scholarly journals Fusarium graminearum Tri12p Influences Virulence to Wheat and Trichothecene Accumulation

2012 ◽  
Vol 25 (11) ◽  
pp. 1408-1418 ◽  
Author(s):  
Jon Menke ◽  
Yanhong Dong ◽  
H. Corby Kistler
Keyword(s):  
Fusarium Graminearum ◽  
Fluorescent Protein ◽  
Toxin Production ◽  
Major Facilitator Superfamily ◽  
Induction Medium ◽  
Wild Type ◽  
In Planta ◽  
Major Facilitator ◽  
Green Fluorescent ◽  
Self Protection

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.

scholarly journals Multidrug Transporters CaCdr1p and CaMdr1p of Candida albicans Display Different Lipid Specificities: both Ergosterol and Sphingolipids Are Essential for Targeting of CaCdr1p to Membrane Rafts

2007 ◽  
Vol 52 (2) ◽  
pp. 694-704 ◽  
Author(s):  
Ritu Pasrija ◽  
Sneh Lata Panwar ◽  
Rajendra Prasad
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Membrane Lipid ◽  
Major Facilitator Superfamily ◽  
Pathogenic Yeast ◽  
Atp Binding Cassette Transporter ◽  
Major Facilitator ◽  
Green Fluorescent

ABSTRACT In this study, we compared the effects of altered membrane lipid composition on the localization of two membrane drug transporters from different superfamilies of the pathogenic yeast Candida albicans. We demonstrated that in comparison to the major facilitator superfamily multidrug transporter CaMdr1p, ATP-binding cassette transporter CaCdr1p of C. albicans is preferentially localized within detergent-resistant membrane (DRM) microdomains called ‘rafts.’ Both CaCdr1p and CaMdr1p were overexpressed as green fluorescent protein (GFP)-tagged proteins in a heterologous host Saccharomyces cerevisiae, wherein either sphingolipid (Δsur4 or Δfen1 or Δipt1) or ergosterol (Δerg24 or Δerg6 or Δerg4) biosynthesis was compromised. CaCdr1p-GFP, when expressed in the above mutant backgrounds, was not correctly targeted to plasma membranes (PM), which also resulted in severely impaired drug resistance. In contrast, CaMdr1p-GFP displayed no sorting defect in the mutant background and remained properly surface localized and displayed no change in drug resistance. Our data clearly show that CaCdr1p is selectively recruited, over CaMdr1p, to the DRM microdomains of the yeast PM and that any imbalance in the raft lipid constituents results in missorting of CaCdr1p.

scholarly journals The Stress-Activated Protein Kinase FgOS-2 Is a Key Regulator in the Life Cycle of the Cereal Pathogen Fusarium graminearum

2012 ◽  
Vol 25 (9) ◽  
pp. 1142-1156 ◽  
Author(s):  
Thuat Van Nguyen ◽  
Wilhelm Schäfer ◽  
Jörg Bormann
Keyword(s):  
Protein Kinase ◽  
Fusarium Graminearum ◽  
Fluorescent Protein ◽  
Microscopic Analysis ◽  
Deletion Mutants ◽  
Wild Type ◽  
In Planta ◽  
Head Blight ◽  
Food And Feed

Fusarium graminearum is one of the most destructive pathogens of cereals and a threat to food and feed production worldwide. It is an ascomycetous plant pathogen and the causal agent of Fusarium head blight disease in small grain cereals and of cob rot disease in maize. Infection with F. graminearum leads to yield losses and mycotoxin contamination. Zearalenone (ZEA) and deoxynivalenol (DON) are hazardous mycotoxins; the latter is necessary for virulence toward wheat. Deletion mutants of the F. graminearum orthologue of the Saccharomyces cerevisiae Hog1 stress-activated protein kinase, FgOS-2 (ΔFgOS-2), showed drastically reduced in planta DON and ZEA production. However, ΔFgOS-2 produced even more DON than the wild type under in vitro conditions, whereas ZEA production was similar to that of the wild type. These deletion strains are dramatically reduced in pathogenicity toward maize and wheat. We constitutively expressed the fluorescent protein dsRed in the deletion strains and the wild type. Microscopic analysis revealed that ΔFgOS-2 is unable to reach the rachis node at the base of wheat spikelets. During vegetative growth, ΔFgOS-2 strains exhibit increased resistance against the phenylpyrrole fludioxonil. Growth of mutant colonies on agar plates supplemented with NaCl is reduced but conidia formation remained unchanged. However, germination of mutant conidia on osmotic media is severely impaired. Germ tubes are swollen and contain multiple nuclei. The deletion mutants completely fail to produce perithecia and ascospores. Furthermore, FgOS-2 also plays a role in reactive oxygen species (ROS)-related signaling. The transcription and activity of fungal catalases is modulated by FgOS-2. Among the genes regulated by FgOS-2, we found a putative calcium-dependent NADPH-oxidase (noxC) and the transcriptional regulator of ROS metabolism, atf1. The present study describes new aspects of stress-activated protein kinase signaling in F. graminearum.

scholarly journals The transporter CefM involved in translocation of biosynthetic intermediates is essential for cephalosporin production

2009 ◽  
Vol 418 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Fernando Teijeira ◽  
Ricardo V. Ullán ◽  
Susana M. Guerra ◽  
Carlos García-Estrada ◽  
Inmaculada Vaca ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Gene Replacement ◽  
Major Facilitator Superfamily ◽  
Cephalosporin C ◽  
Protein Fusion ◽  
Reading Frame ◽  
Peroxisomal Targeting ◽  
Major Facilitator ◽  
Green Fluorescent ◽  
Targeted Inactivation

The cluster of early cephalosporin biosynthesis genes (pcbAB, pcbC, cefD1, cefD2 and cefT of Acremonium chrysogenum) contains all of the genes required for the biosynthesis of the cephalosporin biosynthetic pathway intermediate penicillin N. Downstream of the cefD1 gene, there is an unassigned open reading frame named cefM encoding a protein of the MFS (major facilitator superfamily) with 12 transmembrane domains, different from the previously reported cefT. Targeted inactivation of cefM by gene replacement showed that it is essential for cephalosporin biosynthesis. The disrupted mutant accumulates a significant amount of penicillin N, is unable to synthesize deacetoxy-, deacetyl-cephalosporin C and cephalosporin C and shows impaired differentiation into arthrospores. Complementation of the disrupted mutant with the cefM gene restored the intracellular penicillin N concentration to normal levels and allowed synthesis and secretion of the cephalosporin intermediates and cephalosporin C. A fused cefM-gfp gene complemented the cefM-disrupted mutant, and the CefM–GFP (green fluorescent protein) fusion was targeted to intracellular microbodies that were abundant after 72 h of culture in the differentiating hyphae and in the arthrospore chains, coinciding with the phase of intense cephalosporin biosynthesis. Since the dual-component enzyme system CefD1–CefD2 that converts isopenicillin N into penicillin N contains peroxisomal targeting sequences, it is probable that the epimerization step takes place in the peroxisome matrix. The CefM protein seems to be involved in the translocation of penicillin N from the peroxisome (or peroxisome-like microbodies) lumen to the cytosol, where it is converted into cephalosporin C.

scholarly journals Fusarium graminearum arabinanase (Arb93B) Enhances Wheat Head Blight Susceptibility by Suppressing Plant Immunity

2019 ◽  
Vol 32 (7) ◽  
pp. 888-898 ◽  
Author(s):  
Guixia Hao ◽  
Susan McCormick ◽  
Martha M. Vaughan ◽  
Todd A. Naumann ◽  
Hye-Seon Kim ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Cell Walls ◽  
Fluorescent Protein ◽  
Plant Immunity ◽  
Defense Responses ◽  
Plant Cell Walls ◽  
Plant Defense Responses ◽  
Wild Type ◽  
Head Blight ◽  
Green Fluorescent

Fusarium head blight (FHB) of wheat and barley caused by the fungus Fusarium graminearum reduces crop yield and contaminates grain with mycotoxins. In this study, we investigated two exo-1,5-α-L-arabinanases (Arb93A and Arb93B) secreted by F. graminearum and their effect on wheat head blight development. Arabinan is an important component of plant cell walls but it was not known whether these arabinanases play a role in FHB. Both ARB93A and ARB93B were induced during the early stages of infection. arb93A mutants did not exhibit a detectable change in ability to cause FHB, whereas arb93B mutants caused lower levels of FHB symptoms and deoxynivalenol contamination compared with the wild type. Furthermore, virulence and deoxynivalenol contamination were restored to wild-type levels in ARB93B complemented mutants. Fusion proteins of green fluorescent protein (GFP) with the predicted chloroplast peptide or the mature protein of Arb93B were not observed in the chloroplast. Reactive oxygen species (ROS) production was reduced in the infiltrated zones of Nicotiana benthamiana leaves expressing ARB93B-GFP. Coexpression of ARB93B-GFP and Bax in N. benthamiana leaves significantly suppressed Bax-programmed cell death. Our results indicate that Arb93B enhances plant disease susceptibility by suppressing ROS-associated plant defense responses.

scholarly journals Identification of a Novel System for Boron Transport: Atr1 Is a Main Boron Exporter in Yeast

2009 ◽  
Vol 29 (13) ◽  
pp. 3665-3674 ◽  
Author(s):  
Alaattin Kaya ◽  
Huseyin C. Karakaya ◽  
Dmitri E. Fomenko ◽  
Vadim N. Gladyshev ◽  
Ahmet Koc
Keyword(s):  
Fluorescent Protein ◽  
Efflux Pump ◽  
Major Facilitator Superfamily ◽  
Boron Tolerance ◽  
Cellular Processes ◽  
Dna Library ◽  
Highly Sensitive ◽  
Genomic Dna Library ◽  
Major Facilitator ◽  
Green Fluorescent

ABSTRACT Boron is a micronutrient in plants and animals, but its specific roles in cellular processes are not known. To understand boron transport and functions, we screened a yeast genomic DNA library for genes that confer resistance to the element in Saccharomyces cerevisiae. Thirty boron-resistant transformants were isolated, and they all contained the ATR1 (YML116w) gene. Atr1 is a multidrug resistance transport protein belonging to the major facilitator superfamily. C-terminal green fluorescent protein-tagged Atr1 localized to the cell membrane and vacuole, and ATR1 gene expression was upregulated by boron and several stress conditions. We found that atr1Δ mutants were highly sensitive to boron treatment, whereas cells overexpressing ATR1 were boron resistant. In addition, atr1Δ cells accumulated boron, whereas ATR1-overexpressing cells had low intracellular levels of the element. Furthermore, atr1Δ cells showed stronger boron-dependent phenotypes than mutants deficient in genes previously reported to be implicated in boron metabolism. ATR1 is widely distributed in bacteria, archaea, and lower eukaryotes. Our data suggest that Atr1 functions as a boron efflux pump and is required for boron tolerance.

scholarly journals Mechanism of Aurora-B Degradation and Its Dependency on Intact KEN and A-Boxes: Identification of an Aneuploidy-Promoting Property

2005 ◽  
Vol 25 (12) ◽  
pp. 4977-4992 ◽  
Author(s):  
Hao G. Nguyen ◽  
Dharmaraj Chinnappan ◽  
Takeshi Urano ◽  
Katya Ravid
Keyword(s):  
Chromosome Segregation ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Degradation Pathway ◽  
Aurora B ◽  
Stable Form ◽  
Wild Type ◽  
Green Fluorescent ◽  
Critical Regions

ABSTRACT The kinase Aurora-B, a regulator of chromosome segregation and cytokinesis, is highly expressed in a variety of tumors. During the cell cycle, the level of this protein is tightly controlled, and its deregulated abundance is suspected to contribute to aneuploidy. Here, we provide evidence that Aurora-B is a short-lived protein degraded by the proteasome via the anaphase-promoting cyclosome complex (APC/c) pathway. Aurora-B interacts with the APC/c through the Cdc27 subunit, Aurora-B is ubiquitinated, and its level is increased upon treatment with inhibitors of the proteasome. Aurora-B binds in vivo to the degradation-targeting proteins Cdh1 and Cdc20, the overexpression of which accelerates Aurora-B degradation. Using deletions or point mutations of the five putative degradation signals in Aurora-B, we show that degradation of this protein does not depend on its D-boxes (RXXL), but it does require intact KEN boxes and A-boxes (QRVL) located within the first 65 amino acids. Cells transfected with wild-type or A-box-mutated or KEN box-mutated Aurora-B fused to green fluorescent protein display the protein localized to the chromosomes and then to the midzone during mitosis, but the mutated forms are detected at greater intensities. Hence, we identified the degradation pathway for Aurora-B as well as critical regions for its clearance. Intriguingly, overexpression of a stable form of Aurora-B alone induces aneuploidy and anchorage-independent growth.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

scholarly journals Detection of Oil Palm Root Penetration by Agrobacterium-Mediated Transformed Ganoderma boninense, Expressing Green Fluorescent Protein

2017 ◽  
Vol 107 (4) ◽  
pp. 483-490 ◽  
Author(s):  
Nisha Govender ◽  
Mui-Yun Wong
Keyword(s):  
Oil Palm ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Early Stage ◽  
Marker Gene ◽  
Induction Medium ◽  
Root Penetration ◽  
Ganoderma Boninense ◽  
Agrobacterium Strain ◽  
Green Fluorescent

A highly efficient and reproducible Agrobacterium-mediated transformation protocol for Ganoderma boninense was developed to facilitate observation of the early stage infection of basal stem rot (BSR). The method was proven amenable to different explants (basidiospore, protoplast, and mycelium) of G. boninense. The transformation efficiency was highest (62%) under a treatment combination of protoplast explant and Agrobacterium strain LBA4404, with successful expression of an hyg marker gene and gus-gfp fusion gene under the control of heterologous p416 glyceraldehyde 3-phosphate dehydrogenase promoter. Optimal transformation conditions included a 1:100 Agrobacterium/explant ratio, induction of Agrobacterium virulence genes in the presence of 250 μm acetosyringone, co-cultivation at 22°C for 2 days on nitrocellulose membrane overlaid on an induction medium, and regeneration of transformants on potato glucose agar prepared with 0.6 M sucrose and 20 mM phosphate buffer. Evaluated transformants were able to infect root tissues of oil palm plantlets with needle-like microhyphae during the penetration event. The availability of this model pathogen system for BSR may lead to a better understanding of the pathogenicity factors associated with G. boninense penetration into oil palm roots.

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