Legume-rhizobia interactions in a complex microbiome

Ming-Dao Chia, Anna Simonsen, Andrew Almonte, Justin Borevitz

Why?

Rhizobia (nitrogen-fixing bacterial plant symbionts) are a vital source of bioavailable nitrogen, particularly in poor soils. Nutrients from legumes (rhizobia host plants) are important for agriculture and ecological regeneration.
Many rhizobia species are well studied in single strain inoculation experiments, in culture, and in relation to their plant hosts.
Less is known about their ecology in a complex microbiome, with closely related strains and uncultivated microbes.
Recent developments in metagenomic methods allows studying abundance of rhizobia strains in nodules and free living environments with minimal culture or amplification bias.

Observing patterns of rhizobia strain abundance changes in legume nodules and associated soil.
Observing patterns of rhizobia strain abundance changes in soil in the absence of a legume host.
Identification of horizontal gene transfer events in known strains throughout the samples sequenced.

Study species: legume Acacia acuminata with rhizobial symbiont Bradyrhizobium japonicum (4 strains).
Plants are grown from surface sterilized seeds on autoclaved sand media for 8 weeks inside a growth chamber.
A complex microbiome is inoculated from an external soil sample. Strains are inoculated at once as a balanced culture mix.
DNA is extracted from sand and pots with a soil extraction kit.
Long read sequencing (8-100 kb reads) is conducted with Oxford Nanopore MinION, allowing for strain-level identification and potential for observing horizontal gene transfer in single reads.
Abundances of strains are measured through counting reads.
Potential horizontal gene transfer events are identified with bioinformatics.

Nanopore sequencing consistently yields some multigene metagenomic reads (>50 kb) with our samples.
Combining long and short reads yields single contig reference genomes.
Direct gDNA sequencing of sand and nodules produces reads that can be mapped to references.

Link specific rhizobia genes or variants to abundance patterns
Find potential co-inoculants to maintain stable free living rhizobia populations
Create assays to identify stable inoculants for a variety of hosts and soil conditions
Observe distribution between and within nodules of the same plant
Correlate host genomic variance with abundance patterns