Parallel evolution of dominant pistil-side self-incompatibility suppressors in Arabidopsis

Selfing is a frequent evolutionary trend in angiosperms, and is a suitable model for studying the recurrent patterns underlying adaptive evolution. Many plants avoid self-fertilization by physiological processes referred to as self-incompatibility (SI). In the Brassicaceae, direct and specific interactions between the male ligand SP11/SCR and the female receptor kinase SRK are required for the SI response. Although Arabidopsis thaliana acquired autogamy through loss of these genes, molecular evolution contributed to the spread of self-compatibility alleles requires further investigation. We show here that in this species, dominant SRK silencing genes have evolved at least twice. Different inverted repeat sequences were found in the relic SRK region of the Col-0 and C24 strains. Both types of inverted repeats suppress the functional SRK sequence in a dominant fashion with different target specificities. It is possible that these dominant suppressors of SI contributed to the rapid fixation of self-compatibility in A. thaliana

Action of Enzymes from Clostridium tertium A on the Group A Antigenic Determinant of Human Erythrocytes

It is well known that Clostridium tertium A produces Adecomposing enzymes. We investigated the changes in ABO-antigenicity of human erythrocytes caused by the culture filtrate from this bacteria. Group A erythrocytes were specifically affected, and a decrease in A-activity and an enhancement of H-activity were observed. No change was observed on group B and O cells. There was a significant difference between the agglutinabilities of group A1 and A2 erythrocytes treated with Cl. tertium culture filtrate. A2 erythrocytes completely lost their agglutinability against undiluted anti-A sera. In contrast, A1 erythrocytes preserved a low Aantigenicity. It was clear that this difference reflected the heterogenous nature of A-antigenicity on group A cell membranes. On the other hand, it was suggested that the A-decomposing enzymes in the culture filtrate also had a heterogenous nature. Our crude enzyme preparation was completely inhibited by D-galactosamine, and weakly by N-acetyl-Dgalactosamine or D-galactose. Further enzyme purification was required to elucidate the differences in the antigenic determinants or the structural basis between A1 and A2 erythrocytes

SNP-Ratio Mapping (SRM): identifying lethal alleles and mutations in complex genetic backgrounds by next-generation sequencing

We present a generally applicable method allowing rapid identification of causal alleles in mutagenized genomes by next-generation sequencing. Currently used approaches rely on recovering homozygotes or extensive backcrossing. In contrast, SNP-ratio mapping allows rapid cloning of lethal and/or poorly transmitted mutations and second-site modifiers, which are often in complex genetic/transgenic backgrounds

TURAN and EVAN mediate pollen tube reception in Arabidopsis Synergids through protein glycosylation

Pollen tube (PT) reception in flowering plants describes the crosstalk between the male and female gametophytes upon PT arrival at the synergid cells of the ovule. It leads to PT growth arrest, rupture, and sperm cell release, and is thus essential to ensure double fertilization. Here, we describe TURAN (TUN) and EVAN (EVN), two novel members of the PT reception pathway that is mediated by the FERONIA (FER) receptor-like kinase (RLK). Like fer, mutations in these two genes lead to PT overgrowth inside the female gametophyte (FG) without PT rupture. Mapping by next-generation sequencing, cytological analysis of reporter genes, and biochemical assays of glycoproteins in RNAi knockdown mutants revealed both genes to be involved in protein N-glycosylation in the endoplasmic reticulum (ER). TUN encodes a uridine diphosphate (UDP)-glycosyltransferase superfamily protein and EVN a dolichol kinase. In addition to their common role during PT reception in the synergids, both genes have distinct functions in the pollen: whereas EVN is essential for pollen development, TUN is required for PT growth and integrity by affecting the stability of the pollen-specific FER homologs ANXUR1 (ANX1) and ANX2. ANX1- and ANX2-YFP reporters are not expressed in tun pollen grains, but ANX1-YFP is degraded via the ER-associated degradation (ERAD) pathway, likely underlying the anx1/2-like premature PT rupture phenotype of tun mutants. Thus, as in animal sperm-egg interactions, protein glycosylation is essential for the interaction between the female and male gametophytes during PT reception to ensure fertilization and successful reproduction

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

<i>tun</i> and <i>evn</i> ovules display pollen tube overgrowth and increased callose accumulation at the filiform apparatus.

<p>(A–C) Aniline Blue staining of callose in PT cell walls 2 d after pollination (DAP). (A) PT reception in a wild-type FG. Arrowhead indicates site of PT growth arrest. (B–C) PT overgrowth in <i>tun-1</i> (B) and <i>evn-1</i> mutant FGs (C). Asterisks indicate PT overgrowth. (D–F) β-glucuronidase (GUS) staining of synergid marker ET2634 2 d after emasculation (DAE) in wild-type (D), <i>tun-1</i> (E), and <i>evn-1</i> mutant FGs (F). Arrow indicates abnormal structure at the FA. (G–I) Chloral hydrate clearings of ovules 2 DAE in wild-type (G), <i>tun-1</i> (H), and <i>evn-1</i> mutants (I). Arrows indicate abnormal structure at the FA. (J–L) Aniline Blue staining of callose in 6 μm sections of wild-type (J), <i>tun-1</i> (K), and <i>evn-1</i> ovules 2 DAE (L). Boxes represent close-ups of indicated regions, whereby mutant close-ups in (K) and (L) were captured with reduced exposure time compared to the wild type (J). Scale bars in A–F and J–L = 20 μm; scale bars in G–I = 10 μm.</p

Actin Dynamics in Papilla Cells of Brassica rapa during Self- and Cross-Pollination1[W]

The self-incompatibility system of the plant species Brassica is controlled by the S-locus, which contains S-RECEPTOR KINASE (SRK) and S-LOCUS PROTEIN11 (SP11). SP11 binding to SRK induces SRK autophosphorylation and initiates a signaling cascade leading to the rejection of self pollen. However, the mechanism controlling hydration and germination arrest during self-pollination is unclear. In this study, we examined the role of actin, a key cytoskeletal component regulating the transport system for hydration and germination in the papilla cell during pollination. Using rhodamine-phalloidin staining, we showed that cross-pollination induced actin polymerization, whereas self-pollination induced actin reorganization and likely depolymerization. By monitoring transiently expressed green fluorescent protein fused to the actin-binding domain of mouse talin, we observed the concentration of actin bundles at the cross-pollen attachment site and actin reorganization and likely depolymerization at the self-pollen attachment site; the results correspond to those obtained by rhodamine-phalloidin staining. We further showed that the coat of self pollen is sufficient to mediate this response. The actin-depolymerizing drug cytochalasin D significantly inhibited pollen hydration and germination during cross-pollination, further emphasizing a role for actin in these processes. Additionally, three-dimensional electron microscopic tomography revealed the close association of the actin cytoskeleton with an apical vacuole network. Self-pollination disrupted the vacuole network, whereas cross-pollination led to vacuolar rearrangements toward the site of pollen attachment. Taken together, our data suggest that self- and cross-pollination differentially affect the dynamics of the actin cytoskeleton, leading to changes in vacuolar structure associated with hydration and germination