A protocol for functional study of genes in Brassica juncea by Agrobacterium-mediated transient expression: applicability in other Brassicaceae
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Agrobacterium-mediated transient expression in plant organs is a quick and reliable method for studying gene functions. Due to the significance of transient transformation, substantial efforts have been dedicated to developing such protocols in various plants including the model Arabidopsis thaliana. Despite the importance, a reliable protocol is still lacking in Brassicaceae due to their recalcitrance towards Agrobacterium-mediated transient transformation. We have developed protocols for transient expression in Brassica juncea (PI 211000) and tested three other Brassica sp. for the suitability of the protocol. Co-infiltration of a bacteria-derived avirulence protein AvrPto1 significantly improved expression in B. juncea cotyledonary leaves. The protocol was used successfully in studying protein localization, protein–protein interaction by co-immunoprecipitation assay and transient silencing in B. juncea indicating it to be an excellent model system for transient expression. The efficiency of the protocol varied between Brassica sp. and depended highly on the Agrobacterium strain used. The protocol would be useful in designing functional analyses of genes using transient expression in Brassicaceae, including Arabidopsis and enable inclusion of mutant lines for such studies.
KeywordsAgrobacterium Brassica juncea Transient expression
Green fluorescent protein
Cyan fluorescent protein
Brassica juncea tetratricopeptide
Brassica juncea high-chlorophyll fluorescence
Brassica juncea natural resistance associated macrophage protein
We thank Prof. Fumiaki Katagiri, University of Minnesota Twin Cities, for providing us the AvrPto1 clone. The pMDC vectors were obtained from ABRC. We also thank Dr. Naveen Bisht, NIPGR, India for providing us the B. nigra, B. rapa and B. napus seeds. We thank Dr. Ronita Nag for her assistance with A. thaliana work. We acknowledge the Department of Biotechnology—Interdisciplinary Program in Life Sciences, the University of Calcutta for the confocal microscopy facility and Mr. Arijit Pal and Mr. Souvik Roy jointly for their technical assistance. The organellar markers in pCMU vectors were gifts from Prof. M. J. Harrison and obtained from Dr. Senjuti Sinha Roy, NIPGR, India. We thank Prof. Daisuke Miki and Prof. Ko Shimamoto for providing us with the pANDA35HK vector. Madhumanti Das acknowledges Council of Scientific and Industrial Research (CSIR) and Haraprasad Naiya acknowledges University Grants Commission (UGC) for their fellowships. The work was funded by Council of Scientific and Industrial Research (CSIR) [Project No. 38(1276)/10/EMR-II] and Department of Science and Technology (DST) [SERB/SR/SO/PS/19/2012], Govt. of India.
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Conflict of interest
The authors declare that they have no conflict of interest.
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