Tagging pathogenicity genes in the interaction of barley and the fungal pathogen, Rhynchosporium secalis.

Abstract

The purpose of this study was to identify pathogenicity genes in the fungal pathogen of cultivated barley, Rhynchosporium secalis. Pathogenicity genes are described as genes that are critical for the successful invasion and colonisation of the host plant but not necessary for life cycle completion in culture. To identify genes a pool of insertion mutants was generated. Insertional mutants were generated by two methods, restriction enzyme-mediated integration (REMI) and Agrobacterium tumefaciens-mediated transformation (ATMT). A detailed REMI study showed circular pAN7-1 vector produced higher transformation efficiencies than linear vector at all enzyme levels tested. Fungal strain 5, in combination with 20 units of the restriction enzyme BamHI produced the highest observed transformation efficiency with approximately 40% of these mutants producing simple, single integrations based on interpreted Southern data. The addition of BamHI increased transformation efficiency at all enzyme levels tested with the exception of the highest enzyme concentration: 200 units of enzyme/transformation reaction. In comparison to REMI, the ATMT protocol proved more efficient than REMI and the binary vector backbone pPZP200 produced >50% simple single copy integrations, interpreted from Southern data. This study is the first ATMT protocol for R. secalis and was successfully adapted from other fungal species. In total, 534 BamHI and HindIII REMI mutants of R. secalis fungal strain UK7 (83) and strain 5 (453) were screened on the universally susceptible barley cultivar Sloop yielding 10 non-pathogenic mutants, eight from strain 5 and two from UK7, respectively. During screening experiments strain 5 mutants failed to produce enough spores for a spore suspension to be prepared and inoculated. Strain 5 loses the ability to sporulate after four generations, or successive subculture steps. The inability to sporulate was not correlated to an observable, macroscopic loss in fungal biomass. Starvation experiments utilising carbon and nitrogen sources did not alter sporulation in the sporulating strain 5 sample or reverse the loss of sporulation. However, an overall trend was observed in the sporulation of strain UK7 where sporulation decreased with increasing nitrogen and increased with increasing carbon. Genomic sequence flanking the integration site was isolated and analysed from six of the ten non-pathogenic mutants. Four putative genes were identified with integrations located in their putative promoter sequences. Sequence similarity searches showed three of these putative genes had similarities to amino acid permeases, cytochrome p450 and rhomboid-like genes. The two putative genes with similarities to amino acid permease and cytochrome p450 genes were selected for targeted gene disruption studies using homologous recombination (HR). ATMT was used as the delivery system for the HR construct in an attempt to generate a disruption mutant and prove gene function. Over 200 mutants transformed with the two knock out vectors were screened. However, gene disruption experiments failed and could not be repeated due to a lack of resources and time. In conclusion, this study has demonstrated that the REMI transformation technique is feasible for gene disruption studies in R. secalis. Furthermore, ATMT is a viable alternative transformation method that, for future studies, would be the preferable technique.