3. Results
3.2 The GCKs SmKIN3 and SmKIN24
3.2.3 Deletion of Smkin3 or Smkin24 impairs vegetative growth but only ΔSmkin24 is sterile 102
In order to investigate the functional role of Smkin3 and Smkin24, we generated single and double deletion strains ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 (Figure 41). The respective genes were replaced by homologous recombination in the S. macrospora Δku70 (Pöggeler & Kück, 2006) strain (Figure 41A). The Δku70 background was removed afterwards by crossing the homokaryotic deletion strains with S. macrospora spore-color mutant r2 (Teichert et al., 2014) and the obtained single-spore isolates were verified by PCR (Figure 41B) and Southern blotting (Figure 41C).
A n ti-FLAG A n ti-HA
Figure 41. Generation of ΔSmkin3 and ΔSmkin24 deletion strains. (A) Schematic illustration of the Smkin3 and Smkin24 loci before and after homologous integration of the respective deletion cassette.
Primers used for verification of the deletion strains are indicated by arrows. Sizes of PCR fragments and probes used for Southern hybridization are given. (B) Verification of the respective deletion using PCR.
Sizes of amplicons and positions of the primers as indicated in (A). (C) Integration of the deletion cassette was verified by Southern hybridization (Sambrook et al., 2001). Positions of the respective probes are indicated in (A). ΔSmkin3 (left) was verified using a hygromycin specific probe that only binds within the deletion cassette. ΔSmkin24 (right) was verified using a probe binding at the 3’ region of Smkin24. In this case, the successful integration is represented by a band shift.
h p h
∆Smkin3 wt ∆Smkin3 wt ∆Smkin3 wt ∆Smkin24 wt ∆Smkin24 wt ∆Smkin24 wt
5 kb
Microscopic analysis of the knockout strains revealed that only ΔSmkin3 was fertile and able to generate fruiting bodies with ascospores within 7 days (Figure 42).
Figure 42. Macroscopic and microscopic analysis of the sexual development of wt, ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24. The wt strain produces ascogonia after 3 days which develop to unpigmented protoperithecia at day 4 and pigmented protoperithecia at day 5. After 7 days mature perithecia with asci and ascospores are formed. Like the wt, ΔSmkin3 completes the lifecycle within 7 days and produces germinable ascospores. Development of ΔSmkin24 and the double-deletion strain ΔSmkin3/ΔSmkin24 is arrested at the stage of late protoperithecia formation. Scale bars as indicated.
2 5 µm
5 0 µm
6 0 0 µm
3 0 0 µm 2 5 µm
wt ∆Smkin3 ∆Smkin24 ∆Smkin3/∆Smkin24
3 d 7 d
4 d
5 d
7 d
7 d No mature perithecia
No ascospores
104
The deletion strain ΔSmkin24 was halted during late protoperithecia maturation and thus failed to develop mature fruiting bodies (Figure 42). Additionally, ΔSmkin3 produced more aerial hyphae on solid SWG fructification medium compared to wt (Figure 42).
Figure 43. Macroscopic and microscopic analysis of the sexual development of wt, complemented ΔSmkin3 (ΔSmkin3+), complemented ΔSmkin24 (ΔSmkin24+) and partially complemented ΔSmkin3/ΔSmkin24. The wt strain produces ascogonia after 3 days which develop to unpigmented protoperithecia at day 4 and pigmented protoperithecia at day 5. After 7 days mature perithecia with asci and ascospores are formed. Similar to the wt ΔSmkin3+ and ΔSmkin24+ and ΔSmkin3/ΔSmkin24 + Smkin24 completed the lifecycle within 7 days and produced mature ascospores. Development of ΔSmkin3/ΔSmkin24 + Smkin3 is arrested at stage of late protoperithecia formation, similar to ΔSmkin24.
Scale bars as indicated.
25 µm
25 µm
50 µm
600 µm
300 µm
wt ∆Smkin3+ ∆Smkin24+
∆Smkin3/∆Smkin24 +Smkin3 +Smkin24
3 d 7 d
4 d
5 d
7 d
7 d No
ascospores
105
The double-deletion strain was generated by crossing the single deletion strains, and ΔSmkin3/ΔSmkin24 exhibited a phenotype that was a combination of the phenotypes of both single deletion strains (sterility and increased aerial hyphae on solid SWG medium). Each of the described phenotypes could be complemented by inserting a copy of the deleted gene ectopically as shown in Figure 43.
Sterility of STRIPAK mutants pro11, pro22 and ∆Smmob3 is accompanied by defects in hyphal fusion (Bernhards & Pöggeler, 2011, Bloemendal et al., 2012). However, in contrast with these previously characterized mutants, fusion of vegetative hyphae was not affected in ∆Smkin3 and
∆Smkin24 (Figure 44). Furthermore, deletion of Smkin3 and Smkin24 impaired vegetative growth of S. macrospora: Growth velocity experiments revealed that ΔSmkin3 (1.70 ± 0.24 cm/d), ΔSmkin24 (1.82 ± 0.41 cm/d) and ΔSmkin3/ΔSmkin24 (1.54 ± 0.28 cm/d) grew more slowly than wt (3.09 ± 0.37 cm/d) (Figure 44).
Figure 44. Microscopic investigation of hyphal fusion in wt, ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24. ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 are capable of hyphal fusion.
Hyphal fusion events are highlighted with circles. Pictures of hyphal fusion events were taken at subperiphal regions 10 mm behind the growth front. Hyphal-fusion was investigated 2-3 days past inoculation.
Furthermore, the deletion of Smkin3 and Smkin24 impairs vegetative growth of S. macrospora.
Measuring growth velocity in race tubes over ten days revealed that ΔSmkin3 (1.70 ± 0.24 cm/d), ΔSmkin24 (1.82 ± 0.41 cm/d) and ΔSmkin3/ΔSmkin24 (1.54 ± 0.28 cm/d) display a reduced growth velocity than wt (3.09 ± 0.37 cm/d).
50 µm
wt ∆Smkin3
∆Smkin24 ∆Smkin3/∆Smkin24
106
3.2.4 SmKIN3 and SmKIN24 localize to septa and influence septum formation
In order to determine the localization of SmKIN3 and SmKIN24 in vivo, fluorescence microscopy was performed. Genes coding N-terminally eGFP-tagged full-length SmKIN3 or SmKIN24 were expressed in the respective S. macrospora deletion strains, which show that both kinases were localized at the septa (Figure 45). However SmKIN3 was localized mainly at the outer part of the septum, whereas SmKIN24 was localized at the middle (Figure 45) and this was verified by co-staining with calcofluor white (Figure 45).
Figure 45. Localization of SmKIN3-eGFP and SmKIN24-eGFP in S. macrospora. SmKIN3-eGFP and SmKIN24-eGFP localize to septa. For visualization of cell walls and septa, hyphae were co-stained with calcofluor white. Scale bars as indicated.
These findings lead us to quantify septum formation in the ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 strains by staining with calcofluor white after 18 or 32 h of growth (Figure 46).
SmKIN3-eGFP
SmKIN24-eGFP
DIC eGFP
Calcofluor
white merged
50 µm 50 µm
107
Figure 46. Analysis of septal development in wt, ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 and complemented mutants. Distribution of septa was investigated under the microscope after 18 h and 32 h past inoculation. Septa were stained with calcofluor white.
Furthermore, we quantified the distances between adjacent septa in wt and deletion mutants after 24 h of growth (Figure 47). Septa were distributed in a uniform manner in wt with hyphal compartment length between 31-70 µm (Figure 47). In contrast ΔSmkin3 exhibited larger distances between adjacent septa (>71 µm), although this effect begun to revert after 24 h of growth (Figure 46). Strikingly, ΔSmkin24 developed numerous closely-packed septal bundles of abnormal shape, with much smaller hyphal compartments of 0-30 µm (Figure 47). ΔSmkin3 produced about half the total number of septa within a distance of 18 mm than wt, while ΔSmkin24 produced 20% more than wt after 24 h of growth. With respect to septation, the double deletion mutant ΔSmkin3/ΔSmkin24 displayed a similar phenotype to the ΔSmkin24 single deletion strain. Ectopic integration of the respective wt gene into deletion mutants restored septal formation to wt level (Figure 46 and Figure 47).
wt
∆Smkin3
∆Smkin3
∆Smkin3+
∆Smkin24
∆Smkin24
∆Smkin24+
∆Smkin3/
∆Smkin24
∆Smkin3/
∆Smkin24
18 h 18 h 32 h
18 h 18 h
18 h 18 h 32 h
32 h 50 µm
108
Figure 47. Quantitative analysis of septal development in wt, ΔSmkin3, ΔSmkin24 and ΔSmkin3/ΔSmkin24 and complemented mutants. For quantification, distances between adjacent septa were measured over a distance of 18 mm per strain 24 h past inoculation. The total distance of 18 mm was divided into 30 segments of 600 µm. Measurements were binned into five different compartment lengths: 0-30 µm, 31-50 µm, 51-70, 71-100 mm, and more than 100 µm. The number of analysed compartments was normalized to 100%. Error bars (SD) are given as indicated (n=30). ∆Smkin3+,
∆Smkin24+ complemented mutants carrying an ectopic copy of the respective wt gene.