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The Roots of Plant-Microbe Collaborations

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Science  09 Apr 2004:
Vol. 304, Issue 5668, pp. 234-236
DOI: 10.1126/science.304.5668.234

Genetic studies are decoding the language plants and microbes use to negotiate the symbioses that help feed the world

Bacterial or fungal invasions do not always cause disease. Some invasions—for instance, when microbes break into the cells of plant roots—are decidedly beneficial. By providing essential nutrients, the microbes help both their host plants and the world's agricultural systems.

In one such symbiotic interaction, the roots of many plants are infected by certain fungi that help them acquire phosphate from the soil. In the other key agricultural symbiosis, rhizobial bacteria set up housekeeping in the roots of legumes, such as peas, soybeans, and alfalfa, where they produce the form of nitrogen needed for plant growth. Recently, plant biologists have begun to dissect out the genes needed to establish these life-sustaining relations.

They've found that genes from both plants and microbes contribute to symbiosis. The two partners engage in a complex molecular conversation that allows the microbes to infect the plant cells and then entice the cells to undergo the developmental changes necessary for establishing the symbioses. “You have a bacterium and a plant that are completely different but have to be wired so that they can recognize one another and coordinate their activities,” says Sharon Long, whose lab at Stanford University is among the leaders in studying rhizobia-plant interactions.

In addition to clarifying the molecular underpinnings of these symbioses, the findings are shedding light on plant evolution. The fungal-plant symbioses are much older than rhizobia-legume associations. Detection of fungi in fossilized plants indicates that the associations date back to the first land plants, some 400 million years ago. The symbiosis-forming fungi, which were preserved in the plant root cells, “are among the few fungi in the fossil record,” says Maria Harrison of the Boyce Thompson Institute for Plant Research in Ithaca, New York.

Legumes, however, are a mere 70 million years old. This has long led plant researchers to suspect that rhizobia might have made use of some of the same plant machinery used by the fungi to establish their symbioses—a suspicion supported by the recent molecular findings showing that some of the same plant genes involved in rhizobia invasions are needed for establishment of the fungal infections.

Legume root invasion.

At 4 days after infection (right), a curled root hair surrounds a colony (green) of S. meliloti bacteria; by 7 days (left), the bacteria are making their way through an infection thread into the root.

CREDIT: E. LIMPENS ET AL., SCIENCE 302, 630 (2003)

The work might also have practical implications. Ever since gasoline shortages hit the United States 3 decades ago, researchers have hoped that they might be able to induce nonlegume crop plants, such as corn, to form symbioses with rhizobia. If so, the bacteria could “fix” nitrogen—convert the unusable nitrogen of the air to ammonia—for the cereals just as they do for legumes, thus reducing the need for costly and polluting synthetic nitrogen fertilizers. Identifying the genes needed for rhizobial symbiosis means “we can at least begin to think about how it can be done,” says Douglas Cook of the University of California, Davis.

The plant speaks first

Although the fungal symbioses are the more venerable of the two types of microbe-plant root interactions, researchers are further ahead in their understanding of how rhizobia infect their legume hosts, partly because the energy crises of the 1970s sparked a considerable investment in nitrogen-fixation research. The fungal associations are also harder to study because these microbes grow only on plant roots. Because they can't be cultured independently, mutational studies are much more difficult.

Rhizobial bacteria can live independently, but because they fix nitrogen only after they have entered legume roots and formed structures called nodules, researchers realized early on that in order to make progress, they had to study the two symbiotic partners together. “The basic idea was that if you wanted to scour for genes [expressed] in symbiosis, you didn't want to look at bacteria in a petri dish,” Long recalls.

By the early 1980s, the field had seen two big advances. In one, Frederick Ausubel's team at Harvard University cloned the first rhizobial nitrogen-fixation genes, which encode proteins needed for the chemical conversion of nitrogen to ammonia.

Before that reaction can take place, however, the bacteria have to induce nodule formation. This is a fairly complicated process that begins when the root hairs, small projections from the root surface, curl around the infecting bacteria. Once encapsulated this way, the bacteria burrow into a root hair cell, where they trigger the formation of a so-called infection thread that will carry them into the root proper. There they elicit the formation of a new structure—the nodule—in which they will reside, producing fixed nitrogen while receiving energy from the plant. Nodulation requires a second set of bacterial genes, the nodulation (nod) genes, and Long, then a postdoc in Ausubel's group, cloned the first of those, also in the early 1980s.

Next, Long and other investigators wanted to find out how the nod genes get switched on. In the mid- to late 1980s, they found that the switches are chemicals called flavonoids that are secreted by legume plant roots, with each legume species producing its own particular cocktail of the compounds. In this way, the plant initiates the molecular dialogue with the specific bacterium needed for their symbiotic interaction.

The fact that the various legumes make different flavonoids also contributes to one of the distinctive characteristics of legume-Rhizobium symbioses: A particular rhizobial species has a very limited host range, usually infecting members of only one or a few genera of legumes. For example, Sinorhizobium meliloti infects alfalfa but not peas, whereas Rhizobium leguminosarum bv. viciae infects peas but not alfalfa.

Subsequent work showed that the bacteria also contribute to this specificity. In the early 1990s, two teams in Toulouse, France, coordinated by Jean Dénarié of the INRA-CNRS Laboratory of Plant-Microorganism Interactions, identified the chemical that S. meliloti uses to alert alfalfa to its presence and initiate infection. This turned out to be a carbohydrate consisting of four linked units of the sugar N-acetyl glucosamine with a fatty acid attached on one end and a sulfate group on the other. Since then, researchers in several labs have identified these so-called Nod factors from several rhizobial species. All slightly different, each evokes a nodulation response only in the legumes that host the particular Rhizobium that produces the Nod factor.

Continuing the conversation

This specificity in rhizobial signal and legume response strongly suggests that legume root cells carry receptors that recognize and bind the appropriate Nod factors. “These molecules carry information for [nodule] development in the plant,” Cook says. “There must be receptors.” Much recent work has focused on identifying those receptors and working out the signaling pathways that spark the developmental changes that enable bacteria to induce nodule formation in the plant roots. Researchers usually go about this by inducing mutations in the plants, looking in particular for those that upset the earliest stages of Nod factor responses and thus rhizobial infection.

In one such study, a collaborative effort carried out about 4 years ago by Cook's and Dénarié's teams, the researchers treated plants of the model legume Medicago truncatula with a chemical mutagen. Analysis of the defects in the resulting plants turned up four genes, all of which are required for early responses to Nod factors.

At this point, the researchers found direct evidence that signaling to the plant by both the rhizobia and the fungi works at least partly through the same path. Three of the genes, called DMI (for Does Not Make Infections) −1, −2, and −3, also turned out to be needed for M. truncatula infection by a fungus that forms an arbuscular mycorrhiza, the technical term for the fungal-plant symbiosis involved in phosphate uptake. “Legumes form symbiosis with bacteria and with completely different fungi, but the same genes are involved in both,” says Martin Parniske of the John Innes Centre in Norwich, U.K.

Going nodal.

To prevent the energy drain of excessive nitrogen fixation, normal soybeans (left) restrict nodule growth, but some mutations disrupt that control (right).

CREDIT: I. R. SEARLE ET AL., SCIENCE 299, 109 (2003)

At that time, however, the plant genes were known only by the effects produced by their inactivation, such as blocking root hair curling and infection thread formation. Then, about 2 years ago, György Kiss and colleagues at the Biological Research Center of the Hungarian Academy of Sciences in Szeged cloned the M. sativa version of DMI2. At the same time, a multinational team led by Parniske and Jens Stougaard of the University of Aarhus, Denmark, cloned the Lotus japonicus version of DMI2.

The gene's structure indicates that the protein it encodes is a so-called receptorlike kinase. These proteins are embedded in the cell membrane with one region extending to the outside of the cell where it can bind signal molecules such as growth factors, and an interior segment that appears to be a kinase, that is, an enzyme that regulates other proteins by adding phosphate groups to them. This raised the possibility that the DMI2 protein might be part of the recognition machinery for Nod factors. In fact, the Szeged researchers originally called what turned out to be the DMI2 gene NORK (for Nodulation Receptor Kinase), and the Parniske-Stougaard team called its Lotus gene SYMRK (for Symbiosis Receptor-Like Kinase).

Some findings countered the idea that DMI2 serves as part of the Nod receptor, however. For one, although the roots of Lotus or Medicago plants carrying SYMRK/DMI2 mutations do not form infection threads or nodules when exposed to rhizobial bacteria, their root hairs become deformed. This suggests that they still sense the presence of the bacteria and their Nod factors. “Root hair swelling and deformation are the first indication that the root hair recognizes the bacterium,” Stougaard says.

Furthermore, the protein DMI2 is also needed for initiation of phosphate-providing arbuscular mycorrhizae, but researchers consider it unlikely that the fungal equivalents of rhizobial bacteria's Nod factors, which have yet to be identified, interact with the same receptors on plant roots. The fungi trigger different responses in the roots; they don't cause root hair changes, for example. And unlike rhizobial bacteria, the fungi show little specificity for their plant targets.

Indeed, about a year later, Stougaard's group and also that of Ton Bisseling at Wageningen University in the Netherlands came up with better candidates for Nod factor receptors. Stougaard and his colleagues looked at Lotus mutants that didn't show the early root hair deformations. They found that mutations in either of two genes, which they called NFR1 and NFR5, could cause that defect.

Both genes encode receptor kinases, but even more intriguingly, the portions of the proteins that would be on the outside on the root hair cell membrane resemble protein segments that bind chemical structures similar to those of Nod factors. Genetic analysis also showed that these putative Nod factor receptors come into play before the SYMRK/DMI2 kinase, as would be expected if they are involved in the first step: recognizing specific rhizobia.

The Bisseling team cloned its Nod receptor candidates, called LYKs, from M. truncatula. Although the LYK structures are similar to those of the NFRs found in Lotus, the proteins may have somewhat different functions. Mutations in the LYK genes don't prevent root hair deformation but do prevent the formation of infection threads and thus entry of the bacteria into roots. Because Nod factors induce many changes in legume roots, nitrogen-fixation researchers have long debated whether they might have more than one receptor. These results suggest that they do, although additional confirmation is needed.

Oscillating message

Although the function of DMI2 remains unknown, the roles of DMI1 and DMI3 have become clearer with their recent cloning. Some clues already existed from the earlier work in which the genes were identified and also from studies of the physiological changes that take place in root hair cells when they are stimulated by Nod factors.

In the mid-1990s, Long and her colleagues found that within the first few minutes of stimulation by Nod factors, the root hair cell membranes become depolarized, allowing calcium ions to move into the cells. Then about 10 minutes later, a wave of calcium “spiking” occurs, in which the calcium ion concentrations inside the cell rapidly oscillate up and down.

In other systems, calcium spiking has been linked to changes in gene expression. That may be true here as well, because Nod factors activate the expression of many plant genes needed for nodule development. But whatever its role in the plant, calcium spiking has also proved helpful to researchers, serving as a kind of landmark to help them deduce the relative order of action of the genes they've identified.

The earlier studies showed, Dénarié says, that “DMI1 and −2 are absolutely required for calcium spiking.” Thus, their protein products must come into play before spiking. In contrast, DMI3 mutations do not affect spiking, so its product works downstream of the calcium oscillations.

The structure of the DMI1 gene from M. truncatula, which was described in the 27 February issue of Science (p. 1364) by a large research team including Long, Dénarié, and Cook, suggests that it encodes a channel that allows positively charged ions to move into or out of cells. If so, then it might be involved in the movements of potassium or calcium ions that occur in root cells in response to Nod factor stimulation.

In the same Science issue (p. 1361), Dénarié's team, this time in collaboration with Bisseling and his colleagues, described the cloning and structure of DMI3. This gene closely resembles genes encoding a group of kinases known to be regulated by calcium ions in conjunction with another protein called calmodulin. “It appears that DMI3 can interpret the calcium signal,” says Cook. How it does that remains to be established, but functional DMI3 is apparently needed for the gene activity that occurs in response to Nod factor stimulation. Although calcium spiking occurs in DMI3 mutants, it doesn't induce a change in gene activation.

Fungal friend.

A fungus invades a M. truncatula root (left; the gold globe is a spore). Once inside the root cells, the fungus forms branched structures called arbuscules (right) that help the plant acquire phosphate.

CREDIT: M. J. HARRISON, G. R. DEWBRE, J. LIU, PLANT CELL 14, 2413 (2002)

End of discussion

In addition to getting a better understanding of how nodulation is turned on in legumes, researchers are beginning to learn how it can be turned off. The plants have at least two ways of limiting nodulation. This may be necessary to protect against the severe energy drain that would be imposed by having too many nitrogen-fixing nodules.

About 7 years ago, Cook and R. Varma Penmetsa, who were then at Texas A&M University in College Station, discovered a M. truncatula mutant that develops 10 times the normal number of nodules when inoculated with S. meliloti. Further analysis showed that the mutant plants are insensitive to ethylene, a plant hormone that promotes growth and other plant activities. “The plant is exquisitely sensitive to Nod factor in the absence of ethylene,” Cook says.

The hormone apparently exerts its suppressive effects within the signaling pathway in which the DMI genes operate. Long's group found that ethylene inhibits early Nod factor responses, including calcium spiking.

About 2 years ago, two teams, one led by Stougaard and the other by Masayoshi Kawaguchi of Niigata University in Japan, cloned a gene from Lotus and pea that is also involved in regulating nodule numbers. Although mutations in the gene, which they called HAR1 (for Hypernodulation Aberrant Root 1), also result in a marked increase in nodulation, it's unrelated to the mutant gene conferring ethylene insensitivity. Instead, HAR1's structure showed that it is the Lotus and pea equivalent of a gene called Clavata1, which regulates growth in the model plant Arabidopsis thaliana. Shortly thereafter, Peter Gresshoff's team at the University of Queensland, Australia, identified the soybean equivalent, and Cook and his colleagues came up with the corresponding gene in M. truncatula. “The [regulatory] pathway is highly conserved across the legumes,” Cook says.

Despite the recent progress, researchers have a long way to go to get a complete picture of the genetic interplay involved in rhizobial symbiosis. “We have only a small fraction of the components identified by mutants,” says Long. “But we're getting there.” The genomes of M. truncatula and L. japonicus are expected to be sequenced by 2006 and should be a big help to the effort, just as the Arabidopsis sequence is for researchers studying that plant. Unfortunately for symbiosis studies, Arabidopsis is not a legume, and it's one of the 20% of land plants that do not form arbuscular mycorrhizae.

Studies of these fungal symbioses may soon get a boost as well, as sequencing of one of the fungal partners is under way. In addition, the rhizobial work has surged ahead, partly because researchers were able to identify the chemical signals that legumes and rhizobial bacteria use to communicate. That work provided simpler study systems by making it possible to use Nod factors, rather than the bacteria themselves, to tweak the plants. Those signals haven't yet been identified for mycorrhizal associations, but results reported last month in Plant Physiology by Dénarié, Guillaume Bécard of the University of Toulouse, and colleagues may point to a solution.

They showed that a variety of arbuscular mycorrhiza fungi produce small, diffusible factors that trigger activity of one of the same genes activated by Nod factors in the outer cell layer of M. truncatula roots. This suggests that the fungal factors may play an analogous role, and Dénarié and his colleagues are now trying to purify them. If they do come up with the fungal equivalent of Nod factors, it should help provide a clearer picture of the molecular underpinnings of the fungal associations and how they compare to those involving rhizobia bacteria. Detangling either system could be a boon to the world's breadbaskets.

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