title: High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens.
abstract: Bipolaris sorokiniana, the causal agent of spot blotch of wheat, significantly reduces grain yield worldwide. In order to study pathogenic mechanisms of the fungus, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. To study different stages of hyphal fusion and pathogenic mechanisms of the fungus, two fluorescence markers viz. the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constitutively expressed. Southern hybridizations confirmed the presence of T-DNA in all hygromycin B or geneticin resistant transformants, and also showed random and single copy integration. Fluorescence microscopy suggested the high level expression of both DsRed and EGFP fluorescent proteins in spores and mycelia. The results signify that DsRed and EGFP can be used as efficient reporter gene for monitoring B. sorokiniana hyphal fusion as well as colonization in the host tissues. This work will be useful to develop methodologies for understanding the mechanisms of Bipolaris-wheat interaction and functional genomics of B. sorokiniana for various applications including insertional mutagenesis, targeted disruption of specific genes, ectopic complementation of loss-of-function strains and over-expression.
authors.affiliation: National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India.
authors.forename: Shadab
authors.initials: S
authors.lastname: Nizam
authors.forename: Sandhya
authors.initials: S
authors.lastname: Verma
authors.forename: Kunal
authors.initials: K
authors.lastname: Singh
authors.forename: Rashmi
authors.initials: R
authors.lastname: Aggarwal
authors.forename: Krishna Dutt
authors.initials: KD
authors.lastname: Srivastava
authors.forename: Praveen K
authors.initials: PK
authors.lastname: Verma
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001203:Ascomycota / Q000235:genetics*; D015139:Blotting, Southern; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010935:Plant Diseases / Q000382:microbiology; D014170:Transformation, Genetic; D014908:Triticum / Q000382:microbiology*
chemical_list: D008164:Luminescent Proteins