Tài liệu Báo cáo khoa học: Direct identification of hydrophobins and their processing in Trichoderma

Direct identification of hydrophobins and their processing

in Trichoderma using intact-cell MALDI-TOF MS

Torsten Neuhof1

, Ralf Dieckmann1,*, Irina S. Druzhinina2

, Christian P. Kubicek2

,

Tiina Nakari-Seta¨la¨

3

, Merja Penttila¨

3 and Hans von Do¨ hren1

1 TU Berlin, Institut fu¨r Chemie, FG Biochemie und Molekulare Biologie, Berlin, Germany

2 FB Gentechnik und Angewandte Biochemie, Institut fu¨r Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften, TU Wien,

Vienna, Austria

3 VTT Technical Research Centre of Finland, Espoo, Finland

Hydrophobins are small proteins thought to be ubi￾quitous in filamentous fungi. They are usually present

on the outer surfaces of cell walls of hyphae and coni￾dia. Here, they mediate interactions between the fun￾gus and the environment, such as surface recognition

during pathogenic interactions with plants, insects or

other fungi, and also in symbiosis. The size of hydro￾phobins ranges from approximately 75 to 400 amino

acid residues; they contain eight positionally conserved

cysteine residues, and can be divided into two classes

according to their hydropathy profiles and spacing

between the conserved cysteines [1].

The anamorphic fungal genus Trichoderma (Hypocre￾ales, Ascomycota) contains cosmopolitan soil-borne

fungi with economic importance as biocontrol agents

and producers of beneficial metabolites and enzymes. In

addition, Trichoderma spp. have recently been reported

to occur as endophytes, eliciting positive plant responses

against potential pathogens [2]. Hydrophobins are likely

to play a role in this process, and a hydrophobin gene

has in fact recently been isolated that leads to overpro￾duction of hydrophobins during endophytic interactions

between Trichoderma asperellum and cucumber roots

[3]. However, hydrophobins may also be involved in the

Keywords

fungal biomarker; hydrophobin; intact-cell

MS; MALDI-TOF MS; Trichoderma

Correspondence

H. von Do¨hren, TU Berlin, Institut fu¨r

Chemie, FG Biochemie und Molekulare

Biologie, Franklinstr. 29, 10587 Berlin,

Germany

Fax: +49 30 314 24783

Tel: +49 30 314 22697

E-mail: [email protected]

*Present address

AnagnosTec, Gesellschaft fu¨r Analytische

Biochemie und Diagnostik mbH, Potsdam￾Golm, Germany

(Received 19 September 2006, revised 27

November 2006, accepted 6 December

2006)

doi:10.1111/j.1742-4658.2007.05636.x

Intact-cell MS (ICMS) was applied for the direct detection of hydropho￾bins in various species and strains of Hypocrea ⁄Trichoderma. In both myce￾lia and spores, dominating peaks were identified as hydrophobins by

detecting mass shifts of 8 Da of reduced and unreduced forms, the analysis

of knockout mutants, and comparison with protein databases. Strain-speci￾fic processing was observed in the case of Hypocrea jecorina (anamorph

Trichoderma reesei). An analysis of 32 strains comprising 29 different spe￾cies of Trichoderma and Hypocrea showed hydrophobin patterns that were

specific at both at the species and isolate (subspecies) levels. The method

therefore permits rapid and direct detection of hydrophobin class II com￾positions and may also provide a means to identify Trichoderma (and other

fungal) species and strains from microgram amounts of biomass without

prior cultivation.

Abbreviations

HFB, hydrophobin; ICMS, intact-cell MALDI-TOF MS.

FEBS Journal 274 (2007) 841–852 ª 2007 The Authors Journal compilation ª 2007 FEBS 841