Passwords and eviction notices: How do plants keep their bacterial partners honest?

The nodule-y roots of a soybean plant. (Flickr: Pro-Soil AG Solutions)

ResearchBlogging.orgNitrogen is one of the elemental building blocks of life as we know it—it’s a basic component of amino acids, which are in turn the building blocks of proteins, which form the building blocks and moving parts of every living cell. The nitrogen interwoven in our tissues originated as part of the atmosphere we breathe, but the path from atmosphere to living flesh is far less direct than drawing a breath. Atmospheric nitrogen becomes useful to us animals only via an intimate relationship between a plant and bacterial growing in its roots.

The bacteria, called rhizobia, have the rare ability to “fix” free-floating nitrogen into biologically useable form. In return for this nitrogen source, the host plant allows the rhizobia to infect a specialized knob of root tissue, a root nodule, which it supplies with sugar for the benefit of its nitrogen-fixing guests. The plant uses the fixed nitrogen to make proteins for its own use, and anything that eats the plant afterwards benefits.

If all this sounds familiar, it’s because the interaction between plants and rhizobia is the focus of my developing postdoctoral research, and I’ve been writing about it as I’ve done more reading about it. Specifically, I’ve been interested in how plants might be able to make sure their root nodules house helpful bacteria rather than freeloaders, who enjoy the sugar supply inside the nodule without fixing nitrogen in return.

I’ve discussed a couple of different mathematical models that suggest some options. However, models are really just formal ways to follow through the implications of a particular idea, not necessarily descriptions of what actually transpires between a plant and the rhizobia inside its roots. So I thought it might make sense to step back and survey what we presently know about what goes on inside those root nodules.

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Choosing your partner is only as helpful as the partners you have to choose from

Picking teammates. Original photo by humbert15.

ResearchBlogging.orgWhen you need partners for some sort of cooperative activity—say, teammates for a game of kickball—you’d probably like to have a choice among several candidates. That lets you weigh considerations about kicking strength and running speed—and who promised to give you his dessert at lunch period—to build a winning team. However, if the other team captain snaps up the good players first, the fact that you have a choice among the others might not make much difference.

Plants and animals looking for mutualists face a similar situation. Being able to choose among possible partners should allow the chooser to work with helpful partners and avoid unhelpful ones, but a new study suggests that in one widespread mutualism the process of choosing between partners can leave the chooser worse off than if it had no choice at all [$a].

Coauthors Erol Akçay and Ellen Simms focus on the effects of partner choice in the mutualism between plants and nitrogen-fixing bacteria—the interaction I’m studying in my current postdoc position, as it happens. All living things need nitrogen, but only some strains of bacteria are able to collect nitrogen from the atmosphere and “fix” it into a form that other organisms can use. Many plants, particularly members of the big and diverse bean family, have evolved to allow nitrogen-fixing bacteria to infect their roots—the plants form a nodule of root tissue around the infection and supply the tissue with sugar for the bacteria to feed on as they fix nitrogen. Eventually the nodule dries up and dies off, and the bacteria are freed into the soil, having multiplied many times over thanks to the food supply from the host plant.

A plant’s root nodules, some cut open to show the interior. Photo by pennstatelive.

To see how this choice might work in practice, Akçay and Simms construct a mathematical model of a plant with two nodules. Each nodule produces some level of nitrogen, and recieves some level of sugar from the plant. The plant negotiates with the two nodules in what’s called a “war of attrition” game: whichever partner wants a better deal cuts off the exchange of services, and holds out until the cost of losing the service it recieves is greater than the benefit it hopes to gain in the war of attrition.

Rather like ant-defended plants, plants that host nitrogen-fixing bacteria don’t seem to screen potential mutualistic bacteria before allowing them to infect their roots. However, after root nodules are established, the success of the mutualism from the perspective of both partners depends on the genetics of each [PDF], and when host plants receive supplemental nitrogen, they put fewer resources into growing nodules [PDF]. Host plants have been observed with different strains of bacteria in different nodules, and some nodules could contain diligent nitrogen fixers while others are full of freeloaders. This may be the point at which the plant has a choice of partners—it can potentially direct sugar to helpful nodules, and cut off unhelpful ones.

Because the plant has two nodules to choose from, it can potentially outlast an uncooperative nodule by relying on the other one. This works if the plant can shunt more resources to the cooperative nodule and recieve more nitrogen from it in return. However, the success of this strategy depends on two traits of the bacteria inhabiting the nodules—how readily they ramp up nitrogen production in response to more sugar, and how stubborn they are in the war of attrition game.

If both nodules are stubborn but responsive to extra sugar, the plant can negotiate with one nodule by giving the other more sugar and receiving extra nitrogen. This lets the plant hold out longer in the war of attrition. On the other hand, nodules that are not responsive to extra sugar but also not very stubborn yield quickly in the war of attrition even though they don’t help much in negotiations. In either of these two cases, the negotiations find an equilibrium in which the plant receives a benefit about intermediate between what it would recieve if both nodules were infected by the same strain of bacteria.

However, if the plant hosts a stubborn-responsive bacterial strain in one nodule and a yielding-unresponsive strain in the other, it finds itself in a trap: the yielding-unresponsive strain is no help in negotiation against the stubborn-responsive strain, and the help provided by the stubborn-responsive strain isn’t an advantage in negotiating with the yielding-unresponsive strain. Over successive negotiations, the stubborn-responsive strain can ratchet up the sugar it extracts from the plant, and the plant ends up worse off than it would be if the two nodules were identical.

Just like humans haggling in a marketplace, the outcome of the interaction depends strongly on whether the other party plays along as expected.

Akçay and Simms find a way out of this trap by adding another wrinkle to the model. Much like the contract-theory models of mutualism I’ve discussed before, they modify the model to allow cooperative nodules to benefit from being cooperative. This makes a good deal of intuitive sense—if a nodule provides a better deal to the plant, the plant can potentially grow more leaves to produce more sugar, which would allow it to offer a better deal to the bacteria it hosts. Akçay and Simms call this “partner fidelity feedback,” and they find that, if it is sufficiently strong, it can allow the plant to out-negotiate a stubborn strain of bacteria.

Although it has a good deal of intuitive appeal, the model presented by Akçay and Simms does a fair bit of speculating in the absence of data. This is also a problem for the contract-theory model, and really all models of this widespread and important interaction. We know a great deal about the chemical details of plants’ interaction with nitrogen fixing bacteria. However, we don’t have a good sense of whether and how plants can redirect resources among nodules to haggle with the bacteria they host, and we don’t know whether and how bacteria could adjust their behavior to haggle with the plant. Akçay and Simms devote a big section of their online appendix [$a] to discussing just this point.

To figure out what’s going on inside those nodules, we need to determine how different models of interaction between plants and their bacterial mutualists may shape patterns in things that are easier to observe—both in the compatibility between plant genotypes and bacterial strains in greenhouse tests, and in the broader population genetics of both partners.


Akçay, E., & Simms, E. (2011). Negotiation, sanctions, and context dependency in the legume-rhizobium mutualism. The American Naturalist, 178 (1), 1-14 DOI: 10.1086/659997

Heath, K. (2010). Intergenomic epistasis and coevolutionary constraint in plants and rhizobia. Evolution DOI: 10.1111/j.1558-5646.2009.00913.x

Heath, K.D., Stock, A.J., & Stinchcombe, J.R. (2010). Mutualism variation in the nodulation response to nitrate Journal of Evolutionary Biology, 23 (11), 2494-2500 DOI: 10.1111/j.1420-9101.2010.02092.x

Evolving from pathogen to symbiont

This post was chosen as an Editor's Selection for ResearchBlogging.orgRecently the open-access PLoS Biology published a really cool study in experimental evolution, in which a disease-causing bacterium was converted to something very like an important plant symbiont. The details of the process are particularly interesting, because the authors actually used natural selection to identify the evolutionary change that makes a pathogen into a mutualist.

Life as we know it needs nitrogen – it’s a key element in amino acids, which mean proteins, which mean structural and metabolic molecules in every living cell. Conveniently for life as we know it, Earth’s atmosphere is 78% nitrogen by weight. Inconveniently, that nitrogen is mostly in a biologically inactive form. Converting that inactive form to biologically useful ammonia is therefore extremely important. This process is nitrogen fixation, and it is best known as the reason for one of the most widespread mutualistic interactions, between bacteria capable of fixing nitrogen and select plant species that can host them.

Clover roots, with nodules visible (click through to the original for a nice, close view. Photo by oceandesetoile.

In this interaction, nitrogen-fixing bacteria infect the roots of a host plant. In response to the infection, the host roots form specialized structures called nodules, which provide the bacteria with sugars produced by the plant. The bacteria produce excess ammonia, which the plant takes up and puts to its own uses. The biggest group of host plants are probably the legumes, which include the clover pictured to the right, as well as beans – this nitrogen fixation relationship is the reason that beans are the best source of vegetarian protein, and why crop rotation schemes include beans or alfalfa to replenish nitrogen in the soil.

For the nitrogen-fixation mutualism to work, free-living bacteria must successfully infect newly forming roots in a host plant, and then induce them to form nodules. The chemical interactions between bacteria and host plant necessary for establishing the mutualism are pretty well understood, and in fact genes for many of the bacterial traits, including nitrogen-fixation and nodule-formation proteins thought to be necessary to make it work are conveniently packaged on a plasmid, a self-contained ring of DNA separate from the rest of the bacterial genome, which is easily transferred to other bacteria.

This is exactly what the new study’s authors did. They transplanted the symbiosis plasmid from the nitrogen-fixing bacteria Cupriavidus taiwanensis into Ralstonia solanacearum, a similar, but disease-causing, bacterium. With the plasmid, Ralstonia fixed nitrogen and produced the protein necessary to induce nodule formation – but host plant roots infected with the engineered Ralstonia didn’t form nodules. Clearly there was more to setting up the mutualism than the genes encoded on the plasmid.

Wild-type colonies of Ralstonia (tagged with fluorescent green) are unable to enter root hairs (A), but colonies with inactivated hrcV genes are able to enter and form “infection threads,” like symbiotic bacteria (B). Detail of Marchetti et al. (2010), figure 2.

This is where the authors turned to natural selection to do the work for them. They generated a genetically variable line of plasmid-carrying Ralstonia, and used this population to infect host plant roots. If any of the bacteria in the variable population bore a mutation (or mutations) necessary for establishing mutualism, they would be able to form nodules in the host roots where others couldn’t. And that is what happened: three strains out of the variable population successfully formed nodules. The authors then sequenced the entire genomes of these strains to find regions of DNA that differed from the ancestral, non-nodule-forming strain.

This procedure identified one particular region of the genome associated with virulence – the disease-causing ability to infect and damage a host – that was inactivated in the nodule-forming mutant strains. As seen in the figure I’ve excerpted above, plasmid-bearing Ralstonia with this mutation were able to form infection threads, an intermediate step to nodule-formation, where plasmid-bearing Ralstonia without the mutation could not. Clever use of experimental evolution helped to identify a critical step in the evolution from pathogenic bacterium to nitrogen-fixing mutualist.


Amadou, C., Pascal, G., Mangenot, S., Glew, M., Bontemps, C., Capela, D., Carrere, S., Cruveiller, S., Dossat, C., Lajus, A., Marchetti, M., Poinsot, V., Rouy, Z., Servin, B., Saad, M., Schenowitz, C., Barbe, V., Batut, J., Medigue, C., & Masson-Boivin, C. (2008). Genome sequence of the  beta-rhizobium Cupriavidus taiwanensis and comparative genomics of rhizobia. Genome Research, 18 (9), 1472-83 DOI: 10.1101/gr.076448.108

Gitig, D. (2010). Evolving towards mutualism. PLoS Biology, 8 (1) DOI: 10.1371/journal.pbio.1000279

Marchetti, M., Capela, D., Glew, M., Cruveiller, S., Chane-Woon-Ming, B., Gris, C., Timmers, T., Poinsot, V., Gilbert, L., Heeb, P., Médigue, C., Batut, J., & Masson-Boivin, C. (2010). Experimental evolution of a plant pathogen into a legume symbiont. PLoS Biology, 8 (1) DOI: 10.1371/journal.pbio.1000280