During sexual reproduction in flowering plants such as Arabidopsis, a tip-growing pollen tube (PT) is guided to the synergid cells of the female gametophyte, where it bursts and releases the two sperm. Here we show that PT reception and powdery mildew (PM) infection, which involves communication between a tip-growing hypha and a plant epidermal cell, share molecular components. NORTIA (NTA), a member of the MLO family originally discovered in the context of PM resistance, and FERONIA (FER), a receptor-like kinase, both control PT reception in synergids. Homozygous fer mutants also display PM resistance, revealing a new function for FER and suggesting that conserved components, such as FER and distinct MLO proteins, are involved in both PT reception and PM infection.

Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components.To cope with the great number of potential pathogens, plants evolved specialized pattern recognition receptors (PRRs) 5 through which they detect pathogen-associated molecular patterns (PAMPs) at the cell surface (1). Within seconds to minutes after PAMP perception manifold intracellular responses occur, including ion fluxes across the plasma membrane (PM), increase of cytosolic Ca 2ϩ levels, production of reactive oxygen species (ROS) and protein phosphorylation. At later time points profound transcriptional changes, stomatal closure as well as local cell wall reinforcement take place (2).The best characterized plant PAMP perception system is the recognition of bacterial flagellin and its elicitor-active epitope, flg22, by the Arabidopsis PRR FLS2 (flagellin sensitive 2; (2)). FLS2 undergoes flg22-induced complex formation with BRl1-associated receptor kinase 1 (BAK1), which precedes and is required for FLS2 endocytosis (2, 3). Indeed, ligand-induced reduction in lateral membrane mobility of FLS2 has been observed in protoplasts (4), which could be explained by either ligand-dependent interactions of FLS2 with e.g. BAK1, the confinement of FLS2 to less mobile membrane compartments, or a combination of both. To ensure adequate perception of PAMPs and tightly regulated downstream signaling, the PM must be spatially highly organized and dynamic. In this context, the recruitment of FLS2 to specialized membrane domains seems crucial to enable ligand-induced endocytosis (5).During the past years, lateral compartmentalization has become a well-recognized topic in plant membrane research (6). The membrane raft hypothesis provides a plausible explanation for the spatial and temporal organization of biological membranes based on t...

In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca 2+ export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca 2+ -ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca 2+ transport systems suggested that ACA13 indeed functions as an autoinhibited Ca 2+ transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca 2+ export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca 2+ to the compatible pollen tube, which promotes successful fertilization.

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.

We present a generally applicable method allowing rapid identification of causal alleles in mutagenized genomes by nextgeneration 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. FORWARD genetic screens are powerful in uncovering novel gene functions in genetic model organisms. While some mutant screens can be quick to perform, the identification of the causative mutation by map-based cloning is extremely labor-intensive. Large F 2 mapping populations of .1000 mutant individuals are required (Lukowitz et al. 2000;Jander et al. 2002) to fine-map a chromosomal region harboring a causative mutation. This number of mutant individuals can be difficult to obtain, especially when working with phenotypic traits that (i) are difficult to score, (ii) are weakly transmitted, or (iii) are in organisms that are hard to propagate. The recent development of next-generation sequencing (NGS) platforms has made sequencing of whole genomes quick and affordable. One application of NGS is to replace map-based cloning by the sequencing of mutagenized genomes to quickly identify causative mutations, a method successfully applied in many model organisms (Sarin et al. Here, we describe a generally applicable method, SNPratio mapping (SRM), which allows the rapid identification of lethal and/or poorly transmitted mutations and secondsite modifiers by NGS. It is based on the distinct segregation ratio of the causative (and linked) single-nucleotide polymorphism(s) (SNPs) from that of unlinked SNPs. SRM allows the mapping of lethal mutations after only two rounds of backcrossing via NGS. After backcrossing twice to the non-mutagenized parent, any unlinked SNP created by ethyl methanesulfonate (EMS) mutagenesis segregates 1:3 in a pool of individuals. By selecting only mutant individuals in the F 1 generation of the second backcross (BC2), the causative SNP is enriched and segregates 1:1 in a pool of mutant BC2 individuals (Figure 1). Thus, calculating the SNP/non-SNP segregation ratio allows the quick identification of the causative mutation. The method is applicable to any model organism and mutagen causing mostly point mutations or small indels. SRM is the method of choice when working with (i) lethal mutations, (ii) hard-to-score phenotypes, (iii) mutations with low transmission, and (iv) second-site modifiers in complex genetic/ transgenic backgrounds. Here, we demonstrate the power of