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The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

Gruber S, Zeilinger S - PLoS ONE (2014)

Bottom Line: However, the transcription factors acting downstream of Tmk1 are hitherto unknown.Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases.Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

View Article: PubMed Central - PubMed

Affiliation: Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Wien, Austria.

ABSTRACT
Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

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Impact of Ste12 on the expression of mycoparasitism-related cell wall-degrading enzymes.(A) Extracellular N-acetyl-glucosaminidase (NAGase) and endochitinase activities in the Δste12 mutant (black bars) and the parental strain (white bars). After pre-cultivation on 1% glycerol, mycelial biomass was transferred to 1% N-acetyl-glucosamine-containing media for inducing NAGases and to 1% colloidal chitin-containing media for induction of endochitinases. Culture filtrates were harvested at the indicated time points and determined enzyme activities related to intracellular total protein. (B) Relative transcription ratios of the chitinase-encoding nag1 and ech42 genes and the prb1 protease-encoding gene in the Δste12 mutant (black bars) and the parental strain (white bars). RT-qPCR analyses were performed 5, 14, and 24 hours after transfer to N-acetyl-glucosamine (nag1) and 14, 24, 36, and 48 hours after transfer to colloidal chitin (ech42, prb1) using act1 as reference gene. Un-induced samples of the parental strain harvested after pre-cultivation in glycerol-containing media were arbitrarily assigned the factor 1. Asterisks indicate significantly different (p≤0.05; calculated by REST software) transcription ratios of the mutant compared to the parental strain. (C) Relative transcription ratios of nag1, ech42 and prb1 in Δste12 (white bars) and Δtmk1 (grey bars) mutants and the parental strain (black bars) upon direct confrontation with R. solani. Samples were collected from a control (co) where Trichoderma was confronted with itself and from the early overgrowth stage in the confrontation with R. solani (Rs) and subject to RT-qPCR using sar1 as reference gene. The control sample of the parental strain was arbitrarily assigned the factor 1 and those samples which show significant differences (p≤0.05; calculated by REST software) to this control are marked with an asterisk. Results shown are means ±SD (n = 3).
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pone-0111636-g006: Impact of Ste12 on the expression of mycoparasitism-related cell wall-degrading enzymes.(A) Extracellular N-acetyl-glucosaminidase (NAGase) and endochitinase activities in the Δste12 mutant (black bars) and the parental strain (white bars). After pre-cultivation on 1% glycerol, mycelial biomass was transferred to 1% N-acetyl-glucosamine-containing media for inducing NAGases and to 1% colloidal chitin-containing media for induction of endochitinases. Culture filtrates were harvested at the indicated time points and determined enzyme activities related to intracellular total protein. (B) Relative transcription ratios of the chitinase-encoding nag1 and ech42 genes and the prb1 protease-encoding gene in the Δste12 mutant (black bars) and the parental strain (white bars). RT-qPCR analyses were performed 5, 14, and 24 hours after transfer to N-acetyl-glucosamine (nag1) and 14, 24, 36, and 48 hours after transfer to colloidal chitin (ech42, prb1) using act1 as reference gene. Un-induced samples of the parental strain harvested after pre-cultivation in glycerol-containing media were arbitrarily assigned the factor 1. Asterisks indicate significantly different (p≤0.05; calculated by REST software) transcription ratios of the mutant compared to the parental strain. (C) Relative transcription ratios of nag1, ech42 and prb1 in Δste12 (white bars) and Δtmk1 (grey bars) mutants and the parental strain (black bars) upon direct confrontation with R. solani. Samples were collected from a control (co) where Trichoderma was confronted with itself and from the early overgrowth stage in the confrontation with R. solani (Rs) and subject to RT-qPCR using sar1 as reference gene. The control sample of the parental strain was arbitrarily assigned the factor 1 and those samples which show significant differences (p≤0.05; calculated by REST software) to this control are marked with an asterisk. Results shown are means ±SD (n = 3).

Mentions: While secreted NAGase activities in N-acetyl-glucosamine-induced cultures were decreased upon ste12 deletion at all time points tested, the Δste12 mutant showed elevated extracellular endochitinase activities compared to the parental strain upon induction with colloidal chitin (Fig. 6 A). Further analysis at the transcript level confirmed the enhanced transcription of the ech42 gene in the Δste12 mutant after cultivation on colloidal chitin for 36 hours and, unexpectedly, also revealed enhanced nag1 mRNA levels compared to the parental strain upon cultivation in the presence of N-acetyl-glucosamine for 14 and 24 hours (Fig. 6 B). Similar to ech42, the prb1 gene, which encodes a subtilisin-like serine protease whose over-expression has been shown to improve the biocontrol activity of T. atroviride[44], can be induced by chitin. prb1 expression was highest after 36 hours of cultivation in chitin-containing media in both the Δste12 mutant and the parental strain with prb1 mRNA levels being ∼2-fold enhanced in the mutant.


The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

Gruber S, Zeilinger S - PLoS ONE (2014)

Impact of Ste12 on the expression of mycoparasitism-related cell wall-degrading enzymes.(A) Extracellular N-acetyl-glucosaminidase (NAGase) and endochitinase activities in the Δste12 mutant (black bars) and the parental strain (white bars). After pre-cultivation on 1% glycerol, mycelial biomass was transferred to 1% N-acetyl-glucosamine-containing media for inducing NAGases and to 1% colloidal chitin-containing media for induction of endochitinases. Culture filtrates were harvested at the indicated time points and determined enzyme activities related to intracellular total protein. (B) Relative transcription ratios of the chitinase-encoding nag1 and ech42 genes and the prb1 protease-encoding gene in the Δste12 mutant (black bars) and the parental strain (white bars). RT-qPCR analyses were performed 5, 14, and 24 hours after transfer to N-acetyl-glucosamine (nag1) and 14, 24, 36, and 48 hours after transfer to colloidal chitin (ech42, prb1) using act1 as reference gene. Un-induced samples of the parental strain harvested after pre-cultivation in glycerol-containing media were arbitrarily assigned the factor 1. Asterisks indicate significantly different (p≤0.05; calculated by REST software) transcription ratios of the mutant compared to the parental strain. (C) Relative transcription ratios of nag1, ech42 and prb1 in Δste12 (white bars) and Δtmk1 (grey bars) mutants and the parental strain (black bars) upon direct confrontation with R. solani. Samples were collected from a control (co) where Trichoderma was confronted with itself and from the early overgrowth stage in the confrontation with R. solani (Rs) and subject to RT-qPCR using sar1 as reference gene. The control sample of the parental strain was arbitrarily assigned the factor 1 and those samples which show significant differences (p≤0.05; calculated by REST software) to this control are marked with an asterisk. Results shown are means ±SD (n = 3).
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pone-0111636-g006: Impact of Ste12 on the expression of mycoparasitism-related cell wall-degrading enzymes.(A) Extracellular N-acetyl-glucosaminidase (NAGase) and endochitinase activities in the Δste12 mutant (black bars) and the parental strain (white bars). After pre-cultivation on 1% glycerol, mycelial biomass was transferred to 1% N-acetyl-glucosamine-containing media for inducing NAGases and to 1% colloidal chitin-containing media for induction of endochitinases. Culture filtrates were harvested at the indicated time points and determined enzyme activities related to intracellular total protein. (B) Relative transcription ratios of the chitinase-encoding nag1 and ech42 genes and the prb1 protease-encoding gene in the Δste12 mutant (black bars) and the parental strain (white bars). RT-qPCR analyses were performed 5, 14, and 24 hours after transfer to N-acetyl-glucosamine (nag1) and 14, 24, 36, and 48 hours after transfer to colloidal chitin (ech42, prb1) using act1 as reference gene. Un-induced samples of the parental strain harvested after pre-cultivation in glycerol-containing media were arbitrarily assigned the factor 1. Asterisks indicate significantly different (p≤0.05; calculated by REST software) transcription ratios of the mutant compared to the parental strain. (C) Relative transcription ratios of nag1, ech42 and prb1 in Δste12 (white bars) and Δtmk1 (grey bars) mutants and the parental strain (black bars) upon direct confrontation with R. solani. Samples were collected from a control (co) where Trichoderma was confronted with itself and from the early overgrowth stage in the confrontation with R. solani (Rs) and subject to RT-qPCR using sar1 as reference gene. The control sample of the parental strain was arbitrarily assigned the factor 1 and those samples which show significant differences (p≤0.05; calculated by REST software) to this control are marked with an asterisk. Results shown are means ±SD (n = 3).
Mentions: While secreted NAGase activities in N-acetyl-glucosamine-induced cultures were decreased upon ste12 deletion at all time points tested, the Δste12 mutant showed elevated extracellular endochitinase activities compared to the parental strain upon induction with colloidal chitin (Fig. 6 A). Further analysis at the transcript level confirmed the enhanced transcription of the ech42 gene in the Δste12 mutant after cultivation on colloidal chitin for 36 hours and, unexpectedly, also revealed enhanced nag1 mRNA levels compared to the parental strain upon cultivation in the presence of N-acetyl-glucosamine for 14 and 24 hours (Fig. 6 B). Similar to ech42, the prb1 gene, which encodes a subtilisin-like serine protease whose over-expression has been shown to improve the biocontrol activity of T. atroviride[44], can be induced by chitin. prb1 expression was highest after 36 hours of cultivation in chitin-containing media in both the Δste12 mutant and the parental strain with prb1 mRNA levels being ∼2-fold enhanced in the mutant.

Bottom Line: However, the transcription factors acting downstream of Tmk1 are hitherto unknown.Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases.Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

View Article: PubMed Central - PubMed

Affiliation: Research Area Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Wien, Austria.

ABSTRACT
Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

Show MeSH
Related in: MedlinePlus