A laser capture microdissection transcriptome ofM. truncatularoots responding to rhizobia reveals spatiotemporal tissue expression patterns of genes involved in nodule signaling and organogenesis

Abstract

We report a public resource for examining the spatiotemporal RNA expression of 54,893 M. truncatula genes during the first 72 hours of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of over 100 plants in a single media container, we harvested the same segment of each root responding to rhizobia in the initial inoculation over a time course, collected individual tissues from these segments with laser capture microdissection, and created and sequenced RNA libraries generated from these tissues. We demonstrate the utility of the resource by examining the expression patterns of a set of genes induced very early in nodule signaling, as well as two gene families (CLE peptides and nodule specific PLAT-domain proteins) and show that despite similar whole root expression patterns, there are tissue differences in expression between the genes. Using a rhizobial response data set generated from transcriptomics on intact root segments, we also examined differential temporal expression patterns and determined that, after nodule tissue, the epidermis and cortical cells contained the most temporally patterned genes. We circumscribed gene lists for each time and tissue examined and developed an expression pattern visualization tool. Finally, we explored transcriptomic differences between the inner cortical cells that become nodules and those that do not, confirming that the expression of ACC synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division.

The Single-Cell Transcriptome Program of Nodule Development Cellular Lineages inMedicago truncatula

“…In addition, we evaluated the expression of genes specific to each cluster (Moran's I test, q-value < 0.05) in a laser capture microdissection (LCM, Schnabel et al, 2023) dataset that investigated the response of M. truncatula to rhizobia infection at similar time points as the ones used in the present study (see Methods; Figures S2-S7). Based on the annotation approach described above, we identified nuclei derived from six types of root cells: the epidermal cells (clusters 1, 3, 14, and 22), root hair (clusters 12), cortex (clusters 2, 4, 9, 11, 13, and 16), endodermis (clusters 7 and 24), pericycle (cluster 8) and stele (excluding pericycle; clusters 10, 18, 23 and 26) (Figure 1B-C).…”