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 Peroxisome Function Is Required for Virulence and Survival of Fusarium graminearum

2012 ◽  
Vol 25 (12) ◽  
pp. 1617-1627 ◽  
Author(s):  
Kyunghun Min ◽  
Hokyoung Son ◽  
Jungkwan Lee ◽  
Gyung Ja Choi ◽  
Jin-Cheol Kim ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fusarium Graminearum ◽  
Protein Import ◽  
Fluorescent Proteins ◽  
Deletion Mutants ◽  
In Planta ◽  
Peroxisomal Protein ◽  
Peroxisomal Targeting ◽  
Signal Type

Peroxisomes are organelles that are involved in a number of important cellular metabolic processes, including the β-oxidation of fatty acids, biosynthesis of secondary metabolites, and detoxification of reactive oxygen species (ROS). In this study, the role of peroxisomes was examined in Fusarium graminearum by targeted deletion of three genes (PEX5, PEX6, and PEX7) encoding peroxin (PEX) proteins required for peroxisomal protein import. PEX5 and PEX7 deletion mutants were unable to localize the fluorescently tagged peroxisomal targeting signal type 1 (PTS1)- and PTS2-containing proteins to peroxisomes, respectively, whereas the PEX6 mutant failed to localize both fluorescent proteins. Deletion of PEX5 and PEX6 resulted in retarded growth on long-chain fatty acids and butyrate, while the PEX7 deletion mutants utilized fatty acids other than butyrate. Virulence on wheat heads was greatly reduced in the PEX5 and PEX6 deletion mutants, and they were defective in spreading from inoculated florets to the adjacent spikelets through rachis. Deletion of PEX5 and PEX6 dropped survivability of aged cells in planta and in vitro due to the accumulation of ROS followed by necrotic cell death. These results demonstrate that PTS1-dependent peroxisomal protein import mediated by PEX5 and PEX6 are critical to virulence and survival of F. graminearum.

scholarly journals Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

1999 ◽  
Vol 277 (6) ◽  
pp. C1202-C1209 ◽  
Author(s):  
Robert S. Haworth ◽  
James Sinnett-Smith ◽  
Enrique Rozengurt ◽  
Metin Avkiran
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Phorbol Ester ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Wild Type ◽  
Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

scholarly journals A sterol C-14 reductase encoded by FgERG24B is responsible for the intrinsic resistance of Fusarium graminearum to amine fungicides

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1665-1675 ◽  
Author(s):  
Xin Liu ◽  
Jing Fu ◽  
Yingzi Yun ◽  
Yanni Yin ◽  
Zhonghua Ma
Keyword(s):  
Fusarium Graminearum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ergosterol Biosynthesis ◽  
Deletion Mutants ◽  
Wild Type ◽  
Head Blight ◽  
Intrinsic Resistance ◽  
In The Wild ◽  
Increased Sensitivity

Fusarium graminearum, the causal agent of wheat head blight, shows intrinsic resistance to amine fungicides. It is commonly accepted that the amines target sterol C-14 reductase and sterol Δ8–Δ7 isomerase of ergosterol biosynthesis, encoded by the genes ERG24 and ERG2, respectively. Analysis of the genome sequence of F. graminearum revealed that the fungus contains two paralogous FgERG24 genes (FgERG24A and FgERG24B), which are homologous to the ERG24 of Saccharomyces cerevisiae. In this study, we disrupted FgERG24A and FgERG24B in F. graminearum. Compared to the wild-type strain HN9-1, FgERG24A and FgERG24B deletion mutants did not show recognizable phenotypic changes in mycelial growth on potato dextrose agar or in virulence on wheat heads. HPLC analysis showed that the amount of ergosterol in FgERG24A or FgERG24B deletion mutants was not significantly different from that in the wild-type strain. These results indicate that neither of the two genes is essential for growth, pathogenicity or ergosterol biosynthesis in F. graminearum. FgERG24B deletion mutants exhibited significantly increased sensitivity to amine fungicides, including tridemorph, fenpropidin and spiroxamine, but not to non-amine fungicides. In contrast, FgERG24A deletion mutants did not show changed sensitivity to any amine tested. The resistance of the FgERG24B deletion mutant to amines was restored by genetic complementation of the mutant with wild-type FgERG24B. These results indicate that FgERG24B controls the intrinsic resistance of F. graminearum to amines. The finding of this study provides new insights into amine resistance in filamentous fungi.

scholarly journals Phosphoprotein Enriched in Astrocytes-15 kDa Expression Inhibits Astrocyte Migration by a Protein Kinase Cδ-dependent Mechanism

2006 ◽  
Vol 17 (12) ◽  
pp. 5141-5152 ◽  
Author(s):  
François Renault-Mihara ◽  
Frédéric Beuvon ◽  
Xavier Iturrioz ◽  
Brigitte Canton ◽  
Sophie De Bouard ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fluorescent Protein ◽  
Constitutive Expression ◽  
Mitogen Activated Protein Kinase ◽  
Wild Type ◽  
Bryostatin 1 ◽  
Calcium Calmodulin Dependent ◽  
Mitogen Activated Protein ◽  
Green Fluorescent

Phosphoprotein enriched in astrocytes-15 kDa (PEA-15), a phosphoprotein enriched in astrocytes, inhibits both apoptosis and proliferation in normal and cancerous cells. Here, analysis of PEA-15 expression in glioblastoma organotypic cultures revealed low levels of PEA-15 in tumor cells migrating away from the explants, regardless of the expression levels in the originating explants. Because glioblastomas are highly invasive primary brain tumors that can originate from astrocytes, we explored the involvement of PEA-15 in the control of astrocyte migration. PEA-15−/− astrocytes presented an enhanced motility in vitro compared with their wild-type counterparts. Accordingly, NIH-3T3 cells transfected by green fluorescent protein-PEA-15 displayed a reduced migration. Reexpression of PEA-15 restored PEA-15−/− astrocyte motility to wild-type levels. Pharmacological manipulations excluded a participation of extracellular signal-regulated kinase/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and calcium/calmodulin-dependent protein kinase II in this effect of PEA-15. In contrast, treatment by bisindolylmaleimide, Gö6976, and rottlerin, and chronic application of phorbol 12-myristate 13-acetate and/or bryostatin-1 indicated that PKCδ mediated PEA-15 inhibition of astrocyte migration. PEA-15−/− astrocytes constitutively expressed a 40-kDa form of PKCδ that was down-regulated upon PEA-15 reexpression. Together, these data reveal a new function for PEA-15 in the inhibitory control of astrocyte motility through a PKCδ-dependent pathway involving the constitutive expression of a catalytic fragment of PKCδ.

Endophytic fungi as a promising biocontrol agent to protect wheat from Fusarium graminearum head blight

Plant Disease ◽  
2021 ◽  
Author(s):  
Zachary Albert Noel ◽  
Ludmilla Roze ◽  
Mikaela Breunig ◽  
Frances Trail
Keyword(s):  
Microbial Community ◽  
Fusarium Graminearum ◽  
Endophytic Fungi ◽  
Seed Weight ◽  
Biocontrol Agent ◽  
Fungal Endophytes ◽  
In Planta ◽  
Head Blight ◽  
Species Definitions

The search for beneficial endophytes that can be part of a constructed microbial community has increased in recent years. We characterized three endophytic fungi previously isolated from wheat for their in vitro and in planta antagonism toward the Fusarium head blight pathogen, Fusarium graminearum. The endophytes were phylogenetically characterized and shown to be Alternaria destruens, Fusarium commune, and Fusarium oxysporum. Individual fungal endophytes significantly increased seed weight and lowered the accumulation of the mycotoxin deoxynivalenol compared to F. graminearum infected wheat heads without endophyte pretreatment. Investigation into the mechanism of competition in vitro showed that endophytes competitively excluded F. graminearum by pre-emptive colonization and possible inhibition over a distance. Investigations on the use of these endophytes in the field are in progress. Identification of these three endophytes highlights a common quandary in searching for beneficial microbes to use in agriculture: species definitions often do not separate individual isolates’ lifestyles. A greater understanding of the risks in using intraspecies variants for biocontrol is needed and should be examined in the context of the ecology of the individuals being investigated.

scholarly journals Toxigenic Capacity and Trichothecene Production by Fusarium graminearum Isolates from Argentina and Their Relationship with Aggressiveness and Fungal Expansion in the Wheat Spike

2014 ◽  
Vol 104 (4) ◽  
pp. 357-364 ◽  
Author(s):  
I. Malbrán ◽  
C. A. Mourelos ◽  
J. R. Girotti ◽  
P. A. Balatti ◽  
G. A. Lori
Keyword(s):  
Fusarium Graminearum ◽  
Vascular Tissue ◽  
Close Correlation ◽  
Field Trials ◽  
In Planta ◽  
Head Blight ◽  
Variable Intensity ◽  
Polymerase Chain

At least 20 epidemics of Fusarium head blight (FHB) of wheat have been registered in the last 50 years in Argentina, with variable intensity. Damage induced by the disease is further aggravated by the presence of mycotoxins in affected grains that may cause health problems to humans and animals. The trichothecene chemotype was analyzed for 112 isolates of Fusarium graminearum from Argentina by polymerase chain reaction and two field trials were conducted to study the aggressiveness of a subsample of 14 representative isolates and to analyze deoxynivalenol (DON) production in planta and in vitro. All isolates belonged to the 15-acetyl-DON chemotype. Significant differences were observed in both the symptom severity induced in wheat spikes and the in vivo DON production, and a close correlation was found between these two variables. However, in vitro toxigenic potential was not correlated with the capacity of F. graminearum isolates to produce DON under natural conditions. The progress of infection in the rachis of inoculated wheat spikes was analyzed and the pathogen presence verified in both symptomatic and symptomless spikes. Even isolates with a limited capacity to induce symptoms were able to colonize the vascular tissue and to produce considerable amounts of DON in planta.

scholarly journals The Fusarium graminearum t-SNARE Sso2 Is Involved in Growth, Defense, and DON Accumulation and Virulence

2020 ◽  
Vol 33 (7) ◽  
pp. 888-901
Author(s):  
Sean P. O’Mara ◽  
Karen Broz ◽  
Marike Boenisch ◽  
Zixuan Zhong ◽  
Yanhong Dong ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Deletion Mutant ◽  
Expression Profiles ◽  
Pathogenic Fungus ◽  
Gene Expression Profiles ◽  
Plant Diseases ◽  
In Planta ◽  
Head Blight ◽  
Double Deletion

The plant-pathogenic fungus Fusarium graminearum, causal agent of Fusarium head blight (FHB) disease on small grain cereals, produces toxic trichothecenes that require facilitated export for full virulence. Two potential modes of mycotoxin transport are membrane-bound transporters, which move toxins across cellular membranes, and N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-mediated vesicular transport, by which toxins may be packaged as cargo in vesicles bound for organelles or the plasma membrane. In this study, we show that deletion of a gene (Sso2) for a subapically localized t-SNARE protein results in growth alteration, increased sensitivity to xenobiotics, altered gene expression profiles, and reduced deoxynivalenol (DON) accumulation in vitro and in planta as well as reduced FHB symptoms on wheat. A double deletion mutant generated by crossing the ∆sso2 deletion mutant with an ATP-binding cassette transporter deletion mutant (∆abc1) resulted in an additive reduction in DON accumulation and almost complete loss of FHB symptoms in planta. These results suggest an important role of Sso2-mediated subapical exocytosis in FHB progression and xenobiotic defense and are the first report of an additive reduction in F. graminearum DON accumulation upon deletion of two distinct modes of cellular export. This research provides useful information which may aid in formulating novel management plans of FHB or other destructive plant diseases.

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 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 Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

2004 ◽  
Vol 48 (11) ◽  
pp. 4154-4162 ◽  
Author(s):  
Thomas Herget ◽  
Martina Freitag ◽  
Monika Morbitzer ◽  
Regina Kupfer ◽  
Thomas Stamminger ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
High Efficiency ◽  
Fluorescent Protein ◽  
Human Fibroblasts ◽  
Growth Factor Receptor ◽  
Protein Kinase Activity ◽  
Hcmv Replication

ABSTRACT Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC50s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC50s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 μM, respectively) were in the range of the IC50 of ganciclovir (1.2 ± 0.2 μM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

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