Analysis of the Aspergillus nidulans thaumatin-like cetA gene and evidence for transcriptional repression of pyr4 expression in the cetA-disrupted strain

Greenstein, Shulamit; Shadkchan, Yona; Jadoun, Jeries; Sharon, C.-A. Chen; Markovich, Sarit; Osherov, Nir

doi:10.1016/j.fgb.2005.10.001

Cited by 26 publications

(31 citation statements)

References 31 publications

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“…However, L. edodes tlg1 was not transcribed in vegetative mycelium grown in liquid culture and sawdust culture (data not shown), suggesting that it is induced under stress conditions or that it might not act as an antifungal agent. On the other hand, it was reported that the TL protein-encoding gene cetA from A. nidulans is significantly enriched in mature conidia and involved in germination (Greenstein et al, 2006). This suggests that fungal TL proteins play roles that are different from those of their plant TL protein counterparts.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, Grenier et al (2000) reported that several fungi, such as Rhizoctonia solani and L. edodes, have TL proteins. More recently, TL proteinencoding genes have been found in Aspergillus nidulans (Osherov et al, 2002;Greenstein et al, 2006). Homology between the N-terminal amino acid sequences of fungal and plant TL proteins includes three amino acid residues (Asn, Cys, and Trp) that are highly conserved (Grenier et al, 2000).…”

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confidence: 99%

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Lentinula edodes tlg1 Encodes a Thaumatin-Like Protein That Is Involved in Lentinan Degradation and Fruiting Body Senescence

et al. 2006

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Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising b-1,3-glucan with b-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited b-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Lentinula edodes tlg1 Encodes a Thaumatin-Like Protein That Is Involved in Lentinan Degradation and Fruiting Body Senescence

et al. 2006

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“…The pellet was washed three times with 90% methanol buffer (90% methanol, 50 mM Tris-HCl, pH 7.8) and lyophilized. Protein was resuspended in urea sample-buffer prior to SDS-PAGE analysis as described previously (18). Western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Western blot analysis. Western blot analysis was performed as described previously (18). cspA-encoded myc-tagged protein was detected following incubation of the blot with mouse-derived anti-c-myc monoclonal antibody (9E10) (Santa Cruz Biotechnology Inc., CA) for 1 h followed by two washes in TBST (2.5 mM Tris-HCl [pH 7.4], 15 mM NaCl, 0.005% Tween 20) for 15 min each.…”

Section: Methodsmentioning

confidence: 99%

The Aspergillus fumigatus cspA Gene Encoding a Repeat-Rich Cell Wall Protein Is Important for Normal Conidial Cell Wall Architecture and Interaction with Host Cells

et al. 2010

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cspA (for cell surface protein A) encodes a repeat-rich glycophosphatidylinositol (GPI)-anchored cell wall protein (CWP) in the pathogenic fungus Aspergillus fumigatus. The number of repeats in cspA varies among isolates, and this trait is used for typing closely related strains of A. fumigatus. We have previously shown that deletion of cspA is associated with rapid conidial germination and reduced adhesion of dormant conidia. Here we show that cspA can be extracted with hydrofluoric acid (HF) from the cell wall, suggesting that it is a GPI-anchored CWP. The cspA-encoded CWP is unmasked during conidial germination and is surface expressed during hyphal growth. Deletion of cspA results in weakening of the conidial cell wall, whereas its overexpression increases conidial resistance to cell wall-degrading enzymes and inhibits conidial germination. Double mutant analysis indicates that cspA functionally interacts with the cell wall protein-encoding genes ECM33 and GEL2. Deletion of cspA together with ECM33 or GEL2 results in strongly reduced conidial adhesion, increased disorganization of the conidial cell wall, and exposure of the underlying layers of chitin and ␤-glucan. This is correlated with increasing susceptibility of the ⌬cspA, ⌬ECM33, and ⌬cspA ⌬ECM33 mutants to conidial phagocytosis and killing by human macrophages and hyphal damage induced by neutrophils. However, these strains did not exhibit altered virulence in mice with infected lungs. Collectively, these results suggest a role for cspA in maintaining the strength and integrity of the cell wall.

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“…In the effort to avoid working with auxotrophic strains, the marker is often left at the manipulated locus (20). However, even though this arrangement results in a prototroph, the expression level of the marker at the ectopic location may differ from that of the wild type; thus, the marker contributes to the overall phenotype and complicates further analysis (8,10). For example, when a ura3 mutation in Candida albicans was complemented by integrating a Saccharomyces cerevisiae URA3 gene at different loci, strains displaying various levels of virulence were obtained as the result of differing URA3 expression levels (12).…”

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Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus

Nielsen

Jongh

Meijer

et al. 2007

Appl Environ Microbiol

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Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of pyrG. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed, pyrG function can be rapidly returned to wild type by a simple selection scheme.

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Analysis of the Aspergillus nidulans thaumatin-like cetA gene and evidence for transcriptional repression of pyr4 expression in the cetA-disrupted strain

Cited by 26 publications

References 31 publications

Lentinula edodes tlg1 Encodes a Thaumatin-Like Protein That Is Involved in Lentinan Degradation and Fruiting Body Senescence

Lentinula edodes tlg1 Encodes a Thaumatin-Like Protein That Is Involved in Lentinan Degradation and Fruiting Body Senescence

The Aspergillus fumigatus cspA Gene Encoding a Repeat-Rich Cell Wall Protein Is Important for Normal Conidial Cell Wall Architecture and Interaction with Host Cells

Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus

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