Characterization of the SP11/SCR high-affinity binding site involved in self/nonself recognition in brassica self-incompatibility

In Brassica self-incompatibility, the recognition of self/nonself pollen grains, is controlled by the S-locus, which encodes three highly polymorphic proteins: S-locus receptor kinase (SRK), S-locus protein 11 (SP11; also designated S-locus Cys-rich protein), and S-locus glycoprotein (SLG). SP11, located in the pollen coat, determines pollen S-haplotype specificity, whereas SRK, located on the plasma membrane of stigmatic papilla cells, determines stigmatic S-haplotype specificity. SLG shares significant sequence similarity with the extracellular domain of SRK and is abundant in the stigmatic cell wall, but its function is controversial. We previously showed that SP11 binds directly to its cognate SRK with high affinity (K(d) = 0.7 nM) and induces its autophosphorylation. We also found that an SLG-like, 60-kD protein on the stigmatic membrane forms a high-affinity binding site for SP11. Here, we show that the 60-kD stigmatic membrane protein is a truncated form of SRK containing the extracellular domain, transmembrane domain, and part of the juxtamembrane domain. A transiently expressed, membrane-anchored form of SRK exhibits high-affinity binding to SP11, whereas the soluble SRK (eSRK) lacking the transmembrane domain exhibits no high-affinity binding, as is the case with SLG. The different binding affinities of the membrane-anchored SRK and soluble eSRK or SLG will be significant for the specific perception of SP11 by SRK.     

植物孢子体自交不亲和信号转导功能分子研究进展

孢子体自交不亲和(SSI)是许多植物采取的一种抵制近亲繁殖的重要措施,受S位点复等位基因控制。近年来,参与其信号转导的许多功能分子及它们的编码基因被分离并得到了充分研究：当自花授粉时,SPlI／SCR与SRK特异识别,造成后的Ser／Thr激酶的磷酸化,引发了一系列由SLG、ARC1及水孔蛋白等因子参与的SSI信号转导途径,最终产生自交不亲和的结果。     

A pollen coat protein, SP11/SCR, determines the pollen S-specificity in the self-incompatibility of Brassica species

Many flowering plants have evolved self-incompatibility (SI) systems to prevent inbreeding. In the Brassicaceae, SI is genetically controlled by a single polymorphic locus, termed the S-locus. Pollen rejection occurs when stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S-locus Cys-rich (SP11/SCR). SRK encodes a polymorphic membrane-spanning protein kinase, which is the sole female determinant of the S-haplotype specificity. SP11/SCR encodes a highly polymorphic Cys-rich small basic protein specifically expressed in the anther tapetum and in pollen. In cauliflower (B. oleracea), the gain-of-function approach has demonstrated that an allele of SP11/SCR encodes the male determinant of S-specificity. Here we examined the function of two alleles of SP11/SCR of B. rapa by the same approach and further established that SP11/SCR is the sole male determinant of SI in the genus Brassica sp. Our results also suggested that the 522-bp 5'-upstream region of the S9-SP11 gene used to drive the transgene contained all the regulatory elements required for the unique sporophytic/gametophytic expression observed for the native SP11 gene. Promoter deletion analyses suggested that the highly conserved 192-bp upstream region was sufficient for driving this unique expression. Furthermore, immunohistochemical analyses revealed that the protein product of the SP11 transgene was present in the tapetum and pollen, and that in pollen of late developmental stages, the SP11 protein was mainly localized in the pollen coat, a finding consistent with its expected biological role.     

Immunohistochemical studies on translocation of pollen S-haplotype determinant in self-incompatibility of Brassica rapa

The self-incompatibility system in Brassica is controlled by the S-locus, which contains S-receptor kinase (SRK) and S-locus protein 11 (SP11). SRK and SP11 control stigma and pollen S-haplotype specificity, respectively. SP11 binding to SRK induces the autophosphorylation of SRK, which triggers the signaling cascade that results in the rejection of self-pollen. The localization of SP11 protein during pollen development and pollination, however, have never been demonstrated. In this study, we examined the localization of S(8)-SP11 protein in the anther or pollinated stigma by immuno-electron microscopy. The immunostaining suggested that S(8)-SP11 was secreted from the tapetal cell into the anther locule as a cluster and translocated to the pollen surface at the early developmental stage of the anther. During the pollination process, SP11 was translocated from the pollen surface to the papilla cell, and then penetrated the cuticle layer of the papilla cell to diffuse across the pectin cellulose layer. Furthermore, SP11 protein could only penetrate the cuticle layer of the papilla cell in the presence of pollen grains, and could not penetrate on its own. This suggests that another factor from the pollen grain is needed for SP11 protein to penetrate the papilla cell wall.     

芸薹属自交不亲和基因的分子生物学及进化模式

芸薹属的自交不亲和性是受单基因座、复等位基因控制的孢子体控制型。自交不亲和基因座位（S-locus）是由多个基因组成的复杂区域,称之为S多基因家族,其大多数成员分布于芸薹属的整个染色体组。目前已鉴定出100多个S等位基因,它们的起源分化始于一千万年前。S-座位上存在的多基因有3种：SRK,SLG和SCR/SP11;SRK和SLG在柱头中表达,SCR/SP11在雄蕊中表达。SRK蛋白在识别同类花粉的过程中起主要作用,而SLG蛋白增强了这种自交不亲和反应。SLG与SRK基因中编码S-结构域的核苷酸序列相似性程度高达85％～98%。基因转换可能是SLG和SRK的高度同源性能够得以保持的原因。SRK,SLG和SCR基因紧密相连,并表现出高水平的序列多样性。SRK与SLG基因间的距离很近,在20～25 kb之间。在柱头和花粉中,自交不亲和等位基因之间的共显性关系要比显性和隐性关系更加普遍,这是芸薹属自交不亲和性的一大特点。自交不亲和基因的进化模式存在两种假说：双基因进化模式和中性变异体进化模式;可能存在几种不同的进化方式,它们共同在自然群体中新的S等位基因进化过程中起作用。     

Molecular mechanisms of self-incompatibility in Brassica

In Brassica species, self-incompatibility has been mapped genetically to a single chromosomal location. In this region several closely linked genes have been identified. One of them, S-locus receptor kinase (SRK), determines S haplotype specificity of the stigma and it's the key protein for SI reaction. The role of the S locus glycoprotein (SLG) gene remains unclear. In the last decade approximately 15 additional genes linked to S-locus have been found. Recently, a gene has been identified (SCR) that encodes a small cysteine-rich protein which is a candidate for the pollen ligand. In addition to S locus linked genes there are unlinked SLRgenes (S-locus related genes). In this review, we discuss the role of these genes and the current view on the self-incompatibility mechanism in Brassica.     

The S15 self-incompatibility haplotype in Brassica oleracea includes three S gene family members expressed in stigmas.

Self-incompatibility in Brassica is controlled by a single, highly polymorphic locus that extends over several hundred kilobases and includes several expressed genes. Two stigma proteins, the S locus receptor kinase (SRK) and the S locus glycoprotein (SLG), are encoded by genes located at the S locus and are thought to be involved in the recognition of self-pollen by the stigma. We report here that two different SLG genes, SLGA and SLGB, are located at the S locus in the class II, pollen-recessive S15 haplotype. Both genes are interrupted by a single intron; however, SLGA encodes both soluble and membrane-anchored forms of SLG, whereas SLGB encodes only soluble SLG proteins. Thus, including SRK, the S locus in the S15 haplotype contains at least three members of the S gene family. The protein products of these three genes have been characterized, and each SLG glycoform was assigned to an SLG gene. Evidence is presented that the S2 and S5 haplotypes carry only one or the other of the SLG genes, indicating either that they are redundant or that they are not required for the self-incompatibility response.     

S cysteine-rich (SCR) binding domain analysis of the Brassica self-incompatibility S-locus receptor kinase

Brassica self-incompatibility, a highly discriminating outbreeding mechanism, has become a paradigm for the study of plant cell-cell communications. When self-pollen lands on a stigma, the male ligand S cysteine-rich (SCR), which is present in the pollen coat, is transmitted to the female receptor, S-locus receptor kinase (SRK). SRK is a membrane-spanning serine/threonine receptor kinase present in the stigmatic papillar cell membrane. Haplotype-specific binding of SCR to SRK brings about pollen rejection. The extracellular receptor domain of SRK (eSRK) is responsible for binding SCR. Based on sequence homology, eSRK can be divided into three subdomains: B lectin-like, hypervariable, and PAN. Biochemical analysis of these subdomains showed that the hypervariable subdomain is responsible for most of the SCR binding capacity of eSRK, whereas the B lectin-like and PAN domains have little, if any, affinity for SCR. Fine mapping of the SCR binding region of SRK using a peptide array revealed a region of the hypervariable subdomain that plays a key role in binding the SCR molecule. We show that residues within the hypervariable subdomain define SRK binding and are likely to be involved in defining haplotype specificity.     

In vivo imaging of the S-locus receptor kinase,the female specificity determinant of self-incompatibility,in transgenic self-incompatible Arabidopsis thaliana

Background and Aims The S-locus receptor kinase (SRK), which is expressed in stigma epidermal cells, is responsible for the recognition and inhibition of ‘self’ pollen in the self-incompatibility (SI) response of the Brassicaceae. The allele-specific interaction of SRK with its cognate pollen coat-localized ligand, the S-locus cysteine-rich (SCR) protein, is thought to trigger a signalling cascade within the stigma epidermal cell that leads to the arrest of ‘self’ pollen at the stigma surface. In addition to the full-length signalling SRK receptor, stigma epidermal cells express two other SRK protein species that lack the kinase domain and whose role in the SI response is not understood: a soluble version of the SRK ectodomain designated eSRK and a membrane-tethered form designated tSRK. The goal of this study was to describe the sub-cellular distribution of the various SRK protein species in stigma epidermal cells as a prelude to visualizing receptor dynamics in response to SCR binding.Methods The Arabidopsis lyrata SRKb variant was tagged with the Citrine variant of yellow fluorescent protein (cYFP) and expressed in A. thaliana plants of the C24 accession, which had been shown to exhibit a robust SI response upon transformation with the SRKb–SCRb gene pair. The transgenes used in this study were designed for differential production and visualization of the three SRK protein species in stigma epidermal cells. Transgenic stigmas were analysed by pollination assays and confocal microscopy.Key Results and Conclusions Pollination assays demonstrated that the cYFP-tagged SRK proteins are functional and that the eSRK is not required for SI. Confocal microscopic analysis of cYFP-tagged SRK proteins in live stigma epidermal cells revealed the differential sub-cellular localization of the three SRK protein species but showed no evidence for redistribution of these proteins subsequent to incompatible pollination.     

Recognition specificity of self-incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.     

SRK-SCR转基因拟南芥自交不亲和性的研究进展

十字花科植物自交不亲和性（SI）受墨位点（S-locus）编码的sRK和sCR控制,它们分别是柱头和花粉中的sI特异识别因子。野生型拟南芥不具有sI,而近来通过转基因手段将外源艘K—scR基因转入野生型拟南芥可以使其表现sI,由此建立了一个可用于十字花科sI研究的新型模式植物。本文综述了利用这种转基因拟南芥在SI机制及进化方面取得的进展,包括sI新基因的挖掘、候选基因功能分析和拟南芥生殖模式的转变等。     

Structure of the male determinant factor for Brassica self-incompatibility

Many flowering plants possess a self-incompatibility system to prevent inbreeding. In Brassica rapa, self/non-self recognition in mating is established through S-haplotype-specific interactions between stigma receptors and S-locus protein 11 (SP11, also called S-locus cysteine-rich protein) that is encoded at the highly polymorphic S-locus. Here we describe the solution structure of the SP11 protein of the S8-haplotype (S8-SP11), which specifically binds to the stigma factor of the same haplotype. It folds into an alpha/beta sandwich structure that resembles those of plant defensins. Residues important for structural integrity are highly conserved among the allelic SP11s, suggesting the existence of a common folding pattern. Structure-based sequence alignment and homology modeling of allelic SP11 identified a hyper-variable (HV) region, which is thought to form a loop that bulges out from the body of the protein that is amenable to solvent exposure. We suggest that the HV region could serve as a specific binding site for the stigma receptor.     

Coevolution of the S-locus genes SRK,SLG and SP11/SCR in Brassica oleracea and B. rapa

Brassica self-incompatibility (SI) is controlled by SLG and SRK expressed in the stigma and by SP11/SCR expressed in the anther. We determined the sequences of the S domains of 36 SRK alleles, 13 SLG alleles, and 14 SP11 alleles from Brassica oleracea and B. rapa. We found three S haplotypes lacking SLG genes in B. rapa, confirming that SLG is not essential for the SI recognition system. Together with reported sequences, the nucleotide diversities per synonymous and nonsynonymous site (pi(S) and pi(N)) at the SRK, SLG, and SP11 loci within B. oleracea were computed. The ratios of pi(N):pi(S) for SP11 and the hypervariable region of SRK were significantly >1, suggesting operation of diversifying selection to maintain the diversity of these regions. In the phylogenetic trees of 12 SP11 sequences and their linked SRK alleles, the tree topology was not significantly different between SP11 and SRK, suggesting a tight linkage of male and female SI determinants during the evolutionary course of these haplotypes. Genetic exchanges between SLG and SRK seem to be frequent; three such recent exchanges were detected. The evolution of S haplotypes and the effect of gene conversion on self-incompatibility are discussed.     

Site-Specific N-Glycosylation of the S-Locus Receptor Kinase and Its Role in the Self-Incompatibility Response of the Brassicaceae

The S-locus receptor kinase SRK is a highly polymorphic transmembrane kinase of the stigma epidermis. Through allele-specific interaction with its pollen coat-localized ligand, the S-locus cysteine-rich protein SCR, SRK is responsible for recognition and inhibition of self pollen in the self-incompatibility response of the Brassicaceae. The SRK extracellular ligand binding domain contains several potential N-glycosylation sites that exhibit varying degrees of conservation among SRK variants. However, the glycosylation status and functional importance of these sites are currently unclear. We investigated this issue in transgenic Arabidopsis thaliana stigmas that express the Arabidopsis lyrata SRKb variant and exhibit an incompatible response toward SCRb-expressing pollen. Analysis of single- and multiple-glycosylation site mutations of SRKb demonstrated that, although five of six potential N-glycosylation sites in SRKb are glycosylated in stigmas, N-glycosylation is not important for SCRb-dependent activation of SRKb. Rather, N-glycosylation functions primarily to ensure the proper and efficient subcellular trafficking of SRK to the plasma membrane. The study provides insight into the function of a receptor that regulates a critical phase of the plant life cycle and represents a valuable addition to the limited information available on the contribution of N-glycosylation to the subcellular trafficking and function of plant receptor kinases.     

Genomic organization of the S locus: Identification and characterization of genes in SLG/SRK region of S(9) haplotype of Brassica campestris (syn. rapa).

In Brassica, two self-incompatibility genes, encoding SLG (S locus glycoprotein) and SRK (S-receptor kinase), are located at the S locus and expressed in the stigma. Recent molecular analysis has revealed that the S locus is highly polymorphic and contains several genes, i.e., SLG, SRK, the as-yet-unidentified pollen S gene(s), and other linked genes. In the present study, we searched for expressed sequences in a 76-kb SLG/SRK region of the S(9) haplotype of Brassica campestris (syn. rapa) and identified 10 genes in addition to the four previously identified (SLG(9), SRK(9), SAE1, and SLL2) in this haplotype. This gene density (1 gene/5.4 kb) suggests that the S locus is embedded in a gene-rich region of the genome. The average G + C content in this region is 32.6%. An En/Spm-type transposon-like element was found downstream of SLG(9). Among the genes we identified that had not previously been found to be linked to the S locus were genes encoding a small cysteine-rich protein, a J-domain protein, and an antisilencing protein (ASF1) homologue. The small cysteine-rich protein was similar to a pollen coat protein, named PCP-A1, which had previously been shown to bind SLG.     

Molecular aspects of self-incompatibility in Brassica species.

Many flowering plants possess self-incompatibility (SI) systems to prevent inbreeding. SI in Brassica species is controlled by a single S locus with multiple alleles. In recent years, much progress has been made in determining the male and female S determinant in Brassica species. In the female, a gain-of-function experiment clearly demonstrated that SRK was the sole S determinant, and that SLG enhanced the SI recognition process. By contrast, the male S determinant (termed SP11/SCR) was identified in the course of genome analysis of S locus to be a small cysteine-rich protein, which was classified as a pollen coat protein. This SP11/SCR may function as a ligand for the S domain of SRK in the SI recognition reaction of Brassica species.     

Characterization of expressed genes in the SLL2 region of self-compatible Arabidopsis thaliana.

Self-incompatibility in Brassica species is regulated by a set of S-locus genes: SLG, SRK, and SP11/SCR. In the vicinity of the S-locus genes, several expressed genes, SLL2 and SP2/ClpP, etc., were identified in B. campestris. Arabidopsis thaliana is a self-compatible Brassica relative, and its complete genome has been sequenced. From comparison of the genomic sequences between B. campestris and A. thaliana, microsynteny between gene clusters of Arabidopsis and Brassica SLL2 regions was observed, though the S-locus genes, SLG, SRK, and SP11/SCR were not found in the region of Arabidopsis. Almost all genes predicted in this region of Arabidopsis were expressed in both vegetative and reproductive organs, suggesting that the genes in the SLL2 region might not be related to self-incompatibility. Considering the recent speculation that the S-locus genes were translocated as a single unit between Arabidopsis and Brassica, the translocation might have occurred in the region between the SLL2 and SP7 genes.