Transient Overexpression of E2Fb to Induce Cell Divisions in Nicotiana benthamiana Pavement Cells

The plasma membrane (PM) is at the interface of plant–pathogen interactions and, thus, many bacterial type-III effector (T3E) proteins target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E HopZ1a is a host cell PM-localized effector protein that has several immunity-associated host targets but also activates effector-triggered immunity in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated PM-associated HopZ1a target protein has been identified until now. Here, we show that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 relocalizes to membrane nanodomains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense-related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase avrPphB-susceptible 1 (PBS1) and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a–NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed. [Formula: see text]Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

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Genome-Wide Identification Reveals That Nicotiana benthamiana Hypersensitive Response (HR)-Like Lesion Inducing Protein 4 (NbHRLI4) Mediates Cell Death and Salicylic Acid-Dependent Defense Responses to Turnip Mosaic Virus

Frontiers in Plant Science ◽

10.3389/fpls.2021.627315 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xinyang Wu ◽

Yuchao Lai ◽

Shaofei Rao ◽

Lanqing Lv ◽

Mengfei Ji ◽

...

Keyword(s):

Cell Death ◽

Salicylic Acid ◽

Mosaic Virus ◽

Hypersensitive Response ◽

Nicotiana Benthamiana ◽

Defense Responses ◽

Turnip Mosaic Virus ◽

Genome Wide ◽

Transient Overexpression ◽

Basal Immunity

Hypersensitive response (HR)-like cell death is an important mechanism that mediates the plant response to pathogens. In our previous study, we reported that NbHIR3s regulate HR-like cell death and basal immunity. However, the host genes involved in HR have rarely been studied. Here, we used transcriptome sequencing to identify Niben101Scf02063g02012.1, an HR-like lesion inducing protein (HRLI) in Nicotiana benthamiana that was significantly reduced by turnip mosaic virus (TuMV). HRLIs are uncharacterized proteins which may regulate the HR process. We identified all six HRLIs in N. benthamiana and functionally analyzed Niben101Scf02063g02012.1, named NbHRLI4, in response to TuMV. Silencing of NbHRLI4 increased TuMV accumulation, while overexpression of NbHRLI4 conferred resistance to TuMV. Transient overexpression of NbHRLI4 caused cell death with an increase in the expression of salicylic acid (SA) pathway genes but led to less cell death level and weaker immunity in plants expressing NahG. Thus, we have characterized NbHRLI4 as an inducer of cell death and an antiviral regulator of TuMV infection in a SA-mediated manner.

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Mp10 and Mp42 from the Aphid Species Myzus persicae Trigger Plant Defenses in Nicotiana benthamiana Through Different Activities

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-13-0156-r ◽

2014 ◽

Vol 27 (1) ◽

pp. 30-39 ◽

Cited By ~ 40

Author(s):

Patricia A. Rodriguez ◽

Remco Stam ◽

Tim Warbroek ◽

Jorunn I. B. Bos

Keyword(s):

Subcellular Localization ◽

Myzus Persicae ◽

Nicotiana Benthamiana ◽

Green Peach Aphid ◽

Phytophthora Capsici ◽

Functional Characterization ◽

Aphid Species ◽

Broad Host Range ◽

In Planta ◽

Transient Overexpression

Aphids are phloem-feeding insects that, like other plant parasites, deliver effectors inside their host to manipulate host responses. The Myzus persicae (green peach aphid) candidate effectors Mp10 and Mp42 were previously found to reduce aphid fecundity upon intracellular transient overexpression in Nicotiana benthamiana. We performed functional analyses of these proteins to investigate whether they activate defenses through similar activities. We employed a range of functional characterization experiments based on intracellular transient overexpression in N. benthamiana to determine the subcellular localization of Mp10 and Mp42 and investigate their role in activating plant defense signaling. Mp10 and Mp42 showed distinct subcellular localization in planta, suggesting that they target different host compartments. Also, Mp10 reduced the levels of Agrobacterium-mediated overexpression of proteins. This reduction was not due to an effect on Agrobacterium viability. Transient overexpression of Mp10 but not Mp42 activated jasmonic acid and salicylic acid signaling pathways and decreased susceptibility to the hemibiotrophic plant pathogen Phytophthora capsici. We found that two candidate effectors from the broad-host-range aphid M. persicae can trigger aphid defenses through different mechanisms. Importantly, we found that some (candidate) effectors such as Mp10 interfere with Agrobacterium-based overexpression assays, an important tool to study effector activity and function.

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A Sec-Dependent Secretory Protein of the Huanglongbing-Associated Pathogen Suppresses Hypersensitive Cell Death in Nicotiana benthamiana

Frontiers in Microbiology ◽

10.3389/fmicb.2020.594669 ◽

2020 ◽

Vol 11 ◽

Author(s):

Chao Zhang ◽

Peixiu Du ◽

Hailin Yan ◽

Zongcai Zhu ◽

Xuefeng Wang ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

Pseudomonas Syringae ◽

Nicotiana Benthamiana ◽

Transcriptome Profiling ◽

Secretory Protein ◽

Hypersensitive Cell Death ◽

Challenge Inoculation ◽

Transient Overexpression ◽

Shock Proteins

“Candidatus Liberibacter asiaticus” (CLas) is a phloem-restricted Gram-negative bacterium that is the causal agent of citrus huanglongbing (HLB). In this study, we identified a CLas-encoded Sec-dependent secretory protein CLIBASIA_04405 that could contribute to the pathogenicity of this bacterium. The gene expression level of CLIBASIA_04405 was significantly higher in citrus than in psyllids. Transient overexpression of the mature CLIBASIA_04405 protein (m4405) in Nicotiana benthamiana leaves could suppress hypersensitive response (HR)-based cell death and H2O2 accumulation triggered by the mouse BAX and the Phytophthora infestans INF1. An alanine-substitution mutagenesis assay revealed the essential of amino acid clusters EKR45–47 and DE64–65 in cell death suppression. Challenge inoculation of the transgenic N. benthamiana-expressing m4405 with Pseudomonas syringae DC3000ΔhopQ1-1 demonstrated the greatly reduced bacterial proliferation. Remarkably, transcriptome profiling and RT-qPCR analysis disclosed that the gene expression of six small heat shock proteins (sHSPs), a set of plant defense regulators, were significantly elevated in the transgenic m4405 lines compared with those in wild-type N. benthamiana. In addition, the transgenic m4405 lines displayed phenotypes of dwarfism and leaf deformation. Altogether, these data indicated that m4405 was a virulence factor of CLas.

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A remorin from Nicotiana benthamiana interacts with the Pseudomonas type-III effector protein HopZ1a and is phosphorylated by the immune-related kinase PBS1

10.1101/409235 ◽

2018 ◽

Author(s):

Philip Albers ◽

Suayib Üstün ◽

Katja Witzel ◽

Max Kraner ◽

Frederik Börnke

Keyword(s):

Plasma Membrane ◽

Pseudomonas Syringae ◽

Nicotiana Benthamiana ◽

Effector Proteins ◽

Effector Protein ◽

Type Iii Effector ◽

Defense Signaling ◽

Type Iii ◽

Transient Overexpression

AbstractThe plasma membrane is at the interface of plant-pathogen interactions and thus many bacterial type-III effector proteins (T3Es) target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E is a host cell plasma membrane (PM)-localized effector protein that has several immunity associated host targets but also activates effector triggered immunity (ETI) in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated plasma membrane-associated HopZ1a target protein has been identified until now. We show here, that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 re-localizes to membrane sub-domains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase PBS1 and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a/NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed.

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Hydroponic Treatment of Nicotiana benthamiana with Kifunensine Modifies the N-glycans of Recombinant Glycoprotein Antigens to Predominantly Man9 High-Mannose Type upon Transient Overexpression

Frontiers in Plant Science ◽

10.3389/fpls.2018.00062 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 8

Author(s):

Sugata Roychowdhury ◽

Young J. Oh ◽

Hiroyuki Kajiura ◽

Krystal T. Hamorsky ◽

Kazuhito Fujiyama ◽

...

Keyword(s):

Nicotiana Benthamiana ◽

Recombinant Glycoprotein ◽

High Mannose ◽

Transient Overexpression

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An improved Nicotiana benthamiana strain for aphid and whitefly research

10.1101/2020.08.04.237180 ◽

2020 ◽

Author(s):

Honglin Feng ◽

Lucia Acosta-Gamboa ◽

Lars H. Kruse ◽

Alba Ruth Nava Fereira ◽

Sara Shakir ◽

...

Keyword(s):

Gene Expression ◽

Nicotiana Benthamiana ◽

Plant Virus ◽

Transient Gene Expression ◽

Model System ◽

Plant Insect Interactions ◽

Transient Overexpression ◽

Insect Interactions ◽

Mutant Lines ◽

Virus Interactions

AbstractNicotiana benthamiana is used extensively as a platform for transient gene expression and as a model system for studying plant-virus interactions. However, many tobacco-feeding insects, e.g. Myzus persicae (green peach aphids) and Bemisia tabaci (whiteflies), grow poorly on N. benthamiana, limiting its utility for research on plant-insect interactions. Using CRISPR/Cas9, we generated knockout mutations in two N. benthamiana acylsugar acyltransferase genes, ASAT1 and ASAT2, which contribute to the biosynthesis of insect-deterrent acylsucroses. Whereas ASAT1 mutations reduced the abundance of two predominant acylsucroses, ASAT2 mutations caused almost complete depletion of foliar acylsucroses. Both M. persicae and B. tabaci survived and reproduced significantly better on asat2 mutant plants than on wildtype N. benthamiana. Furthermore, ASAT1 and ASAT2 mutations reduced the water content and increased the temperature of leaves, indicating that foliar acylsucroses can protect against desiccation. Improved aphid and whitefly performance on ASAT2 mutants will make it possible to use the efficient transient overexpression and gene expression silencing systems that are available for N. benthamiana to study plant-insect interactions. Additionally, the absence of acylsugars in ASAT2 mutant lines will simplify transient expression assays for the functional analysis of acylsugar biosynthesis genes from other Solanaceae.

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