skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


This content will become publicly available on April 1, 2026

Title: A novel toolbox of GATEWAY-compatible vectors for rapid functional gene analysis in soybean composite plants
The generation of transgenic plants is essential for plant biology research to investigate plant physiology, pathogen interactions, and gene function. However, producing stable transgenic plants for plants such as soybean is a laborious and time-consuming process, which can impede research progress. Composite plants consisting of wild-type shoots and transgenic roots are an alternative method for generating transgenic plant tissues that can facilitate functional analysis of genes-ofinterest involved in root development or root-microbe interactions. In this report, we introduce a novel set of GATEWAYcompatible vectors that enable a wide range of molecular biology uses in roots of soybean composite plants. These vectors incorporate in-frame epitope fusions of green fluorescent protein, 3x-HA, or miniTurbo-ID, which can be easily fused to a gene-of-interest using the GATEWAY cloning system. Moreover, these vectors allow for the identification of transgenic roots using either mCherry fluorescence or the RUBY marker. We demonstrate the functionality of these vectors by expressing subcellular markers in soybean, providing evidence of their effectiveness in generating protein fusions in composite soybean plants. Furthermore, we show how these vectors can be used for gene function analysis by expressing the bacterial effector, AvrPphB in composite roots, enabling the identification of soybean targets via immunoprecipitation followed by mass spectrometry. Additionally, we demonstrate the successful expression of stable miniTurbo-ID fusion proteins in composite roots. Overall, this new set of vectors is a powerful tool that can be used to assess subcellular localization and perform gene function analyses in soybean roots without the need to generate stable transgenic plants.  more » « less
Award ID(s):
2017314
PAR ID:
10650865
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ;
Publisher / Repository:
Springer-Verlag GmbH Germany
Date Published:
Journal Name:
Plant Cell Reports
Volume:
44
Issue:
4
ISSN:
0721-7714
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; (, Frontiers in Plant Science)
    null (Ed.)
    The ERECTA (ER) family of genes, encoding leucine-rich repeat receptor-like kinase (RLK), influences complex morphological and physiological aspects of plants. Modulation of ER signaling leads to abiotic stress tolerance in diverse plant species. However, whether the gain in stress tolerance is accompanied with desirable agronomic performance is not clearly known. In this study, soybean plants potentially suppressed in ER signaling were evaluated for the phenotypic performance and drought response in the greenhouse. These plants expressed a dominant-negative Arabidopsis thaliana ER ( AtER ) called Δ Kinase to suppress ER signaling, which has previously been linked with the tolerance to water deficit, a major limiting factor for plant growth and development, directly compromising agricultural production. With the aim to select agronomically superior plants as stress-tolerant lines, transgenic soybean plants were subjected to phenotypic selection and subsequently to water stress analysis. This study found a strong inverse correlation of Δ Kinase expression with the agronomic performance of soybean plants, indicating detrimental effects of expressing Δ Kinase that presumably led to the suppression of ER signaling. Two lines were identified that showed favorable agronomic traits and expression of Δ Kinase gene, although at lower levels compared with the rest of the transgenic lines. The drought stress analysis on the progenies of these lines, however, showed that these plants were more susceptible to water-deficit stress as compared with the non-transgenic controls. The selected transgenic plants showed greater stomata density and conductance, which potentially led to higher biomass, and consequently more water demand and greater susceptibility to the periods of water withholding. 
    more » « less
  2. ; ; ; ; ; ; ; (, Plant Direct)
    Abstract Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The class III GRX gene family is unique to land plants, andArabidopsis thalianahas 21 class III GRXs, which remain largely uncharacterized. About 80% ofA. thalianaclass III GRXs are transcriptionally regulated by nitrate, and several recent studies have suggested roles for these GRXs in nitrogen signaling. Our objective was to functionally characterize two nitrate‐induced GRX genes,AtGRXS5andAtGRXS8, defining their roles in signaling and development in theA. thalianaroot. We demonstrated thatAtGRXS5andAtGRXS8are primarily expressed in root and shoot vasculature (phloem), and that the corresponding GRX proteins display nucleo‐cytosolic subcellular localization. Ectopic expression ofAtGRXS8in transgenic plants caused major alterations in root system architecture: Normal primary root development, but a near absence of lateral roots. RNA sequencing demonstrated that the roots ofAtGRXS8‐overexpressing plants show strongly reduced transcript abundance for many primary nitrate response genes, including the major high‐affinity nitrate transporters. Correspondingly, high‐affinity nitrate uptake and the transport of nitrate from roots to shoots are compromised inAtGRXS8‐overexpressing plants. Finally, we demonstrated that the AtGRXS8 protein can physically interact with the TGA1 and TGA4 transcription factors, which are central regulators of early transcriptional responses to nitrate inA. thalianaroots. Overall, these results suggest thatAtGRXS8acts to quench both transcriptional and developmental aspects of primary nitrate response, potentially by interfering with the activity of the TGA1 and TGA4 transcription factors. 
    more » « less
  3. ; ; ; ; ; ; ; ; ; (, New Phytologist)
    Summary Central metabolism produces amino and fatty acids for protein and lipids that establish seed value. Biosynthesis of storage reserves occurs in multiple organelles that exchange central intermediates including two essential metabolites, malate, and pyruvate that are linked by malic enzyme. Malic enzyme can be active in multiple subcellular compartments, partitioning carbon and reducing equivalents for anabolic and catabolic requirements. Prior studies based on isotopic labeling and steady‐state metabolic flux analyses indicated malic enzyme provides carbon for fatty acid biosynthesis in plants, though genetic evidence confirming this role is lacking. We hypothesized that increasing malic enzyme flux would alter carbon partitioning and result in increased lipid levels in soybeans.Homozygous transgenic soybean plants expressing Arabidopsis malic enzyme alleles, targeting the translational products to plastid or outside the plastid during seed development, were verified by transcript and enzyme activity analyses, organelle proteomics, and transient expression assays. Protein, oil, central metabolites, cofactors, and acyl‐acyl carrier protein (ACPs) levels were quantified overdevelopment.Amino and fatty acid levels were altered resulting in an increase in lipids by 0.5–2% of seed biomass (i.e. 2–9% change in oil).Subcellular targeting of a single gene product in central metabolism impacts carbon and reducing equivalent partitioning for seed storage reserves in soybeans. 
    more » « less
  4. ; ; ; ; ; ; ; ; ; ; et al (, Development)
    ABSTRACT Transgenesis is an essential technique for any genetic model. Tol2-based transgenesis paired with Gateway-compatible vector collections has transformed zebrafish transgenesis with an accessible modular system. Here, we establish several next-generation transgenesis tools for zebrafish and other species to expand and enhance transgenic applications. To facilitate gene regulatory element testing, we generated Gateway middle entry vectors harboring the small mouse beta-globin minimal promoter coupled to several fluorophores, CreERT2 and Gal4. To extend the color spectrum for transgenic applications, we established middle entry vectors encoding the bright, blue-fluorescent protein mCerulean and mApple as an alternative red fluorophore. We present a series of p2A peptide-based 3′ vectors with different fluorophores and subcellular localizations to co-label cells expressing proteins of interest. Finally, we established Tol2 destination vectors carrying the zebrafish exorh promoter driving different fluorophores as a pineal gland-specific transgenesis marker that is active before hatching and through adulthood. exorh-based reporters and transgenesis markers also drive specific pineal gland expression in the eye-less cavefish (Astyanax). Together, our vectors provide versatile reagents for transgenesis applications in zebrafish, cavefish and other models. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, Frontiers in Microbiology)
    Oomycete and fungal pathogens cause billions of dollars of damage to crops worldwide annually. Therefore, there remains a need for broad-spectrum resistance genes, especially ones that target pathogens but do not interfere with colonization by beneficial microbes. Motivated by evidence suggesting that phosphatidylinositol-3-phosphate (PI3P) may be involved in the delivery of some oomycete and fungal virulence effector proteins, we created stable transgenic soybean plants that express and secrete two different PI3P-binding proteins, GmPH1 and VAM7, in an effort to interfere with effector delivery and confer resistance. Soybean plants expressing the two PI3P-binding proteins exhibited reduced infection by the oomycete pathogen Phytophthora sojae compared to control lines. Measurements of nodulation by nitrogen-fixing mutualistic bacterium Bradyrhizobium japonicum , which does not produce PI3P, revealed that the two lines with the highest levels of GmPH1 transcripts exhibited reductions in nodulation and in benefits from nodulation. Transcriptome and plant hormone measurements were made of soybean lines with the highest transcript levels of GmPH1 and VAM7 , as well as controls, following P. sojae - or mock-inoculation. The results revealed increased levels of infection-associated transcripts in the transgenic lines, compared to controls, even prior to P. sojae infection, suggesting that the plants were primed for increased defense. The lines with reduced nodulation exhibited elevated levels of jasmonate-isoleucine and of transcripts of a JAR1 ortholog encoding jasmonate-isoleucine synthetase. However, lines expressing VAM7 transgenes exhibited normal nodulation and no increases in jasmonate-isoleucine. Overall, together with previously published data from cacao and from P. sojae transformants, the data suggest that secretion of PI3P-binding proteins may confer disease resistance through a variety of mechanisms. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email