Inducible Biosynthetic Nanoscaffolds as Recruitment Platforms for Detecting Molecular Target Interactions inside Living Cells

We present a novel phenotypic readout using inducible, biosynthetic nanoscaffolds to directly visualize dynamic molecular interactions within living cells at the single-cell level with high sensitivity and selectivity. Labeled ferritin is used to form biological nanoparticles inside cells. Specific supramolecular assembly of ferritin-derived nanoparticles induces highly clustered nanoscaffolds. These inducible biosynthetic nanoscaffolds are used as the artificial recruitment/redistribution platform for monitoring interactions of a small molecule with its target protein(s) inside living cells

Inducible Biosynthetic Nanoscaffolds as Recruitment Platforms for Detecting Molecular Target Interactions inside Living Cells

We present a novel phenotypic readout using inducible, biosynthetic nanoscaffolds to directly visualize dynamic molecular interactions within living cells at the single-cell level with high sensitivity and selectivity. Labeled ferritin is used to form biological nanoparticles inside cells. Specific supramolecular assembly of ferritin-derived nanoparticles induces highly clustered nanoscaffolds. These inducible biosynthetic nanoscaffolds are used as the artificial recruitment/redistribution platform for monitoring interactions of a small molecule with its target protein(s) inside living cells

Inducible Biosynthetic Nanoscaffolds as Recruitment Platforms for Detecting Molecular Target Interactions inside Living Cells

We present a novel phenotypic readout using inducible, biosynthetic nanoscaffolds to directly visualize dynamic molecular interactions within living cells at the single-cell level with high sensitivity and selectivity. Labeled ferritin is used to form biological nanoparticles inside cells. Specific supramolecular assembly of ferritin-derived nanoparticles induces highly clustered nanoscaffolds. These inducible biosynthetic nanoscaffolds are used as the artificial recruitment/redistribution platform for monitoring interactions of a small molecule with its target protein(s) inside living cells

Inducible Biosynthetic Nanoscaffolds as Recruitment Platforms for Detecting Molecular Target Interactions inside Living Cells

We present a novel phenotypic readout using inducible, biosynthetic nanoscaffolds to directly visualize dynamic molecular interactions within living cells at the single-cell level with high sensitivity and selectivity. Labeled ferritin is used to form biological nanoparticles inside cells. Specific supramolecular assembly of ferritin-derived nanoparticles induces highly clustered nanoscaffolds. These inducible biosynthetic nanoscaffolds are used as the artificial recruitment/redistribution platform for monitoring interactions of a small molecule with its target protein(s) inside living cells

Effect of exogenous chemical treatments (SA, MeJA, or COR) on the flg22-triggered oxidative burst.

<p>(A-C) Arabidopsis seedlings were pre-incubated with various concentrations of chemicals for the indicated time periods (0 and 24 h) before the start of ROS measurements. Flg22 (1 µM) was added at zero time. Error bars represent the SD of five independent samples (n = 10) and similar results were obtained in three independent experiments.</p

Generating <i>In Vivo</i> Cloning Vectors for Parallel Cloning of Large Gene Clusters by Homologous Recombination

<div><p>A robust method for the <i>in vivo</i> cloning of large gene clusters was developed based on homologous recombination (HR), requiring only the transformation of PCR products into <i>Escherichia coli</i> cells harboring a receiver plasmid. Positive clones were selected by an acquired antibiotic resistance, which was activated by the recruitment of a short ribosome-binding site plus start codon sequence from the PCR products to the upstream position of a silent antibiotic resistance gene in receiver plasmids. This selection was highly stringent and thus the cloning efficiency of the GFPuv gene (size: 0.7 kb) was comparable to that of the conventional restriction-ligation method, reaching up to 4.3 × 10⁴ positive clones per μg of DNA. When we attempted parallel cloning of GFPuv fusion genes (size: 2.0 kb) and carotenoid biosynthesis pathway clusters (sizes: 4 kb, 6 kb, and 10 kb), the cloning efficiency was similarly high regardless of the DNA size, demonstrating that this would be useful for the cloning of large DNA sequences carrying multiple open reading frames. However, restriction analyses of the obtained plasmids showed that the selected cells may contain significant amounts of receiver plasmids without the inserts. To minimize the amount of empty plasmid in the positive selections, the <i>sacB</i> gene encoding a levansucrase was introduced as a counter selection marker in receiver plasmid as it converts sucrose to a toxic levan in the <i>E. coli</i> cells. Consequently, this method yielded completely homogeneous plasmids containing the inserts via the direct transformation of PCR products into <i>E. coli</i> cells.</p> </div

Restored antibiotic resistance and GFPuv expression by homologous recombination.

<p>(A) <i>E. coli</i> JM109 cells containing the pRMT plasmid were sensitive to 25 μg/l of chloramphenicol in LB broth because the chloramphenicol resistance gene is silent in the receiver plasmid. (B) GFPuv expression is observed in the positive colonies. (C) The effect of the amount of DNA insert on <i>in </i><i>vivo</i> cloning. The DNA inserts were transformed into the cells containing the pRMT vector by electroporation. The colony number increases with higher concentrations of the insert used for electroporation. (D) The effect of the homologous sequence length on the pRMT system. </p

The Activated SA and JA Signaling Pathways Have an Influence on flg22-Triggered Oxidative Burst and Callose Deposition

<div><p>The first line of defense in plants against pathogens is induced by the recognition of microbe-associated molecular patterns (MAMP). Perception of bacterial flagellin (flg22) by the pattern recognition receptor flagellin-sensing 2 (FLS2) is the best characterized MAMP response, although the underlying molecular mechanisms are not fully understood. Here we studied the relationship between salicylic acid (SA) or jasmonic acid (JA) signaling and FLS2-mediated signaling by monitoring flg22-triggered responses in known SA or JA related mutants of <i>Arabidopsis thaliana</i> (L.) Heynh. The <i>sid2</i> mutant, impaired in SA biosynthesis, had less basal <i>FLS2</i> mRNA accumulation than the wild type, which correlated with suppression of early flg22 responses such as ROS production and induction of marker genes, <i>WRKY29</i> and <i>FRK1</i>. The JA-signaling mutants, <i>jar1</i> and <i>coi1,</i> exhibited an enhanced flg22-triggered oxidative burst and more callose accumulation than the wild type, and pretreatment with SA or coronatine (COR), a structural mimic of JA-isoleucine, altered these flg22-induced responses. Nonexpressor of pathogenesis-related genes 1 (NPR1) acted downstream of SID2 and required SA-dependent priming for the enhanced flg22-triggered oxidative burst and callose deposition. Activation of JA signaling by COR pretreatment suppressed the flg22-triggered oxidative burst and callose accumulation in a coronatine insensitive 1 (COI1) dependent manner. COR had a negative effect on flg22 responses but only the flg22-triggered oxidative burst depended on SA-JA/COR signaling antagonism. Thus the activated SA and JA signaling pathways have an influence on flg22-triggered oxidative burst and callose deposition. These results may explain how SA and JA signaling are cross talked for regulation of flg22-triggered responses.</p></div

The homogeneity problem and the solution using a counter selection marker, <i>sacB</i>.

<p>(A) Shown are the genetic structure and scheme of the pRMT and pRMT-sacB plasmids when the GFPuv gene was recombined into both plasmids. (B) Shown is the restriction analysis of the target plasmids from selected hits. upper and lower panels represent the electrophoresis results from the pRMT and pRMT-sacB plasmids, respectively, digested with <i>Bgl</i>II and <i>Sca</i>I. The C- and C+ represent the indicator bands of one without and one with the insert, respectively while the GFPuv gene was used as a target gene.</p