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  1. Free, publicly-accessible full text available July 1, 2023
  2. Abstract

    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.

  3. 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,more »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.« less
  4. Abstract

    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.

  5. 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.