Senjuti Sinharoy

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

This work reports a plant transcription factor, RSD, that is required for symbiosome development and symbiotic nitrogen fixation (SNF) in the model legume Medicago truncatula. RSD represses the expression of the secretory pathway gene VAMP721a, which suggests that alteration in this pathway is important for SNF. 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.

PlantTFcat: an online plant transcription factor and transcriptional regulator categorization and analysis tool

BackgroundPlants regulate intrinsic gene expression through transcription factors (TFs), transcriptional regulators (TRs), chromatin regulators (CRs), and the basal transcription machinery. An understanding of plant gene regulatory mechanisms at a systems level requires the identification of these regulatory elements on a genomic scale.ResultsHere, we present PlantTFcat, a high-performance web-based analysis tool that is designed to identify and categorize plant TF/TR/CR genes from genome-scale protein and nucleic acid sequences by systematically analyzing InterProScan domain patterns in protein sequences. The comprehensive prediction logics that are included in PlantTFcat are based on relationships between gene families and conserved domains from 108 published plant TF/TR/CR families. These prediction logics effectively distinguish TF/TR/CR families with common conserved domains. Our systematic performance evaluations indicate that PlantTFcat annotates known TF/TR/CR families with high coverage and sensitivity.ConclusionsPlantTFcat provides an analysis tool to identify and categorize plant TF/TR/CR genes on a genomic scale. PlantTFcat is freely available to the public at http://plantgrn.noble.org/PlantTFcat/.

MtSWEET11, a Nodule-Specific Sucrose Transporter of Medicago truncatula

SWEET11 is a nodule-specific sucrose transporter of Medicago truncatula. Optimization of nitrogen fixation by rhizobia in legumes is a key area of research for sustainable agriculture. Symbiotic nitrogen fixation (SNF) occurs in specialized organs called nodules and depends on a steady supply of carbon to both plant and bacterial cells. Here we report the functional characterization of a nodule-specific Suc transporter, MtSWEET11 from Medicago truncatula. MtSWEET11 belongs to a clade of plant SWEET proteins that are capable of transporting Suc and play critical roles in pathogen susceptibility. When expressed in mammalian cells, MtSWEET11 transported sucrose (Suc) but not glucose (Glc). The MtSWEET11 gene was found to be expressed in infected root hair cells, and in the meristem, invasion zone, and vasculature of nodules. Expression of an MtSWEET11-GFP fusion protein in nodules resulted in green fluorescence associated with the plasma membrane of uninfected cells and infection thread and symbiosome membranes of infected cells. Two independent Tnt1-insertion sweet11 mutants were uncompromised in SNF. Therefore, although MtSWEET11 appears to be involved in Suc distribution within nodules, it is not crucial for SNF, probably because other Suc transporters can fulfill its role(s).

Autophosphorylation of gatekeeper tyrosine by symbiosis receptor kinase

Plant 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.

A Medicago truncatula Cystathionine-β-Synthase-like Domain-Containing Protein Is Required for Rhizobial Infection and Symbiotic Nitrogen Fixation

A Cystathionine-β-Synthase like protein, exclusively expressed during Medicago-Rhizobium symbiosis, is required for infection thread propagation and bacterial endocytosis. The symbiosis between leguminous plants and soil rhizobia culminates in the formation of nitrogen-fixing organs called nodules that support plant growth. Two Medicago truncatula Tnt1-insertion mutants were identified that produced small nodules, which were unable to fix nitrogen effectively due to ineffective rhizobial colonization. The gene underlying this phenotype was found to encode a protein containing a putative membrane-localized domain of unknown function (DUF21) and a cystathionine-β-synthase domain. The cbs1 mutants had defective infection threads that were sometimes devoid of rhizobia and formed small nodules with greatly reduced numbers of symbiosomes. We studied the expression of the gene, designated M. truncatula Cystathionine-β-Synthase-like1 (MtCBS1), using a promoter-β-glucuronidase gene fusion, which revealed expression in infected root hair cells, developing nodules, and in the invasion zone of mature nodules. An MtCBS1-GFP fusion protein localized itself to the infection thread and symbiosomes. Nodulation factor-induced Ca2+ responses were observed in the cbs1 mutant, indicating that MtCBS1 acts downstream of nodulation factor signaling. MtCBS1 expression occurred exclusively during Medicago-rhizobium symbiosis. Induction of MtCBS1 expression during symbiosis was found to be dependent on Nodule Inception (NIN), a key transcription factor that controls both rhizobial infection and nodule organogenesis. Interestingly, the closest homolog of MtCBS1, MtCBS2, was specifically induced in mycorrhizal roots, suggesting common infection mechanisms in nodulation and mycorrhization. Related proteins in Arabidopsis have been implicated in cell wall maturation, suggesting a potential role for CBS1 in the formation of the infection thread wall.

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.

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

BackgroundGenetic crossing is an essential tool in both forward and reverse genetic approaches to understand the biological functions of genes. For Medicago truncatula (barrel medic) various crossing techniques have been used which differ in the methods used to dissect the female parent’s unopened flower bud to remove immature anthers for prevention of self-pollination. Previously described methods including front, side or back incision methods may damage the flower bud, impeding successful fertilization and/or seed development because they may allow pollen to dislodge and floral organs to desiccate after crossing, all of which diminish the success rates of crossing.ResultsWe report the keel petal incision method for genetic crossing in M. truncatula ecotype R108 and demonstrate successful crosses with two other M. truncatula ecotypes, A17 and A20. In the method presented here, an incision is made along the central line of the keel petal from the bottom 1/3rd of the female parent’s flower bud to its distal end. This allows easy removal of anthers from the flower bud and access for cross-pollination. After pollination, the stigma and the deposited pollen from the male donor are covered by the keel petal, wing petals and standard petal, forming a natural pouch. The pouch prevents dislodging of deposited pollen from the stigma and protects the internal floral organs from drying out, without using cling-film or water-containing chambers to maintain a humid environment. The keel petal incision method showed an approximate 80% success rate in the M. truncatula R108 ecotype and also in other ecotypes including Jemalong A17 and A20.ConclusionsOur keel petal incision protocol shows marked improvement over existing methods with respect to the ease of crossing and the percentage of successful crosses. Developed for the M. truncatula R108 ecotype, the protocol has been demonstrated with A17 and A20 ecotypes and is expected to work with other ecotypes. Investigators of varying experience have achieved genetic crosses in M. truncatula using this method.

Global analysis of the MATE gene family of metabolite transporters in tomato

BackgroundSpecies in the Solanaceae family are known for producing plethora of specialized metabolites. In addition to biosynthesis pathways, a full comprehension of secondary metabolism must also take into account the transport and subcellular compartmentalization of substances. Here, we examined the MATE (Multidrug and Toxic Compound Extrusion, or Multi-Antimicrobial Extrusion) gene family in the tomato (Solanum lycopersicum) genome with the objective of better understanding the transport of secondary metabolites in this model species. MATE membrane effluxers encompass an ancient gene family of secondary transporters present in all kingdoms of life, but with a remarkable expansion in plants. They mediate the transport of primary and secondary metabolites using the proton motive force through several membrane systems of the cell.ResultsWe identified 67 genes coding for MATE transporters in the tomato genome, 33 of which are expressed constitutively whereas 34 are expressed in specific cell types or environmental conditions. Synteny analyses revealed bona fide paralogs and Arabidopsis orthologs. Co-expression analysis between MATE and regulatory genes revealed 78 positive and 8 negative strong associations (ρ≥|0.8|). We found no evidence of MATE transporters belonging to known metabolic gene clusters in tomato.ConclusionsAltogether, our expression data, phylogenetic analyses, and synteny study provide strong evidence of functional homologies between MATE genes of tomato and Arabidopsis thaliana. Our co-expression study revealed potential transcriptional regulators of MATE genes that warrant further investigation. This work sets the stage for genome-wide functional analyses of MATE transporters in tomato and other Solanaceae species of economic relevance.

A high-throughput RNA interference (RNAi)-based approach using hairy roots for the study of plant-rhizobia interactions.

Legumes are major contributors to sustainable agriculture; their key feature is their ability to fix atmospheric nitrogen through symbiotic nitrogen fixation. Legumes are often recalcitrant to regeneration and transformation by Agrobacterium tumefaciens; however, A. rhizogenes-mediated root transformation and composite plant generation are rapid and convenient alternatives to study root biology, including root nodule symbiosis. RNA interference (RNAi), coupled with A. rhizogenes-mediated root transformation, has been very successfully used for analyses of gene function by reverse genetics. Besides being applied to model legumes (Medicago truncatula and Lotus japonicus), this method has been adopted for several other legumes due to the ease and relative speed with which transgenic roots can be generated. Several protocols for hairy root transformation have been published. Here we describe an improved hairy root transformation protocol and the methods to study nodulation in Medicago. We also highlight the major differences between our protocol and others, and key steps that need to be adjusted in order to translate this method to other legumes.