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Allele-Specific Receptor-Ligand Interactions in Brassica Self-Incompatibility

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Science  07 Sep 2001:
Vol. 293, Issue 5536, pp. 1824-1826
DOI: 10.1126/science.1062509

Abstract

Genetic self-incompatibility in Brassica is determined by alleles of the transmembrane serine-threonine kinase SRK, which functions in the stigma epidermis, and of the cysteine-rich peptide SCR, which functions in pollen. Using tagged versions of SRK and SCR as well as endogenous stigma and pollen proteins, we show that SCR binds the SRK ectodomain and that this binding is allele specific. Thus, SRK and SCR function as a receptor-ligand pair in the recognition of self pollen. Specificity in the self-incompatibility response derives from allele-specific formation of SRK-SCR complexes at the pollen-stigma interface.

In self-incompatible Brassicaplants, self-pollinations and crosses between genetically related individuals are nonproductive because self-related pollen grains are inhibited upon contact with the epidermal cells of the stigma, a structure that caps the female reproductive organ. Specificity in this self-incompatibility (SI) response is determined by haplotypes of the polymorphic S locus. The self-recognition molecules encoded by this locus include the single-pass transmembrane receptor–like serine-threonine kinase SRK, which functions in the stigma epidermis (1–3) and becomes phosphorylated upon self-pollination (4), and the cysteine-rich peptide SCR, which functions in pollen (5,6). These two molecules are highly polymorphic, with allelic forms of SRK and SCR exhibiting 10 to 30% and >60% divergence, respectively (1, 5–8). Views of SRK as a ligand-activated receptor kinase and SCR as its ligand are consistent with the predicted molecular properties of these molecules and the rapidity of the SI response (1,9). The SCR peptide is localized on the surface of pollen grains (10). During self-pollination, SCR is predicted to bind the receptor domain of its cognate SRK, thereby triggering an intracellular phosphorylation cascade that leads to inhibition of pollen hydration and germination. Specificity in the SI response is thought to result from haplotype-specific activation of SRK by SCR. Here, we describe experiments that demonstrate a physical and haplotype-specific interaction between SCR and the ectodomain of SRK.

To investigate the SRK-SCR interaction, we generated tagged versions of the two proteins. Recombinant eSRK6, consisting of the ectodomain of SRK6 (from the S6haplotype) and carrying a COOH-terminal FLAG epitope tag, was expressed as a soluble secreted glycoprotein in Nicotiana benthamianaleaves using the potato virus X expression system (11). eSRK6 protein migrated as two molecular mass forms of ∼63 and 70 kD on SDS–polyacrylamide gel electrophoresis (SDS-PAGE), which presumably reflect differential glycosylation of eSRK6-FLAG in Nicotiana leaves. SCR6 and SCR13 (the SCRs of the S6 andS13 haplotypes, respectively) were expressed in bacteria as secreted periplasmic proteins carrying a COOH-terminal myc-His6 tag (11). They exhibited expected masses of ∼8 and 9 kD, respectively, but they migrated as doublets, possibly due to inefficient cleavage of the periplasmic signal peptide in bacteria.

Recombinant SCR-myc-His6 was shown to be biologically active in pollination bioassays (12). Pretreatment of stigmas with purified “self” SCR protein (i.e.,S6S6 stigmas with SCR6-myc-His6 orS13S13 stigmas with SCR13-myc-His6) mixed with pollen-coat protein carrier (12) caused these stigmas to inhibit the germination of normally compatible “non-self” pollen (Table 1, Fig. 1A). In contrast, “non-self” pollen could develop on stigmas treated with “non-self” SCR protein (e.g., S6S6 stigmas treated with SCR13-myc-His6) (Table 1, Fig. 1B). Thus, recombinant SCR-myc-His6 activates the SI response specifically in stigmas that express the cognate SRK.

Figure 1

Effect of purified recombinant SCR protein on cross-pollen tube development.S6S6 stigmas (A) andS2S2 stigmas (B) were treated with SCR6-myc-His6 and pollinated withS13 pollen (12). Addition of “self” SCR6-myc-His6 triggers inhibition of normally compatible S13 pollen on S6S6 but not onS2S2 stigmas.

Table 1

Inhibition of normally cross-compatible pollen on stigmas pretreated with self recombinant SCR protein. Purified SCR-myc-His6 proteins were added to the stigma before pollination (12), except where indicated by (–). In the Pollen coat protein column, “+” indicates that pollen coat protein was added as carrier; “–” indicates that no pollen coat protein was added. Pollen tube development data represent absence (no) or presence (yes) of pollen tubes. Experimental treatments used two different SCR protein preparations and were done in four independent trials, each consisting of two to four pollinated stigmas.

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SRK and SCR were shown to interact in vitro by “pull-down” assays (Fig. 2A) in which eSRK6 immobilized on FLAG-affinity agarose was treated with increasing amounts of SCR6-myc-His6, and the complexes were subjected to immunoblot analysis (13). In addition, an enzyme-linked immunosorbent assay (ELISA) (14) showed that eSRK6 bound SCR6 in a concentration-dependent manner (Fig. 2B), with a dissociation constant (Kd) of 0.4 × 10−10 M. In contrast, eSRK6 bound poorly to SCR13, even at high concentrations of SCR13-myc-His6 (Fig. 2B). The ∼10-fold stronger binding of eSRK6 to self SCR6 than to non-self SCR13 demonstrates the specificity of the in vitro SRK-SCR interaction and provides a molecular basis for haplotype-specificity in the SI response.

Figure 2

Binding of SCR to the SRK ectodomain. (A) Protein blot analysis of the interaction between eSRK6-FLAG and SCR6-myc-His6. eSRK6-FLAG (750 ng) immobilized on affinity agarose was incubated with increasing amounts of SCR6-myc-His6 [50 ng (lane 3), 100 ng (lane 4), 500 ng (lane 5), 1 μg (lane 6), and 2 μg (lane 7)]. Immunoblots of bead-protein complexes were treated with antibodies to myc (top panel) to detect bound SCR or with MabH8 (lower panel) to confirm that all bead complexes contained equivalent amounts of eSRK6-FLAG. SCR6-myc-His6 (1 μg), used as positive control (lane 1), did not bind anti-FLAG agarose (lane 2). (B) ELISA of SRK-SCR binding. eSRK6-FLAG (0.5 μg) exhibited high affinity for SCR6-myc-His6(triangles) but weak affinity for SCR13-myc-His6 (circles). Values represent an average of three experiments. (C) Interaction of eSRK6-FLAG with pollen SCR6. eSRK6-FLAG-agarose was incubated withS6 pollen coat proteins and subjected to nonreducing SDS-PAGE (13). Anti-SCR6 serum detects SCR6 as an ∼16-kD band (arrowhead) and two minor 6- to 8-kD bands that probably correspond to SCR monomers in bacterial extracts (lane 1) and as an ∼16-kD band in pollen coat protein extracts (lane 3). The serum also cross-reacts nonspecifically with several bacterial proteins that are also detected with preimmune serum (lane 2) and with three background bands (circles) in FLAG-agarose lacking eSRK6 (lane 4). Incubation of eSRK6-FLAG-agarose with increasing amounts ofS6 pollen coat protein [50 ng (lane 4), 100 ng (lane 5), 500 ng (lane 6), 1 μg (lane 7), 2 μg (lane 8), and 5 μg (lane 9)] demonstrates concentration-dependent binding of SCR6 above the background of nonspecific cross-reactive bands. (D) SCR6-myc-His6 pull-down assays. SCR6-myc-His6 was immobilized on Ni-NTA agarose and incubated with increasing amounts of either eSRK6-FLAG (eSRK6), microsomal extracts fromS6S6 (SRK6) andS13S13 (SRK13) stigmas, or soluble extracts from S6S6 (SLG6) and S2S2 (SLG2 and SLR1) stigmas. Untreated eSRK6-FLAG (250 ng) served as positive control (lane 1), and untreated stigma proteins, which did not bind Ni-NTA agarose, served as negative control (lane 2). The amounts of eSRK6-FLAG used were 50 ng (lane 3), 100 ng (lane 4), 250 ng (lane 5), and 500 ng (lane 6). The amounts of stigma microsomal or soluble proteins used were 250 ng (lane 3), 500 ng (lane 4), 1 μg (lane 5), and 2 μg (lane 6). Bound proteins were visualized with specific antibodies (13). The two bands inS6S6 stigma microsomal fractions that bind SCR6-myc-His6 represent SRK6 (lower band, arrowhead), and SLG6oligomers (upper band) often detected in stigma extracts (18).

To assess whether eSRK6 also bound endogenous SCR6 from pollen, pollen coat proteins were extracted fromS6 pollen (15) and incubated with eSRK6 beads, and the resulting complexes were subjected to electrophoresis under nonreducing conditions (13). Under these conditions, purified SCR6-myc-His6molecules migrated as a ∼16-kD band, likely consisting of homodimers, with only a small fraction migrating as monomers (Fig. 2C, lane 1). The antibodies to SCR6 also reacted with ∼16-kD molecules in pollen coat extracts (Fig. 2C, lane 2), and these cross-reactive molecules were bound by eSRK6 in a concentration-dependent manner (Fig. 2C, lanes 4 through 9). It is not known whether these SCR homodimers represent the native state of SCR in pollen and whether homodimerization is required for the binding of the SCR to SRK in vivo. These experiments demonstrate that the SRK ectodomain interacts with endogenous pollen SCR and provide biochemical evidence that SCR is a component of the pollen coat, as suggested by the expression pattern of the SCR gene (5, 6,16) and the immunolocalization of SCR to the pollen surface (10).

The SRK-SCR interaction was confirmed by reversing the pull-down assay. Recombinant SCR6 immobilized on Ni-agarose beads bound eSRK6 from N. benthamiana leaves (Fig. 2D). Furthermore, these SCR6 beads bound endogenous SRK6 in S6S6 stigma microsomal fractions (17, 18) but did not bind SRK13 from S13S13 stigma microsomes (Fig. 2D). Similarly, recombinant SCR13immobilized on Ni-agarose beads bound to stigma SRK13but not to SRK6. Thus, according to our in vitro binding assays, SCR can discriminate between allelic forms of SRK that share a substantial amount (∼90%) of amino acid sequence identity. The SCR6 beads also bound—albeit poorly—to SLG6, an abundant glycoprotein component of the cell wall of stigma epidermal cells that exhibits 89% sequence identity with the SRK6 ectodomain and, like SRK, is encoded by the Slocus and exhibits extensive S haplotype-associated polymorphism (19, 20). SLG has been shown to migrate on SDS-PAGE as a cluster of molecular mass forms (Fig. 2D) (21). However, only one of these SLG6 forms bound to SCR6 beads, suggesting a degree of specificity in the observed SLG6-SCR6 binding. Additional evidence for specificity is provided by the finding that treatment ofS2S2 stigma extracts with SCR6beads failed to pull down detectable levels of SLG2or of the S-locus related SLR1 andSLR2 gene products (Fig. 2D), all of which exhibit only 65% amino acid sequence identity to SLG6. SLG is thought to function, at least in some cases, as an accessory molecule that enhances the SRK-mediated SI response (3), possibly by contributing to the stabilization and proper maturation of SRK (18). The interaction observed between SCR and SLG suggests that some forms of SLG might also function in ligand binding. However, the physiological importance of this relatively weak interaction remains to be determined.

Our results demonstrate that SCR interacts with the ectodomain of SRK. Apparently, the SRK-SCR interaction does not require additional components specific to the stigma and pollen surfaces, because the interaction was observed between recombinant proteins purified fromNicotiana leaves and bacteria. The data indicate that specificity in the SI response results from Shaplotype-specific molecular interaction of SCR and SRK, which would selectively trigger activation of self SRK and a pollen-inhibitory chain of events. Analysis of receptor-ligand interactions demonstrated by SRK-SCR and by CLV1-CLV3 of Arabidopsis thaliana(22) should provide useful paradigms for the study of transmembrane receptor signaling and of the function and regulation of small diffusible peptide ligands in plants.

  • * Present Address: SunGene GmbH, Corrensstrasse 3, D-06466, Gatersleben, Germany.

  • To whom correspondence should be addressed. E-mail: jnb2{at}cornell.edu

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