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High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens

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Title High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens
 
Creator Nizam, Shadab
Verma, Sandhya
Singh, Kunal
Aggarwal, Rashmi
Srivastava, Krishna Dutt
Verma, Praveen K.
 
Subject Bipolaris sorokiniana
Spot blotch
ATMT
Anastomosis
DsRed
EGFP
 
Description 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.
This work was supported by research grant provided by Depart-
ment of Biotechnology, Government of India. Authors acknowledge
Dr. Hitoshi Nakayashiki, Laboratory of Plant Pathology, Kobe University, Japan for providing pSilent-Dual1. SN acknowledges University
Grants Commission for the fellowship. SV and KS acknowledge Council of Scientific and Industrial Research for their fellowships.
 
Date 2015-10-27T10:41:01Z
2015-10-27T10:41:01Z
2012
 
Type Article
 
Identifier J. Microbiol. Methods, 88: 386-392
0167-7012
http://www.sciencedirect.com/science/article/pii/S016770121200005X
http://172.16.0.77:8080/jspui/handle/123456789/266
 
Language en_US
 
Publisher Elsevier