A WSU scientist explores the ecological and evolutionary power of symbiosis
Some two billion years ago, a dining experience went sideways. An early nucleated eukaryotic cell engulfed a cyanobacterium—but instead of digestion, co-housekeeping was the result of the union. This ancient endosymbiotic event brought together the mobility of eukaryotes with the photosynthetic ability of cyanobacteria, an evolutionary win-win that resulted in the creation of a new type of organelle—the chloroplast—precipitating the ascendance of plant life on Earth.
The Greek historian and travel writer Herodotus wrote 2,500 years ago of an altogether different sort of dining experience. A plover perches in the gaping mouth of a Nile crocodile, feasting on the leeches that, in turn, are feasting on the croc’s blood. Keen on extracting moral guidance from natural phenomena, Herodotus said that we should learn friendship from the cooperation between the unharmed bird and the predatory reptile.
The example of the plover and the crocodile has served as a paradigm of symbiotic relationships clear through the nineteenth century. But around the time of Darwin, scientists began to look at symbiosis with new eyes. Thanks to their work, we now know that some of the most important evolutionary innovations in the long history of life on Earth—organelles within eukaryotic cells, such as the chloroplast and the mitochondria—are due to symbiosis.
Symbiosis occurs when two or more species live together in close physical contact and, strictly speaking, need not be mutually beneficial. Mutualism, where species cooperate to their shared benefit, is a type of symbiosis. Some researchers argue that bees and flowers, as interdependent ecological niche mates, are symbionts. Similarly, humans and our food plants and animals are in symbiotic relationships: we need to eat them, so we
give them what they need to grow to an edible
As biologists realize that cooperation is as important as competition or predation in the evolution and maintenance of ecological roles and niches, they are also seeing that microbes are foundational partners in the success of a huge number of life forms.
The microbe connection
For microbiologist Stephanie Porter, an assistant professor at Washington State University Vancouver who studies the evolution of cooperation and plant-microbe symbiosis, “The microbiome is the set of all microbes that live in and on plants and animals. Understanding the complex and often positive role the microbiome plays in the health of plants and animals has precipitated a real renaissance in biology. There’s been a blossoming of ideas due to new genomic tools for understanding this microbiome.
“But there’s also been a shift in our thinking about microbes. We’ve moved from microbes being viewed strictly as the cause of diseases or that they are at best harmless, to thinking they have a lot of positive effects on plants and animals. They can help plants and animals resist diseases or tolerate environmental stress and we didn’t previously recognize this. It’s an opening of our eyes to this whole world of complexity that didn’t exist before this paradigm shift in the field.”
Porter and her team of collaborators, which includes other scientists as well as WSU Vancouver undergraduate research interns and graduate students, design experiments that tease out the genetics of cooperation—experiments that at the same time shed light on big evolutionary questions. Her lab is itself a kind of symbiotic relationship. Individuals from varying backgrounds bring hypotheses and design solutions to the table as they all seek answers, Porter says, to “fundamental questions about why plants and microbes cooperate.” Among other things, Porter’s group is keenly interested in understanding how cooperation lasts when, as she says, “the temptation to cheat might turn partners into enemies?”
These are questions that are critical to the resiliency of the human food system threatened by a changing climate that drastically increases stresses. Problems that become more frequent under climate change, Porter says, include soil salinization, drought events, extreme temperature, insect herbivores, and insect-borne pathogens. Researchers in Spain recently conducted experiments suggesting that a two-degree Celsius increase in temperature results in a tripling of soil pathogens. Maintaining a healthy food supply, while minimizing the environmental and economic costs of agriculture, are motivating forces in Porter’s lab.
“Fungi and bacteria that live in plant roots perform functions that we expect, like providing nutrients to the plant, or taking up space and therefore preventing pathogens from invading the plant. But they also manipulate the hormonal and metabolic profiles of plants to make them resist all kinds of different stresses,” Porter says.
She and her team are also investigating symbiosis from the microbe’s point of view. As Porter points out, there has been lots of work on that relationship from the perspective of crop plants (including at WSU), but understanding why a microbe would go to work for a plant is a new frontier.
Even before humans had any clue that there were organisms they couldn’t see, we knew there was something in soil that made plants grow.
Take alfalfa, a legume grown in Greece for livestock fodder since at least the time of Herodotus. When Europeans colonized the Americas, they brought livestock and their fodder with them. But alfalfa wouldn’t grow here. Turns out, the plant was missing its ancient partner, a kind of bacteria called rhizobia.
Alfalfa, and other legumes in the pea family, depend on root-dwelling bacteria to convert nitrogen from the air into a form the plant can use. These bacteria essentially fertilize the plant. European colonists “had to bring soil from Europe to seed their fields” to colonize the soil with compatible rhizobial bacteria and other microbes, Porter says. “They shipped trainloads of soil from their successful alfalfa fields to new areas of cultivation as colonization there proceeded. That’s an early example of manipulating the plant microbiome to make agriculture successful.”
But why would the relationships between legumes and their nitrogen-fixing associates persist for such a long time?
“When you cooperate, you are giving valuable resources to someone else instead of your own offspring,” she says. “Darwin considered cooperation to be a mystery and a problem for the study of evolution. Natural selection should select for traits that benefit your own offspring. So how is it stable over the long term to give resources away—why wouldn’t an unrelated partner just cheat you instead of reciprocating?”
As Porter and others have learned, cheating can be a winning strategy. But biologists have drawn on a theory from economics called partner choice to explain why cheaters don’t prosper in most cooperative interactions.
“In an economic market,” she explains, “partners can see who is going to benefit them and preferentially allocate resources to them.” In a symbiotic relationship, “if there is exploitation going on”—as when a non-nitrogen-fixing bacterium sneaks into a legume—one partner can “stop cooperating before it makes a bad investment.” Partner choice explains some forms of associations, but “cooperation has many forms. It is deep and challenging, and one hypothesis doesn’t work across all these different forms.”
Cooperation sometimes evolves when the “interests of both partners are intrinsically aligned,” Porter says. One example of a stable symbiotic relationship is between the Hawaiian bobtail squid and its bacterial symbiont, a bioluminescent bacterium called Vibrio fischeri. The bacteria live in the squid’s light organ. In exchange for sugars and amino acids, it glows blue at night—providing the squid with protection from predators as it nocturnally feeds close to the ocean surface. Without the bacteria’s glow, the squid would present a dark silhouette against the moonlit surface, making it easy pickings for hungry predators looking up from the depths below.
Porter says “the squid is exquisitely poised to maintain cooperation in these bacteria because the same genetic capacity that makes these bacteria glow allows them to detoxify chemicals the squid fills its light organ with. So the key trait of the bacteria that helps the squid is also the same one that allows it to live in the squid’s organ. It can’t cheat, because if it didn’t have the genetic capacity to make light, it wouldn’t be able to live there anyway.”
Another method symbionts use to ensure a good fit is called screening. Basically, the host organism sets up an entrance exam that only the most beneficial organisms can pass. Acacia trees and ants are a good example.
“The ants live in special swollen thorns on the acacia tree, and feed on nectar and lipid-rich bodies the tree produces. Ants bite any insect or animal that tries to eat the tree, defending the tree. Acacias that put out really great food bring in the most aggressive ant colonies, because they’re the ones that can outcompete all the other ants for this great feast. More aggressive ants are better at warding off animals that try to eat the tree. The big reward allows the tree to screen for the best defenders.”
Just as humans do, plants have microbiomes critical to their health. But there appears to be a catch. Agricultural crops get nutrients and pest control from humans, making microbes less valuable. Porter and her colleagues have found that crop plants often have microbiomes of reduced species diversity that may not provide the same level of benefit as do the microbiomes of wild plants.
“We have domesticated many crop species that now depend on us,” she says. “Corn can’t grow in the wild, and many of our animals can’t survive without us—and we can’t without them. It is possible that elements of plant-human relationship have replaced microbial services to plants. If that’s the case, there’s a lot of potential to benefit from reintroducing beneficial microbes to crop plants.”
Since microbes are known to provide plants with defenses against pathogens and, in some cases, nutrients, encouraging crop microbiome associations to mimic those of wild plants might reduce agrochemical use.
But that’s a big maybe. As Porter points out, “We are far from understanding whether restoring wild-type microbiomes to crop plants would help make agriculture more sustainable because we don’t know what the costs might be. Maybe being better at managing symbionts uses a lot of energy” that might otherwise go to producing a larger yield, or to having some other benefit.
“We have to do the science first! We have to find which aspects of the microbiome changed during crop domestication and which did not.” It’s possible that some crops are better off not relying on their microbiomes. “There are lots of steps to figure out before providing products to farmers.”
Those products, called inoculants, would be applied to seeds, or sprayed on fields after sowing.
“The Holy Grail,” Porter continues, “is identifying the ways in which crops are deficient in their ability to control symbionts,” and comparing the genetics of those plants with wild relatives that manage the relationship well by making sure cheaters don’t prosper.
“We could then introgress those traits back into crops and reduce our dependence on agrochemical inputs and irrigation.” Introgression is a process of moving a gene from one organism to another using hybridization and backcrossing. The process is often slow, requiring many generations of backcrossing to successfully move a trait from one species to another—but it is more acceptable to consumers than genetic engineering, where a gene is artificially introduced into the target plant’s genome.
The recipient of a prestigious National Science Foundation Early Career grant, Porter’s group is seeking to answer the question, “Have we compromised our crops’ ability to benefit from microbes through domestication?”
In looking for the genetic controls of symbiosis, they hope to “select variants that allow the plant to have optimal symbiosis and optimal outcomes with its microbes.”
Together with WSU plant pathologist Maren Friesen, Porter analyzed 87 studies in which plant-microbe relationships were experimentally manipulated. Across the studies, they saw that fungal symbionts were more important for ameliorating stress than bacteria, though both are critical for plant health. The message from that study, Porter says, “is really about the potential for improving plant health via its microbiome, especially under stress.”
In a 2014 paper, a group of scientists described hundreds of studies in which introgressed wild genes enabled domesticated crops to resist pathogens, insects, and improved seed nutrition. One notable example of targeted introgression, the researchers write, comes from the common bean, “Phaseolus vulgaris. Breeders have successfully introgressed genes conferring resistance to insects … and pathogens … , as well as higher nitrogen, iron, and calcium seed content from existing collections of wild Phaseolus. These efforts have contributed to both higher yields and improved nutritional quality and have also lessened the environmental impact of crop production by facilitating reduced pesticide, herbicide, and fertilizer use.”
Porter’s work builds on this to investigate whether genes from wild relatives could boost symbiotic abilities in our crops.
The nitrogen fixers
Among others, Porter studies nitrogen-fixing rhizobial bacteria, which, she says, “have an amazing ability” to insert or eject from their genomes the genes that confer symbiotic ability. The genes for symbiosis are on a large plasmid (a usually circular ring of DNA that can replicate independently of chromosomal DNA). Angeliqua Montoya (’18 Biol.), a graduate student working with Porter, calls it the “symbiosis island. It can be lost or gained—they can just kick it out if they’re feeling stressed.” When the bacterium ejects the island, it gives up on cooperation and can be ejected in turn by the plant. The bacterium is then left to dine on decaying organic matter in the soil.
“We need to understand the potential for these strains of mesorhizobium to give up on cooperation,” Montoya adds, “especially in conditions such as high fertilizer situations where relaxed selection on the plant seems to make the microbes become less cooperative,” and less willing to fix nitrogen in the plant’s root system.
Since nitrogen is a limiting factor in plant growth, “the fact that legumes can get nitrogen from rhizobial bacteria gives them an advantage,” Porter says. “Legumes can be pests but they can also greatly benefit humans. It’s a powerful symbiotic relationship that has been harnessed in agriculture for millennia. It’s a really rich area for both understanding evolution but also doing work that has really practical implications for how we can improve food security and reduce pests.”
As weeds, leguminous plants such as scotch broom and kudzu have “these little engines in their roots pumping out nitrogen,” Porter says. Their microbial associates give them a serious advantage when competing with other vegetation, to the extent that legumes can exclude other plants from the environment.
Rhizobial bacteria give some legumes another advantage, too: the ability to thrive in serpentine soils. Such soils are formed by the weathering of certain mineral-rich rocks. The soils are high in toxic heavy metals like nickel or cobalt, and low in nitrogen and phosphorus, which are essential for plant growth.
While serpentine soils are usually on steep inclines and are shallow due to erosion, industrial sites often have a similar set of environmental conditions: high heavy metal content and low organic matter. Could legumes and their rhizobial partners be the first step in a succession of plants that remediate such soils?
Yes, says Porter, but cautions that “we’re still at the starting block.” She and her team have collected samples of mesorhizobia and their leguminous associates that live in serpentine soils from southern California north into Oregon. From the samples, they extracted “these super tolerant bacteria. We’re unlocking the genetic mechanisms by which they can tolerate heavy metals. They alter their enzymes to function at high levels of heavy metals and they pump them out.”
Miles Roberts (’20 Biol.), an undergraduate working in Porter’s lab until his recent graduation, investigated the heavy metal-tolerant mesorhizobia before heading off to graduate school in Michigan. From the samples, he and his lab mates found that some of the bacteria are “adapted to these soils while other populations are adapted to nearby metal-poor soils.”
Roberts was testing a hypothesis about bacterial speciation: that bacteria adapt to specific local conditions by trading off the ability to live in diverse ecological niches. While Roberts didn’t find direct evidence of trade-offs, Porter had previously done so. In a 2012 paper, she and a colleague write that “trade-offs and adaptive divergence may be important factors maintaining the tremendous diversity within natural assemblages of bacteria.”
But, she cautions, trade-offs are not likely the single explanation for the diversity of life. “One of the great mysteries of ecology, for any organism, is what maintains diversity at any level of organization.” And with bacteria, it gets really tricky. Bacteria have a variety of ways of exchanging genetic information. One organism can, for instance, simply pass, or horizontally transfer, a set of genes to another. “You can have incredible ecological variety in a single species,” to the extent that the concept of species breaks down.
“Even in pathogenic organisms you may have cholera that is highly infectious and deadly, or that doesn’t infect anyone. In terms of the ecological impact of those two different strains of the same species, they’re fundamentally different and yet they may only differ in small portions of their genome.”
Porter suggests that, rather than species, bacteria might be organized in terms of their ecological roles and niches. Eschewed as too ambiguous, the word “species” is replaced by some researchers with terms such as “biovar” or “ecotype.”
“Environments are highly variable and finely structured,” Roberts says. “Stand in one spot in a forest: there’s a dry patch over here, there’s a sunny patch over there” and each little patch will likely have its locals-only population of specially adapted microorganisms.
On a global scale, the myriad microbial eco-niches do form a kind of metaphorical forest—and it’s only recently that we’ve begun to discern the fine structure of the individual trees. And what we now see is that, from root to crown, these “trees,” whether humans, legumes, squids, or real trees, are engaged in struggles to survive. Struggles that, strange as it may seem to the old idea of “survival of the fittest,” are in fact often a matter of cooperation.
A lens for thinking: Research experiences for undergraduates and Porter’s planned Science Scholars web portal that will connect students with researchers looking for interns