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Conserved Molecular Components for Pollen Tube Reception and Fungal Invasion

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Science  12 Nov 2010:
Vol. 330, Issue 6006, pp. 968-971
DOI: 10.1126/science.1195211

Abstract

During sexual reproduction in flowering plants such as Arabidopsis, a tip-growing pollen tube (PT) is guided to the synergid cells of the female gametophyte, where it bursts and releases the two sperm. Here we show that PT reception and powdery mildew (PM) infection, which involves communication between a tip-growing hypha and a plant epidermal cell, share molecular components. NORTIA (NTA), a member of the MLO family originally discovered in the context of PM resistance, and FERONIA (FER), a receptor-like kinase, both control PT reception in synergids. Homozygous fer mutants also display PM resistance, revealing a new function for FER and suggesting that conserved components, such as FER and distinct MLO proteins, are involved in both PT reception and PM infection.

Successful pollination and fertilization depend on interactions between the male gametophyte (pollen) and female tissues (1). Pollen lands on a stigma and hydrates, and the pollen tubes (PTs) grow toward the ovary, where they exit the transmitting tissue and one PT is attracted to each ovule. Until Amici discovered in the 19th century that PTs enter the ovule through the micropyle and play a role in reproduction (2), these tip-growing cells were considered parasites. The final stage of maternal control over male gamete delivery is the arrival of the PT at the female gametophyte (embryo sac) and the release of the two sperm to effect double fertilization, which forms the zygote and the endosperm. In angiosperms, the synergids control the final steps of PT guidance and reception (3). They excrete PT attractants such as the LURE polypeptides (4), and, upon PT arrival at the micropyle, the receptive synergid starts to degenerate (5) and signals the PT to stop its growth and rupture to release the sperm (6, 7). Over 100 years have passed since the discovery of double fertilization (3), but we still know little about the underlying molecular mechanisms behind the female control of PT behavior.

In Arabidopsis, the FERONIA (FER) CrRLK1L-type receptor-like kinase (RLK) controls PT reception. It is localized to the filiform apparatus, a membrane-rich region at the micropylar end of the synergids (8). In fer (which is allelic to sirène) mutants, PTs enter the synergid cell but continue to grow instead of bursting to release the sperm (68). In our search for additional components of this signaling pathway, we identified nortia (nta), which like fer was named after an Etruscan goddess of fertility (9). Like fer, nta is a female gametophytic mutant and displays reduced fertility and PT overgrowth in the synergids (Fig. 1, A to D). Consequently, in reciprocal crosses with wild-type individuals, the nta phenotype is detected only when the mutant is used as the female (fig. S1). In contrast to fer/FER mutants, which have close to 50% unfertilized ovules because of a failure in PT reception and are rarely transmitted through the female gametophyte (68), nta mutant alleles are transmitted normally through male gametophytes and at frequencies of 60 to 80% through female gametophytes (table S1), allowing for the isolation of homozygous mutants. nta-1/nta-1 individuals have 22% (n = 1506 ovules) unfertilized ovules with PT overgrowth, compared to 4% (n = 1076) unfertilized ovules (without PT attraction) in the wild type.This indicates that nta embryo sacs differentiate normally, as evidenced by examination of egg- and synergid-specific β-glucuronidase (GUS) markers and microscopic examination of nta mutant embryo sacs (fig. S2).

Fig. 1

NTA is an MLO protein involved in PT reception at the female gametophyte. (A) Wild-type silique with fertilized ovules developing into seeds. (B) nta/nta silique with unfertilized (shrunken white ovules, arrow) and fertilized ovules. (C and D) Aniline blue staining of callose in PT cell walls at 2 days after pollination. (C) Fertilized wild-type ovule with normal PT reception. (D) Unfertilized nta/nta ovule with PT overgrowth (arrow). (E) Gene structure of NTA with exons (boxes) and introns (lines). Mutations are indicated by inverted triangles: Insertions are dashed and deletions are solid. bp, base pair. (F) Predicted domains in the NTA protein (yellow, signal peptide; gray, transmembrane domains; red, calmodulin-binding domain). Black bars indicate insertion and deletion sites. a.a., amino acid. Scale bars in (A) and (B), 1 mm; in (C) and (D), 100 μm.

Map-based cloning identified the NTA gene as At2g17430, or AtMLO7, a member of the plant-specific Mildew resistance locus o (MLO) family of proteins originally discovered to be required for powdery mildew (PM) susceptibility in barley (10). MLO proteins are predicted to have a signal peptide, seven transmembrane domains, and a calmodulin-binding domain in the C-terminal intracellular domain (Fig. 1, E and F). The nta-1 allele has a 20–base pair deletion at the exon 13/intron 13 boundary, leading to failed splicing and the introduction of an early STOP codon, predicted to truncate the protein to 436 rather than the expected 543 amino acids. Two additional nta alleles with transfer DNA insertions in the first exon (nta-2) and the last exon (nta-3) had the same PT overgrowth phenotype as nta-1 at similar frequencies (fig. S3), indicating that the correct gene had been identified and that the low expressivity of the nta phenotype was not caused by expression of a truncated protein. Genetic redundancy could play a role in the weak phenotype seen in nta mutants. The Arabidopsis genome has 15 members of the MLO gene family (11), and double or triple mutants in some of these genes have stronger phenotypes than single mutants (12, 13). In addition to NTA, transcripts of phylogenetically related AtMLO8 and AtMLO10 are expressed in synergids (14), but mutants in these genes have no PT overgrowth phenotypes (table S2).

The expression pattern of NTA was examined with translational fusions of the NTA genomic sequence to genes encoding GUS and green fluorescent protein (GFP) under control of the native NTA 5′-regulatory sequences. In stable NTA-GUS transformants, expression was detected only in the synergids (Fig. 2A). A previous study on the expression of Arabidopsis MLO genes had detected NTA only in pollen, based on a promoter::GUS fusion lacking the introns and coding sequence of NTA (11). Using reverse transcription polymerase chain reaction on immature flowers, we detected NTA transcript in pistils and immature anthers (up to stage 12), but not in mature pollen (fig. S4), suggesting that not all of the elements regulating NTA expression are present in the promoter. The NTA-GFP fusion construct complemented the nta phenotype in 37 independent transformants (table S3), indicating that the fusion protein is fully functional. The NTA protein has multiple predicted transmembrane domains and is expected to localize to the plasma membrane. Transient expression of an NTA-GFP fusion protein under the constitutive 35S promoter showed NTA-GFP fluorescence at the plasma membrane in onion cells (fig. S5). However, in stable Arabidopsis transformants with the native NTA promoter, NTA-GFP fluorescence was detected in a punctate pattern throughout the cytoplasm of both synergids in unfertilized, mature female gametophytes, possibly reflecting its association with endomembrane compartments (Fig. 2, B and C, and fig. S5, E and F). Limited pollinations were performed to compare NTA-GFP localization in ovules with and without PTs at 5 to 7 hours after pollination, which is the time when PT reception occurs. NTA-GFP became polarly localized to the basal half of the synergids upon PT arrival at the micropyle (Fig. 2, D to F). The redistribution of NTA-GFP to the filiform apparatus upon PT arrival indicates that relocalization of NTA may be important for signaling during PT reception.

Fig. 2

NTA is expressed in the synergids of the female gametophyte and becomes polarly localized upon arrival of the PT. (A) Expression of pNTA::NTA-GUS is detected only in synergids (blue signal). (B) Differential interference contrast (DIC) image of the female gametophyte (white, synergids; yellow, egg cell; red, central cell). (C) NTA-GFP (green signal) is found throughout the synergids in unfertilized ovules. (D) DIC image of an ovule with a PT arriving. (E) NTA-GFP (green signal) is redistributed to the micropylar end of the synergids at PT arrival (red signal, arrow). (F) Percentage of synergid length occupied by NTA-GFP signal before and after PT arrival (white bar, no PT; green bar, after PT arrival). Scale bars, 50 μm.

Because mutations in NTA and FER have similar phenotypes and they are both expressed in synergids, we wondered if their expression and/or localization patterns are interdependent. Expression of a functional pFER::FER-GFP fusion protein was examined in nta-1 homozygous versus wild-type plants. In both cases, FER-GFP was localized to the filiform apparatus (Fig. 3, A to D), indicating that NTA is not involved in the polar localization of FER. Likewise, a pNTA::NTA-GFP fusion protein showed the same distribution in all ovules of fer/FER plants before pollination, indicating that the fer mutation does not affect NTA-GFP expression and localization in mature, unfertilized embryo sacs (Fig. 3, E and F). However, in contrast to the polar localization of NTA-GFP seen in the wild type upon PT arrival (Fig. 2, D to F), in fer mutant embryo sacs, NTA-GFP did not become polarly localized (Fig. 3, G to H). This indicates that the FER pathway plays a role in controlling the redistribution of NTA protein upon PT arrival.

Fig. 3

NTA has no effect on FER localization, but FER controls polar localization of NTA at PT arrival. (A), (C), (E), and (G) Overlayed DIC and fluorescent marker images of FER-GFP and NTA-GFP localization. (B), (D), (F), and (H) Synergids from the boxed regions of ovules shown in (A) to (C). (A and B) FER-GFP (green signal) is localized at the filiform apparatus at the micropylar end of the synergids in wild-type ovules. (C and D) Normal localization of FER-GFP at the micropylar end of an nta ovule. (E and F) NTA-GFP (green signal) in fer ovules shows normal localization throughout the synergids before fertilization. (G and H) NTA-GFP is not polarly localized in a mutant fer embryo sac with PT overgrowth (red signal). Scale bars in (A), (C), (E), and (G), 30 μm; in (B), (D), (F), and (H), 10 μm.

The identification of NTA as a member of the MLO gene family provides an interesting link between PT reception and fungal invasion. PT reception and fungal invasion of plant cells share some common features. First, both fungal hyphae and PTs show tip growth and must communicate with and penetrate into another cell. Second, polar localization of MLO proteins is correlated with the entry processes of both fungal hyphae (15) and PTs. Finally, the control of PT behavior by synergids is an angiosperm innovation (16), and PM has not been reported to infect plants outside of the angiosperm lineage (17).

The first member of the MLO gene family described in plants was the barley (Hordeum vulgare) HvMlo gene, involved in PM resistance (10). In contrast to resistance (R) gene–mediated resistance, in which a pathogen avirulence factor is recognized by a plant R protein, which triggers an immune response in a gene-for-gene manner (18), mlo-based PM resistance is due to mutation of a “susceptibility” gene (10, 19). During fungal invasion, HvMLO and other membrane-associated proteins colocalize at the site of fungal infection (15), creating a pathogen-triggered membrane microdomain that might be related to exosomal transport processes. Recessive mutations in the Arabidopsis HvMlo ortholog AtMLO2 (but not in AtMLO7/NTA) (12) also lead to partial PM resistance that is enhanced in double and triple mutants with AtMLO6 and/or AtMLO12, indicating that the function of MLO proteins in PM invasion is ancient and has been conserved since the monocot/dicot divergence, estimated at around 200 million years ago (20).

Because MLO proteins are involved in both PT reception and PM invasion, we wondered if another component of the PT reception mechanism, the FER RLK, is also involved in PM invasion of plant cells. Unlike NTA, whose expression is restricted to the synergids, FER is expressed not only in the filiform apparatus but in all plant tissues except mature pollen (8). In addition to its role in PT reception (8), FER has recently been reported to cooperate with two other CrRLK1Ls—THESEUS (THE) and HERCULES RECEPTOR KINASE 1 (HERK1)—to control cell elongation (21). Indeed, fer/fer plants are smaller than wild-type siblings but have normal floral development except for an almost complete failure of PT reception, with very few seeds developing in each silique (fig. S6).

PM infection begins when a fungal spore lands on an epidermal cell. Subsequently, the spore hydrates, germinates, and penetrates the plant cell wall to establish a feeding structure (haustorium) within the host cell. The vegetative life cycle of the fungus is completed by the release of asexual spores from the conidiophores, which can spread the infection (22). fer/fer plants were tested for susceptibility to infection by the PM Golovinomyces (syn. Erysiphe) orontii, which is virulent on Arabidopsis accession Landsberg erecta (Ler), by measuring the incidence of entry of PM spores and the production of conidiophores per PM colony. When compared to wild-type plants, fer/fer mutant plants were resistant to PM infection, with somewhat lower entry rates and significantly fewer conidiophores produced per fungal colony (Fig. 4 and fig. S7). This phenotype was rescued in a transgenic line with a functional FER-GFP fusion protein expressed in the fer/fer background (Fig. 4). Thus, like MLO, a wild-type copy of FER seems to be necessary for PM susceptibility. The fer-mediated PM resistance is not caused by a general resistance to pathogens, because fer plants have normal susceptibility to the oomycete Hyaloperonospora arabidopsidis and the ascomycete Colletotrichum higginsianum (fig. S8).

Fig. 4

Homozygous fer mutants are resistant to PM infection. (A, C, and E) Photographs of PM-infected plants 10 days after inoculation. PM infection (white fungal mycelium and conidiophores on leaves) is evident in wild-type Ler (A) and the complemented mutant (E), whereas the fer/fer plant (C) appears to be resistant. (B, D, and F) Close-ups of individual leaves of the plants shown in the respective left panels. Leaves shown in (B) and (F) are enlarged twofold, whereas the leaf shown in (D) is enlarged fourfold compared to (A), (C), and (E). (G) Quantitative assessment of fungal pathogenesis based on host cell entry (light gray bars) and conidiation (dark gray bars). Values in the graph indicate mean ± SD based on four (host cell entry) and three (conidiation) experiments, respectively. Asterisks indicate statistically significant differences from the wild type according to Student’s t test (*P < 0.05, **P < 0.01); the hatch mark signifies a statistically significant difference from the complementation line (P < 0.05).

fer and mlo mutants share several phenotypic similarities suggesting that FER and various MLO genes function together to control PM invasion and PT reception. In fer and nta mutants, PTs exhibit normal entry into the receptive synergid but do not complete their “invasion” process, failing to burst and release the sperm. Likewise, the PM resistance phenotype in fer plants is predominantly manifested at the post-invasive stage: Host cell entry is not affected as severely as the establishment of successful colonies that produce conidiophores (Fig. 4G and fig. S7), a phenotype reminiscent of the Atmlo2 resistance phenotype (12). Disease resistance in plants is often mediated through up-regulation of hormone signaling pathways, followed by a hypersensitive response (HR), in which cells undergo cell death (18, 23). mlo mutants in barley and Arabidopsis exhibit stochastic cell death of mesophyll cells accompanied by increased production of H2O2 in the absence of any pathogen (12, 24). Unchallenged fer leaves have a similar phenotype of spontaneous cell death and H2O2 production (fig. S9). Salicylic acid signaling does not seem to be affected in fer mutants; however, the ethylene/jasmonic acid defense pathways could be involved in both mlo- and fer-mediated resistance (supporting online text and fig. S10).

The mechanism for mlo-mediated resistance remains unclear, but MLO proteins seem to modulate SNARE-dependent and vesicle transport–associated processes at the plasma membrane (15, 25). Thus, MLO could be involved in delivering cargo such as regulatory proteins to the plasma membrane. A similar mechanism can be proposed for the control of PT behavior by FER and NTA. FER would sense the arrival of the PT and initiate a signal transduction cascade leading to relocalization of NTA-containing vesicles to the filiform apparatus. Recently, the maize defensin ZmES4, required for PT rupture, was shown to relocalize to the filiform apparatus at PT arrival (26). A homologous Arabidopsis defensin would be a candidate for cargo being delivered to the filiform apparatus in response to a FER/NTA-mediated signal.

Because FER is ubiquitously expressed in all plant tissues except for mature pollen (8), whereas the 15 AtMLO genes are expressed in different tissues and in response to various stimuli (11, 13), FER might cooperate with diverse members of the MLO family in a tissue-specific manner. The ancestral function of FER and MLO proteins is an open question, but the presence of genes with high sequence-relatedness to FER and MLO in bryophytes (27, 28) suggests that these proteins were either originally implicated in processes distinct from PT reception and PM susceptibility, or that their cooperativity developed later in the course of evolution. As both proteins are required for compatible interactions with either PTs or fungal hyphae, their original role may have been in mediating susceptibility to symbiotic organisms such as mycorrhizal fungi, an interaction that was important for land plant evolution but no longer occurs with Arabidopsis. Because FER is necessary for successful fertilization, deleterious mutations that would also lead to PM resistance would be selected against. Other players in PT reception (29, 30) may also be involved in PM infection, or, alternatively, different accessory molecules may be used in conjunction with FER and the MLOs in distinct response pathways.

Successful seed production and durable resistance to pathogens are two of the most important agronomic traits sought after today. The work presented here has unveiled unforeseen similarities between these processes at the molecular level. As we continue to unravel the signal transduction processes involved in intercellular communication during PT reception, we may also gain important insights into PM resistance mechanisms and vice versa.

Supporting Online Material

www.sciencemag.org/cgi/content/full/330/6006/968/DC1

Materials and Methods

SOM Text

Figs. S1 to S11

Tables S1 to S3

References

  • § Present address: Laboratoire de Reproduction et Développement des Plantes, 46 Allée d'Italie, 69364 Lyon Cedex 07, France.

References and Notes

  1. We thank A. Boisson-Dernier for providing pLat52:DsRED and for critical reading of this manuscript; S. Schauer for providing plasmids; A. Reinstädler for technical assistance with pathogen assays; and J. M. Escobar-Restrepo, H. Lindner, M. Bayer, C. Jäger-Baroux, S. Pien, V. Gagliardini, and members of the Grossniklaus lab for helpful discussions. This work was supported by the University of Zürich, the Max Planck Society, fellowships of the Human Frontiers in Science Program (to S.A.K.), the Forschungskredit of the University of Zürich (to H.S.-A.), the International Max Planck Research School (to N.F.K.), the Royal Society of London (to G.I.), and grants from the Deutsche Forschungsgemeinschaft (SFB670 to R.P.) and the Swiss National Science Foundation (to U.G.).
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