Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus Ascochyta rabiei
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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.
KeywordsCicer arietinum Ascochyta rabiei ATMT DsRed-Express EGFP1
This work is supported by research grant provided by Department of Biotechnology, Government of India. Authors acknowledge Dr. L. Mikkelsen, The Royal Veterinary and Agricultural University, Denmark, for providing pPgpdA-DsRed, Dr. T. Sone, Hokkaido University, Japan, for providing pDESTR vector and Dr. U. Kück, Ruhr-University Bochum, Germany, for providing pIG1783 vector. The Ascochyta rabiei, (Delhi isolate, D11) was provided by Dr. S.C. Dube, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi. S.N. and K.S. acknowledge University Grant Commission and Council of Scientific and Industrial Research for the fellowships, respectively.
Conflict of interest
We have no conflict of interest.
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