A Synthetic Multidomain Peptide That Drives a Macropinocytosis-Like Mechanism for Cytosolic Transport of Exogenous Proteins into Plants

Direct delivery of proteins into plants represents a promising alternative to conventional gene delivery for probing and modulating cellular functions without the risk of random integration of transgenes into the host genome. This remains challenging, however, because of the lack of a protein delivery tool applicable to diverse plant species and the limited information about the entry mechanisms of exogenous proteins in plant cells. Here, we present the synthetic multidomain peptide (named dTat-Sar-EED4) for cytosolic protein delivery in various plant species via simple peptide-protein coincubation. dTat-Sar-EED4 enabled the cytosolic delivery of an active enzyme with up to ∼20-fold greater efficiency than previously described cell-penetrating peptides in several model plant systems. Our analyses using pharmacological inhibitors and transmission electron microscopy revealed that dTat-Sar-EED4 triggered a unique endocytic mechanism for cargo protein internalization. This endocytic mechanism shares several features with macropinocytosis, including the dependency of actin polymerization, sensitivity to phosphatidylinositol-3 kinase activity, and formation of membrane protrusions and large intracellular vesicles (>200 nm in diameter), even though macropinocytosis has not been identified to date in plants. Our study thus presents a robust molecular tool that can induce a unique cellular uptake mechanism for the efficient transport of bioactive proteins into plants

Design of an Artificial Peptide Inspired by Transmembrane Mitochondrial Protein for Escorting Exogenous DNA into the Mitochondria to Restore their Functions by Simultaneous Multiple Gene Expression

新規ミトコンドリア膜貫通ペプチドによる遺伝子送達 --ミトコンドリア内部で効率的な多重遺伝子発現を達成--. 京都大学プレスリリース. 2023-11-02.Mitochondria are vital organelles regulating essential cellular functions. Human mitochondrial DNA (mtDNA) consists of 37 genes, 13 of which encode mitochondrial proteins, and the remaining 24 genes encode two ribosomal RNAs and 22 transfer RNAs needed for the translation of the mtDNA-encoded 13 proteins. However, mtDNA often impairs the expression and function of these genes due to various mutations, ultimately causing mitochondrial dysfunction. To recover from this desperate condition, developing the technology to supply all mitochondrial proteins encoded by mtDNA at once is an urgent task, but there is no established strategy for this purpose. In this study, a simple yet effective mitochondrial gene delivery system is proposed comprising an artificial peptide inspired by a transmembrane mitochondrial membrane protein. The designed mitochondria-targeting peptides presented on the carrier surface effectively guide the encapsulated plasmid to the mitochondria, facilitating mitochondrial uptake and gene expression. The developed system successfully delivers exogenous mtDNA to mtDNA-depleted cells and leads to simultaneous multigene expression, ultimately restoring mitochondrial functions, including the mitochondrial respiration rate. The established multiple gene expression system in each mitochondrion is a game-changing technology that can accelerate the development of mitochondrial engineering technologies as well as clinical applications for mitochondrial diseases

Current Immunotherapeutic Approaches in Pancreatic Cancer

Pancreatic cancer is a highly aggressive and notoriously difficult to treat. As the vast majority of patients are diagnosed at advanced stage of the disease, only a small population is curative by surgical resection. Although gemcitabine-based chemotherapy is typically offered as standard of care, most patients do not survive longer than 6 months. Thus, new therapeutic approaches are needed. Pancreatic cancer cells that develop gemcitabine resistance would still be suitable targets for immunotherapy. Therefore, one promising treatment approach may be immunotherapy that is designed to target pancreatic-cancer-associated antigens. In this paper, we detail recent work in immunotherapy and the advances in concept of combination therapy of immunotherapy and chemotherapy. We offer our perspective on how to increase the clinical efficacy of immunotherapies for pancreatic cancer

Evolution of an assembly factor-based subunit contributed to a novel NDH-PSI supercomplex formation in chloroplasts

The chloroplast NDH complex interacts with Photosystem I to form the NDH-PSI supercomplex. Here the authors show that Arabidopsis NDF5 shares a common ancestor with the NDH subunit PnsB2 and acts as an NDH assembly factor initiating the assembly of PnsB2 and the evolutionarily distinct PnsB3

Suppression of Repeat-Mediated Gross Mitochondrial Genome Rearrangements by RecA in the Moss Physcomitrella patens[C][W]

RecA and its ubiquitous homologs are crucial components in homologous recombination. Besides their eukaryotic nuclear counterparts, plants characteristically possess several bacterial-type RecA proteins localized to chloroplasts and/or mitochondria, but their roles are poorly understood. Here, we analyzed the role of the only mitochondrial RecA in the moss Physcomitrella patens. Disruption of the P. patens mitochondrial recA gene RECA1 caused serious defects in plant growth and development and abnormal mitochondrial morphology. Analyses of mitochondrial DNA in disruptants revealed that frequent DNA rearrangements occurred at multiple loci. Structural analysis suggests that the rearrangements, which in some cases were associated with partial deletions and amplifications of mitochondrial DNA, were due to aberrant recombination between short (<100 bp) direct and inverted repeats in which the sequences were not always identical. Such repeats are abundant in the mitochondrial genome, and interestingly many are located in group II introns. These results suggest that RECA1 does not promote but rather suppresses recombination among short repeats scattered throughout the mitochondrial genome, thereby maintaining mitochondrial genome stability. We propose that RecA-mediated homologous recombination plays a crucial role in suppression of short repeat-mediated genome rearrangements in plant mitochondria

Non-transgenic Gene Modulation via Spray Delivery of Nucleic Acid/Peptide Complexes into Plant Nuclei and Chloroplasts

【研究成果】スプレーで植物を改変 --簡便な非遺伝子組換え植物改変法の開発--. 京都大学プレスリリース. 2022-02-24.Genetic engineering of economically important traits in plants is an effective way to improve global welfare. However, introducing foreign DNA molecules into plant genomes to create genetically engineered plants not only requires a lengthy testing period and high developmental costs but also is not well-accepted by the public due to safety concerns about its effects on human and animal health and the environment. Here, we present a high-throughput nucleic acids delivery platform for plants using peptide nanocarriers applied to the leaf surface by spraying. The translocation of sub-micrometer-scale nucleic acid/peptide complexes upon spraying varied depending on the physicochemical characteristics of the peptides and was controlled by a stomata-dependent-uptake mechanism in plant cells. We observed efficient delivery of DNA molecules into plants using cell-penetrating peptide (CPP)-based foliar spraying. Moreover, using foliar spraying, we successfully performed gene silencing by introducing small interfering RNA molecules in plant nuclei via siRNA-CPP complexes and, more importantly, in chloroplasts via our CPP/chloroplast-targeting peptide-mediated delivery system. This technology enables effective nontransgenic engineering of economically important plant traits in agricultural systems

DataSheet_1_Functional peptide-mediated plastid transformation in tobacco, rice, and kenaf.pdf

In plant engineering, plastid transformation is more advantageous than nuclear transformation because it results in high levels of protein expression from multiple genome copies per cell and is unaffected by gene silencing. The common plastid transformation methods are biolistic bombardment that requires special instruments and PEG-mediated transformation that is only applicable to protoplast cells. Here, we aimed to establish a new plastid transformation method in tobacco, rice, and kenaf using a biocompatible fusion peptide as a carrier to deliver DNA into plastids. We used a fusion peptide, KH-AtOEP34, comprising a polycationic DNA-binding peptide (KH) and a plastid-targeting peptide (AtOEP34) to successfully deliver and integrate construct DNA into plastid DNA (ptDNA) via homologous recombination. We obtained transformants in each species using selection with spectinomycin/streptomycin and the corresponding resistance gene aadA. The constructs remained in ptDNA for several months after introduction even under non-selective condition. The transformants normally flowered and are fertile in most cases. The offspring of the transformants (the T1 generation) retained the integrated construct DNA in their ptDNA, as indicated by PCR and DNA blotting, and expressed GFP in plastids from the integrated construct DNA. In summary, we successfully used the fusion peptide method for integration of foreign DNA in tobacco, rice, and kenaf ptDNA, and the integrated DNA was transmitted to the next generations. Whereas optimization is necessary to obtain homoplasmic plastid transformants that enable stable heterologous expression of genes, the plastid transformation method shown here is a novel nanomaterial-based approach distinct from the conventional methods, and we propose that this easy method could be used to target a wide variety of plants.</p

RECG Maintains Plastid and Mitochondrial Genome Stability by Suppressing Extensive Recombination between Short Dispersed Repeats

<div><p>Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss <i>Physcomitrella patens</i>. In this study, we studied a newly identified <i>P</i>. <i>patens</i> homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. <i>RECG</i> partially complements <i>recG</i> deficiency in <i>Escherichia coli</i> cells. A knockout (KO) mutation of <i>RECG</i> caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the <i>RECA1</i> KO mutant. The <i>RECG</i> KO cells showed heterogeneity in these phenotypes. Analyses of <i>RECG</i> KO plants showed that mitochondrial genome was destabilized due to a recombination between 8–79 bp repeats and the pattern of the recombination partly differed from that observed in the <i>RECA1</i> KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic <i>RECG</i> KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of <i>RECG</i> KO plants. Some of the induced recombination caused efficient genomic rearrangements in <i>RECG</i> KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the <i>RECG</i> KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12–63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.</p></div

Mitochondrial transcripts in <i>RECG</i> KO plants.

<p><b>A</b> and <b>B</b>. Detailed qRT-PCR analysis of <i>nad7</i> (A) and <i>nad9</i> (B) transcripts. Positions of the primers used in the qPCR are schematically represented in the upper parts of the panels. Coding regions are represented by grey boxes. Positions and directions of the primers are shown by arrows. Positions of the repeats involved in the rearrangements between <i>nad7</i> and <i>nad9</i> are indicated by triangles. Relative levels of segments of mitochondrial transcripts from <i>nad7</i> and <i>nad9</i> were normalized to reference of nuclear gene ST-P 2a transcript. WT was given a value of 1. Slight or no amplification was observed in no reverse-transcription controls (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005080#pgen.1005080.s005" target="_blank">S5G Fig.</a>). The data represent mean of three replicates ± SD. *p<0.01 (versus WT). <b>C</b>. RT-PCR analysis of <i>nad7-nad9</i> chimeric transcripts. <i>nad7-nad9</i> chimeric transcripts were amplified using cDNA from WT and <i>RECG</i> KO lines at cycles indicated on the left of the picture. Actin was amplified as an internal control.</p

Complementation of the <i>E</i>. <i>coli recG</i> defect by <i>RECG</i>.

<p><i>E</i>. <i>coli recG</i>-deficient cells harboring a plasmid carrying the <i>P</i>. <i>patens RECG</i> (+Pp <i>RECG</i>, triangle), <i>E</i>. <i>coli recG</i> (+Ec <i>recG</i>, circle) or empty vector (Δ<i>recG</i>, square) were subjected to UV irradiation, and the surviving fraction was calculated as described in Materials and Methods. Data from three independent experiments are expressed as mean ± SD. *p<0.01 (versus Δ<i>recG</i>).</p