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Do legume hosts benefit from suppressing rhizobial reproduction?

This week's paper is by my PhD student Ryoko Oono, with major contributions from Imke Schmitt (University of Minnesota faculty) and Janet Sprent, who was an expert on legume-rhizobium evolution long before I started working on the problem.

"Multiple evolutionary origins of legume traits leading to extreme rhizobial differentiation" has been published on-line in New Phytologist.

Rhizobia are soil bacteria, but a lucky few accept invitations from legume plants to infect their roots, multiply a million-fold or more inside a nodule, and then convert ("fix") atmospheric nitrogen into a form that the plant can use. When the plant dies (or sometimes sooner), an unknown fraction of the rhizobia in each nodule escape back into the soil.

Below left is what rhizobia look like in the soil and in the nodules of some legume hosts, including soybean. In other hosts, including pea, they swell up and/or change their shape (below right, same scale) as they differentiate into the nitrogen-fixing bacteroid form. The swollen form is apparently nonreproductive (like worker bees), but copies of their genes can still end up back in the soil. This is because some of their clonemates in the same nodule haven't become bacteroids yet and so retain the ability to reproduce, like queen bees.
The extreme differentiation shown above right is imposed by the legume host. But why? Are swollen bacteroids somehow more beneficial to the plant? Or are bacteroid swelling and their losing the ability to reproduce side-effects of some other process that may or may not benefit the plant?

Ryoko reasoned that, if a plant trait has evolved repeatedly over the course of evolution, then it is probably beneficial to the plant. On the other hand, a trait that has been abandoned repeatedly is probably harmful. But has either of these happened?

To find out, she studied 40 different legume species (wild and domesticated) and determined whether bacteroids were swollen in each. She used light microscopy, as in the above images, and also flow cytometry. With the flow cytometer, she could analyze thousands of rhizobia per nodule, to see whether they came in two distinct size classes: swollen nitrogen-fixing bacteroids and nonswollen reproductives. Janet Sprent contributed electron micrographs for a number of less-widespread species that Ryoko couldn't find.

Then, Ryoko worked with Imke Schmidt on ancestral-state reconstruction. I last mentioned this approach in discussing the evolution of transfer-RNA. The figure below shows a subset of Ryoko's results. The most-recent common ancestor of these legumes didn't cause swollen bacteroids, as indicated by the filled circle at left. This was also true of the more-distant ancestor (not shown) that these legume species share with peanut, soybean, and various wild species. But, some time after Cicer (chickpea) branched off, a mutant that caused bacteroids to swell arose, and passed this trait to its descendants, including Medicago (alfalfa), Pisum (pea), and Vicia (vetch), as indicated by the open circles.
Looking at a more complete family tree (not shown), Ryoko concluded that the ability to make bacteroids swell has evolved at least five times. (The infinity signs indicate species with indeterminate nodule growth, whose correlation with bacteroid swelling is less consistent than I had thought.)

If host-imposed bacteroid swelling has evolved repeatedly, maybe it benefits the host. But how? This question may be more complicated than it seems, because a trait that provides a long-term benefit to the legume species (perhaps by making rhizobia evolve to be more beneficial) won't evolve unless it also benefits individual plants with the trait. We discussed these issues in a review article published last year. One possibility is that swollen rhizobia might somehow be more efficient, fixing more nitrogen relative to their carbon cost to the plant.

So Ryoko has been measuring the efficiency of nitrogen fixation, comparing the same strain of rhizobia in hosts where bacteroids are versus are not swollen, using a method developed by the late great John Witty, with whom I spent a brief but fun sabbatical twenty years ago. She's getting some very interesting results, which you can hear about if you go to her talk at the Evolution meetings in Portland, in June.

This material is based upon work supported by the National Science Foundation under Grant No. NSF/IOS-0918986.

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