Abstract
A forward genetics approach was applied in order to investigate the molecular basis of morphological transition in the wheat pathogenic fungus Zymoseptoria tritici. Z. tritici is a dimorphic plant pathogen displaying environmentally regulated morphogenetic transition between yeast-like and hyphal growth. Considering the infection mode of Z. tritici, the switching to hyphal growth is essential for pathogenicity allowing the fungus the host invasion through natural openings like stomata. We exploited a previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) to generate a mutant library by insertional mutagenesis including more than 10,000 random mutants. To identify genes involved in dimorphic switch, a plate-based screening system was established. With this approach eleven dimorphic switch deficient random mutants were recovered, ten of which exhibited a yeast-like mode of growth and one mutant predominantly growing filamentously, producing high amount of mycelium under different incubation conditions. Using genome walking approach previously established, the T-DNA integration sites were recovered and the disrupted genomic loci of corresponding mutants were identified and validated within reverse genetics approach. As prove of concept, two of the random mutants obtained were selected for further investigation using targeted gene inactivation. Both genes deduced were found to encode known factors, previously characterized in other fungi: Ssk1p being constituent of HOG pathway and Ade5,7p involved in de novo purine biosynthesis. The targeted mutant strains defective in these genes exhibit a drastically impaired virulence within infection assays on whole wheat plants. Moreover exploiting further physiological assays the predicted function for both gene products could be confirmed in concordance with conserved biological role of homologous proteins previously described in other fungal organisms.
Highlights
The mutagenesis program was conducted by Agrobacterium tumefaciens-mediated transformation (ATMT), representing a stable and reliable transformation method, which was previously established and approved in different reverse genetics studies with Z. tritici [31]. pCAMB-hygromycin B phosphotransferase (HPT)(SalI) was used as a transformation vector (Fig 1C), containing hygromycin phosphotransferase gene (HPT) as a resistance marker derived from Escherichia coli under control of upstream trpC promoter from Aspergillus nidulans and downstream NOS terminator from Agrobacterium tumefaciens [31]
The suitability of random mutagenesis was proved by randomly selecting of 100 transformants and performing a Southern Blot analysis to verify the genomic integration of T-DNA and to assess the copy number of T-DNA
This analysis revealed no bias towards multiple integrations of the T-DNA and confirmed a likely “randomized” distribution of integration sites, especially in view of the potential occurrence of so-called “hot spots”
Summary
Septoria wheat diseases have increased in incidence over the last few decades despite the deployment of fungicide treatments and Z. tritici is consistently the most destructive foliar pathogen of wheat in Europe [1,2,3,4]. STB has a substantial economic impact leading to annual yield losses of up to 50%, representing a significant threat to global food production. In Europe annual losses from STB are estimated to $400 million dollars [5,6]. The increased prevalence of these diseases is considered to be due to the more frequent use of high yielding semi-dwarf rust-resistant cultivars, increased nitrogen application rates and changes to post harvest practices [7,8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
