Abstract
The subcellular localization of proteins is a fundamental aspect of protein functions. Determining the subcellular localization is important for understanding the biological functions of proteins. Here, we developed a set of rice organelle marker lines, in which the expressing fluorescent organelle markers could be used as comparative standards in determining the subcellular localization of the protein of interest. We constructed green fluorescent protein (GFP)- and/or Discosoma sp. red fluorescent protein (DsRed)-tagged organelle markers targeted to the endoplasmic reticulum (ER), mitochondria, Golgi apparatus, peroxisome, actin cytoskeleton, plastid, tonoplast, plasma membrane, and nucleus, respectively. The utility of the rice marker lines for protein subcellular localization studies was demonstrated by detecting a nucleus-localized OsWRKY45 and a mitochondria-associated NbHxk1 in protoplasts of the GFP-OsH2B and the ScCOX4-DsRed lines, respectively. Using a sheath-inoculation method, followed by a live-cell imaging, we detected co-localization of a Magnaporthe oryzae PWL2:mCherry : NLS fusion with the nucleus marker in the GFP-OsH2B rice epidermal cells, confirming the translocation of the M. oryzae effector PWL2 into host cells, and further demonstrating the feasibility of using the organelle marker lines for studying dynamics of proteins in rice cells in the interactions between rice and pathogens. The set of organelle marker lines developed in the present study, provides a valuable resource for protein subcellular localization studies in rice.
Highlights
Eukaryotic cells are highly compartmentalized and various biological processes are implemented within specialized subcellular compartments (Nelson et al, 2007)
As for developing markers targeted to the plastid, tonoplast, plasma membrane, FIGURE 1 | List of green fluorescent protein (GFP)- and Discosoma sp. red fluorescent protein (DsRed)-tagged organelle markers developed in this study
We developed a set of plant expression cassettes for GFP- and DsRed-fusion organelle markers
Summary
Eukaryotic cells are highly compartmentalized and various biological processes are implemented within specialized subcellular compartments (Nelson et al, 2007). Transgenic lines stably expressing FP-tagged organelle markers have been generated in Arabidopsis, M. truncatula, maize, and rice (Geldner et al, 2009; Luo and Nakata, 2012; Kim et al, 2013; Wu et al, 2013; Krishnakumar et al, 2015; Wu et al, 2016) These transgenic marker lines provided valuable resources for protein subcellular localization studies, especially for studying the dynamics of proteins or organelles in developmental or environmental contexts (Geldner et al, 2009; Kim et al, 2013; Wu et al, 2016)
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