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Hidden gene clusters in soil bacteria may hold key to bigger legumes

In a new study, scientists used nearly every tool in their toolkit—genomics, transcriptomics, greenhouse experiments and advanced statistical methods—to gain new insight into the complex chemical interactions that take place in underground root nodules, where legumes like soybeans exchange vital nutrients with soil microbes called rhizobia.

Reported in the Proceedings of the National Academy of Sciences, their study that appear to move rapidly through bacterial populations and drive greater plant biomass in the host plants. Understanding the interplay of host and bacterial genomes will help efforts to optimize plant growth by improving the rhizosphere, the researchers said.

"Just like us, plants are full of microbes, and some form these tightly co-evolved symbioses where a lot of evolutionary history has shaped a very intimate interaction," said Katy Heath, a professor of plant biology at the University of Illinois Urbana-Champaign who led the study with Illinois plant biology professor Amy Marshall-Colón.

"Legumes like soybeans, peas or peanuts develop these special relationships with rhizobia."

Rhizobial bacteria "fix" nitrogen from the atmosphere by converting it into a form the plants can use, Heath said. In exchange, the legumes give the rhizobia carbon-rich sugars, "which is what plants make when they do photosynthesis."

Rather than exploring the role of one or two genes at a time, Heath and her colleagues wanted to get a more global sense of the variation in these exchanges.

They turned to a model system for studying such interactions, pairing the legume Medicago truncatula, a close relative of alfalfa that looks like clover, with the rhizobial bacterium Sinorhizobium meliloti.

In a greenhouse experiment, the team inoculated each M. truncatula plant with one of 20 strains of the rhizobium. The S. meliloti strains differ from one another genetically but still belong to the same species. Some of the strains consistently resulted in greater plant growth, Heath said. Once the plants and microbes formed root nodules, the site of exchange, the researchers plucked off the nodules and froze them for further analysis.

The team analyzed the "transcriptome" of each nodule. Transcriptomes contain all the RNA produced by an organism—or in this case, two organisms—offering a clear picture of every gene that is being expressed.

Once the researchers determined which plant and bacterial genes were being expressed at higher levels in nodules associated with the most vigorous plant growth, they sequenced high-quality reference genomes of each bacterial strain.

Interpreting the data was a formidable task, Heath said.

"Bacteria have genetic processes that are different from ours," she said. "We think a lot in classic genetics about that vertical line of inheritance from parents to offspring—and they do that, too. But then they are also swapping genes horizontally when they bump into other bacteria—within the same species or between different species. The complexity of horizontal gene transfer is massive."

S. meliloti have two sources of DNA: a large primary chromosome, which is inherited from a "parent" bacterium when it divides; and two giant plasmids, each containing roughly half as many genes as the chromosome.

Plasmids are circular chunks of DNA that are more mobile than chromosomal DNA and are the site of horizontal gene transfer, allowing bacteria to acquire new genes from their neighbors. Horizontal gene transfer even allows bacteria to pick up the genes required for them to become rhizobia, Heath said.

Marshall-Colón and postdoctoral researcher Rizwan Riaz conducted detailed statistical analyses and gene network modeling to identify which rhizobial genes correlated with more robust plant growth. The reference genomes were useful for understanding which genes were present and where they were located in the chromosomal or plasmid DNA. This resulted in the discovery that many of the genes of interest were clustered together in plasmids.

Further experiments, led by North Dakota State microbiological sciences professor and study co-author Barney Geddes, involved deleting the specified genes. U. of I. microbiology graduate student Ivan Sosa Marquez tested the effects of these deletions on plant growth, confirming that the identified genes were important for enhanced plant growth.

"We're not trying to say these are the important genes in all rhizobia in all the legumes," Heath said. "But we're gaining an understanding of the level of variation on which natural selection acts."

The study offers a broad picture of one set of S. meliloti genes, "which only some strains have and which appear to boost the growth of one legume species. The genes themselves are less universally applicable than the approach we've developed, which will likely be applicable to many other fields," Heath said.

"These aspects of microbial genetics that we're tapping into are the ones that matter for agricultural productivity, for livestock growth and for human health," she said.

"It's these genes that are moving around and we don't know why. And they're working with the rest of the genome in really complicated ways."

Heath and Marshall-Colón are affiliates of the Carl R. Woese Institute for Genomic Biology at the U. of I.