Abstract
Crop health and yield is linked to microbial activity in the soil, but the influence of the microbial community and its effects on plant growth is still poorly understood. We here present an investigation of the direct interaction between wheat (Tricitum aestivus) and its resident microbiome, by means of stable isotope labelling, with the intent to obtain the soil metagenome, metaproteome and metabolome. This will identify root exudates and key microbes involved in the transformation of these into secondary metabolites, and elucidate interactions between plant and soil microbes.
Wheat plants were grown in controlled environments with 13CO2 (5% and 100%) enriched atmospheres. 13C incorporated into plant constituents, and in time was expelled by the wheat roots as 13C-labelled root exudates. These exudates were then metabolized and incorporated by rhizosphere microbes, labelling molecules from plant tissue to secondary soil metabolites.
Soil was sampled after 21 days, flash frozen with liquid nitrogen and lyophilized. These samples were split into batches for nucleic acid, peptide and metabolite extraction.
DNA and RNA was co-extracted using a NucleoBond Soil RNA Mini kit (Macherey-Nagel), and metagenomes of the soil samples were sequenced on a MinIon (ONT) using a ligation sequencing kit. The sequences were basecalled with Guppy, assembled with Flye-meta and annotated with Prokka, then analysed using Geneious and R. CD-HIT was then used to identify the number of unique peptide fragments in the translated metagenome.
Peptides were extracted using an optimised TCA-FASP protocol and then captured using an HPLC-MS orbitrap system. A custom workflow using the newly generated metagenome as a reference was applied using Proteome Discoverer (Thermo) and the peptides were identified and mapped to the metagenome. CALIS-P will be used to identify labelled peptides.
Metabolites were extracted using an optimized protocol, and captured using HLPC-MS/MS. Compound Discoverer (Thermo) will be used to identify labelled metabolites and other metabolites of interest.
Combining the metagenome, proteome and metabolome will thus provide an accurate overview of the wheat-microbe-soil interactome, through the tracking of labelled compounds from root exudate origins to microbial proteins and biotransformation products. The goal is identification of proteins and metabolites containing the label, and by extension, any loci and genetic pathways associated with these. This will provide a novel and robust overview of key microbial species related to root exudate biotransformation in the wheat rhizosphere. This knowledge allows strategic manipulation of plant-associated microbial communities to support high plant productivity.
Wheat plants were grown in controlled environments with 13CO2 (5% and 100%) enriched atmospheres. 13C incorporated into plant constituents, and in time was expelled by the wheat roots as 13C-labelled root exudates. These exudates were then metabolized and incorporated by rhizosphere microbes, labelling molecules from plant tissue to secondary soil metabolites.
Soil was sampled after 21 days, flash frozen with liquid nitrogen and lyophilized. These samples were split into batches for nucleic acid, peptide and metabolite extraction.
DNA and RNA was co-extracted using a NucleoBond Soil RNA Mini kit (Macherey-Nagel), and metagenomes of the soil samples were sequenced on a MinIon (ONT) using a ligation sequencing kit. The sequences were basecalled with Guppy, assembled with Flye-meta and annotated with Prokka, then analysed using Geneious and R. CD-HIT was then used to identify the number of unique peptide fragments in the translated metagenome.
Peptides were extracted using an optimised TCA-FASP protocol and then captured using an HPLC-MS orbitrap system. A custom workflow using the newly generated metagenome as a reference was applied using Proteome Discoverer (Thermo) and the peptides were identified and mapped to the metagenome. CALIS-P will be used to identify labelled peptides.
Metabolites were extracted using an optimized protocol, and captured using HLPC-MS/MS. Compound Discoverer (Thermo) will be used to identify labelled metabolites and other metabolites of interest.
Combining the metagenome, proteome and metabolome will thus provide an accurate overview of the wheat-microbe-soil interactome, through the tracking of labelled compounds from root exudate origins to microbial proteins and biotransformation products. The goal is identification of proteins and metabolites containing the label, and by extension, any loci and genetic pathways associated with these. This will provide a novel and robust overview of key microbial species related to root exudate biotransformation in the wheat rhizosphere. This knowledge allows strategic manipulation of plant-associated microbial communities to support high plant productivity.
| Originalsprog | Engelsk |
|---|---|
| Publikationsdato | 11 nov. 2021 |
| Status | Udgivet - 11 nov. 2021 |