Search

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.

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. [ABSTRACT FROM AUTHOR]

In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins. [ABSTRACT FROM AUTHOR]