Identifying Genes Aiding Rhizobium Infection in Lotus Japonicus via Single-Cell Analysis

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Summary

In their study, researchers investigated the selective process of rhizobium colonization in legumes during symbiotic nitrogen fixation. They discovered that only specific root hair cells allow the formation of infection threads, leading to the creation of functional nodules in certain cortical layers. Utilizing single-cell analysis, they identified specific cell populations in the epidermal and cortical layers that are key to rhizobium infection. The study highlighted particular nodulation genes, notably SYMRKL1, which is crucial for normal infection thread development and shares similarities with SYMRK. This research unraveled the complex cellular interactions and gene expressions involved in symbiosis, previously blurred by inadequate cellular resolution, thus enriching our understanding of these biological interactions.

Research Criteria

The authors identify genes that are specifically expressed in plant root cells that react to infection by nitrogen-fixing rhizobia using single-cell analysis. They demonstrate that SYMRKL1, one of these genes, is necessary for the typical course of infection.

Sample Type

The samples used for the single-cell RNA sequencing experiments were wild-type seedlings of Lotus japonicus (Lotus), both untreated and treated with rhizobia (M. loti), at different post-inoculation time points.

Result—Single Cell Transcriptomic Analysis of Lotus Roots Infected with Rhizobium

In this study, over 25,000 cells from rhizobium-inoculated and control Lotus roots were analyzed using single-cell RNA sequencing. This revealed about 2,859.5 unique molecular identifiers and 1,500 transcripts per cell. The research employed Seurat44 for clustering, identifying 32 clusters through marker gene information. All Lotus root tissues were present in both sample types, indicating effective protoplasting. The study observed notable transcriptional changes in most tissues in response to rhizobium, with some like phloem and xylem less affected. Additionally, 655 genes were identified as influenced by protoplasting. This research offers new insights into the cellular response of Lotus roots to rhizobial infection, enhancing our understanding of plant-microbe interactions.

Fig.1 A cellular atlas of infection caused by Rhizobium in Lotus root¹.

Result—Recognizing Bacteroid-Containing and Nodule-Containing Infected Cells

Researchers have identified specific cell types in Lotus roots that respond to rhizobial infection, focusing on three distinct groups: infected cells harboring rhizobia, nodule cells integral to the nodule structure but not infected, and bacteroid cells where nitrogen fixation occurs. By reanalyzing rhizobium-inoculated samples, they pinpointed infected cells in both root hairs and cortex, using the expression of the nodulation gene NPL24 and other infection-related genes as markers. Nodule cells were identified by their specific expression of the CARBONIC ANHYDRASE47 gene, and bacteroid cells were distinguished through leghemoglobin genes. This intricate mapping sheds light on the cellular dynamics within Lotus roots during rhizobial infection, revealing the sophisticated interactions between plant cells and symbiotic bacteria.

Fig.2 10 days after inoculation, infected, nodule, and bacteroid-containing cells in Lotus roots¹.

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Reference

1. Frank, Manuel, et al. "Single-cell analysis identifies genes facilitating rhizobium infection in Lotus japonicus." Nature Communications 14.1 (2023): 7171.