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The Plant Journal (2014) 80, 1151–1163 doi: 10.1111/tpj.12706 TECHNICAL ADVANCE A set of fluorescent protein-based markers expressed from constitutive and arbuscular mycorrhiza-inducible promoters to label organelles, membranes and cytoskeletal elements in Medicago truncatula Sergey Ivanov and Maria J. Harrison* Boyce Thompson Institute for Plant Research, Tower Road, Ithaca, NY 14853, USA Received 26 June 2014; revised 1 October 2014; accepted 15 October 2014; published online 20 October 2014. *For correspondence (e-mail [email protected]). SUMMARY Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans-Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis-specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub-cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens-shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology. Keywords: mCherry, GFP, periarbuscular membrane, symbiosis, root, legume, technical advance. INTRODUCTION The Leguminosae is the third largest family of flowering plants (Gepts et al., 2005) and most of its members form symbiotic associations with both arbuscular mycorrhizal (AM) fungi and nitrogen-fixing rhizobium bacteria. Medicago truncatula (barrel medic), is one of three species, the other two being Lotus japonicus and Glycine max, that is widely used as a model for studies of these symbioses, as well as general aspects of legume biology (VandenBosch and Stacey, 2003). Many genomic and genetic resources have been developed for M. truncatula; a genome sequence and SNP resources (Young et al., 2011; © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd Stanton-Geddes et al., 2013), extensive transcriptome datasets that cover plant development and responses to biotic and abiotic stress (Benedito et al., 2008; Gomez et al., 2009; Li et al., 2009; Hogekamp et al., 2011; Gaude et al., 2012; Limpens et al., 2013), transformation systems (Chabaud et al., 1996; Trinh et al., 1998; Boisson-Dernier et al., 2001) and large insertion mutant populations (d’Erfurth et al., 2003; Tadege et al., 2008; Pislariu et al., 2012) provide a wide range of possibilities for studies in M. truncatula. However, a comprehensive set of fluorescent protein fusion constructs that mark individual organelles, cytoskeletal 1151 1152 Sergey Ivanov and Maria J. Harrison components, membranes and membrane compartments is currently missing for M. truncatula. Furthermore, such a resource is not available for either L. japonicus or G. max. Such a cell biology resource can assist in providing insights into sub-cellular changes within a cell which is particularly important in studies of endosymbioses, where the root cells are inhabited a fungal or bacterial endosymbiont. In addition, as researchers try to determine the function of the many genes of unknown function, knowledge of the sub-cellular location of the encoded protein can be helpful in assigning a biological role. Direct, non-invasive visualization of a candidate protein tagged with a fluorescent protein reporter is a widely used approach (Dixit et al., 2006) but this requires the comparison of the distribution of the candidate protein fusion with known fluorescent markers of sub-cellular components (Nelson et al., 2007). In Arabidopsis and maize, sets of fluorescent protein fusion constructs that mark different sub-cellular compartments have been developed and provide a useful resource for co-localization of unknown proteins as well as cell biology studies (Nelson et al., 2007; Mohanty et al., 2009; Kim et al., 2013; Tanz et al., 2013; Wu et al., 2013). The Arabidopsis fluorescent protein markers have been used in other dicot plant species. For example, markers that label the endoplasmic reticulum, the Golgi apparatus, the plasma membrane, the tonoplast, peroxisomes, mitochondria and plastids have been tested in M. truncatula either in transiently transformed roots (Genre et al., 2005; Pumplin and Harrison, 2009) or in stable transgenic plant lines (Luo and Nakata, 2012). However, these marker constructs are under the control of the Cauliflower Mosaic Virus 35S promoter (Nelson et al., 2007) and this promoter is not optimal for certain cell types, particularly colonized cortical cells during AM symbiosis (Pumplin et al., 2012) and also nodule tissues during rhizobium-legume symbiosis (Auriac and Timmers, 2007). Consequently, we developed a comprehensive set of fluorescent protein fusion constructs expressed from constitutive and AM symbiosisspecific promoters to assist cell biology and localization studies in M. truncatula but potentially also suitable for use in other legume species. To illustrate the utility of these markers we used a selection of them to further investigate cellular changes in M. truncatula root cells during arbuscular mycorrhizal symbiosis. During this mutualistic endosymbiosis, the AM fungus colonizes the root and grows extensively in the root cortex, both in the apoplastic spaces where it grows as a linear hypha, and also in the cortical cells, where the hypha differentiates to form a highly branched structure referred to as an arbuscule. The arbuscule fills most of the cell lumen and an extension of plant plasma membrane, called the periarbuscular membrane (PAM) surrounds the arbuscule and separates it from the root cell cytoplasm. The PAM and intervening space between the PAM and the arbuscule, called the periarbuscular space (PAS), forms the interface where nutrient exchange between the two symbionts takes place. Arbuscule formation is accompanied by tremendous changes in the root cortical cells and requires reorganization of the cell cytoplasm, including modulation of the cytoskeleton, organelles and a considerable increase in membrane (Bonfante and Genre, 2010; Harrison, 2012; Gutjahr and Parniske, 2013). Some of these cellular changes have been examined previously using antibodies and/or fluorescent protein fusion markers (Genre et al., 2005, 2008, 2012; Pumplin and Harrison, 2009; Bonfante and Genre, 2010; Harrison, 2012; Gutjahr and Parniske, 2013) but there are still many aspects of the biology of these cells that are unknown. Here we use the fluorescent protein-based markers to investigate re-positioning of the nucleus, cytoskeletal alterations, changes in plastid morphology, tonoplast identity and continuity of the periarbuscular space during AM symbiosis. RESULTS AND DISCUSSION Generation of a set of markers that label organelles, membranes, sub-cellular compartments and elements of the cytoskeleton To establish the fluorescence protein marker resource, we built on knowledge obtained from Arabidopsis and M. truncatula and developed markers of the nucleus, endoplasmic reticulum, Golgi apparatus, trans-Golgi network, plasma membrane, apoplast, late endosome/MVB, transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, PAM and PAS. Information about each marker construct is listed in Table 1. To generate the constructs, Gateway-compatible entry vectors containing the promoters, marker genes, fluorescent proteins and transcription terminators were created (Table S1) and recombined with a destination binary expression vector suitable for plant transformation. Most of the markers are fusions with the mCherry fluorescent protein which emits in the red spectrum and is useful for co-localization with other commonly used fluorescent proteins such as CFP, GFP and YFP (Shaner et al., 2005). For each marker, there are two options for expression: the Arabidopsis thaliana UBQ10 gene promoter (AtUBQ10p) which results in constitutive expression and the M. truncatula Blue Copper Binding Protein 1 promoter (MtBCP1p), which results in AM symbiosis-specific expression. The AtUBQ10 promoter has been used widely in Arabidopsis and tobacco where it provides a moderate level of gene expression which is uniform throughout the plant (Norris et al., 1993; Geldner et al., 2009; Grefen et al., 2010; Dyachok et al., 2014). It has also been demonstrated to be active in roots and root nodules of M. truncatula, L. japonicus and Parasponia andersonii (Markmann et al., 2008; Limpens et al., 2009; den Camp © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 Mitochondria Plastids Peroxisomes Autophagosomes Plasmodesmata PM/trunk and branch periarbuscular membrane (PAM) PM/trunk PAM pCMU-MITr; pCMB-MITr pCMU-PLAr; pCMB-PLAr pCMU-PERr; pCMB-PERr pCMU-AUTr; pCMB-AUTr pCMU-PDESr; pCMB-PDESr pCMB-TMEr; a BCPsp-GFP-BCP1 mCherry-SKL mCherry-LC3 PDLP1-mCherry BCPsp-mCherry-BCP1 COX4-mCherry RUB1sp-mCherry TIP1-1-mCherry FIM1-ABD2-mCherry LifeAct-mCherry; LifeAct-GFP mCherry -MAP4-MBD mCherry-RAB7 mCherry-RAB5 mCherry-SYP41 VHA-mCherry PIP2a-mCherry BCPsp-mCherry MAN49-mCherry mCherry-SYP61 mCherry-HDEL NLS-mCherry Marker name € hler et al. (1997) Ko Dabney-Smith et al. (1999); present study Reumann (2004) Present study Thomas et al. (2008) Pumplin and Harrison (2009); present study Marc et al. (1998) Saito et al. (2002) Sheahan et al. (2004) Riedl et al. (2008) AtTIP1-1, c-tonoplast aquaporin Actin-binding domain2 AtFIMBRIN1 LifeActin (17 aa of ScAbp140) Microtubule binding domain of mammalian MAP4 Signal peptide (29 aa) of ScCOX4 Target signal peptide (80 aa) of small subunit of rubisco MtRubisco1 C-terminal SKL signal peptide of AtPTS1 MtLC3(Atg8), Plasmodesmata-located protein AtPDLP1 Medicago blue copper protein MtBCP1 Limpens et al. (2009) Limpens et al. (2009) Dettmer et al. (2006) Cutler et al. (2000) Pumplin and Harrison (2009); present study Limpens et al. (2009) Saint-Jore-Dupas et al. (2006) Pumplin et al. (2012) Gomord et al. (1997); He et al. (1999) Grebenok et al. (1997) References to markers MtRAB7A2, small GTPase MtSYP41, q-SNARE syntaxin AtVHA-a1, H+-ATPase AtPIP2a, plasma membrane aquaporin Medicago blue copper protein MtBCP1 signal peptide (23 aa) MtRAB5A2, small GTPase N-terminal 15 aa (nuclear localization signal) of tobacco c2 polypeptide, C-terminal GUS N-terminal 29 aa of AtWAK2 signal C-terminal HDEL signal peptide N-terminal 49 aa of GmMAN1 MtSYP61, q-SNARE syntaxin Marker gene or signal peptide CM stands for Cellular Markers, U stands for AtUBQ10 promoter, B for MtBCP1 promoter, r for mCherry, g for eGFP. pCMB-TMEg Microtubules Late endosome/multivesicular bodies (MVB) Transitory late endosome/ Tonoplast Tonoplast Actin microfilaments Plasma membrane (PM) Apoplast pCMU-TPr; pCMB-TPr pCMU-ACTFr; pCMB-ACTFr pCMU-ACTLr; pCMB-ACTLr; pCMU-ACTLg; pCMB-ACTLg pCMU-MTUBr; pCMB-MTUBr pCMU-TLEr; pCMB-TLEr pCMU-LEr; pCMB-LEr pCMU-TGN41r; pCMB-TGN41r pCMU-TGNVHAr; pCMB-TGNVHAr pCMU-PMr; pCMB-PMr pCMU-APr; pCMB-APr Golgi apparatus trans-Golgi network/ Early endosome pCMU-GAr; pCMB-GAr pCMU-TGN61r; pCMB-TGN61r Nucleus Cell compartment Endoplasmic reticulum a pCMU-ERr; pCMB-ERr pCMU-NUCr; pCMB-NUCr Construct name Table 1 Marker genes and target signal peptides for cellular compartments Fluorescent protein markers for cell biology 1153 1154 Sergey Ivanov and Maria J. Harrison et al., 2011a,b) and is constitutively active in the roots of several other plant species including squash (Cucurbita pepo), the parasitic plant Triphysaria versicolor (Tomilov et al., 2007; Ilina et al., 2012) and is even active in onion cells (Eschen-Lippold et al., 2012). Consistent with the previous studies, we found that in M. truncatula, the AtUBQ10 promoter provided uniform expression in cells throughout the root (Figure S1a, b). The MtBCP1 promoter drives AM symbiosis-specific gene expression specifically in regions of the root cortex colonized by AM fungi (Hohnjec et al., 2005; Pumplin and Harrison, 2009) (Figure S1a). This promoter has the advantage of being expressed in cortical cells during hyphal penetration and throughout arbuscule formation but also in neighboring non-colonized cortical cells and thus provides a unique opportunity for comparisons of cellular organization in adjacent colonized and non-colonized cells in the same root. Currently, the MtBCP1 promoter has been tested only in M. truncatula but there is evidence that another M. truncatula AM symbiosis-inducible promoter shows the appropriate activity in potato (Karandashov et al., 2004), so it is possible that the MtBCP1 promoter will function appropriately in other dicot species. Each marker construct was expressed transiently in transgenic M. truncatula roots and the cellular location in cortical cells was assessed by confocal microscopy. All markers showed the predicted sub-cellular localization and the labeled structures had the expected appearance as reported previously (Schmit, 2002; Dixit and Cyr, 2004; Dettmer et al., 2006; Saint-Jore-Dupas et al., 2006; Nelson et al., 2007; Thomas et al., 2008; Limpens et al., 2009; Pumplin and Harrison, 2009). Negative effects of marker expression on root growth or cellular appearance were not observed. The transgenic roots obtained from Agrobacterium rhizogenes transformation arise from independent transformation events and therefore there is variation in the intensity of the fluorescent signals between independent transformants. Roots with moderately intense signals were selected for imaging and representative images are shown in Figures 1 and S2. The major membranes, organelles and components of the cytoskeleton in M. truncatula root cells have the expected appearance as reported in other plant species (Nelson et al., 2007; Mohanty et al., 2009). For the cytoskeletal markers, it was noticeable that LifeActin-mCherry (Riedl et al., 2008) gave a brighter signal than the FIM1-ABD2mCherry (Sheahan et al., 2004) possibly due to the tendency to label thicker actin filaments and cables as has been observed in Arabidopsis (Dyachok et al., 2014). The microtubule marker, mCherry-MAP4-MBD (Marc et al., 1998) revealed microtubules bundles and in addition, punctate signals, potentially the sites of microtubule organizing centers (Schmit, 2002; Dixit and Cyr, 2004), were detected around nucleus. The markers of the cis-Golgi apparatus and three markers of the trans-Golgi network (Dettmer et al., 2006; Saint-Jore-Dupas et al., 2006; Limpens et al., 2009; Pumplin et al., 2012) showed the expected punctate distribution although the intensity of fluorescence from the mCherry-MtSYP41 and AtVHA-a1-mCherry markers was weak. The late endosome/MVB marker, mCherry-MtRAB5, labeled small, punctate structures and bigger aggregates of puncta (Limpens et al., 2009) while the transitory late endosome marker, mCherry-MtRAB7, labeled small punctate structures and to a lesser extent, the tonoplast. This is consistent with previously reported localization of MtRAB5A2 and MtRAB7A2 GFP fusions in M. truncatula (Limpens et al., 2009). The plasmodesmata-located protein AtPDLP1mCherry fusion (Thomas et al., 2008) showed the expected punctate labeling of the cell wall and illustrates the density of plasmodesmal connections between the root cortical cells. As is apparent from Figures 1 and S2, many of the cellular organelles or endosomal compartments have a small punctate or small spherical, dot-like appearance and, in the absence of a marker, it is almost impossible to distinguish these compartments from each other; this further exemplifies the importance of specific markers to assign sub-cellular location. The fluorescent markers expressed from the symbiosisspecific MtBCP1 promoter were analyzed in mycorrhizal Figure 1. Markers of the nucleus (NLS-mCherry), plasma membrane (PIP2a-mCherry), actin microfilament (LifeAct-mCherry) and plastids (RUB1sp-mCherry) expressed from the AtUBQ10 promoter in transgenic roots of M. truncatula. Images shown are from the elongation zone (nuclei) and root cortex (plasma membrane, actin microfilaments and plastids). Root cortical cells contain a large central vacuole which restricts the localization of plastids to cell periphery. Scale bars, 10 lm. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 Fluorescent protein markers for cell biology 1155 roots and the arrangement of the endoplasmic reticulum (ER), Golgi bodies, tonoplast and peroxisomes (Figures S3–S6) confirmed the findings of a previous study in which constitutively expressed constructs were evaluated (Pumplin and Harrison, 2009). In addition, as outlined below, we tested several markers that had not been evaluated previously in mycorrhizal roots. DELLA proteins are not required for signaling leading to re-positioning and enlargement of the nucleus in cortical cells During AM symbiosis, the nuclei in cortical cells which are in contact with intercellular hyphae assume positions towards the site of contact with hypha (Genre et al., 2008). This re-positioning is accompanied by the nuclear enlargement (Cox and Sanders, 1974; Genre et al., 2008) which in turn has been linked to endoreduplication of nuclear content (Bainard et al., 2011). To further examine changes in the size and position of the nucleus during AM symbiosis, we generated M. truncatula roots expressing MtBCP1p::NLS-mCherry and imaged the roots after inoculation with Glomus versiforme. The MtBCP1p::NLS-mCherry construct provides an excellent means of identifying infected regions of the roots as the promoter is active only in cortical cells of colonized root areas and the intense fluorescent signals from the nuclei are readily apparent even with relatively low magnification (Figure S1a). Cells that contained arbuscules clearly showed enlarged nuclei relative to non-colonized cortical cells but in addition, cells which were undergoing fungal invasion also showed enlarged nuclei (Figure 2b, c). Quantitative analysis of nuclear diameter in 150 cells confirmed that nuclear enlargement precedes arbuscule development (Table S2). DELLA proteins are required for arbuscule development and in a della1/della 2 double mutant, fungal hyphae rarely penetrate cells and arbuscules are not formed (Floss et al., 2013). In a della1/della 2 mutant expressing MtBCP1p::NLSmCherry we detected the mCherry signal in nuclei of cortical cells which were in contact with the linear, intercel- (a) (b) lular hyphae as well as in cells adjacent to those in contact with a hypha. Furthermore, the nuclei were located at the side of the cell in contact with the hypha and they exhibited an increase in size (Figure S7a, b) as occurred in wild type colonized roots (Table S2). Re-positioning and enlargement of the nuclei are part of a larger pre-penetration response that includes cytoplasmic aggregation and the development of a pre-penetration apparatus (PPA) (Genre et al., 2005, 2008). PPA formation requires an intact symbiosis signaling pathway (Genre et al., 2009) and the cortical cells of a gain-of-function mutant of a calcium- and calmodulindependent kinase (CCaMK/DMI3) show PPA-like structures and nuclear enlargement in the absence of fungal infection (Takeda et al., 2012). Our data indicate that DELLA proteins, which have been placed downstream of CCaMK/DMI3 (Floss et al., 2013) are not required for nuclear enlargement; consequently signaling downstream of CCaMK for this response must occur through an alternate downstream branch of the symbiosis signaling pathway. Localization of endosomal Rab GTPases reflect changes in vacuole biogenesis Rab GTPases coordinate vesicle trafficking in cells and as they associate with distinct membrane compartments they also serve as useful markers (Stenmark, 2012). Rab GTPases cycle between an active, GTP-bound, membrane-localized state and an inactive GDP-bound cytosolic state (Saito and Uedat, 2009). Here we examined MtBCPp::mCherry-RAB5 and MtBCPp::mCherry-RAB7, markers of late endosome/ MVB and transient late endosome/tonoplast compartments (Limpens et al., 2009) (Table 1 and Figure S2) to determine if the membrane-bound state of either marker is altered during AM symbiosis. In cells containing arbuscules, mCherryRAB5 maintained its localization as small puncta or clusters of puncta (Figure 3) as seen in non-colonized cells (Figure S2). The puncta were generally present in the spaces between the arbuscule branches. Based on the punctate appearance, we infer that the membrane-bound, active state is maintained and that vesicle traffic to late (c) Figure 2. Expression of NLS-mCherry under the control of the AM symbiosis-specific MtBCP1 promoter in M. truncatula transgenic roots colonized by G. versiforme. (a, b) Fluorescence from NLS-mCherry marks the colonized regions of the root including cells containing arbuscules (asterisk) and adjacent non-colonized cells (dot). (b, c) The nuclei are enlarged in cells containing arbuscules (filled arrowhead) in comparison to adjacent non-colonized cells (open arrowhead). The arrow marks a cell in which an arbuscule is developing. The inset in (a) is enlarged in (b). Scale bars: 50 lm in (a, b) and 20 lm in (c). © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 1156 Sergey Ivanov and Maria J. Harrison (a) (a) (b) (b) Figure 3. Endosomal markers mCherry-Rab5 and mCherry-Rab7 expressed from the MtBCP1 promoter. (a) mCherry-Rab5 is a marker of late endosomes/MVB and marks small puncta (filled arrowhead) or larger aggregates of puncta (open arrowhead) in a cell containing an arbuscule (asterisk). (b) mCherry-Rab7 is a marker of the transitory late endosome and tonoplast. In a cell containing an arbuscule (asterisk), this marker is mainly detected in the cytoplasm (open arrow). In an adjacent non-colonized cell (dot), this marker is visible on the tonoplast (filled arrow). Scale bars, 10 lm. endosomes/MVB is likely not altered in colonized cells. However, the fluorescent signal from mCherry-RAB7 was detected mainly in the cytoplasm and only in rare cases on the tonoplast which enveloped the arbuscular branches. In contrast, adjacent non-infected cells showed mCherryRAB7 localized to the tonoplast (Figure 3). During arbuscule development, the vacuole of the invaded plant cell becomes convoluted and partially fragmented to small vacuole compartments (Figure S5); however, the mechanisms underlying this process are currently unknown (Cox and Sanders, 1974; Toth and Miller, 1984; Pumplin and Harrison, 2009). The cytoplasmic localization of RAB7 in colonized cells suggests that vesicle traffic to the tonoplast may be reduced during arbuscule development which might be part of the cellular process that results in vacuole fragmentation. The differential appearance of GFP-tagged and mCherrytagged MtBCP1 and the restricted location of MtBCPspmCherry suggest that the PAS around the arbuscule branches may not be in continuity with PAS around the arbuscule trunk The membrane protein composition of the PAM defines the existence of two broad domains of PAM: the region around the arbuscule trunk, referred to as the ‘trunk domain’ and the region around the arbuscular branches, referred to as Figure 4. MtBCP1 localizes on the PAM around the trunk and arbuscule branches. (a) MtBCP1-GFP expressed from the native MtBCP1 promoter in a cell containing an arbuscule. The GFP signal is visible on plasma membrane (open arrowhead) and trunk domain of the PAM (filled arrowhead). (b) MtBCP1-mCherry expressed from the native MtBCP1 promoter in a cell containing an arbuscule. The mCherry signal is visible on plasma membrane (open arrowhead), the trunk domain of the PAM (filled arrowhead) and also on the branch domain of the PAM (open arrow). Asterisk, arbuscule. Scale bars, 10 lm. the ‘branch domain’ (Harrison et al., 2002; Pumplin and Harrison, 2009; Pumplin et al., 2012). A GFP fusion of the secreted, GPI-anchored protein, MtBCP1, showed signal in the plasma membrane of the cell and in the trunk domain of the PAM but not in the branch domain. Consequently MtBCP1 was attributed to show domain-specific localization (Pumplin and Harrison, 2009). Here, we created fusions of MtBCP1 with mCherry and in contrast to the previous GFPtagged construct (Figure 4a), G. versiforme colonized roots expressing MtBCP1p::MtBCP1sp-mCherry-BCP1 showed a fluorescent signal on the plasma membrane, and the membrane around the arbuscule trunk and arbuscule branches of developing and mature arbuscules (Figure 4b). Hence, these data indicate that MtBCP1 localizes on the entire PAM © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 Fluorescent protein markers for cell biology 1157 and does not have domain-specific localization. This is not unexpected given the recent data and model for membrane targeting to the periarbuscular membrane, which predicts that membrane or membrane-anchored proteins expressed during arbuscule development will be localized throughout the PAM (Pumplin et al., 2012). The apparent difference in the GFP- and mCherry- MtBCP1 fusions in the branch domain of the PAM may be attributed to the properties of two fluorescent proteins. GFP and mCherry differ in their sensitivity to acidic pH with GFP fluorescence being more sensitive and more easily disrupted than mCherry fluorescence (Doherty et al., 2010). Since MtBCP1 is secreted to the apoplast but GPI-anchored to the outer leaflet of plasma membrane the fusion proteins are exposed to acidic environment of the apoplast (Grignon and Sentenac, 1991). The apoplastic space around the periphery of the cell and the arbuscule trunk apparently does not inhibit either GFP or mCherry fluorescence. However, the lack of GFP signal but presence of mCherry signal, from the apoplast around the arbuscule branches may be the result of increased acidity in the PAS (Guttenberger, 2000), probably resulting from the action of the proton ATPase MtHA1 located in the PAM (Krajinski et al., 2014; Wang et al., 2014). These data also raised the question of whether the PAS around the arbuscule branches is continuous with the PAS around the trunk and peripheral apoplast of the cortical cell. As a first step to test this we investigated the location of a secreted mCherry protein (Figure S2), when directed to different regions of the apoplast. Expression of secreted mCherry from the MtBCP1 promoter, which is active in cells before and during arbuscule development, resulted in mCherry secretion to the apoplastic space around the periphery of the cell and to the PAS around the arbuscule trunk and arbuscule branches (Figure 5a). In contrast, expression of secreted mCherry from MtPT4 promoter, directed mCherry to the PAS around the arbuscule branches (Pumplin et al., 2012) and the fluorescent signal was detected only in PAS around the arbuscule branches and not around the trunk or the apoplast around the periphery of the cell (Figure 5b). This location was observed in 32 out of 37 independent cells containing arbuscules that were examined in detail (Table S3). It has been assumed that the PAS around the arbuscule is a continuous apoplastic space. However, these results indicate that the mCherry protein does not move freely from the PAS around the arbuscule branches to the arbuscule trunk. Similar observations have been made in biotrophic fungi- and oomycete pathogen- plant interactions where the pathogens develop structures called haustoria that are considered somewhat analogous to the arbuscules. Haustoria develop within plant cells and are surrounded by a plant membrane called the extrahaustorial membrane. However, the extrahaustorial space, which is the equivalent of the PAS, is sealed by microscopically visible band (a) (b) Figure 5. Secreted mCherry in the periarbuscular space. (a) BCPsp-mCherry expressed from the native MtBCP1 promoter is secreted into the apoplast around the periphery of the cell, the periarbuscular space (PAS) around the trunk (open arrowhead) and PAS around the arbuscule branches (arrow). Filled arrowhead, arbuscule trunk. (b) BCPsp-mCherry expressed from the MtPT4 promoter is secreted to the PAS around arbuscule branches (filled arrow) and there is no visible signal in the PAS around the arbuscule trunk (filled arrowhead). Asterisk, arbuscule. Scale bars, 10 lm. of callose or other extracellular material around the neck of the haustoria. Thus, the extrahaustorial apoplastic compartment is not continuous with the apoplast around the cell (O’Connell and Panstruga, 2006; Yi and Valent, 2013). When fluorescent-labeled effectors are secreted to the extrahaustorial space, they outline haustoria but do not diffuse into the apoplast around the periphery of the cell (Khang et al., 2010; Yi and Valent, 2013) because of the neckband. Currently, there is no microscopical evidence for a neckband or physical barrier between the arbuscule branches and the arbuscule trunk but considering the complexity of the structure, it is likely that such a barrier, if it exists, would be difficult to observe. It is intriguing that mCherry secreted to the PAS around the arbuscule branches is retained in this location. As this observation parallels the observation of the fluorescent signals from secreted effectors in the haustoria, the possibility of a physical barrier in the PAS should not be discounted. Live imaging of the cytoskeleton during arbuscule formation Reorganization of the cytoskeleton of the cortical cells occurs during arbuscule development and has been studied previously in fixed tissues using indirect immunofluo- © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 1158 Sergey Ivanov and Maria J. Harrison rescence (Genre and Bonfante, 1997, 1998; Blancaflor et al., 2001). Here, expressed from the BCP1 promoter, LifeActGFP (Figure 6) and mCherry-MAP4 (Figure S6), permitted live imaging of cytoskeletal changes during symbiosis. LifeAct-GFP gave clear and distinct labeling of actin filaments and improved clarity relative to previous immunolocalization data from fixed tissue (Figure 6). At the stage of hyphal entry into a cortical cell, the nuclei, which were positioned toward the contact site with fungal hyphae, were surrounded by a network of fine actin filaments (Figure 6a and Movie S1). Additionally, in cells where the nucleus had moved back to the center of the cell, we observed a column of fine actin filaments, likely part of the PPA, linking the nucleus and periphery of the cell (Figure 6b and Movie S2). In these cells, a cortical array of actin was still present with thick actin bundles radiating from the nucleus through the cell cortex to the periphery of the cell. Cells with developing arbuscules maintained a peri-nuclear network of fine actin filaments that surrounded the growing arbuscule branches and thick actin filaments connected arbuscule branches to the peripheral cortical actin (Figure 6c and Movie S3). In cells with mature arbuscules, actin was visible as fine filaments in a network around the arbuscule branches and a few thick filaments were present in the peripheral cortical actin network (Figure 6d and Movie S4). Using mCherry-MAP4-MBD, our observations of microtubules in cells containing arbuscules are consistent with previous studies (Blancaflor et al., 2001) and revealed a tight network of microtubules around the developing arbuscule branches (Figure S8). In cells with mature arbuscules the number of thick aggregates was considerably reduced and a tight network of thin microtubules surrounded the arbuscule. Occasionally, we observed bright punctate structures in cytoplasm around the arbuscule (Figure S8) that appeared similar to those observed around the nucleus in non-infected cells (Figure S2). These structures could represent microtubule organizing centers and were not observed in previous analyses (Blancaflor et al., 2001). Thus LifeAct-GFP (mCherry) and mCherry-MAP4MBD provide useful probes for monitoring the cytoskeleton and provide greater detail than was previously attained using indirect immunofluorescence in fixed tissues. Changes in plastid morphology in cortical cells during arbuscule development To study plastid morphology during arbuscule development we generated M. truncatula roots expressing (a) (c) (b) (d) Figure 6. Reorganization of the actin cytoskeleton during the course of arbuscule development. The actin markers, LifeAct-GFP and LifeAct-mCherry expressed from the MtBCP1 promoter. (a) Peri-nuclear networks of fine actin (open arrowhead) around the nuclei (n) which are located towards the contact site with fungal hypha (filled arrowheads) during hyphal penetration of the cells. (b) Subsequently, the nucleus moves across the cell and a column of fine actin (open arrowhead) typical of a PPA is visible between the nucleus (n) and the original site of contact with hypha (filled arrowhead). (c) A network of fine actin (open arrowhead) around the nucleus (n) and arbuscule branches fungal hypha (filled arrowheads) during arbuscule (asterisk) development. Filled arrowhead, fungal hypha. (d) In cells with mature arbuscules (asterisk), fine actin filaments (open arrowhead) are present around the arbuscule branches (filled arrow). Scale bars, 10 lm. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 Fluorescent protein markers for cell biology 1159 MtBCP1p::RUB1sp-mCherry and inoculated them with G. versiforme. In non-infected cells, the plastids are lensshaped in appearance (Figures 1 and 7a), however, cortical cells that harbor intracellular hyphae, showed elongated plastids with thin projections that are referred to as strom€ hler et al., 1997; Osteryoung and Pyke, 2014). ules (Ko These stromulated plastids were located around the nucleus which was in close contact with the invading hypha (Figure 7b). Stromulated plastids increased in numbers in cells with mature arbuscules and were localized in the spaces between arbuscule braches (Figure 7c). As the arbuscules degenerated, the plastids regained their lensshaped structure (Figure 7d). This pattern was observed consistently in over 50 colonized cells (Table S4). Changes in plastid morphology during arbuscule development have been reported previously (Fester et al., 2007) and while networks of tubular plastids were reported in Nicotiana tabacum (Fester et al., 2001), the previous studies of M. truncatula reported that lens-shaped plastids were predominant during arbuscule development whereas stromulated plastids were associated with arbuscule degeneration (Lohse et al., 2006). It was proposed that the lens-shaped plastids were actively involved in biosynthesis of fatty acids and amino acids required during arbuscule development, whereas the stromulated plastids were proposed to be involved in recycling compounds released during arbuscule senescence (Lohse et al., 2006; Fester et al., 2007). However, in previous discussions of stromules in non-green plastids, it was suggested that stromules may enhance the traffic of metabolites such as amino acids and fatty acids, across the cell, particularly in cells under the stress conditions (Hanson and Sattarzadeh, 2013; Osteryoung and Pyke, 2014). In addition, stromules increase the surface area of contact with other organelles and thus could increase the exchange of metabolites (Schattat et al., 2011). Consequently, we suggest that in cells with developing arbuscules where the demand for metabolites is high, stromules may enhance metabolite flow, possibly even via direct contact of stromule membranes with the PAM. In addition, stromule formation is controlled by actin microfilaments and microtubules (Kwok and Hanson, 2003), thus reorganization of the cytoskeleton around the arbuscule branches may also promote stromule formation. Currently, it is unclear why our observations of plastid morphology in M. truncatula during arbuscule development differ from those reported previously, but it may relate to the different biological conditions and the ease with which arbuscules in different stages of development can be distinguished. In summary, we have generated a comprehensive set of fluorescent markers for cellular compartments with the option of expression from either a constitutive or an AM symbiosis-specific promoter. The use of markers expressed from the AM symbiosis-specific promoter for cell biology (a) (b) (c) (d) Figure 7. Changes in plastid morphology during arbuscule development. The plastid marker RUB-mCherry expressed from the MtBCP1 promoter. (a) In non-colonized cells plastids have lens-shaped appearance (open arrowhead). (b) During hyphal entry to the cell (filled arrow), plastids with stromules (filled arrowhead) localize around the nucleus (n). (c) In a cell with a mature arbuscule (asterisk), plastids with stromules (filled arrowhead) are abundant around the arbuscule branches. (d) In cells with collapsed arbuscules (ca), stromules are no longer present and the plastids have a lens-shaped appearance (open arrowhead) similar to non-colonized cells. Scale bars, 10 lm. studies during AM symbiosis has been demonstrated. This resource should be useful for protein localization studies and for analyses of cellular responses in legumes. In © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 1160 Sergey Ivanov and Maria J. Harrison addition, the AtUBQ10 promoter is active in several other species, consequently these marker constructs have the potential to be broadly useful in dicots. EXPERIMENTAL PROCEDURES Plasmid construction Polymerase chain reaction (PCR). mCherry was amplified from the pBIN20 AtPIP2a–mCherry plasmid (Nelson et al., 2007) using forward and reverse primers B3036 and B3037 respectively (B3036/B3037) (Table S5) to create a version of mCherry without a stop codon (ns) and primers B3026/B3027 to create a version with a stop codon (st). eGFP with (st) and without (ns) a stop codon was amplified from pB7WGF2 (Karimi et al., 2005) using the same primers as for mCherry. The sequences of all primers used in this study are shown in Table S5. The following marker genes used to generate the constructs were all amplified from plasmids developed and verified in previous studies (Marc et al., 1998; Sheahan et al., 2004; Dettmer et al., 2006; Nelson et al., 2007; Thomas et al., 2008; Pumplin and Harrison, 2009). A DNA fragment encoding a marker of ER AtWAK4mCherry-HDEL (Nelson et al., 2007) was amplified using primers B2853/B2854. A marker of the Golgi apparatus GmMAN49-mCherry (Nelson et al., 2007) was amplified using primer B2852/B2851. DNA fragments containing the full length coding sequences of the plasma membrane marker AtPIP2a and the tonoplast marker AtTIP1-1 (Nelson et al., 2007) without stop codons were amplified using primers B2857/B2858 and B2859/B2860, respectively. A peroxisome marker, mCherry-SKL (Nelson et al., 2007) was amplified using primers B3036/B3239. An actin microfilament marker, AtFim1-ABD2-ns, (Sheahan et al., 2004) was amplified using primers B3020/B3021 and a microtubule marker, MAP4-MBD, (Marc et al., 1998) was amplified using B3022/B3023. DNA fragments containing the full length coding sequence of plasmodesmata marker without stop codon, AtPDLP1-ns (Thomas et al., 2008) and a transGolgi network marker, AtVHA-a1-ns (Dettmer et al., 2006) without a stop codon, were amplified using B3394/B3395 and B2782/ B2783, respectively. A marker of the plasma membrane and trunk domain of the PAM, MtBCP1sp-GFP-BCP1, (Pumplin and Harrison, 2009) was amplified using primers B3008/B3009. The following marker genes were amplified by PCR using M. truncatula cDNA as a template: MtSYP61 – B3064/B3065, MtSYP41 – B3062/B3063, MtRab5A2 – B4022/B4023, MtRab7A2 – B4024/ B4025 and MtLC3 (ATG8, Medtr2 g104190) – B4020/B4021. All primer sequences included the corresponding attB recombination sites (Table S5). Fusion sequence encoding markers of the plastid (MtRubisco1sp-mCherry), nuclei (NLS-mCherry-GUS), mitochondria (ScCOX4sp-mCherry), actin (LifeActin-mCherry), apoplast (MtBCP1sp-mCherry) and plasma membrane/trunk of PAM (MtBCP1sp-mCherry-BCP1) were created by overlapping PCR. To create MtRubisco1sp-mCherry, the MtRubisco1 signal peptide was amplified from M. truncatula cDNA using primers B2848/B2849. Primer B2849 contained a sequence complementary to 50 -end of mCherry. mCherry was amplified using primers B2850/B2851. Primer B2850 contained a sequence complementary to 30 -end of MtRubisco1sp. The PCR products were purified and used as templates in overlapping PCR to amplify MtRubisco1sp-mCherry using primers B2848/2851 containing recombination attB sites. NLS-mCherry-GUS was generated through overlapping PCR with three fragments. A fragment containing an NLS sequence was amplified using primers B3189/B3190 with B3190 containing sequence complementary to 50 -end of mCherry. mCherry was amplified using primer B3191 which contains a sequence complementary to the 30 -end of NLS and primer B3192 which contains a sequence complementary to the 50 -end of GUS. GUS was amplified using primer B3193 with a sequence complementary to 30 -end of mCherry and primer B3194. The three PCR fragments were purified and used as templates in overlapping PCR with primers B3189/B3194 containing recombination attB sites. To create ScCOX4sp-mCherry the sequence of ScCOX4sp was amplified using primers B2980/B2981 and the product was used as a template in PCR using primer B2977/B2978 with B2978 which contains a sequence complementary to the 50 -end of mCherry. mCherry was amplified using primers B2979/B2851 with B2979 which contains a sequence complementary to the 30 -end of ScCOX4sp. The PCR products were purified and used as templates in overlapping PCR to amplify ScCOX4sp-mCherry using primers B2848/2851 containing recombination attB sites. To create LifeActin-mCherry, LifeActin was amplified using primers B3778/B3777 with B3777 corresponding to the sequence of LifeActin (Riedl et al., 2008) and B3779 containing a sequence complementary to the 50 -end of mCherry. mCherry was amplified using primers B3779/B2851 with B3779 containing a sequence complementary to 30 -end of LifeActin. The PCR products were purified and used as templates in overlapping PCR to amplify LifeActin-mCherry using primers B3778/B2851 containing recombination attB sites. To create MtBCP1sp-mCherry the sequence of MtBCP1sp was amplified from M. truncatula cDNA using primers B3008/B3011 with B3011 containing a sequence complementary to the 50 -end of mCherry. mCherry was amplified using primers B3010/B2851 with B3010 containing a sequence complementary to the 30 -end of MtBCP1sp. The PCR products were purified and used as templates in overlapping PCR to amplify MtBCP1sp-mCherry using primers B3008/B2851 containing recombination attB sites. To create MtBCP1sp-mCherry-BCP1 the sequence of MtBCP1sp-mCherry was amplified using primers B3008/B3013 with B3013 containing a sequence complementary to the 50 -end of BCP1. BCP1 was amplified using primers B3012/B3009 with B3012 a sequence complementary to the 30 -end of mCherry. The PCR products were purified and used as templates in overlapping PCR to amplify MtBCP1sp-mCherry-BCP1 using primers B3008/ B3009 containing recombination attB sites. To amplify MtBCP1sp-GFP-BCP1 we use primers B3008/B3009 containing recombination attB sites and plasmid from previous study (Pumplin and Harrison, 2009) as a template. The AtUBQ10 promoter and terminator were amplified from pNIGEL07 (Geldner et al., 2009). The CaMV35S terminator was amplified from pK7m34GW (Karimi et al., 2005). The MtBCP1 promoter was amplified from M. truncatula genomic DNA (Hohnjec et al., 2005). All amplified fragments were sequence verified. Creation of pENTR clones. PCR fragments of AtWAK4mCherry-HDEL, GmMAN49-mCherry, AtPIP2a-ns, AtTIP1-1-ns, mCherry-SKL, AtFimbrin1-ABD, AtPDLP1-ns, AtVHA-a1-ns, NLS-mCherry-GUS, MtBCPsp-mCherry, ScCOXsp-mCherry, MtRubisco1sp-mCherry, LifeActin-mCherry (GFP), MtBCP1sp-mCherryBCP1 and MtBCP1sp-GFP-BCP1 with flanking attB1 and attB2 sites were recombined with pDONR221 by BP reaction (Invitrogen, http://www.lifetechnologies.com/us/en/home/brands/invitrogen.html) to obtain pENTR2 L1-L2 clones of the corresponding markers (Table S1). PCR fragments of MAP4-MBD, MtSYP61, MtSYP41, MtRab5A2, MtRab7A2, MtLC3, AtUBQ10 terminator and CaMV35S terminator with flanking attB2 and attB3 sites were recombined © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 1151–1163 Fluorescent protein markers for cell biology 1161 with pDONR-P2RP3 to obtain pENTR3 R2-L3 clones (Table S1). PCR fragments of AtUBQ10 and MtBCP1 promoters with flanking attB4 and attB1 sites were recombined with pDONR P4P1R to obtain pENTR1 L4-R1 clones (Table S1). Creation of expression clones. pKm43GW, pK7m34GW, pBm34GW and pB7m34GW destination vectors (Karimi et al., 2005) were used in three fragment recombination by LR reaction (Invitrogen) (Table S1). The final expression vectors are named pCM (CellularMarkers). pCMU- indicates that it contains the AtUBQ10 promoter and pCMB- indicates that it contains the MtBCP1 promoter (Table S1). Plant material, transformation, growth conditions M. truncatula truncatula accession Jemalong A17 was used for all experiments. To obtain composite plants with transgenic roots expressing the cell markers, A. rhizogenes strain ARqual mediated hairy-root transformation was used (Boisson-Dernier et al., 2001). Composite plants were planted into 20.5 cm plastic cones filled with a sterile mixture of play sand/filter sand/gravel in ratio 2:2:1 with 300 sterile spores of G. versiforme placed at a depth of 4 cm below the surface. Plants were grown in a growth chamber under a 16 h light/25°C and 8 h dark/22°C regime and fertilized with halfstrength Hoagland’s solution containing full-strength nitrogen and 20 lM potassium phosphate twice a week. Plants were harvested 3 weeks after planting. Confocal microscopy Transgenic roots showing constitutive fluorescence from the AtUBQ10 promoter or fluorescence associated with fungal colonization from the MtBCP1 promoter were excised to short pieces approximately 3–5 mm in length. The root pieces were then cut longitudinally with a double-edged razor blade and placed on a glass slide with a drop of water with cut surface facing upwards and covered by a cover slip as described previously (Pumplin and Harrison, 2009). Roots sections were observed and fluorescence was imaged using Leica TCS-SP5 confocal microscope (Leica Microsystems, http://www.leica-microsystems.com/) with a 209 or 639 water-immersion objectives. GFP was excited with the argon ion laser (488 nm) and emitted fluorescence was collected from 505 to 545 nm; mCherry was excited with the Diode-Pumped Solid State laser at 561 nm and emitted fluorescence was collected from 605 to 630 nm. Differential interference contrast (DIC) images were collected simultaneously with the fluorescence. All fluorescent markers were assessed in a minimum of six independent transgenic root systems. Images were processed using Leica LAS-AF software versions 2.6.0 (Leica Microsystems), Image J (National Institutes of Health, http://imagej.nih.gov/ij/) and Adobe Photoshop CS5 version 12.0.1 (Adobe Systems Inc., http://www.adobe.com/). ACKNOWLEDGEMENTS Financial support was provided by the National Science Foundation Plant Genome Program, Grant IOS-1127155. Microscopes in the Boyce Thompson Insitute (BTI) Plant Cell Imaging Center used in this study were purchased with a National Science Foundation Instrumentation Grant, NSF DBI-0618969. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. The activities of the constitutive AtUBQ10 promoter and AM symbiosis-specific MtBCP1 promoter in M. truncatula mycorrhizal roots. . Figure S2. Localization of cellular markers in transgenic roots of M. truncatula. Figure S3. Localization of endoplasmic reticulum (mCherry-HDEL) and Golgi apparatus (MAN49-mCherry) markers in arbuscule-containing cells expressed from the MtBCP1 promoter . Figure S4. Localization of the plasma membrane (PIP2a-mCherry) marker in M. truncatula root cortical cells containing arbuscules. Figure S5. Localization of the tonoplast (TIP1-1-mCherry) marker in in root cells containing arbuscules. Figure S6. Localization of mitochondria (COX-mCherry) and peroxisomes (mCherry-SKL) markers in cells containing arbuscules. Figure S7. Nuclear enlargement in the della1 della2 mutant. Figure S8. Localization of a microtubule (mCherry-MAP4-MBD) marker in cells containing arbuscules. mCherry-MAP4-MBD was expressed from the MtBCP1 promoter. Table S1. List of plasmids used to create the fluorescent protein marker constructs for cellular compartments Table S2. 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