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Applied and Environmental Microbiology, May 2009, p. 3222-3229, Vol. 75, No. 10
0099-2240/09/$08.00+0     doi:10.1128/AEM.01764-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Differential Regulation and Posttranslational Processing of the Class II Hydrophobin Genes from the Biocontrol Fungus Hypocrea atroviridis

Marianna Mikus,¹ Lóránt Hatvani,^1,2 Torsten Neuhof,³ Monika Komo-Zelazowska,¹ Ralf Dieckmann,³ Torsten Schwecke,⁵ Irina S. Druzhinina,¹ Hans von Döhren,⁴ and Christian P. Kubicek^1*

FB Gentechnik und Angewandte Biochemie, Institut für Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, TU Wien, Getreidemarkt 9-166, 1060 Vienna, Austria,¹ Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Közép fasor 52, H-6726 Szeged, Hungary,² Anagnostec GmbH, Biotechnologiepark TGZ II, 14943 Luckenwalde, Germany,³ TU Berlin, Institut für Chemie, FG Biochemie und Molekulare Biologie, Franklinstr. 29, 10587 Berlin, Germany,⁴ Robert-Koch-Institut Berlin, Nordufer 20, D-13353 Berlin, Germany⁵

Received 31 July 2008/ Accepted 16 March 2009

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Hydrophobins are small extracellular proteins, unique to and ubiquitous in filamentous fungi, which mediate interactions between the fungus and environment. The mycoparasitic fungus Hypocrea atroviridis has recently been shown to possess 10 different class II hydrophobin genes, which is a much higher number than that of any other ascomycete investigated so far. In order to learn the potential advantage of this hydrophobin multiplicity for the fungus, we have investigated their expression patterns under different physiological conditions (e.g., vegetative growth), various conditions inducing sporulation (light, carbon starvation, and mechanical injury-induced stress), and confrontation with potential hosts for mycoparasitism. The results show that the 10 hydrophobins display different patterns of response to these conditions: one hydrophobin (encoded by hfb-2b) is constitutively induced under all conditions, whereas other hydrophobins were formed only under conditions of carbon starvation (encoded by hfb-1c and hfb-6c) or light plus carbon starvation (encoded by hfb-2c, hfb-6a, and hfb-6b). The hydrophobins encoded by hfb-1b and hfb-5a were primarily formed during vegetative growth and under mechanical injury-provoked stress. hfb-22a was not expressed under any conditions and is likely a pseudogene. None of the 10 genes showed a specific expression pattern during mycoparasitic interaction. Most, but not all, of the expression patterns under the three different conditions of sporulation were dependent on one or both of the two blue-light regulator proteins BLR1 and BLR2, as shown by the use of respective loss-of-function mutants. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of mycelial solvent extracts provided sets of molecular ions corresponding to HFB-1b, HFB-2a, HFB-2b, and HFB-5a in their oxidized and processed forms. These in silico-deduced sequences of the hydrophobins indicate cleavages at known signal peptide sites as well as additional N- and C-terminal processing. Mass peaks observed during confrontation with plant-pathogenic fungi indicate further proteolytic attack on the hydrophobins. Our study illustrates both divergent and redundant functions of the 10 hydrophobins of H. atroviridis.

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Hydrophobins are unique and ubiquitous small proteins, characterized by the presence of eight positionally conserved cysteine residues, and present in all multicellular asco- and basidiomycetes. According to their hydropathy profiles and spacing between the conserved cysteines (37), they are divided into two classes (class I and class II). Hydrophobins are secreted proteins, found on the outer surfaces of the cell walls of hyphae and conidia, where they mediate interactions between the fungus and the environment (18, 24, 37), such as surface recognition during pathogenic interaction with plants, insects, or other fungi, but also in symbiosis (38). In addition, they also influence cell wall composition (33). Because of these manifold roles, it is less surprising that the expression of hydrophobin genes is subject to complex patterns of signals, including those that are related to the triggering of conidiogenesis or indicating the presence of a plant host.

Many species of the fungal genus Hypocrea/Trichoderma are known as mycoparasites, and several of them are therefore applied as biocontrol agents (6, 7, 36). In addition, Trichoderma spp. have recently been reported to occur as endophytes and to be able to elicit positive plant responses against potential pathogens (17). Because of the reasons given above, hydrophobins would be candidate proteins playing a role in this process, and in fact a class I hydrophobin gene has recently been reported to be overproduced during endophytic interaction of Trichoderma asperellum and cucumber roots (35). In addition, other hydrophobins may be involved in the mechanism of mycoparasitism itself as well as the colonization of decaying wood.

Our information about the roles of hydrophobins in the physiology of Trichoderma as well as other ascomycetous fungi is mostly derived from reversed genetics of a few major members (3, 4, 19-22). In Hypocrea jecorina (= Trichoderma reesei), two major class II hydrophobins (HFB-1 and HFB-2) have been studied in detail (4) and shown to be formed under different physiological conditions (29). However, the genome sequence of H. jecorina contains six class II hfb genes (27), and the roles of HFB-3, HFB-4, HFB-5, and HFB-6 are yet unknown. In the biocontrol fungus Hypocrea atroviridis (formerly called "Trichoderma harzianum"), only a single hydrophobin gene has been characterized so far (srh1 [28]) and shown to be expressed mainly under conditions of sporulation. Consequently, very little is known about hydrophobins and their regulation in Trichoderma.

We have recently reported that two species of the Trichoderma/Hypocrea genus, Hypocrea virens and Hypocrea atroviridis, have an exceptional high number of class II hydrophobin genes (i.e., 11 and 10 phylogenetically different genes, respectively [22]). Therefore, the objective of this work was to investigate whether all of them are in fact expressed and, if so, under which conditions. We thereby put emphasis on vegetative growth, mycoparasitic interaction, and different triggers of sporulation and on learning whether the sporulation- and stress-regulating proteins BLR1 and BLR2 (10, 15) play a role in this process.

In addition, we used matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry to detect the respective proteins and to learn their mode of processing. It has previously been shown that direct solvent extraction of mycelia and spores of Ascomycetes in the process of sample preparation provides a small set of protein peaks in the range of 5,000 to 10,000 Da representing the hydrophobin inventory (27). Structural studies of hydrophobins from H. jecorina (2, 20, 30, 31), Schizophyllum commune (13), and Agaricus bisporus (26) have shown expected signal peptide cleavage but also unusual processing patterns, including cleavage after Arg and Pro, as well as C-terminal modification.

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Reagents and standards.
-Cyano-4-hydroxycinnamic acid from Sigma Chemicals was used as the matrix for MALDI-TOF mass spectrometry experiments. Trifluoroacetic acid, ethanol, acetonitrile, and methanol from Merck were used as solvents. The hydrophobins HFBI and HFBII from H. jecorina (= T. reesei) were used as reference proteins and purified by two-phase separation and reverse-phase high-performance liquid chromatography purification as described previously (29).

Microbial strains and cultivations.
H. atroviridis IMI 206040 and two mutants derived from it, blr1 and bl2r mutants (which are impaired in the formation of the BLR1 and BLR2 proteins, respectively [8]), were used throughout this work. All cultures were maintained on plates containing malt extract agar (MEA) (30 g liter^–1 malt extract and 20 g liter^–1 agar in distilled water) at room temperature and subcultured weekly. The strains were grown on MEA either under constant illumination with white light (1,800 lx) or in darkness (8) for up to 3 days, respectively. For injury-induced conidiation (9), fungal colonies were grown on MEA in total darkness for 3 days, cut into strips with a scalpel, and incubated for an additional 2 days in darkness. To induce conidiation by carbon starvation, fungal colonies were grown in darkness for 2 days on minimal medium (MM) (1.66 mM MgSO₄, 5.16 mM K₂HPO₄, 2.68 mM KCl, 12.5 mM NH₄NO₃, 7.19 µM FeSO₄, 6.95 µM ZnSO₄, 10.1 µM MnCl₂ in distilled water) supplemented with 111 mM glucose (9), then transferred to glucose-free MM, and allowed to grow for a further 3 days in either complete darkness or under illumination. For growth under conditions of confrontation with a potential mycoparasitic host, strains of H. atroviridis and either Botrytis cinerea or Rhizoctonia solani were placed on MEA 4 cm apart. The plates were incubated in the absence of light for 2 and 1.5 days, respectively. All inoculations were performed by placing mycelium-covered agar discs (5 mm in diameter) on the plates covered with a sterile cellophane membrane, and the plates were incubated at 28°C.

RNA isolation and analysis of hydrophobin gene expression.
For RNA extraction, mycelia were harvested from the surface of the cellophane membranes covering the plates. Total RNA was extracted as described previously (12) under a red safety light. Hydrophobin gene transcripts were quantified by reverse transcription-PCR (RT-PCR). To this end, the RNA was treated with DNase I (Fermentas) and purified with the RNeasy MinElute Cleanup kit (Qiagen). Five micrograms of each RNA sample was then reverse transcribed using the RevertAid H minus first-strand cDNA synthesis kit (Fermentas) and the oligo(dT)₁₈ primer. RT-PCR was carried out in a T3 Thermocycler system (Biometra), using 25 cycles. Primers for individual hydrophobin genes were designed with the aid of the programs Gene Runner and Oligo Analyzer and are given in Table 1.

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TABLE 1. Primers for RT-PCR used in this study

To rule out contamination of any other PCR components, one reaction was always set up without a template. In addition, controls with chromosomal DNA were included to ensure that the PCR products were due to amplification of the cDNA. All the class II hydrophobins of H. atroviridis contain three introns, thus rendering the genes about 0.2 kb larger than the cDNAs, and the cDNAs could thus easily be differentiated from the chromosomal DNAs on the gels.

Extraction and preparation of mycelia for MALDI-TOF mass spectrometry analysis.
A few micrograms of fungal mycelia were suspended in acetonitrile-methanol-water (1:1:1), and 1 µl of the suspension was directly spotted onto target wells of a 100-position sample plate and immediately mixed with 1 µl of matrix solution (10 mg/ml 2,5-dihydroxybenzoic acid [DHB] in acetonitrile-methanol-water [1:1:1] and 0.3% trifluoroacetic acid). The sample matrix mixture was allowed to air dry prior to analysis. Alternatively, freeze-dried mycelium obtained from shaken cultures or fungi grown on plates was homogenized in 60% ethanol and centrifuged (note that no class I hydrophobins [if any are present] will be extracted by this treatment). One microliter of the protein solution was spotted on a MALDI target plate and mixed with matrix.

MALDI-TOF mass spectrometry analysis.
Mass spectrometry measurements were performed on a Voyager De-Pro-time of flight mass spectrometer from Applied Biosystems (Foster City, CA). Mass spectra were acquired in linear delayed extraction mode using an acceleration voltage of 20 kV and a low mass gate of 1,500 Da. For desorption of the components, a nitrogen laser beam ( = 337 nm) was focused on the template. The laser power is set to just above the threshold of ionization. Spectra for individual specimens were compiled, averaging results from at least 100 shots taken across the width of the specimen for m/z values from 2,000 to 20,000.

Bioinformatic analysis.
In order to correlate these m/z values with individual hydrophobins, we first used SignalP (14) to identify corresponding signal peptide cleavage sequences in the hydrophobins. We then used PROTPARAM (14) to predict the molecular weights of these mature forms and also of various further maturation products resulting from potential subsequent processing after export from the endoplasmic reticulum (1). This approach has been fully described previously (31).

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Hydrophobin gene expression during vegetative growth.
In order to learn which of the 10 hydrophobin genes of H. atroviridis are expressed during vegetative growth, we first cultivated the fungus in darkness and tested their expression during the phase of linear mycelial growth. mRNA was isolated after 24 h of growth (a time when growth was still in the nutrient-unlimited phase, and its gene expression should therefore be typical of growth under these conditions). Therefore, expression is shown only for single time points. Figure 1 shows that at this time, four of the hydrophobin genes (hfb-1b, hfb-2a, hfb-2b, and hfb-5a) were strongly expressed in the wild-type strain. Traces of the hfb-2c transcript were also recorded. The other five genes remained below the level of detection under these conditions.

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FIG. 1. Expression of H. atroviridis hydrophobin genes during cultivation in darkness and in the presence of constant white light in carbon-limited MM (see Materials and Methods). Data are shown for mRNAs isolated from fungal cells after 24 h of growth. The wild-type (WT) parent strain H. atroviridis IMI 206040 and blr1 mutant strain (lanes 1) and blr2 mutant strain (lanes 2) are shown. tef1 specifies the loading control with the housekeeping gene encoding translation elongation factor 1-alpha.

In accordance with its role as a light sensor, a blr1 mutant (which suffered a loss of function in BLR1) did not impair the expression of any of these five genes in the dark. Interestingly, however, the expression of hfb-1b and hfb-2a was impaired in the blr2 mutant strain, indicating a role of BLR2 in hydrophobin gene expression under conditions of vegetative growth in the absence of light.

Hydrophobin gene expression during light-induced conidiation.
The presence of light is known to stimulate conidiation in H. atroviridis (7), and it has also been reported to stimulate the expression of hydrophobin genes in Neurospora crassa (24) and H. jecorina (30). We have therefore investigated which of the 10 hydrophobin genes of H. atroviridis would be expressed if cultivation were performed in the presence of light. mRNA was isolated after 3 days of growth, which was also still in the linear phase of growth under this condition, although the cultures had already started to conidiate. The results (Fig. 1) show that all of the genes, which were already expressed under darkness, were also expressed in the presence of light. However, the abundance of the transcripts of most of these genes was significantly increased. Only hfb-2b remained at the same high constitutive level of expression displayed in darkness. In addition, two hydrophobin genes which were not detectable in darkness (hfb-6a and hfb-6b) were clearly detectable in the presence of light.

The increase in transcript abundance in light was, however, significantly impaired for most genes in the blr1 and blr2 strains (Fig. 1). Expression was not completely eliminated, however, for hfb-1b, hfb-2a, hfb-2c, and hfb-5a. We also observed that this impairment of expression was stronger in the blr2 strain for hfb-1b and hfb-2a.

We should also like to note that we did not observe any changes in the size of the various hfb mRNAs, particularly hfb-2a mRNA. A light-dependent differential splicing has been claimed for an orthologue of this gene in Trichoderma viride (34).

Peptidomics analysis of hydrophobin gene expression and posttranslational modification during vegetative growth and light-induced conidiation.
In order to correlate the results from the expression analysis with the formation of the respective proteins, we have employed direct MALDI-TOF mass spectrometry of mycelia of the different growth conditions, in addition to expression profiles of posttranslational processing, to investigate this. As has been shown for well-characterized hydrophobins of H. jecorina, solvent treatment during sample preparation releases the processed hydrophobic peptides, which can be detected as molecular ions (31). Thus, mass peaks in the region of 2 to 12 kDa have been scanned, and m/z values correlated with sequence data of the expressed hydrophobins. For data interpretation, masses have been calculated for predicted signal cleavage sites using SignalP (14), and additional sites were identified, including a non-Kex2 type Arg cleavage close to the signal peptide site (20, 30), a similar Pro cleavage site (13, 26), and a C-terminal Phe degradation (31). Most, but not all, signals could be correlated, indicating the involvement of additional proteases. Hydrophobins and their processing sites, deduced as described above, are given in Table 2.

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TABLE 2. Processing of H. atroviridis hydrophobins as predicted from MALDI dataa

During vegetative growth and light-induced conidiation, the signal at m/z 7,500 ± 3 corresponded to HFB-2a (= SHR1) processed at the predicted most likely signal cleavage site at Ala₁₅ with four obligatory disulfide bonds (7,499 Da for HFB-2a-1), and the signal at m/z 9,116 ± 3 likewise has been related to HFB-5a (9,115 Da for HFB-5a-1, with a less likely cleavage site within the signal peptide region). In H. jecorina HFB-2, a C-terminal cleavage at F has been verified (31), and by analogy we thus correlate the signal at m/z 7,351 ± 2 with the oxidized HFB-2a, processed at A₁₅ by signal peptide cleavage, from which the C-terminal F had been removed, yielding a predicted mass of 7,352 Da (HFB-2a-3). In the blr2 mutant, the HFB-2a-1 signal is strongly reduced, corresponding to the extremely weak transcriptional signal (Fig. 1). A smaller peak at m/z 7,225 likewise is missing in the blr2 mutant, and this size correlates well with HFB-2a cleaved at the second most likely signal peptide site Ser₁₈ (HFB-2a-2, 7,225 Da).

Other dominating mass signals were found at m/z values of 6,908 ± 1, 7,522, 7,557 ± 1, 7,701 ± 1, and 8,583 ± 3. The signal at m/z 7,702 ± 2 can be tentatively correlated with HFB-2b N-terminally cleaved at R₂₂, the only basic residue preceding the disulfide-bridged hydrophobin core, resulting in a mass of 7,701 Da (HFB-2b-1). Similar cleavage reactions have previously been verified for HFB-1 and HFB-3 from H. jecorina (20, 30). Likewise, the signal at 7,557 corresponds to HFB-1b cleaved at the homologous R25 (HFB-1b-1, calculated 7,553 Da). Interestingly, while these processings of HFB-1b and HFB-2b occur at a monobasic proprotein cleavage site typically found also in other Trichoderma secretory proteins (28), HFB-2a lacks such a second proteolytic cleavage site after the signal peptide, and signal cleavage occurred predominantly as predicted by SignalP. All the other hydrophobins, whose expression was detected (see above), have not been identified by their possible predicted masses, indicating different processing modes.

Hydrophobin gene expression upon carbon starvation.
The changes in hydrophobin gene expression observed under light and darkness could be due to the association of these genes/proteins with sporulation or, alternatively, they could be due to direct stimulation by light. In order to discriminate between these two hypotheses, we used carbon starvation, a condition that has previously been shown to lead to sporulation in H. atroviridis (9). Under carbon starvation in darkness, hfb-2b was the major expressed hydrophobin gene, and traces of hfb-5a and even lower amounts of hfb-2a and hfb-2c were also recorded (Fig. 2). No changes in this pattern were observed in the two blr mutants, with the interesting exception that hfb-2a accumulated in the blr2 strain. In contrast, all hydrophobin genes, but hfb-22a, were expressed under conditions of carbon starvation and in the presence of light, and this expression was completely dependent on BLR1 and BLR2 for hfb-1b, hfb-1c, hfb-2a, hfb-6a, hfb-6b, and hfb-6c. hfb-2b, however, was again expressed at a high level throughout and not influenced by carbon starvation, and a similar behavior was seen for hfb-2c, whose expression was very low however. Impairment of the expression of hfb-2a and hfb-5a was incomplete in the blr2 strain. These data suggest that light, rather than light-induced sporulation, is the major trigger for the expression of most H. atroviridis hydrophobin genes. Controls, using transfer to medium with sufficient carbon (111 mM glucose), essentially led to the same expression pattern as shown in Fig. 1 and are therefore not shown separately.

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FIG. 2. Expression of H. atroviridis hydrophobin genes during cultivation in darkness and in the presence of constant white light in carbon-limited medium (see Materials and Methods). Data are shown for mRNAs from fungal cells isolated after 24 h of growth. For other details, see the legend to Fig. 1.

Hydrophobin peptidomics analysis of hydrophobin gene expression upon carbon starvation.
Under these conditions, the main signals detected are m/z values of 6,908 ± 1, 7,350 ± 1, 7,412 ± 1, 7500 ± 2, 7,523 ± 2, 7,700 ± 2, 8,585 ± 2, and 9,406 ± 2. The mass of 7,701 Da for HFB-2b-1 is expected from the transcription data. The signal of m/z 7,351 ± 1 in the wild type is consistent with HFB-2a (7,352 Da for HFB-2a-3). However, the dominating mass peaks at m/z 6,908 and 7,500 have no clear correlation with known processings applied to HFB-2b, as the other detected masses also have not been assigned to any of the expressed hydrophobins.

Hydrophobin gene expression and protein formation during stress-induced sporulation.
Mechanical injury can induce sporulation of H. atroviridis even in the dark (8). However, the expression patterns of three of the hydrophobin genes (hfb-2b, hfb-2c, and hfb-5a) showed little differences compared to these genes in cells showing vegetative growth in the dark (Fig. 3). The only exception to this was the complete inhibition of expression of hfb-1b, and the reduced expression of hfb-2a and its dependence on BLR1, but not BLR2, under this stress-induced conidiation. Also, this data set supports the interpretation that sporulation per se is not sufficient to trigger the expression of most of the H. atroviridis hydrophobin genes.

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FIG. 3. Expression of H. atroviridis hydrophobin genes during cultivation in darkness (control) and in darkness after mechanical injury (see details in Materials and Methods). For other details, see the legend to Fig. 1.

The MALDI MS patterns exhibit main signals under these conditions with m/z values of 7,500 ± 2, 7,701 ± 3, 7,723 ± 3, and 7,765 ± 3. These correlate with HFB-2a-1 (7,499 Da) and HFB-2b-1 (7,701 Da), while the latter masses could be interpreted as possible Na⁺ adducts of HFB-2b-1.

Hydrophobin gene expression and protein formation during confrontation with other fungi.
In order to identify which class II hydrophobins would be expressed when H. atroviridis is grown in confrontation with other fungi, we used Botrytis cinerea and Rhizoctonia solani as host models. RNA was extracted from H. atroviridis at the time when its hyphae almost touched the hosts, i.e., when they were only 1 to 2 mm distant from them. The data are presented in Fig. 4: four of the hydrophobin genes (i.e., hfb-2a, hfb-2b, hfb-2c, and hfb-6c) were shown to be expressed under these conditions, hfb-2c being the major one. hfb-2b and hfb-6c appeared to be less strongly expressed in confrontation with S. solani. No significant differences in the expression of these four genes were observed between the wild-type and blr1 and blr2 mutant strains, respectively. When the expression pattern was compared to a control in which H. atroviridis was confronted with itself, hfb-1b, hfb-2c, and hfb-6a were found to be downregulated, whereas the expression of hfb-6c, although weak, was upregulated by the presence of the host.

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FIG. 4. Expression of H. atroviridis hydrophobin genes during confrontation with B. cinerea or R. solani. mRNA was extracted from the mycelia facing the host (see scheme) at a time when the two fungi were only 3 mm apart. For a control, H. atroviridis (HA) was confronted with itself, and mRNA was extracted at the same time as described above. For other details, see the legend to Fig. 1.

To our surprise, none of the predicted HFB-2b fragments were observed in the mass spectra. The dominating peaks occur at m/z values of 6,057 ± 1, 6,123 ± 1, 6,710 ± 2, 6,731 ± 1, 7,345 ± 2, 8,090 ± 1, and 8,614 ± 1, which were not yet observed in the analyses above. The major mass peak at a m/z value of 6,710 would imply cleavage close to or at the hydrophobin core, while smaller peptides indicate cleavage within a disulfide-bridged region. Additional experiments with confrontations against Fusarium provided similar patterns and m/z values (data not shown).

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The results from this study show that transcriptional regulation of class II hydrophobin genes of H. atroviridis can be grouped into four categories: one gene, hfb-2b, was abundantly expressed under all conditions tested, and its expression is therefore constitutive (at least under the conditions applied in this work). Phylogenetically it is the H. atroviridis hydrophobin most closely related to H. jecorina HFB-1 and HFB-2 (22). However, expression of the hfb-1 and hfb-2 genes is divergent and different from the expression of hfb-2b: expression of hfb-1 in H. jecorina is observed only in vegetative cultures on glucose-containing and sorbitol-containing media, whereas H. jecorina hfb-2 was strongly induced by N and C starvation, by light, and in conidiating cultures. Nakari-Setälä et al. (29) concluded that HFBI has a role in hyphal development and HFBII in sporulation. Our results suggest that HFB-2b may fulfill both roles in H. atroviridis.

The second group of genes included hydrophobin genes that were expressed in darkness and that were upregulated in the presence of light. These genes were hfb-1b, hfb-2a, and hfb-5a. Phylogenetically, these three genes belonged to different clades. HFB-2a is identical to the previously described "T. harzianum" SRH1, and the data reported here confirm its regulation during sporulation (24). Interestingly, they were expressed in darkness during vegetative growth and under conditions of mechanical-stress-induced conidiation, but their transcripts were not seen in darkness with carbon starvation. Also, expression under the former two conditions required the function of the blue-light receptor protein BLR2, but not BLR1, whereas upregulation in light always required both of them. HFB-1b has recently been identified to belong to a phylogenetic clade of class II hydrophobins (the "HFB-4 clade") whose members exhibit characteristic amino acid differences leading to partially altered hydrophobicity (22). However, this potentially different function is not reflected in an expression pattern different from that of hfb-2a and hfb-5a.

The third group of genes was composed of genes that were expressed only in the presence of light (hfb-6a, hfb-6b, and hfb-6c; the latter only during carbon starvation in light). Their expression was absolutely dependent on BLR1 and BLR2. Finally, hfb-22a was the only gene for which we could not detect expression under any condition. While it is still possible that it may be formed under conditions not tested in this paper, we consider it likely that hfb-22a is a pseudogene. The presence of pseudogenes supports the recently postulated birth-and-death mechanism of evolution of these genes (22).

The data discussed above illustrate that light, rather than sporulation, is the major trigger for expression of most of the hydrophobin genes of H. atroviridis. We have recently discussed that the presence of light may signal to the fungus to be above soil (15). Increased surface hydrophobicity may be beneficial for vegetative growth in the presence of air.

Hydrophobins from various fungi, particularly plant-pathogenic fungi, have been implicated in the process of contact with the hosts (25, 38). If this would also apply for mycoparasitism by H. atroviridis, we would expect to see enhanced expression of one or more of its hydrophobin genes closely before contact. Interestingly, such a pattern was seen only for hfb-6c and it was weak, whereas three other genes (hfb-1b, hfb-2c, and hfb-6a) appeared to become downregulated by the presence of the host. Whether this is due to direct influence by the host or beneficial for the attack by H. atroviridis remains to be further investigated. However, in view of the multiplicity and redundancy of the H. atroviridis hydrophobins, further research of this area will be a demanding task.

The expression of some class I and II hydrophobin genes has been reported to be stimulated by light in some fungi including Trichoderma reesei (29), and this stimulation has been linked to the circadian clock (5). Involvement of the light receptor genes in mediating this stimulation has, however, not yet been shown. Here we show that expression of those H. atroviridis hydrophobin genes that are stimulated by light (hfb-1b, hfb-6a, and hfb-6b) (also hfb-1c and hfb-6c strains under carbon starvation) also depends on the function of the light regulator genes blr1 and blr2. Interestingly, this dependence was not absolute, because a low transcript level was also seen in the blr1 and blr2 mutant strains (e.g., for hfb-2a and hfb-5a). Even more interesting, however, was the fact that expression of hfb-1b and hfb-2a in darkness also depended on blr2 (but not blr1), thus clearly extending the function of BLR2 to physiological conditions other than light. BLR2 (and its N. crassa counterpart WC-2) is characterized by having only a single PAS domain, whereas WC-1 contains three PAS domains (A to C), while otherwise being structurally similar. For the perception of light, WC-1 and WC-2 form complexes through the interaction of the PAS C domain of WC-1 and the PAS domain of WC-2 (10, 11). We therefore speculate that the dependence of hydrophobin gene expression for BLR2 in the dark may be due to an interaction of its PAS domain with other regulatory ligands. In fact, PAS domains can accomplish ligand-dependent switching of a variety of partner domains, including histidine kinase, phosphodiesterase, and basic helix-loop-helix DNA-binding modules and have been demonstrated to do so in all kingdoms of life and under quite diverse physiological roles (16). The genome sequence database of H. atroviridis contains several putative PAS domain-containing proteins of unknown functions, which represent candidates for this interaction (C. P. Kubicek, unpublished data).

Using the MALDI-TOF mass spectrometric approach, we were able to identify 4 of the 11 hydrophobins at the protein level. Although we admit that this prediction of processing sites is in silico only and solid verification would require N-terminal sequencing of the actual proteins, we should like to note that this approach has recently resulted in correct predictions in a similar study (31). While the thereby deduced processing of HFB-2a follows known and thus predicted signal peptide cleavage sites, the less likely cleavage site of HFB-5a is still within a plausible cleavage region: while the classical Chou-Fasman prediction implies that the cleavage region must form a beta-turn which is required for transport, Laforet and Kendall (23) demonstrated that either a beta-turn- or alpha-helix-fostering sequence in the cleavage region can also function indistinguishably from the Chou-Fasman archetype.

Because of the role of hydrophobins in interactions between organisms (38), we also investigated whether any of the H. atroviridis hydrophobins would be specifically induced during confrontation of the fungus with other plant-pathogenic fungal hosts. From the present data, we conclude that none of the class II hydrophobins are specifically needed for this purpose and if hydrophobins do play a role, it is fulfilled by the hydrophobins already expressed during vegetative growth. However, the peptidomics approach showed that the pattern of m/z signals was significantly different from that observed under the other physiological conditions. The fact that all these m/z values were significantly lower than those of the HFB proteins otherwise observed suggests proteolytic processing. The fact that we did not detect even HFB-2b, whose transcript was most abundant under these conditions, supports this hypothesis. Hydrophobins are considered very stable proteins (37), and their proteolytic degradation is virtually unknown. If our interpretation (that the hosts degrade the H. atroviridis hydrophobins) is indeed correct, it may be a part of the defense reactions of the hosts. If so, this would represent a new target for developing agents protecting against these plant-pathogenic fungi.

 
This study was supported by the Fifth (EC) Framework program (Quality of Life and Management of Living Resources; project EUROFUNG 2; QLK3-1999-00729) to C.P.K. and H.V.D., by a grant from the Austrian Science Foundation to C.P.K. (P-17325-FWF), and by a fellowship from the Deutsche Forschungsgemeinschaft (Do270/10) to H.V.D.

We are grateful to A. Herrera-Estrella (Irapuato, Mexico) for providing the blr1 and blr2 mutant strains used in this study.

 
* Corresponding author. Mailing address: Section of Microbial Biochemistry and Gene Technology, Institute of Chemical Engineering, Technische Universität Wien, Getreidemarkt 9-166, Vienna A-1060, Austria. Phone: 43 1 58801 17250. Fax: 43 1 58801 17299. E-mail: ckubicek{at}mail.zserv.tuwien.ac.at

Published ahead of print on 27 March 2009.

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Applied and Environmental Microbiology, May 2009, p. 3222-3229, Vol. 75, No. 10
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