The nationwide experiment will initially include around 100,000 volunteers.
Plants and fungi can use conserved RNA interference machinery to regulate each other’s gene expression—and scientists think they can make use of this phenomenon to create a new generation of pesticides.
February 1, 2017|
Plants, silent as they are to our ears, are in constant conversation with their environment. As scientists have developed ever-more-sensitive tools to eavesdrop on this molecular chatter, they’ve discovered not only dialogue among the cells of an individual plant and with the plant’s immediate surroundings, but between different individuals, sometimes of different species and even different kingdoms. The alphabet of this lingua franca is A, C, G, and U.
Noncoding RNAs are well known for their ability to control gene expression in cells. And as scientists have demonstrated repeatedly, protein production can be affected not just by RNAs made in the same individual, but by RNAs from altogether different organisms. In recent years, researchers have taken advantage of the ability to traffic RNA between distantly related taxa to selectively inhibit the expression of genes in fungi important for their growth, an approach they say might lead to the development of disease-resistant crops. Scientists have also shown in the lab that this cross-kingdom RNA transfer can go both ways: fungi are also sending RNA dispatches to their plant hosts, and the covert operation could be aiding their invasion.
In this conversation between plants and fungi, the organisms rely on a well-worn mechanism of gene-expression regulation that has stood the test of evolutionary time: RNA interference (RNAi). Listening in on the RNA crosstalk between plants and their pathogens could reveal previously unknown facets of basic plant biology, and point the way toward a successful strategy to fend off crop pathogens. Yet, scientists’ manipulation of cross-kingdom RNAi using plants predates their full understanding of exactly how it works or how often it happens in nature.
RNAi is a widely conserved mechanism used during development, in routine cellular processes, and in response to foreign invaders—especially viruses—entering a cell. The cell produces small RNAs that are then integrated into an aggregation of proteins called the RNA-induced silencing complex (RISC), which targets messenger RNA molecules (mRNAs) containing the small RNA’s complementary sequence. RISC then chops up bound transcripts, thereby tamping down gene expression.
Over the past decade, scientists have demonstrated RNAi’s ability to protect numerous plants against nonviral pathogenic foes. In 2007, for instance, Monsanto endowed corn with the ability to fend off western corn rootworm by providing the crop with a gene for an RNA that targeted transcripts of an essential gene in the insect. The transgenic plants suffered less damage, presumably because the insects ingested the interfering RNAs and died.1 Around the same time, research groups showed that the approach—called host-induced gene silencing (HIGS)—could also ward off parasitic worms, and since then, laboratory experiments with transgenic plants have produced an ever-expanding list of animal pests susceptible to engineered RNAi.
I wouldn’t be surprised if there is small-RNA traffic between fungi and plants outside of laboratory experiments. They do everything else in their war against each other.—John Pitkin,
In recent years, genetic engineers have successfully applied HIGS to combat pathogenic fungi. In 2010, a team based at the Leibniz Institute of Plant Genetics and Crop Plant Research in Germany showed that the powdery mildew fungus (Blumeria graminis) was susceptible to RNAi perpetrated by small RNAs engineered into its host plant’s cells. A few years later, Karl-Heinz Kogel’s group at Justus Liebig University in Germany demonstrated that HIGS against fungi was possible in whole plants. His team engineered Arabidopsis and barley plants to produce small RNAs that inhibited the expression of certain genes in the fungus Fusarium graminearum, which causes head blight.2 The products of these fungal genes are the very proteins upon which conventional fungicides work. “We didn’t use a chemical to target the protein; we used small RNAs to target the gene,” says Kogel. “And it is highly effective.”
Tweaking that methodology, a number of groups have now shown that HIGS can protect lab-grown plants from several fungal diseases, including potato late blight, downy mildew (which affects lettuce), cereal rust, and wheat leaf rust. All of these experiments rely on naturally occurring RNAi machinery already present in both the plant and the pest, including RISC and proteins that process the precursors into small RNAs. Hailing Jin, who studies plant immunity at the University of California, Riverside, says success with HIGS in the lab suggests the technique could be an effective fungicide in the field “because those RNAs can be specifically designed to target the pathogens you want. Since the design is relatively easy, you have the potential to target multiple pathogens [at once].”
Researchers have already completed a field experiment that demonstrates the effectiveness of engineering plants to send interfering RNAs to fight off fungal pathogens. In 2015, a team in China designed transgenic wheat plants that produced RNAs targeting essential genes in two fungal pathogens, Fusarium head blight (FHB) and Fusarium seedling blight. Mutant plants grown in the field in Wuhan (“among the most severe wheat FHB epidemic regions in China,” the authors noted in their study) were resistant to the disease.3
“This is an area we’re definitely watching,” says John Pitkin, the Global Disease Management Lead at Monsanto. “It clearly looks like there’s movement of RNA between plants and fungi through the expression of transgenes. Whether those reach the level of commercial efficacy is still in question.” (See “Using RNAi to Protect Crops” below.)
© MESA SCHUMACHERDespite progress on the biotechnology front, scientists haven’t been able to say definitively that the phenomena they can encourage in the lab occur in nature. While the actions of RNAi against viral pathogens within the plant cell have been appreciated for years, it’s unclear whether plants in the wild send RNA mercenaries into fungi and other invading eukaryotic pathogens.
A few months ago, scientists reported perhaps the first evidence that small-RNA transfer between plants and fungi does indeed occur without the intervention of genetic engineers. Hui-Shan Guo at the State Key Laboratory of Plant Genomics at the Chinese Academy of Sciences in Beijing discovered that cotton plants ramp up the production of certain small RNAs after infection by a fungal pest, Verticillium dahliae. Not only do these plant RNAs tamp down the expression of two essential genes in the pathogen, but mutating the fungus’s genes to be resistant to the RNAi made the pest more virulent.4 “Our works are the first direct experimental evidence of the mobility of . . . RNA molecules from plants to fungal cells and inducing target gene silencing in fungal cells,” Guo wrote in an email to The Scientist.
Although Guo’s study stands alone as evidence that plants use RNAi to punch back at pests in the field, plant biologists are nevertheless convinced it’s a widespread defense strategy. “Assuming there’s a mechanism for transferring RNAs from host to pathogen, it makes sense this would be one avenue nature could exploit to kill the pathogen,” says Phillip Zamore, who studies RNAi at the University of Massachusetts Medical School. “It’s a case of nature got there first.”
“I wouldn’t be surprised if there is small-RNA traffic between fungi and plants” outside of laboratory experiments, says Pitkin. “They do everything else in their war against each other.”
USING RNAi TO PROTECT CROPS
© ISTOCK.COM/JEVTIC; GRAHAM RAWLINGS/FLICKRField experiments testing the use of genetically engineered RNA interference against eukaryotic pathogens, an approach called host-induced gene silencing (HIGS), are just now embarking on the long road to commercialization. But even if HIGS is successful at warding off disease-causing insects or fungi, cost may be an insurmountable hurdle. John Pitkin, Global Disease Management Lead at Monsanto, says the commercialization of a transgenic crop costs on the order of $130 million to $140 million. “Finding one single disease that reaches that bar for a HIGS approach is a pretty daunting task,” he says. In other words, can the financial burden of a pathogen justify the mammoth expense of getting a transgenic, RNAi-protected crop on the market?
Then there’s the public’s discomfort with genetically modified crops, particularly in Europe, notes Karl-Heinz Kogel, a plant biologist who uses HIGS at Justus Liebig University in Germany. To get around genetic manipulation, he and others have tested the possibility of an RNA spray. Rather than introducing small RNAs via transgenes in the plant, it may be possible to just apply the interfering molecules directly to the crop, an approach called spray-induced gene silencing (SIGS). Several months ago, Kogel’s team reported on its experiments spraying barley plants with a long noncoding double-stranded RNA—a precursor to the small RNAs used in RNAi—targeting the same genes critical for Fusarium graminearum survival that he attacked using HIGS in 2013. It worked: the plants suffered far less disease (PLOS Pathog, 12: e1005901, 2016).
Interestingly, the researchers found that the RNA was taken up by the plant and transferred into the fungus—results that add to another finding that RNAs could directly enter the pathogen as well. “Not only does the fungus take it up from the surface and is then killed, but the plant takes it up, transports it through the plant body, and then the fungus takes it up again,” says Kogel. He adds that direct RNA intake by the fungus is much less efficient, and thus the uptake and transfer by plants may be necessary to elicit the protective effect of the spray.
For more than a decade, Jin at UC Riverside has been investigating the ways small RNAs come to plants’ defense. Knowing that RNAi machinery is conserved among eukaryotes, Jin decided to look at the other side of the equation: Do plant pests, as part of their attack strategy, produce small RNAs that target specific host genes?
The bane of many a farmer, the fungus Botrytis cinerea, also known as gray mold, covers plants in a coat of fuzz that rots leaves, stems, flowers, and fruits. Jin’s group infected Arabidopsis thaliana and tomato plants with Botrytis and profiled the expressed RNAs within the cells of the plants’ leaves. “We found a lot of Botrytis small RNAs are enriched after infection,” she says. “One obvious but very exciting hypothesis would be that those small RNAs would have the potential to target host genes.”
To find out, she enlisted a bioinformatician to predict the Botrytis RNAs’ target genes in the hosts. She set the bar high, including only those small RNAs that matched up with both Arabidopsis and tomato genes. “Even then, we got 70-something Botrytis small RNAs that can have very good Arabidopsis and tomato targets,” Jin says.
To see if these had any functional significance, she and her colleagues selected three of the more abundant fungal RNAs and expressed them in Arabidopsis. Not only were the plant’s target genes subsequently suppressed, the plants were more susceptible to infection, supporting the idea that the fungus is tamping down its host’s immunity by transferring small RNAs.5 “This demonstrated the first example of cross-kingdom RNAi used as a virulence mechanism in fungal pathogens,” says Jin.
Kogel calls Jin’s discovery “sudden and unexpected. . . The small RNAs in the fungus are very, very similar to the mRNA structure of genes in the plant, and they inhibit the gene function by degrading these plant RNAs.” Just how the fungus gets its RNAs inside the plant leaves remains a mystery, notes Kogel. “This is a black box.”
Jin recently put a clever twist on the cross-kingdom RNAi story—using the gene-suppressing mechanism against itself. Her team engineered Arabidopsis and tomato plants to produce RNAs to destroy Botrytis’s RNAi machinery—specifically, to silence a key RNA-processing enzyme called Dicer-like protein 1. Because the fungal enzyme must trim the RNAs with Dicer-like protein 1 before their transfer to plants, it was like having the plant host lob a bomb at the fungus’s bomb factory. And it worked. The fungus didn’t grow as well on the transgenic plants.6
© SCIMAT/SCIENCE SOURCE; © BIOPHOTO ASSOCIATES/SCIENCE SOURCE
Cross-taxa RNAi isn’t limited to plants and the organisms they interact with. It occurs among a variety of organisms, including humans. In 2012, for instance, researchers reported that microRNAs from human blood cells could transfer into the malaria parasite Plasmodium falciparum, target a particular transcript, and tamp down gene expression.7
Nematode parasites can also pass RNAs to their mammalian hosts. A few years ago, Amy Buck, who studies RNA at the University of Edinburgh, and her colleagues discovered that Heligmosomoides polygyrus, a helminth parasite that lives in the guts of mice, secreted exosomes containing, among other cargo, microRNAs that suppressed the expression of certain mouse genes.8 The RNA-carrying exosomes secreted by the worms also included the protein Argonaute, the member of RISC that chops mRNA, suggesting these cellular blebs come fully loaded to interfere with the recipient’s gene expression.
Buck’s results implicate extracellular vesicles as the vehicles for the transfer of RNAi between organisms. “The vesicles are functional and get into [host] cells,” says Buck. “My sense is that vesicles moving cargo between cells or even between organisms is ubiquitous and ancient, and we just haven’t appreciated it.” Jin suspects that something similar is occurring between plants and fungi.
Buck says one of the questions about the transfer of RNAs within exosomes is whether the concentration of RNA molecules is sufficient to actually have an effect on the host. Just because one organism can produce small RNAs and ferry them over to another organism for interference doesn’t mean that they are indeed having a meaningful influence on the recipient. “You need an enormous number” to have a function, says Zamore—one interfering RNA for every target mRNA. “You can’t beat the laws of thermodynamics.”
Other mechanisms of RNA uptake may be at play. A few months ago, Jin’s group published evidence that Botrytis can bring in from the environment both small RNAs and longer double-stranded RNAs from which they are derived.6 And Kogel, too, has found that cultured fungi could absorb externally applied RNAs. However, like the mechanism of RNA transfer between plant and fungus, how this transfer happens is still unclear.
Despite lingering questions about the function of cross-kingdom RNAi, its use in the lab is becoming a powerful tool for experimentation. The mere ability to control the gene expression of pest organisms with transgenic plants has opened up research opportunities previously closed to scientists. Hans Thordal-Christensen, who studies plant immunity at the University of Copenhagen, says the fungus he works with isn’t amenable to genetic transformation. But with HIGS, he can manipulate fungal gene expression, especially of the effector genes that the pathogen secretes upon infection. “It’s opened up for us being able to study these things that otherwise we really couldn’t.”
JIM WESTWOODDodder, also known as strangleweed (genus Cuscuta), grows in many parts of the globe, sapping nutrients from other plants. It wraps its leafless, yellow-orange stem around its victim and burrows in, forming direct contact with the host’s vasculature. Dodder’s root then dies back, and the parasite sends off new shoots to find other plants to sink their haustoria into. “It looks like a mat of tangled coils and webs of tissue,” says Jim Westwood, who studies strangleweed at Virginia Tech. “It looks like somebody has thrown a bunch of straw out into a patch.”
All manner of material pass from the host to the dodder, including, Westwood has found, RNA. A decade ago he captured and sequenced messenger RNAs passing from tomato and pumpkin plants into the parasite (C. pentagona Engelm.), and found them to represent a handful of genes from each host genome, totaling about 20 in number (Plant Physiol, 143:1037-43, 2008). Shortly after, another group led by Neelima Sinha of the University of California, Davis, also found a few mobile transcripts transferred from tomato to dodder (New Phytol, 179:1133-41, 2008), but the full extent of what was going on was far from appreciated.
A few years later, as next-generation sequencing was becoming more affordable, Westwood’s team grew dodder on tomato and Arabidopsis plants. Westwood anticipated that there would be many more mobile mRNAs that hadn’t been identified by the earlier work, and perhaps he could nab a couple hundred more in this go round. But when the results came back, “it was not a few hundred. It was thousands.”
Most surprisingly, Westwood says, mRNAs were passing bidirectionally—also moving from dodder to host (Science, 345:808-11, 2014). “It suggested RNA movement is much more common than we had thought,” he says. But the effect of these incoming messages on the plants or their relationship “is a big unknown,” he adds. “We don’t have any evidence for an effect of that in the host-parasite interaction.”
February 8, 2017
"Nature got there first!" Is it not a tautology? Unworthy of a scientist or any logical person...
February 8, 2017
Natures' mystry opens new vistas for better understanding host-parasite relationship.
February 8, 2017
Natures' mystery opens new vistas for better understanding host-parasite relationship.
February 8, 2017
The mfd gene was originally identified as the gene required for reducing the rate of UV mutagenesis in E. coli cells held under conditions of amino acid starvation (9, 10).
This refutation of neo-Darwinian pseudoscintific nonense is buried in the rhetoric of an aritcle that links the virucidal anti-entropic energy of sunlight from hydrogen-atom transfer in DNA base pairs in solution to all biophysically constrained biodiversity via the physiology of reproduction in all liviing genera. Endogenous RNA interference is clearly the key to healthy longevity.
Until someone else links chirality to autophagy and healthy longevity via the creation of nucleic acids and RNA-mediated amino acid substitutions in all cell types of all living genera, claims about "RNA Interference Between Kingdoms" should continue to be viewed in the context of what is known to all serious scientists about supercoiled DNA. For example, it protects all organized genomes from virus-driven entropy.
February 8, 2017
It would be great if someone who believes in mutation-driven evolution would comment on their model of how entropy is linked from the bonding of atoms and the arrangement of gas particles in stars to the design of our self-organized brains.
February 9, 2017
We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain ageing.
Is anyone else willing to concede to the facts that link energy-dependent endogenous interference to all healthy longevity via the physiology of reproduction in species from microbes to humans? If not, are you willing to keep ignoring the fact that virus-driven energy theft causes all pathology, including the pathology that now must be attributed to exophers?