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 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.

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 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 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.

Functional studies of the kidney of living animals using multicolor two-photon microscopy

2002 ◽  
Vol 283 (3) ◽  
pp. C905-C916 ◽  
Author(s):  
Kenneth W. Dunn ◽  
Ruben M. Sandoval ◽  
Katherine J. Kelly ◽  
Pierre C. Dagher ◽  
George A. Tanner ◽  
...  
Keyword(s):  
Intravital Microscopy ◽  
Cell Biology ◽  
Fluid Transport ◽  
Biological Tissues ◽  
Two Photon Microscopy ◽  
Functional Studies ◽  
Two Photon ◽  
Cell Vitality ◽  
The Way

Optical microscopy, when applied to living animals, provides a powerful means of studying cell biology in the most physiologically relevant setting. The ability of two-photon microscopy to collect optical sections deep into biological tissues has opened up the field of intravital microscopy to high-resolution studies of the brain, lens, skin, and tumors. Here we present examples of the way in which two-photon microscopy can be applied to intravital studies of kidney physiology. Because the kidney is easily externalized without compromising its function, microscopy can be used to evaluate various aspects of renal function in vivo. These include cell vitality and apoptosis, fluid transport, receptor-mediated endocytosis, blood flow, and leukocyte trafficking. Efficient two-photon excitation of multiple fluorophores permits comparison of multiple probes and simultaneous characterization of multiple parameters and yields spectral information that is crucial to the interpretation of images containing uncharacterized autofluorescence. The studies described here demonstrate the way in which two-photon microscopy can provide a level of resolution previously unattainable in intravital microscopy, enabling kinetic analyses and physiological studies of the organs of living animals with subcellular resolution.

scholarly journals The Centrosomal Swiss Army Knife: A combined in silico and in vivo approach to the structure-function annotation of SPD-2 provides mechanistic insight into its functional diversity

2021 ◽  
Author(s):  
Mikaela Murph ◽  
Shaneen Singh ◽  
Mara Schvarzstein
Keyword(s):  
Structure Function ◽  
Cell Biology ◽  
Asymmetric Cell Division ◽  
Tumor Development ◽  
Coiled Coil ◽  
Immunoglobulin Superfamily ◽  
Major Sperm Protein ◽  
Developmental Abnormalities ◽  
Centriole Duplication

AbstractCentrosomes are organelles that function as hubs of microtubule nucleation and organization, with key roles in organelle positioning, asymmetric cell division, and ciliogenesis. Aberrant centrosome structure or function is linked to neurodegenerative diseases, developmental abnormalities, ciliopathies, and tumor development. A major regulator of centrosome biogenesis and function in C. elegans is the highly conserved protein Spindle-defective protein 2 (SPD-2), a homolog of the human CEP-192 protein. CeSPD-2 is required for centrosome maturation, centriole duplication, spindle assembly and cell polarity establishment. Despite its importance, the specific molecular mechanism of CeSPD-2 function is poorly understood. To address this gap in knowledge, we combined computational analysis with cell biology approaches to uncover structure-function relationships of CeSPD-2 that may shed mechanistic light on its function. Domain prediction analysis corroborated and refined previously identified coiled-coils and ASH (Aspm-SPD-2 Hydin) domains and identified new domains and motifs: an additional coiled-coil, a GEF domain, an Ig-like domain, and a PDZ-like domain. Our findings suggest that ASH domain belongs to the same superfold as PapD chaperone domains and Major Sperm Protein (MSP) domains within the larger Immunoglobulin superfamily. We have identified a large novel basic region in the CeSPD-2 ASH domain that harbors most of the predicted protein and nucleic acid contact residues in the ASH domain. In vivo, ASH::GFP localized to centrosomes and centrosome associated microtubules, and forms aggregates in the cytosol when overexpressed. This study lays the groundwork for designing rational hypothesis-based experiments for future analyses to further elaborate the mechanisms of CeSPD-2 function in vivo.

Scanning Tunneling Microscopy and Atomic Force Microscopy of Enzymes and Enzyme Complexes

1990 ◽  
Vol 48 (3) ◽  
pp. 174-175
Author(s):  
Ronald D. Edstrom ◽  
Xiuru Yang ◽  
Mary E. Gurnack ◽  
Marcia A. Miller ◽  
Rui Yang ◽  
...  
Keyword(s):  
Cell Biology ◽  
Inorganic Ions ◽  
Bulk Liquid ◽  
Soluble Form ◽  
Scanning Tunneling ◽  
Tunneling Microscopy ◽  
Metabolic Channeling ◽  
Simplistic Notion ◽  
Soluble Enzymes

Many of the questions in biochemistry and cell biology are concerned with the relationships of proteins and other macromolecules in complex arrays which are responsible for carrying out metabolic sequences. The simplistic notion that the enzymes we isolate in soluble form from the cytoplasm were also soluble in vivo is being replaced by the concept that these enzymes occur in organized systems within the cell. In this newer view, the cytoplasm is organized and the “soluble enzymes” are in fact fixed in the cellular space and the only soluble components of the cell are small metabolites, inorganic ions etc. Further support for the concept of metabolic organization is provided by the evidence of metabolic channeling. It has been shown that for some metabolic pathways, the intermediates are not in free diffusion equilibrium with the bulk liquid in the cell but are passed along, more or less directly, from one enzyme to the next.

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