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A protocol for functional study of genes in Brassica juncea by Agrobacterium-mediated transient expression: applicability in other Brassicaceae

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Abstract

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.

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Abbreviations

Avr:

Avirulence

GUS:

β-Glucuronidase

GFP:

Green fluorescent protein

CFP:

Cyan fluorescent protein

BjTTP:

Brassica juncea tetratricopeptide

BjHCF:

Brassica juncea high-chlorophyll fluorescence

BjNRAMP:

Brassica juncea natural resistance associated macrophage protein

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Acknowledgements

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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  1. Madhumanti Das and Haraprasad Naiya have contributed equally to this work.

Authors and Affiliations

  1. Department of Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India

    Madhumanti Das, Ananya Marik, Gairik Mukherjee & Anindita Seal

  2. ICAR-Indian Institute of Natural Resins and Gums, Namkum, Ranchi, Jharkhand, 834010, India

    Haraprasad Naiya

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  1. Madhumanti Das
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Correspondence to Anindita Seal.

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Figure S1

Expression of pCAMBIA1305.1-GUS containing catalase intron in different time point. GUS-plus gene was expressed transiently in B. juncea leaves 48hpi and 72hpi. n=15 (2 leaves/plant) (TIFF 7105 kb)

Figure S2

A scheme showing agroinfiltration of B. juncea leaves. B. juncea (or other Brassica sp) leaves were pricked at the abaxial side and infiltrated with desired constructs mixed with AvrPto1 containing Agrobacterium culture. Gene/protein expression was checked 72 hours post infiltration (hpi) (TIFF 66 kb)

Figure S3

Localization study with negative controls. (a) The untransformed leaf in the wavelength range of mCherry (upper panel) and CFP (middle panel). Leaf transformed with empty GFP (pMDC45) vector (lower panel). (b) The untransformed root in the wavelength range of mCherry (upper panel) and CFP (lower panel). B.F. bright field (Scale 20 µm), (each in 3 experimental replicates) (TIFF 841 kb)

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Das, M., Naiya, H., Marik, A. et al. A protocol for functional study of genes in Brassica juncea by Agrobacterium-mediated transient expression: applicability in other Brassicaceae. J. Plant Biochem. Biotechnol. 29, 368–379 (2020). https://doi.org/10.1007/s13562-019-00543-x

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