Abstract
Natural resistance associated macrophage proteins (NRAMPs) are evolutionarily conserved metal transporters involved in the transport of essential and nonessential metals in plants. Fifty protein interactors of a Brassica juncea NRAMP protein was identified by a Split-Ubiquitin Yeast-Two-Hybrid screen. The interactors were predicted to function as components of stress response, signaling, development, RNA binding and processing. BjNRAMP4.1 interactors were particularly enriched in proteins taking part in photosynthetic or light regulated processes, or proteins predicted to be localized in plastid/chloroplast. Further, many interactors also had a suggested role in cellular redox regulation. Among these, the interaction of a photosynthesis-related thioredoxin, homologous to Arabidopsis HCF164 (High-chlorophyll fluorescence164) was studied in detail. Homology modeling of BjNRAMP4.1 suggested that it could be redox regulated by BjHCF164. In yeast, the interaction between the two proteins was found to increase in response to metal deficiency; Mn excess and exogenous thiol. Excess Mn also increased the interaction in planta and led to greater accumulation of the complex at the root apoplast. Network analysis of Arabidopsis homologs of BjNRAMP4.1 interactors showed enrichment of many protein components, central to chloroplastic/cellular ROS signaling. BjNRAMP4.1 interacted with BjHCF164 at the root membrane and also in the chloroplast in accordance with its proposed function related to photosynthesis, indicating that this interaction occurred at different sub-cellular locations depending on the tissue. This may serve as a link between metal homeostasis and chloroplastic/cellular ROS through protein–protein interaction.
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Acknowledgments
We thank Prof. Wolf B. Frommer for providing us the split-ubiquitin system and Prof. Jane Parker for the pJ2B-3XHA-GW and pXCSG-strep vectors. The BiFC (Gelvin, Tzfira, and Citovsky), pMDC vectors, pEarleyGate101, 102 and the organelle marker were obtained from ABRC. We thank Dr. Mathieu Cellier and Mr. Sandipan Chakraborty for their help during NRAMP modeling, Dr. Fumiaki Katagiri for the AvrPto1 clone and Dr. Claus-Peter Witte and Dr. Geetanjali Sundaram for useful discussions. We thank the Centre for Modern Biology for software support in the bioinformatics work. We acknowledge the Department of Biotechnology- Interdisciplinary Program in Life Sciences, University of Calcutta for the confocal microscopy facility and Ms. Boney Halder and Mr. Joydeep Das jointly for their technical assistance. We are grateful to Mr. Jamie Shirlaw for kindly editing the manuscript. Different parts of the work were funded by All India Council for Technical Education (AICTE) [8023/BOR/RID/RPS-40/2007-08], 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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Ananya Marik and Haraprasad Naiya created the constructs, performed the Y2H studies, Co-IP, participated in BiFC/localization studies and helped in preparation of the manuscript. Madhumanti Das and Gairik Mukherjee participated in the BiFC studies. M.D also participated in the FLIM study. Soumalee Basu was involved in the modeling of BjNRAMP4.1. Chinmay Saha cloned the full length BjNRAMP4.1. Rajdeep Chowdhury and Dr. Kankan Bhattacharyya performed the FLIM-FRET and analyzed the data. Anindita Seal conceived the project, planned the experiments, participated in the modeling studies and wrote the manuscript.
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Ananya Marik and Haraprasad Naiya have contributed equally to this study.
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Marik, A., Naiya, H., Das, M. et al. Split-ubiquitin yeast two-hybrid interaction reveals a novel interaction between a natural resistance associated macrophage protein and a membrane bound thioredoxin in Brassica juncea . Plant Mol Biol 92, 519–537 (2016). https://doi.org/10.1007/s11103-016-0528-x
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DOI: https://doi.org/10.1007/s11103-016-0528-x