Green Fluorescent Protein- and Discosoma sp. Red Fluorescent Protein-Tagged Organelle Marker Lines for Protein Subcellular Localization 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.

inTRODUcTiOn Eukaryotic cells are highly compartmentalized and various biological processes are implemented within specialized subcellular compartments (Nelson et al., 2007). In general, the subcellular localization of proteins is closely associated with functions related to the biological processes carried out in the corresponding organelles (Lunn, 2007;Mathur, 2007). Thus, information on the subcellular localization is helpful in understanding the biological functions of proteins (Martin et al., 2009;Tanz et al., 2013).
Protein subcellular localization analysis has been routinely employed as an experimental methodology for gene functional studies. Over the past decades, fluorescent protein (FP) tagging has become the dominant approach for determining the subcellular localization of proteins in living cells in different organisms, including plants (Chudakov et al., 2010;Tanz et al., 2013). Based on fusion of FPs with the well-established targeting proteins or motifs (Mathur, 2007), numerous sets of fluorescent organelle markers have been developed for Arabidopsis (Nelson et al., 2007;Geldner et al., 2009;Kim et al., 2013), Nicotiana benthamiana (Martin et al., 2009), Medicago truncatula (Luo and Nakata, 2012), maize (Wu et al., 2013;Krishnakumar et al., 2015), and rice (Wu et al., 2016;Dangol et al., 2017). Currently, the common strategy utilized to detect localization of FP-tagged proteins has been based on transient expression of fusion constructs in protoplasts through polyethylene glycol (PEG)mediated transfection (Chen et al., 2006;Zhang et al., 2011), or in epidermal cells through biolistic bombardment (Dangol et al., 2017) or agro-infiltration (Lunn, 2007;Nelson et al., 2007). Although transient expression systems provide convenient and rapid assays for subcellular localization, the approaches have limitations in monitoring processes in a developmental context or under particular environmental conditions, such as in response to abiotic stresses, or pathogen attack (Geldner et al., 2009). Recently, 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).
Rice is one of the most important food crops in the world, as well as a monocot model plant. More recently, sets of fluorescent organelle markers have been developed for protein subcellular localization studies in rice (Wu et al., 2016;Dangol et al., 2017). However, only green fluorescent protein (GFP)based organelle marker lines have been generated in rice (Wu et al., 2016). In the present study, to provide more flexible combinations of organelle marker lines, we developed transgenic rice plants stably expressing GFP-and/or Discosoma sp. red fluorescent protein (DsRed)-tagged organelle markers. We demonstrated the utility of the marker lines for co-localization studies by detecting a nucleus-localized rice OsWRKY45 and a mitochondria-associated N. benthamiana hexokinase NbHxk1 in protoplasts of the GFP-OsH2B and the ScCOX4-DsRed rice lines, respectively. In addition, we demonstrated the feasibility of using the organelle marker lines for studying dynamics of proteins in rice cells in the interactions between rice and pathogens. Using a sheath-inoculation method, followed by a live-cell imaging (Khang et al., 2010;Park et al., 2012), 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. The organelle markers and marker lines developed in this study, along with previously developed organelle markers and marker lines (Wu et al., 2016;Dangol et al., 2017), therefore, provide valuable resources for protein localization studies in rice.

Generation of Organelle Marker constructs
The sGFP(S65T) gene (Chiu et al., 1996) and the DsRed gene (Goodin et al., 2002) were used for the construction of fluorescent organelle marker constructs. GFP-or DsRed-fusion fragments were generated by overlapping PCR and were cloned into a plant binary vector, pCXSN (Chen et al., 2009) in which the GFPor DsRed-fusions were driven by the CaMV 35S promoter. All constructs were confirmed by sequencing. Primers used for PCR or overlapping PCR were listed in Supplementary Table S1.

agro-infiltration assay in Nicotiana benthamiana
The organelle marker constructs were introduced into the Agrobacterium tumefaciens strain GV3101. Transformed GV3101 bacteria were cultured in liquid yeast extract peptone media supplemented with kanamycin (50 ug/ml) and rifampicin (50 ug/ml). Suspensions of transformed GV3101 bacteria were adjusted to an OD 600 of 0.7 in the infiltration buffer (10 mM MES, 10 mM MgCl 2 , and 150 μM acetosyringone), and were maintained at room temperature for 2 h. The suspension cultures were infiltrated into leaves of 4-week-old N. benthamiana plants grown in a growth chamber at 25°C under 16/8 h light/dark cycle.

Rice Transformation
The organelle marker constructs were introduced into A. tumefaciens LBA4404 by electroporation. Rice calli induced from the embryos of mature seeds of cv. Nipponbare were used for transformation via the Agrobacterium-mediated method as described previously (Hiei et al., 1994). Regenerated transgenic plants and their self-pollinated progeny were grown in a greenhouse.

Rice Protoplast isolation and Transfection
Protoplasts were isolated from 2-week-old rice seedlings of transgenic rice organelle marker lines or wild type rice cv. Nipponbare. PEG-mediated protoplast transfection was performed as described previously (Chen et al., 2006). Protoplasts were incubated under dark at room temperature for 16-24 h.

Magnaporthe oryzae
A transgenic M. oryzae isolate carrying a PWL2:mCherry : NLS expression cassette (Khang et al., 2010) was used for rice sheath inoculation. Rice leaf sheath inoculation with M. oryzae was performed as described previously (Khang et al., 2010;Park et al., 2012). In brief, leaf sheaths were detached from 4-week-old rice seedlings, and were cut into 5-6 cm segments. Inoculation was performed by incubating fungal spores (1 × 10 5 conidial/ml) in inner interior of leaf sheaths. The inoculated sheaths were placed in a petri dish and were incubated under high humidity (95%) and at 26°C for about 2 days before microscopic inspection.

Subcellular Localizations of Rice OscTP1, Osγ-TiP, OsPiP2, and Osh2B in Nicotiana benthamiana
To validate the subcellular localization patterns of the four rice ortholog organelle markers, GFP-fusions of rice OsCTP1, Osγ-TIP, OsPIP2, and OsH2B were transiently expressed and investigated in N. benthamiana leaves, respectively. Microscopy of leaf epidermal cells showed that, while N. benthamiana leaves infiltrated with the 35S-GFP expression cassette displayed green fluorescence distributed uniformly throughout the whole cells ( Figures 2B, C), the expression of OsCTP1-GFP, Osγ-TIP-GFP, GFP-OsPIP2, or GFP-OsH2B showed specific fluorescence patterns as expected: green fluorescence emitted from OsCTP1-GFP accumulated at the granular plastids (Figures 2E, F); Osγ-TIP-GFP signal appeared in the shape of small rings surrounds the vacuole (Figures 2H, I); fluorescent signal of GFP-OsPIP2 located at the periphery of the cells (Figures 2K, L); and GFP-OsH2B produced green fluorescence targeted to the nucleus of the epidermal cells (Figures 2N, O). These results also suggested that the four rice orthologs, OsCTP1, Osγ-TIP, OsPIP2, and OsH2B had conserved subcellular localization patterns across dicot and monocot species.

Generation of Stable Rice Lines expressing Green Fluorescent Protein/ Discosoma sp. Red Fluorescent Protein-Fusion Organelle Markers
The binary vectors harboring expression cassettes for GFP/ DsRed-fusion organelle markers were introduced into the rice cultivar Nipponbare by Agrobacterium-mediated transformation, and more than 20 independent T 0 plants were generated for each construct. T 0 plants were screened by detecting fluorescent signals from the sliced-sheath tissues, and T 1 lines derived from T 0 plants with good fluorescent signal were used for detailed analyses.
To rule out the possibility that the DsRed-AtCCASP, GFP-mTn, and DsRed-OsH2B cassettes were not working for expression, we further transiently evaluated the three expression cassettes in N. benthamiana leaf cells and in rice protoplasts. Transient expression assays showed that the DsRed-AtCCASP, GFP-mTn, and DsRed-OsH2B fusions could be expressed in N. benthamiana leaves and rice protoplasts, producing fluorescent signals targeted to the Golgi apparatus, actin cytoskeleton, and nucleus, respectively (Supplementary Figure S1). These results indicated that the DsRed-AtCCASP, GFP-mTn, and DsRed-OsH2B cassettes were working for expression. Thus, it remains to be determined whether the three fusion proteins had harmful effects or there are other reasons that resulted in the failure to obtain transgenic rice lines stably expressing DsRed-AtCCASP, GFP-mTn, or DsRed-OsH2B.

Organelle Marker Lines for co-Localization Studies
To test the utility of the GFP/DsRed-fusion organelle marker lines for protein co-localization studies, we selected a rice transcriptional factor OsWRKY45 (Shimono et al., 2007) and a N. benthamiana mitochondria-associated hexokinase NbHxk1 (Kim et al., 2006) for proof-of-concept testing. OsWRKY45 fused with a mCherry and NbHxk1 fused with a GFP, were expressed in protoplasts of the GFP-OsH2B and ScCOX4-DsRed rice lines, respectively. The results showed that the red fluorescence of OsWRKY45-mCherry was merged with the nucleus GFP-OsH2B marker, and the fluorescent signal of NbHxk1-GFP was partly merged with the mitochondria ScCOX4-DsRed marker ( Figure 5). Overall, these experiments demonstrated that using protoplasts of the organelle marker lines can facilitate protein co-localization studies in rice cells.  (Takemoto et al., 2003;Koh et al., 2005;Lu et al., 2012). In this study, we performed sheath-inoculation of the GFP-OsH2B rice plants with a transgenic M. oryzae isolate expressing a PWL2 effector fused with mCherry and a nuclear localization signal (PWL2:mCherry : NLS) (Khang et al., 2010). Our result confirmed that PWL2:mCherry : NLS was translocated into rice cells, as indicated by co-localization with the nucleus GFP-OsH2B marker (Figure 6). This experiment also demonstrated that the organelle marker lines will be useful resources for studying dynamics of proteins or organelles in rice cells in the interactions between rice and pathogens.

DiScUSSiOn
In this study, we developed a set of plant expression cassettes for GFP-and DsRed-fusion organelle markers. The markers designed to target ER, mitochondria, Golgi apparatus, peroxisome, or actin cytoskeleton were based on fusion of GFP or DsRed with the well-established targeting proteins or motifs (Mathur, 2007), and the plastid, tonoplast, plasma membrane, and nucleus markers were constructed by fusing GFP or DsRed to rice orthologs of the OsCTP1 transit peptide, Osγ-TIP, OsPIP2, and OsH2B, respectively. The binary constructs of the organelle markers were used to generate stable transgenic rice plants. Microscopic analysis of sheath cells and protoplasts of transgenic rice plants showed that most of the GFP-or DsRed-fusions could be stably expressed in rice cells. In addition, we observed no significant difference in morphological traits between wild-type and the organelle marker lines (data not shown). However, we were unable to obtain transgenic plants expressing fluorescent signals of DsRed-AtCCASP, GFP-mTn, or DsRed-OsH2B, although the DsRed-AtCCASP, GFP-mTn, and DsRed-OsH2B fusions could be transiently expressed in N. benthamiana leaf epidermal cells and in rice protoplasts (Supplementary Figure S1). Indeed, we also generated another set of expression cassettes of DsRed-AtCCASP, GFP-mTn, and DsRed-OsH2B, in which the fusion organelle marker genes were driven by a strong maize ubiquitin-1 promoter (Christensen et al., 1992;Chen et al., 2009). Similarly, no transgenic plants expressing obvious fluorescent signals of DsRed-AtCCASP, GFP-mTn, or DsRed-OsH2B were obtained with multiple tries (data not shown).  While the reason for failure to obtain the DsRed-AtCCASP, GFP-mTn, or DsRed-OsH2B marker lines remains to be investigated, in the future, we will try to develop rice marker lines expressing green fluorescence targeted to the actin cytoskeleton, and lines expressing red fluorescence targeted to the Golgi apparatus and nucleus, respectively, using mCherry (Shaner et al., 2004) or other FP variants as reporters.
The availability of organelle marker lines will facilitate protein subcellular localization studies. Both our results and previous report (Wu et al., 2016) demonstrated that protoplasts from the rice organelle marker lines were suitable for transfection with fusion constructs of the protein of interest with FPs, and the markers could be used as comparative standards in determining the subcellular localization of the protein of interest. The use of the transgenic organelle marker lines also has an advantage over transient expression assays in studying the dynamics of proteins or organelles in developmental or environmental contexts (Geldner et al., 2009), because the transgenic lines are able to provide consistent, intact tissues for investigation under complicated conditions. For example, the Arabidopsis organelle marker lines have been used to study the interactions of Arabidopsis with oomycete (Takemoto et al., 2003;Lu et al.,2012), and powdery mildew fungus (Koh et al., 2005). In the present study, we presented an example application of using the leaf sheaths of the GFP-OsH2B marker line for detecting translocation of a M. oryzae PWL2 effector into rice cells, demonstrating the feasibility of the rice organelle marker lines as useful tools for dissection of protein dynamics or protein reorganization involved in rice-pathogen interactions. In addition, we generated both GFP-and/or DsRedtagged organelle markers lines. These lines will allow more flexible design of FP combinations for co-localization studies in rice.
In summary, we developed a set of rice lines stably expressing GFP-and/or DsRed-tagged organelle markers, and demonstrated the utility of the marker lines for protein subcellular localization studies, especially for studying dynamics of proteins in rice cells in the interaction between rice with blast fungus. These marker lines, along with previously established organelle markers and marker lines (Wu et al, 2016;Dangol et al., 2017), provide important resources for the rice research community.

DaTa aVaiLaBiLiTY STaTeMenT
The datasets generated for this study are available on request to the corresponding author.
aUThOR cOnTRiBUTiOnS ZW and SC conceived and designed the experiments. ZQC, WZ, LC, CL, TL, ZJC, HX, YH, LK, and XZ performed the experiments. ZQC, WZ, LC, CL, FW, and SC analyzed the data. ZQC, ZW, and SC wrote and revised the manuscript. All authors commented on the manuscript.

FUnDinG
This work was supported by grants from National Natural Science Foundation of China (U1405212, 31171808, 31601596) and grant from Fujian Provincial Science and Technology Program (2017R1019-1).