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American Society of Plant Biologists A Cysteine-Rich Extracellular Protein, LAT52, Interacts with the Extracellular Domain of the Pollen Receptor Kinase LePRK2Plant Gene Expression Center, United States Department of Agriculture/Agricultural Research Service, and Department of Plant and Microbial Biology, University of California at Berkeley, 800 Buchanan Street, Albany, California 94710 3 To whom correspondence should be addressed. E-mail sheilamc{at}nature.berkeley.edu; fax 510-559-5678
Pollen germination and pollen tube growth are thought to require extracellular cues, but how these cues are perceived and transduced remains largely unknown. Pollen receptor kinases are plausible candidates for this role; they might bind extracellular ligands and thereby mediate cytoplasmic events required for pollen germination and pollen tube growth. To search for pollen-expressed ligands for pollen receptor kinases, we used the extracellular domains of three pollen-specific receptor kinases of tomato (LePRK1, LePRK2, and LePRK3) as baits in a yeast two-hybrid screen. We identified numerous secreted or plasma membrane–bound candidate ligands. One of these, the Cys-rich protein LAT52, was known to be essential during pollen hydration and pollen tube growth. We used in vivo coimmunoprecipitation to demonstrate that LAT52 was capable of forming a complex with LePRK2 in pollen and to show that the extracellular domain of LePRK2 was sufficient for the interaction. Soluble LAT52 can exist in differently sized forms, but only the larger form can interact with LePRK2. We propose that LAT52 might be a ligand for LePRK2.
Pollen germination and pollen tube growth are not only crucial for the successful fertilization of plants but also offer an excellent model for studying cell signaling (reviewed by Wheeler et al., 2001  ). In the process of pollination, mature pollen grains are released from the anther in a partially dehydrated state and rehydrate after they contact the female pistil. The pollen tube emerges from one of the apertures and grows by tip growth through the extracellular matrix of the pistil. The pollen tube eventually reaches the ovule and delivers the sperm cells to the embryo sac.
This complicated and tightly controlled process presumably involves signaling between the pollen tube and the pistil: pollen tubes may adhere to the extracellular matrix and follow guidance cues as they traverse different types of tissues en route to an ovule. Recent evidence suggests that, in the pistil, the arabinogalactan protein TTS (Cheung et al., 1995
Extracellular signaling molecules from pollen, if they exist, also might contribute to the pollen tube–pollen tube adhesion sometimes seen in vitro (Lush et al., 1997
Leu-rich repeat receptor kinases (LRR kinases) constitute a large gene family in higher plants (Shiu and Bleecker, 2001
In tomato, three pollen-specific receptor kinases (LePRK1, LePRK2, and LePRK3) localize on the pollen tube wall, with distinct but overlapping patterns (Muschietti et al., 1998
It is typical that ligands are produced and secreted by cells other than those that express the receptor. However, in autocrine signaling systems, both the ligand and its receptor are produced by the same cell (DeWitt et al., 2001
One of the small Cys-rich proteins, LAT52, was of special interest. Previously, we showed that LAT52 is essential during pollen hydration and pollen tube growth, because pollen expressing antisense LAT52 RNA hydrates and germinates abnormally and cannot achieve fertilization (Muschietti et al., 1994
Yeast Two-Hybrid Screens Identify Extracellular Binding Partners for LePRKs To identify pollen proteins that interact with the extracellular domains of the pollen kinases, we used a yeast two-hybrid screen. Although the yeast two-hybrid screen requires that proteins interact in the yeast nucleus, this approach has been successful occasionally (Ozenberger and Young, 1995 ; Zhu and Kahn, 1997; Park et al., 2001) in identifying interacting proteins that are located normally in the plasma membrane or the extracellular space. A cDNA library prepared from mRNA of mature pollen of tomato was fused to the GAL4 activation domain. This library was screened separately against three bait constructs, each encoding an extracellular domain (ECD) of a LePRK fused to the GAL4 DNA binding domain. Perhaps surprisingly, a remarkable portion ( 60%) of the positive cDNA clones from each screen encoded bona-fide secreted proteins or proteins containing a predicted N-terminal signal peptide and therefore were candidate ligands (see supplementary data online for details).
These candidate ligands fall into five groups. The first group comprises diverse Cys-rich proteins (molecular mass ranging from 6 to 16 kD). This group was especially interesting because another small Cys-rich protein, SCR, found on the pollen coat in Brassica species, was shown recently to be the ligand for the stigma-expressed S-locus receptor kinase SRK (Schopfer et al., 1999
The third group is represented by a protein containing LRRs. Because LRRs mediate protein–protein interactions (Kobe and Deisenhofer, 1994
The previously characterized LAT52 gene (Twell et al., 1989
To determine whether LAT52 could interact with the other LePRKs, the full-length LAT52 in the prey vector (pAD-LAT52) or the empty prey vector (pAD) were retransformed into yeast cells harboring bait plasmids (pBD) of the extracellular domains of LePRK1, LePRK2, or LePRK3 or the cytoplasmic domain of LePRK2 (control). Table 1 shows that only the transformants harboring both pAD-LAT52 and pBD-ECD2 grew on selection plates. The
In Vivo Coimmunoprecipitation Demonstrates That LAT52 and LePRK2 Are in the Same Complex in Mature Pollen Extracts Because pollen expressing antisense LAT52 RNA hydrates and germinates abnormally (Muschietti et al., 1994 ), LAT52 seemed the most promising candidate ligand for further characterization. We had antibodies (Figure 1A)
for both LAT52 (Muschietti et al., 1994) and LePRK2 (Muschietti et al., 1998), so we were able to test whether LAT52 and LePRK2 interact with each other in pollen, using a coimmunoprecipitation assay. When equivalent amounts of total protein (S10) extracts from mature pollen were immunoprecipitated with anti-LePRK2 or with normal mouse serum, LAT52 protein was detected only in the immunopellet of anti-LePRK2 (Figure 1B). This result shows that LAT52 and LePRK2 are in the same complex in total protein extracts from mature pollen and confirms that LAT52 and LePRK2 interact with each other in pollen.
In the yeast two-hybrid system, LAT52 interacted specifically with LePRK2 but not with LePRK1 or LePRK3. We tested whether this specificity could be demonstrated in pollen extracts, using antibodies raised against the extracellular domains of the LePRKs (Figure 2A) . In three different experiments, equivalent amounts of total protein extracts from mature pollen were immunoprecipitated separately with antibodies against the extracellular domains of LePRK1, LePRK2, and LePRK3. In all three experiments, a significant amount of LAT52 protein was present in the immunopellet of anti-LePRK2 (Figure 2B), but LAT52 protein was not present in the immunopellet of anti-LePRK3.
In one of three experiments, a small amount of LAT52 was present in the immunopellet of anti-LePRK1 (Figure 2B, experiment B), whereas in the other two experiments, no LAT52 was present in the anti-LePRK1 immunopellet (Figure 2B, experiment A). Thus, in pollen, LAT52 does interact with LePRK2, does not interact with LePRK3, and does not interact significantly with LePRK1. We could not perform the reciprocal coimmunoprecipitation assay with anti-LAT52 because it does not recognize native LAT52 protein in pollen extracts, although it recognizes denatured LAT52 protein and is useful for immunoblot analysis (Muschietti et al., 1994 ).
The Presence of EDTA Abolishes the Interaction between LAT52 and LePRK2
To determine what component of the two buffers might account for the difference in coimmunoprecipitation success, we tested modified PGM buffers. Figure 3 shows that changing the pH did not affect the amount of coimmunoprecipitated LAT52 significantly. The addition of DTT to PGM buffer did not affect coimmunoprecipitation, suggesting that disulfide bonds are not required for the interaction. Nor did the removal of BA and K+ from PGM buffer reduce the amount of coimmunoprecipitated LAT52. However, the addition of EDTA and the omission of MgSO4 and Ca(NO3)2 completely prevented the coimmunoprecipitation of LAT52. Because EDTA is a chelator for metal ions such as Ca2+ and Mg2+, this result suggests that a metal ion (Ca2+ and/or Mg2+) is required for the LAT52–LePRK2 interaction.
The Extracellular Portion of LePRK2 Is Sufficient for Its Interaction with LAT52 The Escherichia coli–expressed His-ECD2 fusion protein was purified under native conditions (Figure 4A) . Total protein extracts from mature pollen were incubated with the His-ECD2 protein, and then LAT52 was coimmunoprecipitated. The purified ECD2 can be recognized by both anti-His and anti-LePRK2 antibodies, but the endogenous LePRK2 can be recognized only by the anti-LePRK2 antibody. We first determined the amounts of LAT52 and LePRK2 in the soluble (S100) and membrane-associated (P100) fractions of mature pollen extracts by immunoblot analysis (Figure 4B). The LAT52 protein was found mostly in the soluble protein fraction. The residual amount of LAT52 detected in the membrane-associated fraction might be attributable to interaction with membrane-associated components or to incomplete fractionation.
As we already knew, LePRK2 can be detected only in the membrane-associated fraction. Consistent with these locations, Figure 4C (lanes 1 and 2) shows that LAT52 was coimmunoprecipitated only with anti-LePRK2 antibody from total protein extracts (S10) from mature pollen, but not from the S100 fraction in which LAT52 was enriched but from which the endogenous LePRK2 was depleted. In the semi-in vivo immunoprecipitation assay, the LAT52 in the mature pollen S100 fraction was able to be coimmunoprecipitated with anti-His antibody if exogenous His-ECD2 protein was added (Figure 4C, compare lanes 3 and 4). When the His-ECD2 protein was added to total protein extracts (S10) of mature pollen, LAT52 was coimmunoprecipitated (Figure 4C, lane 6), but the signal was much weaker than if the coimmunoprecipitation was from a similar amount of S100 fraction (Figure 4C, lane 9). We presume that this difference is caused by competition from the endogenous LePRK2 that was present in the S10 extract. The results showed that LAT52 in pollen extracts also could form a complex with the His-ECD2 protein (Figure 4C). Thus, the ECD of LePRK2 is sufficient for this interaction, and the cytoplasmic portion of LePRK2, including the kinase domain, is not necessary for binding to LAT52. This experiment also indicates that the LAT52 protein in the mature pollen S100 fraction, although temporarily not in the same complex with LePRK2, remains capable of forming a complex with LePRK2. We used the His-tagged fusion proteins (Figure 5A) to determine if LAT52 and the extracellular domains of the other two LePRKs could interact. Figure 5B shows that LAT52 was not precipitated with anti-His antibody when His-ECD3 was incubated with pollen extracts; it also shows that a small amount of LAT52 was detected in the precipitate after His-ECD1 was incubated with pollen extracts. These results are essentially consistent with the in vivo coimmunoprecipitation results (Figure 2).
Pollen Germination or Heating Abolishes the Interaction of LAT52 with LePRK2 We know that LAT52 and LePRK2 can be found in the same complex when protein extracts are prepared from mature pollen under conditions appropriate for pollen germination. We determined if LAT52 and LePRK2 also interact with each other after pollen germination. Both LAT52 and LePRK2 were present in pollen that had been germinated in vitro (Figure 6A) , and as shown previously (Muschietti et al., 1998 ), the amount of LePRK2 was increased dramatically after germination. To our surprise, LAT52 was not coimmunoprecipitated with anti-LePRK2 from pollen that had been germinated in vitro for 4 h (data not shown) or 16 h (Figure 6B, left).
We surmised that either LAT52 or LePRK2 had changed after germination, although no obvious size difference for either protein was apparent from immunoblot analysis (Figure 6A). Because we knew that LAT52 in the S100 fraction from mature pollen remains capable of forming a complex with LePRK2 (Figure 4C), we tested whether LAT52 or LePRK2 had changed after germination. We added the S100 fraction from mature pollen before germination, which contains LAT52, to either the S10 or P100 protein that had been extracted from pollen germinated in vitro, which contains LePRK2. Figure 6B (right) shows that under these conditions, LAT52 was coimmunoprecipitated with anti-LePRK2 antibody. Therefore, LAT52 before germination can form a complex with LePRK2 after germination. This result indicates that it is LAT52, and not LePRK2, that is changed after germination.
We showed previously that LAT52 is a heat-stable protein (Muschietti et al., 1994
Thus, there are two conditions under which LAT52 cannot interact with LePRK2: after heat treatment and after pollen germination. To help determine what might account for this difference, we used crude size fractionation. The LAT52 gene encodes a protein of 161 amino acids, but after processing of the signal peptide, the calculated molecular mass of the mature LAT52 protein is 16 kD. Because LAT52 is glycosylated (Muschietti et al., 1994
We conclude that the soluble LAT52 extracted from mature pollen in PGM buffer exists in a form whose molecular mass obviously is larger than that of the LAT52 monomer. Because the soluble fraction is depleted for most of the membrane-associated LePRK2, this complex does not include LePRK2 (Figure 6A). We then used the YM100 filter to determine the size of LAT52 in the heat-treated S100 protein faction from mature pollen and in the S100 fraction from pollen germinated in vitro. Figures 7B and 7C show that LAT52 was not retained on the YM100 filter in either case, and after heat treatment it was not retained on the YM50 filter (data not shown). These results indicate that there are at least two differently sized forms possible for soluble LAT52. Furthermore, when LAT52 cannot be retained on the YM100 filter, it also is not able to interact with LePRK2. When 10 mM DTT was added to mature pollen extracts, LAT52 was retained on the YM100 filter (Figure 7D), and we showed (Figure 3) that DTT did not prevent the coimmunoprecipitation of LAT52 with anti-LePRK2 from mature pollen. Furthermore, 1 mM EDTA prevented LAT52 from being retained on the YM100 filter (Figure 7E) and prevented it from interacting with LePRK2 (Figure 3). Together, these results suggest that the larger form is required for LAT52 to interact with LePRK2.
Despite anecdotal reports that yeast two-hybrid screens for extracellular proteins are fraught with difficulty, our results show that screens with the extracellular domains of the LePRKs were successful in identifying several interacting proteins that also are extracellular. In this regard, it might be significant that the extracellular domains of the LePRKs are small ( 200 amino acids, with five to six LRRs). By contrast, the extracellular domains of many other LRR receptor kinases (Shiu and Bleecker, 2001) are large (20 or more LRRs) and might not be well expressed or might not fold properly in yeast. Even though we identified numerous extracellular proteins, they still might be false positives. It is always necessary to use independent biochemical assays to confirm candidates obtained from two-hybrid screens. Therefore, we used coimmunoprecipitation from pollen extracts to demonstrate that one of these extracellular proteins, LAT52, does interact with the extracellular domain of LePRK2 in pollen. It will require further work to determine if LAT52 is in fact a ligand whose binding activates the receptor.
Of the many interacting proteins obtained from the yeast two-hybrid screen, we focused on characterizing the LAT52–LePRK2 interaction, because we had shown previously a role for LAT52 during pollen germination (Muschietti et al., 1994
LAT52 is highly expressed in mature pollen, and LAT52 transcripts still are detected after 18 h of in vitro germination (Ursin et al., 1989 The complex containing both LAT52 and LePRK2 was detected in the total proteins of mature pollen extracted in PGM buffer (Figures 1 to 3). In fact, when placed in buffer, pollen hydrated immediately, so it is more accurate to say that our coimmunoprecipitation results represent interactions in hydrated pollen, the beginning stage of pollen germination. Because LAT52 and LePRK2 are in a complex at this first stage, the abnormality in hydration/germination in the antisense LAT52–expressing pollen could be attributable to the absence of this complex. However, we have shown that the LAT52–LePRK2 complex cannot be detected in the total extracts of pollen germinated in vitro (Figure 6B), suggesting that LAT52 and LePRK2 dissociate as in vitro germination proceeds. In the course of determining why the LAT52–LePRK2 interaction is different after in vitro germination, we found that LAT52 can exist in differently sized forms (Figure 7) and that soluble LAT52 from pollen germinated in vitro does not exist in a large form (Figure 7B). Because we could reconstitute the LAT52–LePRK2 complex using LePRK2 from germinated pollen and soluble LAT52 from mature pollen (Figure 6B), the binding characteristics of LePRK2 do not change after germination. This finding suggests that only the large form of soluble LAT52 interacts with LePRK2.
In some receptor–ligand interactions in animals, ligand multimers are required for receptor binding. For example, ephrin must dimerize (Toth et al., 2001
Why would pollen receptors bind to extracellular molecules produced by pollen itself? Autocrine signaling, in which cells secrete soluble ligands that bind to receptors on their own surfaces, has been characterized for several animal growth factors and their receptors. This type of intracellular signaling allows the cell to monitor itself, and in some cases, how the ligand is delivered can have significant effects on the response. For example, when epidermal growth factor (EGF) was delivered to the EGF receptor from the same epithelial cell expressing the receptor, cell migration was prolonged and directional, whereas when EGF was delivered exogenously, cells migrated randomly (Maheshwari et al., 2001
The demonstration that LAT52 interacts with LePRK2 is only a first step toward understanding the signaling pathway mediated by pollen receptor kinases and their extracellular binding partner(s). Interactions of receptors with extracellular binding partners often cause receptor dimerization and phosphorylation, and we know that LePRK1 and LePRK2 can form heterodimers in pollen and when expressed in yeast (I. Valsecchi, M.L. Cabanas, D. Wengier, W. Tang, S. McCormick, and J. Muschietti, unpublished data). Furthermore, the phosphorylation status of LePRK2 changes upon incubation with style extracts (Muschietti et al., 1998
It is worth noting that the failure of the smaller form of LAT52 to interact with LePRK2 does not exclude the possibility of their continued interaction, perhaps with the participation of other molecules from the pistil. In fact, on the pistil, the pollen expressing antisense LAT52 showed a less severe phenotype than when germinated in vitro (Muschietti et al., 1994
There are at least three LePRKs expressed at similar stages and locations (Kim et al., 2002 Although all three LePRKs have extracellular domains composed of LRRs, LePRK3 did not interact with LAT52 under any condition we tested (Figure 2). The weaker interaction between LePRK1 and LAT52 might be the result of the heterodimerization of LePRK1 and LePRK2 or of a low affinity of LAT52 for LePRK1. The apparent higher affinity of LAT52 for LePRK2 rather than LePRK1 illustrates the potential complexity of signaling: LePRKs may form homodimers or heterodimers and interact with different extracellular binding partners along the path of pollen tube growth through the pistil. The challenge now is to test this hypothesis by demonstrating that additional candidates do in fact bind in vivo to the LePRKs and by demonstrating that the binding of LAT52 (or other candidates) initiates the signal transduction pathways required for pollen germination and pollen tube growth.
Plant Material Mature pollen from tomato (Lycopersicon esculentum cv VF36) was obtained by vibrating the anthers of open flowers and was stored immediately at -80°C or used directly for germination experiments. Pollen was germinated in vitro for up to 16 h in pollen germination medium [20 mM Mes, pH 6.0, 3 mM Ca(NO3)2, 1 mM KCl, 0.8 mM MgSO4, 1.6 mM boric acid, 2.5% (w/v) Suc, and 24% (w/v) polyethylene glycol 4000], as described by Muschietti et al. (1994) .
Yeast Two-Hybrid Screens
The yeast strain PJ69-4A was used as the host strain (James et al., 1996
Yeast DNA was extracted and transformed into Escherichia coli XL1Blue to amplify pAD-GAL4 plasmids with cDNA inserts. The plasmids then were retransformed separately into PJ69-4A cells harboring the pBD-GAL4, pBD-ECD1, pBD-ECD2, or pBD-ECD3 plasmid. All transformants then were plated on SC medium supplemented with 3 mM 3-amino-1,2,4-triazole and lacking Leu, Trp, Ade, and His.
DNA Sequence Analysis
Protein Extraction Pollen homogenates were centrifuged at 10,000g for 20 min at 4°C, and the supernatants (S10, or total protein extracts) were used directly in immunoprecipitation experiments or reserved for further fractionation. The S10 was fractionated by centrifugation at 100,000g for 3 h at 4°C. The resulting supernatant was the S100 (soluble) protein fraction. The pellet was resuspended in PGM buffer with the addition of 0.1% Triton X-100 to yield the P100 (membrane-associated) protein fraction. Protein samples were frozen quickly in liquid nitrogen and stored at -80°C. The protein concentration of each sample was determined with the bicinchoninic acid protein assay kit (Pierce).
Expression of Extracellular Domains of LePRKs and Polyclonal Antibodies
Immunoprecipitations After brief centrifugation to precipitate the agarose beads, immunopellets were washed three times with 1 mL of wash buffer (50 mM Tris-HCl, pH 7.6, 0.2% Nonidet P-40, and 100 mM NaCl). The pellet was resuspended in 30 µL of 1 x Laemmli SDS-PAGE loading buffer, mixed vigorously (using a Vortex mixer, Scientific Industries, Bohemia, NY) for 1 min, and boiled for 1 min. After brief centrifugation, the immunoprecipitated proteins were separated by 15% SDS-PAGE, transferred to a nitrocellulose membrane, and subjected to immunoblot analysis with anti-LAT52 antibody. As a control, duplicate samples were subjected to immunoblot analysis with anti-ECD2 antibody.
Semi-In Vivo Immunoprecipitation
SDS-PAGE and Immunoblot Analysis
Protein Partitioning under Native Conditions
The sample reservoir was inverted and recentrifuged briefly to recover the retentate, and the volume was measured. The Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
We thank Robyn Cotter for protocols and advice for the yeast two-hybrid screens, Roy Nattiv, a University of California, Berkeley, undergraduate apprentice, for help in characterizing the many positive yeast clones, and David Hantz for excellent care of the greenhouse plants. We thank Michele Engel, Jenn Fletcher, and Paul Herzmark for comments on the manuscript, and we thank all members of our laboratory for useful discussions throughout the course of this work. This work was supported by U.S. Department of Agriculture Grant CRIS 5335-21000-011-00D.
Online version contains Web-only data.  Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003103.
1 Current address: Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-751 23 Uppsala, Sweden.
2 Current address: Instituto de Ingeniería Genética y Biología Molecular, University of Buenos Aires, Vuelta de Obligado 2490, Buenos Aires (1428), Argentina. Received March 26, 2002; accepted May 19, 2002.
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Abstract
INTRODUCTION
-galactosidase activity of yeast cells harboring both pAD-LAT52 and pBD-ECD2 was 10 times higher than that of any other combination. These results showed that in the yeast two-hybrid system, LAT52 interacts reproducibly and specifically with ECD2 but not with ECD1 or ECD3. Additional candidate ligands also were confirmed in the yeast two-hybrid specificity test (see supplementary data online for details). 
