Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea.
BACKGROUND
Filamentous fungi produce small cysteine rich surface active amphiphilic hydrophobins on the outer surface of cell walls that mediate interactions between the fungus and the environment. The role of hydrophobins in surface hydrophobicity, sporulation, fruit body formation, recognition and adhesion to host surface and virulence have been reported. The aim of the present study was to characterize the biological function of hydrophobins in the fungal biocontrol agent Clonostachys rosea in order to understand their potential roles in biocontrol mechanisms.


RESULTS
Based on the presence of hydrophobin domains, cysteine spacing patterns and hydropathy plots, we identified three class II hydrophobin genes in C. rosea. Gene expression analysis showed basal expression of Hyd1, Hyd2 and Hyd3 in all conditions tested with the exception of induced Hyd1 expression in conidiating mycelium. Interestingly, up-regulation of Hyd1, Hyd2 and Hyd3 was found during C. rosea self interaction compared to interactions with the fungal plant pathogens Botrytis cinerea or Fusarium graminearum in dual culture assays. Phenotypic analysis of C. rosea deletion and complementation strains showed that Hyd1 and Hyd3 are jointly required for conidial hydrophobicity, although no difference in mycelia hydrophobicity was found between wild type (WT) and mutant strains. Interestingly, mutant strains showed increased growth rates, conidiation and enhanced tolerances of conidia to abiotic stresses. Antagonism tests using in vitro dual culture and detached leaf assays showed that the mutant strains were more aggressive towards B. cinerea, F. graminearum or Rhizoctonia solani, and that aggression was partly related to earlier conidial germination and enhanced tolerance of mutant strains to secreted fungal metabolites. Furthermore, in vitro Arabidopsis thaliana root colonization assays revealed reduced root colonization ability of the ΔHyd3 strain, but not for the ΔHyd1 strain. Furthermore, enhanced root colonization ability for the ΔHyd1ΔHyd3 strain was found in comparison to WT.


CONCLUSIONS
These results show a role for hydrophobins in conidial hydrophobicity, control of conidial germination under stress conditions, and in root colonization in C. rosea. However, functional studies of Hyd2 remains to be performed in order to fully assess the role of hydrophobins in C. rosea.
Uppsala BioCenter, Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Box 7026, 75007 Uppsala, Sweden. mukesh.dubey@slu.se.
Mukesh K
MK
Dubey
Dan Funck
DF
Jensen
Magnus
M
Karlsson
D017360:Arabidopsis / Q000382:microbiology; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D020869:Gene Expression Profiling; D005816:Genetic Complementation Test; D057927:Hydrophobic and Hydrophilic Interactions; D006999:Hypocreales / Q000737:chemistry* / Q000254:growth & development*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D056265:Microbial Interactions; D008969:Molecular Sequence Data; D018517:Plant Roots / Q000382:microbiology*; D017422:Sequence Analysis, DNA; D013172:Spores, Fungal / Q000737:chemistry* / Q000254:growth & development*
D005656:Fungal Proteins; D008565:Membrane Proteins