RNA Interference highlights the role of CCaMK in dissemination of endosymbionts in the aeschynomeneae legume arachis

In legume–rhizobia symbiosis, Ca2+/Calmodulin-dependent Protein Kinase (CCaMK) is essential for rhizobial invasion through infection threads in the epidermis and nodule organogenesis in the cortex. Though CCaMK is actively transcribed in the infected zone of nodules, its role in the later stages of nodule development remain elusive because of the epidermal arrest of “loss-of-function” mutants. In Aeschynomeneae legumes such as Arachis hypogea, rhizobia directly access the cortex, where nodule organogenesis as well as endosymbiont dissemination take place by multiplication of infected cortical cells. We characterized CCaMK (GI:195542474) from A. hypogea and downregulated the kinase through RNA interference (RNAi) to understand its role during organogenesis of its characteristic aeschynomenoid nodules. In CCaMK downregulated plants, the inception of nodules was delayed by approximately 4 weeks and nodulation capacity was decreased (>90%). The infected zones of the RNA interference nodules were scattered with uninfected or binucleated cells as opposed to the homogeneous infection zone in empty-vector-transformed nodules. Symbiosomes in RNAi nodules were pleomorphic with diverse geometrical shapes or arrested during division in the final stages of their fission as opposed to uniform-sized, spherical symbiosomes in empty-vector-transformed nodules. Together, our results reveal CCaMK to be essential for development of functional aeschynomenoid nodules, with a critical role in rhizobial dissemination during nodule organogenesis

A Snapshot of Functional Genetic Studies in Medicago truncatula

In the current context of food security, increase of plant protein production in a sustainable manner represents one of the major challenges of agronomic research, which could be partially resolved by increased cultivation of legume crops. Medicago truncatula is now a well-established model for legume genomic and genetic studies. With the establishment of genomics tools and mutant populations in M. truncatula, it has become an important resource to answer some of the basic biological questions related to plant development and stress tolerance. This review has an objective to overview a decade of genetic studies in this model plant from generation of mutant populations to nowadays. To date, the three biological fields, which have been extensively studied in M. truncatula, are the symbiotic nitrogen fixation, the seed development, and the abiotic stress tolerance, due to their significant agronomic impacts. In this review, we summarize functional genetic studies related to these three major biological fields. We integrated analyses of a nearly exhaustive list of genes into their biological contexts in order to provide an overview of the forefront research advances in this important legume model plant.

Transformed Hairy Roots of Arachis hypogea: A Tool for Studying Root Nodule Symbiosis in a Non–Infection Thread Legume of the Aeschynomeneae Tribe

Arachis hypogea is a non–“infection thread” (IT) legume where rhizobial entry or dissemination in the nodules never involves IT. Rhizobia invade through epidermal “cracks” and directly access the cortical cells to develop the characteristic aeschynomenoid nodules. For investigating these nonclassical nodulation features in Arachis spp., we developed an efficient procedure for Agrobacterium rhizogenes R1000-mediated transformation of this plant. In this study, we optimized the induction of hairy roots and nodulation of composite Arachis hypogea plants in the presence of Bradyrhizobium sp. (Arachis) strain NC92. 35S promoterdriven green fluorescent protein and β-glucuronidase expression indicated transformation frequency to be above 80%. The transformed roots had the characteristic rosettetype root hairs and had normal level of expression of symbiosis- related genes SymRK and CCaMK. The transgenic nodules resembled the wild-type nodules with an exception of 2 to 3%, where they structurally deviated from the wildtype nodules to form nodular roots. A 16S rRNA profile of an infected-zone metagenome indicated that identical populations of bradyrhizobia invaded both composite wild-type plants grown in natural soil. Our results demonstrate that Arachis hairy root is an attractive system for undertaking investigations of the nonclassical features associated with its nitrogen-fixing symbiotic interaction

A snapshot of functional genetic studies in Medicago truncatula

In the current context of food security, increase of plant protein production in a sustainable manner represents one of the major challenges of agronomic research, which could be partially resolved by increased cultivation of legume crops. Medicago truncatula is now a well-established model for legume genomic and genetic studies. With the establishment of genomics tools and mutant populations in M. truncatula, it has become an important resource to answer some of the basic biological questions related to plant development and stress tolerance. This review has an objective to overview a decade of genetic studies in this model plant from generation of mutant populations to nowadays. To date, the three biological fields, which have been extensively studied in M. truncatula, are the symbiotic nitrogen fixation, the seed development, and the abiotic stress tolerance, due to their significant agronomic impacts. In this review, we summarize functional genetic studies related to these three major biological fields. We integrated analyses of a nearly exhaustive list of genes into their biological contexts in order to provide an overview of the forefront research advances in this important legume model plant

Transformed hairy roots of Aachis hypogea: a tool for studying root nodule symbiosis in a non–infection thread legume of the Aeschynomeneae tribe

Arachis hypogea is a non–“infection thread” (IT) legume where rhizobial entry or dissemination in the nodules never involves IT. Rhizobia invade through epidermal “cracks” and directly access the cortical cells to develop the characteristic aeschynomenoid nodules. For investigating these nonclassical nodulation features in Arachis spp., we developed an efficient procedure for Agrobacterium rhizogenes R1000-mediated transformation of this plant. In this study, we optimized the induction of hairy roots and nodulation of composite Arachis hypogea plants in the presence of Bradyrhizobium sp. (Arachis) strain NC92. 35S promoter-driven green fluorescent protein and β-glucuronidase expression indicated transformation frequency to be above 80%. The transformed roots had the characteristic rosette-type root hairs and had normal level of expression of symbiosis-related genes SymRK and CCaMK. The transgenic nodules resembled the wild-type nodules with an exception of 2 to 3%, where they structurally deviated from the wild-type nodules to form nodular roots. A 16S rRNA profile of an infected-zone metagenome indicated that identical populations of bradyrhizobia invaded both composite wild-type plants grown in natural soil. Our results demonstrate that Arachis hairy root is an attractive system for undertaking investigations of the nonclassical features associated with its nitrogen-fixing symbiotic interactions

Autophosphorylation of gatekeeper tyrosine by symbiosis receptor kinase

AbstractPlant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as ‘gatekeeper’. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs

PLANT METHODS METHODOLOGY Open Access

Keel petal incision: a simple and efficient metho

Keel petal incision: a simple and efficient method for genetic crossing in Medicago truncatula

Article on keel petal incision and a simple and efficient method for genetic crossing in Medicago truncatula

The C2H2 Transcription Factor REGULATOR OF SYMBIOSOME DIFFERENTIATION Represses Transcription of the Secretory Pathway Gene VAMP721a and Promotes Symbiosome Development in Medicago truncatula.

Transcription factors (TFs) are thought to regulate many aspects of nodule and symbiosis development in legumes, although few TFs have been characterized functionally. Here, we describe REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD) of Medicago truncatula, a member of the Cysteine-2/Histidine-2 (C2H2) family of plant TFs that is required for normal symbiosome differentiation during nodule development. RSD is expressed in a nodule-specific manner, with maximal transcript levels in the bacterial invasion zone. A tobacco (Nicotiana tabacum) retrotransposon (Tnt1) insertion rsd mutant produced nodules that were unable to fix nitrogen and that contained incompletely differentiated symbiosomes and bacteroids. RSD protein was localized to the nucleus, consistent with a role of the protein in transcriptional regulation. RSD acted as a transcriptional repressor in a heterologous yeast assay. Transcriptome analysis of an rsd mutant identified 11 genes as potential targets of RSD repression. RSD interacted physically with the promoter of one of these genes, VAMP721a, which encodes vesicle-associated membrane protein 721a. Thus, RSD may influence symbiosome development in part by repressing transcription of VAMP721a and modifying vesicle trafficking in nodule cells. This establishes RSD as a TF implicated directly in symbiosome and bacteroid differentiation and a transcriptional regulator of secretory pathway genes in plants