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A Membrane-Anchored Protein Kinase Involved in Brassica Self-Incompatibility Signaling

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Science  05 Mar 2004:
Vol. 303, Issue 5663, pp. 1516-1519
DOI: 10.1126/science.1093586

Abstract

Self-incompatibility (SI) response in Brassica is initiated by haplotype-specific interactions between the pollen-borne ligand S locus protein 11/SCR and its stigmatic S receptor kinase, SRK. This binding induces autophosphorylation of SRK, which is then thought to trigger a signaling cascade that leads to self-pollen rejection. A recessive mutation of the modifier (m) gene eliminates the SI response in stigma. Positional cloning of M has revealed that it encodes a membrane-anchored cytoplasmic serine/threonine protein kinase, designated M locus protein kinase (MLPK). Transient expression of MLPK restores the ability of mm papilla cells to reject self-pollen, suggesting that MLPK is a positive mediator of Brassica SI signaling.

Self-incompatibility (SI) is a genetic system in flowering plants that promotes outcrossing by rejecting self-related pollen (1). In Brassica, recognition of pollen is controlled by S haplotypes (designated S1, S2,... Sn), each of which consists of the pistil determinant gene, the S receptor kinase (SRK) (2, 3), and the pollen determinant gene, the S locus protein 11 (SP11, also called SCR) (46). SP11/SCR interacts with the SRK of the same S haplotype and activates its kinase domain (7, 8). This activation is thought to elicit a signaling cascade within the stigmatic papilla cell, leading to rejection of self-pollen. One candidate for the signaling component is ARC1, an arm repeat-containing protein with E3 ubiquitin ligase activity (9, 10). Stone et al. showed that down-regulation of the ARC1 gene by its antisense transgene results in appreciable seed set upon self-pollination, demonstrating that ARC1 functions in the SI response (11). However, the breakdown of SI in transgenic plants was only partial, suggesting that it could be attributed to the incomplete suppression of ARC1 or the presence of another signaling pathway (11). The recessive mutation modifier (m) was identified from a cross between a self-incompatible B. rapa S8 homozygote (S8S8MM) and the self-compatible B. rapa variant Yellow Sarson C634 (Sf2Sf2mm), cultivated in India (12). The M locus is independent of and epistatic to the S locus, and S8S8mm plants exhibit absence of SI response in the stigma but not in the pollen (Fig. 1A) (12), suggesting that M encodes a key effector acting downstream of SRK. It has previously been proposed that M is an aquaporin-like gene MIP-MOD (13), but recent observations contradict this hypothesis, and the true identity of the M gene product has remained unknown (14, 15).

Fig. 1.

Positional cloning of Brassica M gene. (A) Brassica mm plants show defective SI response. Pollen tube growth 6 hours after self-pollination in S8S8MM (left) and S8S8mm (right) plants. Arrow indicates penetrated pollen tubes. Bar indicates 100 μm. (B) Low-resolution genetic map of the M locus. A3 to A82, AFLP markers. (C) Genetic map of the M locus. Dotted line indicates deleted region of the m1-4 mutant. (D) Physical map of M locus based on BAC contig. B1 to B7, sequence-based markers derived from BAC end sequences or a BAC 251-22 sequence. Number of recombinants is shown below each marker, restricting the M locus between B3 and B6 markers. (E) Genomic organization of the delimited M locus. Feature of ORFs predicted by BLAST search: ORF A, GST; B, serine/threonine protein phosphatase PP1; C, D, E, and F, tropinone reductase; G, protein kinase; H, hypothetical protein; I, retroelement pol polyprotein; J, RING finger protein; K, Ac-like transposase; L, histone H4. (F) Gene structure of MLPK. Arrow indicates the position of the m mutation. Arrowheads indicate the location of introns in the genomic sequence. GenBank accession number AB121973.

To identify the M gene by positional cloning, we first generated 28 markers around the M locus by amplified fragment length polymorphism (AFLP) using self-pollinated progeny from S8S8Mm plants (16). A series of genetic mappings based on 100 and 485 gametes (Fig. 1, B and C) placed the M locus at an interval of 0.8 centimorgan (cM) between the A23 and A40 AFLP markers. From these mappings, we obtained nine recombinants whose MIP-MOD/mip-mod genotype was not consistent with their M/m phenotype, indicating that MIP-MOD is not M. To further analyze the M locus, we generated the M locus deletion mutant m1-4 by γ irradiation. However, genomic analysis of m1-4 revealed a large chromosomal deletion spanning the M locus to MIP-MOD (Fig. 1C). Because of this large deletion, we did not further analyze the m1-4 mutant.

Next, we constructed a physical map of the M locus with the use of the A23 and A40 markers as chromosome landing entry points. About 500 kilobase pairs (kbp) of B. rapa bacterial artificial chromosome (BAC) clone (17) contiguous sequence (contig) was built by multiple rounds of chromosome walking, and molecular markers for fine mapping were generated with the use of BAC end sequences (Fig. 1D). Further recombination analysis (2157 gametes in total) placed the M locus on BAC 251-22 (Fig. 1D). Therefore, we shotgun sequenced BAC 251-22 and generated additional sequence-based markers (Fig. 1E). Finally, the M locus was delimited to a 50-kbp region between markers B3 and B6. BLAST searches of this genomic region identified 12 putative open reading frames (ORFs) (Fig. 1E).

Because the M gene product is thought to function downstream of SRK, we first focused our attention on ORF-G, which we expected to encode a protein kinase (Fig. 1E). Comparison of genomic sequence and cDNA sequence from reverse transcription polymerase chain reaction predicted that the gene was composed of eight exons and seven introns encoding a 404–amino acid protein kinase, designated M locus protein kinase (MLPK). The deduced MLPK amino acid sequence contains the typical plant N-myristoylation motif, Met-Gly-XXX-Ser/Thr(Arg), where X is any amino acid [MGXXXS/T(R)] (18), a 30–amino acid serine-rich (33%) domain, and 11 protein kinase subdomains (19) (Fig. 1F). Database searches revealed that MLPK belongs to the receptor-like cytoplasmic kinase VII (RLCK VII) subfamily, which has a common monophyletic origin with receptor-like kinases but has no apparent signal sequence or transmembrane domain (20). Among 46 members of the RLCK VII subfamily in Arabidopsis, MLPK is most similar to APK1b (76% amino acid identity) (21), which is thought to be an Arabidopsis ortholog of MLPK on the basis of the genomic synteny between the Brassica M locus and Arabidopsis chromosome 2 APK1b region.

Northern blot analysis revealed that, among the tissues we examined, MLPK was predominantly expressed in stigma (Fig. 2A). MLPK expression in stigma was at basal levels during early developmental stages, but rapidly increased from 2 to 3 days before flowering (6- to 8-mm buds) and reached its highest level on the day of anthesis (Fig. 2A). This expression pattern of MLPK is similar to that of SRK (2) and is consistent with the stage at which the stigma acquires SI.

Fig. 2.

Characterization of MLPK gene products. (A) Northern blot analysis of MLPK transcripts. RNA (20 μg) from stigma (4- to 10-mm buds), anther, stem, leaf, and differential stages (0 to 2, 2 to 4, 4 to 6, 6 to 8, 8 to 10 mm, and open flower) of stigma was loaded in each lane. Top, MLPK cDNA probe; bottom, ethidium bromide staining. (B) In vitro autophosphorylation of MLPK. Top, autoradiogram of the autophosphorylated wild-type (WT) and mutant (G194R) recombinant MLPK proteins. Bottom, Coomassie Brilliant Blue staining. (C) Northern blot analysis of MLPK mRNA in wild-type (WT) and mm (m) stigmas. Stigma RNA (10 μg) isolated from open flowers was loaded in each lane. Top, full-length MLPK cDNA probe; bottom, ethidium bromide staining. (D) Western blot analysis of MLPK protein in wild-type (WT) and mm (m) stigmas. Stigma extracts equivalent to one stigma were loaded in each lane. MLPK was detected by polyclonal antibody to MLPK. (E) Membrane localization of MLPK. Cytosolic (C), microsomal (M), and plasma membrane (P) protein fractions (6 μg) from stigma were loaded in each lane and detected with polyclonal antibody against MLPK. Asterisk, cross-reactive protein. (F) Subcellular localization of the GFP fusion MLPK in tobacco BY-2 cells. Control GFP (left), MLPK-GFP (middle), and G2A-GFP (right) constructs were transiently expressed after polyethylene glycol-mediated transformation. Bar, 10 μm.

Comparison of the MLPK genomic sequences between the MM and mm strains revealed the presence of a single nucleotide change in the ORF of the MLPK sequence in the mm strain, causing a single amino acid substitution (Gly to Arg) at position 194 (Fig. 1F). Gly194 is a highly conserved amino acid residue in subdomain VIa of the protein kinases (19). To determine whether this single amino acid substitution influences protein kinase activity, we expressed the wild-type and mutant (G194R) forms of MLPK as glutathione S-transferase (GST) fusion proteins in Escherichia coli and examined their autophosphorylation activities in vitro. Wild-type MLPK exhibited autophosphorylation activity (Fig. 2B) and specifically phosphorylated its serine and threonine residues (22), whereas the G194R form of MLPK exhibited no autophosphorylation activity (Fig. 2B). Furthermore, whereas MLPK mRNA was detected in stigmas of both wild-type and mm plants by Northern blot analysis (Fig. 2C), the G194R form of the MLPK protein was not detected in mm plants by Western blot analysis (Fig. 2D) with antibody raised against recombinant MLPK protein (16) (fig. S1). The absence of detectable MLPK protein in mm plants could be because of the instability of the G194R form of the protein, as observed in some proteins with single point mutations (23, 24). Both the loss of kinase activity and the loss of protein expression suggested that the G194R form of MLPK is dysfunctional in mm plants.

Because N-myristoylated proteins are often translocated to the membrane, we examined subcellular localization of MLPK. Western blot analysis revealed that MLPK is present in the microsomal fraction but not in the cytosolic fraction (Fig. 2E). Subsequent aqueous two-phase partitioning showed enrichment of MLPK in the plasma membrane fraction (Fig. 2E). To confirm plasma membrane localization of MLPK, we transiently expressed a green fluorescent protein (GFP) fusion of MLPK in protoplasts prepared from tobacco BY-2 cells. Cells expressing the MLPK-GFP fusion showed GFP signal predominantly at the plasma membrane, whereas cells expressing free GFP (control) exhibited signal around the nucleus and in the cytosol (Fig. 2F), suggesting plasma membrane localization of MLPK. Furthermore, when the putative N-myristoylation site of Gly at position 2 was replaced by Ala, the mutant form (G2A) displayed the same cytoplasmic localization as the control (Fig. 2F), suggesting involvement of N-myristoylation in the plasma membrane localization of MLPK.

To investigate whether the MLPK gene complements the m mutation, we developed a single papilla cell transient assay system. In this assay, S locus glycoprotein (SLG) promoterdriven MLPK and red fluorescent protein (RFP) marker genes were delivered together into papilla cells of S8S8mm or S9S9mm plants by particle bombardment. After 18 hours, RFP-expressing cells were selected under a fluorescent microscope and self- or cross-pollinated with the use of a micromanipulator. Cells were then observed for pollen germination and pollen tube growth. In control experiments in which only the RFP gene was introduced into S8S8mm plants, 58% of self-pollen grains germinated and penetrated into papilla cells (Table 1 and Fig. 3). When RFP and MLPK genes were co-introduced, there was a significant reduction in the number of penetrated self-pollen tubes, to 31% (Table 1 and Fig. 3). This reduction was specific for self-pollen but not for cross-pollen (Table 1 and Fig. 3). S9S9mm plants also rejected self-pollen because of MLPK introduction (Table 1). Restoration of rejection of self-pollen indicates the restoration of SI in mm papilla cells, suggesting that MLPK is sufficient to complement the m mutation.

Fig. 3.

Functional complementation of the m mutation by MLPK gene. Typical pollination phenotype of transiently transformed papilla cells (S8S8mm, 2 hours after pollination). (Left) Light microscopy. Arrows indicate penetrated pollen tubes. (Right) Fluorescent microscopy under RFP filter. Bar, 50 μm.

Table 1.

Effect of MLPK gene expression in mm papilla cells.

Stigma Transgene Pollen Number of penetrated pollen tubes/tests (%)
S 8 S 8 mm RFP Self 28/48 (58)
Cross 32/49 (65)
RFP + MLPK Self 14/45 (31)View inline
Cross 32/48 (67)
S 9 S 9 mm RFP Self 32/50 (65)
Cross 31/48 (65)
RFP + MLPK Self 1/39 (3)View inline
Cross 27/47 (57)
  • View inline* Self-pollinated co-introduced RFP and MLPK (experimental) compared with self-pollinated with only RFP introduced (control): S8S8mm, χ2 = 6.95, P < 0.01; S9S9mm, χ2 = 35.4, P < 0.001.

  • Several combinations of receptor kinases and their corresponding ligands have been characterized in plants (25, 26), but little is known about their downstream effectors. In Arabidopsis, 610 receptor kinases have been identified; 24% of them are RLCKs. Our results suggest that MLPK, a member of the RLCK family, is a mediator of SRK signaling. The plasma membrane localization of MLPK suggests that it may function in the vicinity of SRK. In animal cells, members of the membrane-anchored Src family of protein tyrosine kinases interact with activated receptor tyrosine kinases and mutually stimulate each other's catalytic activity, thereby strengthening and prolonging the signal (27). MLPK, acting within the SRK receptor complex, is an attractive model for the future study of SI signaling.

    Supporting Online Material

    www.sciencemag.org/cgi/content/full/303/5663/1516/DC1

    Materials and Methods

    Fig. S1

    References

    References and Notes

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