Transcriptome analysis ofMedicago truncatulaAutoregulation of Nodulation mutants reveals that disruption of the SUNN pathway causes constitutive expression changes in a small group of genes, but the overall response to rhizobia resembles wild type, including induction ofTML1andTML2

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 the annual medicMedicago 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-functionsunn-4mutant and included therdn1-2autoregulation mutant for comparison. We identified constitutively altered expression of small groups of genes insunn-4roots, including higher levels of transcription factorNF-YA2, and insunn-4shoots. All genes with verified roles in nodulation that were induced in wild type roots during the establishment of nodules were also induced insunn-4, including, surprisingly, autoregulation genesTML2andTML1. Among all genes with a differential response to rhizobia in wild type roots, only an isoflavone-7-O-methyltransferase gene (Medtr7g014510) was found to be unresponsive insunn-4. In shoot tissues of wild type, eight rhizobia-responsive genes were identified, including a MYB family transcription factor gene (Medtr3111880) which remained at a baseline level insunn-4; three genes were found to be induced by rhizobia in shoots ofsunn-4but not wild type. We also 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 ofTMLgenes in roots, a key factor in inhibiting nodulation in response to autoregulation signals, is also triggered insunn-4in the section of roots analyzed suggests that the mechanism ofTMLregulation inM. truncatulamay be more complex than published models.

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

“…To capture signaling events in early nodule development, we developed a harvesting procedure described in (Schnabel et al, 2023). The procedure involved simultaneous inoculation of all plants in an aeroponic system (Cai, et al 2023) and harvest of the developing nodule zone tracked via root growth (summarized in Supplemental Figure 1).…”

“…The data from this experiment (Supplemental Data Set 1) as well as data from (Schnabel et al, 2023) in which an analysis of gene expression changes in root segments during the same time period, were deposited at ePlant and display options created in ePlant (https://bar.utoronto.ca/eplant_medicago/) to allow analysis of the 4,893 individual genes in our dataset. Figure 2C is a representative visualization created by loading the example gene Medtr8g043970 ( ERN1 ) into ePlant, selecting the tissue and experiment viewer (microscope icon), and then clicking on the Root Component view (root cross section) to generate the image.…”

“…Note the scale is calculated to a local maximum, indicated in red, which varies by the gene. D) Average expression, with error bars of the same genes in B in whole root segments under the same conditions (from data in Schnabel, et al 2023).…”

“…Figure 2C shows the expression of the same genes in 2B in whole root segments in inoculated and uninoculated wild type plants grown in the same system and protocol, as well as in the sunn-4 and rdn1-2 hypernodulation mutants. These graphs were generated from data in (Schnabel et al, 2023).…”

“…Because the LCM data set was generated from inoculated roots, we expected that in addition to genes involved in nodulation, genes involved in root development but not nodulation would also change expression over time. In order to focus on nodulation regulatory genes when examining temporal patterns, we used the data from (Schnabel et al, 2023) to identify genes showing an increase in expression in root segments responding to rhizobia in wild type and/or in the autoregulation of nodulation (AON) mutants sunn-1 and rdn1-2 when compared to uninoculated A17 controls; we termed this collection of genes “rhizobial response genes” (n=1932; Supplemental Data Set 2A). All but two of the genes identified as differentially expressed in wild type root segments responding to rhizobia were also identified as differentially expressed in AON mutants responding to rhizobia; thus the AON set appears to be an extension of the wild-type set (Supplemental Figure 2).…”