The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

Temporal change in chromatin accessibility predicts regulators of nodulation in Medicago truncatula.

BACKGROUND: Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula, in response to rhizobial signals that trigger the formation of root nodules. RESULTS: We profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis-regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia. CONCLUSIONS: Our transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.

Single-nuclei transcriptome analysis of the shoot apex vascular system differentiation in Populus.

Differentiation of stem cells in the plant apex gives rise to aerial tissues and organs. Presently, we lack a lineage map of the shoot apex cells in woody perennials - a crucial gap considering their role in determining primary and secondary growth. Here, we used single-nuclei RNA-sequencing to determine cell type-specific transcriptomes of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of the epidermis, leaf mesophyll and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we applied a pipeline for interspecific single-cell gene expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the principles underlying cell division and differentiation conserved between herbaceous and perennial species while also allowing us to examine species-specific differences at single-cell resolution.

Functional and comparative genomics reveals conserved noncoding sequences in the nitrogen-fixing clade.

Nitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root-nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical, but undetermined, evolutionary event at the base of this clade. To identify putative regulatory sequences implicated in the evolution of RNS, we evaluated the genomes of 25 species capable of nodulation and identified 3091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade (NFC). We show that the chromatin accessibility of 452 CNS correlates significantly with the regulation of genes responding to lipochitooligosaccharides in Medicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream of MtCRE1, Cytokinin Response Element 1, required to activate a suite of downstream transcription factors necessary for nodulation in M. truncatula. Genetic complementation of an Mtcre1 mutant showed a significant decrease of nodulation in the absence of the five CNS, when they are driving the expression of a functional copy of MtCRE1. CNS identified in the NFC may harbor elements required for the regulation of genes controlling RNS in M. truncatula.

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

A robust method of nuclei isolation for single-cell RNA sequencing of solid tissues from the plant genus Populus.

Single-cell transcriptome analysis has been extensively applied in humans and animal models to uncover gene expression heterogeneity between the different cell types of a tissue or an organ. It demonstrated its capability to discover key regulatory elements that determine cell fate during developmental programs. Single-cell analysis requires the isolation and labeling of the messenger RNA (mRNA) derived from each cell. These challenges were primarily addressed in mammals by developing microfluidic-based approaches. For plant species whose cells contain cell walls, these approaches have generally required the generation of isolated protoplasts. Many plant tissues' secondary cell wall hinders enzymatic digestion required for individual protoplast isolation, resulting in an unequal representation of cell types in a protoplast population. This limitation is especially critical for cell types located in the inner layers of a tissue or the inner tissues of an organ. Consequently, single-cell RNA sequencing (scRNA-seq) studies using microfluidic approaches in plants have mainly been restricted to Arabidopsis roots, for which well-established procedures of protoplast isolation are available. Here we present a simple alternative approach to generating high-quality protoplasts from plant tissue by characterizing the mRNA extracted from individual nuclei instead of whole cells. We developed the protocol using two different plant materials with varying cellular complexity levels and cell wall structure, Populus shoot apices, and more lignified stems. Using the 10× Genomics Chromium technology, we show that this procedure results in intact mRNA isolation and limited leakage, with a broad representation of individual cell transcriptomes.

In-depth genome characterization of a Brazilian common bean core collection using DArTseq high-density SNP genotyping.

BACKGROUND: Common bean is a legume of social and nutritional importance as a food crop, cultivated worldwide especially in developing countries, accounting for an important source of income for small farmers. The availability of the complete sequences of the two common bean genomes has dramatically accelerated and has enabled new experimental strategies to be applied for genetic research. DArTseq has been widely used as a method of SNP genotyping allowing comprehensive genome coverage with genetic applications in common bean breeding programs. RESULTS: Using this technology, 6286 SNPs (1 SNP/86.5 Kbp) were genotyped in genic (43.3%) and non-genic regions (56.7%). Genetic subdivision associated to the common bean gene pools (K = 2) and related to grain types (K = 3 and K = 5) were reported. A total of 83% and 91% of all SNPs were polymorphic within the Andean and Mesoamerican gene pools, respectively, and 26% were able to differentiate the gene pools. Genetic diversity analysis revealed an average H E of 0.442 for the whole collection, 0.102 for Andean and 0.168 for Mesoamerican gene pools (F ST = 0.747 between gene pools), 0.440 for the group of cultivars and lines, and 0.448 for the group of landrace accessions (F ST = 0.002 between cultivar/line and landrace groups). The SNP effects were predicted with predominance of impact on non-coding regions (77.8%). SNPs under selection were identified within gene pools comparing landrace and cultivar/line germplasm groups (Andean: 18; Mesoamerican: 69) and between the gene pools (59 SNPs), predominantly on chromosomes 1 and 9. The LD extension estimate corrected for population structure and relatedness (r2SV) was ~ 88 kbp, while for the Andean gene pool was ~ 395 kbp, and for the Mesoamerican was ~ 130 kbp. CONCLUSIONS: For common bean, DArTseq provides an efficient and cost-effective strategy of generating SNPs for large-scale genome-wide studies. The DArTseq resulted in an operational panel of 560 polymorphic SNPs in linkage equilibrium, providing high genome coverage. This SNP set could be used in genotyping platforms with many applications, such as population genetics, phylogeny relation between common bean varieties and support to molecular breeding approaches.

SNP discovery in common bean by restriction-associated DNA (RAD) sequencing for genetic diversity and population structure analysis.

Researchers have made great advances into the development and application of genomic approaches for common beans, creating opportunities to driving more real and applicable strategies for sustainable management of the genetic resource towards plant breeding. This work provides useful polymorphic single-nucleotide polymorphisms (SNPs) for high-throughput common bean genotyping developed by RAD (restriction site-associated DNA) sequencing. The RAD tags were generated from DNA pooled from 12 common bean genotypes, including breeding lines of different gene pools and market classes. The aligned sequences identified 23,748 putative RAD-SNPs, of which 3357 were adequate for genotyping; 1032 RAD-SNPs with the highest ADT (assay design tool) score are presented in this article. The RAD-SNPs were structurally annotated in different coding (47.00 %) and non-coding (53.00 %) sequence components of genes. A subset of 384 RAD-SNPs with broad genome distribution was used to genotype a diverse panel of 95 common bean germplasms and revealed a successful amplification rate of 96.6 %, showing 73 % of polymorphic SNPs within the Andean group and 83 % in the Mesoamerican group. A slightly increased He (0.161, n = 21) value was estimated for the Andean gene pool, compared to the Mesoamerican group (0.156, n = 74). For the linkage disequilibrium (LD) analysis, from a group of 580 SNPs (289 RAD-SNPs and 291 BARC-SNPs) genotyped for the same set of genotypes, 70.2 % were in LD, decreasing to 0.10 %in the Andean group and 0.77 % in the Mesoamerican group. Haplotype patterns spanning 310 Mb of the genome (60 %) were characterized in samples from different origins. However, the haplotype frameworks were under-represented for the Andean (7.85 %) and Mesoamerican (5.55 %) gene pools separately. In conclusion, RAD sequencing allowed the discovery of hundreds of useful SNPs for broad genetic analysis of common bean germplasm. From now, this approach provides an excellent panel of molecular tools for whole genome analysis, allowing integrating and better exploring the common bean breeding practices.