A Medicago truncatula Autoregulation of Nodulation Mutant Transcriptome Analysis Reveals Disruption of the SUNN Pathway Causes Constitutive Expression Changes in Some Genes, but Overall Response to Rhizobia Resembles Wild-Type, Including Induction of TML1 and TML2

Abstract

Nodule number regulation in legumes is controlled by a feedback loop that integrates nutrient and rhizobia symbiont status signals to regulate nodule development. Signals from the roots are perceived by shoot receptors, including a CLV1-like receptor-like kinase known as SUNN in Medicago truncatula. In the absence of functional SUNN, the autoregulation feedback loop is disrupted, resulting in hypernodulation. To elucidate early autoregulation mechanisms disrupted in SUNN mutants, we searched for genes with altered expression in the loss-of-function sunn-4 mutant and included the rdn1-2 autoregulation mutant for comparison. We identified constitutively altered expression of small groups of genes in sunn-4 roots and in sunn-4 shoots. All genes with verified roles in nodulation that were induced in wild-type roots during the establishment of nodules were also induced in sunn-4, including autoregulation genes TML2 and TML1. Only an isoflavone-7-O-methyltransferase gene was induced in response to rhizobia in wild-type roots but not induced in sunn-4. In shoot tissues of wild-type, eight rhizobia-responsive genes were identified, including a MYB family transcription factor gene that remained at a baseline level in sunn-4; three genes were induced by rhizobia in shoots of sunn-4 but not wild-type. We cataloged the temporal induction profiles of many small secreted peptide (MtSSP) genes in nodulating root tissues, encompassing members of twenty-four peptide families, including the CLE and IRON MAN families. The discovery that expression of TML2 in roots, a key factor in inhibiting nodulation in response to autoregulation signals, is also triggered in sunn-4 in the section of roots analyzed, suggests that the mechanism of TML regulation of nodulation in M. truncatula may be more complex than published models.

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.…”

“…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.…”

“…Scale bar = 2 cm. (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.…”

“…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).…”