how were they being exposed to the prions? Perhaps knocked into the air as dust from the hooves of a herd walking over the area? Corpses that died in or near a water source would be a more easily understood vector for the prions and would explain somewhat how they can transfer to other cervids that drink from the same source. In such a way, the same herd of deer would be expected to all be exposed at some point due to this all at once and any others that consume the water within a reasonably short time frame.
Researchers suggested that perhaps many such diseased deer ended up dying at “communal hotspots” where many other herds roamed and passed through at some point, thus helping spread the infectious prions to herds that would wander to farther locations. They also couldn’t rule out them being spread by a predatory or insect vector as well that just hadn’t yet been discovered. But all the available research showed that an interspecies barrier prevented this from happening through any sort of oral exposure and that cross-species infection only happened when scientists went to the extent of an intracerebral injection of deer prions. Not something that is likely to happen in the wild.
Even as recently as 2012, a combined effort from scientists at the University of Nebraska, Lincoln and Creighton University were unable to do more than offer suggestions on possible transfer mechanisms of CWD and also give recommendations to help prevent further spread. But news not long after that jump-started research in the area thanks to a presentation at the National Wildlife Health Center in Wisconsin.
A New Revelation
The meeting was meant to showcase all of the research thus summarized on CWD in the past five years and one such presenter outlined his evidence on how soil not just acts as a disease reservoir for prions, but that the uptake of proteins by surrounding plants has included those same prions. Using a laser microscopy setup, he was able to visually see plants being able to take up misfolded proteins through their roots. A biochemical assay confirmed their presence in the plant tissues and, when the isolated source was injected into mice, it proved just as infectious. This meant that plants were a previously unconsidered source of exposure to prion proteins.
A followup experiment was tested in Canada that same year and published in February of 2014 that directly showed how plants take up proteins from the soil, including prions. In the meantime, other soil-based studies were able to find that prions can bind to the soil and, in some cases, this may lead to an increase in their infectivity. However, it does take a month to a month and a half for this process to reach completion. Flowing surface or groundwater may inhibit or outright prevent this binding from occurring, leaving the prions unbound in the soil for extended periods. It is this latter case that is of particular importance.
There are a variety of nutrients that plants obtain from their surroundings in addition to water. Organic nitrogen is one such macronutrient that is vital for plant growth and is obtained from the soil in the form of loose amino acids, peptides, and indeed, full proteins. How beneficial proteins themselves are to plants from this uptake is debated, but the fact that they do take them up is not at question thanks to current experimentation.
That aforementioned Canadian study ran a simple test involving wheat plants being exposed to mouse prion proteins in different forms. An insightful piece of information they found is that normal, unmodified prions in their non-active state were not meaningfully interactable with plant roots. But if the prions had been mixed with protease digestion enzymes, which are able to slightly modify one end of the prions on their otherwise chemically resistant structure, the roots eagerly uptook them.
The prion type that was used for the test was the original prion proteins without the infectious misfolding, though the researchers noted that this misfolding also naturally had the lost end structure, meaning they would likely be taken up easily by plant roots as well. Microbial activity, especially in compost, was a frequent reason for the end cut on infectious prions and it is likely that the soil around a decomposing body would have similar activity leading to a wide number of misfolded, truncated prions ready for plant roots to interact with. Some amount of competition would be expected between soil binding and root binding, but that the roots would be molecularly favored by the prions.
Expanding The Research
The following year in 2015, another study took a more definitive look at the process and explored all the options on how plants can uptake prions. The main improvement of this study over its predecessors is that it not only investigated the uptake process of plants, but also tested the infectious route to animals via oral ingestion of said plants. For experimental purposes, as it would have been difficult to have cervid test subjects for the study, hamsters were used instead, along with hamster prions of the same form. The positive controls for the test were set up to be hamsters administered directly with prion-infected brain matter.
All of the experimental groups exposed to prions in any fashion went on to develop the normally expected diseases associated with them. The groups that ingested the plants took longer to have the condition express itself, but even this time difference was not significant. 147 days for the group with direct prion exposure, 159 days for the group that ingested prion-infected roots, and 164 days for the group that ingested prion-infected leaves, with a 10 or so day deviation error to consider. The negative control group that was exposed to brain matter that wasn’t prion-infected did not develop the disease, as one would hope unless something went truly wrong.
Further tests on plant uptake capabilities with different species-based prions, including ones from cervids and from humans, found that plants do not appear to differentiate or discriminate in the proteins they uptake, with all forms of the prions having the same rates. The use of feces and urine from infected animals did prove to increase the total amount of prions that were taken up, enough that they began being expressed in the leaves as well and not just the roots. Time length tests also showed that the prions stay stable in the plants for several weeks and can be infectious if consumed during that entire time period.
They concluded that plants are highly capable and efficient at taking up even small quantities of proteins in the soil and expressing them in their tissues, making plants a primary vector for the spread of prion diseases like CWD and are also a concern for possible zoonotic transmission of such diseases from animals to humans, much like the oft spoken about mad cow disease once did.
Lastly, a more recent study in 2016 discussed the research that has been ongoing on the native plants in the ranges of the deer herds. Four types of plants, both leaf and grass-based, were considered and assessed for prion levels, along with using rat models fed the plants to see if those same prions were infectious. All evidence from the study appeared to point to a yes to both the presence of and infectivity of said prions, though the testing is still ongoing.
It is apparent that there is still much to investigate when it comes to CWD and prion uptake in plants. The possibility of the infection transferring to other species as well cannot be ignored, making this area of research one that should hopefully be given a high priority by disease investigators until the risk can be quantified.
Now that we’ve looked into this segment of plant physiology, there is yet another manner in which prions play a role in plants, wholly separate from disease concerns. The research is highly contemporary and may prove to completely change how we view plant response mechanisms and prions themselves. Let’s go into that topic below.
Prion-Like Proteins and Plant Memory
The History of Yeast Prions
In the previous decade, much talk was had about prions and several discoveries in yeast and how those self-replicating proteins could be used as a heritable transfer mechanism of semi-genetic information. The evolutionary implications were intriguing, especially since this sort of inheritance would completely bypass the normal expected mechanisms of the central dogma, where information generally flows only one way in form.
Retroviruses had first shown a flaw in the dogma’s mentality with their ability to move backwards from RNA to DNA, But the process of reverse translation, that is from protein back to some nucleic acid form, was seen as impossible. This is due to information loss and redundancy when going from a nucleic acid form to proteins. The 3D structure of proteins also puts a crimp into this idea.
However, prions that were capable of being the inheritable units of information from one generation to the next completely bypassed this issue. There is no need for reverse translation if proteins can just encode the information onto other proteins and pass it down the generations that way.
This was an important discovery at the time and the evidence that these yeast prions focused on memory formation itself led to more curiosities. They also appeared heavily involved in RNA regulation and processing, this being another realm of potentially transmitting information or at least the conditions for appropriate genetic expression. Because of this, these memory prions are able to directly pass on trait configurations onto daughter cells in yeast.
Both a positive and a negative of this process is that the prions do not feature normal genetic mechanisms like mutations, since they are proteins, and so the traits do not change over time. If detrimental in the long run, this could wipe out an entire strain of the yeast, allowing other prion configurations in other strains to rise to dominance as an evolutionary benefit.
Vernalization and Inheritable Response
For plants, it has long been known that they have some sort of memory within their epigenetic expression that can record previous environmental conditions, especially extreme stressors. This can even play a critical role in the plant life cycle, like how the process of vernalization uses the memory of a strong, cold winter in order to promote flowering on time in the spring. This memory in the genome is a part of every cell, so a callus grown from cells of a plant will still remember the winter exposure and flower on time even if it specifically has never been exposed to the winter.
But, beyond the known genetic and epigenetic influences, the full methods of this memory mechanism has never been fully understood. In a 2016 study, researchers at MIT and the Whitehead Institute decided to look into the model organism Arabidopsis thaliana and see if they could identify any suspect proteins that might function similar to those prions found in yeast. In a sleight of hand trick, they used a computational algorithm originally designed to identify those yeast prions and it worked just like they wanted.
In total, they found around 500 Arabidopsis proteins that seemed to have domains in their structure that are similar to that of prions, so-called “prion-like” domains. They narrowed down their results to only those proteins that had a connection to the flowering pathway and that left them with three: : Luminidependens (LD), Flowering Locus PA (FPA), and Flowering Locus CA (FCA). To see if these proteins indeed had prion-like activity, they took a clue once more from the research in yeast.
Prions, at least what had been seen in yeast, have a variety of similar conformations that are nonetheless very specific and distinct from each other. They are referred to as prion strains in order to consider them separately. These strains in turn have several heritable elements that cause phenotypic responses in an organism and these are passed on, as discussed just above, without the involvement of genetic material. Furthermore, since prions in an active state convert all the non-active normal form of their protein into themselves, the active prions are normally the dominant form of the two found in an organism.
Based on all of these changes from the norm, along with how these prion proteins respond strongly to changes on the homeostasis of the general protein environment, they can be directly tested for one by one. Though, of course, doing these tests in the Arabidopsis plants themselves would be many scales harder than doing so in yeast was, so the scientists sidestepped that by fusing the proteins to green fluorescent protein and expressed the fusion in yeast cells.
They were then able to show that Luminidependens is indeed a protein with prion-like behavior. The normal non-active form of LD is used in the flowering pathway, while the higher order active form can change the function of other associated protein domains to its own. And, in doing so, it is able to accomplish the same processes discovered in yeast. That is, the production of protein-based molecular memories.
LD was also found to be highly unique and unlike the other known amyloid prions. Instead of having a high molecular weight in its amyloid form like the others, it creates a low weight oligomer rather than an amyloid and appears to have no interaction with the heat shock protein (Hsp) 104. Since memory-based prions normally interact with this particular Hsp, clearly something different was going on here. Other research had previously shown that at least one fungal prion was dependent on Hsp70 and didn’t form amyloids, so LD may be a part of this little explored alternative group of prion strains.
Going back to the topic of vernalization and winter memory, one issue or concern with the process has always been how does a plant distinguish between one extremely cold night and an entire winter season? If there was a day of unseasonably cold weather outside of winter, might that not throw off the flowering timing of plants? But that doesn’t happen, so there must be some deeper process keeping track of true winter conditions.
Conformational changes to stable, shape-dependent proteins like prions might be the answer. LD and the other two proteins are believed to be able to bind to nucleic acids and the other two are known to be able to bind to RNA with their recognition sites and to control RNA processing and termination of transcription through this. Perhaps changes to their conformations from a cold winter cause them to then influence and make changes to the bundled genetic chromatin, thus making the epigenetic alterations already known to play a part in the system.
Similarly, the form-shifting of prions may perfectly explain how vernalization functions. Especially if the prions are indeed chromatin remodelers. If the normal protein form is inactive during the winter conditions and then converts to an active protein during following better temperatures, that would then lead to its becoming an active prion state once expression of the protein increases during those conditions. That would lead the prions to modify the epigenetic expression and enact the flowering genes. Of course, there could be any number of other variations of this example that is the true way vernalization works.
The authors of the paper noted that all of this is merely hypothetical and that even before investigating these possibilities, LD must first be confirmed to have prion-like behaviors in plants themselves. Just showcasing that it does in yeast cells isn’t good enough, as expression behavior may be different between the two.
A Historical Sidenote
A funny final side comment about history that even the study authors point out at the end is the relationship all of this has to the former Soviet Union and the often, appropriately, maligned set of ideas known as Lysenkoism. In many ways, Trofin Lysenko was a hack of a scientist, if one can even deign to use that job descriptor. His purpose in the claims he pushed was political dominance over his competition. And, sure, he likely believed much of what he preached, but there was no evidence of the scientific method in his practices.
The primary claim he pushed was meant to overturn the genetics view of the rest of the world, destroy the Mendelian inheritance claims that he and the USSR government as a whole wanted to reject. To support his ideas of heritability of traits outside of genes, Lysenko used vernalization as a direct example. Since this was before epigenetic control over gene activation was a known process, there truly was no genetic evidence for how this process occurred.
Using vernalization to increase crop outputs was a success for him and it supported his rejection of the chromosomal theory. He then claimed that it was cellular components that are the true carrier of traits and that were inherited during cell division. This idea resulted in the abolishment and illegalization of Mendelian-based research in the Soviet Union for more than two decades. It was only reinstated after Lysenko’s later failures to extend his claims beyond just vernalization.
Ironically, this was one of the few systems that Lysenko could have chosen where he was actually somewhat correct in his assumptions. But it would be 70 years before any of that would come to fruition in modern day. Even Nikolay Vavilov, who we’ve discussed before here, stated that Lysenko might actually be on to something when it came to vernalization. If only Lysenko had followed that line of inquiry with proper scientific rigor and not through the incorporation of vapid hypotheses and pseudoscience.
Thus far, there have been no follow-up studies published on Luminidependens or the other proteins discovered in Arabidopsis with prion-like domains, so we have yet to see official confirmation of their activities. But there does appear to be a far more complex system of switches and gears in plants than was previously thought, with trait and expression memory only partially having to do with the genome at all.
The Opening of a New Field
These two characteristics of plants, both their capability to uptake prions from their environment and express them in their tissues and the possible use of prion-like proteins in their own cells to control phenotypic expression, are quite likely just the beginning of what we will be finding out about plants and about prions in the near future.
While prion diseases have had a decent amount of research dedicated to them, very little external study has been given to the mechanisms of prions themselves separate from this and whether their unique structural conformations can be found elsewhere and in other uses outside of disease causation. That has been slowly changing thanks to studies like those discussed above, which have highlighted the importance that prions likely play in far more than just the horrors we normally find them engaged in.
Studies that were confined only to yeast and related microorganisms are now being expanded to consider the influence and impact prion-like proteins have on larger organisms and how they fit into the evolutionary history of life on this planet. It should be fully expected that within the next decade, there will be several revolutionary discoveries in the field of prion research and we will continue to unlock the complicated secrets of life itself.
References
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Photo CCs: Creek and old-growth forest-Larch Mountain from Wikimedia Commons
