Table 1.
Strains used in this study.
Figure 1.
Slants containing Vogel’s sucrose medium were inoculated with different strain isolates and grown for 4 days. Strains shown in the top panel from left to right include wild type (WT), Δham-6, Δham-6 transformed with HA-ham-6, Δham-7, Δham-7 transformed with HA-ham-7, Δham-8, Δham-8 transformed with HA-ham-8, Δham-8 transformed with ham-8-GFP, and Δham-8 transformed with RFP-ham-8. The bottom panel shows Δham-9, Δham-9 transformed with HA-ham-9, Δham-10, Δham-10 transformed with RFP-ham-10, Δamph-1, Δamph-1 transformed with HA-amph-1, Δamph-1 transformed with RFP-amph-1, Δwhi-2, and Δwhi-2 transformed with HA-whi-2.
Figure 2.
Complementation of CAT fusion activities by different HA, GFP and RFP tagged proteins.
A) The levels of CAT fusion activity for the gene deletion mutants and for transformants expressing a tagged version of the deleted gene are shown as a percentile of the cell fusion activity for wild type CATs. B) Photograph of CAT fusion activities in wild type (WT), Δham-8, and Δham-8 transformed with HA-ham-8. Arrows point to examples of CAT fusion in the wild-type and Δham-8 transformed with HA-ham-8 panels.
Figure 3.
Western blot analyses of HA-tagged proteins’ expression patterns.
Western blot analyses using anti-HA antibody were performed to detect HA-HAM-6, HA-HAM-7, HA-HAM-8, HA-HAM-9, HA-AMPH-1 and HA-WHI-2 protein levels in four hour germlings (CATs lane) and vegetative hyphae (Hyphae lane). The HA-tagged cell fusion proteins were regulated by their own promoters. Protein samples from wild type germ tubes/CATs were loaded as negative control (WT lane) for each Western blot analysis.
Figure 4.
Localization of RFP-HAM-10 and RFP-AMPH-1 in germ tubes/CATs.
Confocal microscopic images were taken for CATs expressing RFP-HAM-10 (top row of panels) and RFP-AMPH-1 (bottom row of panels). Images shown from left to right are DIC images, RFP fluorescent images, and merged images.
Figure 5.
Immunofluorescent localization images for HA-tagged proteins.
Anti-HA primary antibody and Alexa Fluor 488-conjugated secondary antibody were used to label HA-tagged protein in fixed germ tubes/CATs. Typical DIC images (left), fluorescent images (middle), and merged images (right) are shown. Images are shown for Wild type (WT) control (top row), HA-ham-7 transformant germ tubes (row 2), and CATs (row 3), HA-ham-8 transformant germ tube (row 4) and CATs (row 5), HA-amph-1 transformant germ tube (row 6) and CATs (row 7), HA-whi-2 transformant germ tube (row 8) and CATs (bottom row).
Figure 6.
MAK-2-GFP localization in wild type and mutant germ tubes/CATs.
MAK-2-GFP expressing wild type (WT) and mutant conidia were grown under CAT induction conditions for 4 hours. DIC images (left column), GFP fluorescent images (middle column), and merged images (right column) are shown. The images show germ tubes/CATs for wild type (WT) (row 1), Δham-6 (row 2), Δham-7 (row 3), Δham-8 (row 4), Δham-9 (row 5), Δham-10 (row 6), Δamph-1 (row 7), and Δwhi-2 (row 8). The arrows in the WT GFP fluorescent image point to the localization of MAK-2-GFP at the sites of cell fusion.
Figure 7.
Fusion between MAK-2-GFP-expressing cell fusion mutants and RFP-expressing wild type cells.
Conidia samples containing equal number of RFP-expressing wild type conidia and MAK-2-GFP-expressing wild type or mutant conidia were grown under CAT induction conditions for 4 hours. DIC images, GFP fluorescent images, RFP fluorescent images, and merged images for each combination of conidia types are shown in the columns from left to right respectively. Each row shows the images for RFP-expressing wild type conidia mixed with MAK-2-GFP-expressing wild type (WT) (row 1), Δham-6 (row 2), Δham-7 (row 3), Δham-8 (row 4), Δham-9 (row 5), Δham-10 (row 6), Δamph-1 (row 7), and Δwhi-2 (row 8) conidia. Wild type conidia frequently engaged in cell fusion, while Δamph-1 and Δwhi-2 conidia engaged in cell fusion with w conidia at a low frequency.
Figure 8.
MAK-1 and MAK-2 phosphorylation status in germ tubes/CATs.
Western blot analysis using Phospho-p44/42 MAPK antibody was performed to determine MAK-1 and MAK-2 phosphorylation status in wild type (WT) and mutant (Δham-6, Δham-7, Δham-8, Δham-9, Δham-10, Δamph-1, Δwhi-2, Δmik-1, and Δnrc-1) germ tubes/CATS. The positions of the phosphorylated MAK-1 (p-MAK-1) and phosphorylated MAK-2 (p-MAK-2) in the Western blot are noted in the left margin of the figure. B) The relative MAK-1 phosphorylation status in mutant germ tubes/CATs relative to the MAK-1 phosphorylation status in wild type germ tubes/CATs (WT value is set at 100%). C) The relative MAK-2 phosphorylation status in mutant germ tubes/CATs compared to the MAK-2 phosphorylation status in wild type germ tubes/CATs (WT value is set at 100%).
Figure 9.
Peroxidase activation of MAK-1 and MAK-2 pathways in cell fusion mutant vegetative hyphal cells.
Western blot analyses using Phospho-p44/42 MAPK antibody were performed to evaluate MAK-1 and MAK-2 activation in wild type (WT) and mutant vegetative hyphal cells in response to peroxidase treatments. Quantitative analyses of the Western blots were performed to determine the levels of phosphorylated MAK-1 and MAK-2 in non-stressed and oxidative-stressed samples (wild type, Δham-6, Δham-7, Δham-8, Δham-9, Δham-10, Δamph-1, and Δwhi-2). A) MAK-1 activation in wild type and mutants in response to peroxidase treatment. B) MAK-2 activation in wild type and mutants in response to peroxidase treatment. The levels of MAK-1 and MAK-2 in the non-stressed wild type sample were set as 100% for the quantitative analysis.
Figure 10.
Schematic model for the regulatory network involved in CAT fusion.
PP-1, ADV-1, SNF-5, and RCO-1/RCM-1 are transcription factors required for CAT fusion. MIK-1/MEK-1/MAK-1 and NRC-1/MEK-2/MAK-2 are two MAP kinase pathways required for CAT fusion. HAM-2/HAM-3/HAM-4/MOB-3/PP2A/PPG-1 form the STRIPAK complex that regulates MAK-1 nuclear accumulation. HAM-1/SO and MAK-2 engage in Ping-Pong signaling behavior during CAT fusion. HAM-6/HAM-7/HAM-8 are required at the plasma membrane/cell wall for MAK-1 pathway activation. HAM-10 may regulate vesicular trafficking and could potentially respond to calcium signaling during cell fusion. AMPH-1 regulates vesicular trafficking and endocytosis during cell fusion. WHI-2 may regulate the MAP kinase pathways through a general stress response pathway. The role of HAM-9 during CAT fusion remains to be determined.