Project description:Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia, respectively. Microbotryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae. To assess this hypothesis, an asexual mutant without a gynoecium or stamen was infected with M. lychnidis-dioicae. A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of flower bud development as well as in the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of gynoecium development was not released. M. lychnidis-dioicae would have a function similar to stamen-promoting factor (SPF), since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. An infection experiment also revealed that a deletion on the Y chromosome of the asexual mutant eliminated genes for maturation of tapetal cells because the tapetal cells did not mature in the asexual mutant infected with M. lychnidis-dioicae.
Project description:When Microbotryum lychnidis-dioicae infects a male Silene latifolia, M. lychnidis-dioicae smut spores develop in the pollen sac instead of pollen. In contrast, when M. lychnidis-dioicae infects a female S. latifolia, the female flowers become male-like, promoting stamen formation. However, it is unclear when and how M. lychnidis-dioicae invades the anther. It is important to investigate not only whether hyphae exist when the apical meristem tissue differentiates into flowers and anthers, but also whether hyphae exist when stamen filaments form. We used Grocott's methenamine silver stain and lectin stain, which stain chitin in the fungal cell wall, to search for M. lychnidis-dioicae in flower tissues. A few M. lychnidis-dioicae hyphae were observed intercellularly in the center of the connective of vascular bundles at the early anther developmental stage. Subsequently, large numbers of deeply stained M. lychnidis-dioicae hyphae were observed intercellularly in the cells surrounding the pollen sac, as well as in the center of the pollen sac. Hyphae stained with lectin were observed intercellularly in all of the stamen filaments at flower development stages. Hyphae were observed in the peduncle connecting the flower and stem. It is thought that M. lychnidis-dioicae invaded the anther via the stamen filament over a long period. Additionally, in total, 163 sections of connective were obtained, and the cell structure of each anther was colored and subjected to three-dimensional reconstruction. The M. lychnidis-dioicae hyphae observed in the connective were mainly old hyphae with large vacuoles or dead hyphae (S1 Fig). These hyphae branched out, towards the pollen sac, while growing between the cells. We also observed that the host cells that collapsed near the hyphae had thick cell walls and teliospores. Cell wall collapse and cell degeneration were observed only around hyphae with thick cell walls.
Project description:Sex chromosomes in plants and animals and fungal mating-type chromosomes often show exceptional genome features, with extensive suppression of homologous recombination and cytological differentiation between members of the diploid chromosome pair. Despite strong interest in the genetics of these chromosomes, their large regions of suppressed recombination often are enriched in transposable elements and therefore can be challenging to assemble. Here we show that the latest improvements of the PacBio sequencing yield assembly of the whole genome of the anther-smut fungus, Microbotryum lychnidis-dioicae (the pathogenic fungus causing anther-smut disease of Silene latifolia), into finished chromosomes or chromosome arms, even for the repeat-rich mating-type chromosomes and centromeres. Suppressed recombination of the mating-type chromosomes is revealed to span nearly 90% of their lengths, with extreme levels of rearrangements, transposable element accumulation, and differentiation between the two mating types. We observed no correlation between allelic divergence and physical position in the nonrecombining regions of the mating-type chromosomes. This may result from gene conversion or from rearrangements of ancient evolutionary strata, i.e., successive steps of suppressed recombination. Centromeres were found to be composed mainly of copia-like transposable elements and to possess specific minisatellite repeats identical between the different chromosomes. We also identified subtelomeric motifs. In addition, extensive signs of degeneration were detected in the nonrecombining regions in the form of transposable element accumulation and of hundreds of gene losses on each mating-type chromosome. Furthermore, our study highlights the potential of the latest breakthrough PacBio chemistry to resolve complex genome architectures.
Project description:Microbotryum lychnidis-dioicae is an obligate biotrophic parasite of the wildflower species Silene latifolia. This dikaryotic fungus, commonly known as an anther smut, requires that haploid, yeast-like sporidia of opposite mating types fuse and differentiate into dikaryotic hyphae that penetrate host tissue as part of the fungal life cycle. Mating occurs under conditions of cool temperatures and limited nutrients. Further development requires host cues or chemical mimics, including a variety of lipids, e.g. phytols. To identify global changes in transcription associated with developmental shifts, RNA-Seq was conducted at several in vitro stages of fungal propagation, i.e. haploid cells grown independently on rich and nutrient-limited media, mated cells on nutrient-limited media as well as a time course of such mated cells exposed to phytol. Comparison of haploid cells grown under rich and nutrient-limited conditions identified classes of genes probably associated with general nutrient availability, including components of the RNAi machinery. Some gene enrichment patterns comparing the nutrient-limited and mated transcriptomes suggested gene expression changes associated with the mating programme (e.g. homeodomain binding proteins, secreted proteins, proteins unique to M. lychnidis-dioicae¸ multicopper oxidases and RhoGEFs). Analysis for phytol treatment compared with mated cells alone allowed identification of genes likely to be involved in the dikaryotic switch (e.g. oligopeptide transporters). Gene categories of particular note in all three conditions included those in the major facilitator superfamily, proteins containing PFAM domains of the secretory lipase family as well as proteins predicted to be secreted, many of which have the hallmarks of fungal effectors with potential roles in pathogenicity.
Project description:BACKGROUND:The genus Microbotryum includes plant pathogenic fungi afflicting a wide variety of hosts with anther smut disease. Microbotryum lychnidis-dioicae infects Silene latifolia and replaces host pollen with fungal spores, exhibiting biotrophy and necrosis associated with altering plant development. RESULTS:We determined the haploid genome sequence for M. lychnidis-dioicae and analyzed whole transcriptome data from plant infections and other stages of the fungal lifecycle, revealing the inventory and expression level of genes that facilitate pathogenic growth. Compared to related fungi, an expanded number of major facilitator superfamily transporters and secretory lipases were detected; lipase gene expression was found to be altered by exposure to lipid compounds, which signaled a switch to dikaryotic, pathogenic growth. In addition, while enzymes to digest cellulose, xylan, xyloglucan, and highly substituted forms of pectin were absent, along with depletion of peroxidases and superoxide dismutases that protect the fungus from oxidative stress, the repertoire of glycosyltransferases and of enzymes that could manipulate host development has expanded. A total of 14% of the genome was categorized as repetitive sequences. Transposable elements have accumulated in mating-type chromosomal regions and were also associated across the genome with gene clusters of small secreted proteins, which may mediate host interactions. CONCLUSIONS:The unique absence of enzyme classes for plant cell wall degradation and maintenance of enzymes that break down components of pollen tubes and flowers provides a striking example of biotrophic host adaptation.
Project description:Recombination suppression on sex chromosomes often extends in a stepwise manner, generating evolutionary strata of differentiation between sex chromosomes. Sexual antagonism is a widely accepted explanation for evolutionary strata, postulating that sets of genes beneficial in only one sex are successively linked to the sex-determining locus. The anther-smut fungus Microbotryum lychnidis-dioicae has mating-type chromosomes with evolutionary strata, only some of which link mating-type genes. Male and female roles are non-existent in this fungus, but mating-type antagonistic selection can also generate evolutionary strata, although the life cycle of the fungus suggests it should be restricted to few traits. Here, we tested the hypothesis that mating-type antagonism may have triggered recombination suppression beyond mating-type genes in M. lychnidis-dioicae by searching for footprints of antagonistic selection in evolutionary strata not linking mating-type loci. We found that these evolutionary strata (i) were not enriched in genes upregulated in the haploid phase, where cells are of alternative mating types, (ii) carried no gene differentially expressed between mating types, and (iii) carried no genes displaying footprints of specialization in terms of protein sequences (dN/dS) between mating types after recommended filtering. Without filtering, eleven genes showed signs of positive selection in the strata not linking mating-type genes, which constituted an enrichment compared to autosomes, but their functions were not obviously involved in antagonistic selection. Thus, we found no strong evidence that antagonistic selection has contributed to extending recombination suppression beyond mating-type genes. Alternative hypotheses should therefore be explored to improve our understanding of the sex-related chromosome evolution.