Contrary to these reports, functional characterization of hydrophobins in Fusarium verticilloides does not indicate a role of these proteins in growth, infection or mycelium hydrophobicity [12]. Similar results are reported for Botrytis cinerea where deletion mutants of hydrophobin genes does not display any phenotypic differences compared to the wild type (WT) strain [13]. The fungus Clonostachys rosea is a ubiquitous soil borne ascomycete known for its antagonistic abilities against a wide range of plant pathogens [14–18], and for its entomopathogenic and see more nematophagous behaviour [19–21]. The modes of

action of C. rosea as a biological control agent (BCA) are not fully known, although mycoparasitism, competition for nutrients, and secondary metabolite production are suggested to play significant roles [14, 18, 22]. Furthermore, C. rosea can rapidly Compound Library price colonize outer and inner root surfaces (epidermal and cortical cells) of plants like carrot, barley, cucumber and wheat [23, 24], which results in induced defence responses [25]. Hydrophobins in mycoparasitic Trichoderma spp,

are suggested to be involved in hyphal development, sporulation, and plant root attachment and colonization [26–28]. The current study aims to understand the biological function of hydrophobins in C. rosea with emphasis on its role in fungal growth and development, antagonism, and interactions with plants. Our results showed induced expression of C. rosea Hyd1, Hyd2 and Hyd3 in dual cultures during self interaction in comparison to interaction Inhibitor Library ic50 with the phytopathogenic fungi B. cinerea and F. graminearum. In addition, Hyd1 showed significant upregulation in conidiating mycelium, although a basal expression of C. rosea Hyd1, Hyd2 and Hyd3 was observed in all conditions tested. By generating individual Hyd1 and Hyd3 deletion (ΔHyd1; ΔHyd3), complementation (ΔHyd1+; ΔHyd3+) and Hyd1, Hyd3 double deletion (ΔHyd1ΔHyd3) strains, we probed the roles of two

C. rosea hydrophobins in conidial hydrophobicity and plant root colonization. Results Identification and phylogenetic analysis of C. rosea hydrophobins Blast searches Oxalosuccinic acid against a C. rosea strain IK726 draft genome database using a total of 35 class I, class Ia (the intermediate class) and class II hydrophobin amino acid sequences from Trichoderma spp. [29], identified three genes with an E-value ≤ 1 × 10-5. The presence of additional hydrophobin gene/s in C. rosea genome cannot be excluded, as the short hydrophobin genes may be problematic to predict. Identification of start and stop codons, determination of exon-intron boundaries and open reading frames (ORFs) were done manually, and were further validated by cDNA sequencing. The resulting genes were named Hyd1, Hyd2 and Hyd3. The Hyd1, Hyd2 and Hyd3 sequences are submitted to GenBank with accession numbers KF834267, KF834268, KF834269, respectively.