Abstract Background Plants are naturally associated with root microbiota, which are microbial communities influential to host fitness. Thus, it is important to understand how plants control root microbiota. Epigenetic factors regulate the readouts of genetic information and consequently many essential biological processes. However, it has been elusive whether RNA-directed DNA methylation (RdDM) affects root microbiota assembly. Results By applying 16S rRNA gene sequencing, we investigated root microbiota of Arabidopsis mutants defective in the canonical RdDM pathway, including dcl234 that harbors triple mutation in the Dicer-like proteins DCL3, DCL2, and DCL4, which produce small RNAs for RdDM. Alpha diversity analysis showed reductions in microbe richness from the soil to roots, reflecting the selectivity of plants on root-associated bacteria. The dcl234 triple mutation significantly decreases the levels of Aeromonadaceae and Pseudomonadaceae , while it increases the abundance of many other bacteria families in the root microbiota. However, mutants of the other examined key players in the canonical RdDM pathway showed similar microbiota as Col-0, indicating that the DCL proteins affect root microbiota in an RdDM-independent manner. Subsequently gene analysis by shotgun sequencing of root microbiome indicated a selective pressure on microbial resistance to plant defense in the dcl234 mutant. Consistent with the altered plant-microbe interactions, dcl234 displayed altered characters, including the mRNA and sRNA transcriptomes that jointly highlighted altered cell wall organization and up-regulated defense, the decreased cellulose and callose deposition in root xylem, and the restructured profile of root exudates that supported the alterations in gene expression and cell wall modifications. Conclusion Our findings demonstrate an important role of the DCL proteins in influencing root microbiota through integrated regulation of plant defense, cell wall compositions, and root exudates. Our results also demonstrate that the canonical RdDM is dispensable for Arabidopsis root microbiota. These findings not only establish a connection between root microbiota and plant epigenetic factors but also highlight the complexity of plant regulation of root microbiota.
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Evaluation of Protein Extraction Methods for Metaproteomic Analyses of Root-Associated Microbes
Metaproteomics is a powerful tool for the characterization of metabolism, physiology, and functional interactions in microbial communities, including plant-associated microbiota. However, the metaproteomic methods that have been used to study plant-associated microbiota are very laborious and require large amounts of plant tissue, hindering wider application of these methods. We optimized and evaluated different protein extraction methods for metaproteomics of plant-associated microbiota in two different plant species ( Arabidopsis and maize). Our main goal was to identify a method that would work with low amounts of input material (40 to 70 mg) and that would maximize the number of identified microbial proteins. We tested eight protocols, each comprising a different combination of physical lysis method, extraction buffer, and cell-enrichment method on roots from plants grown with synthetic microbial communities. We assessed the performance of the extraction protocols by liquid chromatography-tandem mass spectrometry–based metaproteomics and found that the optimal extraction method differed between the two species. For Arabidopsis roots, protein extraction by beating whole roots with small beads provided the greatest number of identified microbial proteins and improved the identification of proteins from gram-positive bacteria. For maize, vortexing root pieces in the presence of large glass beads yielded the greatest number of microbial proteins identified. Based on these data, we recommend the use of these two methods for metaproteomics with Arabidopsis and maize. Furthermore, detailed descriptions of the eight tested protocols will enable future optimization of protein extraction for metaproteomics in other dicot and monocot plants. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .
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- Award ID(s):
- 2033621
- NSF-PAR ID:
- 10396296
- Date Published:
- Journal Name:
- Molecular Plant-Microbe Interactions®
- Volume:
- 35
- Issue:
- 11
- ISSN:
- 0894-0282
- Page Range / eLocation ID:
- 977 to 988
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1094/MPMI-05-22-0116-TA
