A receptor heteromer mediates the male perception of female attractants in plants

  • A Corrigendum to this article was published on 11 May 2016

Abstract

Sexual reproduction requires recognition between the male and female gametes. In flowering plants, the immobile sperms are delivered to the ovule-enclosed female gametophyte by guided pollen tube growth. Although the female gametophyte-secreted peptides have been identified to be the chemotactic attractant to the pollen tube1,2,3, the male receptor(s) is still unknown. Here we identify a cell-surface receptor heteromer, MDIS1–MIK, on the pollen tube that perceives female attractant LURE1 in Arabidopsis thaliana. MDIS1, MIK1 and MIK2 are plasma-membrane-localized receptor-like kinases with extracellular leucine-rich repeats and an intracellular kinase domain. LURE1 specifically binds the extracellular domains of MDIS1, MIK1 and MIK2, whereas mdis1 and mik1 mik2 mutant pollen tubes respond less sensitively to LURE1. Furthermore, LURE1 triggers dimerization of the receptors and activates the kinase activity of MIK1. Importantly, transformation of AtMDIS1 to the sister species Capsella rubella can partially break down the reproductive isolation barrier. Our findings reveal a new mechanism of the male perception of the female attracting signals.

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Figure 1: Expression of MDIS1, MDIS2, MIK1 and MIK2 and their mutant phenotype.
Figure 2: MDIS1, MIK1 and MIK2 are LURE1 receptors.
Figure 3: MDIS1 and MIKs synergistically perceive LURE1.
Figure 4: AtMDIS1 breaks down the reproductive isolation between A. thaliana and C. rubella.

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Acknowledgements

We thank L. Qu for technique assistance in the pollen tube guidance assay. We thank Y. Guo, Quantum Design Inc. China, and core facilities of public technology service centre of Institute of Microbiology and Institute of Genetics and Developmental Biology (Chinese Academy of Sciences) for the MST measurement. We thank J. Zhou and Q. Xie for sharing seeds. This work was supported by the Ministry of Science and Technology of China grants 2013CB945103 to W.-C.Y. and 2015CB910202 to H.-J.L. and the National Natural Science Foundation of China 31330053 and 31221063 to W.-C.Y.

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Authors

Contributions

H.-J.L. and W.-C.Y. designed the study, interpreted the results and wrote the paper. T.W. performed most of the experiments. L.L. performed the guidance assay. Y.X. performed the mutants screening. M.-X.Z. performed the LURE1 construction. P.-F.J. performed the cell biology analysis and W.C. performed the qPCR experiments. Y.-C.W. performed mass spectrometry analysis.

Corresponding authors

Correspondence to Hong-Ju Li or Wei-Cai Yang.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Pollen tubes expressing MDIS1DN shows micropylar guidance defect.

a, Phylogenetic tree of the analysed RLKs expressed in pollen (tubes). b, Protein structure of MIK1, MIK2 and MDIS1. Green box, leucine-rich repeats; red, signal peptide and transmembrane domain; yellow, kinase domain; blue, proline-rich domain; purple, linker region. c, Schematic diagram of dominant-negative construct of MDIS1 driven by the pollen-specific promoter LAT52. ECD, ectodomain; TM, transmembrane domain of MDIS1. The kinase domain of MDIS1 was replaced by the dead kinase domain of BRI1 with an AAG-to-GAG site mutation. d, The wild-type pollen tube (arrow) enters the micropyle opening directly. Images are representative of 30 images captured. e, The pollen tube (arrow) from the MDIS1DN transgenic plants exhibits defective micropylar guidance to the wild-type ovules. Images are representative of 30 images captured. Asterisks in d and e represent micropyles. Scale bars, 50 μm. f, Percentage of wild-type ovules with micropylar guidance defect minimally pollinated with pollen from six independent hemizygous and homozygous MDIS1DN transgenic lines. Error bars, s.e.m. of 3 independent replicates; **P < 0.01 (Student’s t-test); n = 300 for each sample. g, Fertilization efficiency of the pollen tubes from the six MDIS1DN hemizygous and homozygous lines. The ratio of numbers of successfully targeted pollen tubes to the pollen tubes in the styles was calculated from 30 minimally pollinated pistils. Error bars, s.e.m. of 3 independent replicates; **P < 0.01 (Student’s t-test); n = 200 for each sample. h, MDIS1 interacts with MIK1 and MIK2 as shown by dual membrane yeast two-hybrid system. Yeasts were co-transformed with bait construct MDIS1-Cub and prey construct MIK1-NubG or MIK2-NubG, and the transformants were grown on selective media.

Extended Data Figure 2 MDIS1, MDIS2, MIK1 and MIK2 are expressed in the pollen tubes.

a, Time-lapse images showing the dynamic distribution of MDIS1–GFP and MDIS2–GFP during pollen tube growth in vitro. Images are representative of 30 images captured. Scale bars, 10 μm. b, c, Histological GUS staining of seedlings transformed with MDIS1- and MDIS2-GUS under the native promoters, respectively. Images are representative of 20 images captured. Scale bars, 5 mm. d, Quantitative PCR (qPCR) showing the expression of MDIS1, MDIS1, MIK1, MIK2 and PXY in pollen, pollen tubes and seedlings. Error bars, s.e.m of 3 independent replicates. e, Specificity test of MIK1 and MIK2 antibodies with Arabidopsis protoplasts expressing Flag-tagged MIK1 and MIK2. Equal amount of Arabidopsis MIK–Flag-transformed (T) or wild-type (untransformed; UT) protoplasts were lysed and subjected to immunoblotting. Anti-MIK1 and anti-MIK2 recognize the corresponding protoplasts-expressed Flag fusion proteins specifically. f, The target protein was recognized by anti-MIK1 and anti-MIK2 in the wild-type pollen, but not in the corresponding mutants. Total protein of the same amount of pollen grains from the wild type and mutants were subjected to SDS–PAGE and immunoblot. Arrows denote target proteins.

Extended Data Figure 3 Pollen performance of the mutants.

a, Schematic representation of gene structure of MDIS1, MDIS2, MIK1 and MIK2 and the T-DNA insertion site. The T-DNA insertion positions are indicated by triangles. Filled boxes, exons; open boxes, untranslated region; lines, introns. b, Expression of the transcripts in the opening flowers of the wild-type and corresponding mutants. c, Representative images of pollen tube length of the corresponding mutants grown in the wild-type pistils at 3, 6 and 8 h after pollination (HAP). Arrows indicate the points the bulk of the pollen tubes reached. Images are representative of 60 images captured. Scale bars, 200 μm. d, Pollen tube length of mdis1 mdis2, mik1, mik2 and mik1 mik2 is comparable to the wild type. n = 60 pistils for each sample; P > 0.1 (Student’s t-test); n.s., not significant. Error bars, s.e.m. of 3 independent measurements. e, The in vitro pollen germination of mdis1 mdis2, mik1, mik2 and mik1 mik2 is normal. Error bars, s.e.m. of 3 independent replicates; P > 0.1 (Student’s t-test); n = 300 for each sample. f, g, The ratio of earlier to later fertilized wild-type ovules targeted by the mik1 mik2 and mik1 mik2/+ pollen tubes is higher than by the wild-type pollen tubes. Approximately 40 pollen tubes were hand-pollinated on the wild-type stigma, which was then subjected to aniline blue staining 30 HAP. Left panel in f represents image of the earlier fertilized ovules; right panel represents image of the later fertilized ovules in the same silique. Arrow denotes the enlarged ovule. Scale bars, 20 μm. g, Statistics of results shown in f. n, numbers of ovules scored. Error bars, s.e.m. of 3 independent replicates; *P < 0.05, ***P < 0.001 (Student’s t-test).

Extended Data Figure 4 Verification of the predicted disulfide bonds by mass spectrometry.

Disulfide bonds of the purified MDIS1ECD, MIK1ECD and MIK2ECD were identified at Cys193–Cys201 of MDIS1, Cys60–Cys67 of MIK1 and Cys683–Cys695 of MIK2. Cys64 of MDIS1, Cys609 and Cys616 of MIK1 were at the oxidized form.

Extended Data Figure 5 LURE1.2 induced the endocytosis and decrease of MDIS1–GFP in the pollen tube tip.

a, Binding affinity between ERECTA and TfLURE2 by MST. Error bars, s.e.m. of 3 independent measurements. be, Confocal images showing the distribution of MDIS1–GFP before LURE1.2 (0.5 μM) treatment (b), and at 0 min (c), 20 min (d) and 60 min (e) after treatment. Images are representative of 63 images captured. Intensity plots along the red lines of each image are shown below. Scale bars, 5 μm. The maximum y-axis values are the same for all intensity plots. The arrows indicate the signal accumulation at the plasma membrane. Scale bars, 5 μm. f, g, His–MIK1ECD and His–MIK2ECD specifically bind GST–MDIS1ECD, but not the GST affinity beads. Full blots are shown in Supplementary Data.

Extended Data Figure 6 LURE1.2 is perceived by the MDIS1–MIK complex.

af, Confocal images of tobacco leaf showing stronger bimolecular fluorescence complementation signal in the presence of LURE1.2–Flag (a, d) as compared with the weak signal in the absence of LURE1.2–Flag (b, e). cf, Quantification of the total fluorescence signal of the same areas. Error bars, s.e.m. of 3 independent replicates; *P < 0.05 (Student’s t-test). Five leaves with positive signal were analysed for each experiment. Scale bars, 50 μm. g, Anti-LURE1 and anti-Flag antibodies recognize the LURE1–Flag fusion protein. h, Endogenous interaction between LURE and MIK1 or MIK2 by LURE antibody with the total crude proteins extracted from the wild-type pollinated flowers (8 HAP), but not with the mik1 mik2 mutant. Arrow denotes target proteins. Full blots are shown in Supplementary Data.

Extended Data Figure 7 Ion-trap MS/MS spectra identifying phosphorylation sites of the kinase domain of MDIS1 and MIK1.

Identification of one phosphorylation site for MDIS1 (Ser663) and eight for MIK1 (Thr741, Thr742, Thr862, Ser864, Thr710, Tyr879, Thr880 and Thr992) by ion-trap liquid chromatography tandem mass spectrometry (LC–MS/MS).

Extended Data Figure 8 Expression pattern of homologues of MDIS1, MIK1 and MIK2 in C. rubella and E. salsugineum by RT–PCR analysis.

a, CrMDIS1, but not CrMIK1 or CrMIK2, is expressed in pollen of C. rubella. b, EsMDIS1, but not EsMIK1 or EsMIK2, is expressed in pollen of E. salsugineum. ACTIN11 transcripts were amplified as controls. Genomic DNA was used as the control for primer specificity.

Extended Data Table 1 Segregation analysis of MDIS1DN represented by segregation ratio of hygromycin resistance (R) to sensitivity (S) with T2 MDIS1DN lines carrying a single T-DNA insertion
Extended Data Table 2 Transmission efficiency test of mdis1 and mdis2 by reciprocal crosses

Supplementary information

Supplementary Information

This file contains full blots of the gels. (PDF 629 kb)

Video 1: Fluorescence dynamics of MDIS1-GFP in the pollen tube

This video shows the time-lapse imaging of the dynamic localization of MDIS1-GFP in the pollen tube. (WMV 700 kb)

Video 2: Fluorescence dynamics of MDIS2-GFP in the pollen tube

This video shows the time-lapse imaging of the dynamic localization of MDIS2-GFP in the pollen tube. (WMV 1427 kb)

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Wang, T., Liang, L., Xue, Y. et al. A receptor heteromer mediates the male perception of female attractants in plants. Nature 531, 241–244 (2016). https://doi.org/10.1038/nature16975

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