VIGS-mediated <i>MPF2</i> silencing phenocopies <i>MPF2</i>-RNAi.

<p>(<b>A</b>) An intact WT flower. (<b>B</b>) An intact <i>MPF2</i>-VIGS flower. Bars = 1 mm. (<b>C</b>) I₂-KI stained pollen from WT. (<b>D</b>) I₂-KI stained pollen from <i>MPF2</i>-VIGS. Active pollen is blue and sterile pollen is tawny. Bars = 100 µm. (<b>E</b>) Floral calyx epidermal cells of WT. (<b>F</b>) Floral calyx epidermal cells of <i>MPF2</i>-VIGS. Bars = 20 µm. (<b>G</b>) Size of calyx surface (gray column) and epidermal cells (white column) of the floral calyx in WT and <i>MPF2</i>-VIGS (“VIGS”). 20 cells and 20 calyces were analyzed for both WT and <i>MPF2</i>-VIGS samples. Mean values and standard deviation are presented. (<b>H</b>) Gene expression analysis of <i>MPF2</i>-RNAi and -VIGS. Expression of <i>MPF2</i> was compared between <i>MPF2</i>-RNAi flowers (R1–R3), <i>MPF2</i>-VIGS flowers (V1–V3) and wild-type (WT) <i>Physalis</i> via qRT-PCR analysis. The severe <i>MPF2</i> residual in VIGS was only 6% of that in the wild-type (WT), while in the RNAi the <i>MPF2</i> residual was 14% of that in the wild-type (WT). <i>PFACTIN</i> was used as an internal control. (<b>I</b>) <i>MPF2</i> expression was evaluated in two floral organs of VIGS flowers. Expression of <i>MPF2</i> in <i>MPF2</i>-RNAi (gray column), <i>MPF2</i>-VIGS (white column) was compared with that in the wild-type (WT, black column). The gene expression in the calyx of the WT was set as 1, and <i>PFACTIN</i> was used as an internal control. The experiments were repeated with three independent biological samples. Mean expression values and standard deviation are presented.</p

VIGS-mediated <i>MPF3</i> silencing phenocopies <i>MPF3</i>-RNAi.

<p>(<b>A</b>) An intact WT small flower bud. (<b>B</b>) An intact WT flower bud. (<b>C</b>) A WT flower. (<b>D</b>) A WT ICS. (<b>E</b>) An intact <i>MPF3</i>-VIGS small flower bud. (<b>F</b>) An intact <i>MPF3</i>-VIGS flower bud. (<b>G</b>) An <i>MPF3</i>-VIGS flower. (<b>H</b>) An <i>MPF3</i>-VIGS ICS. (<b>I</b>) An <i>MPF3</i>-RNAi flower. Bars = 1 mm in <b>A</b>, <b>B</b>, <b>C</b>, <b>E</b>, <b>F</b>, <b>G,</b> and <b>I</b>. Bars = 5 mm in <b>D</b> and <b>H</b>. (<b>J</b>) <i>MPF3</i> was silenced using a VIGS approach. <i>MPF3</i> expression was evaluated in five floral organs of VIGS flowers. (<b>K</b>) <i>MPF3</i> was silenced using an RNAi approach. <i>MPF3</i> expression was evaluated in five floral organs of RNAi flowers. Total RNA from the indicated mutated floral organs was subjected to qRT-PCR. Gene expression in pedicels of WT samples were set as 1, and <i>PFACTIN</i> was used as an internal control. The dark gray column stands for the gene expression in WT organs light gray column indicates the gene expression in the organs of the mutants, as indicated. The experiments were repeated with three independent biological samples. Mean expression values and standard deviation are presented.</p

Efficient Gene Silencing Mediated by Tobacco Rattle Virus in an Emerging Model Plant <i>Physalis</i>

<div><p>The fruit of <i>Physalis</i> has a berry and a novelty called inflated calyx syndrome (ICS, also named the ‘Chinese lantern’). Elucidation of the underlying developmental mechanisms of fruit diversity demands an efficient gene functional inference platform. Here, we tested the application of the tobacco rattle virus (TRV)-mediated gene-silencing system in <i>Physalis floridana</i>. First, we characterized the putative gene of a phytoene desaturase in <i>P. floridana</i> (<i>PfPDS</i>). Infecting the leaves of the <i>Physalis</i> seedlings with the <i>PfPDS</i>-<i>TRV</i> vector resulted in a bleached plant, including the developing leaves, floral organs, ICS, berry, and seed. These results indicated that a local VIGS treatment can efficiently induce a systemic mutated phenotype. qRT-PCR analyses revealed that the bleaching extent correlated to the mRNA reduction of the endogenous <i>PfPDS</i>. Detailed comparisons of multiple infiltration and growth protocols allowed us to determine the optimal methodologies for VIGS manipulation in <i>Physalis</i>. We subsequently utilized this optimized VIGS methodology to downregulate the expression of two MADS-box genes, <i>MPF2</i> and <i>MPF3</i>, and compared the resulting effects with gene-downregulation mediated by RNA interference (RNAi) methods. The VIGS-mediated gene knockdown plants were found to resemble the mutated phenotypes of floral calyx, fruiting calyx and pollen maturation of the RNAi transgenic plants for both <i>MPF2</i> and <i>MPF3</i>. Moreover, the two MADS-box genes were appeared to have a novel role in the pedicel development in <i>P. floridana</i>. The major advantage of VIGS-based gene knockdown lies in practical aspects of saving time and easy manipulation as compared to the RNAi. Despite the lack of heritability and mosaic mutation phenotypes observed in some organs, the TRV-mediated gene silencing system provides an alternative efficient way to infer gene function in various developmental processes in <i>Physalis</i>, thus facilitating understanding of the genetic basis of the evolution and development of the morphological diversities within the Solanaceae.</p></div

Local treatments induce a systemic syndrome.

<p>(<b>A</b>) A wild-type seedling of <i>P. floridana</i>. (<b>B</b>) Phenotypic variation in a seedling after <i>PfPDS</i>-<i>TRV2</i> infection for one week. Bars = 5.0 cm. (<b>C</b>) A 3-month old plant infected with <i>PfPDS</i>-<i>TRV2</i>. Bars = 1.5 cm. (<b>D–G</b>) Floral phenotypic variations. In comparison with wild-type floral bud (<b>D</b>) and mature flower (<b>F</b>), the floral bud (<b>E</b>) and mature flower (<b>G</b>) from the <i>PfPDS</i>-<i>TRV2</i> infected plants are bleached. Bars = 10 mm in <b>D</b> and <b>E</b>, and 25 mm in <b>F</b> and <b>G</b>. (<b>H</b>) ICS from wild-type <i>Physalis</i>. (<b>I</b>) Berry from wild-type <i>Physalis</i>. (<b>J</b>) Mosaic bleached ICS. (<b>K</b>) Completely bleached ICS. (<b>L</b>) Bleached berries with different bleaching degrees from the <i>PfPDS</i>-<i>TRV2</i> infected plants. Bars = 50 mm in <b>H</b>, <b>J</b> and <b>K</b>, and 25 mm in <b>I</b> and <b>L</b>. (<b>M</b>) Wild-type seeds. (<b>N</b>) Bleached seeds. Bars = 5 mm. (<b>O</b>) Expressions of <i>PfPDS</i> in the <i>PfPDS</i>-<i>TRV2</i> infected plants. The black column is for the wild type and the other columns are for the <i>PfPDS</i>-<i>TRV2</i> infected plants with different degrees of bleaching. qRT-PCR was performed using total RNA from the organs indicated. <i>PFACTIN</i> was used as an internal control. The experiments were repeated three times using independent biological samples. Mean expression values and standard deviation are presented.</p

Seedling survival and infected efficiency in different treatments.

<p>Seedling survival and infected efficiency in different treatments.</p

Organization of TRV-VIGS vectors.

<p><i>TRV</i> cDNA clones were placed in between duplicated CaMV 35S promoters (2×35 S) and the nopaline synthase terminator (NOSt) in a T-DNA vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085534#pone.0085534-Liu1" target="_blank">[22]</a>. RdRp: RNA-dependant RNA polymerase; MP: movement protein; 16 K: cysteine rich protein; Rz: self-cleaving ribozyme; CP: coat protein. <i>PfPDS-</i>, <i>MPF2-,</i> and <i>MPF3</i>-specific fragments were inserted separately into <i>TRV</i>2 using <i>Nco</i> I and <i>BamH</i> I restriction sites.</p

<i>MPF3</i> and <i>MPF2</i> regulate pedicel development and pedicel cell length.

<p>Pedicel cells from the WT (<b>A</b>), <i>MPF2</i>-RNAi (<b>B</b>), <i>MPF2</i>-VIGS (<b>C</b>), <i>MPF3</i>-RNAi (<b>D</b>), and <i>MPF3</i>-VIGS (<b>E</b>). Bars = 50 µm. (<b>F</b>) Quantification of the pedicel size (dark gray column) and the respective pedicel cells lengths (light gray column). The number of pedicels analyzed was 20 for each line above. The numbers of cells analyzed were 60 in WT, <i>MPF2</i>-RNAi and <i>MPF2</i>-VIGS, and 50 in <i>MPF3</i>-RNAi and <i>MPF3</i>-VIGS. Mean values and standard deviation are presented in both cases. Two-tailed <i>t</i>-test significance was given as follows: one star for <i>p</i><0.05, and two stars for <i>p</i><0.01.</p

Reduction of ETV1 is Identified as a Prominent Feature of Age-Related Cataract

To identify the inactive genes in cataract lenses and explore their function in lens epithelial cells (LECs). Lens epithelium samples obtained from both age-related cataract (ARC) patients and normal donors were subjected to two forms of histone H3 immunoprecipitation: H3K9ac and H3K27me3 chromatin immunoprecipitation (ChIP), followed by ChIP-seq. The intersection set of “active genes in normal controls” and “repressed genes in cataract lenses” was identified. To validate the role of a specific gene, ETV1, within this set, quantitative polymerase chain reaction (qPCR), western blot, and immunofluorescence were performed using clinical lens epithelium samples. Small interference RNA (siRNA) was utilized to reduce the mRNA level of ETV1 in cultured LECs. Following this, transwell assay and western blot was performed to examine the migration ability of the cells. Furthermore, RNA-seq analysis was conducted on both cell samples with ETV1 knockdown and control cells. Additionally, the expression level of ETV1 in LECs was examined using qPCR under H2O2 treatment. Six genes were identified in the intersection set of “active genes in normal controls” and “repressed genes in ARC lenses”. Among these genes, ETV1 showed the most significant fold-change decrease in the cataract samples compared to the control samples. After ETV1 knockdown by siRNA in cultured LECs, reduced cell migration was observed, along with a decrease in the expression of β-Catenin and Vimentin, two specific genes associated with cell migration. In addition, under the oxidative stress induced by H2O2 treatment, the expression level of ETV1 in LECs significantly decreased. Based on the findings of this study, it can be concluded that ETV1 is significantly reduced in human ARC lenses. The repression of ETV1 in ARC lenses appears to contribute to the disrupted differentiation of lens epithelium, which is likely caused by the inhibition of both cell differentiation and migration processes.</p

Facile Method To Prepare Microcapsules Inspired by Polyphenol Chemistry for Efficient Enzyme Immobilization

In this study, a method inspired by polyphenol chemistry is developed for the facile preparation of microcapsules under mild conditions. Specifically, the preparation process includes four steps: formation of the sacrificial template, generation of the polyphenol coating on the template surface, cross-linking of the polyphenol coating by cationic polymers, and removal of the template. Tannic acid (TA) is chosen as a representative polyphenol coating precursor for the preparation of microcapsules. The strong interfacial affinity of TA contributes to the formation of polyphenol coating through oxidative oligomerization, while the high reactivity of TA is in charge of reacting/cross-linking with cationic polymer polyethylenimine (PEI) through Schiff base/Michael addition reaction. The chemical/topological structures of the resultant microcapsules are simultaneously characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier Transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), <i>etc.</i> The wall thickness of the microcapsules could be tailored from 257 ± 20 nm to 486 ± 46 nm through changing the TA concentration. The microcapsules are then utilized for encapsulating glucose oxidase (GOD), and the immobilized enzyme exhibits desired catalytic activity and enhanced pH and thermal stabilities. Owing to the structural diversity and functional versatility of polyphenols, this study may offer a facile and generic method to prepare microcapsules and other kinds of functional porous materials

Combination of Redox Assembly and Biomimetic Mineralization To Prepare Graphene-Based Composite Cellular Foams for Versatile Catalysis

Graphene-based materials with hierarchical structures and multifunctionality have gained much interest in a variety of applications. Herein, we report a facile, yet universal approach to prepare graphene-based composite cellular foams (GCCFs) through combination of redox assembly and biomimetic mineralization enabled by cationic polymers. Specifically, cationic polymers (e.g., polyethyleneimine, lysozyme, etc.) could not only reduce and simultaneously assemble graphene oxide (GO) into cellular foams but also confer the cellular foams with mineralization-inducing capability, enabling the formation of inorganic nanoparticles (e.g., silica, titania, silver, etc.). The GCCFs show highly porous structure and appropriate structural stability, where nanoparticles are well distributed on the surface of the reduced GO. Through altering polymer/inorganic pairs, a series of GCCFs are synthesized, which exhibit much enhanced catalytic performance in enzyme catalysis, heterogeneous chemical catalysis, and photocatalysis compared to nanoparticulate catalysts