Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungusUstilago maydis

Spellig, Tilman; Bottin, Arnaud; Kahmann, Regine

doi:10.1007/bf02172396

Cited by 124 publications

(45 citation statements)

References 30 publications

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“…This was then used to replace by marker-exchange the Δ UhAvr1 deletion in strain Uh1289, thereby putting the chimer in its original expression site ( Figure S6C ). Confocal microscopy of this constructed strain Uh1353 ( Table S2 ) clearly corroborated the qRT-PCR expression results since no fluorescence was observed in haploid or mated cells at the time of inoculation of barley coleoptiles ( Figure 6A ), whereas bright fluorescence comparable to GFP expressed from the strong U. maydis otef promoter [44] ( Figure 6B ) was apparent after 48 hrs in mated dikaryotic hyphae upon infection ( Figure 6C ) and while extending in the coleoptile later during the infection ( Figure 6D ). GFP fluorescence was seen in growing hyphal tips, possibly in vesicle-like structures ( Figure 6D and E ) and in older hyphae associated with the cell wall.…”

Section: Resultssupporting

confidence: 71%

An Immunity-Triggering Effector from the Barley Smut Fungus Ustilago hordei Resides in an Ustilaginaceae-Specific Cluster Bearing Signs of Transposable Element-Assisted Evolution

et al. 2014

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The basidiomycete smut fungus Ustilago hordei was previously shown to comprise isolates that are avirulent on various barley host cultivars. Through genetic crosses we had revealed that a dominant avirulence locus UhAvr1 which triggers immunity in barley cultivar Hannchen harboring resistance gene Ruh1, resided within an 80-kb region. DNA sequence analysis of this genetically delimited region uncovered the presence of 7 candidate secreted effector proteins. Sequence comparison of their coding sequences among virulent and avirulent parental and field isolates could not distinguish UhAvr1 candidates. Systematic deletion and complementation analyses revealed that UhAvr1 is UHOR_10022 which codes for a small effector protein of 171 amino acids with a predicted 19 amino acid signal peptide. Virulence in the parental isolate is caused by the insertion of a fragment of 5.5 kb with similarity to a common U. hordei transposable element (TE), interrupting the promoter of UhAvr1 and thereby changing expression and hence recognition of UhAVR1p. This rearrangement is likely caused by activities of TEs and variation is seen among isolates. Using GFP-chimeric constructs we show that UhAvr1 is induced only in mated dikaryotic hyphae upon sensing and infecting barley coleoptile cells. When infecting Hannchen, UhAVR1p causes local callose deposition and the production of reactive oxygen species and necrosis indicative of the immune response. UhAvr1 does not contribute significantly to overall virulence. UhAvr1 is located in a cluster of ten effectors with several paralogs and over 50% of TEs. This cluster is syntenous with clusters in closely-related U. maydis and Sporisorium reilianum. In these corn-infecting species, these clusters harbor however more and further diversified homologous effector families but very few TEs. This increased variability may have resulted from past selection pressure by resistance genes since U. maydis is not known to trigger immunity in its corn host.

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Section: Resultssupporting

confidence: 71%

An Immunity-Triggering Effector from the Barley Smut Fungus Ustilago hordei Resides in an Ustilaginaceae-Specific Cluster Bearing Signs of Transposable Element-Assisted Evolution

et al. 2014

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“…To activate the expression of individual CAZyme genes during the biotechnologically relevant yeast-like growth in axenic culture, the native promoters of candidate genes were replaced by two different synthetic promoters, termed P otef (57) and P oma (58,59), using homologous recombination (57, 58) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

Geiser

Reindl

Blank

et al. 2016

Appl Environ Microbiol

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The microbial conversion of plant biomass to valuable products in a consolidated bioprocess could greatly increase the ecologic and economic impact of a biorefinery. Current strategies for hydrolyzing plant material mostly rely on the external application of carbohydrate-active enzymes (CAZymes). Alternatively, production organisms can be engineered to secrete CAZymes to reduce the reliance on externally added enzymes. Plant-pathogenic fungi have a vast repertoire of hydrolytic enzymes to sustain their lifestyle, but expression of the corresponding genes is usually highly regulated and restricted to the pathogenic phase. Here, we present a new strategy in using the biotrophic smut fungus Ustilago maydis for the degradation of plant cell wall components by activating its intrinsic enzyme potential during axenic growth. This fungal model organism is fully equipped with hydrolytic enzymes, and moreover, it naturally produces value-added substances, such as organic acids and biosurfactants. To achieve the deregulated expression of hydrolytic enzymes during the industrially relevant yeast-like growth in axenic culture, the native promoters of the respective genes were replaced by constitutively active synthetic promoters. This led to an enhanced conversion of xylan, cellobiose, and carboxymethyl cellulose to fermentable sugars. Moreover, a combination of strains with activated endoglucanase and ␤-glucanase increased the release of glucose from carboxymethyl cellulose and regenerated amorphous cellulose, suggesting that mixed cultivations could be a means for degrading more complex substrates in the future. In summary, this proof of principle demonstrates the potential applicability of activating the expression of native CAZymes from phytopathogens in a biocatalytic process. IMPORTANCEThis study describes basic experiments that aim at the degradation of plant cell wall components by the smut fungus Ustilago maydis. As a plant pathogen, this fungus contains a set of lignocellulose-degrading enzymes that may be suited for biomass degradation. However, its hydrolytic enzymes are specifically expressed only during plant infection. Here, we provide the proof of principle that these intrinsic enzymes can be synthetically activated during the industrially relevant yeast-like growth. The fungus is known to naturally synthesize valuable compounds, such as itaconate or glycolipids. Therefore, it could be suited for use in a consolidated bioprocess in which more complex and natural substrates are simultaneously converted to fermentable sugars and to value-added compounds in the future. O ne central aim of a sustainable bioeconomy is the switch from fossil-to bio-based production of platform chemicals and other valuable substances. To date, the most promising feedstock for biorefineries is lignocellulosic nonfood plant biomass (1). Lignocellulose is a complex composite mainly consisting of cellulose, hemicelluloses, pectin, and lignin (2). The selective conversion of lignocellulosic biomass comprises different steps: pret...

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“…The pyrithiamine resistance cassette was obtained by Sfi I-restriction of plasmid pSK275. The fhpA DNA flanking fragments and the Sfi I-digested ptrA gene were cloned into plasmid p123 (Spellig et al, 1996) restricted with Xma I/ Xho I. The resulting plasmid was digested with Xma I and Xho I and the deletion cassette was used to transform A. fumigatus CEA17Δ akuB or Δ fhpB to create the Δ fhpA mutant and the Δ fhpA /Δ fhpB double mutant, respectively.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the Aspergillus fumigatus detoxification systems for reactive nitrogen intermediates and their impact on virulence

et al. 2014

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Aspergillus fumigatus is a saprophytic mold that can cause life-threatening infections in immunocompromised patients. In the lung, inhaled conidia are confronted with immune effector cells that attack the fungus by various mechanisms such as phagocytosis, production of antimicrobial proteins or generation of reactive oxygen intermediates. Macrophages and neutrophils can also form nitric oxide (NO) and other reactive nitrogen intermediates (RNI) that potentially also contribute to killing of the fungus. However, fungi can produce several enzymes involved in RNI detoxification. Based on genome analysis of A. fumigatus, we identified two genes encoding flavohemoglobins, FhpA, and FhpB, which have been shown to convert NO to nitrate in other fungi, and a gene encoding S-nitrosoglutathione reductase GnoA reducing S-nitrosoglutathione to ammonium and glutathione disulphide. To elucidate the role of these enzymes in detoxification of RNI, single and double deletion mutants of FhpA, FhpB, and GnoA encoding genes were generated. The analysis of mutant strains using the NO donor DETA-NO indicated that FhpA and GnoA play the major role in defense against RNI. By generating fusions with the green fluorescence protein, we showed that both FhpA-eGFP and GnoA-eGFP were located in the cytoplasm of all A. fumigatus morphotypes, from conidia to hyphae, whereas FhpB-eGFP was localized in mitochondria. Because fhpA and gnoA mRNA was also detected in the lungs of infected mice, we investigated the role of these genes in fungal pathogenicity by using a murine infection model for invasive pulmonary aspergillosis. Remarkably, all mutant strains tested displayed wild-type pathogenicity, indicating that the ability to detoxify host-derived RNI is not essential for virulence of A. fumigatus in the applied mouse infection model. Consistently, no significant differences in killing of ΔfhpA, ΔfhpB, or ΔgnoA conidia by cells of the macrophage cell line MH-S were observed when compared to the wild type.

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Green fluorescent protein (GFP) as a new vital marker in the phytopathogenic fungusUstilago maydis

Cited by 124 publications

References 30 publications

An Immunity-Triggering Effector from the Barley Smut Fungus Ustilago hordei Resides in an Ustilaginaceae-Specific Cluster Bearing Signs of Transposable Element-Assisted Evolution

An Immunity-Triggering Effector from the Barley Smut Fungus Ustilago hordei Resides in an Ustilaginaceae-Specific Cluster Bearing Signs of Transposable Element-Assisted Evolution

Activating Intrinsic Carbohydrate-Active Enzymes of the Smut Fungus Ustilago maydis for the Degradation of Plant Cell Wall Components

Characterization of the Aspergillus fumigatus detoxification systems for reactive nitrogen intermediates and their impact on virulence

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