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Partnership Sprouts Up to Use RNAi to Uncover Genetics Behind Legume Symbioses

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Despite the global prevalence of legumes as crops, many questions about the underlying biology of the plants remain unanswered. Researchers from the University of Minnesota, Twin Cities, in collaboration with a scientist from the Boyce Thompson Institute for Plant Research at Cornell, are looking to answer one of those questions — which genes are involved in the development of bacterial and fungal symbioses in legumes — using RNA interference.

Using the model legume Medicago truncatula, a forage crop closely related to alfalfa, the project is seeking to identify the genes of the legume that allow these kinds of plants to form a symbiotic relationship with rhizobial bacteria.

“Legumes in general form symbiotic relationships with soil bactera that produce what are called nodules on the roots, and the bacteria on the nodules will fix nitrogen — [that is,] convert atmospheric nitrogen to ammonia,” Steve Gantt, associate professor of plant molecular biology at the University of Minnesota, Twin Cities, and leader of the project, told RNAi News. “The plant can then use the ammonia as a nitrogen source.”

According to Gantt, legumes fix so much nitrogen that they are frequently used in crop rotations. “A lot of times what people will do are things like corn/soy bean crop rotations — soy beans are another legume,” he said. “They’ll do it every other year and put nitrogen back in the soil … so it becomes available to next year’s crop.”

The RNAi project is also examining the genetics of the fungal symbiosis in legumes that is responsible for phosphorous acquisition, in collaboration with Maria Harrison from the Boyce Thompson.

“She is interested in the plant genes that are involved in forming another symbiotic relationship … with a fungus, mycorrhizal fungi,” Gantt said. “The primary benefit of that symbiosis to the plant seems to be the uptake of phosphorus from soil.”

In exchange for their help to legumes, Gantt noted, rhizobial bacteria and mycorrhizal fungi — which, incidentally, don’t form symbioses with the common model plant Arabidopsis thaliana — receive sugars and other nutrients from the plant.

To identify the genes that play a role in these symbioses, Gantt and his colleagues are using RNAi to knock down the expression of about 1,500 M. truncatula genes believed to be relevant based on sequence database analysis and gene expression studies. To pinpoint these genes, “we’re using the information that we’ve gained through our previous genome grants on Medicago truncatula,” he said.

This previous work includes a project that resulted in the collection of between 160,000 and 170,000 expressed sequence tags, from which many of the genes that are active in roots of the legume during symbiosis have been identified.

To achieve RNAi, Gantt said he and his colleagues insert a hairpin RNA for a particular gene into a plasmid known as Hell’s Gate, which was developed by Peter Waterhouse and Chris Helliwell, of the Commonwealth Scientific and Industrial Research Organization, using Invitrogen’s Gateway gene cloning and expression technology (Helliwell and Gateway, hence, Hell’s Gate).

“The cells recognizes the double-stranded RNA , which is about 400 base pairs long … and then Dicer goes in” and the RNAi process continues, he said.

The actual gene silencing occurs only in transgenic roots called hairy roots, Gantt said. “These are roots that are transformed — the RNAi-inducing gene is inserted into a cell, and that cell goes on and forms a root.” In these composite plants, the shoot is non-transgenic, but the root is, he explained.

In some of these plants the root, which has had a particular gene silenced via RNAi, is then infected with rhizobial bacteria and the researchers look for changes in nitrogen fixation or nodulation. In others, the root is infected with mycorrhizal fungi and phosphorous uptake is evaluated.

Thus far, the researchers have made about 150 RNAi constructs and are in the process of screening them, Gantt said. “But, we’re still tweaking, trying to figure out the best way to screen these things. How can we do the numbers that we want at the depth we want?”

Still, the researchers have already found a few genes that “were previously unknown to be involved in root development or nodule formation, but look like they are,” he said.

While the information gleaned from the legume symbioses project will undoubtedly help advance researchers’ understanding of the plant, Gantt foresees some very practical benefits, as well.

“The goal that people have talked about for a long time is to try to make nitrogen-fixing non-legume crop plants, so that you don’t have to apply nitrogen fertilizers to them,” he said, adding that these fertilizers are both expensive and toxic. “Some reasonably small amount of the nitrogen that you put on a field is actually used by the plant — a lot of it just runs off into streams and causes pollution in the oceans.”

This nitrogen pollution is such a problem, Gantt said, that it has been linked to an enormous hypoxic area — or dead zone — in the Gulf of Mexico. “It’s [fertilizer] runoff from, mostly, agriculture in the Midwest,” he said. “It goes down the Mississippi River, dumps out in the Gulf of Mexico.” The nitrogen in the fertilizer then stimulates the growth of algae that strips the water of oxygen, making the area hostile to most marine life, Gantt said. “It’s a big problem, and not just there [in the Gulf].”

Additionally, Gantt said that reserves of easily attainable phosphorus are rapidly being depleted. “In the next 40 or 50 years, we’ll have to get phosphorus for fertilizers from sources that are much more difficult to get at, so the cost of the phosphorous is going to shoot up,” he said.

Aside from generating genetic data about legumes, the project also has an educational component. According to the grant’s abstract, “the research team will introduce select undergraduate students to many aspects [of] the research.

“Additionally, collaborators at Gustavus Adolphus College, an undergraduate institution, will develop a laboratory course that follows the general protocols described in the research proposal,” it states. “The outcome of this effort will be the description of a series of experiences that undergraduate students in plant biology, plant molecular biology, or plant physiology can obtain in [a] one semester course.”

Supporting the legume symbioses project is a four-year National Science Foundation grant worth about $2 million. The grant is scheduled to begin Oct. 1, 2004 and run until Sept. 30, 2008. Results of the project are to be made publicly available on the Internet.

— DM

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