Filamentous fungi generally contain multiple hydrophobin genes, which play important roles in fungal growth, development and environmental communication [1, 2, 6, 7]. We identified only 3 class II hydrophobin genes in the genome of the mycoparasite C. rosea. This is in strong contrast with the closely related mycoparasites T. atroviride and T. virens that contain high numbers (10 and 9 respectively) and diversity of class II hydrophobins . This indicate important ecological differences between C. rosea and Trichoderma spp., and emphasize that different mycoparasites may rely on different mechanisms of interaction. The expansion of the hydrophobin gene family in Trichoderma spp. is hypothesized to help the fungus to attach to the hyphae of a broad range of asco- and basidiomycetes .
The high expression of Hyd1 in conidiating mycelia in comparison with germinating conidia indicates that Hyd1 may have a role during conidiophore development. This is consistent with the expression pattern of hyd1 in M. anisoplia where expression is low in germinating conidia and high in mycelium with conidiophores . The expression, but lack of regulation, of Hyd1, Hyd2 and Hyd3 on different nutrient regimes, and between developmental stages of Hyd2 and Hyd3, indicate a constitutive role of the corresponding proteins in C. rosea. Constitutive roles of hydrophobins in fungal growth and development are reported in many species [6, 7, 36]. However, certain hydrophobins from Trichoderma spp. and M. brunneum are regulated by nutritional conditions and between different life cycle stages [5, 11, 28, 37].
Expression levels of Hyd1, Hyd2 and Hyd3 are repressed in C. rosea during interactions with B. cinerea and F. graminearum, which is consistent with the expression pattern of T. atroviride hydrophobin genes hfb-1b, hfb-2c and hfb-6a. This may suggest that Hyd1, Hyd2 and Hyd3 are not involved in protecting hyphae from recognition by other organisms [6, 7]. Alternatively, the data can also be interpreted as an induction during C. rosea self interaction that may suggest a role for Hyd1, Hyd2 and Hyd3 in intraspecific signalling or hyphal fusion. Hydrophobins that are known to be involved in interactions with plant leaves and roots are usually highly expressed during these conditions [8, 9, 28]. Therefore, the low expression of the 3 C. rosea hydrophobin genes during barley root colonization indicates that the corresponding proteins may not be necessary for root adhesion and colonization.
Deletion of hydrophobin genes from different fungal species often results in variable and sometimes contradicting phenotypes. This is a reflection of the birth-and-death type of evolution of the hydrophobin gene family , which results in functionally diverse proteins with many species specific members. This is evident for Hyd1 and Hyd3 in C. rosea as gene deletions results in increased growth rate and sporulation, which is in contrast to the reduced sporulation in T. reesei, M. oryzae and M. brunneum due to deletion of the hydrophobin genes HFB2, MPG1 and MHP1[8, 9] and hyd1, hyd2 and hyd3, respectively. The situation is even more complicated as deletion of HCf-1 and HCf-2 in Cladosporium fulvum, cpph1 in Claviceps purpurea and hfb1 in T. reesei results in no differences in sporulation in comparison with the WT strain.
Deletion of Hyd1 or Hyd3 does not influence mycelial hydrophobicity in C. rosea, which is consistent with previous reports in C. purpurea, M. brunneum, F. verticilloides and B. cinerea[11–13, 38]. However, it seems that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity and dispersal, as the conidia from the double deletion mutant ΔHyd1ΔHyd3 clump together in solution and have lower hydrophobicity index than the WT. Similar phenotypes are repeatedly reported from many different species [8, 9, 11, 12, 34, 39]. Furthermore, deletion of Hyd1 and Hyd3 does not influence the expression levels of Hyd2, which suggests that Hyd2 is subject to different regulatory signals than Hyd1 and Hyd3. Failure to delete Hyd2 despite several trials may suggest an essential function of the corresponding protein.
Hyd1 and Hyd3 do not appear to be involved in protection of the C. rosea mycelium during abiotic stress conditions. In contrast, higher conidial germination rates during abiotic stress conditions in Hyd1 and Hyd3 mutants suggests that these hydrophobins inhibit conidial germination in environments not suitable for mycelial growth. Similar results are shown previously in M. oryzae and the entomopathogenic fungus B. bassiana against thermal stress [9, 10]. Hence, under unfavourable conditions hydrophobins may act as a sensor for the conidial germination signalling pathway and consequently protect the conidia by limiting its germination until favourable conditions are prevail .
The increased growth rate of Hyd1 and Hyd3 deletion strains under normal conditions such as PDA, may explain the faster overgrowth of the fungal prey in plate confrontations and the higher growth rate on plates previously colonized by B. cinerea, F. graminearum or R. solani. Similar results are reported previously in T. asperellum, where deletion of TasHyd1 does not reduce in vitro mycoparasitic ability . Hydrophobins are highly expressed proteins that may account for up to 10% of the total amount of secreted proteins [40, 41]. In C. rosea, deletion of both Hyd1 and Hyd3 results in a reduction of the total amount of secreted proteins. Despite this, no differences in pathogen biomass production in sterile filtered culture filtrates from single and double deletion strains are recorded. This may suggest that Hyd1 and Hyd3 do not exert a direct toxic effect on the fungal prey.
The higher conidial germination rates (under certain conditions) and higher growth rates of Hyd1 and Hyd3 deletion strains may explain the reduced necrotic lesion area, caused by B. cinerea, on A. thaliana leaves preinoculated with the mutant strains in comparison with WT preinoculated leaves. As a consequence, the C. rosea deletion strains may parasitize B. cinerea to a greater extent or simply outcompete it for space or nutrients. Hydrophobins in T. asperellum are reported to influence root surface attachment and intercellular root colonization . Similarly, our results show that Hyd3 is needed for barley root colonization. Unexpectedly, deletion of Hyd1 in a ΔHyd3 background increases the root colonization ability. The exact mechanism responsible for this cannot be discerned based on the current data, but we may speculate that it can be related to the lower conidial hydrophobicity or the lower protein secretion of the double deletion strain compared with the Hyd1 and Hyd3 single gene deletion strains. In the entomopathogenic fungus B. bassiana, reduced virulence is recorded for a Δhyd1 strain, while no effect is observed for a Δhyd2 strain. However, the effect of the Δhyd1Δhyd2 double deletion mutant on virulence is cumulative and lower than for the single Δhyd1 strain .