Whom do cheating bacteria cheat: host plants or other bacteria?
Bacteria known as pseudomonads produce and release chemicals (defensive toxins) that protect plants from fungi that would otherwise attack their roots. In return, the roots release various organic compounds that serve as food for the bacteria.
The "in return" part has always bothered me. Each root system is associated with millions of bacteria. In a 2003 paper (Cooperation in the rhizosphere and the "free rider" problem. Ecology 84, 838-845), we pointed out that this system is a potential tragedy of the commons. Mutant bacteria that don't make root-protecting chemicals free up resources for their own reproduction, so we might expect them to out-compete more-beneficial strains. If these "cheaters" become common enough, the host plant might be killed by fungi, but that would hurt the beneficial strains around that root system just as much as it hurt the cheaters. We suggested that the bacteria make these toxic chemicals to protect themselves, with protection of roots as a side effect. Research by others, including some of the authors of this week's paper, has provided data consistent with this hypothesis. For example, toxins made by pseudomonads protect them from predators.
More recently (in Annual Review of Ecology, Evolution, and Systematics), Toby Kiers and I suggested that cooperation between microbes and plants is better understood as cooperation among microbes. For example, by providing their host plant with the nitrogen it needs to grow, rhizobia (root-nodule bacteria) help all the other rhizobia infecting the same plant.
This week's paper shows that defensive toxin production by pseudomonads is similar. Toxin production by pseudomonads may benefit the plant and may benefit individual cells, but it also benefits other pseudomonads nearby. "Predators promote defence of rhizosphere bacterial populations by selective feeding on non-toxic cheaters" was published in The ISME Journal by Alexandre Jousset and colleagues in Germany and Switzerland. These pseudomonads produce various toxins, especially when there are many of them in close proximity. This dependence of toxin production on bacterial density is an example of quorum sensing. Mutants "defective" in quorum sensing have been shown to grow (i.e., reproduce by dividing) faster, because they save the cost of toxin production. Can these "cheaters" free-load on defensive toxin producers nearby, essentially hiding behind their chemical defenses?
To find out, the researchers gave the two genotypes (toxin-producers and nonproducers) genes for two different fluorescent proteins, so that they could be told apart under a microscope (or on culture plates; see photo). They then mixed the two strain together in different proportions (10:1, 1:1, or 1:10). In their most interesting experiment, these mixtures were applied to the roots of rice plants. They then added predators that eat bacteria (amoebae or nematodes) to some of the roots.
When they started with mostly cheaters, the frequency of cheaters decreased. When they started with mostly cooperators (defensive-toxin makers), cheaters became more common. This kind of result is known as negative-frequency-dependent selection. Under nematode predation, they estimated that the two strains would have equal fitness when the frequency of the cheaters was 20%.
The frequency-dependent selection appeared to result from the interaction of two factors. The predators preferred to eat nontoxic bacteria. By itself, this effect might be strong enough to eliminate the growth advantage of the cheaters, perhaps eliminating them from the population. But when nontoxic mutants were rare, they were protected by toxin produced by others.
One curious result was that they also found negative frequency dependence (although the equilibrium frequency of cheaters was greater) even when no predators were added. Previously, some of these authors have suggested that the toxins may have a role in competition, as well as in protection from predation.
A role in competition might also help explain another thing I wondered about. If toxins protect individual pseudomonad cells from predation (because predators preferentially eat nontoxic individuals), why is toxin production linked to quorum sensing, so that little toxin is produced by isolated individuals? Maybe they use the toxins to kill competitors or to chase them away. Or maybe predators are attracted to groups of bacteria, so that isolated bacteria have less need for protection against predation.
If cheating pseudomonads have an advantage only when they are relatively rare, then maybe we don't need to worry that production of pseudomonad toxins (which protect roots from fungal pathogens) will disappear over the course of evolution. Instead, we should expect some mixture of protective and nonprotective pseudomonads under most conditions. On the other hand, if selection for toxin production depends on how well these toxins protect the pseudomonads from predators, then there is no guarantee that the toxins will also protect plants from every new fungal strain that evolves.
Photo from Alexandre Jousset shows two strains marked with different fluorescent markers, growing together.