S-locus Receptor Kinase Research Articles

A pollen selection system links self and interspecific incompatibility in the Brassicaceae.

Self-incompatibility and recurrent transitions to self-compatibility have shaped the extant mating systems underlying the nonrandom mating critical for speciation in angiosperms. Linkage between self-incompatibility and speciation is illustrated by the shared pollen rejection pathway between self-incompatibility and interspecific unilateral incompatibility (UI) in the Brassicaceae. However, the pollen discrimination system that activates this shared pathway for heterospecific pollen rejection remains unknown. Here we show that Stigma UI3.1, the genetically identified stigma determinant of UI in Arabidopsis lyrata × Arabidopsis arenosa crosses, encodes the S-locus-related glycoprotein 1 (SLR1). Heterologous expression of A. lyrata or Capsella grandiflora SLR1 confers on some Arabidopsis thaliana accessions the ability to discriminate against heterospecific pollen. Acquisition of this ability also requires a functional S-locus receptor kinase (SRK), whose ligand-induced dimerization activates the self-pollen rejection pathway in the stigma. SLR1 interacts with SRK and interferes with SRK homomer formation. We propose a pollen discrimination system based on competition between basal or ligand-induced SLR1-SRK and SRK-SRK complex formation. The resulting SRK homomer levels would be sensed by the common pollen rejection pathway, allowing discrimination among conspecific self- and cross-pollen as well as heterospecific pollen. Our results establish a mechanistic link at the pollen recognition phase between self-incompatibility and interspecific incompatibility.

Genetic Components of Self-Incompatibility in Brassica Vegetables

Brassica vegetables are very important to human beings. Self-incompatibility (SI) is a common phenomenon in Brassica. Breeding by SI lines is an important way to utilize heterosis of Brassica vegetables. It is believed that the SI inheritance in Brassica species is controlled by three linkage genes on the S-locus, including SRK (S-locus receptor kinase), SCR (S-locus cystine-rich protein)/SP11 (S-locus protein 11), and SLG (S-locus glycoprotein). SRK is the female determinant and SCR/SP11 is the pollen S gene. The expression of SLG is necessary for SRK, and it enhances the SRK-mediated SI reaction. In addition to these three S-locus genes, some other functional molecules also have significant regulatory effects on SI, such as ARC1 (arm repeat containing 1), MLPK (M-locus protein kinase), Exo70A1 (exocyst compounds), THLl/THL2 (thioredoxin H-like), MOD (aquaporin), SLR (S-locus-related glycoprotein), BPCI (pollen calcium-binding protein I), etc. SI is also associated with the dominant/recessive relationship between S alleles. Here, the genetic elements and molecular mechanisms of SI, mainly in Brassica vegetables, are reviewed.

Stigma receptors control intraspecies and interspecies barriers in Brassicaceae

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4–6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7–9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12–14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

S haplotype collection in Brassicaceae crops-an updated list of S haplotypes.

Self-incompatibility is the system that inhibits pollen germination and pollen tube growth by self-pollen. This trait is important for the breeding of Brassica and Raphanus species. In these species, self-incompatibility is governed by the S locus, which contains three linked genes (a set called the S haplotype), i.e., S-locus receptor kinase, S-locus cysteine-rich protein/S-locus protein 11, and S-locus glycoprotein. A large number of S haplotypes have been identified in Brassica oleracea, B. rapa, and Raphanus sativus to date, and the nucleotide sequences of their many alleles have also been registered. In this state, it is important to avoid confusion between S haplotypes, i.e., an identical S haplotype with different names and a different S haplotype with an identical S haplotype number. To mitigate this issue, we herein constructed a list of S haplotypes that are easily accessible to the latest nucleotide sequences of S-haplotype genes, together with revisions to and an update of S haplotype information. Furthermore, the histories of the S-haplotype collection in the three species are reviewed, the importance of the collection of S haplotypes as a genetic resource is discussed, and the management of information on S haplotypes is proposed.

Classification and Identification of S Haplotypes in Radish Based on SRK Kinase Domain Sequence Analysis

Radish is a typical self-incompatible crop. The rapid and accurate identification of S haplotypes can circumvent the blindness of the hybrid combination process, which is critical in radish heterosis utilization and the breeding of new varieties. In this study, based on the gene sequence which encodes the S-locus receptor kinase (SRK) of radish, and the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, the S haplotypes were identified among 79 cultivated radish genotypes. The PCR results indicated that 79 radish genotypes could be divided into 48 Class I, 13 Class II, and 17 Class I/II S haplotypes. Sequence alignment confirmed that the Class I materials contained 19 S haplotypes, of which three haplotypes (‘NAU-S53’, ‘NAU-S54’ and ‘NAU-S55’) were identified for the first time in radish. After digestion using the Hinf I restriction endonuclease, the SRK domain of DNA fragments of different genotypes showed high polymorphism. Homozygous materials S haplotypes could be quickly distinguished by the differences in the digested bands. Molecular identification of the S haplotype was highly consistent with the field pollination and pollen tube germination results. These results would provide an important approach for the rapid identification of radish S haplotypes and the efficient utilization of self-incompatibility in heterosis breeding.

FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in Brassica rapa

Most plants in the Brassicaceae evolve self-incompatibility (SI) to avoid inbreeding and generate hybrid vigor. Self-pollen is recognized by the S-haplotype-specific interaction of the pollen ligand S-locus protein 11 (SP11) (also known as S-locus cysteine-rich protein [SCR]) and its stigma-specific S-locus receptor kinase (SRK). However, mechanistically much remains unknown about the signaling events that culminate in self-pollen rejection. Here, we show that self-pollen triggers high levels of reactive oxygen species (ROS) in stigma papilla cells to mediate SI in heading Chinese cabbage (Brassica rapa L. ssp. pekinensis). We found that stigmatic ROS increased after self-pollination but decreased after compatible(CP)- pollination. Reducing stigmatic ROS by scavengers or suppressing the expression of respiratory burst oxidase homologs (Rbohs), which encode plant NADPH oxidases that produce ROS, both broke down SI. On the other hand, increasing the level of ROS inhibited the germination and penetration of compatible pollen on the stigma, mimicking an incompatible response. Furthermore, suppressing a B.rapa FERONIA (FER) receptor kinase homolog or Rac/Rop guanosine triphosphatase (GTPase) signaling effectively reduced stigmatic ROS and interfered with SI. Our results suggest that FER-Rac/Rop signaling-regulated, NADPH oxidase-produced ROS is an essential SI response leading to self-pollen rejection.

Investigation and screening of chrysanthemum resources to identify self-compatible mutants

Chrysanthemum (Chrysanthemum × morifolium) is an important horticultural crop with great ornamental and economic value. Most chrysanthemum cultivars are self-incompatible, which limits the creation of pure chrysanthemum lines which are the material basis for breeding and theoretical research. Here, artificial self-pollination and natural cross-pollination were used to analyze the self-compatibility (SC) and cross-compatibility (CC) of 85 chrysanthemums cultivated by our research group. By conducting a phenotypic study, observing of pollen germination in vitro and in vivo and gene expression analyses, we explored the reasons for the self-compatibility of chrysanthemum and identified self-compatible mutants. It was found that there were wide variations in self-compatibility among different chrysanthemum cultivars and the SC index ranged from 0 to 5.64, which could classify the chrysanthemum cultivars into three types: self-compatibility (SC index ≥ 1), self-incompatibility (SI, 1 > SC index > 0) and high self-incompatibility (SC index = 0). Seven chrysanthemum cultivars with self-pollinated progenies (‘Hongguan’, K183−8, A22, B12, D87, D76 and S8) were selected, and the pollen germination rates in vitro and in vivo were not significantly related to SC. The pollen tube of ‘Hongguan’ could pass through its own stigma and grow toward the embryo sac along the style, and there was no obvious inbreeding depression in self-pollinated progenies of ‘Hongguan’. In addition, we found that one S-locus receptor kinase (SRK), two S-locus cysteine-rich protein (SCR), two exocyst complex component EXo70A1 (Exo70A1) and one thioredoxin h-like 1 (THL1) were important for regulating self-compatibility of C. × morifolium. Our results not only laid a material foundation for creating pure lines through continuous self-pollination and analyzing the regulation mechanism underlying SC variation but also provided the possibility for theoretical research on the important ornamental traits of chrysanthemums in the future.

Molecular cloning and expression analysis of hybrid hazelnut (Corylus heterophylla×Corylus avellana) ChaSRK1/2 genes and their homologs from other cultivars and species.

The self-incompatibility system of Corylus is a sporophytic type that is phenotypically similar to that of Brassica. While the molecular mechanism of sporophytic self-incompatibility (SSI) has been investigated extensively in Brassica (Brassicaceae), little is known about the corresponding mechanism in Corylus (Betulaceae). Here, we discuss the SSI mechanism with respect to S-locus receptor kinase (SRK) gene homologs. To obtain two SRK candidate unigenes, we compared all of the unigenes in a transcriptional protein database from our previous study with BnSRK-1 (AB270767) using BLASTX with a cutoff e-value of 10-5. We then cloned the full-length cDNA of ChaSRK1 and ChaSRK2 genes from Ping'ou hybrid hazelnut (Corylus heterophylla×Corylus avellana) using RACE techniques. Bioinformatics approaches were used to analyze the cDNA sequences, protein sequences, and domains of the encoded proteins. The full-length ChaSRK1 cDNA was 2883 base pairs (bp) with a coding sequence (CDS) of 2,547bp encoding 849 amino acid residues. The full-length ChaSRK2 cDNA was 2,693bp, with a CDS of 2,433bp encoding 811 amino acids. The ChaSRK1/2 proteins contained an S-domain (extracellular domain), a transmembrane domain, a Ser/Thr protein kinase active site (kinase domain), and DUF3660 and/or DUF3403 domains. The lengths of 18 partial SRK homologs ranged from 1347 to 1451bp, and they contained the same structural domains as ChaSRK1 and ChaSRK2. Phylogenetic analysis revealed that all SRK homologs could be divided into two categories that were similar to the classification of SRKs in Brassica. The expression patterns of ChaSRK1 and ChaSRK2 differed: ChaSRK2 was predominantly expressed in mature stigmatic styles, while ChaSRK1 was expressed in other tissues with the highest in the root tips of Corylus. Using dual-color fluorescence in situ hybridization, ChaSRK1/2 expression was found to be localized in papillar cells. Collectively, these results revealed that SRKs from Corylus had similar characteristics to SRKs from Brassica. We therefore speculated that the SSI mechanism in Corylus might be more similar to the Brassica mechanism than to other SSI types.

A Molecular Evolutionary Framework of Self-Incompatibility in the Angiosperms

SUMMARYThe angiosperms are the largest vascular plant group. However, the mechanisms for their rapid expansions in the Cretaceous and Paleogene remain unclear. Here, we find that self-incompatibility (SI) appears to be one central component contributing to their expansions. SI is a genetic barrier to prevent inbreeding widely found in the angiosperms and controlled by the polymorphic S -locus. So far, five molecular types of S -loci have been identified with the first defined by Lolium perenne domain of unknown function 247 ( LpS-DUF247 ), the second S-RNase and S-locus F-box ( SLF s), the third S-locus cysteine-rich protein ( SCR ) and S-locus receptor kinase ( SRK ), the fourth Papaver rhoeas stigma S ( PrsS ) and Papaver rhoeas pollen S ( PrpS ) and the fifth a hemizygous S -locus supergene of Primula . Phylogenomics and functional analyses showed that SI evolved twice in the Cretaceous and around the Cretaceous-Paleogene boundary, respectively. The first evolution involved the origins of the S genes of types 1 and 2 in the ancestors of Poales and eudicots, respectively. Then, the S -locus of type 2 underwent divergent evolution with losses in several ancestral eudicot lineages. The second involved the S -loci of types 3, 4 and 5 in the ancestors of Brassicaceae, Paperveraceae and Primulaceae, respectively. Interestingly, the times of the two evolutions of SI systems coincided with early angiosperm expansions. Furthermore, we find that neofunctionalization and de novo gene formation contributed to the S gene origins. Taken together, our results reveal a molecular evolutionary framework of SI in the angiosperms and its promoting role in their expansions.

Sorting nexins, a component of retromer complex, interact with SRK in Brassica oleracea

Flowering plants have evolved self-incompatibility (SI) system to reject self-pollination and promote outcrossing. To further excavate SI molecular factors in Brassica oleracea, we screened pistil protein profiles. One protein spot was identified as SNX2b (sorting nexin protein 2b), which was down-regulated when expressed in cross-pollinated pistil. After cloning and sequencing SNX2b and its homologous SNX1 (sorting nexin protein 1) and SNX2a (sorting nexin protein 2a) in B. oleracea, we found that all of them contain a PX domain in N-terminal. Compared to BoSNXs (sorting nexin proteins) expression levels in reproductive organs, we found that, BoSNX1 and BoSNX2a were highly expressed in anther; however, BoSNX2b was highly expressed in pistil. qRT-PCR assays revealed that BoSNX1 and BoSNX2a showed similar expression pattern and BoSNX2b showed different expression pattern in both self- and cross-pollination process. In yeast two-hybrid assays, BoSNX1, BoSNX2a, and BoSNX2b interacted with BoSRKj (S-locus receptor kinase protein, kinase domain). Taken together, our results demonstrated that BoSNX1, BoSNX2a and BoSNX2b were differentially expressed in flower organs and in the early pollination process. On the other hand, BoSNX1, BoSNX2a, and BoSNX2b interacted with BoSRKj. Taking together, it can be suggested that BoSNX1 and BoSNX2a share similar functions but BoSNX2b plays a different role in SI response.

Reverse genetics analysis of the 55-kDa B regulatory subunit of 2A serine/threonine protein phosphatase (PP2A) related to self-incompatibility in Chinese cabbage

The type 2A serine/threonine protein phosphatases (PP2As) are key components in regulating signal transduction and controlling cell metabolism. In particular, PP2A has a role in pollen-pistil interaction during pollination in Brassica. In this study, reverse genetics screening was used to obtain a T-DNA-inserted Brassica rapa mutant line with the self-compatible phenotype. The type 2A PP2A 55-kDa B regulatory subunit gene (PR55/B) in this mutant was knocked out by T-DNA insertion, and its expression level was decreased, which led to self-compatible phenotypes, such as seed setting by flower self-pollination. For functional analysis of this self-compatible mutant line, the full-length PR55/B gene related to the self-incompatibility mechanism was isolated and the down-regulation vector (pPPi) was constructed for introduction into Chinese cabbage. Chinese cabbage lines transformed with pPPi showed significantly decreased PR55/B mRNA accumulation, which yielded self-compatible phenotypes. These transgenic lines were found to set pods and seeds by the self-pollination in both buds and flowers. Down-regulation of PP2A, which is related to self-incompatibility signal transduction, led to higher levels of the phosphorylated S-locus receptor kinase (SRK) substrate, which phosphorylated SRK blocks activation of armadillo-repeat-containing 1 (ARC1). Thus, the self-incompatibility response by ARC1 was not activated, and this led to the self-compatible response. Finally, PP2A is proposed to be a significant factor in self-incompatibility of Chinese cabbage.

Genetic and tissue-specific RNA-sequencing analysis of self-compatible mutant TSC28 in Brassica rapa L. toward identification of a novel self-incompatibility factor.

Self-incompatibility (SI) is a sophisticated system for pollen selectivity to prevent pollination by genetically identical pollen. In Brassica, it is genetically controlled by a single, highly polymorphic S-locus, and the male and female S-determinant factors have been identified as S-locus protein 11 (SP11)/S-locus cysteine-rich protein (SCR) and S-locus receptor kinase (SRK), respectively. However, the overall molecular system and identity of factors in the downstream cascade of the SI reaction remain unclear. Previously, we identified a self-compatible B. rapa mutant line, TSC28, which has a disruption in an unidentified novel factor of the SI signaling cascade. Here, in a genetic analysis of TSC28, using an F2 population from a cross with the reference B. rapa SI line Chiifu-401, the causal gene was mapped to a genetic region of DNA containing markers BrSA64 and ACMP297 in B. rapa chromosome A1. By fine mapping using an F2 population of 1,034 plants, it was narrowed down to a genetic region between DNA markers ACMP297 and BrgMS4028, with physical length approximately 1.01 Mbp. In this genomic region, 113 genes are known to be located and, among these, we identified 55 genes that were expressed in the papilla cells. These are candidates for the gene responsible for the disruption of SI in TSC28. This list of candidate genes will contribute to the discovery of a novel downstream factor in the SP11-SRK signaling cascade in the Brassica SI system.

Functional Analysis of M-Locus Protein Kinase Revealed a Novel Regulatory Mechanism of Self-Incompatibility in Brassica napus L.

Self-incompatibility (SI) is a widespread mechanism in angiosperms that prevents inbreeding by rejecting self-pollen. However, the regulation of the SI response in Brassica napus is not well understood. Here, we report that the M-locus protein kinase (MLPK) BnaMLPKs, the functional homolog of BrMLPKs in Brassica rapa, controls SI in B. napus. We identified four paralogue MLPK genes in B. napus, including BnaA3.MLPK, BnaC3.MLPK, BnaA4.MLPK, and BnaC4.MLPK. Two transcripts of BnaA3.MLPK, BnaA3.MLPKf1 and BnaA3.MLPKf2, were generated by alternative splicing. Tissue expression pattern analysis demonstrated that BnaA3.MLPK, especially BnaA3.MLPKf2, is highly expressed in reproductive organs, particularly in stigmas. We subsequently created RNA-silencing lines and CRISPR/Cas9-induced quadruple mutants of BnaMLPKs in B. napus SI line S-70. Phenotypic analysis revealed that SI response is partially suppressed in RNA-silencing lines and is completely blocked in quadruple mutants. These results indicate the importance of BnaMLPKs in regulating the SI response of B. napus. We found that the expression of SI positive regulators S-locus receptor kinase (SRK) and Arm-Repeat Containing 1 (ARC1) are suppressed in bnmlpk mutant, whereas the self-compatibility (SC) element Glyoxalase I (GLO1) maintained a high expression level. Overall, our findings reveal a new regulatory mechanism of MLPK in the SI of B. napus.

Identification and characterization of BoPUB3: a novel interaction protein with S-locus receptor kinase in Brassica oleracea L.

Armadillo repeat containing 1 (ARC1) is phosphorylated by S-locus receptor kinase (SRK) and functions as a positive regulator in self-incompatibility response of Brassica. However, ARC1 only causes partial breakdown of the self-incompatibility response, and other SRK downstream factors may also participate in the self-incompatibility signaling pathway. In the present study, to search for SRK downstream targets, a plant U-box protein 3 (BoPUB3) was identified from the stigma of Brassica oleracea L. BoPUB3 was highly expressed in the stigma, and its expression was increased with the stigma development and reached to the highest level in the mature-stage stigma. BoPUB3, a 76.8-kDa protein with 697 amino acids, is a member of the PUB-ARM family and contains three domain characteristics of BoARC1, including a U-box N-terminal domain, a U-box motif, and a C-terminal arm repeat domain. The phylogenic tree showed that BoPUB3 was close to BoARC1. The synteny analysis revealed that B. oleracea chromosomal region containing BoPUB3 had high synteny with the Arabidopsis thaliana chromosomal region containing AtPUB3 (At3G54790). In addition, the subcellular localization analysis showed that BoPUB3 primarily localized in the plasma membrane and also in the cytoplasm. The combination of the yeast two-hybrid and in vitro binding assay showed that both BoPUB3 and BoARC1 could interact with SRK kinase domain, and SRK showed much higher level of β-galactosidase activity in its interaction with BoPUB3 than with BoARC1. These results implied that BoPUB3 is a novel interactor with SRK, which lays a basis for further research on whether PUB3 participates in the self-incompatibility signaling pathway.

A Flower-Specific Phospholipase D Is a Stigmatic Compatibility Factor Targeted by the Self-Incompatibility Response in Brassica napus

Self-incompatibility (SI) is a genetic mechanism in hermaphroditic flowers that prevents inbreeding byrejection of self-pollen, while allowing cross- or genetically diverse pollen to germinate on the stigma to successfully fertilize the ovules. In Brassica, SI is initiated by the allele-specific recognition of pollen-encoded, secreted ligand (SCR/SP11) by the stigmatic receptor kinase S-locus receptor kinase (SRK), resulting in activation of SRK through phosphorylation [1-3]. Once activated, this phospho-relay converges on intracellular compatibility factors, which are immediately targeted for degradation by the E3 ligase, ARC1, resulting in the pollen rejection response [4, 5]. Through proteomics approach using proteins from SI activated stigmas of Brassica napus, we identified phospholipase D α1 (PLDα1) as one of the candidates that is most likely targeted for degradation after SI [6]. PLDα1 is enriched in the stigmas and functions as a stigmatic compatibility factor as loss of PLDα1 compromised compatible pollination, while overexpression of PLDα1 in self-incompatible stigmas led to breakdown of SI response. PLDα1 can be ubiquitinated by ARC1 and accumulate in ARC1-suppressed lines, confirming PLDα1 as a target of ARC1 during SI response. Addition of phosphatidic acid (PA) to PLDα1-deficient stigmas was sufficient to rescue compatibility, suggesting an essential role for PA generated by PLDα1 for compatible interactions. We propose that PA produced by PLDα1 activity during compatible pollination promotes vesicle fusion at the membrane to facilitate exocytosis necessary for pollen germination to occur, while SI response could abrogate this process by targeting PLDα1 for degradation.

Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis.

Internalization of plasma membrane (PM)-localized ligand-activated receptor kinases and their trafficking to sorting endosomes have traditionally been viewed as functioning primarily in the down-regulation of receptor signaling, but are now considered to be also essential for signaling by some receptors. A major mechanism for internalization of PM proteins is clathrin-mediated endocytosis (CME). CME is mediated by the Adaptor Protein Complex 2 (AP2), which is involved in interaction of the AP2 μ-adaptin subunit with a tyrosine-based Yxxϕ motif located in the cytoplasmic domain of the cargo protein. In this study, we investigated the role of AP2-mediated CME for signaling by the S-locus receptor kinase (SRK), a protein localized in the PM of stigma epidermal cells, which, together with its pollen coat-localized S-locus cysteine-rich (SCR) ligand, functions in the self-incompatibility (SI) response of the Brassicaceae. Using Arabidopsis thaliana plants that were made self-incompatible by transformation with an A. lyrata-derived SRK/SCR gene pair, we tested the effect on SI of site-directed mutations in each of the two Yxxϕ motifs in SRK and of a CRISPR/Cas9-induced null mutation in the AP2 μ-adaptin gene AP2M. Both in vitro SRK kinase activity and the in planta SI response were abolished by substitution of tyrosine in one of the two Yxxϕ motifs, but were unaffected by elimination of either the second Yxxϕ motif or AP2M function. Thus, AP2-mediated CME is considered to be unnecessary for SRK signaling in the SI response.

Sequence diversification and phylogenetic analysis of selfincompatibility specific determinant genes in Brassicaceae

It is a long history of theoretical interest in understanding the genetic causes and consequences of shifts between outcrossing and inbreeding in plant mating systems. Selfincompatibility (SI) systems genetically promote outcrossing, and its transitions to inbreeding potentially changes genetic diversity in the evolution of species. SI in the Brassicaceae family is sporophytically controlled by a single locus (S-locus), which locates the female determinant gene SRK (S-locus receptor kinase) and the male determinant gene SCR (S-locus cysteine rich). Operation of diversifying selection maintains the diversity of SRK and SCR during the evolutionary course of S haplotypes. Here we used deduced amino acid sequences of SRK and/or SCR publicly available from seven species in three genera, Brassica, Arabidopsis and Raphanus, to investigate the sequence diversification and phylogenetic distribution of S haplotypes in the Brassicaceae family. The results reveal that SCR shows higher sequence distance than SRK within or between genera, and also in the interspecific pairs of S haplotypes. Phylogenetic analysis supports the conclusion that SI has a single origin but evolves differently within the Brassicaceae. The genetic mechanisms and consequences of the loss of SI in the Brassicaceae family are discussed.

A Flower-Specific Phospholipase D1 is a Stigmatic Compatibility Factor Targeted by the Self-Incompatibility Response in Brassica Napus

Self-incompatibility (SI) is a genetic mechanism in hermaphroditic flowers that prevents inbreeding by rejection of self-pollen, while allowing cross or genetically diverse pollen to germinate on the stigma to successfully fertilize the ovules. In Brassica, SI is initiated by the allele-specific recognition of pollen-encoded, secreted ligand (SCR/SP11) by the stigmatic receptor kinase S-locus receptor kinase (SRK), resulting in activation of SRK through phosphorylation. Once activated, this phospho-relay converges on intracellular compatibility factors, which are immediately targeted for degradation by the E3 ligase, ARC1, resulting in the pollen rejection response. Through proteomics approach using proteins from SI activated stigmas of canola (Brassica napus), we identified phospholipase D1 (PLD1) as one of the candidates that is likely targeted for degradation following SI. PLD1 is enriched in the stigmas and functions as a stigmatic compatibility factor as loss of PLD1 compromised compatible pollination, while overexpression of PLD1 in self-incompatible stigmas led to breakdown of SI response. PLD1 overexpression was also associated with enhanced membrane activity following SI pollination, mimicking compatible pollination. PLD1 can be ubiquitinated by ARC1 and accumulate in ARC1- suppressed lines confirming PLD1 as a direct target of ARC1 during SI response. Addition of phosphatidic acid (PA) to PLD1 deficient stigmas was sufficient to rescue compatibility suggesting an essential role for PA generated by PLD1 for compatible interactions. We propose that PA produced by PLD1 activity during compatible pollination promotes vesicle fusion at the membrane to facilitate exocytosis necessary for pollen germination to occur, while SI response could abrogate this process by targeting PLD1 for degradation.

Structural basis for specific self-incompatibility response in Brassica.

Self-incompatibility (SI) is a widespread mechanism in flowering plants which prevents self-fertilization and inbreeding. In Brassica, recognition of the highly polymorphic S-locus cysteine-rich protein (SCR; or S-locus protein 11) by the similarly polymorphic S-locus receptor kinase (SRK) dictates the SI specificity. Here, we report the crystal structure of the extracellular domain of SRK9 (eSRK9) in complex with SCR9 from Brassica rapa. SCR9 binding induces eSRK9 homodimerization, forming a 2:2 eSRK:SCR heterotetramer with a shape like the letter "A". Specific recognition of SCR9 is mediated through three hyper-variable (hv) regions of eSRK9. Each SCR9 simultaneously interacts with hvI and one-half of hvII from one eSRK9 monomer and the other half of hvII from the second eSRK9 monomer, playing a major role in mediating SRK9 homodimerization without involving interaction between the two SCR9 molecules. Single mutations of residues critical for the eSRK9-SCR9 interaction disrupt their binding in vitro. Our study rationalizes a body of data on specific recognition of SCR by SRK and provides a structural template for understanding the co-evolution between SRK and SCR.

What causes mating system shifts in plants? Arabidopsis lyrata as a case study

The genetic breakdown of self-incompatibility (SI) and subsequent mating system shifts to inbreeding has intrigued evolutionary geneticists for decades. Most of our knowledge is derived from interspecific comparisons between inbreeding species and their outcrossing relatives, where inferences may be confounded by secondary mutations that arose after the initial loss of SI. Here, we study an intraspecific breakdown of SI and its consequences in North American Arabidopsis lyrata to test whether: (1) particular S-locus haplotypes are associated with the loss of SI and/or the shift to inbreeding; (2) a population bottleneck may have played a role in driving the transition to inbreeding; and (3) the mutation(s) underlying the loss of SI are likely to have occurred at the S-locus. Combining multiple approaches for genotyping, we found that outcrossing populations on average harbour 5 to 9 S-locus receptor kinase (SRK) alleles, but only two, S1 and S19, are shared by most inbreeding populations. Self-compatibility (SC) behaved genetically as a recessive trait, as expected from a loss-of-function mutation. Bulked segregant analysis in SC × SI F2 individuals using deep sequencing confirmed that all SC plants were S1 homozygotes but not all S1 homozygotes were SC. This was also revealed in population surveys, where only a few S1 homozygotes were SC. Together with crossing data, this suggests that there is a recessive factor that causes SC that is physically unlinked to the S-locus. Overall, our results emphasise the value of combining classical genetics with advanced sequencing approaches to resolve long outstanding questions in evolutionary biology.