The self-incompatibility phenotype in brassica is altered by the transformation of a mutant S locus receptor kinase

The self-incompatible (SI) Brassica napus line W1, which carries the 910 S allele, was transformed with an inactive copy of the 910 S locus receptor kinase (SRK) gene. Two transformed lines were analyzed based on their heritable ability to set self-seed. The first line was virtually completely self-compatible (SC), and reciprocal pollinations with the original W1 line demonstrated that only the stigma side of the SI phenotype was altered. An analysis of the expression of endogenous SRK-910 demonstrated that the mechanism of transgene action is via gene suppression. Furthermore, the expression of the S locus glycoprotein gene present in the 910 allele (SLG-910), SLG-A10, which is derived from a nonfunctional S allele, and an S locus-related gene were also suppressed. When the transgene was crossed into another SI line carrying the A14 S allele, it was also capable of suppressing the expression of the endogenous genes and of making this line SC. The second transgenic line studied was only partly SC. In this case as well, only the stigma phenotype was affected, although no gene suppression was detected for endogenous SRK-910 or SLG-910. In this line, the expression of the transgene most likely was causing the change in phenotype, and no effect was observed when this transgene was crossed into the other SI line. Therefore, this work reinforces the hypothesis that the SRK gene is required, but only for the stigma side of the SI phenotype, and that a single transgene can alter the SI phenotype of more than one S allele.     

Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase.

To determine potential targets of the S locus receptor kinase (SRK) during the Brassica self-incompatibility response, a yeast two-hybrid library was screened with the SRK-910 protein kinase domain. Two thioredoxin-h-like clones, THL-1 and THL-2, were found to interact specifically with the SRK-910 protein kinase domain and not to interact with the protein kinase domains from the Arabidopsis receptor-like protein kinases (RLK) RLK4 and RLK5. The interaction between THL-1 and the SRK-910 protein kinase domain was confirmed using coimmunoprecipitation experiments with fusion proteins produced in Escherichia coli. THL-1 has thioredoxin activity based on an insulin reduction assay, and THL-1 is weakly phosphorylated by the SRK-910 protein kinase domain. THL-1 and THL-2 are both expressed in a variety of tissues but show some differences in steady state mRNA levels, with THL-2 being preferentially expressed in floral tissues. This indicates a more general biological function for these thioredoxins in addition to a potential role as effector molecules in the self-incompatibility signal cascade.     

Use of the polymerase chain reaction to isolate an S-locus glycoprotein cDNA introgressed from Brassica campestris into B. napus ssp. oleifera.

Summary A self-incompatible canola-quality Brassica napus ssp. oleifera line (W1) was generated by introgressing the S-locus from a self-incompatible B. campestris plant into the Westar cultivar. Using the polymerase chain reaction (PCR) with primers derived from conserved regions in S-locus glycoprotein (SLG) alleles, the central region of the active SLG gene (910) was obtained. The remaining portions of the cDNA for this 910 gene were subsequently cloned using the PCR-rapid amplification of cDNA ends (RACE) procedure. Sequence analysis revealed that the 910 cDNA show a high degree of sequence similarity to SLG alleles associated with Class I self-incompatible lines. The 910 gene was found to be absent in the original self-compatible cv. Westar (B. napus) and segregated with self-incompatibility in a mixed population generated from a cross between self-incompatible W1 and self-compatible Westar. RNA blot analysis indicated that high levels of 910 mRNAs were present in the stigma as buds approached anthesis. Thus, the SLG allele of W1 transferred from B. campestris via backcrosses to a line of cv. Westar has been identified.     

Expression of the S receptor kinase in self-compatible Brassica napus cv. Westar leads to the allele-specific rejection of self-incompatible Brassica napus pollen

Expression of an S receptor kinase (SRK910) transgene in the self-compatible Brassica napus cv. Westar conferred on the transgenic pistil the ability to reject pollen from the self-incompatible Brassica napus W1 line, which carries the S910 allele. In one of the SRK transgenic lines, 1C, virtually no seeds were produced when the transgenic pistils were pollinated with W1 pollen (Mean number of seeds per pod = 1.22). This response was specific to the W1 pollen since pollen from a different self-incompatible Brassica napus line (T2) and self-pollinations were fully compatible. Westar plants expressing an S locus glycoprotein transgene (SLG910) did not show any self-incompatibility response towards W1 pollen. Transgenic Westar plants resulting from crosses between the 1C SRK transgenic line and three SLG910 transgenic lines were also tested for rejection of W1 pollen. The additional expression of the SLG910 transgene in the SRK910 transgenic plants did not cause any significant further reduction in seed production (Mean seeds/pod = 1.04) or have any detectable effects on the number of pollen grains that adhered to the pistil. Thus, while the allele-specific SLG gene was previously reported to have an enhancing effect on the self-incompatibility response, no evidence for such a role was found in this study.     

Molecular mechanism of self-recognition in Brassica self-incompatibility

In most self-incompatible plant species, recognition of self-pollen is controlled by a single locus, termed the S-locus. In Brassica, genetic dissection of the S-locus has revealed the presence of three highly-polymorphic genes: S-receptor kinase (SRK), S-locus protein 11 (SP11) (also known as S-locus cysteine-rich protein; SCR) and S-locus glycoprotein (SLG). SRK encodes a membrane-spanning serine/threonine kinase that determines the S-haplotype specificity of the stigma. SP11 encodes a small cysteine-rich protein that determines the S-haplotype specificity of pollen. SLG encodes a secreted form of stigma protein similar to the extracellular domain of SRK. Recent biochemical studies have revealed that SP11 functions as the sole ligand for its cognate SRK receptor complex. Their interaction induces the autophosphorylation of SRK, which is expected to trigger the signalling cascade that results in the rejection of self-pollen. This so-called ligand-receptor complex interaction and receptor activation occur in an S-haplotype-specific manner, and this specificity is almost certainly the basis for self-pollen recognition.     

A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L., encodes a functional serine/threonine kinase.

To investigate the catalytic properties of the Brassica oleracea S-locus receptor kinase (SRK), we have expressed the domain that is homologous to protein kinases as a fusion protein in Escherichia coli. Following in vivo labeling of cultures with 32P-labeled inorganic phosphate, we observed phosphorylation of the fusion protein on serine and threonine, but not on tyrosine. In contrast, labeling was not observed when lysine-524, a residue conserved among all protein kinases, was mutated to arginine, thus confirming that SRK phosphorylation was the result of intrinsic serine/threonine kinase activity.     

Production and characterization of the Brassica oleracea self-incompatibility locus glycoprotein and receptor kinase in a baculovirus infected insect cell culture system

Self-incompatibility is a phenomenon that involves recognition of self versus non-self pollen, leading to the rejection of self-related pollen and preventing self-fertilization. In this study, we used a baculovirus-infected insect cell culture system to express two Brassica oleracea stigma-specific proteins required for self-incompatibility: the S-locus glycoprotein, a soluble cell wall-localized glycosylated protein, and the S-locus receptor kinase, a receptor-like integral plasma membrane glycoprotein with serine/threonine kinase activity. Insect cells expressing the S-locus receptor kinase were used in conjunction with immunofluorescence and a whole cell enzyme-linked immunosorbant assay to demonstrate that the receptor is targeted to the cell surface and is oriented with its N-terminal S domain towards the outside of the cell. Received: 20 January 1999 / Revision accepted: 13 April 1999     

A gene encoding a protein serine/threonine kinase is required for normal development of M. xanthus, a gram-negative bacterium.

PCR reactions were carried out on the genomic DNA of M. xanthus, a soil bacterium capable of differentiation to form fruiting bodies, using oligonucleotides representing highly conserved regions of eukaryotic protein serine/threonine kinases. A gene (pkn1) thus cloned contains an ORF of 693 amino acid residues whose amino-terminal domain shows significant sequence similarity with the catalytic domain of eukaryotic protein serine/threonine kinases. The pkn1 gene was overexpressed in E. coli, and the gene product has been found to be autophosphorylated at both serine and threonine residues. The expression of pkn1 is developmentally regulated to start immediately before spore formation. When pkn1 is deleted, differentiation starts prematurely, resulting in poor spore production. These results indicate that the protein serine/threonine kinase plays an important role in the onset of proper differentiation.     

Characterization of a eukaryotic-like tyrosine protein kinase expressed by the Shiga toxin-encoding bacteriophage 933W

The Shiga toxin (Stx)-encoding bacteriophage 933W contains an open reading frame, stk, with amino acid sequence similarity to the catalytic domain of eukaryotic serine/threonine (Ser/Thr) protein kinases (PKs). Eukaryotic PKs are related by a common catalytic domain, consisting of invariant and nearly invariant residues necessary for ATP binding and phosphotransfer. We demonstrate that rather than a Ser/Thr kinase, stk encodes a eukaryotic-like tyrosine (Tyr) kinase. An affinity-purified recombinant Stk (rStk) autophosphorylates and catalyzes the phosphorylation of an artificial substrate on Tyr residues and not on Ser or Thr residues. A change of an invariant lysine within the putative catalytic domain abolishes this kinase activity, indicating that Stk uses a phosphotransfer mechanism similar to the mechanism used by eukaryotic PKs. We provide evidence suggesting that stk is cotranscribed with cI from the phage promoter responsible for maintaining CI expression during lysogeny. The stk gene was identified in prophages obtained from independently isolated Stx-producing Escherichia coli clinical isolates, suggesting that selective pressure has maintained the stk gene in these pathogenic bacteria.     

Molecular characterization of the S locus in two self-incompatible Brassica napus lines.

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region, there are two closely linked genes coding for the S locus glycoprotein (SLG) and S locus receptor kinase (SRK). They appear to comprise the pistil component of the self-incompatibility reaction. SLG and SRK are thought to recognize an unknown pollen component on the incompatible pollen, and the gene encoding this pollen component must also be linked to the SLG and SRK genes. To further our understanding of self-incompatibility, the chromosomal region carrying the SLG and SRK genes has been studied. The physical region between the SLG-910 and the SRK-910 genes in the Brassica napus W1 line was cloned, and a search for genes expressed in the anther revealed two additional S locus genes located downstream of the SLG-910 gene. Because these two genes are novel and are conserved at other S alleles, we designated them as SLL1 and SLL2 (for S locus-linked genes 1 and 2, respectively). The SLL1 gene is S locus specific, whereas the SLL2 gene is not only present at the S locus but is also present in other parts of the genomes in both self-incompatible and self-compatible Brassica ssp lines. Expression of the SLL1 gene is only detectable in anthers of self-incompatible plants and is developmentally regulated during anther development, whereas the SLL2 gene is expressed in anthers and stigmas in both self-incompatible and self-compatible plants, with the highest levels of expression occurring in the stigmas. Although SLL1 and SLL2 are linked to the S locus region, it is not clear whether these genes function in self-incompatibility or serve some other cellular roles in pollen-pistil functions.     

A novel transmembrane serine/threonine protein kinase gene from a rifamycin SV-producing amycolatopsis mediterranei U32.

Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoamino acid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.     

Structural and transcriptional comparative analysis of the S locus regions in two self-incompatible Brassica napus lines

Self-incompatibility (SI) in Brassica is controlled by a single locus, termed the S locus. There is evidence that two of the S locus genes, SLG, which encodes a secreted glycoprotein, and SRK, which encodes a putative receptor kinase, are required for SI on the stigma side. The current model postulates that a pollen ligand recognizing the SLG/SRK receptors is encoded in the genomic region defined by the SLG and SRK genes. A fosmid contig of approximately 65 kb spanning the SLG-910 and SRK-910 genes was isolated from the Brassica napus W1 line. A new gene, SLL3, was identified using a novel approach combining cDNA subtraction and direct selection. This gene encodes a putative secreted small peptide and exists as multiple copies in the Brassica genome. Sequencing analysis of the 65-kb contig revealed seven additional genes and a transposon. None of these seven genes exhibited features expected of S genes on the pollen side. An approximately 88-kb contig of the A14 S region also was isolated from the B. napus T2 line and sequenced. Comparison of the two S regions revealed that (1) the gene organization downstream of SLG in both S haplotypes is highly colinear; (2) the distance between SLG-A14 and SRK-A14 genes is much larger than that between SLG-910 and SRK-910, with the intervening region filled with retroelements and haplotype-specific genes; and (3) the gene organization downstream of SRK in the two haplotypes is divergent. These observations lead us to propose that the SLG downstream region might be one border of the S locus and that the accumulation of heteromorphic sequences, such as retroelements as well as haplotype-unique genes, may act as a mechanism to suppress recombination between SLG and SRK.     

Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.     

Expression cloning of an activin receptor, a predicted transmembrane serine kinase.

Activins are involved in the regulation of multiple biological events, ranging from early development to pituitary function. To characterize the cellular mechanisms involved in these processes, cDNAs coding for an activin receptor were cloned from AtT20 mouse corticotropic cells by screening COS cell transfectants for binding of 125I-activin A. The cDNAs code for a protein of 494 amino acids comprising a ligand-binding extracellular domain, a single membrane-spanning domain, and an intracellular kinase domain with predicted serine/threonine specificity. 125I-activin A binds to transfected COS cells with an affinity of 180 pM and can be competed by activin A, activin B, and inhibin A, but not by transforming growth factor beta 1. The kinase domain, but not the extracellular sequence, of the activin receptor is most closely related to the C. elegans daf-1 gene product, a putative transmembrane serine/threonine-specific protein kinase for which the ligand is not known.     

daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase

The dauer larva is a developmentally arrested, non-feeding dispersal stage normally formed in response to overcrowding and limited food. The daf-1 gene specifies an intermediate step in a hierarchy of genes thought to specify a pathway for neural transduction of environmental cues. Mutations in daf-1 result in constitutive formation of dauer larvae even in abundant food. This gene has been cloned by Tc1-transposon tagging, and it appears to encode a new class of serine/threonine kinase. A daf-1 probe detects a 2.5 kb mRNA of low abundance, and the DNA sequence indicates that the gene encodes a 669 amino acid protein, with a putative transmembrane domain and a C-terminal protein kinase domain most closely related to the cytosolic, raf proto-oncogene family. Hence, the daf-1 product appears to be a cell-surface receptor required for transduction of environmental signals into an appropriate developmental response.     

Identification and characterization of a myristylated and palmitylated serine/threonine protein kinase.

We report the molecular cloning and initial characterization of a novel fatty acid acylated serine/threonine protein kinase. The putative open reading frame is predicted to encode a 305 amino acid protein possessing a carboxy-terminal protein kinase domain and amino-terminal myristylation and palmitylation sites. The protein kinase has been accordingly denoted as the myristylated and palmitylated serine/threonine protein kinase (MPSK). Human and mouse MPSKs share approximately 93% identity at the amino acid level with complete retention of acylation sites. Radiation hybridization localized the human MPSK gene to chromosome 2q34-37. Northern analysis demonstrated that the human MPSK 1.7 kilobase mRNA is widely distributed. Epitope tagged human MPSK was found to be acylated by myristic acid at glycine residue 2 and by palmitic acid at cysteines 6 and/or 8. Palmitylation of MPSK in these experiments was found to require an intact myristylation site. While epitope tagged MPSK in immune complexes or purified human glutathione S transferase-MPSK was found to autophosphorylate at one or more threonine residues, the enzyme was not found to phosphorylate several other common exogenous substrates. Indeed, only PHAS-I was identified as an exogenous substrate which was found to be phosphorylated on threonine and serine residues.     

Cloning of a Ca(2+)/calmodulin-dependent protein kinase gene from the filamentous fungus Arthrobotrys dactyloides

A Ca(2+)/calmodulin-dependent protein kinase (CaMK) gene was cloned and characterized from Arthrobotrys dactyloides, a nematode-trapping fungus. The resulting 373-amino-acid protein, FCaMK, has significant homology to mammalian CaMKs. FCaMK contains a serine/threonine kinase domain followed by a calmodulin-binding domain. The activation loop in FCaMK (amino acids 184-199) contains a phosphorylation site at threonine-188, which could be the target of a kinase activator. Truncated FCaMK mutants revealed that amino acids 296-324 are essential for calmodulin binding. An oligopeptide designed from residues 297-324 formed a stable peptide-calmodulin complex of 1:1 stoichiometry. Southern blot analysis detected a single copy of the fcamk gene, suggesting that FCaMK plays an important role in Ca(2+)/calmodulin signaling in A. dactyloides.     

Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21

The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein consisting of leucine-rich repeats in the putative extracellular domain and a serine/threonine kinase in the putative intracellular domain. The putative XA21 kinase domain was expressed as maltose-binding and glutathione S-transferase fusion proteins in Escherichia coli. The fusion proteins are capable of autophosphorylation. Phosphoamino acid analysis of the glutathione S-transferase fusion protein indicates that only serine and threonine residues are phosphorylated. The relative phosphorylation rate of the XA21 kinase against increasing enzyme concentrations follows a first-order rather than second-order kinetics, indicating an intramolecular phosphorylation mechanism. Moreover, the active XA21 kinase cannot phosphorylate a kinase-deficient mutant of XA21 kinase. The enzymatic activity of the XA21 kinase in a buffer containing Mn(2+) is at least 15 times higher than that with Mg(2+). The K(m) and V(max) of XA21 kinase for ATP are 0.3 microm and 8.4 nmol/mg/min, respectively. Tryptic phosphopeptide mapping reveals that multiple sites on the XA21 kinase are phosphorylated. Finally, our data suggest that the region of XA21 kinase corresponding to the RD kinase activation domain is not phosphorylated, revealing a distinct mode of action compared with the tomato Pto serine/threonine kinase conferring disease resistance.     

Expression cloning of the TGF-beta type II receptor, a functional transmembrane serine/threonine kinase.

A cDNA encoding the TGF-beta type II receptor protein has been isolated by an expression cloning strategy. The cloned cDNA, when transfected into COS cells, leads to overexpression of an approximately 80 kd protein that specifically binds radioiodinated TGF-beta 1. Excess TGF-beta 1 competes for binding of radioiodinated TGF-beta 1 in a dose-dependent manner and is more effective than TGF-beta 2. The predicted receptor structure includes a cysteine-rich extracellular domain, a single hydrophobic transmembrane domain, and a predicted cytoplasmic serine/threonine kinase domain. A chimeric protein containing the intracellular domain of the type II receptor and expressed in E. coli can phosphorylate itself on serine and threonine residues in vitro, indicating that the cytoplasmic domain of the type II receptor is a functional kinase. This result implicates serine/threonine phosphorylation as an important mechanism of TGF-beta receptor-mediated signaling.