The Powdery Mildew Effector CSEP0027 Interacts With Barley Catalase to Regulate Host Immunity

Powdery mildew is one of the most important fungal pathogen diseases. The genome of barley mildew fungus, Blumeria graminis f. sp. hordei (Bgh), encodes a large number of candidate secreted effector proteins (CSEPs). So far, the function and mechanism of most CSEPs remain largely unknown. Here, we identify a Bgh effector CSEP0027, a member of family 41, triggering cell death in Nicotiana benthamiana. CSEP0027 contains a functional signal peptide (SP), verified by yeast secretion assay. We show that CSEP0027 promotes Bgh virulence in barley infection using transient gene expression and host-induced gene silencing (HIGS). Barley catalase HvCAT1 is identified as a CSEP0027 interactor by yeast two-hybrid (Y2H) screening, and the interaction is verified in yeast, in vitro and in vivo. The coexpression of CSEP0027 and HvCAT1 in barley cells results in altered localization of HvCAT1 from the peroxisome to the nucleus. Barley stripe mosaic virus (BSMV)-silencing and transiently-induced gene silencing (TIGS) assays reveal that HvCAT1 is required for barley immunity against Bgh. We propose that CSEP0027 interacts with barley HvCAT1 to regulate the host immunity and likely reactive oxygen species (ROS) homeostasis to promote fungal virulence during barley infection.

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The barley powdery mildew effectors CSEP0139 and CSEP0182 suppress cell death and promote B. graminis fungal virulence in plants

Phytopathology Research ◽

10.1186/s42483-021-00084-z ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Xue Li ◽

Cong Jin ◽

Hongbo Yuan ◽

Wanting Huang ◽

Fang Liu ◽

...

Keyword(s):

Cell Death ◽

Powdery Mildew ◽

Host Plants ◽

Host Immunity ◽

Effector Proteins ◽

Fungal Virulence ◽

Cell Death Pathways ◽

Barley Powdery Mildew ◽

Powdery Mildew Fungi ◽

Transient Overexpression

AbstractThe powdery mildew fungi secrete numerous Candidate Secreted Effector Proteins (CSEPs) to manipulate host immunity during infection of host plants. However, the function of most of these CSEPs in cell death suppression has not yet been established. Here, we identified several CSEPs from Blumeria graminis f. sp. hordei (Bgh) that have the potential to suppress BAX- and NtMEK2DD-triggered cell death in Nicotiana benthamiana. We further characterized two effector candidates, CSEP0139 and CSEP0182, from family six and thirty-two, respectively. CSEP0139 and CSEP0182 contain a functional signal peptide and are likely secreted effectors. Expression of either CSEP0139 or CSEP0182 suppressed cell death triggered by BAX and NtMEK2DD but not by the AVRa13/MLA13 pair in N. benthamiana. Transient overexpression of CSEP0139 or CSEP0182 also inhibited BAX-induced cell death and collapse of cytoplasm in barley cells. Furthermore, overexpression of either CSEPs significantly increased Bgh haustorial formation in barley, whereas host-induced gene silencing (HIGS) of the CSEP genes reduced haustorial formation, suggesting both CSEPs promote Bgh virulence in barley. In addition, expression of CSEP0139 and CSEP0182 reduced size of the lesions caused by the necrotrophic Botrytis cinerea in N. benthamiana. Our findings suggest that CSEP0139 and CSEP0182 may target cell death components in plants to promote fungal virulence, which extends the current understanding of the functions of Bgh CSEPs and provides an opportunity for further investigation of fungal virulence in relation to cell death pathways in host plants.

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A Phytophthora effector recruits a host cytoplasmic transacetylase into nuclear speckles to enhance plant susceptibility

eLife ◽

10.7554/elife.40039 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 13

Author(s):

Haiyang Li ◽

Haonan Wang ◽

Maofeng Jing ◽

Jinyi Zhu ◽

Baodian Guo ◽

...

Keyword(s):

Root Rot ◽

Plant Immunity ◽

Phytophthora Sojae ◽

Host Immunity ◽

Effector Proteins ◽

Nuclear Speckles ◽

Rxlr Effector ◽

Plant Susceptibility

Oomycete pathogens secrete host cell-entering effector proteins to manipulate host immunity during infection. We previously showed that PsAvh52, an early-induced RxLR effector secreted from the soybean root rot pathogen, Phytophthora sojae, could suppress plant immunity. Here, we found that PsAvh52 is required for full virulence on soybean and binds to a novel soybean transacetylase, GmTAP1, in vivo and in vitro. PsAvh52 could cause GmTAP1 to relocate into the nucleus where GmTAP1 could acetylate histones H2A and H3 during early infection, thereby promoting susceptibility to P. sojae. In the absence of PsAvh52, GmTAP1 remained confined to the cytoplasm and did not modify plant susceptibility. These results demonstrate that GmTAP1 is a susceptibility factor that is hijacked by PsAvh52 in order to promote epigenetic modifications that enhance the susceptibility of soybean to P. sojae infection.

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In vivo cell specific gene silencing in the liver using novel siRNA-loaded biodegradable nanohydrogel particles

Zeitschrift für Gastroenterologie ◽

10.1055/s-0036-1586951 ◽

2016 ◽

Vol 54 (08) ◽

Author(s):

D Schuppan

Keyword(s):

Gene Silencing ◽

Specific Gene

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Gene Silencing using siRNA for Preventing Liver Ischaemia-Reperfusion Injury

Current Pharmaceutical Design ◽

10.2174/1381612824666180807124356 ◽

2018 ◽

Vol 24 (23) ◽

pp. 2692-2700 ◽

Cited By ~ 1

Author(s):

H. Susana Marinho ◽

Paulo Marcelino ◽

Helena Soares ◽

Maria Luísa Corvo

Keyword(s):

Gene Silencing ◽

Reperfusion Injury ◽

Therapeutic Potential ◽

Major Complication ◽

Ischaemia Reperfusion Injury ◽

Small Interference Rna ◽

Ischaemia Reperfusion ◽

Promising Therapeutic Approach ◽

Sirna Therapy

Background: Ischaemia-reperfusion injury (IRI), a major complication occurring during organ transplantation, involves an initial ischemia insult, due to loss of blood supply, followed by an inflammation-mediated reperfusion injury. A variety of molecular targets and pathways involved in liver IRI have been identified. Gene silencing through RNA interference (RNAi) by means of small interference RNA (siRNA) targeting mediators of IRI is a promising therapeutic approach. Objective: This study aims at reviewing the use of siRNAs as therapeutic agents to prevent IRI during liver transplantation. Method: We review the crucial choice of siRNA targets and the advantages and problems of the use of siRNAs. Results: We propose possible targets for siRNA therapy during liver IRI. Moreover, we discuss how drug delivery systems, namely liposomes, may improve siRNA therapy by increasing siRNA stability in vivo and avoiding siRNA off-target effects. Conclusion: siRNA therapeutic potential to preclude liver IRI can be improved by a better knowledge of what molecules to target and by using more efficient delivery strategies.

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The Zn-finger of Saccharomyces cerevisiae Rad18 and its adjacent region mediate interaction with Rad5

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab041 ◽

2021 ◽

Author(s):

Orsolya Frittmann ◽

Vamsi K Gali ◽

Miklos Halmai ◽

Robert Toth ◽

Zsuzsanna Gyorfy ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Ubiquitin Ligase ◽

Dna Lesions ◽

Template Switching ◽

Adjacent Region ◽

Yeast Two Hybrid ◽

Replication Complex ◽

Two Hybrid

Abstract DNA damages that hinder the movement of the replication complex can ultimately lead to cell death. To avoid that, cells possess several DNA damage bypass mechanisms. The Rad18 ubiquitin ligase controls error-free and mutagenic pathways that help the replication complex to bypass DNA lesions by monoubiquitylating PCNA at stalled replication forks. In Saccharomyces cerevisiae, two of the Rad18 governed pathways are activated by monoubiquitylated PCNA and they involve translesion synthesis polymerases, whereas a third pathway needs subsequent polyubiquitylation of the same PCNA residue by another ubiquitin ligase the Rad5 protein, and it employs template switching. The goal of this study was to dissect the regulatory role of the multidomain Rad18 in DNA damage bypass using a structure-function based approach. Investigating deletion and point mutant RAD18 variants in yeast genetic and yeast two-hybrid assays we show that the Zn-finger of Rad18 mediates its interaction with Rad5, and the N-terminal adjacent region is also necessary for Rad5 binding. Moreover, results of the yeast two-hybrid and in vivo ubiquitylation experiments raise the possibility that direct interaction between Rad18 and Rad5 might not be necessary for the function of the Rad5 dependent pathway. The presented data also reveal that yeast Rad18 uses different domains to mediate its association with itself and with Rad5. Our results contribute to better understanding of the complex machinery of DNA damage bypass pathways.

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Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts

Plant Cell Reports ◽

10.1007/s00299-021-02671-y ◽

2021 ◽

Author(s):

Johannes W. Debler ◽

Bernadette M. Henares ◽

Robert C. Lee

Keyword(s):

Broad Spectrum ◽

Faba Bean ◽

Fluorescent Protein ◽

Transient Gene Expression ◽

Low Frequency ◽

Effector Proteins ◽

Field Pea ◽

Grain Legume ◽

Pathogen Effectors ◽

Green Fluorescent

Abstract Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

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Evidence for the Involvement of the Glc7-Reg1 Phosphatase and the Snf1-Snf4 Kinase in the Regulation of INO1 Transcription in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/152.1.73 ◽

1999 ◽

Vol 152 (1) ◽

pp. 73-87 ◽

Cited By ~ 6

Author(s):

Margaret K Shirra ◽

Karen M Arndt

Keyword(s):

Rna Polymerase Ii ◽

Glucose Repression ◽

Snf1 Kinase ◽

Glucose Levels ◽

Recessive Mutations ◽

Mutant Strains ◽

Rna Polymerase Ii Transcription ◽

Two Hybrid

AbstractBinding of the TATA-binding protein (TBP) to the promoter is a pivotal step in RNA polymerase II transcription. To identify factors that regulate TBP, we selected for suppressors of a TBP mutant that exhibits promoter-specific defects in activated transcription in vivo and severely reduced affinity for TATA boxes in vitro. Dominant mutations in SNF4 and recessive mutations in REG1, OPI1, and RTF2 were isolated that specifically suppress the inositol auxotrophy of the TBP mutant strains. OPI1 encodes a repressor of INO1 transcription. REG1 and SNF4 encode regulators of the Glc7 phosphatase and Snf1 kinase, respectively, and have well-studied roles in glucose repression. In two-hybrid assays, one SNF4 mutation enhances the interaction between Snf4 and Snf1. Suppression of the TBP mutant by our reg1 and SNF4 mutations appears unrelated to glucose repression, since these mutations do not alleviate repression of SUC2, and glucose levels have little effect on INO1 transcription. Moreover, mutations in TUP1, SSN6, and GLC7, but not HXK2 and MIG1, can cause suppression. Our data suggest that association of TBP with the TATA box may be regulated, directly or indirectly, by a substrate of Snf1. Analysis of INO1 transcription in various mutant strains suggests that this substrate is distinct from Opi1.

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Rho GTPases as Key Molecular Players within Intestinal Mucosa and GI Diseases

Cells ◽

10.3390/cells10010066 ◽

2021 ◽

Vol 10 (1) ◽

pp. 66

Author(s):

Rashmita Pradhan ◽

Phuong A. Ngo ◽

Luz d. C. Martínez-Sánchez ◽

Markus F. Neurath ◽

Rocío López-Posadas

Keyword(s):

Intestinal Mucosa ◽

Rho Gtpases ◽

Current Knowledge ◽

Cell Types ◽

Effector Proteins ◽

Special Focus ◽

Specific Cell ◽

Rho Proteins

Rho proteins operate as key regulators of the cytoskeleton, cell morphology and trafficking. Acting as molecular switches, the function of Rho GTPases is determined by guanosine triphosphate (GTP)/guanosine diphosphate (GDP) exchange and their lipidation via prenylation, allowing their binding to cellular membranes and the interaction with downstream effector proteins in close proximity to the membrane. A plethora of in vitro studies demonstrate the indispensable function of Rho proteins for cytoskeleton dynamics within different cell types. However, only in the last decades we have got access to genetically modified mouse models to decipher the intricate regulation between members of the Rho family within specific cell types in the complex in vivo situation. Translationally, alterations of the expression and/or function of Rho GTPases have been associated with several pathological conditions, such as inflammation and cancer. In the context of the GI tract, the continuous crosstalk between the host and the intestinal microbiota requires a tight regulation of the complex interaction between cellular components within the intestinal tissue. Recent studies demonstrate that Rho GTPases play important roles for the maintenance of tissue homeostasis in the gut. We will summarize the current knowledge on Rho protein function within individual cell types in the intestinal mucosa in vivo, with special focus on intestinal epithelial cells and T cells.

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The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

Journal of Fungi ◽

10.3390/jof7020086 ◽

2021 ◽

Vol 7 (2) ◽

pp. 86

Author(s):

Bilal Ökmen ◽

Daniela Schwammbach ◽

Guus Bakkeren ◽

Ulla Neumann ◽

Gunther Doehlemann

Keyword(s):

Fungal Pathogens ◽

Plant Pathogens ◽

Expression System ◽

Pathogenic Fungi ◽

Effector Proteins ◽

Mating Partner ◽

Fungal Virulence ◽

Functional Studies ◽

Infection Structures ◽

Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

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