(A to F)
A. nidulans but not
N. crassa hyphae pass through the channels. (A) Time series showing a hypha of
A. nidulans (nuclei labeled with GFP) growing into the channel. Kymograph along the growth axis before, in, and after the channel (from Movie S1). The hyphal elongation rates are shown by an arrow. Total 6 h, scale bar: 20 μm. (B) Time series of
A. nidulans (nuclei labeled with GFP) two or three hyphae passing through the same channel (see Movie S1). Each hyphal tip is shown by arrows. Scale bars: 20 μm. (C) Image of the
N. crassa spores germinated to the opposite side of slits. (D) Ratio of direction in germination toward or opposite to channels in
A. nidulans,
A. oryzae, and
N. crassa;
n = 50 each. (E) Time series images of
N. crassa (DIC, left; CHS-1-GFP, right) hyphae growing into a channel (from Movie S8). The arrows indicate the SPK. The elapsed time is given in minutes. Scale bar: 20 μm. (F) The plot profile along the apical membrane indicates the signal peaks of SPK (arrows) at 10, 20, and 30 min, but not at 40 or 50 min. The plot profile along the growth axis (right) indicates the peaks at the apex of hyphae at 10, 20, or 30 min, but at the subapex at 40 or 50 min. (G to J) Increased number of septa and SPK. (G and H) Image sequence of forming septa (arrows) and formed septa (arrow heads) in the depolarized hyphae through the channel (G) and in the hyphae through the channel (H). The elapsed time is given in hours:minutes. Scale bar: 20 μm. (I) Number of septa in 200-μm hyphae around the channel, in the hyphae that passed the channels, or in the depolarized hyphae. Error bars = SD;
n = 5; ***,
P ≤ 0.001. (J) SPK of
N. crassa hypha grown in MM + 0.6 M KCl. Images of the
N. crassa (SPK labeled with GFP) hypha in the channel from Movie S8. The arrow indicates the SPK before, in, and after the channel. The elapsed time is given in hours:minutes. Scale bar: 20 μm. (K to O) Phylogenetic tree and growth on the plates. (K) Phylogenic tree of filamentous fungi used in this study. Maximum likelihood (ML) tree obtained from the ITS1 and ITS2 regions of the fungal strains. The bootstrap consensus inferred from 100 replicates. (L) Correlation between the hyphal elongation rate and depolarized hyphae. Two groups are shown by red or blue ellipses. (M) No correlation between the hyphal width with the growth defect in channels. (N) Colony diameter of
N. crassa,
R. oryzae, and
C. cinerea on MM or MM + 0.6 M KCl plates. (O) Colonies of
A. nidulans (An),
A. oryzae (Ao),
C. cinerea (Cc),
N. crassa (Nc), and
R. oryzae (Ro) growth on minimal medium (MM) plates or MM + 0.6 M KCl plates for 2 to 7 days. (P and Q) Elastic modules measurement by a scanning probe microscope (SPM). The principle of SPM equipment is composed of the following: (i) laser diode and photo detector; (ii) cantilever and holder; and (iii) scanner (
http://www.shimadzu.com/an/surface/spm/faq/index.html). The basis of the force curve measurement is the measurement performed at one point of the sample. As the distance of the probe changes relative to the sample, this distance can be plotted on the horizontal axis, as shown on the graph. Also, it is possible to calculate from the spring constant of the cantilever and plot this on the vertical axis as nN. When the probe and sample distance are far away, the force does not work, hence the vertical axis is ①. When the cantilever touches the sample it is ②. After that, the slope of the graph when the repulsive force acts reflects the hardness of the sample, shown as ③. When a release-curve is observed, often a large attractive area can be seen. This is because the probe is caught by the adsorption layer on the sample surface, shown as ④. From this approach-curve and release-curve, Young’s modulus can be calculated using JKR or Hertz. Therefore, by saving the data at each pixel, a mapping image can be constructed. Fungal cells are grown in a noninvasive manner at high magnifications. Download
FIG S1, TIF file, 2.7 MB.