Clathrin-mediated Endocytosis Facilitates Internalization of Magnaporthe oryzae Effectors into Rice Cells

Many filamentous eukaryotic plant pathogens, such as fungi and oomycetes, deliver effectors into living plant cells to suppress defenses and control plant processes needed for infection. To date, little is known about the mechanism by which these pathogens translocate effector proteins across the plasma membrane into the plant cytoplasm. The blast fungus Magnaporthe oryzae secretes cytoplasmic effectors into a specialized biotrophic interfacial complex (BIC) before translocation. Here we show that cytoplasmic effectors within BICs are packaged into dynamic vesicles that can occasionally be found separated from BICs in the host cytoplasm. Live cell imaging with fluorescently-labelled rice lines, showed that BICs are enriched in plant plasma membrane, actin, and Clathrin Light Chain-1, a marker for clathrin-mediated endocytosis (CME). We report that a novel cytoplasmic effector, Bas83, labels empty membrane vesicles surrounding BICs. Inhibition of CME using VIGS and chemical treatments results in a distinctive swollen BIC phenotype lacking effector vesicles. In contrast, fluorescent marker co-localization, VIGS and chemical inhibitor studies failed to implicate clathrin-independent endocytosis in effector vesicle formation. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by clathrin-mediated endocytosis in BICs and suggests a role for M. oryzae effectors in co-opting plant endocytosis.

Download Full-text

Effectors of biotrophic fungal plant pathogens

Functional Plant Biology ◽

10.1071/fp10072 ◽

2010 ◽

Vol 37 (10) ◽

pp. 913 ◽

Cited By ~ 12

Author(s):

Pamela H. P. Gan ◽

Maryam Rafiqi ◽

Adrienne R. Hardham ◽

Peter N. Dodds

Keyword(s):

Plant Tissue ◽

Fungal Pathogen ◽

Plant Pathogens ◽

Plant Cells ◽

Host Cells ◽

Plant Proteins ◽

Living Plant ◽

Host Cytoplasm ◽

Fungal Plant Pathogens ◽

Biotrophic Fungi

Plant pathogenic biotrophic fungi are able to grow within living plant tissue due to the action of secreted pathogen proteins known as effectors that alter the response of plant cells to pathogens. The discovery and identification of these proteins has greatly expanded with the sequencing and annotation of fungal pathogen genomes. Studies to characterise effector function have revealed that a subset of these secreted pathogen proteins interact with plant proteins within the host cytoplasm. This review focuses on the effectors of intracellular biotrophic and hemibiotrophic fungal plant pathogens and summarises advances in understanding the roles of these proteins in disease and in elucidating the mechanism of fungal effector uptake into host cells.

Download Full-text

Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils

Biochemical Journal ◽

10.1042/bj3140469 ◽

1996 ◽

Vol 314 (2) ◽

pp. 469-475 ◽

Cited By ~ 6

Author(s):

R. Alexander BLACKWOOD ◽

James E. SMOLEN ◽

Ronald J. HESSLER ◽

Donna M. HARSH ◽

Amy TRANSUE

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Membrane Vesicles ◽

Phospholipid Vesicle ◽

Human Neutrophils ◽

Plasma Membrane Vesicles ◽

Fluorescent Marker ◽

Whole Cells ◽

Specific Granules ◽

Neutrophil Degranulation

Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide (‘DPX’) to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 μM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.

Download Full-text

Bridging the Gap: Type III Secretion Systems in Plant-Beneficial Bacteria

Microorganisms ◽

10.3390/microorganisms10010187 ◽

2022 ◽

Vol 10 (1) ◽

pp. 187

Author(s):

Antoine Zboralski ◽

Adrien Biessy ◽

Martin Filion

Keyword(s):

Type Iii Secretion ◽

Plant Pathogens ◽

Plant Immunity ◽

Effector Proteins ◽

Secretion Systems ◽

Beneficial Bacteria ◽

Living Plant ◽

Pseudomonas Spp ◽

Type Iii ◽

Type Iii Secretion Systems

Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines translocating effector proteins into the cytoplasm of eukaryotic cells. They have been intensively studied for their important roles in animal and plant bacterial diseases. Over the past two decades, genome sequencing has unveiled their ubiquitous distribution in many taxa of Gram-negative bacteria, including plant-beneficial ones. Here, we discuss the distribution and functions of the T3SS in two agronomically important bacterial groups: the symbiotic nodule-forming nitrogen-fixing rhizobia and the free-living plant-beneficial Pseudomonas spp. In legume-rhizobia symbiosis, T3SSs and their cognate effectors play important roles, including the modulation of the plant immune response and the initiation of the nodulation process in some cases. In plant-beneficial Pseudomonas spp., the roles of T3SSs are not fully understood, but pertain to plant immunity suppression, biocontrol against eukaryotic plant pathogens, mycorrhization facilitation, and possibly resistance against protist predation. The diversity of T3SSs in plant-beneficial bacteria points to their important roles in multifarious interkingdom interactions in the rhizosphere. We argue that the gap in research on T3SSs in plant-beneficial bacteria must be bridged to better understand bacteria/eukaryotes rhizosphere interactions and to support the development of efficient plant-growth promoting microbial inoculants.

Download Full-text

Bacteriophages to control plant diseases

10.19103/as.2021.0093.18 ◽

2021 ◽

pp. 473-506

Author(s):

Manoj Choudhary ◽

◽

Mathews Paret ◽

Aleksa Obradović ◽

Katarina Gašić ◽

...

Keyword(s):

Plant Pathogens ◽

Uv Light ◽

Control Measure ◽

Management Strategies ◽

Control Plant ◽

Plant Diseases ◽

Bacterial Diseases ◽

Time Of Application ◽

Development Of Resistance ◽

And Control

Crop yield loss due to bacterial plant pathogens need to be reduced to increase global food production demand. Currently available disease management strategies involving copper-based bactericides and antibiotics are losing efficacy due to development of resistance in bacteria. There is long familiar demand of environmentally friendly and sustainable strategies to control bacterial diseases. Bacteriophages are virus that kill target bacteria without affecting another microorganism and environment. Bacteriophage efficiency on phyllosphere is mainly affected by ultraviolet (UV) light. Use of combination of phage, mixture with phage carrier bacteria and optimizing time of application helps in persistence of bacteriophage. There are several bacteriophage products already available in the market to control destructive bacterial diseases. Unlike chemical based traditional control measure, bacteriophage mixture can be easily amended to reduce resistance development in bacteria. In this chapter, the authors discuss from phage isolation to interaction with bacteria and control mechanism of plant diseases.

Download Full-text

Application of a C. trachomatis expression system to identify C. pneumoniae proteins translocated into host cells

Journal of Bacteriology ◽

10.1128/jb.00511-20 ◽

2021 ◽

Author(s):

Izumi Yanatori ◽

Koshiro Miura ◽

Yi-Shan Chen ◽

Raphael H. Valdivia ◽

Fumio Kishi

Keyword(s):

Plasma Membrane ◽

Expression System ◽

Effector Proteins ◽

Host Cells ◽

Cell Protein ◽

Secretion Systems ◽

Cellular Functions ◽

Host Cytoplasm ◽

Pathogen Effector ◽

Specific Proteins

Chlamydia pneumoniae is a Gram-negative, obligate intracellular pathogen that causes community-acquired respiratory infections. C. pneumoniae uses a cell contact-dependent type-III secretion (T3S) system to translocate pathogen effector proteins that manipulate host cellular functions. While several C. pneumoniae T3S effectors have been proposed, few have been experimentally confirmed in Chlamydia. In this study, we expressed 382 C. pneumoniae genes in C. trachomatis as fusion proteins to an epitope tag derived from glycogen synthase kinase 3β (GSK3β) which is the target of phosphorylation by mammalian kinases. Based on the detection of the tagged C. pneumoniae protein with anti-phospho GSK3β antibodies, we identified 49 novel C. pneumoniae-specific proteins that are translocated by C. trachomatis into the host cytoplasm and thus likely play a role as modifiers of host cellular functions. In this manner, we identified and characterized a new C. pneumoniae effector CPj0678 that recruits the host cell protein PACSIN2 to the plasma membrane. These findings indicate that C. trachomatis provides a powerful screening system to detect candidate effector proteins encoded by other pathogenic and endosymbiotic Chlamydia species. Importance Chlamydia injects numerous effector proteins into host cells to manipulate cellular functions important for bacterial survival. Based on findings in C. trachomatis, it has been proposed that between 5-10% of the C. pneumoniae genome, a related respiratory pathogen, encodes secreted effectors. However only a few C. pneumoniae effectors have been identified and experimentally validated. With the development of methods for the stable genetic transformation of C. trachomatis, it is now possible to use C. trachomatis shuttle plasmids to express and explore the function of proteins from other Chlamydia in a more relevant bacterial system. In this study, we experimentally determined that 49 C. pneumoniae-specific proteins are translocated into the host cytoplasm by Chlamydia secretion systems, and identify a novel effector required to recruit the host factor PACSIN2 to the plasma membrane during infection.

Download Full-text