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. 2017 Jul 19;175(1):172–185. doi: 10.1104/pp.17.00523

MILDEW RESISTANCE LOCUS O Function in Pollen Tube Reception Is Linked to Its Oligomerization and Subcellular Distribution1,[OPEN]

Daniel S Jones a, Jing Yuan a,b, Benjamin E Smith a,c, Andrew C Willoughby a, Emily L Kumimoto a, Sharon A Kessler a,b,d,2
PMCID: PMC5580752  PMID: 28724621

MLOs can substitute for NORTIA function in pollen tube reception if they localize to a Golgi compartment prior to pollen tube arrival and retain the ability to homooligomerize.

Abstract

Sexual reproduction in flowering plants requires communication between synergid cells and a tip-elongating pollen tube (PT) for the successful delivery of sperm cells to the embryo sac. The reception of the PT relies on signaling within the synergid cell that ultimately leads to the degeneration of the receptive synergid and PT rupture, releasing the sperm cells for double fertilization. In Arabidopsis (Arabidopsis thaliana), NORTIA, a member of the MILDEW RESISTANCE LOCUS O (MLO) family of proteins, plays a critical role in the communication processes regulating PT reception. In this study, we determined that MLO function in PT reception is dependent on MLO protein localization into a Golgi-associated compartment before PT arrival, indicating that PT-triggered regulation of the synergid secretory system is important for synergid function during pollination. Additionally, a structure-function analysis revealed that MLO homooligomerization, mediated by the amino-terminal region of the protein, and carboxyl-terminal tail identity both contribute to MLO activity during PT reception.

Intercellular communication is central to the development and maintenance of all multicellular organisms. In plants, communication between cells controls processes ranging from organ development and tissue patterning (Chaiwanon et al., 2016; Otero et al., 2016) to defense responses (Yi and Valent, 2013; Stahl and Faulkner, 2016) and reproduction (Higashiyama and Takeuchi, 2015; Bircheneder and Dresselhaus, 2016). The importance of cell-cell communication is evident during reproduction in flowering plants, as the attraction and reception of the male gametophyte (pollen tube [PT]) by the synergid cells of the female gametophyte (also known as the embryo sac) are essential for reproductive success (Beale and Johnson, 2013; Higashiyama and Takeuchi, 2015). The synergid cells have two primary functions in reproduction: PT attraction and intercellular communication during PT reception (Russell, 1992; Higashiyama et al., 1998). Synergid cells are responsible for emitting LURE peptides that are perceived by the approaching PTs to guide them to the embryo sac (Okuda et al., 2009). PT reception then initiates as the PT makes contact with the synergid cells and pauses its growth to communicate with the receptive synergid at the filiform apparatus, a membrane-rich region at the micropylar end of synergid cells (Denninger et al., 2014; Hamamura et al., 2014; Ngo et al., 2014). Signal transduction pathways in the synergid and the PT lead to PT rupture, releasing the sperm cells for double fertilization, and to the degeneration of the receptive synergid.

PT reception is a complex process with key genes expressed in both the synergid cell and the PT facilitating this communication (Kessler and Grossniklaus, 2011; Beale and Johnson, 2013). The synergid FERONIA (FER)/LORELEI receptor complex plays a role early in PT reception (Escobar-Restrepo et al., 2007; Li et al., 2015; Liu et al., 2016), while NORTIA (NTA; also known as AtMLO7), a member of the MILDEW RESISTANCE LOCUS O (MLO) family of proteins, acts downstream of the receptor complex to facilitate PT reception by an unknown mechanism. In nta homozygous mutants, PT-synergid communication is disrupted. Instead of bursting to release the sperm cells, PTs continue to grow within the embryo sac, a phenotype known as PT overgrowth (Kessler et al., 2010). Recently, it was found that calcium oscillations in the synergid cells begin upon the arrival of the PT at the filiform apparatus (Denninger et al., 2014; Hamamura et al., 2014; Ngo et al., 2014). The PT reception components FER and LORELEI are both necessary for the signaling process that initiates these calcium oscillations. In contrast, nta-1 mutant ovules retain these oscillations but at lower amplitudes, suggesting that NTA could play a part in modulating these calcium signals but not in initiating them (Ngo et al., 2014). The incomplete expressivity of the nta mutant phenotype, in which some ovules carrying the mutation are fertilized normally, suggests that a threshold level of calcium (or some other signaling molecule) in synergids is necessary for PT reception.

MLOs are seven-transmembrane proteins first identified as susceptibility factors for the fungal pathogen powdery mildew (Büschges et al., 1997). The MLO family was first described in barley (Hordeum vulgare), where mutations in the mlo locus conferred resistance to powdery mildew (Büschges et al., 1997). MLOs have a conserved calmodulin-binding domain (CaMBD) in their C-terminal cytosolic tail that actively binds calmodulin in a Ca2+-dependent manner both in vitro and in vivo (Kim et al., 2002; Bhat et al., 2005). There are 15 MLO proteins in Arabidopsis (Arabidopsis thaliana), with representatives from five of the seven MLO clades in the plant kingdom (Devoto et al., 2003; Kusch et al., 2016). MLO proteins have been shown to function as a part of dynamic signaling pathways in response to various stimuli. Both NTA and barley MLO (HvMLO) actively redistribute within their respective cell types to the site of invasion/penetration (the filiform apparatus in synergids or powdery mildew penetration sites; Bhat et al., 2005; Kessler et al., 2010). However, the functional significance of such redistribution remains to be elucidated. Similarities between MLO function in pathogen susceptibility and PT reception make determining the role that MLO proteins play in either system invaluable to understanding widely conserved intercellular communication pathways within plant cells.

Taking advantage of the diversity within the Arabidopsis MLO family (Supplemental Fig. S1), we determined that the localization of AtMLO proteins within a Golgi-associated compartment in the synergid cell before PT arrival correlates with but does not completely explain MLO function in PT reception. This suggests that many coincidental characteristics influence MLO function within this pathway. A structure-function analysis via domain-swap experiments revealed that the N-terminal region of NTA is sufficient to confer NTA activity on MLO8 and may play a role in homooligomerization. Additionally, we found that the identity of NTA’s C-terminal tail is important for MLO-mediated PT reception and controls the specificity of function even between closely related MLO proteins.

RESULTS

Expression of MLO Proteins in the Synergids of nta-1 Reveals Differences in Their Ability to Mediate Pollen Tube Reception

Previous work with AtMLO (MLO) proteins involved in powdery mildew infection showed that MLO2, MLO6, and MLO12, all members of the same monophyletic clade, were involved in powdery mildew susceptibility (Consonni et al., 2006). By analogy to the powdery mildew clade, we wondered if MLO10 and MLO8, both part of the same clade as NTA (Fig. 1A, clade III), could have redundant functions within PT reception. We first asked whether these closely related MLOs are expressed in synergid cells as NTA is. Native promoter genomic GUS (Fig. 1, C–E) and GFP (Fig. 1, H–K) fusions of MLO10 and MLO8 were analyzed and compared with NTA to determine their expression patterns in unfertilized ovules. NTA was limited to synergids in both GUS-expressing (Fig. 1C) and GFP-expressing (Fig. 1, H and I) lines, similar to previous reports (Kessler et al., 2010). MLO10 (Fig. 1D) and MLO8 (Fig. 1E) were expressed in ovules, but only MLO8 was expressed near the synergids. Additional analysis with MLOpro::MLO-GFP constructs revealed no GFP accumulation within the synergid cells of either MLO10 (Fig. 1J) or MLO8 (Fig. 1K) expression lines. However, MLO8-GFP was detected just outside of the synergids, in inner integument cells closely associated with the filiform apparatus (Fig. 1K; Supplemental Fig. S2).

Figure 1.

Figure 1.

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Native MLO expression patterns in Arabidopsis ovules. A, Maximum-likelihood phylogenetic tree from an amino acid alignment of the 15 MLO proteins in Arabidopsis generated using MEGA6 (Tamura et al., 2013). Roman numerals refer to previously defined clades within the MLO family (Devoto et al., 2003; Kusch et al., 2016). B, Diagram of an Arabidopsis ovule at PT arrival. An, Antipodals; CC, central cell; EC, egg cell; FA, filiform apparatus; InI, inner integument; OI, outer integument; PT, pollen tube; Syn, synergid. C to G, GUS-stained mature ovules of native promoter genomic MLO fusion proteins. H to M, Mature ovules of native promoter-driven MLO-GFP protein fusion lines labeled with the membrane-localized dye FM464 to reveal the full outline of the synergid cell (I). Bars = 20 μm (C–G) and 10 μm (H–M).

In order to better understand how NTA functions in PT reception, we utilized the sequence diversity found within the MLO family by testing divergent MLOs for their ability to function in MLO-mediated PT reception (Fig. 2A). In addition to NTA, MLO10, and MLO8, we also chose MLO2 (from clade V) and MLO1 (from clade II), as even more distant proteins within the MLO phylogeny with respect to NTA. All MLO proteins tested in our experiment have the same predicted topology, with seven membrane-spanning domains and a calmodulin-binding domain in the C-terminal cytoplasmic tail (Supplemental Fig. S1). MLO2 and MLO1 are both expressed in ovules but not in synergid cells (Fig. 1, F, G, L, and M). All five of these MLOs were then ectopically expressed in the synergid cells of nta-1 using MYB98pro, a synergid-selective promoter (Kasahara et al., 2005), in order to assay their ability to rescue the nta-1 PT reception phenotype. These constructs also incorporated a C-terminal GFP fusion onto each MLO (MYB98pro::MLO-GFP) in order to confirm expression and determine distribution/localization within the synergid cell.

Figure 2.

Figure 2.

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Analysis of MLO proteins expressed in synergids of nta-1. A, Box plot of unfertilized ovule counts in T3 plants homozygous for the indicated MYB98pro::MLO-GFP constructs in nta-1 mutants. Each box plot represents the full range of each data set with median at the center. WS, Wassilewskija. Adjusted P values are as follows: ****, P < 0.0001; *, P = 0.05 to 0.01; and ns, P > 0.05. B to F, MYB98pro::MLO-GFP (green) distribution in synergid cells of unfertilized mature ovules merged with FM464 labeling (magenta). Bars = 10 μm.

In nta-1 mutants, ovules remain unfertilized due to PT overgrowth during PT reception (Kessler et al., 2010); thus, the percentage of unfertilized ovules can be used as a measure for the ability of an introduced construct to complement the nta-1 phenotype. Unfertilized ovule counts revealed that NTA-GFP expressed under the control of MYB98pro could fully rescue nta-1 in comparison with NTA-GFP expressed under the control of its native promoter (Supplemental Fig. S3), indicating that MYB98pro drives expression in the synergid cells at sufficient levels for NTA activity. Both MLO10-GFP and MLO2-GFP significantly rescued nta-1 when expressed in synergids, while MLO8-GFP and MLO1-GFP did not significantly reduce the percentage of unfertilized ovules compared with nta-1 (Fig. 2A). MLO10 has the highest sequence similarity and is the most closely related to NTA according to phylogenetic analysis (Fig. 1A). Interestingly, MLO8 cannot rescue nta-1, although it is in the same subclade as NTA, while MLO2, a clade V MLO, does complement nta-1. This suggests that MLO-mediated PT reception requires specific properties of an MLO and that not all MLO proteins can function in the pathway.

MLOs Differ in their Distribution within Synergids and in Their Localization in the Endomembrane System

We hypothesized that an MLO’s ability to complement the nta-1 phenotype when expressed in synergids may be dependent on whether it is localized within the appropriate subcellular location. To assay MLO distribution in the synergid cell prior to PT arrival, unfertilized ovules from lines transformed with each MLO-GFP construct were imaged using confocal laser scanning microscopy. As reported previously (Kessler et al., 2010), NTA accumulated within the endomembrane system of the synergid cell and was distributed throughout the entire cell but was excluded from the filiform apparatus prior to PT arrival (Fig. 2B). MLO10 and MLO2 distribution was similar to that of NTA; however, some GFP signal also was detected in the filiform apparatus (Fig. 2, C and E). MLO8 was distributed predominantly toward the filiform apparatus, with additional GFP signal detected throughout the rest of the cell (Fig. 2D), while MLO1 was localized primarily to the filiform apparatus (Fig. 2F). Quantification of MLO-GFP distribution within the synergid cell confirmed that MLO10 and NTA are significantly less distributed toward the filiform apparatus than either MLO8 or MLO1 (Supplemental Fig. S4). Interestingly, MLO2 varied in its distribution, with some synergids having GFP distributed toward the filiform apparatus and others being more cytoplasmic (Supplemental Fig. S4, I–K). Overall, the presence of the ectopically expressed MLOs in the filiform apparatus of synergids in unfertilized ovules made it difficult to assess whether active redistribution in response to PT arrival was conserved among the proteins analyzed. However, these results demonstrate that the presence of an MLO protein at the filiform apparatus during PT reception is not sufficient to rescue nta-1 (MLO8 and MLO1) and that either an MLO’s localization within the specific compartments where NTA localizes or the active process of redistribution to the filiform apparatus may be key to MLO function in PT reception.

While MLO distribution within the synergid cell varies, the ability of MLO10, MLO8, and MLO2 to localize within cytoplasmic compartments similar to those seen in NTA-GFP lines may represent an important characteristic for MLO function in this pathway (Fig. 2, B–E). Therefore, determining the identity of the NTA-localized compartment may be important for understanding MLO function. To this end, the subcellular localization of NTA was defined using a stable coexpression assay in synergids with markers for specific organelles. Previously reported markers for Golgi, endoplasmic reticulum, and peroxisomes (Liu et al., 2016) were coexpressed with MYB98pro::NTA-GFP in synergid cells (Fig. 3). NTA colocalized predominantly with the Golgi marker, with additional GFP signal detected outside the range of the mCherry channel (Fig. 3A). This suggests that NTA is localized within Golgi bodies and also may be associated with post-Golgi vesicular trafficking. We then performed a colocalization analysis using the same Golgi marker coexpressed with the MLOs used in this study to determine if the others accumulate in this compartment similar to NTA (Fig. 4). MLO10 (Fig. 4A) partially colocalized with the Golgi marker at similar levels to NTA. MLO2 (Fig. 4C) colocalized with the Golgi marker to a lesser extent than either MLO10 or NTA, while MLO8 (Fig. 4B) and MLO1 (Fig. 4D) showed no overlap with the Golgi marker in synergid cells.

Figure 3.

Figure 3.

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NTA colocalization with secretory markers in synergids. Colocalization of MYB98pro::NTA-GFP (green) is shown with the Golgi (A; Man49-mCherry), endoplasmic reticulum (ER; B; SP-mCherry-HDEL), and peroxisome (PX; C; mCherry-PTS1) markers (magenta) expressed under the control of the synergid selective promoter of LRE (Liu et al., 2016) in the synergid cell. Bars = 10 μm.

Figure 4.

Figure 4.

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MLO colocalization with a Golgi marker in synergids. MYB98pro::MLO-GFP (green) was coexpressed with the Golgi marker (LREpro::Man49-mCherry; magenta) in the synergid cell. Bars = 10 μm.

Since the differences detected in subcellular localization between closely related MLOs correlated well with their ability to rescue nta-1 (neither MLO8 nor MLO1 colocalized with the Golgi marker), we wanted to further elucidate the identity of the MLO8-localized compartment and compare it with NTA. To this end, MYB98pro::SYP61-mCherry, a trans-Golgi network (TGN)-localized marker (Sanderfoot et al., 2001; Viotti et al., 2010), was coexpressed in synergids with NTA-GFP, MLO10-GFP, and MLO8-GFP in order to determine if these closely related MLOs colocalized differentially within TGN-associated compartments (Fig. 5). NTA had no overlap (Fig. 5A), while MLO10 and MLO8 both partially colocalized with the TGN marker in synergids (Fig. 5, B and C). This further demonstrates a distinct difference between NTA and MLO8, revealing that localization within the proper compartment could be functionally significant. Interestingly, the partial colocalization of MLO10 (which fully rescues nta-1) with both Golgi and TGN markers suggests that the presence of an MLO within endomembrane compartments outside the range of NTA does not have a negative impact on function.

Figure 5.

Figure 5.

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NTA, MLO10, and MLO8 colocalization with a TGN marker in synergids. MYB98pro::MLO-GFP (green) was coexpressed with the TGN marker (MYB98pro::SYP61-mCherry; magenta) in the synergid cell. Bars = 10 μm.

These distribution/localization experiments revealed that MLO proteins are trafficked differentially within synergid cells. Multispanning membrane proteins can be initially directed into the membrane through a variety of different mechanisms, typically encoded as signals within the protein (Goder and Spiess, 2001). In an effort to predict the presence of signal peptides in MLO proteins, the sequences of all 15 MLOs were examined using the SignalP 4.0 prediction server (Petersen et al., 2011). Interestingly, we found that, out of the 15 Arabidopsis MLOs, only NTA, MLO10, and MLO8 had significantly predicted cleavable N-terminal signal peptides (Supplemental Fig. S5). This suggests that this subclade (NTA, MLO10, and MLO8) of the clade III MLOs may be type I multispanning membrane proteins (Goder and Spiess, 2001), further differentiating them from the other 12 Arabidopsis MLOs. To determine if NTA’s predicted signal peptide influences its localization and function within PT reception, we cloned NTA without this signal peptide (NORTIA Δsp) and expressed it in the synergid cells of nta-1 under the control of MYB98pro (Supplemental Fig. S6). Removing NTA’s signal peptide appeared to have no effect on its function or distribution in the synergid cell prior to PT arrival (Supplemental Fig. S6, A–D). We then generated 35Spro::NTA-GFP and 35Spro::NORTIA Δsp-GFP constructs and performed a transient coexpression assay in Nicotiana benthamiana epidermal cells with a previously published Golgi-mCherry marker (Nelson et al., 2007). Similar to its localization in synergids, NTA partially colocalized with the Golgi marker (Supplemental Fig. S6, H–J), while NORTIA Δsp-GFP accumulated predominantly in subdomains of the plasma membrane with no overlap with the Golgi marker (Supplemental Fig. S6, E–G). The presence of a signal peptide could help explain the MLO localization/distribution patterns observed within synergid cells; however, this is not enough to account for function in PT reception or for localization within Golgi-associated compartments, as MLO8 does not rescue the nta-1 PT reception phenotype and localization within Golgi was not detected (Figs. 2A and 4B). Additionally, MLO2, which has no predicted signal peptide, and NORTIA Δsp are able to rescue nta-1’s PT reception phenotype and are distributed similarly to full-length NTA in the synergid cell. This suggests that synergids may have cell type-specific mechanisms that determine the localization of this class of membrane proteins.

The N- and C-Terminal Domains of NTA Are Important for Function

MLO8 is closely related to NTA, sharing 61% identity and 73% similarity at the amino acid level. The differential subcellular localization patterns and inability to complement the nta-1 PT reception phenotype make MLO8 a good tool for dissecting regions of the NTA protein that are important for its function in PT reception. In order to identify functionally significant regions in the NTA protein, we performed a targeted structure-function analysis of NTA via domain-swap experiments with MLO8. Regions swapped were chosen based both on sequence dissimilarity between the two MLOs and on domains found previously to be important for MLO function (Elliott et al., 2005; Chen et al., 2009). Chimeric MLOs were expressed in the nta-1 mutant background under the control of MYB98pro, and ovule counts from T2 homozygous transgenic lines were used as a proxy to determine functionality in PT reception. Protein-GFP fusion distribution was analyzed for each line and revealed significant accumulation of all chimeric proteins with varying localization patterns in comparison with the full-length MLOs (Supplemental Figs. S7–S9). Chimeric proteins that were able to complement nta-1 were not confined primarily to the Golgi-associated compartment, as seen with NTA; in addition to Golgi localization, proteins accumulated within the synergid vacuole. Comparable variations in localization have been reported previously in a similar structure-function analysis of MLO family proteins (Chen et al., 2009), indicating that chimeric fusions of MLOs may partially disrupt intramolecular interactions within the chimeric proteins and subsequent regulation of their trafficking within the cell.

An N-terminal swap, including the N terminus through the first extracellular loop (NEL1; Fig. 6B), demonstrated this region to be functionally significant for MLO-mediated PT reception. NTA[NEL1]-MLO8 could partially rescue nta-1 in comparison with full-length NTA, while MLO8[NEL1]-NTA was not significantly different from full-length MLO8 in its ability to complement the nta-1 phenotype (Fig. 6B). This domain was further subdivided into swaps of only intracellular loop 1 (IL1) and extracellular loop 1 (EL1). Interestingly, constructs with either MLO’s EL1 (NTA-MLO8[EL1]-NTA and MLO8-NTA[EL1]-MLO8) were capable of rescuing nta-1 (Fig. 6C), while swaps of IL1 conferred no significant changes from the full-length proteins’ ability to rescue (Supplemental Fig. S9A), indicating that NTA’s EL1 is sufficient to confer NTA activity on MLO8, but the MLO8 EL1 does not compromise NTA’s function unless it is combined with the MLO8 IL1 and N terminus. Additionally, swaps of intracellular loop 2 (IL2), a prominent domain in both size and variability, had no significant impact on either MLO’s function (Supplemental Fig. S9F). In a previous study, it was shown that the NEL1 domain of MLO4 could confer partial function to MLO2 in rescuing the root-curling phenotype of mlo4-1; however, the reciprocal swap placing MLO2’s NEL1 domain onto MLO4 remained functional (Chen et al., 2009). Here, we have identified the NEL1 domain of NTA as necessary for its role in PT reception and sufficient in conferring function to MLO8.

Figure 6.

Figure 6.

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Analysis of domain swaps between MLO8 and NTA. A, Diagram of an MLO protein with the described domains and regions annotated. CaMBD, Calmodulin-binding domain; CTerm, C-terminal intracellular tail; EL, extracellular loops 1 to 3; IL, intracellular loops 1 to 3; N, N-terminal domain; SP, signal peptide; TM, transmembrane domain. B to E, Box plots of unfertilized ovule counts of T2 homozygous lines of MYB98pro-expressed domain-swap constructs in the nta-1 background. B, N terminus through the first extracellular loop [NEL1] domain swap between NTA and MLO8. C, EL1 domain swap between NTA and MLO8. D, C-terminal swap including IL3, EL3, and the C-terminal tail (TM5) between NTA and MLO8. E, C-terminal domain swaps between NTA and MLO8. WS, Wassilewskija. Adjusted P values are as follows: ****, P < 0.0001; ***, P = 0.001 to 0.0001; *, P = 0.05 to 0.01; and ns, = P > 0.05.

The C-terminal tails of MLO proteins have been described as intrinsically disordered, a characteristic that may contribute to specificity in function between closely related MLOs (Kusch et al., 2016). In comparison with NTA, MLO8 has a longer C-terminal tail following the conserved calmodulin-binding domain (CaMBD; Supplemental Fig. S1). To test if their C-terminal intracellular domains provide functional specificity, NTA/MLO8 C-terminal domain swaps were analyzed in nta-1. NTA’s C terminus on MLO8 (MLO8-NTA[CTerm]) rescued nta-1 at similar levels as full-length NTA, whereas MLO8’s C terminus on NTA (NTA-MLO8[CTerm]) rescued at an intermediate level and was significantly different from NTA (Fig. 6E). When this domain swap was expanded, incorporating both the third intracellular loop (IL3) and the third extracellular loop (EL3), similar results were observed in which NTA[TM5]-MLO8 did not fully rescue and MLO8[TM5]-NTA rescued nta-1 compared with full-length NTA (Fig. 6D), demonstrating that there is no change in the effect on function with these additional domains. This suggests that the identity of NTA’s C-terminal tail domain is crucial for its function and that this domain is necessary and sufficient for MLO-mediated PT reception.

In our analysis of full-length MLO proteins expressed in synergid cells, distribution within the synergid cell and colocalization with a Golgi marker correlated well with their ability to function in PT reception. Based upon these observations, we hypothesized that the functional NTA/MLO8 domain swaps were distributed primarily throughout the cell and not toward the filiform apparatus and that they would localize within the Golgi in a similar manner to NTA, MLO10, or MLO2. To test this, we quantified MLO-GFP distribution for the NEL1 and C-terminal domain swaps within the synergid cell and also performed a colocalization analysis with the same Golgi marker expressed in synergid cells (Fig. 7; Supplemental Fig. S8). Interestingly, the domain swaps that failed to fully function in PT reception (MLO8[NEL1]-NTA and NTA-MLO8[CTerm]) were more similar to NTA in their distribution than their functional reciprocal swaps (Supplemental Fig. S8). Additionally, partial colocalization with the Golgi marker was detected with all four swaps analyzed (Fig. 7), and we could not identify MLO8-like exclusion of any of the chimeric protein-GFP fusions from the Golgi-associated compartments. Even MLO chimeric proteins with disrupted function (NTA[NEL1]-MLO8 and NTA-MLO8[CTerm]) partially colocalized with the Golgi marker in synergids. This suggests that an MLO’s ability to function in PT reception is not dependent solely on localization within a specific part of the endomembrane system (although at least some accumulation appears to be required, as MLO1 does not rescue nta-1) but requires some additional characteristic of the protein influenced by their N- and C-terminal domains.

Figure 7.

Figure 7.

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Colocalization of NEL1 and C-terminal swaps with a Golgi marker in synergids. MYB98pro::MLO-GFP (green) was coexpressed with the Golgi marker (LREpro::Man49-mCherry; magenta) in the synergid cell. Bars = 10 μm.

The N-Terminal Domain of NTA Influences Efficient Homooligomerization

The domain-swap constructs that were compromised in their ability to complement nta-1 did not show MLO8-like exclusion from the Golgi compartment, indicating that other molecular properties may have been disrupted in these chimeric proteins. MLO proteins have been shown previously to homooligomerize, and conserved Cys residues residing within EL1 were proposed to play a role in this interaction (Elliott et al., 2005). Since the replacement of the NTA NEL1 domain with MLO8’s NEL1 fully disrupted its ability to function, we hypothesized that homooligomerization was important for NTA’s function and that the NEL1 domain swap inhibited this interaction. Using a Förster resonance energy transfer (FRET)-based assay in N. benthamiana epidermal cells, we tested whether NTA and MLO8 were able to homooligomerize or heterooligomerize in planta and whether the NEL1 chimeric proteins generated could successfully recapitulate this characteristic. FRET was determined using a correlation analysis comparing the fluorescence emission of the donor (mCerulean3 [Cer3]; Markwardt et al., 2011) with the emission of the acceptor (YFP; Nagai et al., 2002) detected after four subsequent rounds of acceptor photobleaching. The fluorescence emission from both channels was compared using a Pearson product-moment correlation analysis in which the more negative the correlation coefficient, the stronger the FRET signal, as this would indicate a proportional increase in donor fluorescence and corresponding decreases in acceptor fluorescence (Supplemental Fig. S10; see “Materials and Methods”). Since NTA localized within Golgi compartments in N. benthamiana epidermal cells (Supplemental Fig. S6, H–J), we generated a positive control for the detection of FRET via acceptor photobleaching in NTA-localized compartments, in which Cer3 and YFP were fused in tandem onto NTA’s C-terminal tail (NTA-Cer3-YFP). This ideal standard had a median donor-acceptor correlation coefficient of −0.9864, confirming that FRET could be detected reproducibly within NTA-associated Golgi and post-Golgi vesicles (Fig. 8). NTA-Cer3 was coexpressed with GUS-YFP and analyzed in each experiment as a negative control (with a median coefficient of 0.5217). FRET was detected at similar levels in test pairs that included NTA-Cer3 + NTA-YFP and NTA[NEL1]-MLO8-Cer3 + NTA[NEL1]-MLO8-YFP (with median coefficients of −0.93405 and −0.8906, respectively). FRET was detected at significantly lower levels from the first group in the test pairs MLO8-Cer3 + MLO8-YFP, NTA-Cer3 + MLO8-YFP, and MLO8[NEL1]-NTA-Cer3 + MLO8[NEL1]-NTA-YFP (with median coefficients of −0.6815, −0.5859, and −0.4126, respectively; Fig. 8). While all pairs examined were significantly different from the negative control, this experiment indicates that MLO8[NEL1]-NTA, which does not rescue nta-1, homooligomerizes less efficiently than full-length NTA under our experimental conditions. Reciprocally, no difference was detected between the homooligomerization efficiencies of NTA or NTA[NEL1]-MLO8, which both rescue nta-1. Taken together, these data suggest that the homooligomerization of NTA could be an intrinsic part of its function in PT reception and that disruption of NTA’s NEL1 domain may reduce the efficiency of this interaction, thus compromising its function.

Figure 8.

Figure 8.

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Detection of the oligomerization of MLO constructs via FRET analysis. Box plots show the correlation coefficients from FRET analysis of homooligomerization and heterooligomerization of the constructs indicated. Groupings of a, b, c, and d are based on significant differences between each data set’s distribution as determined using a Kruskal-Wallis test. See also Supplemental Figure S9. Data sets in all box plots represent the full range of data with median at the center.

DISCUSSION

PT reception requires a chain of signaling events in both the arriving PT and within the receptive synergid cell (Kessler and Grossniklaus, 2011). In this study, we have further defined the role of NTA, an MLO protein involved on the female side of this signaling process. The analysis of MLOs closely related to NTA revealed a divergence in expression patterns within reproductive tissues and in their ability to function within PT reception. Previous studies of Arabidopsis MLOs have identified functional redundancy and cofunctional relationships within discrete monophyletic clades of the MLO family (Consonni et al., 2006; Chen et al., 2009). Our data indicate that, of the MLOs presented in this study, NTA is the only MLO that is expressed specifically within synergid cells and likely functions independently from other closely related MLOs. The ectopic expression of full-length MLO proteins in the synergid cells of nta-1 revealed that specific characteristics of an MLO are required for function in PT reception. The closely related MLO8 (clade III) does not complement nta-1, while the more distantly related MLO2 (clade V) could, further demonstrating this divergence in function.

Our data showed that the ability of other MLO proteins to complement the nta-1 PT reception phenotype was closely correlated with the ability of the synergid-expressed MLO to accumulate in a Golgi-associated compartment before PT arrival. However, analysis of chimeric MLO proteins during a structure-function analysis revealed that localization/retention within this Golgi-associated compartment is not the sole determinant of MLO function in PT reception. Accumulation within the synergid endomembrane system prior to PT arrival is conserved between those MLOs (and chimeric constructs) capable of functioning in this pathway. Previous work showed that NTA redistributes to the filiform apparatus during PT reception, indicating that precise control of the synergid secretory system and polarized transport may be necessary for MLO function in this pathway (Kessler et al., 2010). Instances of enhanced trafficking of vesicles and induction of specialized secretory mechanisms in response to pathogens have been widely reported in plants (Kwon et al., 2008; Nielsen et al., 2012; Ding et al., 2014), including responses specific to powdery mildew infection (An et al., 2006a, 2006b). Polar redistribution events observed in NTA-mediated PT reception and in MLO-mediated fungal penetration suggest a conservation of signal-induced MLO transport. Additionally, the SNARE proteins ROR2 (barley) and PEN1 (Arabidopsis) are required for mlo pathogen resistance, providing evidence that MLO-mediated responses require activation of the secretory pathway, with reported focal accumulation of ROR2 occurring at the site of infection (Bhat et al., 2005; Hückelhoven and Panstruga, 2011). NTA seems to follow a similar paradigm, with NTA’s redistribution dependent on signaling via FER, a member of the CrRLK1L-type family of receptor-like kinases and a key player in the early stages of PT reception (Escobar-Restrepo et al., 2007; Kessler et al., 2010; Ngo et al., 2014).

Secretory pathway regulation also has been shown to be important for controlling intercellular signaling during compatibility responses in Brassica spp. (Samuel et al., 2009; Safavian and Goring, 2013). In early pollen recognition events, an induction of secretory activity in stigmatic papillae was observed during compatible pollinations, while an active inhibition of trafficking was noted in incompatible pollinations (Safavian and Goring, 2013). This increase in secretory activity occurs in a polarized manner toward the pollen grain upon successful signal transduction within the receptive papilla cell and is associated directly with the hydrating pollen grain. The parallels between the secretory regulation of polarization during pollen-stigma and PT-synergid interactions suggest that precise control of the secretory pathway in response to extracellular signals may be an important characteristic of multiple levels of pollen-pistil interactions during pollination. It will be interesting to determine if a similar inhibition of trafficking activity occurs during instances of PT overgrowth, as described during incompatible pollinations, and if MLO-mediated processes are involved in both.

Our experiments expressing full-length MLO proteins in synergid cells show that one important determinant of MLO functionality in PT reception is the ability to localize within an endomembrane-associated compartment prior to PT arrival and, thus, presumably before FER-mediated signaling occurs. While distribution and localization within the synergid cell varied between MLOs, the accumulation of MLO proteins involved in PT reception within endomembrane compartments of the synergid cell could be required for multiple reasons. One hypothesis is that proper timing of polar redistribution to the filiform apparatus is critical for PT reception and that the retention of MLOs within either the Golgi or the TGN prevents this redistribution until proper signaling is achieved as a PT communicates with the receptive synergid. This suggests that the presence of an MLO (or an MLO-associated compartment) at the filiform apparatus prior to PT arrival is not ideal and may even be inhibitory to the synergid cell by functioning too early. However, the data presented here do not support this hypothesis, since MLO2 and several chimeric MLO fusions rescued nta-1 even though instances of MLO-GFP distribution toward the filiform apparatus prior to PT arrival were observed. A second possibility is that NTA retention facilitates its colocalization and/or interaction with some unknown cofactor required for MLO-mediated PT reception. This cofactor could be involved in forming a complex with NTA and/or other components of the PT reception machinery or could be a secreted signaling molecule that needs to be escorted to the filiform apparatus for communication with the arriving PT. Further dissection of the required components involved in MLO-mediated processes will help elucidate the exact mechanism of MLO function.

The differences observed in MLO distribution within the synergid cell and localization between multiple cell types (NTA versus NORTIA Δsp localization in the synergid compared with N. benthamiana epidermal cells) may represent cell type-specific processes regulating the incorporation and retention of MLOs within the required endomembrane compartments of the synergid cell; these data support the second hypothesis proposed above. Mechanisms regulating membrane protein polarization in plant cells have been well described, especially related to the asymmetric distribution of Arabidopsis PIN-FORMED-type auxin transport proteins (Luschnig and Vert, 2014). However, it is unclear just how conserved such polarization mechanisms are within the synergid cell. Synergids are highly polarized cells that have two purposes in plant reproduction. Before PT arrival, the synergid cells secrete small LURE peptides in order to attract the PT to the micropyle of the ovule (Higashiyama, 2002; Okuda et al., 2009). The second purpose of synergids is to recognize when a PT has arrived and communicate with it, facilitating the release of sperm cells so that double fertilization can occur. These distinct purposes necessitate a change in synergid cell biology from an attractant state, with a high level of protein secretion, to a signaling state, where calcium oscillations and FER-mediated signal transduction cascades prepare the synergid for PT reception (Kessler and Grossniklaus, 2011; Hamamura et al., 2014; Ngo et al., 2014; Higashiyama and Takeuchi, 2015). Interestingly, no PT reception mutants have been reported as having defects in PT attraction, providing evidence for distinct processes regulating both pathways (Huck et al., 2003; Escobar-Restrepo et al., 2007; Kessler et al., 2010; Tsukamoto et al., 2010; Lindner et al., 2015). This suggests that a change in secretory mechanisms occurs as the PT arrives and FER-mediated PT reception initiates. Overall, more work must be done to determine the conservation of mechanisms regulating membrane polarization and protein secretion that govern these distinct roles of the synergid cell.

Homooligomerization and heterooligomerization have been demonstrated to be functionally important in various multispanning membrane protein families, where oligomerization is critical for processes such as signal transduction (Maurel et al., 2008), pore or channel formation (Musil and Goodenough, 1993; Véry and Sentenac, 2002), as well as subcellular localization within the endomembrane system (Weisz et al., 1993). While the homooligomerization of MLO proteins was established previously (Elliott et al., 2005), the functional significance of this interaction and the domains contributing to dimerization were not described. Here, we have shown the homooligomerization of NTA in planta with a FRET-based interaction assay and demonstrated a reduction of this dimerization upon disruption of an N-terminal domain (NEL1). Further confirmation of the NEL1 domain’s importance in the effective dimerization of NTA was established in the reciprocal detection of efficient homooligomerization of the NTA[NEL1]-MLO8 chimeric protein, which fully complements the nta-1 unfertilized ovule phenotype. It should be noted that, since this experiment was based on a transient assay in N. benthamania epidermal cells and not in the synergid cells of Arabidopsis, where NTA functions in PT reception, care must be taken in the interpretation of these interaction data. However, the similar localization of NTA-GFP within the Golgi of both cell types demonstrates some conservation of its native characteristics in our assay. Furthermore, the data presented in this study suggest a reduction in the efficiency of interactions between those MLOs incapable of functioning in PT reception (MLO8 and MLO8[NEL1]-NTA), and, as such, more validation is required to fully elucidate the role of homooligomerization in MLO-mediated PT reception.

Calcium is an important signaling molecule in plants, with reported roles in systemic responses, cell-cell communication, and intracellular signaling (Dodd et al., 2010; Gilroy et al., 2014). Oscillation of calcium in the synergid cell upon PT arrival is dependent on FER-mediated signaling during PT reception (Hamamura et al., 2014; Ngo et al., 2014). In nta-1 mutants, PT reception is not impaired in every ovule. Incomplete penetrance of the nta-1 phenotype suggests that some ovules have normal fertilization if calcium oscillations within the synergid cell can reach a certain threshold (Ngo et al., 2014). The calmodulin-binding capacity of NTA may in some way modulate calcium signaling during PT reception. In this study, we found that swapping the C-terminal tail (which contains the conserved MLO CaMBD) of NTA with MLO8’s partially disrupted its ability to function in this pathway. However, NTA’s C-terminal tail on MLO8 was able to function in MLO-mediated PT reception; therefore, we speculate that this domain may confer the ability to modulate calcium signaling within the synergid cell above this threshold when it is present in a closely related MLO. The MLO C-terminal tail was described recently as intrinsically disordered, suggesting that this domain influences functional specificity throughout the family (Kusch et al., 2016). These data further support the hypothesis that fundamental differences affecting function exist in the C-terminal domains of MLO proteins.

While this study contributes significantly to our understanding of MLO-mediated PT reception, the exact mechanism of NTA’s function throughout gametophytic interaction remains to be determined. NTA itself could be involved in signaling with the PT to trigger release of the sperm cells; alternatively, other factors contained in the NTA-associated compartments could be delivered to the filiform apparatus along with NTA to assist in communication with the PT. Our domain-swap experiments have indicated that both homooligomerization, influenced by the N-terminal domain of NTA, and C-terminal tail identity are important for NTA function in PT reception. Future work on NTA’s active redistribution during PT reception and its relationship to calcium oscillations within the synergid cell will provide more insight into how MLO proteins function during cell-cell communication.

MATERIALS AND METHODS

Plant Growth Conditions

Full-length MLOs and domain-swap constructs were transformed into homozygous nta-1 insertion mutants in the Wassilewskija (Ws) background of Arabidopsis (Arabidopsis thaliana). Wild-type Ws and nta-1 were used as the controls for all ovule-count experiments. Native promoter GUS and GFP fusion constructs were transformed into the Columbia-0 (Col-0) ecotype. All Arabidopsis plants were grown at 22°C in long-day conditions (16 h of light and 8 h of dark) in a custom-built grow room. Seeds were sterilized and plated on one-half-strength Murashige and Skoog (MS) plates with transgenic lines either grown on one-half-strength MS plates supplemented with 20 mg L−1 hygromycin (full-length MLO and domain-swap ovule-count lines and native promoter lines) or planted directly on soil (endomembrane marker coexpression lines) and kept in the dark at 4°C for 2 d for cold stratification. Five- to 7-d-old seedlings were transplanted from plates to soil (ProMix Flex supplemented with 40 g of MARATHON pesticide and Peter’s 20:20:20 [NPK] fertilizer at recommended levels) and grown until flowering for further analysis. Seeds sown directly on soil were treated with BASTA herbicide spray for 3 successive days starting at 5 d after germination, and resistant plants were maintained for analysis. Nicotiana benthamiana was grown on soil in long-day conditions at 22°C in a Conviron ATC13 growth chamber.

Cloning and Generation of Transgenic Lines

PCR with PHUSION High-Fidelity Polymerase (NEB M0535S) was utilized to generate all constructs in this study. Genes were amplified with primers that had attB1 and attB2 sites for recombination via BP reaction into the Gateway-compatible entry vector pDONR207 (Supplemental Table S1). Native promoter constructs were amplified from Col-0 genomic DNA, while full-length MLO and domain-swap constructs were amplified from cDNA derived from rosette leaves or flowers of Col-0. Native promoter entry vectors were recombined via LR reactions into pMDC107 (GFP) and pMDC163 (GUS; Curtis and Grossniklaus, 2003) to generate reporter fusion expression vectors. Full-length MLO cDNA entry vectors were recombined via LR reactions into the pMDC83 backbone with the MYB98pro promoter (Müller et al., 2016) to drive synergid-selective expression with a C-terminal GFP fusion. Domain-swap and site-directed mutagenesis constructs were made using primers listed in Supplemental Table S1 to generate overlapping fragments of specific regions from the full-length NTA and MLO8 entry vector cDNA template described above. AttB sites were included on the ends of the chimeric constructs, and fragments were pieced together using a PCR-pasting protocol. Amplified chimeras were recombined via BP reaction into pDONR207, and entry vectors were subsequently recombined via LR reaction into the modified pMDC83 backbone described previously. All entry vectors were sequenced to ensure that swaps were present within each construct.

The MYB98pro::SYP61-mCherry construct was generated in a single reaction using a three-fragment Gibson Assembly, with Gibson Assembly Master Mix NEB E2611S (Gibson et al., 2009). SYP61 was amplified from Col-0 flower cDNA, and MYB98pro was amplified using the pMDC83 backbone described previously (Lindner et al., 2015). Fragments were assembled into the pEarlyGate301 backbone following digestion with PacI (NEB R0547S) and BamHI (NEB R3136S) and subsequent gel purification. Primers with overlaps used for amplification are listed in Supplemental Table S1.

Constructs for transient expression in N. benthamiana were generated using entry vectors of full-length MLOs recombined via LR reaction into pMDC83, which has a 2× 35S viral promoter and C-terminal GFP fusion (Curtis and Grossniklaus, 2003). Gateway-compatible destination vectors with C-terminal mCer3, C-terminal YFP, and N-terminal YFP were used for all FRET analyses (Myers et al., 2016). Entries described above were recombined into their respective FRET vectors via LR reaction.

nta-1 Complementation Assays

Expression vectors were transformed into the Agrobacterium tumefaciens strain GV3101 and subsequently transformed into nta-1 via the floral dip method (Bent, 2006). The resulting seeds were plated on one-half-strength MS plates supplemented with 20 mg L−1 hygromycin for selection of transgenic plants. Three to four different insertion lines for each construct were taken to the T2 generation, and homozygous plants were identified using fluorescence microscopy to ensure synergid expression of GFP in every ovule. Fertilization rates of self-pollinated flowers were counted in at least three plants from each line. Counts of unfertilized versus fertilized ovules were performed and compared with controls in order to determine the complementation status of each construct in the nta-1 background. Aniline Blue staining of PTs in pollinated pistils was used to confirm that unfertilized ovules in the complementation lines were the result of nta mutant-induced PT overgrowth and not other problems with female gametophyte development or PT attraction. Data were graphed and statistically analyzed using Prism (www.graphpad.com). Significance was determined using ANOVA and Dunnett’s multiple comparison test. Full-length MLO complementation lines were compared with both nta-1 and Ws unfertilized ovule counts, resulting in the same significance with both tests (constructs that complement the nta-1 phenotype were significantly different from the nta-1 control but not significantly different from the Ws control, but only statistical tests with nta-1 comparisons are shown in the figures for simplicity). Domain-swap complementation lines were compared with full-length NTA and MLO8 unfertilized ovule counts.

GUS Staining of Ovules

Flowers from two to three T1 plants of each native promoter line were emasculated. At 2 d after emasculation, pistils were slit down the septum prior to being fixed for 45 min in 90% (v/v) acetone at −20°C. Exposed ovules were vacuum infiltrated and incubated overnight in GUS staining solution (10 mm EDTA, 0.1% [v/v] Triton X-100, 2 mm Fe2+CN, 2 mm Fe3+CN, and 1 mg mL−1 5-bromo-4-chloro-3-indolyl-β-glucuronic acid [GoldBio] in 100 mm sodium phosphate buffer, pH 7.2) at 37°C. Ovules were dissected out of the pistils into chloral hydrate:glycerol:water solution (8:1:2, w/v/v) and imaged using differential interference contrast optics on a Nikon Eclipse Ni-U compound microscope.

Transient Expression in N. benthamiana

Leaves from 4- to 6-week-old N. benthamiana were coinfiltrated with A. tumefaciens strain GV3101 harboring either an MLO-GFP construct and the mCherry secretory marker discussed or Cer3- and YFP-fused proteins for analysis of FRET, along with the A. tumefaciens strain C58C1 containing the viral silencing suppressor helper complex pCH32 (Hamilton, 1997). For colocalization experiments, A. tumefaciens overnight cultures were pretreated as described (Myers et al., 2016). For FRET analysis, A. tumefaciens overnight cultures were centrifuged for 5 min at 4,000g and resuspended/washed in 10 mm MgCl2 twice. A final suspension of each A. tumefaciens in 10 mm MgCl2 was then coinfiltrated into the leaf. Plants were maintained in the growth chamber, and all further analyses took place 2 to 4 d after infiltration.

Microscopy

Young flowers from lines with NTA-GFP driven by NTA’s native promoter and MYB98pro were dissected out in water on a clean microscope slide. Ovules were imaged using a 40× dry objective (numerical aperture = 0.75) on a Nikon Eclipse Ni-U compound epifluorescence microscope equipped with an X-Cite 120 light-emitting diode fluorescent lamp and narrow-band eGFP filter cube. Bright-field images were taken using differential interference contrast optics and overlaid with fluorescence to determine where NTA-GFP was detected.

Ovules from MLO-GFP lines were dissected out 2 d after emasculating the flower and were removed but retained on the septum for analysis of GFP localization and distribution on a confocal scanning laser microscope. Freshly dissected ovules were incubated on a slide in 20 μm FM464 (Molecular Probes; T3166) in PIPES buffer (50 mm PIPES, 5 mm EGTA, and 1 mm MgSO4 at pH 6.8) for 10 min on ice in a humid chamber (pipette tip box with ice in the bottom and moist KimWipes under the slide on the top). Coverslips were placed right before the samples were taken to the microscope, and excessive staining solution was removed with a clean KimWipe. Images were taken on a Leica TCS SP8 confocal scanning laser microscope using an HC PL APO 40× water-immersion objective (numerical aperture = 1.1) with GFP excited by a 488-nm argon laser and FM464 excited by a 561-nm diode laser in sequential mode. Fluorescent signals were detected using two HyD detectors in photon-counting mode (single sections) and normal mode (z-series).

Colocalization of secretory markers in mature ovules used similar settings but a more narrow detection window for mCherry fluorescence. Ovules were dissected out but retained on the septum, placed in PIPES buffer, and imaged immediately. For transient colocalization experiments in N. benthamiana, leaf discs were collected near the site of infiltration and placed in PIPES buffer, and GFP and mCherry signals were imaged in cells expressing both constructs.

FRET Detection via Acceptor Photobleaching

Due to the rapid trafficking of NTA- and MLO8-localized compartments within the epidermal cells of N. benthamiana, leaf discs were treated with 20 μg mL−1 cytochalasin B (Sigma-Aldrich; C6762) in PIPES buffer for 10 min at room temperature prior to acceptor photobleaching. Epidermal cells coexpressing the two constructs to be assayed were chosen with mCer3 (donor) excited at 458 nm and detected using a HyD detector from 459 to 512 nm, while YFP (acceptor) was excited at 514 nm and detected with a photomultiplier tube (PMT) from 512 to 562 nm, while using a 458/514 notch filter. Three images of both mCer3 and YFP were taken before photobleaching and between each subsequent period of photobleaching (5 s of high-intensity 514 nm). After four rounds of photobleaching, a final set of three images was taken of both mCer3 and YFP fluorescence.

The overall drift of punctate fluorescent compartments also made the analysis of FRET via a calculation of FRET efficiency (as in previous studies [Karpova et al., 2003; Bhat et al., 2005]) inaccurate, as increases in fluorescence intensity could be the result of axial shifts of the sample being imaged. Thus, all data gathered were analyzed through the use of a custom macro in Fiji (Schindelin et al., 2012). The fluorescence of mCer3 and YFP in the photobleached region was graphed and compared by determining their Pearson product-moment correlation coefficients. This enabled the accurate detection of mCer3 fluorescence signal changes over time in response to acceptor photobleaching. The distributions of the correlation coefficients from all assayed pairs within each experiment were compared using the nonparametric Kruskal-Wallis test with false discovery rate-corrected multiple pairwise comparisons determined using the Dunn test. Statistical analyses for FRET data were all carried out in R version 3.1.2 (R Core Team, 2014) using the packages dplyr (Wickham and Francois, 2015) and DescTools (Signorell, 2015).

Figure Construction and Image Processing

All images were edited using Fiji (Schindelin et al., 2012). MLO protein and ovule diagrams were made in Inkscape version 0.91. All graphs were generated in Prism (www.graphpad.com), and figures were constructed in Gimp version 2.8.14.

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. Ravishankar Palanivelu and Dr. Xunliang Liu (University of Arizona) for the LREpro::Golgi, endoplasmic reticulum, and peroxisome-mCherry constructs and Dr. Ben F. Holt, III, and Zachary A. Myers (University of Oklahoma) for the Gateway-compatible mCer3 and YFP destination vectors for FRET analysis and for helpful discussions; we also thank Patrick Day, Nic Cejda, and Arianna Dino for technical assistance and Jody Banks, Noel Lucca, and Thomas Davis for commenting on the article.

Glossary

PT

pollen tube

TGN

trans-Golgi network

FRET

Förster resonance energy transfer

Col-0

Columbia-0

MS

Murashige and Skoog

Ws

Wassilewskija

Footnotes

1

This work was supported by the National Science Foundation (grant no. IOS-1457098) and the Oklahoma Center for the Advancement of Science and Technology (grant no. PS14-008).

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References

  1. An Q, Ehlers K, Kogel KH, van Bel AJ, Hückelhoven R (2006a) Multivesicular compartments proliferate in susceptible and resistant MLA12-barley leaves in response to infection by the biotrophic powdery mildew fungus. New Phytol 172: 563–576 [DOI] [PubMed] [Google Scholar]
  2. An Q, Hückelhoven R, Kogel KH, van Bel AJ (2006b) Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus. Cell Microbiol 8: 1009–1019 [DOI] [PubMed] [Google Scholar]
  3. Beale KM, Johnson MA (2013) Speed dating, rejection, and finding the perfect mate: advice from flowering plants. Curr Opin Plant Biol 16: 590–597 [DOI] [PubMed] [Google Scholar]
  4. Bent A. (2006) Arabidopsis thaliana floral dip transformation method. Methods Mol Biol 343: 87–103 [DOI] [PubMed] [Google Scholar]
  5. Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R (2005) Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proc Natl Acad Sci USA 102: 3135–3140 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bircheneder S, Dresselhaus T (2016) Why cellular communication during plant reproduction is particularly mediated by CRP signalling. J Exp Bot 67: 4849–4861 [DOI] [PubMed] [Google Scholar]
  7. Büschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, van Daelen R, van der Lee T, Diergaarde P, Groenendijk J, et al. (1997) The barley Mlo gene: a novel control element of plant pathogen resistance. Cell 88: 695–705 [DOI] [PubMed] [Google Scholar]
  8. Chaiwanon J, Wang W, Zhu JY, Oh E, Wang ZY (2016) Information integration and communication in plant growth regulation. Cell 164: 1257–1268 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen Z, Noir S, Kwaaitaal M, Hartmann HA, Wu MJ, Mudgil Y, Sukumar P, Muday G, Panstruga R, Jones AM (2009) Two seven-transmembrane domain MILDEW RESISTANCE LOCUS O proteins cofunction in Arabidopsis root thigmomorphogenesis. Plant Cell 21: 1972–1991 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Consonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, Westphal L, Vogel J, Lipka V, Kemmerling B, Schulze-Lefert P, et al. (2006) Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat Genet 38: 716–720 [DOI] [PubMed] [Google Scholar]
  11. Curtis MD, Grossniklaus U (2003) A Gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133: 462–469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Denninger P, Bleckmann A, Lausser A, Vogler F, Ott T, Ehrhardt DW, Frommer WB, Sprunck S, Dresselhaus T, Grossmann G (2014) Male-female communication triggers calcium signatures during fertilization in Arabidopsis. Nat Commun 5: 4645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Devoto A, Hartmann HA, Piffanelli P, Elliott C, Simmons C, Taramino G, Goh CS, Cohen FE, Emerson BC, Schulze-Lefert P, et al. (2003) Molecular phylogeny and evolution of the plant-specific seven-transmembrane MLO family. J Mol Evol 56: 77–88 [DOI] [PubMed] [Google Scholar]
  14. Ding Y, Robinson DG, Jiang L (2014) Unconventional protein secretion (UPS) pathways in plants. Curr Opin Cell Biol 29: 107–115 [DOI] [PubMed] [Google Scholar]
  15. Dodd AN, Kudla J, Sanders D (2010) The language of calcium signaling. Annu Rev Plant Biol 61: 593–620 [DOI] [PubMed] [Google Scholar]
  16. Elliott C, Müller J, Miklis M, Bhat RA, Schulze-Lefert P, Panstruga R (2005) Conserved extracellular cysteine residues and cytoplasmic loop-loop interplay are required for functionality of the heptahelical MLO protein. Biochem J 385: 243–254 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Escobar-Restrepo JM, Huck N, Kessler S, Gagliardini V, Gheyselinck J, Yang WC, Grossniklaus U (2007) The FERONIA receptor-like kinase mediates male-female interactions during pollen tube reception. Science 317: 656–660 [DOI] [PubMed] [Google Scholar]
  18. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA III, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343–345 [DOI] [PubMed] [Google Scholar]
  19. Gilroy S, Suzuki N, Miller G, Choi WG, Toyota M, Devireddy AR, Mittler R (2014) A tidal wave of signals: calcium and ROS at the forefront of rapid systemic signaling. Trends Plant Sci 19: 623–630 [DOI] [PubMed] [Google Scholar]
  20. Goder V, Spiess M (2001) Topogenesis of membrane proteins: determinants and dynamics. FEBS Lett 504: 87–93 [DOI] [PubMed] [Google Scholar]
  21. Hamamura Y, Nishimaki M, Takeuchi H, Geitmann A, Kurihara D, Higashiyama T (2014) Live imaging of calcium spikes during double fertilization in Arabidopsis. Nat Commun 5: 4722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hamilton CM. (1997) A binary-BAC system for plant transformation with high-molecular-weight DNA. Gene 200: 107–116 [DOI] [PubMed] [Google Scholar]
  23. Higashiyama T. (2002) The synergid cell: attractor and acceptor of the pollen tube for double fertilization. J Plant Res 115: 149–160 [DOI] [PubMed] [Google Scholar]
  24. Higashiyama T, Kuroiwa H, Kawano S, Kuroiwa T (1998) Guidance in vitro of the pollen tube to the naked embryo sac of Torenia fournieri. Plant Cell 10: 2019–2032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Higashiyama T, Takeuchi H (2015) The mechanism and key molecules involved in pollen tube guidance. Annu Rev Plant Biol 66: 393–413 [DOI] [PubMed] [Google Scholar]
  26. Huck N, Moore JM, Federer M, Grossniklaus U (2003) The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception. Development 130: 2149–2159 [DOI] [PubMed] [Google Scholar]
  27. Hückelhoven R, Panstruga R (2011) Cell biology of the plant-powdery mildew interaction. Curr Opin Plant Biol 14: 738–746 [DOI] [PubMed] [Google Scholar]
  28. Karpova TS, Baumann CT, He L, Wu X, Grammer A, Lipsky P, Hager GL, McNally JG (2003) Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J Microsc 209: 56–70 [DOI] [PubMed] [Google Scholar]
  29. Kasahara RD, Portereiko MF, Sandaklie-Nikolova L, Rabiger DS, Drews GN (2005) MYB98 is required for pollen tube guidance and synergid cell differentiation in Arabidopsis. Plant Cell 17: 2981–2992 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kessler SA, Grossniklaus U (2011) She’s the boss: signaling in pollen tube reception. Curr Opin Plant Biol 14: 622–627 [DOI] [PubMed] [Google Scholar]
  31. Kessler SA, Shimosato-Asano H, Keinath NF, Wuest SE, Ingram G, Panstruga R, Grossniklaus U (2010) Conserved molecular components for pollen tube reception and fungal invasion. Science 330: 968–971 [DOI] [PubMed] [Google Scholar]
  32. Kim MC, Panstruga R, Elliott C, Müller J, Devoto A, Yoon HW, Park HC, Cho MJ, Schulze-Lefert P (2002) Calmodulin interacts with MLO protein to regulate defence against mildew in barley. Nature 416: 447–451 [DOI] [PubMed] [Google Scholar]
  33. Kusch S, Pesch L, Panstruga R (2016) Comprehensive phylogenetic analysis sheds light on the diversity and origin of the MLO family of integral membrane proteins. Genome Biol Evol 8: 878–895 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kwon C, Bednarek P, Schulze-Lefert P (2008) Secretory pathways in plant immune responses. Plant Physiol 147: 1575–1583 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Li C, Yeh FL, Cheung AY, Duan Q, Kita D, Liu MC, Maman J, Luu EJ, Wu BW, Gates L, et al. (2015) Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis. eLife 4: 06587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lindner H, Kessler SA, Müller LM, Shimosato-Asano H, Boisson-Dernier A, Grossniklaus U (2015) TURAN and EVAN mediate pollen tube reception in Arabidopsis synergids through protein glycosylation. PLoS Biol 13: e1002139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Liu X, Castro C, Wang Y, Noble J, Ponvert N, Bundy M, Hoel C, Shpak E, Palanivelu R (2016) The role of LORELEI in pollen tube reception at the interface of the synergid cell and pollen tube requires the modified eight-cysteine motif and the receptor-like kinase FERONIA. Plant Cell 28: 1035–1052 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Luschnig C, Vert G (2014) The dynamics of plant plasma membrane proteins: PINs and beyond. Development 141: 2924–2938 [DOI] [PubMed] [Google Scholar]
  39. Markwardt ML, Kremers GJ, Kraft CA, Ray K, Cranfill PJ, Wilson KA, Day RN, Wachter RM, Davidson MW, Rizzo MA (2011) An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PLoS ONE 6: e17896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Maurel D, Comps-Agrar L, Brock C, Rives ML, Bourrier E, Ayoub MA, Bazin H, Tinel N, Durroux T, Prézeau L, et al. (2008) Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat Methods 5: 561–567 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Müller LM, Lindner H, Pires ND, Gagliardini V, Grossniklaus U (2016) A subunit of the oligosaccharyltransferase complex is required for interspecific gametophyte recognition in Arabidopsis. Nat Commun 7: 10826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Musil LS, Goodenough DA (1993) Multisubunit assembly of an integral plasma membrane channel protein, gap junction connexin43, occurs after exit from the ER. Cell 74: 1065–1077 [DOI] [PubMed] [Google Scholar]
  43. Myers ZA, Kumimoto RW, Siriwardana CL, Gayler KK, Risinger JR, Pezzetta D, Holt BF III (2016) NUCLEAR FACTOR Y, subunit C (NF-YC) transcription factors are positive regulators of photomorphogenesis in Arabidopsis thaliana. PLoS Genet 12: e1006333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A (2002) A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20: 87–90 [DOI] [PubMed] [Google Scholar]
  45. Nelson BK, Cai X, Nebenführ A (2007) A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J 51: 1126–1136 [DOI] [PubMed] [Google Scholar]
  46. Ngo QA, Vogler H, Lituiev DS, Nestorova A, Grossniklaus U (2014) A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery. Dev Cell 29: 491–500 [DOI] [PubMed] [Google Scholar]
  47. Nielsen ME, Feechan A, Böhlenius H, Ueda T, Thordal-Christensen H (2012) Arabidopsis ARF-GTP exchange factor, GNOM, mediates transport required for innate immunity and focal accumulation of syntaxin PEN1. Proc Natl Acad Sci USA 109: 11443–11448 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Okuda S, Tsutsui H, Shiina K, Sprunck S, Takeuchi H, Yui R, Kasahara RD, Hamamura Y, Mizukami A, Susaki D, et al. (2009) Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells. Nature 458: 357–361 [DOI] [PubMed] [Google Scholar]
  49. Otero S, Helariutta Y, Benitez-Alfonso Y (2016) Symplastic communication in organ formation and tissue patterning. Curr Opin Plant Biol 29: 21–28 [DOI] [PubMed] [Google Scholar]
  50. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8: 785–786 [DOI] [PubMed] [Google Scholar]
  51. R Core Team (2014) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna [Google Scholar]
  52. Russell SD. (1992) Double fertilization. Int Rev Cytol 140: 357–388 [Google Scholar]
  53. Safavian D, Goring DR (2013) Secretory activity is rapidly induced in stigmatic papillae by compatible pollen, but inhibited for self-incompatible pollen in the Brassicaceae. PLoS ONE 8: e84286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Samuel MA, Chong YT, Haasen KE, Aldea-Brydges MG, Stone SL, Goring DR (2009) Cellular pathways regulating responses to compatible and self-incompatible pollen in Brassica and Arabidopsis stigmas intersect at Exo70A1, a putative component of the exocyst complex. Plant Cell 21: 2655–2671 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sanderfoot AA, Kovaleva V, Bassham DC, Raikhel NV (2001) Interactions between syntaxins identify at least five SNARE complexes within the Golgi/prevacuolar system of the Arabidopsis cell. Mol Biol Cell 12: 3733–3743 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, et al. (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9: 676–682 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Signorell A. (2015) DescTools: Tools for Descriptive Statistics. R package version 0.99 15 November 13, 2015, https://CRAN.R-project.org/package=DescTools
  58. Stahl Y, Faulkner C (2016) Receptor complex mediated regulation of symplastic traffic. Trends Plant Sci 21: 450–459 [DOI] [PubMed] [Google Scholar]
  59. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30: 2725–2729 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Tsukamoto T, Qin Y, Huang Y, Dunatunga D, Palanivelu R (2010) A role for LORELEI, a putative glycosylphosphatidylinositol-anchored protein, in Arabidopsis thaliana double fertilization and early seed development. Plant J 62: 571–588 [DOI] [PubMed] [Google Scholar]
  61. Véry AA, Sentenac H (2002) Cation channels in the Arabidopsis plasma membrane. Trends Plant Sci 7: 168–175 [DOI] [PubMed] [Google Scholar]
  62. Viotti C, Bubeck J, Stierhof YD, Krebs M, Langhans M, van den Berg W, van Dongen W, Richter S, Geldner N, Takano J, et al. (2010) Endocytic and secretory traffic in Arabidopsis merge in the trans-Golgi network/early endosome, an independent and highly dynamic organelle. Plant Cell 22: 1344–1357 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weisz OA, Swift AM, Machamer CE (1993) Oligomerization of a membrane protein correlates with its retention in the Golgi complex. J Cell Biol 122: 1185–1196 [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Wickham H, Francois R (2015) dplyr: A Grammar of Data Manipulation. R package version 0.4 1: 20 February 10, 2015, https://CRAN.R-project.org/package=dplyr
  65. Yi M, Valent B (2013) Communication between filamentous pathogens and plants at the biotrophic interface. Annu Rev Phytopathol 51: 587–611 [DOI] [PubMed] [Google Scholar]

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