scholarly journals The Cytospora chrysosperma Virulence Effector CcCAP1 Mainly Localizes to the Plant Nucleus To Suppress Plant Immune Responses

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Zhu Han ◽  
Dianguang Xiong ◽  
Zhiye Xu ◽  
Tingli Liu ◽  
Chengming Tian
Keyword(s):  
Cell Death ◽  
Transient Expression ◽  
Nuclear Localization ◽  
Nicotiana Benthamiana ◽  
Plant Immunity ◽  
Host Cells ◽  
Inhibiting Activity ◽  
Content Type ◽  
Cytospora Chrysosperma ◽  
Plant Nucleus

ABSTRACT Canker disease is caused by the fungus Cytospora chrysosperma and damages a wide range of woody plants, causing major losses to crops and native plants. Plant pathogens secrete virulence-related effectors into host cells during infection to regulate plant immunity and promote colonization. However, the functions of C. chrysosperma effectors remain largely unknown. In this study, we used Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana and confocal microscopy to investigate the immunoregulation roles and subcellular localization of CcCAP1, a virulence-related effector identified in C. chrysosperma. CcCAP1 was significantly induced in the early stages of infection and contains cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily domain with four cysteines. CcCAP1 suppressed the programmed cell death triggered by Bcl-2-associated X protein (BAX) and the elicitin infestin1 (INF1) in transient expression assays with Nicotiana benthamiana. The CAP superfamily domain was sufficient for its cell death-inhibiting activity and three of the four cysteines in the CAP superfamily domain were indispensable for its activity. Pathogen challenge assays in N. benthamiana demonstrated that transient expression of CcCAP1 promoted Botrytis cinerea infection and restricted reactive oxygen species accumulation, callose deposition, and defense-related gene expression. In addition, expression of green fluorescent protein-labeled CcCAP1 in N. benthamiana showed that it localized to both the plant nucleus and the cytoplasm, but the nuclear localization was essential for its full immune inhibiting activity. These results suggest that this virulence-related effector of C. chrysosperma modulates plant immunity and functions mainly via its nuclear localization and the CAP domain. IMPORTANCE The data presented in this study provide a key resource for understanding the biology and molecular basis of necrotrophic pathogen responses to Nicotiana benthamiana resistance utilizing effector proteins, and CcCAP1 may be used in future studies to understand effector-triggered susceptibility processes in the Cytospora chrysosperma-poplar interaction system.

scholarly journals Core N-Glycan Structures Are Critical for the Pathogenicity of Cryptococcus neoformans by Modulating Host Cell Death

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Eun Jung Thak ◽  
Su-Bin Lee ◽  
Shengjie Xu-Vanpala ◽  
Dong-Jik Lee ◽  
Seung-Yeon Chung ◽  
...  
Keyword(s):  
Cell Death ◽  
Fungal Infection ◽  
Host Cell ◽  
Host Cells ◽  
Glycan Structure ◽  
Macrophage Cell ◽  
Content Type ◽  
Host Cell Death ◽  
Intact Core

ABSTRACT Cryptococcus neoformans is a human-pathogenic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised individuals. To investigate the roles of N-glycan core structure in cryptococcal pathogenicity, we constructed mutant strains of C. neoformans with defects in the assembly of lipid-linked N-glycans in the luminal side of the endoplasmic reticulum (ER). Deletion of ALG3 (alg3Δ), which encodes dolichyl-phosphate-mannose (Dol-P-Man)-dependent α-1,3-mannosyltransferase, resulted in the production of truncated neutral N-glycans carrying five mannose residues as a major species. Despite moderate or nondetectable defects in virulence-associated phenotypes in vitro, the alg3Δ mutant was avirulent in a mouse model of systemic cryptococcosis. Notably, the mutant did not show defects in early stages of host cell interaction during infection, including attachment to lung epithelial cells, opsonic/nonopsonic phagocytosis, and manipulation of phagosome acidification. However, the ability to drive macrophage cell death was greatly decreased in this mutant, without loss of cell wall remodeling capacity. Furthermore, deletion of ALG9 and ALG12, encoding Dol-P-Man-dependent α-1,2-mannosyltransferases and α-1,6-mannosyltransferases, generating truncated core N-glycans with six and seven mannose residues, respectively, also displayed remarkably reduced macrophage cell death and in vivo virulence. However, secretion levels of interleukin-1β (IL-1β) were not reduced in the bone marrow-derived dendritic cells obtained from Asc- and Gsdmd-deficient mice infected with the alg3Δ mutant strain, excluding the possibility that pyroptosis is a main host cell death pathway dependent on intact core N-glycans. Our results demonstrated N-glycan structures as a critical feature in modulating death of host cells, which is exploited by as a strategy for host cell escape for dissemination of C. neoformans. IMPORTANCE We previously reported that the outer mannose chains of N-glycans are dispensable for the virulence of C. neoformans, which is in stark contrast to findings for the other human-pathogenic yeast, Candida albicans. Here, we present evidence that an intact core N-glycan structure is required for C. neoformans pathogenicity by systematically analyzing alg3Δ, alg9Δ, and alg12Δ strains that have defects in lipid-linked N-glycan assembly and in in vivo virulence. The alg null mutants producing truncated core N-glycans were defective in inducing host cell death after phagocytosis, which is triggered as a mechanism of pulmonary escape and dissemination of C. neoformans, thus becoming inactive in causing fatal infection. The results clearly demonstrated the critical features of the N-glycan structure in mediating the interaction with host cells during fungal infection. The delineation of the roles of protein glycosylation in fungal pathogenesis not only provides insight into the glycan-based fungal infection mechanism but also will aid in the development of novel antifungal agents.

scholarly journals SseK1 and SseK3 Type III Secretion System Effectors Inhibit NF-κB Signaling and Necroptotic Cell Death in Salmonella-Infected Macrophages

2017 ◽  
Vol 85 (3) ◽  
Author(s):  
Regina A. Günster ◽  
Sophie A. Matthews ◽  
David W. Holden ◽  
Teresa L. M. Thurston
Keyword(s):  
Cell Death ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Host Cells ◽  
Macrophage Infection ◽  
Content Type ◽  
Type Iii ◽  
Tnf Α ◽  
Arginine Residues

ABSTRACT Within host cells such as macrophages, Salmonella enterica translocates virulence (effector) proteins across its vacuolar membrane via the SPI-2 type III secretion system. Previously, it was shown that when expressed ectopically, the effectors SseK1 and SseK3 inhibit tumor necrosis factor alpha (TNF-α)-induced NF-κB activation. In this study, we show that ectopically expressed SseK1, SseK2, and SseK3 suppress TNF-α-induced, but not Toll-like receptor 4- or interleukin-induced, NF-κB activation. Inhibition required a DXD motif in SseK1 and SseK3, which is essential for the transfer of N-acetylglucosamine to arginine residues (arginine-GlcNAcylation). During macrophage infection, SseK1 and SseK3 inhibited NF-κB activity in an additive manner. SseK3-mediated inhibition of NF-κB activation did not require the only known host-binding partner of this effector, the E3-ubiquitin ligase TRIM32. SseK proteins also inhibited TNF-α-induced cell death during macrophage infection. Despite SseK1 and SseK3 inhibiting TNF-α-induced apoptosis upon ectopic expression in HeLa cells, the percentage of infected macrophages undergoing apoptosis was SseK independent. Instead, SseK proteins inhibited necroptotic cell death during macrophage infection. SseK1 and SseK3 caused GlcNAcylation of different proteins in infected macrophages, suggesting that these effectors have distinct substrate specificities. Indeed, SseK1 caused the GlcNAcylation of the death domain-containing proteins FADD and TRADD, whereas SseK3 expression resulted in weak GlcNAcylation of TRADD but not FADD. Additional, as-yet-unidentified substrates are likely to explain the additive phenotype of a Salmonella strain lacking both SseK1 and SseK3.

scholarly journals XopQ induced stromule formation in Nicotiana benthamiana is causally linked to ETI signaling and depends on ADR1 and NRG1

2021 ◽  
Author(s):  
Jennifer Prautsch ◽  
Jessica L. Erickson ◽  
Sedef Özyürek ◽  
Rahel Gormannns ◽  
Lars Franke ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Nicotiana Benthamiana ◽  
Double Mutant ◽  
Plant Immunity ◽  
Signaling Cascade ◽  
Nuclear Dynamics ◽  
Hypersensitive Cell Death ◽  
Host Cell Death ◽  
Dependent Cell

In Nicotiana benthamiana, expression of the Xanthomonas effector XopQ triggers ROQ1-dependent ETI responses and in parallel accumulation of plastids around the nucleus and the formation of stromules. Both processes were proposed to contribute to ETI-related hypersensitive cell death and thereby to plant immunity. Whether these reactions are directly connected to ETI signaling events has not been tested. Here we utilized transient expression experiments to determine whether XopQ-mediated plastid reactions are a result of XopQ perception by ROQ1 or a consequence of XopQ virulence activity. We find that N. benthamiana mutants lacking ROQ1, both RNLs (NRG1 and ADR1) or EDS1, fail to elicit XopQ-dependent host cell death and stromule formation. Mutants lacking only NRG1 lost XopQ-dependent cell death but retained some stromule induction that was abolished in the RNL double mutant. This analysis aligns XopQ-induced stromules with the ETI signaling cascade but not to host programmed cell death. Furthermore, data reveal that XopQ-triggered plastid clustering is not strictly linked to stromule formation during ETI. Our data suggest that stromule formation, in contrast to chloroplast peri-nuclear dynamics, is an integral part of the N. benthamiana ETI response and that both RNL sub-types play a role in this ETI response.

scholarly journals Type III Secretion System-Dependent Translocation of Ectopically Expressed Yop Effectors into Macrophages by Intracellular Yersinia pseudotuberculosis

2011 ◽  
Vol 79 (11) ◽  
pp. 4322-4331 ◽  
Author(s):  
Yue Zhang ◽  
Galina Romanov ◽  
James B. Bliska
Keyword(s):  
Cell Death ◽  
Type Iii Secretion ◽  
Yersinia Pseudotuberculosis ◽  
Bacterial Pathogen ◽  
Type Iii Secretion System ◽  
Inducible Promoter ◽  
Secretion System ◽  
Host Cells ◽  
Content Type ◽  
Type Iii

ABSTRACTYersinia pseudotuberculosisis a Gram-negative bacterial pathogen. Virulence inY. pseudotuberculosisrequires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected withY. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete andY. pseudotuberculosisis internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently whenYersiniais extracellular. However, it is not clear whether the T3SS can be utilized by intracellularY. pseudotuberculosisto translocate Yops. This possibility was investigated here usingY. pseudotuberculosisstrains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viableY. pseudotuberculosisin macrophages. Collectively, our results suggest that the Ysc T3SS ofY. pseudotuberculosiscan function within macrophage phagosomes to translocate Yops into the host cytosol.

scholarly journals Overexpression of a Novel Biotrophy-Specific Colletotrichum truncatum Effector, CtNUDIX, in Hemibiotrophic Fungal Phytopathogens Causes Incompatibility with Their Host Plants

2012 ◽  
Vol 12 (1) ◽  
pp. 2-11 ◽  
Author(s):  
Vijai Bhadauria ◽  
Sabine Banniza ◽  
Albert Vandenberg ◽  
Gopalan Selvaraj ◽  
Yangdou Wei
Keyword(s):  
Cell Death ◽  
Signal Peptide ◽  
Plant Pathogens ◽  
Grain Legumes ◽  
Host Cells ◽  
Nudix Hydrolase ◽  
Colletotrichum Truncatum ◽  
Content Type ◽  
Cell Plasma ◽  
Fungal Phytopathogens

ABSTRACT The hemibiotrophic fungus Colletotrichum truncatum causes anthracnose disease on lentils and a few other grain legumes. It shows initial symptomless intracellular growth, where colonized host cells remain viable (biotrophy), and then switches to necrotrophic growth, killing the colonized host plant tissues. Here, we report a novel effector gene, CtNUDIX , from C. truncatum that is exclusively expressed during the late biotrophic phase (before the switch to necrotrophy) and elicits a hypersensitive response (HR)-like cell death in tobacco leaves transiently expressing the effector. CtNUDIX homologs, which contain a signal peptide and a Nudix hydrolase domain, may be unique to hemibiotrophic fungal and fungus-like plant pathogens. CtNUDIX lacking a signal peptide or a Nudix motif failed to induce cell death in tobacco. Expression of CtNUDIX:eGFP in tobacco suggested that the fusion protein might act on the host cell plasma membrane. Overexpression of CtNUDIX in C. truncatum and the rice blast pathogen, Magnaporthe oryzae , resulted in incompatibility with the hosts lentil and barley, respectively, by causing an HR-like response in infected host cells associated with the biotrophic invasive hyphae. These results suggest that C. truncatum and possibly M. oryzae elicit cell death to signal the transition from biotrophy to necrotrophy.

scholarly journals Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System

2021 ◽  
Vol 12 ◽  
Author(s):  
Tanapati Phakham ◽  
Christine Joy I. Bulaon ◽  
Narach Khorattanakulchai ◽  
Balamurugan Shanmugaraj ◽  
Supranee Buranapraditkun ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Mammalian Cells ◽  
Expression System ◽  
Sodium Dodecyl ◽  
Production Costs ◽  
Plant Production ◽  
Production Platform

The striking innovation and clinical success of immune checkpoint inhibitors (ICIs) have undoubtedly contributed to a breakthrough in cancer immunotherapy. Generally, ICIs produced in mammalian cells requires high investment, production costs, and involves time consuming procedures. Recently, the plants are considered as an emerging protein production platform due to its cost-effectiveness and rapidity for the production of recombinant biopharmaceuticals. This study explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody (mAb), Pembrolizumab, in Nicotiana benthamiana. The transient expression of this mAb in wild-type N. benthamiana accumulated up to 344.12 ± 98.23 μg/g fresh leaf weight after 4 days of agroinfiltration. The physicochemical and functional characteristics of plant-produced Pembrolizumab were compared to mammalian cell-produced commercial Pembrolizumab (Keytruda®). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results demonstrated that the plant-produced Pembrolizumab has the expected molecular weight and is comparable with the Keytruda®. Structural characterization also confirmed that both antibodies have no protein aggregation and similar secondary and tertiary structures. Furthermore, the plant-produced Pembrolizumab displayed no differences in its binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction with the Keytruda®. In vitro efficacy for T cell activation demonstrated that the plant-produced Pembrolizumab could induce IL-2 and IFN-γ production. Hence, this proof-of-concept study showed that the plant-production platform can be utilized for the rapid production of functional mAbs for immunotherapy.

scholarly journals Agromonas: a rapid disease assay for Pseudomonas syringae growth in agroinfiltrated leaves

2020 ◽  
Author(s):  
Pierre Buscaill ◽  
Nattapong Sanguankiattichai ◽  
Yoon Joo Lee ◽  
Jiorgos Kourelis ◽  
Gail Preston ◽  
...  
Keyword(s):  
Transient Expression ◽  
Pseudomonas Syringae ◽  
Nicotiana Benthamiana ◽  
Function Analysis ◽  
Plant Immunity ◽  
Comprehensive Analysis ◽  
Immune Gene ◽  
Proof Of Concept ◽  
Easy Method ◽  
Lengthy Process

SUMMARYThe lengthy process to generate transformed plants is a limitation in current research on the interactions of the model plant pathogen Pseudomonas syringae with plant hosts. Here we present an easy method called agromonas, where we quantify P. syringae growth in agroinfiltrated leaves of Nicotiana benthamiana using a cocktail of antibiotics to select P. syringae on plates. As a proof of concept, we demonstrate that transient expression of PAMP receptors reduces bacterial growth and that transient depletion of a host immune gene and transient expression of a T3 effector increase P. syringae growth in agromonas assays. We show that we can rapidly achieve structure-function analysis of immune components and test the function of immune hydrolases. The agromonas method is easy, fast and robust for routine disease assays with various Pseudomonas strains without transforming plants or bacteria. The agromonas assay offers reliable opportunity for further comprehensive analysis of plant immunity.One sentence summaryAgromonas is a rapid and robust disease assay to monitor Pseudomonas syringae growth in agroinfiltrated leaves expressing immune components and their suppressors.

scholarly journals The GHKL ATPase MORC1 Modulates Species-Specific Plant Immunity in Solanaceae

2015 ◽  
Vol 28 (8) ◽  
pp. 927-942 ◽  
Author(s):  
Patricia Manosalva ◽  
Murli Manohar ◽  
Karl-Heinz Kogel ◽  
Hong-Gu Kang ◽  
Daniel F. Klessig
Keyword(s):  
Cell Death ◽  
Transient Expression ◽  
Plant Immunity ◽  
Terminal Region ◽  
Transcriptional Gene Silencing ◽  
Basal Resistance ◽  
Specific Effects ◽  
Oomycete Pathogen ◽  
Species Specific

The microrchidia (MORC) proteins, a subset of the GHKL ATPase superfamily, were recently described as components involved in transcriptional gene silencing and plant immunity in Arabidopsis. To assess the role of MORC1 during resistance to Phytophthora infestans in solanaceous species, we altered the expression of the corresponding MORC1 homologs in potato, tomato, and Nicotiana benthamiana. Basal resistance to P. infestans was compromised in StMORC1-silenced potato and enhanced in overexpressing lines, indicating that StMORC1 positively affects immunity. By contrast, silencing SlMORC1 expression in tomato or NbMORC1 expression in N. benthamiana enhanced basal resistance to this oomycete pathogen. In addition, silencing SlMORC1 further enhanced resistance conferred by two resistance genes in tomato. Transient expression of StMORC1 in N. benthamiana accelerated cell death induced by infestin1 (INF1), whereas SlMORC1 or NbMORC1 suppressed it. Domain-swapping and mutational analyses indicated that the C-terminal region dictates the species-specific effects of the solanaceous MORC1 proteins on INF1-induced cell death. This C-terminal region also was required for homodimerization and phosphorylation of recombinant StMORC1 and SlMORC1, and its transient expression induced spontaneous cell death in N. benthamiana. Thus, this C-terminal region likely plays important roles in both determining and modulating the biological activity of MORC1 proteins.

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