title: Libraries for two-hybrid screening of yeast and hyphal growth forms in Zymoseptoria tritici.
abstract: Pathogenic fungi are constantly emerging resistance to anti-fungal treatments. Therefore, identification of new fungicide targets is important. Good candidates are essential fungal proteins and their regulators. An efficient way to reveal the molecular environment of an essential protein is the search for interacting factors. Here, we establish three yeast two-hybrid libraries, covering yeast and hyphal stages of the wheat pathogen Zymoseptoria tritici. No detectable genomic DNA was present in any of the 3 libraries. Random amplification revealed that the libraries include cDNA fragments of up to 2000bp, suggesting that small-to-medium sized proteins are represented therein. Indeed, full-length cDNAs of five proteins were found in all libraries. The full-length cDNA of large chitin synthase gene mcs1 (5742bp with introns; 5568bp without introns) could not be amplified, but its 5' and 3' regions were represented, suggesting that even larger genes are covered in all libraries. Finally, we tested for the expected interaction of the autophagy proteins ZtAtg4 and ZtAtg8 in Z. tritici, and then used ZtAtg4 to screen one of the two-hybrid libraries. Indeed, we found ZtAtg8 as a positive interaction partner, confirming that interacting proteins can be identified. Thus, these molecular tools promise to be useful in identifying novel fungicide target proteins.
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK.
authors.forename: W
authors.initials: W
authors.lastname: Ma
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK. Electronic address: S.Kilaru@exeter.ac.uk.
authors.forename: S
authors.initials: S
authors.lastname: Kilaru
authors.affiliation: Shannon Applied Biotechnology Centre, Limerick Institute of Technology, Moylish Park, Limerick, Ireland(2).
authors.forename: C
authors.initials: C
authors.lastname: Collins
authors.affiliation: Syngenta Crop Protection AG, Schaffhauserstrasse, 4332 Stein, Switzerland.
authors.forename: M
authors.initials: M
authors.lastname: Courbot
authors.affiliation: Biosciences, University of Exeter, Exeter EX4 4QD, UK. Electronic address: G.Steinberg@exeter.ac.uk.
authors.forename: G
authors.initials: G
authors.lastname: Steinberg
mesh_terms: D001203:Ascomycota / Q000166:cytology* / Q000235:genetics* / Q000254:growth & development; D015723:Gene Library; D005800:Genes, Fungal; D005820:Genetic Testing / Q000379:methods*; D025301:Hyphae / Q000235:genetics / Q000254:growth & development; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques
keywords: Mycosphaerella graminicola; Pathogenic fungi; Protein–protein interaction; Septoria tritici blotch; Wheat disease