Six Hydrophobins Are Involved in Hydrophobin Rodlet Formation in <i>Aspergillus nidulans</i> and Contribute to Hydrophobicity of the Spore Surface

<div><p>Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In <i>Aspergillus nidulans</i> two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter. Here we analyzed four more hydrophobins, DewB-E, in <i>A. nidulans</i> and found that all six hydrophobins contribute to the hydrophobic surface of the conidiospores but only deletion of <i>rodA</i> caused loss of the rodlet structure. Analysis of the rodlets in the <i>dewB-E</i> deletion strains with atomic force microscopy revealed that the rodlets appeared less robust. Expression of DewA and DewB driven from the <i>rodA</i> promoter and secreted with the RodA secretion signal in a strain lacking RodA, restored partly the hydrophobicity. DewA and B were able to form rodlets to some extent but never reached the rodlet structure of RodA. The rodlet-lacking <i>rodA</i>-deletion strain opens the possibility to systematically study rodlet formation of other natural or synthetic hydrophobins.</p></div

Figure 9

<p>(<b>A</b>) Analysis of the capability of hydrophobins DewA and DewB to form rodlets on the spore surface of a RodA lacking strain. The <i>ΔrodA</i> strain was used as expression platform. DewA and B were expressed from the <i>rodA</i> promoter and secreted with the signal peptide of RodA. As a control <i>rodA</i> was expressed in the same way. Strains: SAGR14, SAGR06, and 07. Scale bars, 100 nm. (<b>B</b>) Comparison of the number of rodlets per bundle. The number of rodlets was counted in 20 bundles. The mean value is displayed and the standard deviation indicated. (<b>C</b>) Water contact angle measurement of the <i>rodA-</i>expressing strain (SAGR14), the <i>rodA</i>-deletion strain and the Δ<i>rodA</i> strain transformed with the other two hydrophobins. The mean of ten independent measurements is displayed with the standard deviation. (<b>D</b>) Expression of <i>rodA, dewA</i> and <i>dewB</i> in the corresponding strains quantified by real time RT PCR. Strains were grown on the surface of liquid minimal medium for 24 h. Expression was normalized to histone 2B minus the normalized expression of the two hydrophobins in a <i>rodA</i>-deletion strain (RMS019). RodA was then set as one. In this way the value reflects the expression due to the activity of the <i>rodA</i> promoter. The mean of two technical and three biological replicates is shown with the standard error.</p

Phase and amplitude images of the spore surface of the wild type TN02A3 taken with atomic force microscopy (AFM) in tapping mode.

<p>The ordered rodlet structure is clearly visible. For TN02A3 phase as well as amplitude images show very good quality. Scale bars, 100</p

Alignment and structure of the six <i>A. nidulans</i> hydrophobins.

<p>(<b>A</b>) Alignment of RodA with the other five hydrophobins. Sequences were aligned manually. The blue box shows the N-terminal signal peptide, 8 characteristic cysteines of each hydrophobin (red), which appear in the characteristic pattern 1-2-1-1-2-1. The different colors represent the homology between the hydrophobin open reading frames. Blue indicates 100% identity, pink 71% and green 57% homology. Besides the conserved cysteine residues, overall sequence similarity of the hydrophobins is very low. (<b>B</b>) Assignment of hydrophobin proteins to hydrophobin classes. The analyzed hydrophobins all feature the common eight cysteine-motif and are members of the Class-I hydrophobin family with the exception of DewD.</p

Characterization of all hydrophobin deletion strains.

<p>(<b>A</b>) AFM amplitude images of the spore surfaces. Whereas the <i>rodA</i> deletion strain (RMS019) does not show any rodlets, the dewA-E deletion strains (TMS027, STT01, STT02, SAGR01, SAGR12) display rodlets. Scale bars, 100 nm. (<b>B</b>) Determination of the number of rodlets per bundle. For each strain 20 bundles were analyzed and the mean value displayed. The error bars represent the standard deviation. Strains see Figure legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094546#pone-0094546-g005" target="_blank">Fig. 5</a>.</p

Expression patterns of <i>A. nidulans</i> hydrophobins (rodA, dewA-E) during development.

<p>TN02A3 was grown in liquid medium over night before shifting to solid minimal medium. RNA was isolated and the mRNA of the corresponding genes quantified by real time PCR. The expression was normalized to histone H2b. Error bars represent the standard error (SEM).</p

Water contact angle measurement of all hydrophobin deletion strains and their corresponding re-complementations in comparison to wild type.

<p>150 μl of a detergent solution (0.2% SDS, 50 mM EDTA) were dropped on the surface of a lawn. The angle (degree) between the surface and the drop was measured. The mean of 10 measurements is displayed. The error bar represents the standard deviation.</p

Localization of mRFP-tagged hydrophobins.

<p>Constructs (as indicated) were transformed into GR5 or TN02A3. Strains: SCOS170-SCOS175, SAGR19a, and STT08. Scale bar, 2 μm in <b>A</b> and 5 μm in <b>B</b>.</p

Pairwise alignments for all hydrophobin sequences in comparison to RodA.

<p>(<b>A</b>) Up: Hydrophobicity of the alignment of RodA with DewA. In comparison with DewB, DewA does not share as much similarity in the hydrophobicity pattern with RodA. The two hydrophobic unstructured loops are conserved; DewA features a stronger hydrophobic region towards the N-Terminus. Middle: Predicted disorder for RodA/DewA. Similar to RodA, intrinsically disordered regions are predicted for two large loops towards the C-Termini. (<b>B</b>) Up: Hydrophobicity of the alignment of RodA with DewB. Due to their similarity in sequence RodA and DewB share similar hydropathicity patterns. A large gap is observed in the sequence of DewB towards the N-Terminus. Middle: Predicted disorder for RodA/DewB. Both proteins share a very similar predicted disorder pattern. The RodA sequence aligned to the gap in DewB is predicted to be largely unstructured. The two unstructured loops between the cysteines towards the C-Terminus can be clearly discerned. (<b>C</b>) Up: Hydrophobicity of the alignment of RodA with DewC. Especially towards the C-Terminus DewC exhibits a hydrophobicity pattern unlike the one from RodA. Middle: Predicted disorder for RodA/DewC. A large unordered region was predicted for the DewC sequence. Like all other analyzed hydrophobins except for DewD, the large unstructured loop (alignment positions 100-150) is conserved. (<b>D</b>) Up: Hydrophobicity of the alignment of RodA with DewD. DewD exhibits a hydrophobicity pattern unlike all the other hydrophobins. Especially the characteristic loop between alignment positions 125 and 150 is missing. Middle: Predicted disorder for RodA/DewD. DewD is predicted to exhibit a very high amount of intrinsic disorder. (<b>E</b>) Up: Hydrophobicity of the alignment of RodA with DewE. The hydrophobicity pattern of DewE is unlike that of RodA with a second hydrophobic region in the middle of the sequence. Middle: Predicted disorder for RodA/DewE. The two short unordered loops of DewE are shifted in comparison with RodA. For all images: Down: Sequence Alignment Blue/Red RodA/Gaps, Green/Yellow Other/Gaps</p

Hydrophobicity tests of wild type, all hydrophobin deletion strains (left) and their corresponding re-complementations (right).

<p>Strains were inoculated as spore suspensions (10⁶ per plate) using glass beads to obtain very homogenous lawns and grown for 40 h at 37°C. The bigger pictures show the aspect of the entire agar plate covered with a lawn of the corresponding strain and with a droplet in the middle of the plate. The small pictures show enlarged droplets. 10 μl of a detergent solution (0.2% SDS, 50 mM EDTA) were dropped onto the surface of a colony. Lower hydrophobicity is indicated by soaking of the liquid into the colony. Besides change of the contact angle, the effect is easily visible through the brown color. This is due to the color of conidiophore stalks and vesicles. If the droplet does not soak into the colony, the brown color is hidden by the yellow spore color. Strains: TN02A3, RMS019, TMS027, STT01, STT02, SAGR01, SAGR12, SAGR02, 03, 04, 05, 11, 13.</p