Laser Capture Microdissection Transcriptome Reveals Spatiotemporal Tissue Gene Expression Patterns of Medicago truncatula Roots Responding to Rhizobia

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.

TML1ANDTML2SYNERGISTICALLY REGULATE NODULATION BUT NOT ARBUSCULAR MYCORRHIZA INMEDICAGO TRUNCATULA

“…Following expression in undissected root segments over time (Figure 5B), both genes are induced by rhizobia in wild type (green line versus blue line) over 72 hours, as well as in the hypernodulation mutant sunn-4 (orange versus gray line). However, as reported in (Schnabel et al, 2023) TML1 expression does not begin to decrease over this time frame in the sunn-4 mutant, while TML2 does. Examined at the level of single cell RNASeq over a time course of combined experiments harvested at time points at 0, 48 and 96 hpi, a tissue heatmap of the combined transcriptomics data of individual cells shows both TML genes are expressed maximally in the vascular bundle cells and moderately in the pericycle cells, but only TML1 is expressed in cells identified as nodule and cortex (Figure 5C).…”

“…The current AON model is highly simplified, involving signal transduction in both space and time based on evidence gathered by multiple groups in multiple systems and species. What appear to be differences in MtTML expression results between our work and others can explained by differences in timing and set up between experiments – the decreased MtTML expression data in (Gautrat et al, 2020) was collected at 5 dpi in whole roots on plates versus the increase of MtTML1 in (Schnabel et al, 2023) at 3 dpi in nodulating segments of roots in an aeroponic chamber. Additionally, we do not know the half-life of the MtTML proteins, which would affect the length of time between detecting decreased mRNA and the action of the MtTML proteins in halting nodulation; split root analysis suggests the AON systemic signal has an effect on nodule number within 72 hpi in M. truncatula (Kassaw et al, 2015) but no decreased TML expression is detected during this time.…”

“…Using the ePlant resource for early nodulation (Schnabel et al, 2023) and past transcriptomics work in our lab (Schnabel et al, 2023) we examined the expression of the two MtTML genes in root segments during the first 72 hours responding to rhizobia (Figure 5). The genes are expressed in wild type plants at low levels at the 0 hr timepoint, which is 4-day old N starved plants in our experimental system, but expression rises in the vascular tissue for both genes at 12 and 24 hpi (Figure 5A).…”

“…(A) Diagrams produced from ePlant resource (Schnabel et al, 2023) with gradient display adjusted to identical color representation to allow comparison of the expression levels as well as localization of TML1 and TML2 across time in wild type roots responding to rhizobia up to 72 hpi. (B) Transcriptomics data re-graphed from (Schnabel et al, 2023) showing TML expression in whole root segments under the same conditions. The average FPKMs of three libraries are shown for five time points for uninoculated and inoculated (wild type A17 and sunn-4 ) root segments.…”

“…Both signals are transduced through miR2111 levels dropping and TML message levels rising, signaling competence to nodulate (Moreau et al, 2021; Laffont and Frugier, 2023). When competent plants begin to nodulate, TML levels continue to rise as nodulation begins along with CLE13/SUNN signaling (Schnabel et al, 2023) and fall as nodulation proceeds and miR2111 levels rise, a result of the CLE13/SUNN signal AON feedback.…”