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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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Analysis of conidial anastomosis tube (CAT) formation and hyphal fusion in Δste12 and Δtmk1 mutants.Microscopic analyses of germlings of Δste12 and Δtmk1 mutants, the parental strain and the complemented strains ste12-C1 and ste12-C2 16 hours after inoculation of conidia in potato dextrose broth. CATs and fusion bridges between germ tubes of the parental strain are marked with arrows. The scale bar represents 10 µm.
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pone-0111636-g007: Analysis of conidial anastomosis tube (CAT) formation and hyphal fusion in Δste12 and Δtmk1 mutants.Microscopic analyses of germlings of Δste12 and Δtmk1 mutants, the parental strain and the complemented strains ste12-C1 and ste12-C2 16 hours after inoculation of conidia in potato dextrose broth. CATs and fusion bridges between germ tubes of the parental strain are marked with arrows. The scale bar represents 10 µm.

Mentions: Despite the fact that T. atroviride Δste12 and Δtmk1 mutants lost negative hyphal autotropism in the colony periphery which resulted in the observed hyphal aggregation, we were not able to detect distinct fusions between aggregated hyphae. To further analyze a putative role of Tmk1 and Ste12 in regulating fusion processes, the behavior of Δste12 and Δtmk1 germlings was assessed microscopically. Conidial anastomosis tubes as well as fusion bridges between germ tubes were frequently observed in the parental and the complemented strain (Fig. 7). In contrast, CATs could not be detected in the Δste12 and Δtmk1 mutants and also fusions between germ tubes were only rarely observed despite frequent contacts between the germlings. It is worth mentioning that Δtmk1 conidia showed delayed germ tube formation and extensive aggregation of Δtmk1 germlings occurred (Fig. 7). Recent studies in the fungal model N. crassa led to the identification of target genes being required for cell fusion which are under control of the Mak-2 MAPK and the Ste12 homolog PP-1 [45]. To further substantiate our above findings of Tmk1 and Ste12 playing key roles in fusion processes in T. atroviride, mycelia of Δste12 and Δtmk1 mutants and the parental strain were harvested from the internal as well as peripheral zones of the fungal colony. For gene expression analyses, the respective T. atroviride orthologues (Ta300768, tmk2, hex1, Ta302802, Ta294940) of the N. crassa fusion genes ham-7 (encoding a GPI-anchored protein required for activation of the cell wall integrity MAPK MAK-1), mak-1 (MAPK), hex-1 (involved in septal plugging), nox-1 (NADPH oxidase), and ham-9 (pleckstrin domain protein) were identified in the T. atroviride genome database (http://genome.jgi-psf.org/Triat2/Triat2.home.html) by BLAST searches. Moreover, the glycosyl-hydrolase 18 (GH18) subgroup C chitinase-encoding gene tac6 (Ta348129), which plays a role in hyphal network formation in T. atroviride[24], was included in the study.


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)

Analysis of conidial anastomosis tube (CAT) formation and hyphal fusion in Δste12 and Δtmk1 mutants.Microscopic analyses of germlings of Δste12 and Δtmk1 mutants, the parental strain and the complemented strains ste12-C1 and ste12-C2 16 hours after inoculation of conidia in potato dextrose broth. CATs and fusion bridges between germ tubes of the parental strain are marked with arrows. The scale bar represents 10 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214791&req=5

pone-0111636-g007: Analysis of conidial anastomosis tube (CAT) formation and hyphal fusion in Δste12 and Δtmk1 mutants.Microscopic analyses of germlings of Δste12 and Δtmk1 mutants, the parental strain and the complemented strains ste12-C1 and ste12-C2 16 hours after inoculation of conidia in potato dextrose broth. CATs and fusion bridges between germ tubes of the parental strain are marked with arrows. The scale bar represents 10 µm.
Mentions: Despite the fact that T. atroviride Δste12 and Δtmk1 mutants lost negative hyphal autotropism in the colony periphery which resulted in the observed hyphal aggregation, we were not able to detect distinct fusions between aggregated hyphae. To further analyze a putative role of Tmk1 and Ste12 in regulating fusion processes, the behavior of Δste12 and Δtmk1 germlings was assessed microscopically. Conidial anastomosis tubes as well as fusion bridges between germ tubes were frequently observed in the parental and the complemented strain (Fig. 7). In contrast, CATs could not be detected in the Δste12 and Δtmk1 mutants and also fusions between germ tubes were only rarely observed despite frequent contacts between the germlings. It is worth mentioning that Δtmk1 conidia showed delayed germ tube formation and extensive aggregation of Δtmk1 germlings occurred (Fig. 7). Recent studies in the fungal model N. crassa led to the identification of target genes being required for cell fusion which are under control of the Mak-2 MAPK and the Ste12 homolog PP-1 [45]. To further substantiate our above findings of Tmk1 and Ste12 playing key roles in fusion processes in T. atroviride, mycelia of Δste12 and Δtmk1 mutants and the parental strain were harvested from the internal as well as peripheral zones of the fungal colony. For gene expression analyses, the respective T. atroviride orthologues (Ta300768, tmk2, hex1, Ta302802, Ta294940) of the N. crassa fusion genes ham-7 (encoding a GPI-anchored protein required for activation of the cell wall integrity MAPK MAK-1), mak-1 (MAPK), hex-1 (involved in septal plugging), nox-1 (NADPH oxidase), and ham-9 (pleckstrin domain protein) were identified in the T. atroviride genome database (http://genome.jgi-psf.org/Triat2/Triat2.home.html) by BLAST searches. Moreover, the glycosyl-hydrolase 18 (GH18) subgroup C chitinase-encoding gene tac6 (Ta348129), which plays a role in hyphal network formation in T. atroviride[24], was included in the study.

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