Publications of NIPGR Scientists

Permanent URI for this communityhttps://ndkr-library.nipgr.ac.in/handle/123456789/1

Browse

Author: search.filters.author.Singh, KunalSubject: search.filters.subject.ATMT

Search Results

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens
    (Elsevier, 2012) Nizam, Shadab; Verma, Sandhya; Singh, Kunal; Aggarwal, Rashmi; Srivastava, Krishna Dutt; Verma, Praveen K.
    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-Ex- press) 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 Bipo- laris–wheat interaction and functional genomics of B. sorokiniana for various applications including insertion- al mutagenesis, targeted disruption of specific genes, ectopic complementation of loss-of-function strains and over-expression.
  • Thumbnail Image
    Item
    Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus Ascochyta rabiei
    (Springer, 2010) Nizam, Shadab; Singh, Kunal; Verma, Praveen K.
    Ascochyta blight caused by the ascomycete fungus Ascochyta rabiei, is a major biotic constraint of chickpea (Cicer arietinum L.), resulting in disastrous crop losses worldwide. To study early stages of development and pathogenic mechanisms of the fungus, two binary vectors for the constitutive expression of the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constructed. Furthermore, we have developed an improved and highly reproducible Agrobacterium tumefaciens-mediated transformation protocol for A. rabiei. Transformation events were confirmed through Southern hybridizations that suggest single-copy integration of reporter genes in majority of the transformants. High level expression of both DsRed and EGFP proteins was obtained both in spores and in mycelia as detected by fluorescence microscopy. Intense fluorescence was used as a highly efficient vital marker to visualize early developmental changes of the fungus. The formation of infection structures like appressoria and germ tubes were observed both in vitro and in planta. This work will be useful to develop methodologies for understanding the mechanisms of Ascochyta-chickpea interaction and functional genomics of A. rabiei towards the isolation of virulence genes.