Microscopic and Transcriptomic Analyses of Dalbergoid Legume Peanut Reveal a Divergent Evolution Leading to Nod-Factor-Dependent Epidermal Crack-Entry and Terminal Bacteroid Differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS comes from model legumes that exhibit a root-hair mode of bacterial infection, in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step toward understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin-derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen-scavenging mechanisms in the Papilionoid family. Finally, the absence of cysteine-rich motif-1-containing nodule-specific cysteine-rich peptide (NCR) genes but the recruitment of defensin-like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobia symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Nodule INception-independent epidermal events lead to bacterial entry during nodule development in peanut (Arachis hypogaea).

Legumes can host nitrogen-fixing rhizobia inside root nodules. In model legumes, rhizobia enter via infection threads (ITs) and develop nodules in which the infection zone contains a mixture of infected and uninfected cells. Peanut (Arachis hypogaea) diversified from model legumes c. 50-55 million years ago. Rhizobia enter through 'cracks' to form nodules in peanut roots where cells of the infection zone are uniformly infected. Phylogenomic studies have indicated symbiosis as a labile trait in peanut. These atypical features prompted us to investigate the molecular mechanism of peanut nodule development. Combining cell biology, genetics and genomic tools, we visualized the status of hormonal signaling in peanut nodule primordia. Moreover, we dissected the signaling modules of Nodule INception (NIN), a master regulator of both epidermal infection and cortical organogenesis. Cytokinin signaling operates in a broad zone, from the epidermis to the pericycle inside nodule primordia, while auxin signaling is narrower and focused. Nodule INception is involved in nodule organogenesis, but not in crack entry. Nodulation Pectate Lyase, which remodels cell walls during IT formation, is not required. By contrast, Nodule enhanced Glycosyl Hydrolases (AhNGHs) are recruited for cell wall modification during crack entry. While hormonal regulation is conserved, the function of the NIN signaling modules is diversified in peanut.

An Improvised Hairy Root Transformation Method for Efficient Gene Silencing in Roots and Nodules of Arachis hypogaea.

Peanut (Arachis hypogaea) is a major oilseed crop and is widely cultivated in tropical and subtropical climate zone worldwide. Peanut belongs to the Papilionoid family with an atypical nodule developmental program. In particular, rhizobia enter through developmental cracks and lead to the formation of aeschynomenoid subtype determinate nodules. Peanut nodules are efficient nitrogen-fixers and form swollen bacteroid containing symbiosomes. The allotetraploid genome and recalcitrance to stable transformation used to be the major bottleneck for peanut biologists. Recent genome sequencing of peanut cultivar Tifrunner has opened up a huge opportunity for molecular research. A composite plant contains transformed roots with a non-transformed shoot. The composite plant-based approach has already proven to be a tool of choice for high throughput studies in root biology. The available protocols failed to generate efficient hairy root transformation in the genome sequenced cultivar Tifrunner. Here we describe an efficient hairy root transformation and composite plant generation protocol for the peanut cultivar Tifrunner. Our protocol generated ~92% plant regeneration efficiency with between 21.8% and 58.6% co-transformed root regeneration. We also show that this protocol can be efficiently used for protein localization, promoter GUS analysis, monitoring hormone response, and RNAi mediated knockdown of the genes using genome sequenced cultivar Tifrunner.