Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery

Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidin-desthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice

Overexpressing <i>GarWRKYs Arabidopsis</i> plants at germination stage under saltstress.

<p>(A) Seedlings of the WT and 2 <i>GarWRKY104</i>-overexpressing lines germinated on MS medium adding 150 mM NaCl; (B) Seeds from the WT and 3 <i>GarWRKY17</i>-overexpressing lines were germinated on MS medium; (C) Germination rate of WT and 2 <i>GarWRKY104</i>-overexpressing lines; (D) Germination rate of WT and 3 <i>GarWRKY17</i>-overexpressing lines. The data are mean ± SE of three biological replicates. * and ** indicate statistical significance at the 0.05 and 0.01 probability level, respectively.</p

Heat map of the expression patterns of 28 salt-responsive <i>GarWRKYs</i> from RNA-Seq and qRT-PCR data.

<p>Changes in expression levels are displayed from green (downregulated) to red (upregulated), as shown in the color gradient at the bottom right corner (color figure online). Heat maps were generated and hierarchical clustering was performed using Cluster 3.0 based on log2 fold-change data in response to NaCl stress. The normalized expression values from RNA-Seq data were provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126148#pone.0126148.s004" target="_blank">S4 Table</a>.</p

Characterization of 28 salt-responsive <i>GarWRKY</i> members.

<p>Characterization of 28 salt-responsive <i>GarWRKY</i> members.</p

Phylogenetic relationships and motif compositions of 28 salt-responsive GarWRKY members and their orthologs of <i>Arabidopsis</i>, rice and soybean.

<p>Unrooted phylogenetic tree was constructed by using MEGA4.0. The motifs identified by MEME software are represented by colored boxes. * indicated the genes induced under salt tress.</p

Expression patterns of <i>GarWRKY</i> genes in various tissues.

<p>Semi-quantitative RT-PCR was conducted under normal growth condition. The cotton actin gene was used as an internal reference.</p

Transcriptome-Wide Identification of Salt-Responsive Members of the <i>WRKY</i> Gene Family in <i>Gossypium aridum</i>

<div><p>WRKY transcription factors are plant-specific, zinc finger-type transcription factors. The WRKY superfamily is involved in abiotic stress responses in many crops including cotton, a major fiber crop that is widely cultivated and consumed throughout the world. Salinity is an important abiotic stress that results in considerable yield losses. In this study, we identified 109 WRKY genes (<i>GarWRKYs</i>) in a salt-tolerant wild cotton species <i>Gossypium aridum</i> from transcriptome sequencing data to elucidate the roles of these factors in cotton salt tolerance. According to their structural features, the predicted members were divided into three groups (Groups I–III), as previously described for <i>Arabidopsis</i>. Furthermore, 28 salt-responsive <i>GarWRKY</i> genes were identified from digital gene expression data and subjected to real-time quantitative RT-PCR analysis. The expression patterns of most <i>GarWRKY</i> genes revealed by this analysis are in good agreement with those revealed by RNA-Seq analysis. RT-PCR analysis revealed that 27 <i>GarWRKY</i> genes were expressed in roots and one was exclusively expressed in roots. Analysis of gene orthology and motif compositions indicated that WRKY members from <i>Arabidopsis</i>, rice and soybean generally shared the similar motifs within the same subgroup, suggesting they have the similar function. Overexpression-<i>GarWRKY17</i> and –<i>GarWRKY104</i> in <i>Arabidopsis</i> revealed that they could positively regulate salt tolerance of transgenic <i>Arabidopsis</i> during different development stages. The comprehensive data generated in this study provide a platform for elucidating the functions of WRKY transcription factors in salt tolerance of <i>G</i>. <i>aridum</i>. In addition, <i>GarWRKYs</i> related to salt tolerance identified in this study will be potential candidates for genetic improvement of cultivated cotton salt stress tolerance.</p></div

Phylogenetic analysis of WRKY domains in <i>G</i>. <i>aridum</i>.

<p>WRKY protein name with the suffix ‘N’ or ‘C’ indicates the N-terminal WRKY domains or the C-terminal WRKY domains. The black arcs indicate different groups of WRKY domains. ● represent AtWRKY proteins, ► represent salt-responsive GarWRKY proteins.</p