Getting to the Root of the Plant Microbiota

Upon first glance, plant roots seem distinct from animal guts. However, like the intestines, roots are responsible for nutrient acquisition, they interact with robust microbial communities, and both are split into segments with different functions.^1-3 As roots wind their way through the soil, they create an underground network responsible for anchoring a plant’s above-ground portion, absorbing water, and communicating with the environment. Different root segments, including the differentiation zone, elongation zone, and root tip, are heterogeneous in their architecture, gene expression, immune state, and metabolic profiles, all of which may influence the root microbiota.

Exploring another potential similarity, plant microbiologist Eliza Loo, a group leader under Wolf Frommer at Heinrich Heine University Düsseldorf, studied the spatial colonization of the plant microbiota. In animals, members of the gut microbiota are spatially distributed depending on aspects such as nutrient and chemical gradients.^4,5 In a study recently published in Cell Host & Microbe, Loo and her colleagues discovered that the spatial distribution of a root’s excreted metabolites similarly pattern the microbial community along its length.⁶ These findings shed light on host-microbe interactions and could support plant health initiatives by highlighting how to modulate the microbiota, which has implications for plant immune responses, stress tolerance, and more.

“Extrapolating from the medical field, we know that the microbiome influences almost everything that we do, from the way we think, from what kind of food we eat, from our own responses towards a certain illness,” said Loo. “We show as well that the plant root microbiome is not very different from the human microbiome.”

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To understand root-microbe interactions in detail, the researchers grew Arabidopsis thaliana and performed genomic, transcriptomic, and metabolomic analyses on root segments as small as two centimeters. Loo initially struggled to gently extract the roots from the soil without disturbing the microbes she hoped to identify. “Imagine if you pull a root, you're dislocating the microbiota from the top to the bottom,” Loo said. “We wanted something that opens up really nicely without disturbing the root.” The researchers developed a novel yet simple solution—a growth system within compact disc (CD) cases, which allowed the roots to grow in a single layer enclosed in the thin plastic containers. Once ready to extract, the scientists opened up the cases to access the content within, as if they were about to slip their favorite band’s album into their CD player.

The CD growth system did not completely solve the researchers’ difficulties working with A. thaliana. “The thickness of the root is probably as thick as one strand of my hair,” Loo said. “To isolate a perfect root that is so thin, and to cut that into little pieces and collect enough DNA for microbiota profiling, at the same time making sure that … there's no dislocation of the microbiota, that was extremely difficult.” Yet, the researchers persevered and sequenced the bacteria belonging to each root segment and found that the microbial community was in fact spatially organized along each root’s longitudinal axis.

Next, the researchers tested the roots’ metabolome to see whether the microbiota’s spatial distribution correlated with excreted root metabolites. The various root regions are known to release different compounds, and scientists have postulated that adding sugar into the soil helps plants recruit beneficial microorganisms.^7,8 To better observe root growth and improve sectioning prior to performing metabolomic studies, the researchers needed to use an agar-based plant growth medium. However, to accurately assess metabolites released from the plants, they had to develop a new artificial soil that lacked sugar, which is a common supplement in plant growth media. Using root segments grown in their newly-coined “ArtSoil,” the team performed untargeted gas chromatography-mass spectrometry and found distinct clusters of metabolites, primarily carbohydrates and organic acids, associating with specific root segments in a longitudinal spatial distribution along the root.

Most plants have a number of sugar transporters that distribute carbohydrates.⁹ To determine if they had a role in metabolite spatial organization, Loo analyzed publicly-available plant gene expression data sets and found that certain SWEET (sugars will eventually be transported) sugar transporter genes had specific spatial distribution patterns along the A. thaliana root.¹⁰ Back in her laboratory, Loo found that in plants grown in a sterile medium, SWEET transporters ended up in different places along the root compared to those in plants grown in bacteria-containing ArtSoil. Loo’s team also sequenced the bacterial microbiota within the plants’ internal tissues and found that various A. thaliana harboring SWEET mutations had different community organization compared to wild-type plants. Metabolite abundance was also affected in the mutants.

“This was not clearly shown until now … this micro-niche concept, where we have different exudates … and, of course, different microorganisms that are associated with these different younger and older parts of the roots,” said Alga Zuccaro, a plant-microbe interactions researcher at the University of Cologne, who was not involved in this study. “The biggest strength is that the paper really shows that there is a spatially separated sugar transport event … and this correlates with the presence and absence of different microbiota in these different areas of the roots.”

From now on, we will have to really think about the roots as an organ that has different areas that are colonized in a different way.
– Alga Zuccaro, University of Cologne

The exact mechanism driving the spatial relationship of the microbiota and SWEET transporters is not yet clear. For future experiments, Loo hopes to fine-tune the root protocol—a need that Zuccaro also highlighted. “The next step would be to go for even smaller fractions … and look at single cells,” said Zuccaro. This would allow the researchers to determine the transporters’ spatial distributions not only longitudinally but from the external epidermis through the internal endodermis.

This study is a first step toward gathering information useful for developing new strategies to affect plant health through modulating the microbiota and metabolite excretion. Until then, one immediate outcome of Loo’s work is its influence on how plant-microbe interaction research is conducted. “We have to pay attention to which media we use and the presence and absence of specific nutrients, especially sugars,” said Zuccaro. “From now on, we will have to really think about the roots as an organ that has different areas that are colonized in a different way.”

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