scholarly journals A vector system for fast-forward in vivo studies of the ZAR1 resistosome in the model plant Nicotiana benthamiana

2020 ◽  
Author(s):  
Adeline Harant ◽  
Toshiyuki Sakai ◽  
Sophien Kamoun ◽  
Hiroaki Adachi
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Biology ◽  
Plant Immunity ◽  
Transient Gene Expression ◽  
Coiled Coil ◽  
Experimental System ◽  
In Vivo Studies ◽  
Vector System ◽  
In Planta

ABSTRACTNicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of NLR plant immune receptors has greatly advanced following the discovery of Arabidopsis ZAR1 resistosome. Here, we describe a novel vector system of 52 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. NbZAR1 triggers autoimmune cell death in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source plasmids form an additional experimental system to Arabidopsis for in planta resistosome studies.

A vector system for fast-forward studies of the HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome in the model plant Nicotiana benthamiana

2021 ◽  
Author(s):  
Adeline Harant ◽  
Hsuan Pai ◽  
Toshiyuki Sakai ◽  
Sophien Kamoun ◽  
Hiroaki Adachi
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Biology ◽  
Plant Immunity ◽  
Transient Gene Expression ◽  
Coiled Coil ◽  
Experimental System ◽  
Vector System ◽  
In Planta ◽  
Functional Studies

Abstract Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.

scholarly journals Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns

2015 ◽  
Vol 112 (17) ◽  
pp. 5533-5538 ◽  
Author(s):  
Manuel Benedetti ◽  
Daniela Pontiggia ◽  
Sara Raggi ◽  
Zhenyu Cheng ◽  
Flavio Scaloni ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Plant Immunity ◽  
Inducible Promoter ◽  
Spectrometric Analysis ◽  
In Planta ◽  
Gene Encoding ◽  
Polygalacturonase Inhibiting Protein ◽  
Molecular Patterns ◽  
Damage Associated Molecular Patterns

Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP–PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense.

scholarly journals Non-coding RNAs in cardiovascular cell biology and atherosclerosis

2019 ◽  
Vol 115 (12) ◽  
pp. 1732-1756 ◽  
Author(s):  
Francesca Fasolo ◽  
Karina Di Gregoli ◽  
Lars Maegdefessel ◽  
Jason L Johnson
Keyword(s):  
Animal Models ◽  
Cell Biology ◽  
Therapeutic Targets ◽  
Cell Types ◽  
In Vivo Studies ◽  
Circular Rnas ◽  
Potential Contribution ◽  
Clinicopathological Findings ◽  
Non Coding Rnas

Abstract Atherosclerosis underlies the predominant number of cardiovascular diseases and remains a leading cause of morbidity and mortality worldwide. The development, progression and formation of clinically relevant atherosclerotic plaques involves the interaction of distinct and over-lapping mechanisms which dictate the roles and actions of multiple resident and recruited cell types including endothelial cells, vascular smooth muscle cells, and monocyte/macrophages. The discovery of non-coding RNAs (ncRNAs) including microRNAs, long non-coding RNAs, and circular RNAs, and their identification as key mechanistic regulators of mRNA and protein expression has piqued interest in their potential contribution to atherosclerosis. Accruing evidence has revealed ncRNAs regulate pivotal cellular and molecular processes during all stages of atherosclerosis including cell invasion, growth, and survival; cellular uptake and efflux of lipids, expression and release of pro- and anti-inflammatory intermediaries, and proteolytic balance. The expression profile of ncRNAs within atherosclerotic lesions and the circulation have been determined with the aim of identifying individual or clusters of ncRNAs which may be viable therapeutic targets alongside deployment as biomarkers of atherosclerotic plaque progression. Consequently, numerous in vivo studies have been convened to determine the effects of moderating the function or expression of select ncRNAs in well-characterized animal models of atherosclerosis. Together, clinicopathological findings and studies in animal models have elucidated the multifaceted and frequently divergent effects ncRNAs impose both directly and indirectly on the formation and progression of atherosclerosis. From these findings’ potential novel therapeutic targets and strategies have been discovered which may pave the way for further translational studies and possibly taken forward for clinical application.

scholarly journals MicroRNA-139-5p Inhibits Cell Proliferation and Invasion by Targeting RHO-Associated Coiled-Coil-Containing Protein Kinase 2 in Ovarian Cancer

2018 ◽  
Vol 26 (3) ◽  
pp. 411-420 ◽  
Author(s):  
Yanli Wang ◽  
Jia Li ◽  
Chunling Xu ◽  
Xiaomeng Zhang
Keyword(s):  
Ovarian Cancer ◽  
Protein Kinase ◽  
Coiled Coil ◽  
Figo Stage ◽  
Inhibitory Effect ◽  
In Vivo Studies ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Poor Overall Survival

Increasing evidence indicates that the dysregulation of microRNAs is associated with the development and progression of various cancers. MicroRNA-139-5p (miR-139-5p) has been reported to have a tumor suppressive role in many types of cancers. The role of miR-139-5p in ovarian cancer (OC) is poorly understood. The purpose of the present study was to explore the expression of miR-139-5p and its function in OC. The results showed that miR-139-5p expression was markedly downregulated in OC tissues and cell lines. In addition, underexpression of miR-139-5p was significantly associated with FIGO stage, lymph mode metastasis, and poor overall survival of OC patients. Functional analyses indicated that overexpression of miR-139-5p significantly inhibited proliferation, colony formation, migration, and invasion of OC cells. Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) was identified as a direct target of miR-139-5p using luciferase reporter assays, qualitative real-time reverse transcriptase PCR (qRT-PCR), and Western blot. In addition, ROCK2 expression was upregulated and was inversely correlated with miR-139-5p levels in OC tissues. Rescue experiments showed that overexpression of ROCK2 effectively reversed the inhibitory effect of OC cells induced by miR-139-5p. Most interestingly, in vivo studies indicated that miR-139-5p markedly suppressed the growth of tumors by repressing ROCK2 expression in nude mice. Taken together, these findings demonstrated that miR-139-5p plays an important tumor suppressor role in OC by directly binding to ROCK2, providing a novel target for the molecular treatment of OC.

scholarly journals Insights from In Vivo Studies of Cellular Senescence

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 954
Author(s):  
Luis I. Prieto ◽  
Sara I. Graves ◽  
Darren J. Baker
Keyword(s):  
Cellular Senescence ◽  
Cell Biology ◽  
Senescent Cell ◽  
In Vivo Studies ◽  
Cycle Arrest ◽  
Mammalian Cell Lines ◽  
Age Related ◽  
Cell Accumulation ◽  
Secretory Phenotype

Cellular senescence is the dynamic process of durable cell-cycle arrest. Senescent cells remain metabolically active and often acquire a distinctive bioactive secretory phenotype. Much of our molecular understanding in senescent cell biology comes from studies using mammalian cell lines exposed to stress or extended culture periods. While less well understood mechanistically, senescence in vivo is becoming appreciated for its numerous biological implications, both in the context of beneficial processes, such as development, tumor suppression, and wound healing, and in detrimental conditions, where senescent cell accumulation has been shown to contribute to aging and age-related diseases. Importantly, clearance of senescent cells, through either genetic or pharmacological means, has been shown to not only extend the healthspan of prematurely and naturally aged mice but also attenuate pathology in mouse models of chronic disease. These observations have prompted an investigation of how and why senescent cells accumulate with aging and have renewed exploration into the characteristics of cellular senescence in vivo. Here, we highlight our molecular understanding of the dynamics that lead to a cellular arrest and how various effectors may explain the consequences of senescence in tissues. Lastly, we discuss how exploitation of strategies to eliminate senescent cells or their effects may have clinical utility.

scholarly journals The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling

2017 ◽  
Vol 28 (23) ◽  
pp. 3298-3314 ◽  
Author(s):  
Shruthi Viswanath ◽  
Massimiliano Bonomi ◽  
Seung Joong Kim ◽  
Vadim A. Klenchin ◽  
Keenan C. Taylor ◽  
...  
Keyword(s):  
Cell Biology ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Resonance Energy ◽  
Spindle Pole ◽  
Physical Structure ◽  
Molecular Architecture ◽  
X Ray ◽  
Pole Body

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.

scholarly journals A Polygalacturonase-Inhibiting Protein from Grapevine Reduces the Symptoms of the Endopolygalacturonase BcPG2 from Botrytis cinerea in Nicotiana benthamiana Leaves Without Any Evidence for In Vitro Interaction

2007 ◽  
Vol 20 (4) ◽  
pp. 392-402 ◽  
Author(s):  
Dirk A. Joubert ◽  
Ilona Kars ◽  
Lia Wagemakers ◽  
Carl Bergmann ◽  
Gabré Kemp ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Nicotiana Benthamiana ◽  
Substantial Reduction ◽  
In Vitro Assays ◽  
Physical Interaction ◽  
In Planta ◽  
Polygalacturonase Inhibiting Protein ◽  
Inhibiting Protein

Six endopolygalacturonases from Botrytis cinerea (BcPG1 to BcPG6) as well as mutated forms of BcPG1 and BcPG2 were expressed transiently in leaves of Nicotiana benthamiana using agroinfiltration. Expression of BcPG1, BcPG2, BcPG4, BcPG5, and mutant BcPG1-D203A caused symptoms, whereas BcPG3, BcPG6, and mutant BcPG2-D192A caused no symptoms. Expression of BcPG2 caused the most severe symptoms, including wilting and necrosis. BcPG2 previously has been shown to be essential for B. cinerea virulence. The in vivo effect of this enzyme and the inhibition by a polygalacturonase-inhibiting protein (PGIP) was examined by coexpressing Bcpg2 and the Vvpgip1 gene from Vitis vinifera in N. benthamiana. Coinfiltration resulted in a substantial reduction of the symptoms inflicted by the activity of BcPG2 in planta, as evidenced by quantifying the variable chlorophyll fluorescence yield. In vitro, however, no interaction between pure VvPGIP1 and pure BcPG2 was detected. Specifically, VvPGIP1 neither inhibited BcPG2 activity nor altered the degradation profile of polygalacturonic acid by BcPG2. Furthermore, using surface plasmon resonance technology, no physical interaction between VvPGIP1 and BcPG2 was detected in vitro. The data suggest that the in planta environment provided a context to support the interaction between BcPG2 and VvPGIP1, leading to a reduction in symptom development, whereas neither of the in vitro assays detected any interaction between these proteins.

scholarly journals NCAM2 Regulates Dendritic and Axonal Differentiation through the Cytoskeletal Proteins MAP2 and 14-3-3

2020 ◽  
Vol 30 (6) ◽  
pp. 3781-3799
Author(s):  
Antoni Parcerisas ◽  
Lluís Pujadas ◽  
Alba Ortega-Gascó ◽  
Bartomeu Perelló-Amorós ◽  
Ricardo Viais ◽  
...  
Keyword(s):  
Cell Biology ◽  
Hippocampal Neurons ◽  
Cytoskeletal Proteins ◽  
In Vivo Studies ◽  
Microtubule Associated Proteins ◽  
Neural Cell Adhesion ◽  
Aberrant Phenotype ◽  
Associated Proteins ◽  
And Migration

Abstract Neural cell adhesion molecule 2 (NCAM2) is involved in the development and plasticity of the olfactory system. Genetic data have implicated the NCAM2 gene in neurodevelopmental disorders including Down syndrome and autism, although its role in cortical development is unknown. Here, we show that while overexpression of NCAM2 in hippocampal neurons leads to minor alterations, its downregulation severely compromises dendritic architecture, leading to an aberrant phenotype including shorter dendritic trees, retraction of dendrites, and emergence of numerous somatic neurites. Further, our data reveal alterations in the axonal tree and deficits in neuronal polarization. In vivo studies confirm the phenotype and reveal an unexpected role for NCAM2 in cortical migration. Proteomic and cell biology experiments show that NCAM2 molecules exert their functions through a protein complex with the cytoskeletal-associated proteins MAP2 and 14-3-3γ and ζ. We provide evidence that NCAM2 depletion results in destabilization of the microtubular network and reduced MAP2 signal. Our results demonstrate a role for NCAM2 in dendritic formation and maintenance, and in neural polarization and migration, through interaction of NCAM2 with microtubule-associated proteins.

scholarly journals NRG1 functions downstream of EDS1 to regulate TIR-NLR-mediated plant immunity in Nicotiana benthamiana

2018 ◽  
Vol 115 (46) ◽  
pp. E10979-E10987 ◽  
Author(s):  
Tiancong Qi ◽  
Kyungyong Seong ◽  
Daniela P. T. Thomazella ◽  
Joonyoung Ryan Kim ◽  
Julie Pham ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Nicotiana Benthamiana ◽  
Plant Immunity ◽  
Coiled Coil ◽  
Large Family ◽  
Sequencing Analysis ◽  
Immune Receptors ◽  
Transcriptional Changes ◽  
Effector Triggered Immunity ◽  
Profile Changes

Effector-triggered immunity (ETI) in plants involves a large family of nucleotide-binding leucine-rich repeat (NLR) immune receptors, including Toll/IL-1 receptor-NLRs (TNLs) and coiled-coil NLRs (CNLs). Although various NLR immune receptors are known, a mechanistic understanding of NLR function in ETI remains unclear. The TNL Recognition of XopQ 1 (Roq1) recognizes the effectors XopQ and HopQ1 from Xanthomonas and Pseudomonas, respectively, which activates resistance to Xanthomonas euvesicatoria and Xanthomonas gardneri in an Enhanced Disease Susceptibility 1 (EDS1)-dependent way in Nicotiana benthamiana. In this study, we found that the N. benthamiana N requirement gene 1 (NRG1), a CNL protein required for the tobacco TNL protein N-mediated resistance to tobacco mosaic virus, is also essential for immune signaling [including hypersensitive response (HR)] triggered by the TNLs Roq1 and Recognition of Peronospora parasitica 1 (RPP1), but not by the CNLs Bs2 and Rps2, suggesting that NRG1 may be a conserved key component in TNL signaling pathways. Besides EDS1, Roq1 and NRG1 are necessary for resistance to Xanthomonas and Pseudomonas in N. benthamiana. NRG1 functions downstream of Roq1 and EDS1 and physically associates with EDS1 in mediating XopQ-Roq1–triggered immunity. Moreover, RNA sequencing analysis showed that XopQ-triggered gene-expression profile changes in N. benthamiana were almost entirely mediated by Roq1 and EDS1 and were largely regulated by NRG1. Overall, our study demonstrates that NRG1 is a key component that acts downstream of EDS1 to mediate various TNL signaling pathways, including Roq1 and RPP1-mediated HR, resistance to Xanthomonas and Pseudomonas, and XopQ-regulated transcriptional changes in N. benthamiana.

scholarly journals Two VQ Proteins are Substrates of the OsMPKK6-OsMPK4 Cascade in Rice Defense Against Bacterial Blight

Rice ◽  
2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Na Li ◽  
Zeyu Yang ◽  
Juan Li ◽  
Wenya Xie ◽  
Xiaofeng Qin ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Bacterial Blight ◽  
Plant Immunity ◽  
Signal Transduction Pathway ◽  
Mitogen Activated Protein Kinase ◽  
In Vivo Studies ◽  
Mitogen Activated Protein ◽  
Signaling Components

Abstract Background The plant-specific valine-glutamine (VQ) protein family with the conserved motif FxxxVQxLTG reportedly functions with the mitogen-activated protein kinase (MAPK) in plant immunity. However, the roles of VQ proteins in MAPK-mediated resistance to disease in rice remain largely unknown. Results In this study, two rice VQ proteins OsVQ14 and OsVQ32 were newly identified to function as the signaling components of a MAPK cascade, OsMPKK6-OsMPK4, to regulate rice resistance to Xanthomonas oryzae pv. oryzae (Xoo). Both OsVQ14 and OsVQ32 positively regulated rice resistance to Xoo. In vitro and in vivo studies revealed that OsVQ14 and OsVQ32 physically interacted with and were phosphorylated by OsMPK4. OsMPK4 was highly phosphorylated in transgenic plants overexpressing OsMPKK6, which showed enhanced resistance to Xoo. Meanwhile, phosphorylated OsVQ14 and OsVQ32 were also markedly accumulated in OsMPKK6-overexpressing transgenic plants. Conclusions We discovered that OsVQ14 and OsVQ32 functioned as substrates of the OsMPKK6-OsMPK4 cascade to enhance rice resistance to Xoo, thereby defining a more complete signal transduction pathway for induced defenses.

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