Journal: Frontiers in Microbiology

Article Title: Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes

doi: 10.3389/fmicb.2020.01722

Figure Lengend Snippet: Phenotype of mak2 , mek2 , mik2 , and ham5 deletion mutants. (A) All pheromone response (PR) deletion strains as well as Δnor1 and Δham5Δnor1 show an arrest of fruiting body formation at the stage of unpigmented protoperithecia. Sexual development of deletion strains was observed after 3 (ascogonia), 5 (unpigmented and pigmented protoperithecia) and 7 days (perithecia). All examined strains carry the fus1-1 mutation. (B) Hyphal fusion is impaired in all PR kinase deletion strains as well as in Δnor1 and Δham5Δnor1. Hyphal fusion bridges (arrowheads) were observed in all control (fus and wild type) and complemented strains, but were lacking in the investigated deletion strains (asterisks) after 2 days of growth on cellophane-covered MMS. The hyphal fusion defect was restored in all strains after introduction of the corresponding GFP-fusion constructs, except for Δham5. (C) Expression of gfp-tagged fusion constructs of PR genes and gfp/mCherry -tagged nor1 restored fertility in the corresponding deletion strains. All strains were grown on solid BMM for 7 days. Scale bars indicate 20 μm (black), 100 μm (white), or 2 mm (yellow). Developmental phenotypes of Δham5 were identical in wild type and fus mutant strains.

Article Snippet: We further identified the gene for HAM5 ( SMAC_02471 ), which is a homolog of a PR pathway scaffold protein in the filamentous fungi Aspergillus nidulans and N. crassa ( ; ; ).

Techniques: Mutagenesis, Control, Construct, Expressing

Journal: Frontiers in Microbiology

Article Title: Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes

doi: 10.3389/fmicb.2020.01722

Figure Lengend Snippet: Localization of PR components in hyphae. (A–D) All PR components are recruited to septal pores (white arrowheads). (A) GFP-MAK2 localizes to the cytoplasm near hyphal tips and additionally shows enhanced fluorescence in spherical structures in mature hyphae (red arrowhead). (B) GFP-MEK2 shows faint cytoplasmic localization near hyphal tips and in mature hyphae. (C) GFP-MIK2 displays a cytoplasmic distribution near hyphal tips and localizes to small spots (yellow arrowheads) in mature hyphae. (D) Compared to MEK2 and MIK2, the more abundant GFP-HAM5 spots are larger in size and can be found close to hyphal tips as well as in mature hyphae (yellow arrowhead). All scale bars indicate 10 μm. BF, bright-field (Confocal microscopy).

Article Snippet: We further identified the gene for HAM5 ( SMAC_02471 ), which is a homolog of a PR pathway scaffold protein in the filamentous fungi Aspergillus nidulans and N. crassa ( ; ; ).

Techniques: Fluorescence, Confocal Microscopy

Journal: Frontiers in Microbiology

Article Title: Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes

doi: 10.3389/fmicb.2020.01722

Figure Lengend Snippet: Localization of MIK2 and MEK2 to spindle pole bodies (SPBs). (A) Spots of GFP-MIK2 can be observed in the cytoplasm (blue arrowheads) and in association with nuclei (white arrowheads). (B) Dividing nuclei show punctate GFP-MIK2 fluorescence at both ends (white arrowheads), suggesting a localization at SPBs. (C) Co-expression of GFP-MIK2 with the SPB marker mRFP-GRC1/TUB4-mCherry shows that MIK2 partially accumulates at SPBs (white arrowheads). (D) GFP-MEK2 localizes to nuclei associated spots. (E) Co-labeling with the SPB marker mRFP-GRC1/TUB4-mCherry confirms MEK2 targeting to SPBs. (F) GFP-HAM5 localizes to nuclei-associated spots. (G) Co-labeling of GFP-HAM5 with the SPB marker shows partial localization of HAM5 at SPBs. All scale bars represent 10 μm. BF, bright-field (Confocal microscopy); DIC, differential interference contrast (Conventional fluorescence microscopy).

Article Snippet: We further identified the gene for HAM5 ( SMAC_02471 ), which is a homolog of a PR pathway scaffold protein in the filamentous fungi Aspergillus nidulans and N. crassa ( ; ; ).

Techniques: Fluorescence, Expressing, Marker, Labeling, Confocal Microscopy, Microscopy

Journal: Frontiers in Microbiology

Article Title: Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes

doi: 10.3389/fmicb.2020.01722

Figure Lengend Snippet: Localization of NOR1. (A) NOR1-GFP localizes to the cytoplasm and to cytoplasmic spots in mature hyphae and near the hyphal tip. Co-expression with H2A-mRFP reveals an association of NOR1-GFP spots with nuclei as indicated by white arrowheads. (B) Confocal imaging of the Δnor1 strain. NOR1-GFP and the SPB marker show partial co-localization (white arrowheads). (C) GFP-HAM5 and NOR1-mCherry co-localize in cytoplasmic spots (white arrowheads) of mature hyphae in the double deletion strain Δham5Δnor1. (D) Same as (C) but showing co-localization of HAM5 and NOR1 at the plasma membrane of hyphal tips (white arrowhead). Scale bars indicate 10 μm. BF, bright field; DIC, Differential interference contrast.

Article Snippet: We further identified the gene for HAM5 ( SMAC_02471 ), which is a homolog of a PR pathway scaffold protein in the filamentous fungi Aspergillus nidulans and N. crassa ( ; ; ).

Techniques: Expressing, Imaging, Marker, Clinical Proteomics, Membrane

Journal: Frontiers in Microbiology

Article Title: Crosstalk Between Pheromone Signaling and NADPH Oxidase Complexes Coordinates Fungal Developmental Processes

doi: 10.3389/fmicb.2020.01722

Figure Lengend Snippet: Double deletion of ham5 and nor1 suppresses Δnor1 phenotype during the oxidative stress response and the initiation of fruiting body formation. (A) Vegetative growth tests were performed with wild type, ∆ham5, ∆ham5∆nor1, and ∆nor1. Strains were grown in Petri dishes on solid synthetic Westergaard’s medium (SWG) at 27°C for 2 days. The growth front was marked after 24 and 48 h. The graph shows means and SDs from three biological replicates per strain. (B) Stress-related growth analysis was conducted with strains described in (A) on solid SWG medium containing 0.01% H 2 O 2 at 27°C for 2 days. The growth front was marked after 24 and 48 h. The ratio of the growth rates on SWG and SWG with 0.01% H 2 O 2 is shown in percentage (%). The graph shows means and SDs from three biological replicates per strain. (C) The quantification of ascogonia and protoperithecia of wild type, ∆ham5, ∆ham5∆nor1, and ∆nor1. Graphs represent means and SDs of three biological replicats per strain. For each strain, ascogonia and protoperithecia were counted in an area of 0.5 cm 2 , located 1 cm behind the growth front. Statistical analysis was performed using students t -test. Significant differences ( p ≤ 0.05) to fus (a), Δham5/fus (b 1 ) or Δham5/fus and Δham5Δnor1/fus (b 2 ) are indicated on top of each bar. All examined strains carry the fus1-1 mutation.

Article Snippet: We further identified the gene for HAM5 ( SMAC_02471 ), which is a homolog of a PR pathway scaffold protein in the filamentous fungi Aspergillus nidulans and N. crassa ( ; ; ).

Techniques: Mutagenesis