Cell- and Tissue-Specific Transcriptome Analyses of Medicago truncatula Root Nodules

Abstract

Legumes have the unique ability to host nitrogen-fixing Rhizobium bacteria as symbiosomes inside root nodule cells. To get insight into this key process, which forms the heart of the endosymbiosis, we isolated specific cells/tissues at different stages of symbiosome formation from nodules of the model legume Medicago truncatula using laser-capture microdissection. Next, we determined their associated expression profiles using Affymetrix Medicago GeneChips. Cells were collected from the nodule infection zone divided into a distal (where symbiosome formation and division occur) and proximal region (where symbiosomes are mainly differentiating), as well as infected cells from the fixation zone containing mature nitrogen fixing symbiosomes. As non-infected cells/tissue we included nodule meristem cells and uninfected cells from the fixation zone. Here, we present a comprehensive gene expression map of an indeterminate Medicago nodule and selected genes that show specific enriched expression in the different cells or tissues. Validation of the obtained expression profiles, by comparison to published gene expression profiles and experimental verification, indicates that the data can be used as digital “in situ”. This digital “in situ” offers a genome-wide insight into genes specifically associated with subsequent stages of symbiosome and nodule cell development, and can serve to guide future functional studies.

Root developmental programs shape theMedicago truncatulanodule meristem

“…Nevertheless, comparison of the expression patterns indicates that AtPLT3 and Medtr5g031880 are expressed in the RM, whereas AtPLT7 and Medtr8g068510 are not [Galinha et al, 2007;Prasad et al, 2011; The Medicago truncatula Gene Expression Atlas Project (http:// mtgea.noble.org/v3/)]. Based on these data and to keep in line with the previously designated MtPLT2 (Limpens et al, 2013), we utilize from now on the following nomenclature: Medtr2g098180 (MtPLT1), Medtr4g065370 (MtPLT2), Medtr5g031880 (MtPLT3) and Medtr7g080460 (MtPLT4) ( Table 1). The proposed gene annotations were subsequently used to design primers (supplementary material Table S5) to enable gene expression studies by qPCR.…”

“…Recent studies showed that orthologs of AtPLT genes, named MtPLT2(Medtr4g65370) and MtBBM/PLT4(Medtr7g080460) are expressed in the NM (Boutilier et al, 2002;Hofhuis et al, 2013;Limpens et al, 2013;Roux et al, 2014). We asked whether the other Medicago PLT orthologs are also expressed in the NM and performed reciprocal BLAST searches (in Mt4.0v1) using the AtPLT protein sequences as a query to identify their homologs in Medicago (Table 1; supplementary material Fig.…”

“…The annotation for Medicago PLT1 and PLT2 is arbitrary (but following a previous annotation by Limpens et al, 2013) because Medicago and Arabidopsis PLT1 and PLT2 genes were formed by independent gene duplication events (see supplementary material Fig. S1).…”

“…To demonstrate the effect of reducing gene expression of more than one MtPLT gene in nodules, we conducted RNAi using the MtENOD12 promoter. During nodule ontogenesis this gene is activated in the nodule primordium, the NM and in the infection zone of mature nodules (Limpens et al, 2009(Limpens et al, , 2013. We tested the effect of ENOD12::MtPLT1i,2i, ENOD12::MtPLT3i,4i and ENOD12::MtPLTi in triplicate on nodule growth and development.…”

“…Assuming that DR5::GUS and MtWOX5::GUS colocalize to areas of stem cell activity in nodules, in analogy to the situation in roots, this suggests that stem cells are present in the NM periphery. Recently, the expression of several auxin-responsive genes in the central part of the NM has been reported (Limpens et al, 2013;Breakspear et al, 2015;Roux et al, 2014), suggesting that auxin signaling occurs in this region of the NM. Indeed, upon prolonged incubation (16 h), DR5 activity becomes detectable throughout the vascular bundles and the nodule apex (Fig.…”