Jian-Feng Li

Massachusetts General Hospital, Boston, Massachusetts, United States

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Publications (13)80.57 Total impact

  • Jian-Feng Li, Dandan Zhang, Jen Sheen
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    ABSTRACT: Artificial miRNA (amiRNA) technology offers highly specific gene silencing in diverse plant species. The principal challenge in amiRNA application is to select potent amiRNAs from hundreds of bioinformatically designed candidates to enable maximal target gene silencing at the protein level. To address this issue, we developed the epitope-tagged protein-based amiRNA (ETPamir) screens, in which single or multiple potential target genes encoding epitope-tagged proteins are constitutively or inducibly coexpressed with individual amiRNA candidates in plant protoplasts. Accumulation of tagged proteins, detected by immunoblotting with commercial tag antibodies, inversely and quantitatively reflects amiRNA efficacy in vivo. The core procedure, from protoplast isolation to identification of optimal amiRNA, can be completed in 2-3 d. The ETPamir screens circumvent the limited availability of plant antibodies and the complexity of plant amiRNA silencing at target mRNA and/or protein levels. The method can be extended to verify predicted target genes for endogenous plant miRNAs.
    Nature Protocol 04/2014; 9(4):939-49. · 8.36 Impact Factor
  • Jian-Feng Li, Dandan Zhang
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    ABSTRACT: This unit describes the split firefly luciferase complementation (SFLC) assay, a high-throughput quantitative method that can be used to investigate protein-protein interactions (PPIs) in plant mesophyll protoplasts. In SFLC, the two proteins to be tested for interaction are expressed as chimeric proteins, each fused to a different half of firefly luciferase. If the proteins interact, a functional luciferase can be transitorily reconstituted, and is detected using the cell-permeable substrate D-luciferin. An advantage of the SFLC assay is that dynamic changes in PPIs in a cell can be detected in a near real-time manner. Another advantage is the unusually high DNA co-transfection and protein expression efficiencies that can be achieved in plant protoplasts, thereby enhancing the throughput of the method. Curr. Protoc. Mol. Biol. 107:20.9.1-20.9.8. © 2014 by John Wiley & Sons, Inc.
    Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 01/2014; 107:20.9.1-8.
  • Jian-Feng Li, Dandan Zhang, Jen Sheen
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    ABSTRACT: Targeted modification of plant genome is key to elucidating and manipulating gene functions in plant research and biotechnology. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is emerging as a powerful genome-editing method in diverse plants that traditionally lacked facile and versatile tools for targeted genetic engineering. This technology utilizes easily reprogrammable guide RNAs (sgRNAs) to direct Streptococcus pyogenes Cas9 endonuclease to generate DNA double-stranded breaks in targeted genome sequences, which facilitates efficient mutagenesis by error-prone nonhomologous end-joining (NHEJ) or sequence replacement by homology-directed repair (HDR). In this chapter, we describe the procedure to design and evaluate dual sgRNAs for plant codon-optimized Cas9-mediated genome editing using mesophyll protoplasts as model cell systems in Arabidopsis thaliana and Nicotiana benthamiana. We also discuss future directions in sgRNA/Cas9 applications for generating targeted genome modifications and gene regulations in plants.
    Methods in enzymology. 01/2014; 546C:459-472.
  • Nature Biotechnology 08/2013; 31(8):688-91. · 32.44 Impact Factor
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    ABSTRACT: Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5' coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.
    The Plant Cell 05/2013; · 9.25 Impact Factor
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    Jian-Feng Li, Jen Sheen
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    ABSTRACT: DNA purification is a routine procedure in most plant laboratories. Although different kits are available in the market allowing convenient DNA purification, the cumulative cost of purchasing multiple kits for a laboratory can be staggering. Here, we describe a protocol using homemade silicon dioxide matrix for DNA purification from Escherichia coli and Agrobacterium tumefaciens cells, PCR and restriction digestion mixtures, agarose gel slices and plant tissues. Compared with the commercial kits, this protocol enables easy DNA purification from diverse sources with comparable yield and purity at negligible expenses.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 862:53-9. · 1.29 Impact Factor
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    ABSTRACT: Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.
    PLoS ONE 01/2011; 6(11):e27364. · 3.53 Impact Factor
  • Jian-Feng Li, Andreas Nebenführ
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    ABSTRACT: Genome sequencing has identified a massive number of uncharacterized genes in Arabidopsis thaliana and several other plant species. To decipher these unknown gene functions, several transient expression assays have been developed as rapid and convenient alternatives to the lengthy creation of transgenic plants. As one of these transient assays, Agrobacterium-mediated transformation harnesses the natural capability of Agrobacterium to transfer foreign DNA into plant cells with intact cell walls. However, pioneering applications of Agrobacterium-based transient transformation to Arabidopsis have led to rather limited success with great variability. In this protocol, we describe a Fast Agrobacterium-mediated Seedling Transformation (FAST) technique for transient gene expression analysis in Arabidopsis and other dicot or monocot species. This technique makes use of the cocultivation of young plant seedlings with Agrobacterium in the presence of the surfactant Silwet L-77. The young seedlings can be grown easily and were found to be more susceptible to Agrobacterium transformation compared with adult plants. The surfactant facilitates transformation of plant cells, thus replacing wounding or a device-dependent vacuum step during plant transformation. This protocol provides a quick, efficient, and economical assay for gene function in intact plants with minimal manual handling and without the need for a dedicated device.
    Cold Spring Harbor Protocols 05/2010; 2010(5):pdb.prot5428. · 4.63 Impact Factor
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    Jian-Feng Li, Li Li, Jen Sheen
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    ABSTRACT: Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 mul water, leading to high DNA concentrations (>1 mug/mul) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.
    Plant Methods 01/2010; 6(1):1. · 2.59 Impact Factor
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    ABSTRACT: Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes. We present a novel transient assay based on cocultivation of young Arabidopsis (Arabidopsis thaliana) seedlings with Agrobacterium tumefaciens in the presence of a surfactant which does not require any dedicated equipment and can be carried out within one week from sowing seeds to protein analysis. This Fast Agro-mediated Seedling Transformation (FAST) was used successfully to express a wide variety of constructs driven by different promoters in Arabidopsis seedling cotyledons (but not roots) in diverse genetic backgrounds. Localizations of three previously uncharacterized proteins were identified by cotransformation with fluorescent organelle markers. The FAST procedure requires minimal handling of seedlings and was also adaptable for use in 96-well plates. The high transformation efficiency of the FAST procedure enabled protein detection from eight transformed seedlings by immunoblotting. Protein-protein interaction, in this case HY5 homodimerization, was readily detected in FAST-treated seedlings with Förster resonance energy transfer and bimolecular fluorescence complementation techniques. Initial tests demonstrated that the FAST procedure can also be applied to other dicot and monocot species, including tobacco, tomato, rice and switchgrass. The FAST system provides a rapid, efficient and economical assay of gene function in intact plants with minimal manual handling and without dedicated device. This method is potentially ideal for future automated high-throughput analysis.
    Plant Methods 06/2009; 5:6. · 2.59 Impact Factor
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    Jian-Feng Li, Andreas Nebenführ
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    ABSTRACT: Cytoplasmic streaming is a ubiquitous process in plant cells that is thought to be driven by the active movement of myosin XI motor proteins along actin filaments. These myosin motors bind to organelles through their C-terminal globular tail domain, although recent studies have also suggested a role for the central coiled-coil region during organelle binding. Here we have investigated the relationship between these two protein domains of MYA1, an Arabidopsis myosin XI, in a series of in vivo experiments demonstrating that dimerization of the coiled-coil region stabilizes organelle binding of the globular tail. Surprisingly, yeast two-hybrid assays, bimolecular fluorescence complementation, Förster resonance energy transfer and in vitro pull-down experiments all demonstrated that dimerization of the 174-residue MYA1 coiled coils by themselves was unstable. Furthermore, only the first of the two major coiled-coil segments in MYA1 contributed significantly to dimer formation. Interestingly, dimerization of myosin tail constructs that included the organelle-binding globular tail was stable, although the globular tails by themselves did not interact. This suggests an inter-dependent relationship between dimerization and organelle binding in myosin XI, whereby each process synergistically stimulates the other.
    The Plant Journal 07/2008; 55(3):478-90. · 6.58 Impact Factor
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    Jian-Feng Li, Andreas Nebenführ
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    ABSTRACT: Actin-based organelle movements are driven by the related multifunctional myosin motors of class V in animals and fungi and class XI in plants. The versatility of these motors depends critically on their C-terminal globular tail domain that allows them to bind to a broad variety of cargo molecules. Regulation of this motor-cargo attachment is frequently employed to modulate organelle movement. While the overall structure of the cargo-binding globular tail appears to be conserved between myosin V and XI, it has become apparent that the motor-cargo interactions differ widely even within a single organism and involve protein complexes with different architecture and completely unrelated protein domains. At the same time, indirect evidence suggests that adaptor or receptor dimerization could facilitate efficient myosin capture. Comparison of myosin V and XI across the large evolutionary distance between animals and plants will likely reveal more fundamental insights into these important motors.
    Traffic 04/2008; 9(3):290-8. · 4.65 Impact Factor
  • Jian-Feng Li, Andreas Nebenführ
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    ABSTRACT: Myosin XI are actin-based molecular motors that are thought to drive organelle movements in plants, analogous to myosin V in animals and fungi. Similar domain structure of these myosins suggests that binding to organelles may occur via the globular tail domain in both types of motors, even though sequence similarity is low. To address this hypothesis, we developed a structure homology model for the globular tail of MYA1, a myosin XI from Arabidopsis, based on the known structure of yeast myosin V (Myo2p) globular tail. This model suggested an interaction between two subdomains of the globular tail which was verified by yeast two-hybrid assay and by in vivo bimolecular fluorescence complementation (BiFC). Interface mapping demonstrated that this subdomain interaction depends critically on the C terminus of helix H6 as well as three specific residues in helices H3 and H15, consistent with the structural prediction. The reconstituted globular tails of several Arabidopsis myosin XIs in BiFC assays targeted to peroxisomes in plant cells, identifying this domain as sufficient for cargo binding. Unlike myosin V, either subdomain of myosin XI alone was targeting-competent and responsible for association with different organelles. In addition, our data suggest that organelle binding is regulated by an allosteric interaction between two tail subdomains. We conclude that the globular tail of myosin XI shares a similar structure with that of myosin V, but has evolved plant-specific cargo binding mechanisms.
    Journal of Biological Chemistry 08/2007; 282(28):20593-602. · 4.65 Impact Factor

Publication Stats

245 Citations
80.57 Total Impact Points

Institutions

  • 2012–2014
    • Massachusetts General Hospital
      • • Molecular Biology Laboratory
      • • Department of Molecular Biology
      Boston, Massachusetts, United States
  • 2009–2013
    • Harvard Medical School
      • Department of Genetics
      Boston, MA, United States
  • 2007–2010
    • University of Tennessee
      • Department of Biochemistry and Cellular and Molecular Biology
      Knoxville, TN, United States
  • 2008
    • The University of Tennessee Medical Center at Knoxville
      Knoxville, Tennessee, United States