NGS and root endophyte assembly cues

Cologne and Sanssouci, close to Golm: what effect do the soils at these two historic locations have on the local plant roots?

Highlighted article: Davide Bulgarelli, Matthias Rott, Klaus Schlaeppi, Emiel Ver Loren van Themaat, Nahal Ahmadinejad, Federica Assenza, Philipp Rauf,  Bruno Huettel, Richard Reinhardt, Elmon Schmelzer, Joerg Peplies, Frank Oliver Gloeckner, Rudolf Amann, Thilo Eickhorst, and Paul Schulze-Lefert (2012) Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota Nature 488:91

Background

Although plant-microbe and plant-soil dynamics are widely studied areas of plant science, up until now there has been no broad picture of plant endophytic systems: which phyla are common endophytes; how the populations form; and what affects them. Endophytes colonise plant tissues, where unlike pathogens they do not cause harm or an immune response, and unlike endosymbionts they do not live inside plant cells or have an obvious mutually beneficial relationship with the plant. A recent review on bacterial endophytes is this one by Reinhold-Hurek and Hurek (2011).

Here, Bulgarelli et al. use an Arabidopsis system to shed light on the specifics of below ground plant-bacteria interactions, and set out a methodology for future investigations into other plants and soil types. This study and another article in the same issue of Nature by Lundberg et al. use next generation sequencing (NGS) to show similar cues for assembly of root endophytes.

These are two of many cases where next generation sequencing (NGS) has been used to great effect. GARNet is running a free one day Tools and Technologies to Advance Plant Research workshop in November – find out more here.

Method

As one of the objectives in this experiment was to assess influencers of entophytic microbiota in roots, the starting materials were Arabidopsis ecotypes Shakdara and Landsberg and soils from German cities Potsdam (Golm locality) and Cologne. Cologne soil was rich in clay and silt, while soil from Golm was sand-rich.

The plants were grown in a controlled environment and at a set timepoint, samples were taken from soil, the rhizosphere, and root. The latter two samples were taken by washing, sonicating and centrifuging roots, separating the material into ‘rhizosphere’, soil and bacteria from immediately next to the roots, and the ‘root’.

Bulgarelli et al. extracted DNA from the samples and amplified 400 base pair segments of the bacterial 16S ribosomal RNA gene. The amplicons were sequenced and the team used several databases to identify phyla. This method was not suitable to classify many sequences, so the team clustered the sequences of all compartments and defined operational taxonomic units (OTUs). OTU richness was used as a measure of bacterial diversity.

Results

Bacterial diversity was highest in unplanted soil. The rhizosphere and root compartment showed roughly the same reduced level of diversity, but their populations were significantly different from one another. There was significant difference too between soil and rhizosphere microbiota from different soil types, and from the same experimental set up in different seasons.

Bulgarelli et al. identified three main root-inhabiting phyla in all root samples which were comparatively under-represented in the soil and rhizosphere fractions: Proteobacteria, Actinobacteria, and Bacteroidetes. Although there was diversity within the phyla, each one was represented strongly by a dominant family.

To shed light on what causes and limits the populations of endophytic microbiota, Burgarelli et al. put wooden splinters in the soil alongside the growing Arabidopsis plants. They found that a that a subset of root endophytes also colonized the wooden splinters, suggesting that for these microbiota the presence of plant cell walls is enough for them to colonize biomass. Some endophytes were only present in root tissue, indicating that metabolic activity from living cells induces their growth inside roots. Actinobacteria, a phyla in which antimicrobial compound production is common, dominated this group, perhaps representing a benefit for the host plant.

Interestingly there was a third subset found on the wood splinters in greater abundance than in soil that was not identifiable in root tissue. This third subset points toward a preventative mechanism, separate from the immune response which was not triggered, in roots which stops colonization by certain bacteria.

Bulgarelli et al. used the same analysis on 8 Arabidopsis ecotypes growing naturally in Cologne. The OTU richness of all three compartments was similar to that in the controlled environment, and a large proportion of OTUs were the same. The method used here is a reliable method to assess endophyte and rhizosphere microbiota diversity in natural environments as well as in controlled conditions.

Teaching resources

This new research could be given a low-tech makeover in your classroom by growing bacteria from different soil samples on growth medium. You might need to test the experiment a few times to see which soil or plant samples give visibly different colonies. If you don’t have the facilities to make lab-grade agar plates, here are two websites with instructions for homemade agar plates: Kitchen Pantry Scientist, Mad About Science.

Use different sweets to teach students what classification is and help them think about how microorganisms are classified with this suggestion from e! Science News.

Image credits: Cologne Skyline by Thomas Römer (thoroe) and Sanssouci by Marc Slingerland (vormin) via stock.xchng



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