Search for: All records

Medicago (Medicago truncatula) establishes a symbiosis with the rhizobia Sinorhizobium sp, resulting in the formation of nodules where the bacteria fix atmospheric nitrogen. The loss of immunity repression or early senescence activation compromises symbiont survival and leads to the formation of nonfunctional nodules (fix−). Despite many studies exploring an overlap between immunity and senescence responses outside the nodule context, the relationship between these processes in the nodule remains poorly understood. To investigate this phenomenon, we selected and characterized three Medicago mutants developing fix− nodules and showing senescence responses. Analysis of specific defense (PATHOGENESIS-RELATED PROTEIN) or senescence (CYSTEINE PROTEASE) marker expression demonstrated that senescence and immunity seem to be antagonistic in fix− nodules. The growth of senescence mutants on non-sterile (sand/perlite) substrate instead of sterile in vitro conditions decreased nodule senescence and enhanced defense, indicating that environment can affect the immunity/senescence balance. The application of wounding stress on wild-type (WT) fix+ nodules led to the death of intracellular rhizobia and associated with co-stimulation of defense and senescence markers, indicating that in fix+ nodules the relationship between the two processes switches from opposite to synergistic to control symbiont survival during response to the stress. Our data show that the immune response in stressed WT nodules is linked to the repression of DEFECTIVE IN NITROGEN FIXATION 2 (DNF2), Symbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (SymCRK), and REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD), key genes involved in symbiotic immunity suppression. This study provides insight to understand the links between senescence and immunity in Medicago nodules.

 

Agrobacterium-mediated plant transformation (AMT) is the basis of modern-day plant biotechnology. One major drawback of this technology is the recalcitrance of many plant species/varieties toAgrobacteriuminfection, most likely caused by elicitation of plant defense responses. Here, we develop a strategy to increase AMT by engineeringAgrobacterium tumefaciensto express a type III secretion system (T3SS) fromPseudomonas syringaeand individually deliver theP. syringaeeffectors AvrPto, AvrPtoB, or HopAO1 to suppress host defense responses. Using the engineeredAgrobacterium, we demonstrate increase in AMT of wheat, alfalfa and switchgrass by ~250%–400%. We also show that engineeredA. tumefaciensexpressing a T3SS can deliver a plant protein, histone H2A-1, to enhance AMT. This strategy is of great significance to both basic research and agricultural biotechnology for transient and stable transformation of recalcitrant plant species/varieties and to deliver proteins into plant cells in a non-transgenic manner.

 

Legumes are of great interest for sustainable agricultural production as they fix atmospheric nitrogen to improve the soil. Medicago truncatula is a well-established model legume, and extensive studies in fundamental molecular, physiological, and developmental biology have been undertaken to translate into trait improvements in economically important legume crops worldwide. However, M. truncatula reference genome was generated in the accession Jemalong A17, which is highly recalcitrant to transformation. M. truncatula R108 is more attractive for genetic studies due to its high transformation efficiency and Tnt1-insertion population resource for functional genomics. The need to perform accurate synteny analysis and comprehensive genome-scale comparisons necessitates a chromosome-length genome assembly for M. truncatula cv. R108. Here, we performed in situ Hi-C (48×) to anchor, order, orient scaffolds, and correct misjoins of contigs in a previously published genome assembly (R108 v1.0), resulting in an improved genome assembly containing eight chromosome-length scaffolds that span 97.62% of the sequenced bases in the input assembly. The long-range physical information data generated using Hi-C allowed us to obtain a chromosome-length ordering of the genome assembly, better validate previous draft misjoins, and provide further insights accurately predicting synteny between A17 and R108 regions corresponding to the known chromosome 4/8 translocation. Furthermore, mapping the Tnt1 insertion landscape on this reference assembly presents an important resource for M. truncatula functional genomics by supporting efficient mutant gene identification in Tnt1 insertion lines. Our data provide a much-needed foundational resource that supports functional and molecular research into the Leguminosae for sustainable agriculture and feeding the future. 

ArabidopsisVIRE2-INTERACTINGPROTEIN2 (VIP2) was previously described as a protein with a NOT domain, and Arabidopsisvip2mutants are recalcitrant toAgrobacterium-mediated root transformation. Here we show that VIP2 is a transcription regulator and the C-terminal NOT2 domain of VIP2 interacts with VirE2. Interestingly,AtVIP2overexpressor lines in Arabidopsis did not show an improvement inAgrobacterium-mediated stable root transformation, but the transcriptome analysis identified 1,634 differentially expressed genes compared to wild-type. These differentially expressed genes belonged to various functional categories such as membrane proteins, circadian rhythm, signaling, response to stimulus, regulation of plant hypersensitive response, sequence-specific DNA binding transcription factor activity and transcription regulatory region binding. In addition to regulating genes involved inAgrobacterium-mediated plant transformation,AtVIP2overexpressor line showed differential expression of genes involved in abiotic stresses. The majority of the genes involved in abscisic acid (ABA) response pathway, containing the Abscisic Acid Responsive Element (ABRE) element within their promoters, were down-regulated inAtVIP2overexpressor lines. Consistent with this observation,AtVIP2overexpressor lines were more susceptible to ABA and other abiotic stresses. Based on the above findings, we hypothesize that VIP2 not only plays a role inAgrobacterium-mediated plant transformation but also acts as a general transcriptional regulator in plants.

 

Arbuscular mycorrhizal fungi help their host plant in the acquisition of nutrients, and this association is itself impacted by soil nutrient levels. High phosphorus levels inhibit the symbiosis, whereas high nitrogen levels enhance it. The genetic mechanisms regulating the symbiosis in response to soil nutrients are poorly understood. Here, we characterised the symbiotic phenotypes in fourMedicago truncatula Tnt1‐insertion mutants affected in arbuscular mycorrhizal colonisation. We located theirTnt1insertions and identified alleles for two genes known to be involved in mycorrhization,RAM1andKIN3. We compared the effects of thekin3‐2andram1‐4mutations on gene expression, revealing that the two genes alter the expression of overlapping but not identical gene sets, suggesting thatRAM1acts upstream ofKIN3.Additionally,KIN3appears to be involved in the suppression of plant defences in response to the fungal symbiont.KIN3is located on the endoplasmic reticulum of arbuscule‐containing cortical cells, andkin3‐2mutants plants hosted significantly fewer arbuscules than the wild type.KIN3plays an essential role in the symbiotic response to soil nitrogen levels, as, contrary to wild‐type plants, thekin3‐2mutant did not exhibit increased root colonisation under high nitrogen.

 

Cu⁺‐chaperones are a diverse group of proteins that allocate Cu⁺ions to specific copper proteins, creating different copper pools targeted to specific physiological processes.

Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required.

MtNCC1 is a nodule‐specific Cu⁺‐chaperone encoded in theMedicago truncatulagenome, with a N‐terminus Atx1‐like domain that can bind Cu⁺with picomolar affinities. MtNCC1 is able to interact with nodule‐specific Cu⁺‐importer MtCOPT1.MtNCC1is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation,ncc1mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper‐dependent cytochromecoxidase activity.

A subset of the copper proteome is also affected in thencc1mutant nodules. Many of these proteins can be pulled down when using a Cu⁺‐loaded N‐terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper‐dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.

 

Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants. Floral organ identity is controlled by the well-known ABC model, which has been generalized to many flowering plants. Here, we report a previously uncharacterized MYB-like gene,AGAMOUS-LIKE FLOWER(AGLF), involved in flower development in the model legumeMedicago truncatula. Loss-of-function ofAGLFresults in flowers with stamens and carpel transformed into extra whorls of petals and sepals. Compared with the loss-of-function mutant of the class C geneAGAMOUS(MtAG) inM. truncatula, the defects in floral organ identity are similar betweenaglfandmtag, but the floral indeterminacy is enhanced in theaglfmutant. Knockout ofAGLFin the mutants of the class A geneMtAP1or the class B geneMtPIleads to an addition of a loss-of-C-function phenotype, reflecting a conventional relationship ofAGLFwith the canonical A and B genes. Furthermore, we demonstrate thatAGLFactivatesMtAGin transcriptional levels in control of floral organ identity. These data shed light on the conserved and diverged molecular mechanisms that control flower development and morphology among plant species.

 

Plants being sessile integrate information from a variety of endogenous and external cues simultaneously to optimize growth and development. This necessitates the signaling networks in plants to be highly dynamic and flexible. One such network involves heterotrimeric G‐proteins comprised of Gα, Gβ, and Gγ subunits, which influence many aspects of growth, development, and stress response pathways. In plants such as Arabidopsis, a relatively simple repertoire of G‐proteins comprised of one canonical and three extra‐large Gα, one Gβ and three Gγ subunits exists. Because the Gβ and Gγ proteins form obligate dimers, the phenotypes of plants lacking the soleGβor allGγgenes are similar, as expected. However, Gα proteins can exist either as monomers or in a complex with Gβγ, and the details of combinatorial genetic and physiological interactions of different Gα proteins with the sole Gβ remain unexplored. To evaluate such flexible, signal‐dependent interactions and their contribution toward eliciting a specific response, we have generated Arabidopsis mutants lacking specific combinations ofGαandGβgenes, performed extensive phenotypic analysis, and evaluated the results in the context of subunit usage and interaction specificity. Our data show that multiple mechanistic modes, and in some cases complex epistatic relationships, exist depending on the signal‐dependent interactions between the Gα and Gβ proteins. This suggests that, despite their limited numbers, the inherent flexibility of plant G‐protein networks provides for the adaptability needed to survive under continuously changing environments.