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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Curr Biol. Author manuscript; available in PMC 2012 March 8.
Published in final edited form as:
PMCID: PMC3072602
NIHMSID: NIHMS267779

Signaling in the Arabidopsis shoot meristem stem cell niche correlates with ligand-dependent trafficking of the CLV1 receptor kinase

Summary

Cell numbers in above-ground meristem types of plants are thought to be maintained by a feedback loop driven by perception of the glycopeptide ligand CLAVATA3 (CLV3) by the CLAVATA1 (CLV1) receptor kinase and the CLV2/CORYNE (CRN) receptor-like complex [1]. CLV3 made in the stem cells at the meristem apex limits the expression level of the stem cell-promoting homeodomain protein WUSCHEL (WUS) in the cells beneath, where CLV1 as well as WUS RNA are localized. WUS downregulation nonautonomously reduces stem cell proliferation. High-level overexpression of CLV3 eliminates the stem cells and causes meristem termination [2], and loss of CLV3 function allows meristem overproliferation [3]. There are many open questions regarding the CLV3/CLV1 interaction, including where in the meristem it occurs, how it is regulated, and how it is that a large range of CLV3 concentrations gives no meristem size phenotype [4]. Here we use genetics and live imaging to examine the cell biology of CLV1 in Arabidopsis meristematic tissue. We demonstrate that plasma membrane-localized CLV1 is reduced in concentration by CLV3, which causes trafficking of CLV1 to lytic vacuoles. We find that changes in CLV2 activity have no detectable effects on CLV1 levels. We also find that CLV3 appears to diffuse broadly in meristems, contrary to a recent sequestration model which states that CLV3 is quantitatively bound by CLV1 in the apical regions of the meristem, allowing continued WUS activity in lower regions [5]. This study provides a new model for CLV1 function in plant stem cell maintenance and suggests that downregulation of plasma membrane-localized CLV1 by its CLV3 ligand can account for the buffering of CLV3 signaling in the maintenance of stem cell pools in plants.

Introduction

Post-embryonic growth in higher plants is driven by populations of meristematic cells that are continuously maintained over the life of the plants. In plants like Arabidopsis, above-ground tissues are derived from the shoot apical meristem (SAM), which begins as a vegetative meristem making leaves, then becomes an inflorescence meristem (IM) that bears determinate floral meristems (FM), from which the reproductive organs are derived. These diverse meristematic forms share a common tissue organization composed of a stem cell population called a central zone at the apical meristem tip, above a set of cells sometimes called the organizing center or rib meristem, and flanked by a peripheral zone in which organs form. This identity organization is overlaid upon a tissue pattern consisting of clonal monolayers of L1 and L2 cells overlying the corpus cell population. Mutations in any of the CLV/CRN loci result in an overproliferation of stem cells in shoot meristems. Mutations in the homeodomain protein WUSCHEL result in a premature termination of the stem cell population [6]. CLV3 encodes a small extracellular protein which is processed into a 13 amino acid peptide and modified with β-1,2-linked tri-arabinoside chains into its active in vivo form (CLV3p, [79]). It is thought that the mature CLV3p diffuses from its expression domain in the upper meristem layers into deeper tissue layers where CLV1 is expressed [10]. CLV1 is a transmembrane receptor kinase with extracellular leucine-rich repeats that can bind CLV3p in vitro [10, 11]. Binding is thought to activate CLV1 kinase activity which in turn leads to a downregulation of WUS expression in the rib meristem. WUS can activate CLV3 expression in a cell non-autonomous manner, thereby providing a mechanistic feedback loop which maintains stem cell balance [2, 12]. In addition, a parallel CLV3p sensing pathways involving the receptor-like protein CLV2 and the receptor-like kinase homologue CRN also regulates stem cell proliferation via WUS [13, 14]. CLV2/CRN is expressed broadly and this complex is proposed to act as a receptor for a diverse set of CLV3-related ligands, termed CLE proteins, throughout the plant [1]. In addition, the receptor kinase RPK2/TOAD2 also acts to limit WUS expression in the meristem[15]. Because it is thought that CLV1 and WUS could be expressed in overlapping cell populations in the meristem, it has been proposed that CLV1 may act to prevent the diffusion of CLV3 into the entire WUS domain by sequestering CLV3p [5]. Although high levels of ectopic CLV3 can downregulate WUS, alteration of CLV3 levels over a wide range are buffered by unknown mechanisms in the meristem [4]. In addition, long-term expression of high levels of CLV3 can lead to reactivation of WUS [4], suggesting that strict controls upon the perception of CLV3 exist in plants. What these control mechanisms are and how they relate to the function of the various receptors is unknown.

Our knowledge of how these loci interact to control stem cell function is derived from genetic, biochemical and transient expression imaging studies. However the literature contains several contradictions. For example, past biochemical studies suggested that CLV2 is required for stabilization of CLV1, with a 90% reduction in CLV1 levels seen in clv2-1 null plants [16]. This result does not appear to be compatible with the genetic observation that clv2 clv1 and crn clv1 double mutants are additive [14, 15, 17]. Attempts to image CLV1 and associated proteins in living meristems have until now been unsuccessful.

Here we use a combination of genetic analysis and live confocal imaging of CLV1-reporters to explore the cell biology of CLV1 in living meristematic tissue. We observe that CLV1 accumulates at the plasma membrane (PM) in clv3 plants and is preferentially targeted to the lytic vacuole by CLV3. These results indicate that CLV3 promotes ligand-dependent trafficking of the CLV1 receptor kinase, analogous to the ligand dependent downregulation of receptors in animal systems and at least one plant receptor like kinase (RLK) [18]. Endosomal trafficking and lysosomal degradation of receptors in animal and yeast cells provides a mechanism for downregulation of activated receptors [19]. In some cases receptors signal from endosomal compartments providing strict temporal and spatial regulation of pathway activation [20]. Trafficking of CLV1 likely requires meristem-specific cofactors. We demonstrate that CLV2 is neither required for CLV3-dependent trafficking of CLV1 nor is it required for stabilization of CLV1 in clv3 plants. These results call into question the validity of some biochemical studies, but are consistent with genetic analyses of the loci. Using trafficking of CLV1 as a marker for CLV3 perception we are able to ascertain that CLV3 appears to diffuse broadly in the meristem and that sequestration of CLV3 by CLV1 is therefore unlikely to play a major role in meristem maintenance. These data provide a new model for CLV1/CLV3 interactions and suggest that downregulation of CLV1 by CLV3 could provide the CLV3 buffering function observed in previous work.

Results

CLV1 accumulates at the plasma membrane in clv3 mutants

In order to study the cell biology of CLV1-CLV3 interactions we first created a CLV1 promoter construct to express CLV1-2xGFP for live imaging (Supplemental Experimental Procedures, Supplemental Figure 1). This construct fully complemented the clv1-11 and clv1-101 mutants in all lines examined (n>30) (Supplemental Figure 1 A and B). This fusion protein localizes to the PM in tobacco transient expression experiments (data not shown). In contrast to the results in tobacco we rarely observed PM localization of CLV1-2xmGFP in the IMs in T1 transgenic plants. Instead we observed in most cases a faint GFP signal in the vacuole of L3 cells. In other cases, we observed faint apparently cytosolic background signal (compare Figure 1C and Figure 2A and C, Supplemental Figure 2 for imaging controls). In animal systems, receptors are internalized and degraded in response to ligand binding and we postulated that we might be observing a similar process in the case of CLV1. We introgressed a single insert line of pCLV1::CLV1-2xGFP into the clv1-11 clv3-2 background, which is RNA null for both CLV1 and CLV3 [21, 22]. We compared young lateral IMs as these were the closest in size and shape between the two genotypes. We observed the same weak apparent vacuolar accumulation and lack of PM GFP signal in CLV3 progeny in rib meristem cells as in a wild-type genetic background (Figure 1A and 1C). In clv3-2 progeny, however, we observed GFP signal at the presumed PM in rib meristem cells and a corresponding lack of vacuolar accumulation (Figure 1B and 1D). In both genotypes (clv1-11 clv3-2 and clv1-11 CLV3) we observed punctate structures in the cytosol which are likely Golgi apparatus (see Supplemental Experimental Procedures). We confirmed these results by introgressing a separate clv1-11 pCLV1::CLV1-2xGFP line into the clv1-11 clv3-2 background and repeating the earlier observations (Supplemental Figure 3). This line complements but expresses at a much lower level. In this line we never observed PM accumulation in the IMs in CLV3 plants. These data indicate that in the absence of CLV3, CLV1 accumulates at the PM, while it is found in the vacuole in the presence of CLV3 protein (see Supplemental Material). This suggests that CLV3 drives CLV1 endocytosis from the PM followed by subsequent targeting to the vacuole. Both CLV1 and CLV3 are also expressed in young FMs arising from the flanks of the main IM. In stage 3 FMs, CLV1-2xGFP localization was largely similar to that of the main IM (Supplemental Figures 4 and 6). In general, levels of CLV1-2xGFP appeared higher in FMs compared to IMs when expressed from pCLV1, although this may be due to partial shadowing of the IM by peripheral FMs. On rare occasions (3/20 experiments) we observed weak CLV1-GFP signal at the PM in FMs of CLV3 plants, however CLV1-GFP PM signal was always higher in clv3 mutants in those same experiments. In a separate survey of 21 individual CLV1:CLV1-2xYpet CLV3 transgenic lines we did not detect any PM localization in FMs, further confirming that PM accumulation of CLV1 is strongly reduced in the CLV3 background.

Figure 1
CLV1 accumulates at the plasma membrane in clv3-2 plants
Figure 2
CLV1 traffics to the vacuole in a CLV3-dependent manner

We then expressed the CLV1-2xGFP fusion at high level in all cells of the IM. We generated a pUBQ::CLV1-2xGFP construct and transformed clv1-11, clv3-2 clv1-11, and clv1-101 plants. Constitutive expression of CLV1 complemented the clv1 null phenotype without any other observed effects (Supplemental Figure 1F). We introgressed three individual pUBQ::CLV1-2xGFP lines, two clv1-11 lines and one clv1-101 line, into clv3-2 clv1-11 plants and analyzed progeny in a similar manner to the above pCLV1 experiments. Consistent with the imaging of the pCLV1::CLV1-2xGFP lines, we observed a strong vacuolar accumulation in CLV3 plants and a contrasting strong PM accumulation in clv3-2 plants (Figure 2). The vacuolar signal derived from the pUBQ10 lines was considerably stronger than that seen in most, but not all, pCLV1 imaging attempts. Similar results to those with pCLV1::CLV1-2xGFP were obtained from the introgression of two other independent pUBQ::CLV1-GFP lines, one in the clv1-101 background (Figure 2A and 2B) and another in clv1-11, into the clv3-2 clv1-11 plants (Figure 2C and 2D, detail shown). In very high level expressing IMs, we occasionally noticed weak PM accumulation of CLV1 in the CLV3 background from pUBQ and pCLV1 lines (Figure 2C, data not shown), suggesting that CLV1 may saturate downstream trafficking components at very high levels. Variation at ERECTA did not affect CLV1-2xGFP localization in either clv3 or CLV3 backgrounds (Figure 2, data not shown). These results confirm that CLV3 promotes vacuolar localization of CLV1 at the expense of PM accumulation.

CLV1 traffics to the lytic vacuole in VTI11-dependent manner

We confirmed the localization of CLV1 in CLV3 and clv3 plants using known markers for the PM and lytic vacuole respectively (Suppl. data and methods). To further test the hypothesis that CLV3 alters CLV1 trafficking, we took a genetic approach and crossed the pCLV1::CLV1-2xGFP transgene into a zig-1 mutant background. ZIG/VTI11 encodes a Q-SNARE protein which localizes to the Trans-Golgi Network/Pre-Vacuolar Compartment (TGN/PVC) and is required for transport of cargo to the lytic vacuole [2326]. In zig-1 CLV3 plants the CLV1-2xGFP vacuolar signal was greatly reduced compared to ZIG CLV3 plants in most experiments, indicating that CLV1 is trafficked to the lytic vacuole in a ZIG/VTI11-dependent manner in CLV3 plants (Figure 3A and B). We consistently observed a stronger PM accumulation of CLV1-2xGFP signal in zig-1 CLV3 plants that is rarely seen in ZIG CLV3 plants. We noted that upon prolonged FM4-64 staining in CLV3 plants, internal CLV1-2xGFP was bounded by FM4-64 stained tonoplast (Figure 3C). We also observed colocalization of CLV1-2xGFP signal with two known lytic vacuole markes, LysoTracker Red and the vacuolar RFP marker (VAC-RFP, Figure 3D and E, see Supplemental Experimental Procedures), confirming the vacuolar targeting of CLV1-2xGFP in CLV3 plants. We confirmed the localization of CLV1 in clv3 plants via colocalization with PM targeted mRUBY and FM4-64 staining in cold treated plants (Figure 4, Supplemental Figure 4, Supplemental Experimental Procedures).

Figure 3
CLV1 traffics to the lytic vacuole in a VTI11 dependent manner
Figure 4
CLV1 localizes to the plasma membrane in clv3 plants

Ectopic CLV1 does not sequester CLV3

CLV3 is expressed in the L1, L2 and L3 layers in wild type plants. From there it is believed that the CLV3 pro-peptide is secreted to the extracellular space where it is processed and modified with L-arabinose into its active 13 amino acid glycopeptide form, CLV3p [8]. It is not known where CLV3p diffuses and is active in the shoot apical meristem. Previous attempts to define the sites to which CLV3 diffuses in the IM used a CLV3-GFP fusion [5, 7]. Later studies demonstrated that CLV3p is proteolytically cleaved from its precursor at both its amino-terminal and carboxyl-terminal ends, indicating that the CLV3-GFP in these earlier experiments likely reported only the extracellular movement of the processed GFP and not the active CLV3p. Using UBQ::CLV1-2xGFP lines and vacuolar trafficking of CLV1-2xGFP as a proxy for the CLV3p response of CLV1, we determined that CLV3p diffuses broadly throughout the IM from its site of synthesis (Figure 3 A and B, Figure 5). We observed CLV3-dependent vacuolar accumulation of CLV1-2xGFP throughout the L1 layer, in the peripheral zone and central zone, and also in deeper regions encompassing the rib meristem. We also consistently observed CLV3-dependent vacuole targeting in young flower primordia, suggesting that CLV3 may either diffuse across the boundary regions or be synthesized in low levels in emerging primordia. It has been proposed that CLV1 protects lower regions of the IM from CLV3 by sequestering CLV3 and preventing its diffusion [2]. CLV1 is normally expressed at higher levels in the rib zone of IMs, and its RNA is largely absent from the L1 and L2 layers. We tested this sequestration hypothesis using high level ectopic expression of CLV1, including in the L1, L2 and L3 layers in the pUBQ lines. We found that CLV1 overexpression in L1 and L2 cells did not prevent perception of CLV3p by CLV1 in the lower central zone and in the rib meristem as judged by the vacuolar targeting of CLV1-2xGFP in L3 and L4 cells (Figure 5). In addition, lateral epidermal L1 cells on the flanks of the IM still showed response to CLV3. These cells would be expected to be even further from the site of CLV3 than L3 cells. This observation suggests that functional sequestration of CLV3p by CLV1 doesn't occur to a detectable degree in the L3 layer of wild type meristems. These results are also consistent with the observation that meristem function is unperturbed over a wide range of CLV3 levels [4] and the lack of wus-like phenotypes seen in any combination of clv1 heterozygotes [21, 27].

Figure 5
High levels of CLV1 in L1 and L2 fail to prevent CLV3-dependent trafficking of CLV1 in L3 cells

CLV3 is sufficient to promote CLV1 vacuolar trafficking

We sought to assess whether it was sufficient to induce CLV1-2xGFP trafficking in shoot apical meristems. We generated DEX::CLV3 clv3-2 plants in which constitutive CLV3 expression is inducibly controlled by the application of dexamethasone (DEX) in the clv3-2 background (Supplemental Materials and Methods). In control clv3-2 pUBQ::CLV1-2xGFP plants and untreated clv3-2 pUBQ::CLV1-2xGFP DEX::CLV3 plants we observed the expected PM accumulation of CLV1-2xGFP in L1 cells in young IMs (Figure 6 top row and bottom row). In contrast, clv3-2 pUBQ::CLV1-GFP DEX::CLV3 plants treated with DEX for several days displayed a range of CLV1-2xGFP localizations. While some IMs displayed near normal levels of CLV1-2xGFP at the PM in L1 cells (data not shown), others displayed a strong vacuolar accumulation similar to that seen in the CLV3 pUBQ::CLV1-2xGFP plant lines (Figure 6, upper middle row). Others yet displayed greatly reduced CLV1-2xGFP signal at the PM (Figure 6, lower middle row), yet still expressed CLV1-2xGFP strongly in the epidermis of mature sepals (data not shown). Neither of the latter observations were ever seen in control treated clv3-2 pUBQ::CLV1-GFP DEX::CLV3 plants. These results indicate that CLV3 is sufficient to induce both PM depletion and drive trafficking of CLV1 to the lytic vacuole. Vacuolar accumulation of the CLV1-GFP signal was first weakly detected at 4 hours post DEX application in two independent experiments, suggesting that this response was relatively rapid. These data demonstrate that CLV3 is both necessary and sufficient to drive depletion of CLV1 from the PM in apical meristems, followed by trafficking to the lytic vacuole in a post-transcriptional manner.

Figure 6
CLV3 is sufficient to drive CLV1 downregulation at the plasma membrane and trafficking to the lytic vacuole

We never observed strong vacuolar targeting of CLV1-2xGFP in DEX-treated tissues outside of meristems in pUBQ::CLV1-GFP DEX::CLV3 plants, consistent with the robust PM accumulation of CLV1-2xGFP in the mature sepal epidermis of CLV3 pUBQ::CLV1-GFP plants (data not shown, Supplemental Experimental Procedures). This observation suggests that CLV1 requires meristem-specific cofactors in order to traffic to the vacuole in response to CLV3. Also consistent with this, we observed no changes in CLV1-2xGFP vacuolar targeting in response to either CLV3p or DEX::CLV3 in transient tobacco leaf transformation assays (data not shown).

CLV2 is not required for CLV1 stability or trafficking

Previously, it has been published that CLV2 is required for CLV1 stability, with a reduction in CLV1 levels by 90% in CLV3 clv2 plants relative to wild type [16]. As described in the introduction, several lines of evidence challenge these data and conclusions. We therefore sought to address what role, if any, CLV2 might play in CLV1 levels or cellular location. We crossed both pCLV1::CLV1-2xGFP and pUBQ::CLV1-2xGFP plant lines into a clv1-11 clv2-1 clv3-2 triple mutant, which is protein-null for CLV2 as a result of a stop codon at amino acid position 33 in the CLV2 signal sequence [16] and selected the appropriate F2 genotypes. We observed a similar pattern of CLV1-2xGFP signal in pUBQ lines, with reduced PM accumulation in the pUBQ lines and significant vacuolar targeting in CLV3 CLV2 and CLV3 clv2-1 plants (Figure 7A). These data demonstrate that loss of CLV2 does not impair CLV3-dependent lytic vacuole targeting. Similarily, CLV1 levels and PM targeting appeared unchanged the clv3-2 CLV2 and clv3-2 clv2-1 backgrounds (Figure 7B). . Similar results were also seen in both IMs and stage 3 FMs from pCLV1 lines (Supplemental Figures 5 and 6). We also did not detect a reduction in CLV1-2xGFP levels in the clv2-1 mutant by Western Blot analysis (Supplemental figure 7B). These data indicate that CLV2 is not required for the stability or accumulation of GFP-labeled CLV1.

Figure 7
CLV2 is not required for CLV1 trafficking or stability

Discussion

The experiments reported here suggest a new model for CLV1 function. In the absence of CLV3, CLV1 accumulates at the PM. In this model CLV3p is secreted from L1/L2 and some L3 cells and diffuses into to the CLV1 expression domain. Ligand binding triggers activation of the CLV1 kinase, which in turn causes recruitment of accessory proteins, resulting in CLV1 internalization from the PM followed by VTI11/ZIG-dependent trafficking to the lytic vacuole, where CLV1 is degraded. It seems unlikely that quantitative sequestration of CLV3 by CLV1 occurs, as is currently believed, because CLV3 has an effect on the subcellular localization of CLV1 through much of the IM, even when CLV1 is overexpressed. Although WUS, CLV3 and CLV1 have never been colocalized simultaneously in the same plant, recent in situ hybridizations suggest that CLV3 expression partially overlaps with that of WUS in meristems [28].

We did not observe robust changes in CLV1 protein levels between CLV3 and clv3-2 plants. Similar results have been seen for FLS2 and S-Receptor kinase (S. Robatzek, personal communication, and [29]). It could be that any changes in CLV1 levels are masked by CLV1 levels outside of these cells, for example in vascular tissues, where CLV1 is present. Alternatively the amount of PM CLV1 in clv3 plants might be roughly equivalent to the sum of CLV1 in vesicles in CLV3 plants. Clearly some CLV1 must always be present at the PM in wild-type plants in order to perceive CLV3. Consistent with this in highly expressing IMs and FMs we do observe weak accumulation of CLV1 at the PM in CLV3 plants suggesting that the efficacy of CLV1 trafficking is sensitive to CLV1 levels.

The results reported here demonstrate that CLV1 accumulates at the PM in both shoot and floral meristems, and traffics to the lytic vacuole in a ZIG/VTI11-dependent manner. This appears to differ from the RLK STRUBBELIG, which accumulates at the PM in a domain overlapping with that of CLV1 in IMs [30]. Interestingly, the stem cell limiting RLK RPK2 appears to be post translationally downregulated in IMs similar to CLV1 [15], suggesting it may also undergo ligand dependent trafficking. While trafficking to the vacuole is reduced in CLV3 zig-1 mutants, we also observe a consistent accumulation of CLV1-2xGFP at the PM in CLV3 zig-1 IMs. This may reflect an increase in CLV1 recycling to the PM when vacuolar targeting is reduced. This would be consistent with the known localization of VTI11, its role in lytic vacuole trafficking and the recent observation that the zig-1 phenotype is largely dependent on retromer-mediated resorting [2326, 31]. CLV1 has been shown to interact in the yeast two-hybrid system with a sorting nexin, a critical component of the retromer recycling complex [3234]. In addition, it has been shown that blocking lysosomally targeted cargo in animal systems can also induce sorting into the recycling pathway [35]. It remains to be determined if CLV1 is an actual cargo for retromer or VTI11-dependent trafficking

We would expect that CLV1 undergoes endocytosis from the PM prior to trafficking to the vacuole. Despite several attempts we were unable to visualize any intermediate locations between the PM and vacuole accumulation. It is possible that any endosomes mediating this traffic are transient and smaller in diameter than Golgi vesicles. It should be noted that we were also unable to see any VHAa1-RFP signal, which labels the early endosome/TGN, in the IM of Arabidopsis (data not shown) nor were we able to resolve Rab C1 YFP-labeled compartments beyond a background glow (wave 3, wave collection, [36]), despite robust imaging of these compartments in root tissues (data not shown).

It has been demonstrated that, while transient CLV3 signaling downregulates WUS, sustained CLV3 often results in reactivation of WUS [4]. In addition, CLV3 levels can be manipulated over a wide range and still result in apparently wild type plants [4]. Thus the perception and response to CLV3 is buffered within the growing meristem. Cytokinin regulation of WUS or RLKs which counter act CLV1 may provide buffering activity [37, 38]. Ligand-induced trafficking and degradation of CLV1 could also provide a mechanism for this buffering.

Experimental Procedures

Plant Material

clv1-11 [21], clv2-1 [16], and clv3-2 [22] were described previously and are all in the Landsberg-erecta genetic background. clv1-101 is a fully recessive T-DNA insertion mutant in the Columbia background and was obtained from the from the ABRC (WiscDsLox489-492B1 [15]). The VAC-RFP lines corresponds to the spL-RFP lines described in [39]. See Supplemental Material and Methods for additional methods.

Construction of vectors

Details of vector construction are provided in Supplemental Experimental Proceedures.

Confocal microscopy

Microscopy was performed on freshly detached lateral inflorescences as previously described as in [40]. Young IMs were used in which developing flowers had not yet opened. Tissue from this remains viable for several hours [40] but were imaged immediately following detachment. The tissue was imaged using a 63X dipping lens. GFP and chlorophyll were stimulated using a 488 nm laser at 18.9% activation. A 505–550 bandpass filter for GFP and 585 longpass filter was used for chlorophyll detection. The 505–550 filter was used to maximize GFP signal collection. Additional details, including dye staining are provided in Supplemental Experimental Procedures.

Protein Extraction and Analysis

10 lateral inflorescence meristems were removed and unopened flowers were dissected off. Fresh tissue was ground in extraction buffer (10 mM Hepes pH 7.4, 0.2 mM EDTA, 2mM MgCl2, 0.1 M NaCl, 10% Glycerol, 1% Triton X-100, 1 mM PMSF) containing PhosphoStop and Protease Complete tablets (Roche). Samples were incubated with gentle rotation at 4 °C overnight. Samples were spun 10 minutes, supernatant was quantified using BioRad protein quantification kit and the remaining sample was diluted into 6× Laemmli Buffer. Proteins were separated on a 8% SDS Page gel, blotted and probed with anti-GFP (Roche).

Supplementary Material

01

Acknowledgements

We thank Dr. Philippa Barrell for kindly providing the MOA binary series of vectors. We thank Dr. Natasha Raikhel for providing the zig-1 seed. We thank Dr. Lorenzo Frigerio for providing the VAC-RFP seed line. We thank the ABRC for providing seed stocks. We thank Arnavaz Garda for her excellent technical assistance. We also thank members of the Meyerowitz lab for help with imaging techniques and manuscript comments. This work was funded by NIH National Research Service Award F32 GM080843 to ZLN, NIH National Research Service Award F32 GM075460 to XQ, NIH National Research Service Award F32 GM090534 to PT, and NIH grant 1R01 GM086639 to EMM.

Footnotes

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