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1.
bioRxiv ; 2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38895378

ABSTRACT

The formation of functional epithelial tubules is a central feature of many organ systems. Although the process of tubule formation by epithelial cells is well-studied, the way in which tubules connect with each other (i.e. anastomose) to form functional networks both in vivo and in vitro is not well understood. A key, unanswered question in the kidney is how the renal vesicles of the embryonic kidney connect with the nascent collecting ducts to form a continuous urinary system. We performed a ligand-receptor pair analysis on single cell RNA-seq data from embryonic mouse kidney tubules undergoing anastomosis to select candidates that might mediate this process in vivo. This analysis identified hepatocyte growth factor (HGF), which has known roles in cell proliferation, migration, and tubulogenesis, as one of several possible candidates. To test this possibility, we designed a novel assay to quantitatively examine epithelial tubule anastomosis in vitro using epithelial spheroids with fluorescently-tagged apical surfaces to enable direct visualization of anastomosis. This revealed that HGF is a potent inducer of tubule anastomosis. Tubule anastomosis occurs through a proliferation-independent mechanism that acts through the MAPK signaling cascade and matrix metalloproteinases (MMPs), the latter suggestive of a role in extracellular matrix turnover. Accordingly, treatment of explanted embryonic mouse kidneys with HGF and collagenase was sufficient to induce kidney tubule anastomosis. These results lay the groundwork for investigating how to promote functional interconnections between tubular epithelia, which have important clinical implications for utilizing in vitro grown kidney tissue in transplant medicine.

2.
Plant J ; 119(1): 557-576, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38627952

ABSTRACT

Medicago truncatula is a model legume for fundamental research on legume biology and symbiotic nitrogen fixation. Tnt1, a retrotransposon from tobacco, was used to generate insertion mutants in M. truncatula R108. Approximately 21 000 insertion lines have been generated and publicly available. Tnt1 retro-transposition event occurs during somatic embryogenesis (SE), a pivotal process that triggers massive methylation changes. We studied the SE of M. truncatula R108 using leaf explants and explored the dynamic shifts in the methylation landscape from leaf explants to callus formation and finally embryogenesis. Higher cytosine methylation in all three contexts of CG, CHG, and CHH patterns was observed during SE compared to the controls. Higher methylation patterns were observed in assumed promoter regions (~2-kb upstream regions of transcription start site) of the genes, while lowest was recorded in the untranslated regions. Differentially methylated promoter region analysis showed a higher CHH methylation in embryogenesis tissue samples when compared to CG and CHG methylation. Strong correlation (89.71%) was identified between the differentially methylated regions (DMRs) and the site of Tnt1 insertions in M. truncatula R108 and stronger hypermethylation of genes correlated with higher number of Tnt1 insertions in all contexts of CG, CHG, and CHH methylation. Gene ontology enrichment and KEGG pathway enrichment analysis identified genes and pathways enriched in the signal peptide processing, ATP hydrolysis, RNA polymerase activity, transport, secondary metabolites, and nitrogen metabolism pathways. Combined gene expression analysis and methylation profiling showed an inverse relationship between methylation in the DMRs (regions spanning genes) and the expression of genes. Our results show that a dynamic shift in methylation happens during the SE process in the context of CG, CHH and CHG methylation, and the Tnt1 retrotransposition correlates with the hyperactive methylation regions.


Subject(s)
DNA Methylation , Gene Expression Regulation, Plant , Medicago truncatula , Plant Somatic Embryogenesis Techniques , Retroelements , Medicago truncatula/genetics , Medicago truncatula/metabolism , Retroelements/genetics , Genome, Plant/genetics , Promoter Regions, Genetic/genetics
3.
Antioxidants (Basel) ; 12(6)2023 May 30.
Article in English | MEDLINE | ID: mdl-37371916

ABSTRACT

The development of treatment strategies for human corneal endothelial cells (hCECs) disease is necessary because hCECs do not regenerate in vivo due to the properties that are similar to senescence. This study is performed to investigate the role of a p-Tyr42 RhoA inhibitor (MH4, ELMED Inc., Chuncheon) in transforming growth factor-beta (TGF-ß)- or H2O2-induced cellular senescence of hCECs. Cultured hCECs were treated with MH4. The cell shape, proliferation rate, and cell cycle phases were analyzed. Moreover, cell adhesion assays and immunofluorescence staining for F-actin, Ki-67, and E-cadherin were performed. Additionally, the cells were treated with TGF-ß or H2O2 to induce senescence, and mitochondrial oxidative reactive oxygen species (ROS) levels, mitochondrial membrane potential, and NF-κB translocation were evaluated. LC3II/LC3I levels were determined using Western blotting to analyze autophagy. MH4 promotes hCEC proliferation, shifts the cell cycle, attenuates actin distribution, and increases E-cadherin expression. TGF-ß and H2O2 induce senescence by increasing mitochondrial ROS levels and NF-κB translocation into the nucleus; however, this effect is attenuated by MH4. Moreover, TGF-ß and H2O2 decrease the mitochondrial membrane potential and induce autophagy, while MH4 reverses these effects. In conclusion, MH4, a p-Tyr42 RhoA inhibitor, promotes the regeneration of hCECs and protects hCECs against TGF-ß- and H2O2-induced senescence via the ROS/NF-κB/mitochondrial pathway.

4.
Antioxidants (Basel) ; 12(6)2023 Jun 07.
Article in English | MEDLINE | ID: mdl-37371958

ABSTRACT

Human corneal-endothelial cells (hCEnCs) are located on the inner layer of the cornea. Injury to CEnCs leads to permanent corneal edema, requiring corneal transplantation. NADPH oxidase 4 (NOX4) has been reported to be implicated in the pathogenesis of CEnCs diseases. Thus, we investigated the role of NOX4 in CEnCs in this study. In an animal study, siRNA for NOX4 (siNOX4) or plasmid for NOX4 (pNOX4) was introduced into the corneal endothelium of rats by electroporation, using a square-wave electroporator (ECM830, Havard apparatus) to decrease or increase the expression of NOX4, respectively, and the rat corneas were cryoinjured through contact with a metal rod of 3 mm diameter frozen in liquid nitrogen for 10 min. The immunofluorescence staining of NOX4 and 8-OHdG showed that the levels of NOX4 and 8-OHdG were decreased in the siNOX4 group compared to the siControl, and increased in the pNOX4 group compared to the pControl at one week after treatment. Without cryoinjury, corneal opacity was more severe, and the density of CEnCs was lower, in pNOX4-treated rats compared to pControl. After cryoinjury, the corneas were more transparent, and the CEnC density was higher, in siNOX4-treated rats. The hCEnCs were cultured and transfected with siNOX4 and pNOX4. The silencing of NOX4 in hCEnCs resulted in a normal cell shape, higher viability, and higher proliferation rate than those transfected with the siControl, while NOX4 overexpression had the opposite effect. NOX4 overexpression increased the number of senescent cells and intracellular oxidative stress levels. NOX4 overexpression increased ATF4 and ATF6 levels, and nuclear translocation of XBP-1, which is the endoplasmic reticulum (ER) stress marker, while the silencing of NOX4 had the opposite effect. Additionally, the mitochondrial membrane potential was hyperpolarized by the silencing of NOX4, and depolarized by NOX4 overexpression. The LC3II levels, a marker of autophagy, were decreased by the silencing of NOX4, and increased by NOX4 overexpression. In conclusion, NOX4 plays a pivotal role in the wound-healing and senescence of hCEnCs, by modulating oxidative stress, ER stress, and autophagy. The regulation of NOX4 may be a potential therapeutic strategy for regulating the homeostasis of CEnCs, and treating corneal-endothelial diseases.

5.
Nat Commun ; 13(1): 2581, 2022 05 11.
Article in English | MEDLINE | ID: mdl-35546550

ABSTRACT

Agrobacterium-mediated plant transformation (AMT) is the basis of modern-day plant biotechnology. One major drawback of this technology is the recalcitrance of many plant species/varieties to Agrobacterium infection, most likely caused by elicitation of plant defense responses. Here, we develop a strategy to increase AMT by engineering Agrobacterium tumefaciens to express a type III secretion system (T3SS) from Pseudomonas syringae and individually deliver the P. syringae effectors AvrPto, AvrPtoB, or HopAO1 to suppress host defense responses. Using the engineered Agrobacterium, we demonstrate increase in AMT of wheat, alfalfa and switchgrass by ~250%-400%. We also show that engineered A. tumefaciens expressing a T3SS can deliver a plant protein, histone H2A-1, to enhance AMT. This strategy is of great significance to both basic research and agricultural biotechnology for transient and stable transformation of recalcitrant plant species/varieties and to deliver proteins into plant cells in a non-transgenic manner.


Subject(s)
Plant Cells , Type III Secretion Systems , Agrobacterium tumefaciens/genetics , Agrobacterium tumefaciens/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Plant Cells/metabolism , Plant Diseases/microbiology , Pseudomonas syringae/genetics , Pseudomonas syringae/metabolism , Type III Secretion Systems/genetics , Type III Secretion Systems/metabolism
6.
PLoS One ; 17(5): e0264917, 2022.
Article in English | MEDLINE | ID: mdl-35594245

ABSTRACT

Nonhost disease resistance is the most common type of plant defense mechanism against potential pathogens. In the present study, the metabolic enzyme formate dehydrogenase 1 (FDH1) was identified to associate with nonhost disease resistance in Nicotiana benthamiana and Arabidopsis thaliana. In Arabidopsis, AtFDH1 was highly upregulated in response to both host and nonhost bacterial pathogens. The Atfdh1 mutants were compromised in nonhost resistance, basal resistance, and gene-for-gene resistance. The expression patterns of salicylic acid (SA) and jasmonic acid (JA) marker genes after pathogen infections in Atfdh1 mutant indicated that both SA and JA are involved in the FDH1-mediated plant defense response to both host and nonhost bacterial pathogens. Previous studies reported that FDH1 localizes to mitochondria, or both mitochondria and chloroplasts. Our results showed that the AtFDH1 mainly localized to mitochondria, and the expression level of FDH1 was drastically increased upon infection with host or nonhost pathogens. Furthermore, we identified the potential co-localization of mitochondria expressing FDH1 with chloroplasts after the infection with nonhost pathogens in Arabidopsis. This finding suggests the possible role of FDH1 in mitochondria and chloroplasts during defense responses against bacterial pathogens in plants.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Disease Resistance , Plant Diseases , Arabidopsis/enzymology , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cyclopentanes , Disease Resistance/genetics , Formate Dehydrogenases/genetics , Gene Expression Regulation, Plant , Plant Diseases/genetics , Plant Diseases/microbiology , Pseudomonas syringae/metabolism , Salicylic Acid/metabolism , Nicotiana
7.
Cells ; 12(1)2022 12 22.
Article in English | MEDLINE | ID: mdl-36611829

ABSTRACT

Damage to human corneal endothelial cells (hCECs) leads to bullous keratopathy because these cells cannot be regenerated in vivo. In this study, we investigated the protective role of microRNA (miR)-302a against interferon-γ (IFN-γ)-induced senescence and cell death of hCECs. Cultured hCECs were transfected with miR-302a and treated with IFN-γ (20 ng/mL) to evaluate the protective effect of miR-302a on IFN-γ-induced cell death. Senescence was evaluated by the senescence-associated ß-galactosidase (SA-ß-gal) assay, and the secretion of senescence-associated secretory phenotype (SASP) factors was analyzed. Mitochondrial function and endoplasmic reticulum (ER) stress were assessed. We revealed that miR-302a enhanced the cell viability and proliferation of hCECs and that IFN-γ increased the cell size, the number of SA-ß-gal-positive cells, and SASP factors, and arrested the cell cycle, which was eliminated by miR-302a. miR-302a ameliorated mitochondrial oxidative stress and ER stress levels which were induced by IFN-γ. IFN-γ decreased the mitochondrial membrane potential and promoted autophagy, which was eliminated by miR-302a. The in vivo study showed that regeneration of rat CECs was promoted in the miR-302a group by inhibiting IFN-γ and enhancing mitochondrial function. In conclusion, miR-302a eliminated IFN-γ-induced senescence and cellular damage by regulating the oxidative and ER stress, and promoting the proliferation of CECs. Therefore, miR-302a may be a therapeutic option to protect hCECs against IFN-γ-induced stress.


Subject(s)
Endothelial Cells , MicroRNAs , Humans , Rats , Animals , Endothelial Cells/metabolism , Interferon-gamma/metabolism , Cell Death , Cell Division , MicroRNAs/metabolism
8.
Mol Plant Microbe Interact ; 34(12): 1358-1364, 2021 Dec.
Article in English | MEDLINE | ID: mdl-34615361

ABSTRACT

E3 ubiquitin ligase salt- and drought-induced ring finger 1 (SDIR1) plays a novel role in modulating plant immunity against pathogens. The molecular interactors of SDIR1 during pathogen infection are not known. SDIR1-interacting jasmonate zinc-finger inflorescence meristem domain (JAZ) proteins were identified through a yeast two-hybrid (Y2H) screen. Full-length JAZ9 interacts with SDIR1 only in the presence of coronatine (a bacteria-secreted toxin) or jasmonic acid (JA) in a Y2H assay. The bimolecular fluorescence complementation and pull-down assays confirm the in planta interaction of these proteins. JAZ9 proteins, negative regulators of JA-mediated plant defense, were degraded during the pathogen infection by SDIR1 through a proteasomal pathway causing disease susceptibility against hemibiotrophic pathogens.[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2021.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Repressor Proteins , Ubiquitin-Protein Ligases , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cyclopentanes , Droughts , Gene Expression Regulation, Plant , Oxylipins , Plant Diseases , Plant Immunity , Plant Proteins , Sodium Chloride
9.
Int J Mol Sci ; 22(13)2021 Jul 01.
Article in English | MEDLINE | ID: mdl-34281196

ABSTRACT

Until recently, genes from the iron-sulfur (Fe-S) cluster pathway were not known to have a role in plant disease resistance. The Nitrogen Fixation S (NIFS)-like 1 (NFS1) and Mitochondrial Ferredoxin-1 (MFDX1) genes are part of a set of 27 Fe-S cluster genes induced after infection with host and nonhost pathogens in Arabidopsis. A role for AtNFS1 in plant immunity was recently demonstrated. In this work, we showed that MFDX1 is also involved in plant defense. More specifically, Arabidopsis mfdx1 mutants were compromised for nonhost resistance against Pseudomonas syringae pv. tabaci, and showed increased susceptibility to the host pathogen P. syringae pv. tomato DC3000. Arabidopsis AtMFDX1 overexpression lines were less susceptible to P. syringae pv. tomato DC3000. Metabolic profiling revealed a reduction of several defense-related primary and secondary metabolites, such as asparagine and glucosinolates in the Arabidopsis mfdx1-1 mutant when compared to Col-0. A reduction of 5-oxoproline and ornithine metabolites that are involved in proline synthesis in mitochondria and affect abiotic stresses was also observed in the mfdx1-1 mutant. In contrast, an accumulation of defense-related metabolites such as glucosinolates was observed in the Arabidopsis NFS1 overexpressor when compared to wild-type Col-0. Additionally, mfdx1-1 plants displayed shorter primary root length and reduced number of lateral roots compared to the Col-0. Taken together, these results provide additional evidence for a new role of Fe-S cluster pathway in plant defense responses.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Ferredoxins/genetics , Arabidopsis/immunology , Arabidopsis Proteins/immunology , Disease Resistance , Ferredoxins/immunology , Ferredoxins/metabolism , Glucosinolates/genetics , Glucosinolates/immunology , Iron/metabolism , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Mitochondria/metabolism , Multigene Family , Plant Diseases/genetics , Plant Diseases/immunology , Plant Immunity/genetics , Stress, Physiological/genetics , Sulfur/metabolism
10.
STAR Protoc ; 2(2): 100566, 2021 06 18.
Article in English | MEDLINE | ID: mdl-34159320

ABSTRACT

This protocol describes the analysis of protein cysteine redox status. Redox status is crucial in regulating protein activity, stability, and redox signaling cascades. It is determined by conjugation with 1.24 kDa MM(PEG)24 molecule to each reduced cysteine followed by western blot analysis. This protocol is easy to follow, and most of the reagents and instruments required are of common use in any lab. This protocol can be successfully applied to other biological sources. For complete details on the use and execution of this protocol, please refer to Pant et al. (2020).


Subject(s)
Cysteine/metabolism , Proteins/metabolism , Sulfhydryl Compounds/metabolism , Blotting, Western , Oxidation-Reduction , Proteins/chemistry , Proteomics/methods , Signal Transduction
11.
Mol Plant Microbe Interact ; 34(3): 297-308, 2021 Mar.
Article in English | MEDLINE | ID: mdl-33231502

ABSTRACT

Many plant-encoded E3 ligases are known to be involved in plant defense. Here, we report a novel role of E3 ligase SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1) in plant immunity. Even though SDIR1 is reasonably well-characterized, its role in biotic stress response is not known. The silencing of SDIR1 in Nicotiana benthamiana reduced the multiplication of the virulent bacterial pathogen Pseudomonas syringae pv. tabaci. The Arabidopsis sdir1 mutant is resistant to virulent pathogens, whereas SDIR1 overexpression lines are susceptible to both host and nonhost hemibiotrophic bacterial pathogens. However, sdir1 mutant and SDIR1 overexpression lines showed hypersusceptibility and resistance, respectively, against the necrotrophic pathogen Erwinia carotovora. The mutant of SDIR1 target protein, i.e., SDIR-interacting protein 1 (SDIR1P1), also showed resistance to host and nonhost pathogens. In SDIR1 overexpression plants, transcripts of NAC transcription factors were less accumulated and the levels of jasmonic acid (JA) and abscisic acid were increased. In the sdir1 mutant, JA signaling genes JAZ7 and JAZ8 were downregulated. These data suggest that SDIR1 is a susceptibility factor and its activation or overexpression enhances disease caused by P. syringae pv. tomato DC3000 in Arabidopsis. Our results show a novel role of SDIR1 in modulating plant defense gene expression and plant immunity.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Disease Resistance , Host-Pathogen Interactions , Ubiquitin-Protein Ligases , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Disease Resistance/genetics , Gene Expression Regulation, Plant , Host-Pathogen Interactions/genetics , Pectobacterium carotovorum/physiology , Plant Diseases/microbiology , Pseudomonas syringae/physiology , Nicotiana/enzymology , Nicotiana/microbiology , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism
12.
Cell Rep ; 33(11): 108512, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33326777

ABSTRACT

Global warming and emerging plant diseases challenge agricultural food/feed production. We identify mechanism(s) regulating both plant thermotolerance and disease resistance. Using virus-induced gene silencing (VIGS)-based genetic screening, we identify a thioredoxin-like 1 (TRXL1) gene involved in plant nonhost disease resistance and thermotolerance. TRXL1 is reduced, partly degraded via proteases and proteasome, and alters its chloroplast localization during heat stress. TRXL1 interacts with more than 400 proteins, including chaperonin CPN60A, caseinolytic protease (CLPC1), and NADP-dependent malate dehydrogenase (NADP-MDH). Chaperonin 60A (CPN60A) guards TRXL1 from degradation, whereas CLPC1 degrades TRXL1 during heat stress. TRXL1 regulates NADP-MDH activity, leading to an increase in malate level and inhibition of superoxide radical formation. We show that CPN60A and NADP-MDH positively regulate nonhost resistance, and CPN60A positively and CLPC1 negatively regulate thermotolerance. This study shows an antagonistic post-translational regulation of TRXL1 by CPN60A and CLPC1 and regulation of MDH by TRXL1, leading to plant disease resistance and thermotolerance.


Subject(s)
Chloroplasts/immunology , Plant Diseases/immunology , Disease Resistance , Thermotolerance
13.
Mol Plant Pathol ; 21(11): 1481-1494, 2020 11.
Article in English | MEDLINE | ID: mdl-32964634

ABSTRACT

Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10-silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA-sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two-hybrid assay. Silencing or mutants of genes encoding these RPL10-interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/RPL10 positively regulates the defence and translation-associated genes during nonhost pathogen infection.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Plant Diseases/immunology , Pseudomonas syringae/pathogenicity , Ribosomal Protein L10/metabolism , Ribosomal Proteins/metabolism , Solanum lycopersicum/microbiology , Arabidopsis/immunology , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Disease Resistance/genetics , Gene Expression , Mutation , Plant Diseases/microbiology , Protein Biosynthesis , RNA Interference , Ribosomal Protein L10/genetics , Ribosomal Proteins/genetics , Ribosomes/metabolism , Sequence Analysis, RNA , Nicotiana/genetics , Nicotiana/immunology , Nicotiana/microbiology , Two-Hybrid System Techniques
14.
Plant J ; 103(5): 1924-1936, 2020 08.
Article in English | MEDLINE | ID: mdl-32410353

ABSTRACT

Brachypodium distachyon is an annual C3 grass used as a monocot model system in functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using the tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis, generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed-derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1-5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 3.37 kb, we estimated that 29 613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1-based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon, which will aid in functional genomics studies of other C3 grasses.


Subject(s)
Brachypodium/genetics , Mutagenesis, Insertional , Plant Proteins/genetics , Retroelements/genetics , Chromosomes, Plant/genetics , Mutagenesis, Insertional/methods , Plant Proteins/metabolism , Plants, Genetically Modified
15.
Int J Mol Sci ; 19(7)2018 Jun 30.
Article in English | MEDLINE | ID: mdl-29966336

ABSTRACT

Plant defense responses at stomata and apoplast are the most important early events during plant⁻bacteria interactions. The key components of stomatal defense responses have not been fully characterized. A GTPase encoding gene, NOG1-2, which is required for stomatal innate immunity against bacterial pathogens, was recently identified. Functional studies in Arabidopsis revealed that NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimulus through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. Interestingly, in this study, Jasmonate-ZIM-domain protein 9 (JAZ9) was identified to interact with NOG1-2 for the regulation of stomatal closure. Upon interaction, JAZ9 reduces GTPase activity of NOG1-2. We explored the role of NOG1-2 binding with JAZ9 for COI1-mediated JA signaling and hypothesized that its function may be closely linked to MYC2 transcription factor in the regulation of the JA-signaling cascade in stomatal defense against bacterial pathogens. Our study provides valuable information on the function of a small GTPase, NOG1-2, in guard cell signaling and early plant defense in response to bacterial pathogens.


Subject(s)
Arabidopsis Proteins/metabolism , GTP-Binding Proteins/metabolism , Repressor Proteins/metabolism , Amino Acids/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , GTP-Binding Proteins/genetics , Gene Expression Regulation, Plant , Indenes/metabolism , Plant Immunity/genetics , Plant Immunity/physiology , Repressor Proteins/genetics
16.
Plant Cell ; 29(9): 2233-2248, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28855332

ABSTRACT

Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here, we report GENERAL CONTROL NONREPRESSIBLE4 (GCN4), an AAA+-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis thaliana compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H+-ATPase activity, stomata that are less responsive to pathogen virulence factors such as coronatine (phytotoxin produced by the bacterium Pseudomonas syringae) or fusicoccin (a fungal toxin produced by the fungus Fusicoccum amygdali), reduced pathogen entry, and enhanced drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggests that GCN4 degrades RIN4 and 14-3-3 proteins via a proteasome-mediated pathway and thereby reduces the activity of the plasma membrane H+-ATPase complex, thus reducing proton pump activity to close stomata.


Subject(s)
14-3-3 Proteins/metabolism , Adaptation, Physiological , Arabidopsis Proteins/metabolism , Arabidopsis/immunology , Disease Resistance , Droughts , Nicotiana/immunology , Plant Stomata/physiology , Abscisic Acid/pharmacology , Adaptation, Physiological/drug effects , Arabidopsis/microbiology , Arabidopsis/physiology , Cell Membrane/metabolism , Conserved Sequence , DNA, Complementary/genetics , Gene Silencing/drug effects , Models, Biological , Plant Immunity/drug effects , Plant Stomata/drug effects , Plants, Genetically Modified , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Proton-Translocating ATPases/metabolism , Stress, Physiological , Nicotiana/drug effects , Nicotiana/physiology
17.
Sci Rep ; 7(1): 9260, 2017 08 23.
Article in English | MEDLINE | ID: mdl-28835689

ABSTRACT

Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.


Subject(s)
Monomeric GTP-Binding Proteins/metabolism , Plant Immunity , Plants/immunology , Plants/metabolism , Arabidopsis , Disease Resistance/immunology , Gene Expression Regulation, Plant , Host-Pathogen Interactions/immunology , Models, Biological , Phenotype , Plant Diseases/immunology , Plant Diseases/microbiology , Plants/genetics , Plants/microbiology , Signal Transduction , Stress, Physiological , Transcriptome
18.
Gut Liver ; 10(3): 412-9, 2016 May 23.
Article in English | MEDLINE | ID: mdl-27114436

ABSTRACT

BACKGROUND/AIMS: Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown beneficial effects in experimental colitis models, but the underlying mechanisms are not fully understood. We investigated the long-term effects of BM-MSCs, particularly in mice with chronic colitis. METHODS: Chronic colitis was induced by administering 3% dextran sulfate sodium (DSS) in a series of three cycles. BMMSCs were injected intravenously into DSS-treated mice three times during the first cycle. On day 33, the therapeutic effects were evaluated with clinicopathologic profiles and histological scoring. Inflammatory mediators were measured with real-time polymerase chain reaction. RESULTS: Systemic infusion of BM-MSCs ameliorated the severity of colitis, and body weight restoration was significantly promoted in the BMMSC- treated mice. In addition, BM-MSC treatment showed a sustained beneficial effect throughout the three cycles. Microscopic examination revealed that the mice treated with BM-MSCs had fewer inflammatory infiltrates, a lesser extent of inflammation, and less crypt structure damage compared with mice with DSS-induced colitis. Anti-inflammatory cytokine levels of interleukin-10 were significantly increased in the inflamed colons of BM-MSC-treated mice compared with DSS-induced colitis mice. CONCLUSIONS: Systemic infusion of BM-MSCs at the onset of disease exerted preventive and rapid recovery effects, with long-term immunosuppressive action in mice with repeated DSS-induced chronic colitis.


Subject(s)
Colitis/prevention & control , Mesenchymal Stem Cell Transplantation , Animals , Chronic Disease , Colitis/chemically induced , Cytokines/metabolism , Dextran Sulfate/toxicity , Disease Models, Animal , Female , Irritants/toxicity , Mesenchymal Stem Cells/physiology , Mice , Mice, Inbred C57BL
19.
Mol Carcinog ; 53 Suppl 1: E1-10, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23065858

ABSTRACT

The association between the types of genomic instability and cancer stem cell (CSC) has not been elucidated. We aimed to investigate the expressions of CSC markers with respect to microsatellite instability (MSI) status in human colorectal cancer (CRC). Immunostainings for CD133, CD44, and CD166, and K-ras mutation analysis were performed on 50 MSI-high (MSI-H), and 50 microsatellite stable (MSS) CRC tissues. In 11 MSS and MSI-H CRC cell lines, CD133 expression and DNA methylation statuses of the CD133 promoter were determined. The proportion of CD133 positive cells and the ability of colosphere formation were compared between HCT116 cells and HCT116 + Chr3 cells (hMLH1-restored HCT116 cells). Immunohistochemistry for CSC markers revealed that high CD133 expression was more frequent in MSS cancers than in MSI-H (P < 0.001, 74.0% vs. 28.0%, respectively), and related with short disease-free survival. Neither CD44 nor CD166 expression differed significantly with respect to MSI status. K-ras mutation showed no association with expressions of CD133, CD44, or CD166. CD133 expression was relatively high in the MSS cell lines compared to those in MSI-H, and showed a reverse correlation with DNA methylation of the CD133 promoter. hMLH1-restored HCT116 cells increased proportions of CD133 positive cells and colosphere forming ability, compared to those in HCT116 cells. In conclusion, high levels of CD133 expression were observed more frequently in MSS CRC than in MSI-H, suggesting that differential expression of colon CSC markers may be linked to tumor characteristics dependent on MSI status.


Subject(s)
Antigens, CD/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Colorectal Neoplasms/metabolism , Fetal Proteins/metabolism , Glycoproteins/metabolism , Hyaluronan Receptors/metabolism , Microsatellite Instability , Peptides/metabolism , AC133 Antigen , Blotting, Western , Colorectal Neoplasms/genetics , Colorectal Neoplasms/mortality , Colorectal Neoplasms/pathology , Female , Flow Cytometry , Humans , Immunoenzyme Techniques , Male , Middle Aged , Mutation/genetics , Neoplasm Staging , Prognosis , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Survival Rate , Tumor Cells, Cultured , ras Proteins/genetics
20.
Int J Cancer ; 134(3): 519-29, 2014 Feb 01.
Article in English | MEDLINE | ID: mdl-23852449

ABSTRACT

Cancer stem cells (CSCs) play a pivotal role in cancer relapse or metastasis. We investigated the CSC-suppressing effect of nonsteroidal anti-inflammatory drugs (NSAIDs) and the relevant mechanisms in colorectal cancer. We measured the effect of NSAIDs on CSC populations in Caco-2 or SW620 cells using colosphere formation and flow cytometric analysis of PROM1 (CD133)(+) CD44(+) cells after indomethacin treatment with/without prostaglandin E2 (PGE2) or peroxisome proliferator-activated receptor γ (PPARG) antagonist, and examined the effect of indomethacin on transcriptional activity and protein expression of NOTCH/HES1 and PPARG. These effects of indomethacin were also evaluated in a xenograft mouse model. NSAIDs (indomethacin, sulindac and aspirin), celecoxib, γ-secretase inhibitor and PPARG agonist significantly decreased the number of colospheres formation compared to controls. In Caco-2 and SW620 cells, compared to controls, PROM1 (CD133)(+) CD44(+) cells were significantly decreased by indomethacin treatment, and increased by 5-fluorouracil (5-FU) treatment. This 5-FU-induced increase of PROM1 (CD133)(+) CD44(+) cells was significantly attenuated by combination with indomethacin. This CSC-inhibitory effect of indomethacin was reversed by addition of PGE2 and PPARG antagonist. Indomethacin significantly decreased CBFRE and increased PPRE transcriptional activity and their relative protein expressions. In xenograft mouse experiments using 5-FU-resistant SW620 cells, the 5-FU treatment combined with indomethacin significantly reduced tumor growth, compared to 5-FU alone. In addition, treatment of indomethacin alone or combination of 5-FU and indomethacin decreased the expressions of PROM1 (CD133), CD44, PTGS2 (cyclooxygenase 2) and HES1, and increased PPARG expression. NSAIDs could selectively reduce the colon CSCs and suppress 5-FU-induced increase of CSCs via inhibiting PTGS2 (cyclooxygenase 2) and NOTCH/HES1, and activating PPARG.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Basic Helix-Loop-Helix Transcription Factors/antagonists & inhibitors , Colorectal Neoplasms/pathology , Cyclooxygenase 2/drug effects , Homeodomain Proteins/antagonists & inhibitors , Neoplastic Stem Cells/drug effects , PPAR gamma/agonists , Receptors, Notch/antagonists & inhibitors , Animals , Cell Line, Tumor , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/metabolism , Humans , Male , Mice , Mice, Inbred BALB C , Neoplastic Stem Cells/enzymology , Neoplastic Stem Cells/metabolism , Transcription Factor HES-1 , Xenograft Model Antitumor Assays
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